WO2017040801A2 - Methods for making and using an immunoconjugate for the treatment of cancer - Google Patents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
- A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
- A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
- A61K47/6817—Toxins
- A61K47/6819—Plant toxins
- A61K47/6825—Ribosomal inhibitory proteins, i.e. RIP-I or RIP-II, e.g. Pap, gelonin or dianthin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K4/00—Peptides having up to 20 amino acids in an undefined or only partially defined sequence; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/165—Extraction; Separation; Purification by chromatography mixed-mode chromatography
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin
Definitions
- EpCAM for Epithelial Cell Adhesion Molecule, which also known as 17-1A, KSA, EGP-2 and GA733-2.
- EpCAM is a transmembrane protein that is highly expressed in many solid tumors, including carcinomas of the lung, breast, ovary, colorectum, bladder, and squamous cell carcinoma of the head and neck, but weakly expressed in most normal epithelial tissues.
- HNSCC squamous cell carcinoma of the head and neck
- EpCAM expression correlates with the progression of HNSCC in humans.
- HNSCC is presently the sixth most common cancer in the world.
- HNSCC is a disease that causes significant morbidity, especially with respect to speech and swallowing functions. Removal of tumors in subjects suffering from HNSCC can be difficult or impossible due to their size and location. Surgery, radiation therapy, chemotherapy, or combinations of these are generally available as treatment options.
- Salvage therapy consists of the same treatment options as for first line therapy.
- Bladder cancer is the 7th most common cancer worldwide that results in an estimated 260,000 new cases each year. Carcinomas in the bladder tissue are also associated with increased EpCAM expression, and occur almost entirely within the transitional epithelium, the surface layer of tissue that lines the bladder, as transitional cell carcinomas. At initial diagnosis, 70 to 90% of patients with bladder cancers have superficial disease which involves carcinomas in the superficial urothelial layer that are noninvasive and exhibit papillary (finger-like projections) tumors. Current treatment includes the intravesicular delivery of chemotherapy and immunotherapy with the bacille Calmette-Guerin (BCG) vaccine that involves the additional risk of systemic infection with the tuberculosis bacterium. Despite this aggressive treatment regime, 70% of these superficial papillary tumors will recur over a prolonged clinical course, causing significant morbidity; approximately 4 to 8% will progress to invasive carcinomas.
- BCG Bacille Calmette-Guerin
- the present disclosure relates to novel immunoconjugates, and methods for treating or preventing cancer by administering such immunoconjugates to a subject in need thereof.
- the present disclosure also relates to methods for making novel immunoconjugates.
- the immunoconjugates comprise an antibody fragment that binds to the extracellular domain of human EpCAM and a toxin.
- the present disclosure provides an immunoconjugate comprising an antigen binding domain and a toxin.
- the antigen binding domain is an EpCAM antibody or a fragment thereof.
- the immunoconjugate comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 2 or SEQ ID NO: 21.
- the immunoconjugate comprises SEQ ID NO: 2 or SEQ ID NO: 21.
- the immunoconjugate consists essentially of SEQ ID NO: 2 or SEQ ID NO: 21.
- the immunoconjugate consists of SEQ ID NO: 2 or SEQ ID NO: 21.
- the amino acid sequence is encoded by a non codon- optimized nucleic acid sequence.
- the present disclosure provides a nucleic acid molecule enoding the immunoconjugate comprising SEQ ID NO: 2 or SEQ ID NO: 21.
- the immunoconjugate exhibits cytotoxic activity against EpCAM-expressing cells.
- the immunoconjugate comprises (i) an antigen binding domain comprising a light chain CDR1 having at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 5, a light chain CDR2 having at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 6, a light chain CDR3 having at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 7, a heavy chain CDR1 having at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 12, a heavy chain CDR2 having at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 13, and a heavy chain CDR3 having at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 14; and (ii) a toxin.
- the amino acid sequence of the immunoconjugate does not comprise a His tag.
- the amino acid sequence of the immunoconjugate comprises a single His tag.
- the single His tag is located at the N-terminus of the immunoconjugate.
- the antigen binding domain comprises light chain CDR1, CDR2, and CDR3 comprising an amino acid sequence according to SEQ ID NOs: 5, 6, and 7, respectively; and a heavy chain CDR1, CDR2, and CDR3 according to SEQ ID NOs: 12, 13, and 14, respectively.
- the antigen binding domain comprises a light chain variable region having at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 4 and a heavy chain variable region at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 11.
- the antigen binding domain is an scFv.
- the scFv comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 19.
- the toxin of the immunoconjugates provided herein is
- the toxin comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 18.
- the antigen binding domain is conjugated to the toxin via a linker.
- the linker comprises a sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 16.
- the immunoconjugate is encoded by a non-codon optimized nucleic acid sequence.
- the present disclosure provides a nucleic acid molecule comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 1.
- the present disclosure provides a nucleic acid molecule comprising a sequence according to SEQ ID NO: 1.
- the present disclosure provides a protein encoded by a nucleic acid sequence according to SEQ ID NO: 1.
- the present disclosure provides expression vectors and host cells comprising expression vectors.
- the expression vectors provided herein comprise a nucleic acid molecule according to SEQ ID NO: 1.
- compositions and immunoconjugates provided herein exhibit cytotoxic activity against EpCAM-expressing cells.
- the compositions and immunoconjugates provided herein exhibit cytotoxic activity against EpCAM positive cell lines such as, for example, Cal-27 or TCCSUP cells.
- the present disclosure provides methods for treating or preventing cancer comprising administering a composition provided herein.
- the cancer is head and neck cancer or bladder cancer.
- the cancer is head and neck cancer is squamous cell carcinoma of the head and neck (HNSCC).
- the compositions provided herein are administered to a subject in need thereof.
- the subject is a human.
- the composition is administered directly to the cancer site.
- the composition is administered intratumorally, intravesicularly, or peritumorally.
- the composition is administered systemically.
- the systemic administration is intravenous administration.
- the present disclosure provides methods for treating or preventing cancer comprising administering to a subject in need thereof a composition provided herein, and further administering to the subject one or more additional therapeutic agent.
- the additional therapeutic agent is administered simultaneously, separately, or sequentially with the composition provided herein.
- the additional therapeutic agent is a chemotherapeutic drug or a radiotherapeutic drug.
- the present disclosure provides methods for reducing the size of one or more tumors present in a subject, wherein the method comprises administering a composition provided herein to the subject.
- the present disclosure provides methods for treating cancer comprising administering a composition provided herein followed by surgical removal of one or more tumors present in the subject.
- the present disclosure also provides kits for treating and preventing cancer, the kits comprising an effective amount of the compositions provided herein and directions for the use thereof to treat or prevent the cancer.
- the present disclosure provides methods for producing a protein, the method comprising (i) inserting a non-codon optimized nucleic acid sequence encoding SEQ ID NO: 2 or SEQ ID NO: 21 in a plasmid, (ii) transforming E. coli cells in a cell culture with the plasmid; (iii) inducing expression of the protein encoded by the nucleic acid sequence, and (iv) obtaining the protein from the cell culture supernatant.
- the methods further comprise purifying the protein.
- the present disclosure provides methods for producing and purifying the immunoconjugates and compositions provided herein for in vitro, diagnostic, or clinical use.
- the E. coli cells used in the methods provided herein are E. coli E104 or TGI cells.
- the plasmid used in the methods provided herein is selected from pING-RBS, pING3302, and pSJFI plasmids. In some embodiments,
- the present disclosure provides methods for purifying the proteins provided herein.
- the methods comprise purifying the protein using one or more chromatographic steps.
- the methods comprise contacting the protein with at least one chromatographic material.
- Chromatographic materials that maybe used in the methods provided herein include mixed mode, ion-exchange, affinity, hydrophobic interaction, reverse phase, size exclusion, and adsorption materials.
- the chromatographic material is hydroxy apatite and/or Capto MMC.
- the production and purification methods provided herein comprise concentrating the protein, for example, by diafiltration, ultrafiltration, or tangential flow filtration.
- the methods comprise contacting the protein with a Capto MMC chromatographic material.
- the present disclosure provides methods for producing and purifying an immunoconjugate comprising an EpCAM antibody fragment and a toxin, the methods comprising the following steps: (i) inserting a non-codon optimized nucleic acid sequence encoding SEQ ID NO: 2 or SEQ ID NO: 21 in a plasmid, (ii) transforming E.
- Figure 1A-1C provides the nucleic acid and amino acid sequences of VB4-
- Figure 2 shows a Western blot of VB4-847-CODA expressed from pING3302 in E. coli El 04.
- Supernatants of 3 VB4-847-CODA induced clones (lane 1, 2 and 3) were loaded under non-reducing conditions on a SDS-PAGE gel and immunoblotted with a rabbit anti-PE antibody (1/5000) and sheep anti-rabbit-HRP antibody (1/1000).
- the induced supernatant of VB4-845-CODA-His was loaded on lane 4.
- Figure 3 shows a Western blot of VB4-847-CODA expressed from pING-
- Figure 4 shows a Western blot of VB4-847 expressed from pING-RBS in E. coli E104. Supernatants of VB4-847/pING3302 (lane 1), VB4-847/pING-RBS (lane 2), VB4- 847-CODA/pING3302 (lane 3), VB4-847-CODA/pING-RBS (lane 4), VB4-845- His/pING3302 (land 5) and VB4-845-His/pING-RBS (lane 6) were loaded under non- reducing conditions on a SDS-PAGE gel and immunoblotted with a rabbit anti-PE antibody (1/5000) and sheep anti-rabbit-HRP antibody (1/1000). Lane L corresponds to the ladder.
- Figure 5 shows a Western blot of VB4-847-CODA expressed from pSJFl in
- E. coli TGI E. coli TGI .
- Periplasmic extracts of 2 VB4-847-CODA induced clones (lane 1 and 2) were loaded under non-reducing conditions on a SDS-PAGE gel and immunoblotted with a rabbit anti-PE antibody (1/5000) and sheep anti-rabbit-HRP antibody (1/1000).
- the induced supernatants of VB4-847-CODA and VB4-845-CODA-His were loaded on lanes 3 and 4, respectively.
- Lane L corresponds to the ladder.
- Figures 6A and 6B show the cytotoxic activity of VB4-847 against Cal-27
- (6A) and A-375 (6B) cells measured by MTS assay.
- Cells were incubated with VB4-847 (open circle) or VB4-845-His (filled circle). After 3 days incubation, cell viability was measured and IC5 0 determined.
- the present disclosure provides immunoconjugates comprising a binding protein that binds to EpCAM and a toxin, methods of use thereof, and methods for producing and purifying immunoconjugates comprising a binding protein that binds to EpCAM and a toxin.
- the immunoconjugates do not comprise His tags.
- the immunoconjugates comprise a single His tag.
- the immunoconjugates provided herein advantageously can be effectively purified using the methods provided herein, which do not require the presence of a His tag, or which utilize the presence of a single His tag.
- the immunoconjugates provided herein result in a reduction in toxicity in subjects.
- the nickel column used during purification removes impurities.
- the present disclosure provides methods for purifying proteins lacking a histidine tag, herein referred to as “non-His tagged proteins”. The methods provided herein, comprising, for example, the use of mixed mode and size exclusion columns, provided superior, and quite unexpected results for purifying a non-His tagged protein.
- the present disclosure provides immunoconjugates comprising EpCAM binding proteins.
- the EpCAM binding protein comprises a light chain CDR1 having an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homology to a sequence as set forth in SEQ ID NO: 3
- the EpCAM binding protein comprises a light chain variable region encoded by a nucleic acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homology to a sequence as set forth in SEQ ID NO: 3.
- the binding protein comprises a heavy chain variable region encoded by a nucleic acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homology to a sequence as set forth in SEQ ID NO: 10.
- the binding protein comprises a light- heavy chain linker, wherein the light-heavy chain linker is encoded by a nucleic acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homology to a sequence as set forth in SEQ ID NO: 8.
- the binding protein comprises a light-heavy chain linker encoded by a nucleic acid sequence according to SEQ ID NO: 8.
- the binding protein comprises a light chain variable region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homology to an amino acid sequence as set forth in SEQ ID NO: 4.
- the binding protein comprises a heavy chain variable region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homology to an amino acid sequence as set forth in SEQ ID NO: 11.
- the binding protein is an antibody or a fragment thereof.
- Antibodies and fragments thereof are selected from polyclonal antibodies, monoclonal antibodies, Fab, Fab', (Fab')2, single chain fragments (scFv), disulfide-stabilized fragments (dsFv), single domain antibodies (sdAb), diabodies, and cys-diabodies (cysteine-modified diabodies).
- diabodies include bivalent, bispecific antibodies or antibody fragments.
- diabodies include bivalent antibodies that are made up of the same or different antibody or antibody fragment polypeptides; thus, in some embodiments, the diabodies are homo-multimers or hetero-multimers.
- Antibodies and fragments thereof may comprise an immunoglobulin constant region selected from the group consisting of IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgD, IgE, and IgM.
- the antibody or fragment thereof may be from any species including mice, rats, rabbits, hamsters, and humans.
- the antibody or fragment is chimeric. Chimeric antibodies or fragments thereof are antibody molecules that combine a non-human animal variable region and a constant region or portion of a constant region that is human.
- the immunoconjugates provided herein comprise an scFv having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homology to an amino acid sequence as set forth in SEQ ID NO: 19.
- the antibody fragment may be Fab, and the light chain and the heavy chain are linked by a covalent bond.
- the covalent linkage may be a disulfide bond.
- the covalent linkage may be through chemical crosslinkers, such as dimethyl adipimidate, dimethyl suberimidate, and the like.
- amino acid crosslinkers such as (Gly 4 -Ser) n may be used.
- the sequences of the light chain and the heavy chain described herein may be used to derive scFv, diabodies, tribodies, tetrabodies, and the like.
- Various protein linking strategies may be used to produce bivalent or bispecific Fab and scFvs, as well as bifunctional Fab and scFv fusions.
- the antigen-binding fragments of any of the above-described binding proteins are prepared using means known in the art, for example, by preparing nested deletions using enzymatic degradation or convenient restriction enzymes.
- the humanized antibodies, chimeric antibodies or immunoreactive fragments thereof are screened to ensure that antigen binding has not been disrupted by the humanization, chimerization, or fragmentation of the parent monoclonal antibody. This may be accomplished by any of a variety of means known in the art, including, for example, use of a phage display library.
- the present disclosure provides an immunoconjugate comprising an antibody fragment that binds to the extracellular domain of human EpCAM and a toxin.
- the immunoconjugate comprises more than one toxin.
- the immunoconjugate comprises a diabody comprising more than one toxin.
- the toxin is a molecule that blocks protein synthesis in a target cell, therein leading to cell death.
- the toxin is a cytotoxin.
- Cytotoxins are known in the art and include, for example, Pseudomonas exotoxin A (ETA) or variants thereof; geionin, bouganin, deimmimized bouganin (e.g., a toxin as described in U.S. Patent No. 7,339,031, incorporated by reference herein in its entirety), saporin, ricin, ricin A chain, bryodin, diphtheria toxin, and restrictocin.
- ETA Pseudomonas exotoxin A
- bouganin e.g., a toxin as described in U.S. Patent No. 7,339,031, incorporated by reference herein in its entirety
- saporin ricin, ricin A chain, bryodin, diphtheria toxin,
- the toxin is Pseudomonas exotoxin A (ETA) or a variant thereof.
- the toxin is ETA(252-608).
- ETA(252-608) which is a truncated form of ETA that lacks the cell binding domain, is a single polypeptide fusion protein produced by continuous translation of a single construct.
- the toxin is encoded by a nucleic acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homology to SEQ ID NO: 17. In some embodiments, the toxin is encoded by a nucleic acid sequence according to SEQ ID NO: 17. In some embodiments, the toxin comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homology to SEQ ID NO: 18.
- the toxm comprises an agent that acts to disrupt DNA.
- toxins may comprise, without limitation, enediynes (e.g., calicheamicin and esperamicin) and non-enediyne small molecule agents (e.g., bleomycin, methidiumpropyl-EDTA-Fe(II)).
- Other toxins useful in accordance with the invention include, without limitation, daunorubicin, doxorubicin, distamycin A, cisplatin, mitomycin C, ecteinascidins, duocarmycin/CC-1065, and bleomyem/pepleomyem.
- the toxin comprises an agent that acts to disrupt tubulin.
- toxins may comprise, without limitation, rhizoxin/maytansine, paclitaxel, vincristine and vinblastine, colchicine, auri statin dolastatin 10 MMAE, and peloruside A.
- the toxin may comprise an alkylating agent including, without limitation, Asaiey NSC 167780, AZQ NSC 182986, BCNU NSC 409962, Busulfan NSC 750, carboxyphthaiatoplatinum NSC 271674, CBDCA NSC 241240, CCNU NSC 79037, CHIP NSC 256927, chlorambucil NSC 3088, chlorozotocin NSC 178248, cis- platmum NSC 119875, clomesone NSC 338947, cyanomorpholinodoxorubicin NSC 357704, cyclodisone NSC 348948, dianhydrogalactitol NSC 132313, fluorodopan NSC 73754, hepsulfam NSC 329680, hycanthone NSC 142982, melphaJan NSC 8806, methyl CCNU NSC 95441, mitomycin C NSC 26980,
- the toxin may comprise an antimitotic agent including, without limitation, allocolchicine NSC 406042, Halichondrin B NSC 609395, colchicine NSC 757, colchicine derivative NSC 33410, dolastatin 10 NSC 376128 (NG— auri statin derived), maytansme NSC 153858, rhizoxm NSC 332598, taxol NSC 125973, taxol derivative NSC 608832, thiocolchicine NSC 361792, trityl cysteine NSC 83265, vinblastine sulfate NSC 49842, and vincristine sulfate NSC 67574 [0040]
- the toxin may comprise an topoisomerase I inhibitor including, without limitation, camptothecm NSC 94600, camptothecin, Na salt NSC 100880, aminocamptothecin NSC 603071 , camptothecm derivative NSC 95382, camptothec
- the toxin may comprise an topoisomerase II inhibitor including, without limitation, doxorubicin NSC 123127, amonafide NSC 308847, m-AMSA NSC 249992, anthrap razole derivative NSC 355644, pyrazoloacridine NSC 366140, bisantrene HCL NSC 337766, daunorubicin NSC 82151 , deoxydoxorubicin NSC 267469, mitoxantrone NSC 301739, menogaril NSC 269148, ⁇ , ⁇ -dihenzyl daunomycin NSC 268242, oxanthrazole NSC 349174, rubidazone NSC 164011, VM-26 NSC 122819, and VP-16 NSC 141540.
- an topoisomerase II inhibitor including, without limitation, doxorubicin NSC 123127, amonafide NSC 308847, m-AMSA NSC 249992, an
- the toxin may comprise an RNA or DNA antimetabolite including, without limitation, L-alanosine NSC 153353, 5-azacytidine NSC 102816, 5- fluorouracil NSC 19893, acivicin NSC 163501, aminopterin derivative NSC 132483, aminopterin derivative NSC 184692, ammopterin derivative NSC 134033, an antifoi NSC 633713, an antifoi NSC 623017, Baker's soluble antifoi NSC 139105, dichlorallyl lawsone NSC 126771, brequinar NSC 368390, ftorafur (pro-drug) NSC 148958, 5,6-dihydro-5- azacytidme NSC 264880, methotrexate NSC 740, methotrexate derivative NSC 174121, N- (phosphonoacetyl)-L-aspartate (PALA) NSC 224
- a toxin may be altered to decrease or inhibit binding outside of the context of the immunoconjugate, or to reduce specific types of toxicity.
- the toxin may be altered to adjust the isoelectric point to approximately 7.0 such that liver toxicity is reduced.
- the immunoconjugates useful in the methods of the present disclosure comprise a binding protein that is attached to a toxin by a peptide linker.
- Peptide linkers may be cleavable linkers or non-cleavable linkers.
- the linker is a cleavable linker selected from the group consisting of a furin sensitive linker, a cathepsin sensitive linker, a metalloproteinase linker, or a lysosomal hydrolase sensitive linker.
- the linker is a flexible linker. In some embodiments, the linker is between about 5 and about 50 amino acids in length.
- the linker is between about 10 and about 30 amino acids in length. In further embodiments, the linker is about 20 amino acids in length. In some embodiments, the linker is encoded by a nucleic acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homology to SEQ ID NO: 15. In some embodiments, the linker has at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homology to SEQ ID NO: 16.
- the antibodies or the antibody fragments described herein may be conjugated to the effector molecule by any means.
- the antibody or the antibody fragment may be attached to the toxin by chemical or recombinant means.
- Chemical means for preparing fusions or conjugates are known in the art and can be used to prepare the immunoconjugate.
- the method used to conjugate the antibody or the antibody fragment and toxin must be capable of joining the antibody with the toxin without interfering with the ability of the antibody or the antibody fragment to bind to the target molecule.
- the antibody and toxin are both proteins and can be conjugated using techniques well known in the art.
- the crosslinker is generally chosen based on the reactive functional groups available or inserted on the antibody or toxin.
- a photoactivatible crosslinker can be used.
- Crosslinking agents known to the art include the homobifunctional agents: glutaraldehyde, dimethyladipimidate and bis(diazobenzidine) and the heterobifunctional agents: m- maleimidobenzoyl-N-hydroxysuccinimide and sulfo-m maleimidobenzoyl-N- hy droxy succinimi de.
- Other crosslinkers that may be used to couple an effector molecule to the antibody fragment include TPCH(S-(2- thiopyridyl)-L-cysteine hydrazide and TPMPH ((S- (2-thiopyridyl) mercapto- propionohydrazide).
- TPCH and TPMPH react at the carbohydrate moieties of glycoproteins that have been previously oxidized by mild periodate treatment, thus forming a hydrazone bond between the hydrazide portion of the crosslinker and the periodate generated aldehydes.
- the hetero-bifunctional crosslinkers GMBS (N-gama- malimidobutyryloxy)-succinimide) and SMCC (succinimidyl 4-(N-maleimido- methyl)cyclohexane) react with primary amines, thus introducing a maleimide group onto the component.
- This maleimide group can subsequently react with sulfhydryls on the other component, which can be introduced by previously mentioned crosslinkers, thus forming a stable thioether bond between the components.
- crosslinkers can be used which introduce long spacer arms between components and include derivatives, such as n-succinimidyl-3-(2- pyridyldithio)propionate (SPDP).
- SPDP n-succinimidyl-3-(2- pyridyldithio)propionate
- the immunoconjugate is VB4-847.
- VB4-847 and VB4-845 are both recombinant fusion proteins comprising a humanized single- chain antibody fragment (scFv) specific for EpCAM antigen linked to ETA(252-608).
- VB4- 845 is described in U.S. Patent No. 8,545,840, which is incorporated by reference herein in its entirety.
- VB4-845 is produced in E. coli cells and purified by a process utilizing N- and C- terminal Histidine tags (His tags) on the VB4-845 molecule and including a 2+ ⁇ iminodiacetic (IDA) column for purification.
- IDA iminodiacetic
- VB4-847 does not include His tags in the amino acid sequence. In other embodiments, VB4-847 includes a single His tag. In some embodiments, VB4-847 includes only a C-terminal His tag. In preferred embodiments, VB4- 847 includes only an N-terminal His tag.
- VB4-847 is generated by expressing a plasmid comprising a non-codon optimized DNA sequence in E. coli cells.
- VB4-847 can be produced and purified to optimal levels for clinical use in treatment or diagnostics.
- nucleic acid and amino acid sequences of VB4-847 are provided below in
- TCC ACC AAA TCC CTC CTG CAC TCC AAC GGT ATC ACC
- GGT ACC GAC TTC ACC CTG ACC ATC TCT TCT CTG CAG
- the immunoconjugate comprises a binding protein wherein the heavy chain variable region is encoded by a nucleic acid sequence sharing at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 10.
- the heavy chain variable region is encoded by the nucleic acid sequence according to SEQ ID NO: 10.
- the light chain variable region is encoded by a nucleic acid sequence sharing at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 3.
- the light chain variable region is encoded by a nucleic acid sequence according to SEQ ID NO: 3.
- the heavy chain variable region and the light chain variable region are linked by a linker.
- the linker is encoded by a nucleic acid sequence according to SEQ ID NO: 9.
- the immunoconjugate is encoded by a nucleic acid sequence sharing at least 90% homology to SEQ ID NO: 1. In a further embodiment, the immunoconjugate is encoded by a nucleic acid sequence according to SEQ ID NO: 1. In some embodiments, the immunoconjugate is encoded by nucleic acids 79-1995 of SEQ ID NO: 1. In some embodiments, the immunoconjugate comprises an amino acid sequence sharing at least 90% homology to SEQ ID NO: 2 or SEQ ID NO: 21. In further embodiments, the immunoconjugate comprises an amino acid sequence according to SEQ ID NO: 2 or SEQ ID NO: 21. In some embodiments, the immunoconjugate comprises amino acids 23-635 of SEQ ID NO: 2.
- the present invention contemplates methods of treating or preventing cancer comprising administering an effective amount of said immunoconjugates to a subject in need thereof.
- the immunoconjugates may be used to treat or prevent cancer.
- the immunoconjugates may be used to diagnose cancer in a subject.
- the cancer is head and neck cancer, bladder cancer, lung cancer, gastric cancer, renal cancer, thyroid cancer, breast cancer, ovarian cancer, cervical cancer, colorectal cancer, hepatocellular carcinoma, esophageal cancer, oral squamous cell carcinoma, pancreatic cancer, or prostate cancer. Cancers originating from any epithelial cell may also be targeted by these immunoconjugates.
- the cancer is head and neck cancer.
- the head and neck cancer is HNSCC.
- the immunoconjugates provided herein exhibit decreased liver toxicity in subjects due to the lack of a His tag.
- a VB4-845 (containing a His tag) immunoconjugate may promote liver uptake which could result in liver toxicity.
- the immunoconjugates provided herein, which do not contain a histidine tag (His tag; e.g., VB4-847) lead to a decrease in liver toxicity.
- the cancer is amenable to treatment by direct administration of the immunoconjugate to the cancer site.
- the present disclosure provides methods and immunoconjugates for reducing tumor size.
- a reduction in tumor size can facilitate removal of the tumor.
- the immunoconjugates of the present invention advantageously effectively reduce tumor size, making surgical removal of the tumor or portions of the tumor feasible.
- the present disclosure provides methods for treating cancer in a subject in need thereof comprising administering a composition provided herein, followed by surgical removal of one or more tumors present in the subject.
- the present disclosure provides methods for treating or preventing cancer comprising detecting the presence of a human papilloma virus (HPV) infection in a subject; and administering to the subject a composition provided herein.
- the composition is administered to the subject if the subject tests positive for HPV.
- Methods for testing HPV are known in the art and include detection assays selected from, for example, PCR, in situ hybridization, ELISAs, immunohistochemistry assays, antigen and/or antibody assays for HPV proteins or immunocomplexes, protein chip assays, radioimmunoprecipitation assays, rapid stick immunochromatographic assays, and flow cytometry based assays.
- the methods comprise detecting HPV proteins, or DNA encoding HPV proteins, such as LI, E6, or E7 prior to administration of a composition provided herein.
- kits for diagnosing and/or treating cancer may include an the immunoconjugates provided herein.
- the kits further comprise a reagent containing a labeled anti-Ig antibody, for example, an anti-Ig antibody linked with an enzyme such as alkaline phosphatase or a radiolabeled anti-Ig antibody.
- the immunoconjugate may be attached to a chromophore, a fluorophore or a radiolabelled ligand.
- the kits provided herein may further comprise instructions for use of the immunoconjugates.
- the kits provided herein comprise reagents for HPV detection.
- the immunoconjugates disclosed herein may also be used to detect or monitor cancer in a subject.
- a method of detecting or monitoring cancer in a subject may involve contacting a test sample taken from the subject with an immunoconjugate to form an immunoconjugate-antigen complex; measuring the amount of the immunoconjugate-antigen complex in the test sample; and normalizing the results against a control.
- the test sample may be serum, lymph, ascitic exudate, intercellular fluid, tissue lysate, saliva, tissue sections, cells, biopsy samples, and the like.
- the immunoconjugate- antigen complex may be detected by any means, such as for example, dot-blot method, Western blot method, ELISA method, or sandwich ELISA method.
- the immunoconjugate- antigen complex can be detected by use according to multistage reactions, such as reaction with a biotin-bound anti-Ig antibody and then with an avidin-bound material.
- a method of detecting or monitoring cancer in a subject may involve contacting a test sample taken from the subject with an immunoconjugate to form a complex.
- the immunoconjugates disclosed herein may be used for imaging a tumor in a subject
- the immunoconjugates as described herein are labeled with a radiolabel.
- a radiolabel is typically a substance containing a radioisotope that allows for easy detection and measurement.
- a number of different forms of hydrogen, carbon, phosphorous, sulfur and iodine are commonly used in medical diagnostics.
- the immunoconjugates of the present invention may also be labeled with any fluorescent probes known in the art. Non-limiting examples include fluorescein, amino coumarin acetic acid, tetramethylchodomine isocyanate, Texas Red, Cy 3.0, Cy 5.0, green fluorescent protein, and the like.
- the immunoconjugates as described herein are labeled with a contrast agent.
- a contrast agent is a substance used to increase or modify the contrast of organs, fluids or anatomical structures in the human or animal body.
- the immunoconjugates of the present invention may be labeled with any suitable contrast agent.
- Preferred contrast agents include contrast agents for medical imaging.
- the immunoconjugates of the present invention are labeled with an MRI (magnetic resonance imaging) contrast agent such as a superparamagnetic contrast agent or a paramagnetic contrast agent.
- MRI contrast agents are typically chelated metals or colloids.
- Contrast agents include gadolinium (Gd) based contrast agents such as gadolinium-DTPA, iron oxide based contrast agents such as superparamagnetic Small Particles of Iron Oxide (SPIO) and superparamagnetic Ultrasmall Small Particles of Iron Oxide (USPIO) and paramagnetic contrast agents based on manganese chelates such as Mn-DPDP.
- Gd gadolinium
- SPIO superparamagnetic Small Particles of Iron Oxide
- USPIO superparamagnetic Ultrasmall Small Particles of Iron Oxide
- Mn-DPDP paramagnetic contrast agents based on manganese chelates
- the immunoconjugate of the invention is administered in addition to one or more other therapeutic agents, which may include, without limitation, 2,2',2"trichlorotriethylamine, 6-azauridine, 6-diazo-5-oxo-L-norleucine, 6-mercaptopurine, aceglarone, aclacinomycinsa actinomycin, altretamine, aminoglutethimide, aminoglutethimide, amsacrine, anastrozole, ancitabine, angiogenin antisense oligonucleotide, anthramycin, azacitidine, azaserine, aziridine, batimastar, bcl-2 antisense oligonucleotide, benzodepa, bicalutamide, bisantrene, bleomycin, buserelin, busulfan, cactinomycin, calusterone, carboplatin, carboquone, carmofur, carmus
- compositions comprising one or more cancer therapeutics (e.g., FLAG, CHOP) are also contemplated by the present invention.
- FLAG comprises fludarabine, cytosine arabinoside (Ara-C) and G-CSF.
- CHOP comprises cyclophosphamide, vincristine, doxorubicin, and prednisone.
- the immunoconjugate of the invention may be used in conjunction with radiation therapy or other known cancer therapeutic modalities.
- An immunoconjugate of the present invention can be administered with a cancer therapeutic modality such as an antibody drug conjugate (ADC).
- ADC comprises a monoclonal antibody or antibody fragment, a cytotoxic payload or drug and a stable, chemical linker with labile bonds connecting the payload to the antibody.
- ADCs approved by the FDA and routinely used in the treatment of various cancers include gemtuzumab ozogamicin, ibritumomab tiuxetan, tositumomab, ado-trastuzumab emtansine, and Brentuximab Vedotin.
- An immunoconjugate of the present disclosure can be administered with a cancer therapeutic modality such as immune checkpoint inhibitors.
- checkpoint inhibitor it is meant that the compound inhibits one or more proteins in a number of inhibitory pathways that usually serve to modulate an immune response. The pathways are co-opted by tumors to evade the immune system and proliferate. Proteins in the checkpoint signaling pathways include for example, PD-1, PD-L1, PD-L2, TIM3, LAG3 and CTLA-4.
- Checkpoint inhibitors are known in the art.
- the checkpoint inhibitor can be a small molecule.
- a "small molecule” as used herein, is meant to refer to a composition that has a molecular weight in the range of less than about 5 kD to 50 kD, for example less than about 4 kD, less than about 3.5 kD, less than about 3 kD, less than about 2.5 kD, less than about 2 kD, less than about 1.5 kD, less than about 1 kD, less than 750 daltons, less than 500 daltons, less than about 450 daltons, less than about 400 daltons, less than about 350 daltons, less than 300 daltons, less than 250 daltons, less than about 200 daltons, less than about 150 daltons, less than about 100 daltons.
- Small molecules can be, e.g., nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic or inorganic molecules.
- the checkpoint inhibitor is an antibody or antibody fragment thereof.
- the antibody or antibody fragment thereof is specific to a protein in a checkpoint signaling pathway, such as PD-1, PD-L1, PD-L2, LAG3, TIM3 or CTLA-4.
- methods of treating cancer comprising administering an immunoconjugate in combination with a regimen of radiation therapy.
- the therapy may also comprise surgery and/or chemotherapy.
- the immunoconjugate may be administered in combination with radiation therapy and cisplatin, fluo-rouracil, carboplatin, and/or paclitaxel.
- Treatment with the immunoconjugate may allow use according to lower doses of radiation and/or less frequent radiation treatments, which may for example, reduce the incidence of severe sore throat that impedes swallowing function potentially resulting in undesired weight loss or dehydration.
- methods of treating cancer comprising administering an immunoconjugate in combination with an immunotherapeutic which includes, without limitation, rituxan, rituximab, campath-1, gemtuzumab, and trastuzumab.
- an immunotherapeutic which includes, without limitation, rituxan, rituximab, campath-1, gemtuzumab, and trastuzumab.
- an immunoconjugate is administered in combination with one or more anti- angiogenic agents which include, without limitation, angiostatin, thalidomide, kringle 5, endostatin, Serpin (Serine Protease Inhibitor), anti-thrombin, 29 kDa N-terminal and a 40 kDa C-terminal proteolytic fragments of fibronectin, 16 kDa proteolytic fragment of prolactin, 7.8 kDa proteolytic fragment of platelet factor-4, a 13 amino acid peptide corresponding to a fragment of platelet factor-4 (Maione et al, 1990, Cancer Res.
- anti- angiogenic agents include, without limitation, angiostatin, thalidomide, kringle 5, endostatin, Serpin (Serine Protease Inhibitor), anti-thrombin, 29 kDa N-terminal and a 40 kDa C-terminal proteolytic fragments of fibronectin, 16 kDa
- an immunoconjugate is administered in combination with a cancer vaccine including, without limitation, autologous cells or tissues, non-autologous cells or tissues, carcinoembryonic antigen, alpha-fetoprotein, human chorionic gonadotropin, BCG live vaccine, melanocyte lineage proteins, and mutated, tumor- specific antigens.
- a cancer vaccine including, without limitation, autologous cells or tissues, non-autologous cells or tissues, carcinoembryonic antigen, alpha-fetoprotein, human chorionic gonadotropin, BCG live vaccine, melanocyte lineage proteins, and mutated, tumor- specific antigens.
- an immunoconjugate is administered in association with hormonal therapy.
- Hormonal therapeutics include, without limitation, a hormonal agonist, hormonal antagonist (e.g., flutamide, tamoxifen, leuprolide acetate), and steroid (e.g., dexamethasone, retinoid, betamethasone, Cortisol, cortisone, prednisone, dehydrotestosterone, glucocorticoid, mineralocorticoid, estrogen, testosterone, progestin).
- a hormonal agonist e.g., flutamide, tamoxifen, leuprolide acetate
- steroid e.g., dexamethasone, retinoid, betamethasone, Cortisol, cortisone, prednisone, dehydrotestosterone, glucocorticoid, mineralocorticoid, estrogen, testosterone, progestin.
- an immunoconjugate is administered in association with a gene therapy program to treat or prevent cancer.
- Combination therapy with an immunoconjugate may sensitize the cancer or tumor to administration of an additional cancer therapeutic.
- the present invention contemplates combination therapies for treating, and/or preventing recurrence of cancer comprising administering an effective amount of an immunoconjugate prior to, subsequently, or concurrently with a reduced dose of a cancer therapeutic.
- initial treatment with an immunoconjugate may increase the sensitivity of a cancer or tumor to subsequent challenge with a dose of cancer therapeutic. This dose is near, or below, the low range of standard dosages when the cancer therapeutic is administered alone, or in the absence of an immunoconjugate.
- the immunoconjugate may be administered separately from the cancer therapeutic, and optionally, via a different mode of administration.
- the cancer therapeutic may be selected from, for example, a steroid; a biologic anti-autoimmune drug such as a monoclonal antibody, a fusion protein, or an anti- cytokine; a non-biologic anti-autoimmune drug; an immunosuppressant; an antibiotic; and anti-viral agent; a cytokine; or an agent otherwise capable of acting as an immune-modulator.
- the cancer therapeutic is an immune checkpoint inhibitor, such as a a therapeutic that inhibits signalling from the PD-l/PD-Ll pathway, or CTLA4 pathway.
- the cancer therapeutic may be an antibody or fragment thereof that is specific for PD-1, PD-L1, or CTLA4.
- the cancer therapeutic may be selected from, without limitation, pembrolizumab, pidilizumab, nivolumab, ipilimumab, or tremelimumab.
- compositions for combination therapy may also include, without limitation, antibiotics (e.g., dactinomycin, bleomycin, mithramycin, anthramycin), asparaginase, Bacillus and Guerin, procaine, tetracaine, lidocaine, propranolol, anti-mitotic agents, abrin, ricinA, Pseudomonas exotoxin, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, antihistaminic agents, anti-nausea agents, etc.
- antibiotics e.g., dactinomycin, bleomycin, mithramycin, anthramycin
- asparaginase Bacillus and Guerin
- procaine tetracaine
- lidocaine propranolol
- anti-mitotic agents e.g., dactinomycin, bleomycin, mithramycin, anthramycin
- Clinical outcomes of cancer treatments using an immunoconjugate of the invention are readily discernible by one of skill in the relevant art, such as a physician.
- standard medical tests to measure clinical markers of cancer may be strong indicators of the treatment's efficacy. Such tests may include, without limitation, physical examination, performance scales, disease markers, 12-lead ECG, tumor measurements, tissue biopsy, cytoscopy, cytology, longest diameter of tumor calculations, radiography, digital imaging of the tumor, vital signs, weight, recordation of adverse events, assessment of infectious episodes, assessment of concomitant medications, pain assessment, blood or serum chemistry, urinalysis, CT scan, and pharmacokinetic analysis.
- synergistic effects of a combination therapy comprising the immunoconjugate and another cancer therapeutic may be determined by comparative studies with patients undergoing monotherapy.
- the effective dose of immunotoxin to be administered during a cycle varies according to the mode of administration.
- Direct administration e.g., intratumoral injection
- the effective dose of a specific immunoconjugate construct may depend on additional factors, including the type of cancer, the size of the tumor in the case of HNSCC, the stage of the cancer, the immunoconjugate's toxicity to the patient, the specificity of targeting to cancer cells, as well as the age, weight, and health of the patient.
- the effective dose by direct administration of immunoconjugate may range from about 10 to 3000, 20 to 900, 30 to 800, 40 to 700, 50 to 600, 60 to 500, 70 to 400, 80 to 300, 90 to 200, or 100 to 150 micrograms/tumor/day.
- the dose may range from approximately 10 to 20, 21 to 40, 41 to 80, 81 to 100, 101 to 130, 131 to 150, 151 to 200, 201 to 280, 281 to 350, 351 to 500, 501 to 1000, 1001 to 2000, or 2001 to 3000 micrograms/tumor/day.
- the dose may be at least approximately 20, 40, 80, 130, 200, 280, 400, 500, 750, 1000, 2000, or 3000 mi crograms/tumor/ day .
- the effective dose of immunoconjugate may range from about 100 to 5000, 200 to 4000, 300 to 3000, 400 to 2000, 500 to 1000, 600 to 900, or 700 to 1500 micrograms/tumor/month. In other embodiments, the dose may range from approximately 100 to 199, 200 to 399, 400 to 649, 650 to 999, 1000 to 1799, 1800 to 2499, 2500 to 3499, 3500 to 4999, 5000 to 7499, 7500 to 10000, or 10001 to 20000 micrograms/tumor/month.
- the dose may be at least approximately 100, 200, 400, 650, 1000, 1400, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 7500, 10000, or 20000 micrograms/tumor/month.
- the effective dose of immunoconjugate results in an intratumoral concentration of at least approximately 5, 10, 20, 30, 40, 50, 60, 75, 100, 125, 150, 100, 200, 300, 400, or 500 micrograms/cm 3 of the immunoconjugate.
- the resulting intratumoral concentration of immunoconjugate is approximately 5 to 500, 10 to 400, 15 to 300, 20 to 200, 25 to 100, 30 to 90, 35 to 80, 40 to 70, 45 to 60, or 50 to 55 micrograms/cm 3 .
- the resulting intratumoral concentration of immunoconjugate is approximately 10 to 15, 16 to 20, 21 to 25, 26 to 30, 31 to 35, 36 to 40, 41 to 45, 46 to 50, 51 to 55, 56 to 60, 61 to 65, 66 to 70, 71 to 75, 76 to 80, 81 to 85, 86 to 90, 91 to 95, 96 to 100, or 100 to 200 micrograms/cm 3 .
- the effective dose of immunoconjugate results in a plasma concentration of less than approximately 0.1, 1, 2.5, 5, 7.5, 10, 15, 20, 30, 40, or 50 micrograms/liter.
- the resulting circulating concentration of immunoconjugate is approximately 0.1 to 50, 1 to 40, 2.5 to 30, 5 to 20, or 7.5 to 10 micrograms/liter.
- the resulting circulating concentration of immunoconjugate is approximately 0.1 to 1, 1.1 to 2.4, 2.5 to 5, 5.1 to 7.4, 7.5 to 10, 11 to 15, 16 to 20, 21 to 30, 31 to 40, or 41 to 50 micrograms/liter.
- the effective dose of the immunoconjugate is between about 100 and 3000 micrograms/tumor/month, for example approximately 100, 200, 300, 400, 750, or 1000 micrograms/tumor/month, wherein the patient is administered a single dose per day.
- the single dose is administered approximately every month for approximately 1, 2, 3, 4, 5, or 6 consecutive months. After this cycle, a subsequent cycle may begin approximately 1, 2, 4, 6, or 12 months later.
- the treatment regime may include 1, 2, 3, 4, 5, or 6 cycles, each cycle being spaced apart by approximately 1, 2, 4, 6, or 12 months.
- the effective dose of the immunoconjugate is between about 20 and 1240 micrograms/tumor/day, for example approximately 20, 40, 80, 130, 200, or 280 micrograms/tumor/day or approximately 100, 200, 330, 500, 700, 930, 1240 micrograms/tumor/day, wherein the patient is administered a single dose per day.
- the single dose is administered approximately every day (one or more days may optionally be skipped) for approximately 1, 2, 3, 4, 5, 6 or 7 consecutive days. After this cycle, a subsequent cycle may begin approximately 1, 2, 3, 4, 5, or 6 weeks later.
- the treatment regime may include 1, 2, 3, 4, 5, or 6 cycles, each cycle being spaced apart by approximately 1, 2, 3, 4, 5, or 6 weeks.
- the injection volume preferably is at least an effective amount, which is appropriate to the type and/or location of the tumor.
- the maximum injection volume in a single dose may be between about 25% and 75% of tumor volume, for example approximately one-quarter, one-third, or three-quarters of the estimated target tumor volume. In a specific, non-limiting embodiment, the maximum injection volume in a single dose is approximately 30% of the tumor volume.
- the immunotoxin is administered intratumourally at a total dose per cycle equivalent to, or below the maximum tolerated dose established in a safety trial but the dosage is standardized in relation to the tumor volume.
- subjects will receive between 1 microgram per cm 3 and 500 microgram per cm 3 tumor or a dose sufficient to reach about between 14 picomole and 7 nanomole per cm 3 tumor tissue.
- the dose will be administered in a volume not exceeding about 20-50% of the tumor volume.
- the immunotoxin will be diluted in a suitable salt solution.
- the effective dose of the immunoconjugate is between about 20 and 300 micrograms/tumor/day, for example approximately 20, 40, 80, 130, 200, or 280 micrograms/tumor/day, wherein the patient is administered a single dose per day.
- the maximum injection volume in a single dose may be between about 25% and 75% of tumor volume, for example approximately one-quarter, one-third, or three-quarters of the estimated target tumor volume.
- the single dose is administered every other day for approximately 5, 7, 9, 11 , 13, 15, 17, 19, 21, 23, 25, 27, 29, or 31 consecutive days.
- a subsequent cycle may begin approximately 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 weeks later.
- the treatment regime may include 1, 2, 3, 4, 5, or 6 cycles, each cycle being spaced apart by approximately 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 weeks.
- the effective dose of a specific immunoconjugate construct may depend on additional factors, including the type of cancer, the size of the tumor, the stage of the cancer, the immunoconjugate's toxicity to the patient, the specificity of targeting to cancer cells, as well as the age, weight, and health of the patient.
- the immunoconjugates described herein may be administered to the patient via any suitable route.
- the immunoconjugates may be administered by injection into the vascular system or by injection into an organ.
- Administration routes include parenteral, intravascular, intravenous inj ection, topical, oral, buccal, or ocular routes, or intravaginally, by inhalation, by depot injections, or by implants.
- Parenteral administration includes subcutaneous, intramuscular, intraperitoneal, intracavity, intrathecal, intratumoral, transdermal and intravenous injection.
- the immunoconjugates are administered intravenously as a bolus or by continuous infusion over a period of time.
- the immunoconjugates may be administered directly to the cancer site.
- the immunoconjugate and/or other anticancer agent is delivered to the patient by direct administration.
- the immunoconjugate and/or other anticancer agent may be administered, without limitation, by one or more direct injections into the tumor, by continuous or discontinuous perfusion into the tumor, by introduction of a reservoir of the immunoconjugate, by introduction of a slow-release apparatus into the tumor, by introduction of a slow-release formulation into the tumor, and/or by direct application onto the tumor.
- introduction of the immunoconjugate and/or other anticancer agent to the area of the tumor, or into a blood vessel or lymphatic vessel that substantially directly flows into the area of the tumor is also contemplated.
- the pharmaceutical composition is administered in at least an amount sufficient to achieve the endpoint, and if necessary, comprises a pharmaceutically acceptable carrier.
- the immunoconjugates may be administered intratumorally, whereas any other anticancer agent may be delivered to the patient by other modes of administration (e.g., intravenously).
- the immunoconjugate and one or more of the other anticancer agent may be delivered intratumorally, whereas other anticancer agents may be delivered by other modes of administration (e.g., intravenously and orally).
- a composition may be an immunoconjugate described herein and a pharmaceutically acceptable excipient, carrier, buffer or stabilizer.
- the composition comprises an immunoconjugate in a formulation comprising 20 mM sodium phosphate and 0.1 % Polysorbate 80.
- An immunoconjugate according to the invention may be comprised in a pharmaceutical composition or medicament.
- Pharmaceutical compositions adapted for direct administration include, without limitation, lyophilized powders or aqueous or non-aqueous sterile injectable solutions or suspensions, which may further contain antioxidants, buffers, bacteriostats and solutes that render the compositions substantially isotonic with the blood of an intended recipient.
- compositions include water, alcohols, polyols, glycerin and vegetable oils, for example.
- Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.
- Immunoconjugate may be supplied, for example but not by way of limitation, as a lyophilized powder which is reconstituted with sterile water or saline prior to administration to the patient.
- compositions of the invention may comprise a pharmaceutically acceptable carrier.
- suitable pharmaceutically acceptable carriers include essentially chemically inert and nontoxic compositions that do not interfere with the effectiveness of the biological activity of the pharmaceutical composition.
- suitable pharmaceutical carriers include, but are not limited to, water, saline solutions, glycerol solutions, ethanol, N-(l(2,3-dioleyloxy) propyl)N,N,N-trimethylammonium chloride (DOTMA), diolesylphosphotidyl-ethanolamine (DOPE), and liposomes.
- DOTMA N-(l(2,3-dioleyloxy) propyl)N,N,N-trimethylammonium chloride
- DOPE diolesylphosphotidyl-ethanolamine
- liposomes Such compositions should contain a therapeutically effective amount of the compound, together with a suitable amount of carrier so as to provide the form for direct administration to the patient.
- a pharmaceutical composition comprises an immunoconjugate and one or more additional anticancer agent, optionally in a pharmaceutically acceptable carrier.
- the composition may be in the form of a pharmaceutically acceptable salt which includes, without limitation, those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylarnino ethanol, histidine, procaine, etc.
- the pharmaceutical carrier may include, without limitation, binders, coating, disintegrants, fillers, diluents, flavors, colors, lubricants, glidants, preservatives, sorbents, sweeteners, conjugated linoleic acid (CLA), gelatin, beeswax, purified water, glycerol, any type of oil, including, without limitation, fish oil or soybean oil, or the like.
- Pharmaceutical compositions of the immunoconjugates also can comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as, e.g., polyethylene glycols.
- sequences, vectors, and constructs of the present invention are not codon optimized.
- An exemplary non-codon optimized immunoconjugate sequence is provided in SEQ ID NO: 1. Codon optimization methods are known in the art and can be found, for example, in US2008/0194511, and US2007/0292918, both of which are incorporated herein for all purposes.
- the nucleic acid sequences of the present disclosure may be incorporated in a known manner into an appropriate expression vector which ensures good expression of the proteins encoded thereof.
- Possible expression vectors include, but are not limited to, cosmids, plasmids, or modified viruses (e.g. replication defective retroviruses, adenoviruses and adeno-associated viruses), so long as the vector is compatible with the host cell used.
- the expression vectors are "suitable for transformation of a host cell", which means that the expression vectors contain a nucleic acid molecule of the present application and regulatory sequences selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid molecule.
- Operatively linked is intended to mean that the nucleic acid is linked to regulatory sequences in a manner which allows expression of the nucleic acid.
- the expression vector is a plasmid.
- the plasmid is a pING-RBS, pING3302, or pSJFI plasmid.
- the present application therefore contemplates a recombinant expression vector of the present application containing a nucleic acid molecule of the present application, or a fragment thereof, and the necessary regulatory sequences for the transcription and translation of the inserted protein sequence.
- Suitable regulatory sequences may be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes (For example, see the regulatory sequences described in (Goeddel, 1990), Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). Selection of appropriate regulatory sequences is dependent on the host cell chosen as discussed below, and may be readily accomplished by one of ordinary skill in the art. Examples of such regulatory sequences include: a transcriptional promoter and enhancer or RNA polymerase binding sequence, a ribosomal binding sequence, including a translation initiation signal. Additionally, depending on the host cell chosen and the vector employed, other sequences, such as an origin of replication, additional DNA restriction sites, enhancers, and sequences conferring inducibility of transcription may be incorporated into the expression vector.
- the recombinant expression vectors of the present application may also contain a selectable marker gene which facilitates the selection of host cells transformed or transfected with a recombinant molecule of the present application.
- selectable marker genes are genes encoding a protein such as G418 and hygromycin which confer resistance to certain drugs, (3-galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or an immunoglobulin or portion thereof such as the Fc portion of an immunoglobulin preferably IgG Transcription of the selectable marker gene is monitored by changes in the concentration of the selectable marker protein such as ⁇ -galactosidase, chloramphenicol acetyltransferase, or firefly luciferase.
- selectable marker gene encodes a protein conferring antibiotic resistance such as neomycin resistance transformant cells can be selected with G418. Cells that have incorporated the selectable marker gene will survive, while the other cells die. This makes it possible to visualize and assay for expression of recombinant expression vectors of the present application and in particular to determine the effect of a mutation on expression and phenotype. It will be appreciated that selectable markers can be introduced on a separate vector from the nucleic acid of interest.
- Recombinant expression vectors can be introduced into host cells to produce a transformed host cell.
- the terms “transformed with”, “transfected with”, “transformation” and “transfection” are intended to encompass introduction of nucleic acid (e.g. a vector) into a cell by one of many possible techniques known in the art.
- the term “transformed host cell” as used herein is intended to also include cells capable of glycosylation that have been transformed with a recombinant expression vector of the present application.
- Prokaryotic cells can be transformed with nucleic acid by, for example, electroporation or calcium- chloride mediated transformation.
- nucleic acid can be introduced into mammalian cells via conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran mediated transfection, lipofectin, electroporation or microinjection.
- conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran mediated transfection, lipofectin, electroporation or microinjection.
- Suitable methods for transforming and transfecting host cells can be found in (Sambrook et al, 2001) (Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, 2001), and other laboratory textbooks.
- Suitable host cells include a wide variety of eukaryotic host cells and prokaryotic cells.
- the proteins of the present application may be expressed in bacterial cells, yeast cells or mammalian cells.
- the immunoconjugates of the present disclosure may be expressed in prokaryotic cells, such as Escherichia coli (Zhang et al, 2004), Science 303(5656): 371-3).
- Other suitable host cells include yeast cells or mammalian cells, and can be found in (Goeddel, 1990), Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1991).
- the present disclosure provides a recombinant expression vector comprising one or more of the novel nucleic acid sequences as well as methods and uses of the expression vectors in the preparation of recombinant proteins. Further, the disclosure provides a host cell comprising one or more of the novel nucleic acid sequences or expression vectors comprising one or more of the novel nucleic acid sequences.
- the present disclosure provides methods for purifying an immunoconjugate comprising an EpCAM antibody fragment and a toxin.
- purification refers to steps taken in order to reduce the amounts of foreign elements, such as biological macromolecules (e.g., DNA or protein) that are not the protein of interest and that may be present in a sample of the protein.
- the purification methods provided herein include one or more chromatographic steps in order to purify the immunoconjugate proteins provided herein from a biological source.
- Chromatographic materials and methods are known in the art and described, for example, in Chromatography: 6 th Edition (E. Heftmann, 2004), incorporated herein by reference in its entirety.
- chromatographic materials useful in the present disclosure include, but are not limited to, cation-exchange, ani on-exchange (e.g., Q- Sepharose), affinity, hydrophobic interaction, mixed mode (e.g., hydroxy apatite or Capto MMC, Capto MMC Impres and Nuvia C prime), reversed phase, size exclusion, metal affinity (e.g., Ni2 + column) and adsorption materials.
- the invention also contemplates many support medium, including agarose, cellulose, silica, and poly(stryrene-divinylbenzene) (PSDVB).
- PSDVB poly(stryrene-divinylbenzene)
- multiple chromatographic methods can be used including conventional chromatography, HPLC (High Performance Liquid Chromatography or High pressure Liquid Chromatography), or perfusion chromatography.
- HPLC High Performance Liquid Chromatography or High pressure Liquid Chromatography
- perfusion chromatography perfusion chromatography.
- the size of the column i.e., diameter and length
- centrifugation and/or filtration and/or chromatography steps are used to clarify and/or concentrate the protein.
- One embodiment of the invention involves clarification by precipitation and/or centrifugation and/or filtration (e.g., microfiltration, diafiltration).
- precipitation is carried out with carefully selected concentrations of the chemical agents as this reduces co-precipitation of contaminating proteins.
- Precipitated proteins may be separated from soluble materials by filtration or by centrifugation.
- Capto MMC column which is a mixed mode column
- the primary mechanism of a Capto MMC column is thought to be as a weak cation exchanger.
- the protein to be captured has to be at a lower pH relative to the pi of the molecule.
- the His tagged immunoconjugate molecules disclosed herein precipitate when the pH is dropped below the pi and therefore the same was expected for the non-his tagged version.
- the Capto MMC column (as a positively charged cationic exchanger) would not be expected to be capable of capturing the non-His tagged immunoconjugate at a pH above its pi value based on electrostatic charge.
- the non-His tagged immunoconjugates were successfully captured by the Capto MMC column despite the fact that the protein applied had a higher pH than pi.
- the column may exhibit hydrophobic interactions that lead to the successful capture of the non-His tagged immunoconjugates.
- a hydroxyapatite column removes host cell proteins only, because the Nickel column removes the product-related impurities.
- a non-His tagged protein such as the immunoconjugates disclosed herein, a significant amount of disulfide bonded, product-related impurities must be removed by the hydroxyapatite column.
- the present disclosure provides methods for purifying a non-His tagged protein wherein the method comprises the use of a hydroxy apatite column, and wherein the hydroxyapatite column removes both host cell proteins and product-related impurities.
- the flow through/wash is collected following contact with the hydroxyapatite column.
- impurities are generally removed as a final step in a purification process of a protein by ultrafiltration.
- ultrafiltration did not successfully remove the small molecular weight impurities.
- a size exclusion step which is not generally used in commercial processes due to the added cost and manufacturing time as the final purification step, successfully removed the impurities present non-His tagged proteins disclosed herein.
- immunoconjugate As used herein, the singular forms "a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to an “immunoconjugate” is a reference to one or more immunoconjugates and equivalents thereof known to those skilled in the art, and so forth.
- the term "about” means plus or minus 10% of the numerical value of the number with which it is being used. Therefore, about 50% means in the range of 45%-55%.
- the term “subject” or “patient” refers to any member of the subphylum cordata, including, without limitation, humans and other primates, including non- human primates such as chimpanzees and other apes and monkey species.
- the terms “mammals” and “animals” are included in this definition.
- the term “subject” as used herein refers to a human that has been diagnosed with cancer.
- binding protein includes antibodies and fragments thereof.
- antibody is intended to include monoclonal antibodies, polyclonal antibodies, and chimeric antibodies. The antibody may be from recombinant sources and/or produced in transgenic animals.
- antibody fragment as used herein is intended to include without limitations Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, and multimers thereof, multispecific antibody fragments and Domain Antibodies.
- Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin.
- the resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments.
- Fab, Fab' and F(ab')2, scFv, dsFv, ds- scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques.
- the term "His tag” refers to a stretch of histidine amino acid residues included in a protein sequence.
- the His tag is used for protein purification, for example using a Ni2 + column. While there is no limit on how many histidine residues make up a His tag, there should be as many as is required to effectively bind to, for example, a Ni2 + column.
- the His tag comprises at least 2 histidine residues.
- the His tag comprises at least 3, or at least 4, or at least 5 or at least 6 or at least 7 or at least 8 or at least 9 or at least 10 histidine residues.
- the His tag comprises 6 histidine residues and is also termed a "Hexahistitine tag" or a "HexaHis tag.”
- humanized antibody or antibody fragment means that the antibody or fragment comprises human framework regions.
- the humanization of antibodies from non-human species has been well described in the literature. See for example EP-B1 0 239400 and Carter & Merchant 1997 (Curr Opin Biotechnol 8, 449-454, 1997).
- the term “immunoconjugate” comprises (1) a binding protein attached to (2) a toxin.
- the terms “immunotoxin” and “immunoconjugate” are used interchangeably herein.
- the phrase “treating cancer” refers to inhibition of cancer cell replication, inhibition of cancer spread (metastasis), inhibition of tumor growth, reduction of cancer cell number or tumor growth, decrease in the malignant grade of a cancer (e.g., increased differentiation), or improved cancer-related symptoms.
- the phrase “preventing cancer” refers to prevention of cancer occurrence. In certain instances, the preventative treatment reduces the recurrence of the cancer. In other instances, preventative treatment decreases the risk of a patient from developing a cancer, or inhibits progression of a pre-cancerous state (e.g. a colon polyp) to actual malignancy.
- anticancer agents or “cancer therapeutics” refers to compounds or treatments that are effective in treating or preventing cancer including, without limitation, chemical agents, other immunotherapeutics, cancer vaccines, anti-angiogenic compounds, certain cytokines, certain hormones, gene therapy, radiotherapy, surgery, and dietary therapy.
- the term "effective amount” or “therapeutically effective amount” means an amount effective, at dosages and for periods of time necessary to achieve the desired result. Effective amounts of an immunoconjugate may vary according to factors such as the disease state, age, sex, weight of the animal. Dosage regime may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
- the phrase "the immunoconjugate is administered directly to the cancer site” refers to direct or substantially direct introduction including, without limitation, single or multiple injections of the immunoconjugate directly into the tumor or peritumorally, continuous or discontinuous perfusion into the tumor or peritumorally, introduction of a reservoir into the tumor or peritumorally, introduction of a slow-release apparatus into the tumor or peritumorally, introduction of a slow-release formulation into the tumor or peritumorally, direct application onto the tumor, direct injection into an artery that substantially directly feeds the area of the tumor, direct injection into a lymphatic vessel that substantially drains into the area of the tumor, direct or substantially direct introduction in a substantially enclosed cavity (e.g., pleural cavity) or lumen (e.g., intravesicular).
- a substantially enclosed cavity e.g., pleural cavity
- lumen e.g., intravesicular
- Peritumoral is a term that describes a region, within about 10 cm, preferably within 5 cm, more preferably within 1 cm, of what is regarded as the tumor boundary, such as, but not limited to, a palpable tumor border.
- Direct administration in the context of prevention of occurrence or prevention of recurrence is defined as administration directly into a site at risk for development or recurrence of a cancer. In one embodiment, direct administration is by systemic delivery.
- the phrase "ligand that binds to a protein on the cancer cell” includes any molecule that can selectively target the immunoconjugate to the cancer cell by binding to a protein on the cancer cells.
- the targeted protein on the cancer cell is preferably a tumor associated antigen that is expressed at higher levels on the cancer cell as compared to normal cells.
- linker refers to a short peptide sequence that occurs between protein domains.
- linkers are composed of flexible residues like glycine and serine so that the adjacent protein domains are free to move relative to one another. Longer linkers are used when it is necessary to ensure that two adjacent domains do not sterically interfere with one another. In another embodiment, linkers are rigid and function to prohibit unwanted interactions between discrete protein domains. Fusion proteins or polypeptides can use linkers to connect the regions that do not naturally occur together in nature.
- a furin protease sensitive peptide linker connects, links, joins or fuses a toxin to a binding protein that recognizes one or more tumor associated antigens on the surface of cancer cells.
- a "furin protease sensitive peptide linker”, “furin protease sensitive linker” or “furin linker” comprises a furin cleavage site that is recognized and cleaved by furin, an enzyme which belongs to the subtilisin-like proprotein convertase family. The members of this family are proprotein convertases that process latent precursor proteins into their biologically active products.
- This encoded protein is a calcium-dependent serine endoprotease that can efficiently cleave precursor proteins at their paired basic amino acid processing sites.
- a furin protease sensitive peptide linker fuses a binding protein portion to a toxin portion in an immunoconjugate.
- the toxin is cleaved from the binding protein of the immunoconjugate by a furin enzyme once the immunoconjugate is internalized in a cancer cell, allowing the free toxin to exert its cytotoxic effect.
- polypeptide As used herein, the terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to polymers of amino acid residues of any length.
- the polymer may be linear or branched, it may comprise modified amino acids or amino acid analogs, and it may be interrupted by chemical moieties other than amino acids.
- the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling or bioactive component.
- polynucleotide is a polymeric form of nucleotides of any length, which contain deoxyribonucleotides, ribonucleotides, and analogs in any combination analogs. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The term “polynucleotide” includes double-, single-stranded, and triple-helical molecules.
- any embodiment of the invention described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double stranded form of either the DNA, RNA or hybrid molecules.
- polynucleotides a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
- a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracyl, other sugars and linking groups such as fluororibose and thioate, and nucleotide branches.
- sequence of nucleotides may be interrupted by non-nucleotide components.
- a polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.
- Other types of modifications included in this definition are caps, substitution of one or more of the naturally occurring nucleotides with an analog, and introduction of means for attaching the polynucleotide to proteins, metal ions, labeling components, other polynucleotides, or a solid support.
- the term "recombinant" polynucleotide means a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which either does not occur in nature or is linked to another polynucleotide in a nonnatural arrangement.
- the phrase "pharmaceutically acceptable” refers to general clinical use and/or approval by a regulatory agency of the Federal or state government, listing in the United States Pharmacopoeia, or general acceptance by those skilled in the relevant art.
- physiologic conditions for antibody binding reflect but do not necessarily exactly duplicate the conditions in which a human epidermal growth factor receptor 2-binding polypeptide would encounter a human epidermal growth factor receptor 2 molecule in vivo. Binding under physiologic conditions should be reasonably predictive that binding in vivo will occur.
- a “signal peptide” or “leader sequence” is a short amino acid sequence that directs a newly synthesized protein through a cellular membrane, usually the endoplasmic reticulum in eukaryotic cells, and either the inner membrane or both inner and outer membranes of bacteria. Signal peptides are typically at the N-terminal portion of a polypeptide and are typically removed enzymatically between biosynthesis and secretion of the polypeptide from the cell. The signal peptide is not present in the secreted protein, only during protein production. "Signal peptide” and “leader sequence” are used interchangeably herein.
- a "cell line” or “cell culture” denotes bacterial, plant, insect or higher eukaryotic cells grown or maintained in vitro. The descendants of a cell may not be completely identical (either morphologically, genotypically, or phenotypically) to the parent cell.
- a monoclonal antibody may be produced by a hybridoma or other cell. Methods of making hybridomas, both murine and human, are known in the art.
- the term "host cell” denotes a prokaryotic or eukaryotic cell that has been genetically altered, or is capable of being genetically altered by administration of an exogenous polynucleotide, such as a recombinant plasmid or vector. When referring to genetically altered cells, the term refers both to the originally altered cell, and to the progeny thereof.
- the host cell is a bacterial cell.
- the host cell is an E. coli cell, such as E. coli El 04 or E. coli TGI cells.
- the term "vector” refers to a recombinant DNA or RNA plasmid or virus that comprises a heterologous polynucleotide to be delivered into a target cell, either in vitro or in vivo.
- the heterologous polynucleotide may comprise a sequence of interest for purposes of therapy, and may optionally be in the form of an expression cassette.
- a vector need not be capable of replication in the ultimate target cell or subject.
- the term includes cloning vectors for the replication of a polynucleotide, and expression vectors for translation of a polynucleotide encoding sequence.
- viral vectors which comprise a polynucleotide encapsidated or enveloped in a viral particle.
- VB4-845 is a recombinant fusion protein comprising an anti-Epithelial Cell Adhesion Molecule (EpCAM) single-chain antibody genetically fused with a truncated form of Pseudomonas exotoxin A (252-6 ⁇ 8) (ETA (2 52-608)) and having His tags at the N- and C- terminus of the amino acid sequence.
- VB4-847 is a novel recombinant fusion protein consisting of an EpCAM single-chain antibody fused with ETA(252-608) without any His tags.
- a study was conducted to compare the expression from different constructs of VB4-847 (non-His) to VB4-845 (His) in order to determine the methods for clinical production of VB4-847.
- Biological testing of purified VB4-847 demonstrated that the potency of the molecule is not affected by removal of the His tags. Furthermore, based on the yields obtained in a non-optimized fermentation and purification run, the VB4-847/pING-RBS clone was demonstrated to be a feasible option for producing drug product at the clinical scale.
- CODA was removed by SOE PCR.
- the fragment containing EcoRI-CATA-Met-PelB-V L - Yu-BbvCl was cloned into the pCR 2.1 vector and transformed into 10F E. coli cells for sequencing.
- the pCR2.1 plasmid containing the correct insert was digested with EcoKl- BbvCl and ligated along with the BbvCl-N n-VE-Xhol fragment of VB4-847-CODA into pING/RBS plasmid pre-digested with EcoR ⁇ -Xho ⁇ .
- Chemically competent 10F E. coli cells were transformed with the ligation reaction and a transformed colony grown for plasmid extraction. Plasmid with the 2kb insert was then used to transform E. coli E104 and selected colonies grown for small-scale expression.
- the pCR2.1 plasmids containing the correct inserts were digested with EcoRl-Smal and Narl-Xhol for the 5' and 3' fragments, respectively, and ligated with the Smal-Vn-PE-Narl fragment into the pING3302 plasmid pre-digested with EcoRl-Xhol.
- Chemically competent 10F E. coli cells were transformed with the ligation reaction and a transformed colony grown for plasmid extraction. Plasmid with the 2kb insert was then used to subclone the complete VB4-847 fragment as an EcoRl-Xhol fragment into the pING-RBS plasmid pre-digested with the same enzymes.
- Chemically competent 10F E. coli cells were transformed with the ligation reaction and a transformed colony grown for plasmid extraction. Plasmid with the 2kb insert was then used to transform E. coli E104 and selected colonies grown for small-scale expression.
- CODA was removed by PCR mutagenesis using a 5' primer containing the EcoRl site followed by the initiation methionine codon and PelB sequence and a 3' primer in the VH region.
- the fragment containing i?coRI-Met-PelB-VL-VH- 3 ⁇ 4vCI was cloned into the pCR 2.1 vector and transformed into 10F E. coli cells for sequencing.
- a Hindlll site was introduced between 3' end of PE and the Xhol site by PCR mutagenesis and the fragment containing ApaLl-VE-Hindlll-Xhol was cloned into the pCR 2.1 vector and transformed into 10F E. coli cells for sequencing.
- the pCR2.1 plasmids containing the correct inserts were digested with EcoRl-BbvCl and ApaLl-Xhol for the 5' and 3' fragments, respectively, and ligated with the BbvCl-ApaLl VH-PE fragment into the pING3302 plasmid pre-digested with EcoRl-Xhol.
- Chemically competent 10F E. coli cells were transformed with the ligation reaction and a transformed colony grown for plasmid extraction. Plasmid with the 2kb insert was then used to subclone the complete VB4-847-CODA fragment as an EcoRl-Hindlll fragment into the pSJFI plasmid pre-digested with the same enzymes.
- Chemically competent 10F E. coli cells were transformed with the ligation reaction and a transformed colony grown for plasmid extraction. Plasmid with the 2kb insert was then used to transform E. coli TGI and selected colonies grown for small-scale expression
- Transformed TGI cells containing VB4-847-CODA expressed from the pSJFI plasmid were inoculated into 5 mL 2-YT containing 100 ⁇ g/mL kanamycin and incubated at 37°C with constant shaking at 225 rpm. After 16 hours of incubation, 600 overnight seed culture was inoculated into 30 mL TB (2% inoculum), and incubated at 28°C with constant shaking at 225 rpm for 16 hours. The culture was induced with 30 of 100 mM IPTG (0.1 mM final) and incubated at 28°C with constant shaking at 225 rpm for 10 hours.
- the cells were pelleted by centrifugation and extraction buffer (3 M NaCl, 2 mM EDTA, 1 mM Tris- HC1 pH 8.0) added at a ratio of 1 mL of buffer per gram of cell pellet. Following a 30 minute incubation with mixing, the pellet was harvested and the supernatant transferred to a new tube. Shock buffer was added to the pellet at a ratio of 4 mL per original gram of cell pellet and incubated with mixing for 30 minutes. The supernatant was then harvested and combined with the extraction buffer supernatant to yield the periplasmic extract. The volume was corrected to adjust to the original culture volume for analysis by Western blot.
- extraction buffer 3 M NaCl, 2 mM EDTA, 1 mM Tris- HC1 pH 8.0
- the level of expression was estimated by Western blot analysis. Briefly, 16 ⁇ . of induced culture periplasmic extract or supernatant and 4 ⁇ . LDS sample buffer were loaded onto a NuPAGE 10% Bis-Tris gel. The gel was then transferred to a nitrocellulose membrane at 40V for 1 hour. After blocking and washing the membrane, VB4-847 protein was detected using a rabbit anti-PE (1/5000) followed by a goat anti-rabbit antibody (1/1000) coupled to HRP. The membrane was developed using DAB and the level of expression of the VB4-847 proteins were compared to control VB4-845 as appropriate.
- E. coli E104 cells transformed with VB4-847/pING-RBS were cultivated in glycerol minimal media (GMM) + 25 ⁇ g/mL tetracycline in a 2 L Erlenmeyer flask in a shaker incubator (26°C, 200 rpm).
- GMM glycerol minimal media
- a volume of 150 mL was used to seed ⁇ 15 L of GMM in a 20 L Bioreactor.
- the cells were induced with the addition of L-arabinose for VB4-847 expression for ⁇ 40 hours.
- the supernatant was then collected by centrifugation using a disk stacked continuous centrifuge and further clarified by microfiltration.
- the eluate was then buffered exchanged by ultrafiltration against 20 mM sodium phosphate buffer pH 7.0 and applied to a Capto MMC column previously equilibrated with 20 mM sodium phosphate, pH 7.0. After sample loading, the column was washed with a 20 mM sodium phosphate, 100 mM NaCl pH 7.0 buffer until UV280 baselined.
- the Capto MMC column should not have successfully captured the protein because it generally requires that the protein to be captured has a lower pH than pi. However, the VB4-847 molecule precipitates when the pH falls below the pi. Surprisingly, the Capto MMC column was able to capture the VB4-847 protein despite the higher pH than pi.
- Bound VB4-847 was eluted with 20 mM sodium phosphate, 400 mM NaCl pH 7.0. The fractions containing the product were pooled, diluted to obtain a final concentration of 5 mM sodium phosphate, 200 mM NaCl, pH 7.1 and applied onto a ceramic hydroxy apatite (CHT) column previously equilibrated with 5 mM sodium phosphate, 200 mM NaCl pH 7.1 buffer. After washing to UV2 80 baseline with equilibration buffer, bound VB4-847 was eluted with 10 mM sodium phosphate, 200 mM NaCl pH 7.1 buffer.
- CHT ceramic hydroxy apatite
- the CHT flow through and wash was pooled based purity and was then diluted 2-fold to reduce the salt concentration and pH adjusted to 8.0 for application on a Q-sepharose high performance column.
- the column was equilibrated with 20 mM sodium phosphate, 100 mM NaCl pH 8.0. After sample loading, the column was washed to UV2 80 baseline with equilibration buffer and bound VB4-847 was eluted in 20 mM sodium phosphate, 200 mM NaCl, pH 8.0.
- the peak fractions from the Q-sepharose column contained an approximately 2% impurity in the form of a small molecular weight fragment.
- the present inventors found that ultrafiltration did not remove this impurity; therefore, a size exclusion column was used at this stage to remove the impurity as expected.
- the peak fractions were concentrated and applied onto a size-exclusion column (Sephacryl S200) that was equilibrated in 150 mM NaCl in 20mM sodium phosphate pH 7.5 buffer.
- the VB4-847 peak fractions from the Sephacryl S200 column was then concentrated and formulated to 5 mg/mL in 20 mM sodum phosphate buffer pH 8.0 containing 0.1 % Polysorbate 80.
- Cal-27 and A-375 cells were used to assess the potency of VB4-847.
- the cytotoxicity was measured by MTS assay (Promega) following a 3 day treatment. Briefly, cells were seeded at 5000 cells per well in a 96 well plate and allowed to adhere at 37°C for 2-3 hours. Subsequently, indicated drugs were added to the cells over a range of concentrations. After 3 days, the cell viability was determined and the IC50 was interpolated from the resulting plot.
- the expression of VB4-847 was then evaluated in E104 cells using the pING-RBS plasmid.
- the pING-RBS plasmid is a modification of the pING3302 plasmid in which the nucleotides between the end of the arabinose promoter and the RBS of the PelB leader sequence are deleted.
- the use of the pING/RBS plasmid has previously been shown to increase expression of both VB4-845 and an immunoconjugate comprising an EpCAM scFv and a de-immunized bouganin toxin (VB6-845).
- VB6-845 de-immunized bouganin toxin
- VB4-847/pING-RBS clone in E104 was fermented at the 15 L scale and purification performed as described above. The cytotoxicity of purified VB4-847 was then tested against the EpCAM positive cell line, Cal-27 and the EpCAM negative A-375 cell line. As seen in Figure 6A, VB4-847 was highly potent against Cal-27 cells with an average IC5 0 of 0.385 pM. This was similar to the control VB4-845-His which showed an average IC5 0 of 0.405 pM. As expected, neither VB4-847 or VB4-845-His displayed any potency against the antigen negative A-375 cells (Figure 6B).
- EpCAM-specific immunoconjugate The effect of modifying the RBS in the pING plasmid has been mixed in past experiences, depending on the insert being expressed.
- the modified RBS increased expression, whereas VB4-845-CODA-His expression was negatively affected when the modified RBS was used.
- VB4-845-His expression is greatly increased by the modified RBS.
- a comparison between VB4-845 (which contains His tags) and VB4-847 (no His tags) was conducted.
- VB4-847 was also amenable to periplasmic expression in E. coli. Although the amount of VB4-847-CODA extracted from the periplasm following induction was reasonable, periplasmic extraction is not the ideal process for large scale drug production and the use of a soluble expression system would be preferable.
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Abstract
The present invention relates to immunoconjugates having a ligand that binds to EpCAM and a toxin, and methods for making such immunoconjugates. Also encompassed by the invention are compositions and methods for the prevention or treatment of cancer.
Description
METHODS FOR MAKING AND USING AN IMMUNOCONJUGATE FOR THE
TREATMENT OF CANCER
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Application Serial No.
62/213,315, filed September 2, 2015, and 62/256,388, filed November 17, 2015, each of which is hereby incorporated by reference in its entirety for all purposes.
STATEMENT REGARDING THE SEQUENCE LISTING
[0002] The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is VIVE_034_01US_SeqList_ ST25.txt. The text file is 27 KB, was created on November 17, 2015, and is being submitted electronically via EFS-Web.
BACKGROUND OF THE INVENTION
[0003] Immunotherapy has emerged as a potentially effective new approach to combat cancer. One tumor associated antigen of interest for immunotherapy is EpCAM (for Epithelial Cell Adhesion Molecule, which also known as 17-1A, KSA, EGP-2 and GA733-2). EpCAM is a transmembrane protein that is highly expressed in many solid tumors, including carcinomas of the lung, breast, ovary, colorectum, bladder, and squamous cell carcinoma of the head and neck, but weakly expressed in most normal epithelial tissues.
[0004] One of the cancers associated with increased expression of EpCAM is squamous cell carcinoma of the head and neck ("HNSCC"). EpCAM expression correlates with the progression of HNSCC in humans. HNSCC is presently the sixth most common cancer in the world. HNSCC is a disease that causes significant morbidity, especially with respect to speech and swallowing functions. Removal of tumors in subjects suffering from HNSCC can be difficult or impossible due to their size and location. Surgery, radiation therapy, chemotherapy, or combinations of these are generally available as treatment options. Despite all attempts to cure patients afflicted with HNSCC, recurrence remains the most common cause of failure (in 40%-50% of patients) after head and neck cancer therapy. Salvage therapy consists of the same treatment options as for first line therapy. However, palliative surgery is often difficult and disfiguring. Furthermore, radiation therapy is rarely feasible or beneficial, and chemotherapy does not substantially improve survival rates in
HNSCC patients. Prognosis for these patients remains poor, such that the median survival after recurrence is only approximately six months. In addition, liver toxicity remains a problem for HNSCC patients treated with the available treatment options.
[0005] Bladder cancer is the 7th most common cancer worldwide that results in an estimated 260,000 new cases each year. Carcinomas in the bladder tissue are also associated with increased EpCAM expression, and occur almost entirely within the transitional epithelium, the surface layer of tissue that lines the bladder, as transitional cell carcinomas. At initial diagnosis, 70 to 90% of patients with bladder cancers have superficial disease which involves carcinomas in the superficial urothelial layer that are noninvasive and exhibit papillary (finger-like projections) tumors. Current treatment includes the intravesicular delivery of chemotherapy and immunotherapy with the bacille Calmette-Guerin (BCG) vaccine that involves the additional risk of systemic infection with the tuberculosis bacterium. Despite this aggressive treatment regime, 70% of these superficial papillary tumors will recur over a prolonged clinical course, causing significant morbidity; approximately 4 to 8% will progress to invasive carcinomas.
[0006] There is considerable need for the development of effective tumor-specific therapies for cancers including head and neck and bladder cancers.
SUMMARY OF THE INVENTION
[0007] The present disclosure relates to novel immunoconjugates, and methods for treating or preventing cancer by administering such immunoconjugates to a subject in need thereof. The present disclosure also relates to methods for making novel immunoconjugates. In some embodiments, the immunoconjugates comprise an antibody fragment that binds to the extracellular domain of human EpCAM and a toxin.
[0008] In one aspect, the present disclosure provides an immunoconjugate comprising an antigen binding domain and a toxin. In further embodiments, the antigen binding domain is an EpCAM antibody or a fragment thereof. In some embodiments, the immunoconjugate comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 2 or SEQ ID NO: 21. In further embodiments, the immunoconjugate comprises SEQ ID NO: 2 or SEQ ID NO: 21. In yet further embodiments, the immunoconjugate consists essentially of SEQ ID NO: 2 or SEQ ID NO: 21. In still further embodiments, the immunoconjugate consists of SEQ ID NO: 2 or SEQ ID NO: 21. In some embodiments, the amino acid sequence is encoded by a non codon- optimized nucleic acid sequence. In some embodiments the present disclosure provides a
nucleic acid molecule enoding the immunoconjugate comprising SEQ ID NO: 2 or SEQ ID NO: 21. In some embodiments, the immunoconjugate exhibits cytotoxic activity against EpCAM-expressing cells.
[0009] In some embodiments, the immunoconjugate comprises (i) an antigen binding domain comprising a light chain CDR1 having at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 5, a light chain CDR2 having at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 6, a light chain CDR3 having at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 7, a heavy chain CDR1 having at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 12, a heavy chain CDR2 having at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 13, and a heavy chain CDR3 having at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 14; and (ii) a toxin. In further embodiments, the amino acid sequence of the immunoconjugate does not comprise a His tag. In some embodiments, the amino acid sequence of the immunoconjugate comprises a single His tag. In further embodiments, the single His tag is located at the N-terminus of the immunoconjugate. In some embodiments, the antigen binding domain comprises light chain CDR1, CDR2, and CDR3 comprising an amino acid sequence according to SEQ ID NOs: 5, 6, and 7, respectively; and a heavy chain CDR1, CDR2, and CDR3 according to SEQ ID NOs: 12, 13, and 14, respectively. In some embodiments, the antigen binding domain comprises a light chain variable region having at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 4 and a heavy chain variable region at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 11. In some embodiments, the antigen binding domain is an scFv. In further embodiments, the scFv comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 19.
[0010] In some embodiments, the toxin of the immunoconjugates provided herein is
ETA(252-608). In some embodiments, the toxin comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 18. In some embodiments, the antigen binding domain is conjugated to the toxin via a linker. In further embodiments, the linker comprises a sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 16.
[0011] In some embodiments, the immunoconjugate is encoded by a non-codon optimized nucleic acid sequence. In some embodiments, the present disclosure provides a nucleic acid molecule comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 1. In some embodiments, the present
disclosure provides a nucleic acid molecule comprising a sequence according to SEQ ID NO: 1. In some embodiments, the present disclosure provides a protein encoded by a nucleic acid sequence according to SEQ ID NO: 1. In some embodiments, the present disclosure provides expression vectors and host cells comprising expression vectors. In further embodiments, the expression vectors provided herein comprise a nucleic acid molecule according to SEQ ID NO: 1.
[0012] The compositions and immunoconjugates provided herein, in some embodiments, exhibit cytotoxic activity against EpCAM-expressing cells. For example, in some embodiments, the compositions and immunoconjugates provided herein exhibit cytotoxic activity against EpCAM positive cell lines such as, for example, Cal-27 or TCCSUP cells.
[0013] In one aspect, the present disclosure provides methods for treating or preventing cancer comprising administering a composition provided herein. In some embodiments, the cancer is head and neck cancer or bladder cancer. In further embodiments, the cancer is head and neck cancer is squamous cell carcinoma of the head and neck (HNSCC). In some embodiments, the compositions provided herein are administered to a subject in need thereof. In further embodiments, the subject is a human. In some embodiments, the composition is administered directly to the cancer site. In some embodiments, the composition is administered intratumorally, intravesicularly, or peritumorally. In other embodiments, the composition is administered systemically. In further embodiments, the systemic administration is intravenous administration.
[0014] In some embodiments, the present disclosure provides methods for treating or preventing cancer comprising administering to a subject in need thereof a composition provided herein, and further administering to the subject one or more additional therapeutic agent. In further embodiments, the additional therapeutic agent is administered simultaneously, separately, or sequentially with the composition provided herein. In some embodiments, the additional therapeutic agent is a chemotherapeutic drug or a radiotherapeutic drug.
[0015] In some embodiments, the present disclosure provides methods for reducing the size of one or more tumors present in a subject, wherein the method comprises administering a composition provided herein to the subject. In some embodiments, the present disclosure provides methods for treating cancer comprising administering a composition provided herein followed by surgical removal of one or more tumors present in the subject.
[0016] The present disclosure also provides kits for treating and preventing cancer, the kits comprising an effective amount of the compositions provided herein and directions for the use thereof to treat or prevent the cancer.
[0017] In one aspect, the present disclosure provides methods for producing a protein, the method comprising (i) inserting a non-codon optimized nucleic acid sequence encoding SEQ ID NO: 2 or SEQ ID NO: 21 in a plasmid, (ii) transforming E. coli cells in a cell culture with the plasmid; (iii) inducing expression of the protein encoded by the nucleic acid sequence, and (iv) obtaining the protein from the cell culture supernatant. In some embodiments, the methods further comprise purifying the protein. For example, in some embodiments, the present disclosure provides methods for producing and purifying the immunoconjugates and compositions provided herein for in vitro, diagnostic, or clinical use.
[0018] In some embodiments, the E. coli cells used in the methods provided herein are E. coli E104 or TGI cells. In some embodiments, the plasmid used in the methods provided herein is selected from pING-RBS, pING3302, and pSJFI plasmids. In some embodiments,
[0019] In some embodiments, the present disclosure provides methods for purifying the proteins provided herein. In further embodiments, the methods comprise purifying the protein using one or more chromatographic steps. For example, in some embodiments, the methods comprise contacting the protein with at least one chromatographic material. Chromatographic materials that maybe used in the methods provided herein include mixed mode, ion-exchange, affinity, hydrophobic interaction, reverse phase, size exclusion, and adsorption materials. In some embodiments, the chromatographic material is hydroxy apatite and/or Capto MMC. In some embodiments, the production and purification methods provided herein comprise concentrating the protein, for example, by diafiltration, ultrafiltration, or tangential flow filtration. In particular embodiments, the methods comprise contacting the protein with a Capto MMC chromatographic material.
[0020] In one aspect, the present disclosure provides methods for producing and purifying an immunoconjugate comprising an EpCAM antibody fragment and a toxin, the methods comprising the following steps: (i) inserting a non-codon optimized nucleic acid sequence encoding SEQ ID NO: 2 or SEQ ID NO: 21 in a plasmid, (ii) transforming E. coli cells in a cell culture with the plasmid, (iii) inducing expression of the protein encoded by the nucleic acid sequence, and (iv) obtaining the protein from the cell culture supernatant, (v) clarifying the supernatant by microfiltration; (vi) concentrating and diafiltrating the supernatant containing the protein, (vii) contacting and eluting the protein on a Q-sepharose column,
(viii) contacting the protein with a Capto MMC column, (ix) eluting the protein from the Capto MMC column, (x) contacting the eluted protein with a hydroxy apatite column, (xi) eluting the protein from the hydroxyapatite column, (xii) adjusting the pH of the eluate to about 8.0, (xiii) contacting the eluted protein with a Q-sepharose high performance column, ( xiv) eluting the protein from the Q-sepharose high performance column and (xv) contacting the protein on a Sephacryl S200 size exclusion column prior to buffer exchange by tangential flow filtration using 10 kDa MWCO hydrosart membrane for final formulation.
[0021] Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
DESCRIPTION OF THE DRAWINGS
[0022] Figure 1A-1C provides the nucleic acid and amino acid sequences of VB4-
847 SEQ ID NOs: 1 and 2.
[0023] Figure 2 shows a Western blot of VB4-847-CODA expressed from pING3302 in E. coli El 04. Supernatants of 3 VB4-847-CODA induced clones (lane 1, 2 and 3) were loaded under non-reducing conditions on a SDS-PAGE gel and immunoblotted with a rabbit anti-PE antibody (1/5000) and sheep anti-rabbit-HRP antibody (1/1000). The induced supernatant of VB4-845-CODA-His was loaded on lane 4.
[0024] Figure 3 shows a Western blot of VB4-847-CODA expressed from pING-
RBS in E. coli E104. Supernatants of 2 VB4-847-CODA induced clones (lane 1 and 2) were loaded under non-reducing conditions on a SDS-PAGE gel and immunoblotted with a rabbit anti-PE antibody (1/5000) and sheep anti-rabbit-HRP antibody (1/1000). The induced supernatant of VB4-847-CODA/3302 was loaded on lane 3. Lane L corresponds to the ladder.
[0025] Figure 4 shows a Western blot of VB4-847 expressed from pING-RBS in E. coli E104. Supernatants of VB4-847/pING3302 (lane 1), VB4-847/pING-RBS (lane 2), VB4- 847-CODA/pING3302 (lane 3), VB4-847-CODA/pING-RBS (lane 4), VB4-845- His/pING3302 (land 5) and VB4-845-His/pING-RBS (lane 6) were loaded under non- reducing conditions on a SDS-PAGE gel and immunoblotted with a rabbit anti-PE antibody (1/5000) and sheep anti-rabbit-HRP antibody (1/1000). Lane L corresponds to the ladder.
[0026] Figure 5 shows a Western blot of VB4-847-CODA expressed from pSJFl in
E. coli TGI . Periplasmic extracts of 2 VB4-847-CODA induced clones (lane 1 and 2) were loaded under non-reducing conditions on a SDS-PAGE gel and immunoblotted with a rabbit anti-PE antibody (1/5000) and sheep anti-rabbit-HRP antibody (1/1000). The induced supernatants of VB4-847-CODA and VB4-845-CODA-His were loaded on lanes 3 and 4, respectively. Lane L corresponds to the ladder.
[0027] Figures 6A and 6B show the cytotoxic activity of VB4-847 against Cal-27
(6A) and A-375 (6B) cells measured by MTS assay. Cells were incubated with VB4-847 (open circle) or VB4-845-His (filled circle). After 3 days incubation, cell viability was measured and IC50 determined.
DETAILED DESCRIPTION
[0028] The present disclosure provides immunoconjugates comprising a binding protein that binds to EpCAM and a toxin, methods of use thereof, and methods for producing and purifying immunoconjugates comprising a binding protein that binds to EpCAM and a toxin. In one aspect, the immunoconjugates do not comprise His tags. In another aspect, the immunoconjugates comprise a single His tag. The immunoconjugates provided herein advantageously can be effectively purified using the methods provided herein, which do not require the presence of a His tag, or which utilize the presence of a single His tag. In some embodiments, the immunoconjugates provided herein result in a reduction in toxicity in subjects.
[0029] In the context of a protein that has been engineered to contain a histidine tag, herein referred to as a "His tagged protein", the nickel column used during purification removes impurities. In one aspect, the present disclosure provides methods for purifying proteins lacking a histidine tag, herein referred to as "non-His tagged proteins". The methods provided herein, comprising, for example, the use of mixed mode and size exclusion columns, provided superior, and quite unexpected results for purifying a non-His tagged protein.
EpCAM Binding Proteins
[0030] In one aspect, the present disclosure provides immunoconjugates comprising EpCAM binding proteins. In some embodiments, the EpCAM binding protein comprises a light chain CDR1 having an amino acid sequence having at least about 80%, at least about 85%, at least
about 90%, at least about 95%, or at least about 99% homology to a sequence as set forth in SEQ ID NO: 3
[0031] In some embodiments, the EpCAM binding protein comprises a light chain variable region encoded by a nucleic acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homology to a sequence as set forth in SEQ ID NO: 3. In some embodiments, the binding protein comprises a heavy chain variable region encoded by a nucleic acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homology to a sequence as set forth in SEQ ID NO: 10. In some embodiments, the binding protein comprises a light- heavy chain linker, wherein the light-heavy chain linker is encoded by a nucleic acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homology to a sequence as set forth in SEQ ID NO: 8. In some embodiments, the binding protein comprises a light-heavy chain linker encoded by a nucleic acid sequence according to SEQ ID NO: 8.
[0032] In some embodiments, the binding protein comprises a light chain variable region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homology to an amino acid sequence as set forth in SEQ ID NO: 4. In some embodiments, the binding protein comprises a heavy chain variable region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homology to an amino acid sequence as set forth in SEQ ID NO: 11.
[0033] In some embodiments, the binding protein is an antibody or a fragment thereof. Antibodies and fragments thereof are selected from polyclonal antibodies, monoclonal antibodies, Fab, Fab', (Fab')2, single chain fragments (scFv), disulfide-stabilized fragments (dsFv), single domain antibodies (sdAb), diabodies, and cys-diabodies (cysteine-modified diabodies). In some embodiments, diabodies include bivalent, bispecific antibodies or antibody fragments. In some embodiments, diabodies include bivalent antibodies that are made up of the same or different antibody or antibody fragment polypeptides; thus, in some embodiments, the diabodies are homo-multimers or hetero-multimers. Antibodies and fragments thereof may comprise an immunoglobulin constant region selected from the group consisting of IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgD, IgE, and IgM. The antibody or fragment thereof may be from any species including mice, rats, rabbits, hamsters, and humans. In one embodiment, the antibody or fragment is chimeric. Chimeric antibodies or fragments thereof are antibody molecules that combine a non-human animal variable region and a constant region or portion of a constant region that is human. In some embodiments, the
immunoconjugates provided herein comprise an scFv having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homology to an amino acid sequence as set forth in SEQ ID NO: 19.
[0034] In some embodiments, the antibody fragment may be Fab, and the light chain and the heavy chain are linked by a covalent bond. In some embodiments, the covalent linkage may be a disulfide bond. In some embodiments, the covalent linkage may be through chemical crosslinkers, such as dimethyl adipimidate, dimethyl suberimidate, and the like. In some embodiments, amino acid crosslinkers, such as (Gly4-Ser)n may be used. The sequences of the light chain and the heavy chain described herein may be used to derive scFv, diabodies, tribodies, tetrabodies, and the like. Various protein linking strategies may be used to produce bivalent or bispecific Fab and scFvs, as well as bifunctional Fab and scFv fusions.
[0035] The antigen-binding fragments of any of the above-described binding proteins are prepared using means known in the art, for example, by preparing nested deletions using enzymatic degradation or convenient restriction enzymes. In some embodiments, the humanized antibodies, chimeric antibodies or immunoreactive fragments thereof are screened to ensure that antigen binding has not been disrupted by the humanization, chimerization, or fragmentation of the parent monoclonal antibody. This may be accomplished by any of a variety of means known in the art, including, for example, use of a phage display library.
Toxins
[0036] In one aspect, the present disclosure provides an immunoconjugate comprising an antibody fragment that binds to the extracellular domain of human EpCAM and a toxin. In some embodiments, the immunoconjugate comprises more than one toxin. For example, in some embodiments, the immunoconjugate comprises a diabody comprising more than one toxin.
[0037] In some embodiments, the toxin is a molecule that blocks protein synthesis in a target cell, therein leading to cell death. Thus, in some embodiments, the toxin is a cytotoxin. Cytotoxins are known in the art and include, for example, Pseudomonas exotoxin A (ETA) or variants thereof; geionin, bouganin, deimmimized bouganin (e.g., a toxin as described in U.S. Patent No. 7,339,031, incorporated by reference herein in its entirety), saporin, ricin, ricin A chain, bryodin, diphtheria toxin, and restrictocin. In some embodiments, the toxin is Pseudomonas exotoxin A (ETA) or a variant thereof. In further embodiments, the toxin is ETA(252-608). ETA(252-608), which is a truncated form of ETA
that lacks the cell binding domain, is a single polypeptide fusion protein produced by continuous translation of a single construct.
[0038] In some embodiments, the toxin is encoded by a nucleic acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homology to SEQ ID NO: 17. In some embodiments, the toxin is encoded by a nucleic acid sequence according to SEQ ID NO: 17. In some embodiments, the toxin comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homology to SEQ ID NO: 18.
[0039] In other embodiments, the toxm comprises an agent that acts to disrupt DNA. Thus, toxins may comprise, without limitation, enediynes (e.g., calicheamicin and esperamicin) and non-enediyne small molecule agents (e.g., bleomycin, methidiumpropyl-EDTA-Fe(II)). Other toxins useful in accordance with the invention include, without limitation, daunorubicin, doxorubicin, distamycin A, cisplatin, mitomycin C, ecteinascidins, duocarmycin/CC-1065, and bleomyem/pepleomyem. in some embodiments, the toxin comprises an agent that acts to disrupt tubulin. Such toxins may comprise, without limitation, rhizoxin/maytansine, paclitaxel, vincristine and vinblastine, colchicine, auri statin dolastatin 10 MMAE, and peloruside A. in other nonlimiting embodiments, the toxin may comprise an alkylating agent including, without limitation, Asaiey NSC 167780, AZQ NSC 182986, BCNU NSC 409962, Busulfan NSC 750, carboxyphthaiatoplatinum NSC 271674, CBDCA NSC 241240, CCNU NSC 79037, CHIP NSC 256927, chlorambucil NSC 3088, chlorozotocin NSC 178248, cis- platmum NSC 119875, clomesone NSC 338947, cyanomorpholinodoxorubicin NSC 357704, cyclodisone NSC 348948, dianhydrogalactitol NSC 132313, fluorodopan NSC 73754, hepsulfam NSC 329680, hycanthone NSC 142982, melphaJan NSC 8806, methyl CCNU NSC 95441, mitomycin C NSC 26980, mitozolamide NSC 353451, nitrogen mustard NSC 762, PCNU NSC 95466, piperazine NSC 344007, piperazinedione NSC 135758, pipobroman NSC 25154, porfiromycm NSC 56410, spirohydantoin mustard NSC 172112, teroxirone NSC 296934, tetraplatin NSC 363812, thio-tepa NSC 6396, triethylenemelamine NSC 9706, uracil nitrogen mustard NSC 34462, and Yoshi-864 NSC 102627. In other embodiments, the toxin may comprise an antimitotic agent including, without limitation, allocolchicine NSC 406042, Halichondrin B NSC 609395, colchicine NSC 757, colchicine derivative NSC 33410, dolastatin 10 NSC 376128 (NG— auri statin derived), maytansme NSC 153858, rhizoxm NSC 332598, taxol NSC 125973, taxol derivative NSC 608832, thiocolchicine NSC 361792, trityl cysteine NSC 83265, vinblastine sulfate NSC 49842, and vincristine sulfate NSC 67574
[0040] In embodiments, the toxin may comprise an topoisomerase I inhibitor including, without limitation, camptothecm NSC 94600, camptothecin, Na salt NSC 100880, aminocamptothecin NSC 603071 , camptothecm derivative NSC 95382, camptothecin derivative NSC 107124, camptoihecin derivative NSC 643833, camptothecin derivative NSC 629971, camptothecin derivative NSC 295500, camptothecin derivative NSC 249910, camptothecin derivative NSC 606985, camptothecin derivative NSC 374028, camptothecin derivative NSC 176323, camptothecin derivative NSC 295501, camptothecin derivative NSC 606172, camptothecin derivative NSC 606173, camptothecin derivative NSC 610458, camptothecin derivative NSC 618939, camptothecin derivative NSC 610457, camptothecin derivative NSC 610459, camptothecin denvative NSC 606499, camptothecin derivative NSC 610456, camptothecin derivative NSC 364830, camptotheci derivative NSC 606497, and morpholinodoxorubicin NSC 354646.
[0041] In embodiments, the toxin may comprise an topoisomerase II inhibitor including, without limitation, doxorubicin NSC 123127, amonafide NSC 308847, m-AMSA NSC 249992, anthrap razole derivative NSC 355644, pyrazoloacridine NSC 366140, bisantrene HCL NSC 337766, daunorubicin NSC 82151 , deoxydoxorubicin NSC 267469, mitoxantrone NSC 301739, menogaril NSC 269148, Ν,Ν-dihenzyl daunomycin NSC 268242, oxanthrazole NSC 349174, rubidazone NSC 164011, VM-26 NSC 122819, and VP-16 NSC 141540.
[0042] In other embodiments, the toxin may comprise an RNA or DNA antimetabolite including, without limitation, L-alanosine NSC 153353, 5-azacytidine NSC 102816, 5- fluorouracil NSC 19893, acivicin NSC 163501, aminopterin derivative NSC 132483, aminopterin derivative NSC 184692, ammopterin derivative NSC 134033, an antifoi NSC 633713, an antifoi NSC 623017, Baker's soluble antifoi NSC 139105, dichlorallyl lawsone NSC 126771, brequinar NSC 368390, ftorafur (pro-drug) NSC 148958, 5,6-dihydro-5- azacytidme NSC 264880, methotrexate NSC 740, methotrexate derivative NSC 174121, N- (phosphonoacetyl)-L-aspartate (PALA) NSC 224131, pyrazoiurin NSC 143095, trimetrexate NSC 352122, 3 -HP NSC 95678, 2'-deoxy-5-fluorouridine NSC 27640, 5-HP NSC 107392, alpha-TGDR NSC 71 851 , aphidicolin glycinate NSC 303812, ara-C NSC 63878, 5-aza-2'- deoxycytidme NSC 127716, beta-TGDR NSC 71261, cyclocytidme NSC 145668, guanazole NSC 1 895, hydroxyurea NSC 32065, inosine glycodi aldehyde NSC 118994, macbecm II NSC 330500, pyrazoloimidazole NSC 51 143, thioguanine NSC 752, and thiopurine NSC 755.
[0043] Furthermore, a toxin may be altered to decrease or inhibit binding outside of the context of the immunoconjugate, or to reduce specific types of toxicity. For example, the
toxin may be altered to adjust the isoelectric point to approximately 7.0 such that liver toxicity is reduced.
[0044] In some embodiments, the immunoconjugates useful in the methods of the present disclosure comprise a binding protein that is attached to a toxin by a peptide linker. Peptide linkers may be cleavable linkers or non-cleavable linkers. In some embodiments, the linker is a cleavable linker selected from the group consisting of a furin sensitive linker, a cathepsin sensitive linker, a metalloproteinase linker, or a lysosomal hydrolase sensitive linker. In some embodiments, the linker is a flexible linker. In some embodiments, the linker is between about 5 and about 50 amino acids in length. In further embodiments, the linker is between about 10 and about 30 amino acids in length. In further embodiments, the linker is about 20 amino acids in length. In some embodiments, the linker is encoded by a nucleic acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homology to SEQ ID NO: 15. In some embodiments, the linker has at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homology to SEQ ID NO: 16.
Immunoconjugates
[0045] The antibodies or the antibody fragments described herein may be conjugated to the effector molecule by any means. For example, the antibody or the antibody fragment may be attached to the toxin by chemical or recombinant means. Chemical means for preparing fusions or conjugates are known in the art and can be used to prepare the immunoconjugate. The method used to conjugate the antibody or the antibody fragment and toxin must be capable of joining the antibody with the toxin without interfering with the ability of the antibody or the antibody fragment to bind to the target molecule.
[0046] In one embodiment, the antibody and toxin are both proteins and can be conjugated using techniques well known in the art. There are several hundred crosslinkers disclosed in the art that can conjugate two proteins. The crosslinker is generally chosen based on the reactive functional groups available or inserted on the antibody or toxin. In addition, if there are no reactive groups, a photoactivatible crosslinker can be used. In certain instances, it may be desirable to include a spacer between the antibody and the toxin. Crosslinking agents known to the art include the homobifunctional agents: glutaraldehyde, dimethyladipimidate and bis(diazobenzidine) and the heterobifunctional agents: m- maleimidobenzoyl-N-hydroxysuccinimide and sulfo-m maleimidobenzoyl-N- hy droxy succinimi de.
[0047] Other crosslinkers that may be used to couple an effector molecule to the antibody fragment include TPCH(S-(2- thiopyridyl)-L-cysteine hydrazide and TPMPH ((S- (2-thiopyridyl) mercapto- propionohydrazide). TPCH and TPMPH react at the carbohydrate moieties of glycoproteins that have been previously oxidized by mild periodate treatment, thus forming a hydrazone bond between the hydrazide portion of the crosslinker and the periodate generated aldehydes. The hetero-bifunctional crosslinkers GMBS (N-gama- malimidobutyryloxy)-succinimide) and SMCC (succinimidyl 4-(N-maleimido- methyl)cyclohexane) react with primary amines, thus introducing a maleimide group onto the component. This maleimide group can subsequently react with sulfhydryls on the other component, which can be introduced by previously mentioned crosslinkers, thus forming a stable thioether bond between the components. If steric hindrance between components interferes with either component's activity, crosslinkers can be used which introduce long spacer arms between components and include derivatives, such as n-succinimidyl-3-(2- pyridyldithio)propionate (SPDP). Thus, there is an abundance of suitable crosslinkers that can be used and each of which is selected depending on the effects it has on optimal immunoconjugate production.
VB4-847
[0048] In one aspect of the present disclosure, the immunoconjugate is VB4-847.
VB4-847 and VB4-845 are both recombinant fusion proteins comprising a humanized single- chain antibody fragment (scFv) specific for EpCAM antigen linked to ETA(252-608). VB4- 845 is described in U.S. Patent No. 8,545,840, which is incorporated by reference herein in its entirety. VB4-845 is produced in E. coli cells and purified by a process utilizing N- and C- terminal Histidine tags (His tags) on the VB4-845 molecule and including a 2+~ iminodiacetic (IDA) column for purification. Thus, VB4-845 comprises two His tags. In contrast to VB4-845, in some embodiments VB4-847 does not include His tags in the amino acid sequence. In other embodiments, VB4-847 includes a single His tag. In some embodiments, VB4-847 includes only a C-terminal His tag. In preferred embodiments, VB4- 847 includes only an N-terminal His tag. VB4-847 is generated by expressing a plasmid comprising a non-codon optimized DNA sequence in E. coli cells. Advantageously, VB4-847 can be produced and purified to optimal levels for clinical use in treatment or diagnostics.
[0049] The nucleic acid and amino acid sequences of VB4-847 are provided below in
Table 1.
Table 1. VB4-847 sequences
Description Sequence
GGG AAT TCC ATA ATG AAA TAC CTA TTG CCT ACG GCA
GCC GCT GGA TTG TTA TTA CTC GCT GCC CAA CCA GCG
ATG GCG GAT ATC CAG ATG ACC CAG TCC CCG TCC TCC
CTG AGT GCT TCT GTT GGT GAC CGT GTT ACC ATC ACC
TGC CGT TCC ACC AAA TCC CTC CTG CAC TCC AAC GGT
ATC ACC TAC CTT TAT TGG TAT CAA CAG AAA CCG GGT
AAA GCT CCG AAA CTT CTG ATC TAC CAG ATG TCC AAC
CTG GCT TCC GGT GTT CCG TCT CGT TTC TCC AGT TCT
GGT TCT GGT ACC GAC TTC ACC CTG ACC ATC TCT TCT
CTG CAG CCG GAA GAC TTC GCT ACC TAC TAC TGC GCT
CAG AAC CTG GAA ATC CCG CGT ACC TTC GGT CAG GGT
ACC AAA GTT GAA CTT AAG CGC GCT ACC CCG TCT CAC
AAC TCC CAC CAG GTT CCA TCC GCA GGC GGT CCG ACT
GCT AAC TCT GGA ACT AGT GGA TCC GAA GTA CAG CTG
GTT CAG TCC GGC CCG GGT CTT GTT CAA CCG GGT GGT
TCC GTT CGT ATC TCT TGC GCT GCT TCT GGT TAC ACG
TTC ACC AAC TAC GGC ATG AAC TGG GTC AAA CAG GCT
CCG GGT AAA GGC CTG GAA TGG ATG GGC TGG ATC AAC
ACC TAC ACC GGT GAA TCC ACC TAC GCT GAC TCC TTC
AAA GGT CGC TTC ACT TTC TCC CTC GAC ACA AGT GCT
AGT GCT GCA TAC CTC CAA ATC AAC TCG CTG CGT GCA
GAG GAT ACA GCA GTC TAT TAC TGC GCC CGT TTC GCT
ATC AAA GGT GAC TAC TGG GGT CAA GGC ACG CTG CTG
ACC GTT TCC TCG GAA TTT GGT GGC GCG CCG GAG TTC
CCG AAA CCG TCC ACC CCG CCG GGT TCT TCT GGT TTA
VB4-847 GAG GGC GGC AGC CTG GCC GCG CTG ACC GCG CAC CAG nucleic acid GCC TGC CAC CTG CCG CTG GAG ACT TTC ACC CGT CAT sequence CGC CAG CCG CGC GGC TGG GAA CAA CTG GAG CAG TGC
GGC TAT CCG GTG CAG CGG CTG GTC GCC CTC TAC CTG
GCG GCG CGA CTG TCA TGG AAC CAG GTC GAC CAG GTG
ATC CGC AAC GCC CTG GCC AGC CCC GGC AGC GGC GGC
GAC CTG GGC GAA GCG ATC CGC GAG CAG CCG GAG CAG
GCC CGT CTG GCC CTG ACC CTG GCC GCC GCC GAG AGC
GAG CGC TTC GTC CGG CAG GGC ACC GGC AAC GAC GAG
GCC GGC GCG GCC AGC GCC GAC GTG GTG AGC CTG ACC
TGC CCG GTC GCC GCC GGT GAA TGC GCG GGC CCG GCG
GAC AGC GGC GAC GCC CTG CTG GAG CGC AAC TAT CCC
ACT GGC GCG GAG TTC CTC GGC GAC GGT GGC GAC GTC
AGC TTC AGC ACC CGC GGC ACG CAG AAC TGG ACG GTG
GAG CGG CTG CTC CAG GCG CAC CGC CAA CTG GAG GAG
CGC GGC TAT GTG TTC GTC GGC TAC CAC GGC ACC TTC
CTC GAA GCG GCG CAA AGC ATC GTC TTC GGC GGG GTG
CGC GCG CGC AGC CAG GAT CTC GAC GCG ATC TGG CGC
GGT TTC TAT ATC GCC GGC GAT CCG GCG CTG GCC TAC
GGC TAC GCC CAG GAC CAG GAA CCC GAC GCG CGC GGC
CGG ATC CGC AAC GGT GCC CTG CTG CGG GTC TAT GTG
CCG CGC TCC AGC CTG CCG GGC TTC TAC CGC ACC GGC
CTG ACC CTG GCC GCG CCG GAG GCG GCG GGC GAG GTC
GAA CGG CTG ATC GGC CAT CCG CTG CCG CTG CGC CTG
GAC GCC ATC ACC GGC CCC GAG GAG GAA GGC GGG CGC
CTG GAG ACC ATT CTC GGC TGG CCG CTG GCC GAG CGC
ACC GTG GTG ATT CCC TCG GCG ATC CCC ACC GAC CCG
CGC AAC GTC GGT GGC GAC CTC GAC CCG TCC AGC ATC
CCC GAC AAG GAA CAG GCG ATC AGC GCC CTG CCG GAC TAC GCC AGC CAG CCC GGC AAA CCG CCG AAA GAC GAA CTG TAG TGA CTC GAG
DIQMTQSPSSLSASVGDRVTITCRSTKSLLHSNGITYLYWYQQKPGKAP KLLIYQMSNLASGVPSRFSSSGSGTDFTLTISSLQPEDFATYYCAQNLE IPRTFGQGTKVELKRATPSHNSHQVPSAGGPTANSGTSGSEVQLVQSGP GLVQPGGSVRISCAASGYTFTNYGMNWVKQAPGKGLEWMGWINTYTGES TYADSFKGRFTFSLDTSASAAYLQINSLRAEDTAVYYCARFAIKGDYWG
VB4-847 QGTLLTVSSEFGGAPEFPKPSTPPGSSGLEGGSLAALTAHQACHLPLET immuno- FTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPG conjugate SGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASADWS
LTCPVAAGECAGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWT VERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWR GFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTGL TLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTV VIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPKDEL
GAT ATC CAG ATG ACC CAG TCC CCG TCC TCC CTG AGT
GCT TCT GTT GGT GAC CGT GTT ACC ATC ACC TGC CGT
TCC ACC AAA TCC CTC CTG CAC TCC AAC GGT ATC ACC
Nucleotide TAC CTT TAT TGG TAT CAA CAG AAA CCG GGT AAA GCT sequence of CCG AAA CTT CTG ATC TAC CAG ATG TCC AAC CTG GCT
TCC GGT GTT CCG TCT CGT TTC TCC AGT TCT GGT TCT
GGT ACC GAC TTC ACC CTG ACC ATC TCT TCT CTG CAG
CCG GAA GAC TTC GCT ACC TAC TAC TGC GCT CAG AAC
CTG GAA ATC CCG CGT ACC TTC GGT CAG GGT ACC AAA
GTT GAA CTT AAG CGC
Amino acid
DIQMTQSPSSLSASVGDRVTITCRSTKSLLHSNGITYLYWYQQKPGKAP
sequence of
KLLIYQMSNLASGVPSRFSSSGSGTDFTLTISSLQPEDFATYYCAQNLE IPRTFGQGTKVELKR
VB4-847
RSTKSLLHSNGITYLY LCDR1
VB4-847
QMSNLAS LCDR2
VB4-847
AQNLEIPRT LCDR3
Nucleotide
sequence of GCT ACC CCG TCT CAC AAC TCC CAC CAG GTT CCA TCC VB4-847 GCA GGC GGT CCG ACT GCT AAC TCT GGA ACT AGT GGA light-heavy TCC
chain linker
Amino acid
sequence of
VB4-847 ATPSHNSHQVPSAGGPTANSGTSGS light-heavy
chain linker
GAA GTA CAG CTG GTT CAG TCC GGC CCG GGT CTT GTT CAA CCG GGT GGT TCC GTT CGT ATC TCT TGC GCT GCT
Nucleotide TCT GGT TAC ACG TTC ACC AAC TAC GGC ATG AAC TGG sequence of GTC AAA CAG GCT CCG GGT AAA GGC CTG GAA TGG ATG VH GGC TGG ATC AAC ACC TAC ACC GGT GAA TCC ACC TAC GCT GAC TCC TTC AAA GGT CGC TTC ACT TTC TCC CTC GAC ACA AGT GCT AGT GCT GCA TAC CTC CAA ATC AAC TCG CTG CGT GCA GAG GAT ACA GCA GTC TAT TAC TGC
GCC CGT TTC GCT ATC AAA GGT GAC TAC TGG GGT CAA
GGC ACG CTG CTG ACC GTT TCC TCG
Amino acid EVQLVQSGPGLVQPGGSVRISCAASGYTFTNYGMNWVKQAPGKGLEWMG sequence of WINTYTGESTYADSFKGRFTFSLDTSASAAYLQINSLRAEDTAVYYCAR
1 1
VH FAIKGDYWGQGTLLTVSS
VB4-847 NYGMN 12 HCDR1
VB4-847 WINTYTGESTYADSFKG 13 HCDR2
VB4-847 FAIKGDY 14 HCDR3
VB4-847 GAA TTT GGT GGC GCG CCG GAG TTC CCG AAA CCG TCC
scFv-toxin ACC CCG CCG GGT TCT TCT GGT TTA 15 Linker
VB4-847
scFv-toxin EFGGAPEFPKPSTPPGSSGL 16
Linker
GAG GGC GGC AGC CTG GCC GCG CTG ACC GCG CAC CAG
GCC TGC CAC CTG CCG CTG GAG ACT TTC ACC CGT CAT
CGC CAG CCG CGC GGC TGG GAA CAA CTG GAG CAG TGC
GGC TAT CCG GTG CAG CGG CTG GTC GCC CTC TAC CTG
GCG GCG CGA CTG TCA TGG AAC CAG GTC GAC CAG GTG
ATC CGC AAC GCC CTG GCC AGC CCC GGC AGC GGC GGC
GAC CTG GGC GAA GCG ATC CGC GAG CAG CCG GAG CAG
GCC CGT CTG GCC CTG ACC CTG GCC GCC GCC GAG AGC
GAG CGC TTC GTC CGG CAG GGC ACC GGC AAC GAC GAG
GCC GGC GCG GCC AGC GCC GAC GTG GTG AGC CTG ACC
TGC CCG GTC GCC GCC GGT GAA TGC GCG GGC CCG GCG
GAC AGC GGC GAC GCC CTG CTG GAG CGC AAC TAT CCC
ACT GGC GCG GAG TTC CTC GGC GAC GGT GGC GAC GTC
Nucleotide AGC TTC AGC ACC CGC GGC ACG CAG AAC TGG ACG GTG sequence of GAG CGG CTG CTC CAG GCG CAC CGC CAA CTG GAG GAG
Pseudomon CGC GGC TAT GTG TTC GTC GGC TAC CAC GGC ACC TTC
17 as exotoxin CTC GAA GCG GCG CAA AGC ATC GTC TTC GGC GGG GTG
A (252-608) CGC GCG CGC AGC CAG GAT CTC GAC GCG ATC TGG CGC
GGT TTC TAT ATC GCC GGC GAT CCG GCG CTG GCC TAC
GGC TAC GCC CAG GAC CAG GAA CCC GAC GCG CGC GGC
CGG ATC CGC AAC GGT GCC CTG CTG CGG GTC TAT GTG
CCG CGC TCC AGC CTG CCG GGC TTC TAC CGC ACC GGC
CTG ACC CTG GCC GCG CCG GAG GCG GCG GGC GAG GTC
GAA CGG CTG ATC GGC CAT CCG CTG CCG CTG CGC CTG
GAC GCC ATC ACC GGC CCC GAG GAG GAA GGC GGG CGC
CTG GAG ACC ATT CTC GGC TGG CCG CTG GCC GAG CGC
ACC GTG GTG ATT CCC TCG GCG ATC CCC ACC GAC CCG
CGC AAC GTC GGT GGC GAC CTC GAC CCG TCC AGC ATC
CCC GAC AAG GAA CAG GCG ATC AGC GCC CTG CCG GAC
TAC GCC AGC CAG CCC GGC AAA CCG CCG AAA GAC GAA
CTG
Amino acid
sequence of EGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLA
Pseudonom ARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESER as exotoxin FVRQGTGNDEAGAASADWSLTCPVAAGECAGPADSGDALLERNYPTGA
(252-608) EFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAA QSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNG ALLRVYVPRSSLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGP EEEGGRLETILGWPLAERTWIPSAIPTDPRNVGGDLDPSSIPDKEQAI SALPDYASQPGKPPKDEL
DIQMTQSPSSLSASVGDRVTITCRSTKSLLHSNGITYLYWYQQKPGKAP KLLIYQMSNLASGVPSRFSSSGSGTDFTLTISSLQPEDFATYYCAQNLE
EpCAM IPRTFGQGTKVELKRATPSHNSHQVPSAGGPTANSGTSGSEVQLVQSGP
19 scFv GLVQPGGSVRISCAASGYTFTNYGMNWVKQAPGKGLEWMGWINTYTGES
TYADSFKGRFTFSLDTSASAAYLQINSLRAEDTAVYYCARFAIKGDYWG QGTLLTVSS
PelB leader MKYLLPTAAAGLLLLAAQPAMA 20 sequence
HHHHHHDIQMTQSPSSLSASVGDRVTITCRSTKSLLHSNGITYLYWYQQ KPGKAPKLLIYQMSNLASGVPSRFSSSGSGTDFTLTISSLQPEDFATYY CAQNLEIPRTFGQGTKVELKRATPSHNSHQVPSAGGPTANSGTSGSEVQ LVQSGPGLVQPGGSVRISCAASGYTFTNYGMNWVKQAPGKGLEWMGWIN
VB4-847 TYTGESTYADSFKGRFTFSLDTSASAAYLQINSLRAEDTAVYYCARFAI
immunoKGDYWGQGTLLTVSSEFGGAPEFPKPSTPPGSSGLEGGSLAALTAHQAC
conjugate HLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRN
QGTGNDEAGAA 21 with a single ALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVR
(N-term) His SADWSLTCPVAAGECAGPADSGDALLERNYPTGAEFLGDGGDVSFSTR
tag GTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQD
LDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPG FYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWP LAERTWIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPP KDEL
[0050] In some embodiments, the immunoconjugate comprises a binding protein wherein the heavy chain variable region is encoded by a nucleic acid sequence sharing at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 10. In a particular embodiment, the heavy chain variable region is encoded by the nucleic acid sequence according to SEQ ID NO: 10. In another embodiment, the light chain variable region is encoded by a nucleic acid sequence sharing at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 3. In a particular embodiment, the light chain variable region is encoded by a nucleic acid sequence according to SEQ ID NO: 3.
[0051] In some embodiments, the heavy chain variable region and the light chain variable region are linked by a linker. In another embodiment, the linker is encoded by a nucleic acid sequence according to SEQ ID NO: 9.
[0052] In some embodiments, the immunoconjugate is encoded by a nucleic acid sequence sharing at least 90% homology to SEQ ID NO: 1. In a further embodiment, the immunoconjugate is encoded by a nucleic acid sequence according to SEQ ID NO: 1. In some embodiments, the immunoconjugate is encoded by nucleic acids 79-1995 of SEQ ID NO: 1. In some embodiments, the immunoconjugate comprises an amino acid sequence
sharing at least 90% homology to SEQ ID NO: 2 or SEQ ID NO: 21. In further embodiments, the immunoconjugate comprises an amino acid sequence according to SEQ ID NO: 2 or SEQ ID NO: 21. In some embodiments, the immunoconjugate comprises amino acids 23-635 of SEQ ID NO: 2.
Methods of Use
[0053] Disclosed herein are methods of using immunoconjugates described herein.
The present invention contemplates methods of treating or preventing cancer comprising administering an effective amount of said immunoconjugates to a subject in need thereof.
[0054] In some embodiments, the immunoconjugates may be used to treat or prevent cancer. In some embodiments, the immunoconjugates may be used to diagnose cancer in a subject. In some embodiments, the cancer is head and neck cancer, bladder cancer, lung cancer, gastric cancer, renal cancer, thyroid cancer, breast cancer, ovarian cancer, cervical cancer, colorectal cancer, hepatocellular carcinoma, esophageal cancer, oral squamous cell carcinoma, pancreatic cancer, or prostate cancer. Cancers originating from any epithelial cell may also be targeted by these immunoconjugates. In a preferred embodiment, the cancer is head and neck cancer. In a further embodiment, the head and neck cancer is HNSCC.
[0055] In some embodiments, the immunoconjugates provided herein exhibit decreased liver toxicity in subjects due to the lack of a His tag. For example, in some embodiments, a VB4-845 (containing a His tag) immunoconjugate may promote liver uptake which could result in liver toxicity. However, the immunoconjugates provided herein, which do not contain a histidine tag (His tag; e.g., VB4-847) lead to a decrease in liver toxicity.
[0056] In some embodiments, the cancer is amenable to treatment by direct administration of the immunoconjugate to the cancer site.
[0057] In some embodiments, the present disclosure provides methods and immunoconjugates for reducing tumor size. In further embodiments, a reduction in tumor size can facilitate removal of the tumor. In particular, in head and neck cancers, the immunoconjugates of the present invention advantageously effectively reduce tumor size, making surgical removal of the tumor or portions of the tumor feasible. Thus, in some embodiments, the present disclosure provides methods for treating cancer in a subject in need thereof comprising administering a composition provided herein, followed by surgical removal of one or more tumors present in the subject.
[0058] In some embodiments, the present disclosure provides methods for treating or preventing cancer comprising detecting the presence of a human papilloma virus (HPV)
infection in a subject; and administering to the subject a composition provided herein. In some embodiments, the composition is administered to the subject if the subject tests positive for HPV. Methods for testing HPV are known in the art and include detection assays selected from, for example, PCR, in situ hybridization, ELISAs, immunohistochemistry assays, antigen and/or antibody assays for HPV proteins or immunocomplexes, protein chip assays, radioimmunoprecipitation assays, rapid stick immunochromatographic assays, and flow cytometry based assays. For example, in some embodiments, the methods comprise detecting HPV proteins, or DNA encoding HPV proteins, such as LI, E6, or E7 prior to administration of a composition provided herein.
[0059] In some embodiments, a kit for diagnosing and/or treating cancer may include an the immunoconjugates provided herein. In some embodiments, the kits further comprise a reagent containing a labeled anti-Ig antibody, for example, an anti-Ig antibody linked with an enzyme such as alkaline phosphatase or a radiolabeled anti-Ig antibody. In some embodiments, the immunoconjugate may be attached to a chromophore, a fluorophore or a radiolabelled ligand. The kits provided herein may further comprise instructions for use of the immunoconjugates. In some embodiments, the kits provided herein comprise reagents for HPV detection.
[0060] The immunoconjugates disclosed herein may also be used to detect or monitor cancer in a subject. In some embodiments, a method of detecting or monitoring cancer in a subject may involve contacting a test sample taken from the subject with an immunoconjugate to form an immunoconjugate-antigen complex; measuring the amount of the immunoconjugate-antigen complex in the test sample; and normalizing the results against a control. The test sample may be serum, lymph, ascitic exudate, intercellular fluid, tissue lysate, saliva, tissue sections, cells, biopsy samples, and the like. The immunoconjugate- antigen complex may be detected by any means, such as for example, dot-blot method, Western blot method, ELISA method, or sandwich ELISA method. The immunoconjugate- antigen complex can be detected by use according to multistage reactions, such as reaction with a biotin-bound anti-Ig antibody and then with an avidin-bound material. In other embodiments, a method of detecting or monitoring cancer in a subject may involve contacting a test sample taken from the subject with an immunoconjugate to form a complex. In some embodiments, the immunoconjugates disclosed herein may be used for imaging a tumor in a subject
[0061] In certain embodiments, the immunoconjugates as described herein are labeled with a radiolabel. A radiolabel is typically a substance containing a radioisotope that allows
for easy detection and measurement. A number of different forms of hydrogen, carbon, phosphorous, sulfur and iodine are commonly used in medical diagnostics. The immunoconjugates of the present invention may also be labeled with any fluorescent probes known in the art. Non-limiting examples include fluorescein, amino coumarin acetic acid, tetramethylchodomine isocyanate, Texas Red, Cy 3.0, Cy 5.0, green fluorescent protein, and the like. In one embodiment, the immunoconjugates as described herein are labeled with a contrast agent. A contrast agent is a substance used to increase or modify the contrast of organs, fluids or anatomical structures in the human or animal body. The immunoconjugates of the present invention may be labeled with any suitable contrast agent. Preferred contrast agents include contrast agents for medical imaging. Preferably, the immunoconjugates of the present invention are labeled with an MRI (magnetic resonance imaging) contrast agent such as a superparamagnetic contrast agent or a paramagnetic contrast agent. MRI contrast agents are typically chelated metals or colloids. Contrast agents include gadolinium (Gd) based contrast agents such as gadolinium-DTPA, iron oxide based contrast agents such as superparamagnetic Small Particles of Iron Oxide (SPIO) and superparamagnetic Ultrasmall Small Particles of Iron Oxide (USPIO) and paramagnetic contrast agents based on manganese chelates such as Mn-DPDP.
[0062] In some embodiments, the immunoconjugate of the invention is administered in addition to one or more other therapeutic agents, which may include, without limitation, 2,2',2"trichlorotriethylamine, 6-azauridine, 6-diazo-5-oxo-L-norleucine, 6-mercaptopurine, aceglarone, aclacinomycinsa actinomycin, altretamine, aminoglutethimide, aminoglutethimide, amsacrine, anastrozole, ancitabine, angiogenin antisense oligonucleotide, anthramycin, azacitidine, azaserine, aziridine, batimastar, bcl-2 antisense oligonucleotide, benzodepa, bicalutamide, bisantrene, bleomycin, buserelin, busulfan, cactinomycin, calusterone, carboplatin, carboquone, carmofur, carmustine, carubicin, carzinophilin, chlorambucil, chloraphazine, chlormadinone acetate, chlorozotocin, chromomycins, cisplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, defosfamide, demecolcine, denopterin, diaziquone, docetaxel, doxifluridine, doxorubicin, droloxifene, dromostanolone, edatrexate, eflornithine, elliptinium acetate, emitefur, enocitabune, epirubicin, epitiostanol, estramustine, etoglucid, etoposide, fadrozole, fenretinide, floxuridine, fludarabine, fluorouracil, flutamide, folinic acid, formestane, fosfestrol, fotemustine, gallium nitrate, gemcitabine, goserelin, hexestrol, hydroxyurea, idarubicin, ifosfamide, improsulfan, interferon-alpha, interferon-beta, interferon-gamma, interleukin-2, L-asparaginase, lentinan, letrozole, leuprolide, lomustine, lonidamine,
mannomustine, mechlorethamine, mechlorethamine oxide hydrochloride, medroxyprogesterone, megestrol acetate, melengestrol, melphalan, menogaril, mepitiostane, methotrexate, meturedepa, miboplatin, miltefosine, mitobronitol, mitoguazone, mitolactol, mitomycins, mitotane, mitoxantrone, mopidamol, mycophenolic acid, nilutamide, nimustine, nitracine, nogalamycin, novembichin, olivomycins, oxaliplatin, paclitaxel, pentostain, peplomycin, perfosfamide, phenamet, phenesterine, pipobroman, piposulfan, pirarubicin, piritrexim, plicamycin, podophyllinic acid 2-ethyl-hydrazide, polyestradiol phosphate, porfimer sodium, porfiromycin, prednimustine, procabazine, propagermanium, PSK, pteropterin, puromycin, ranimustine, razoxane, roquinimex, sizofican, sobuzoxane, spirogermanium, streptonigrin, streptozocin, tamoxifen, tegafur, temozolomide, teniposide, tenuzonic acid, testolacone, thiamiprine, thioguanine, Tomudex, topotecan, toremifene, triaziquone, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, trilostane, trimetrexate, triptorelin, trofosfamide, trontecan, tubercidin, ubenimex, uracil mustard, uredepa, urethan, vinblastine, vincristine, zinostatin, and zorubicin, cytosine arabinoside, gemtuzumab, thioepa, cyclothosphamide, antimetabolites (e.g., methotrexate, 6- mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, fludarabine, gemcitabine, dacarbazine, temozoamide), hexamethylmelamine, LYSODREN, nucleoside analogues, plant alkaloids (e.g., Taxol, paclitaxel, camptothecin, topotecan, irinotecan (CAMPTOSAR,CPT- 11), vincristine, vinca alkyloids such as vinblastine.) podophyllotoxin, epipodophyllotoxin, VP- 16 (etoposide), cytochalasin B, gramicidin D, ethidium bromide, emetine, anthracyclines (e.g., daunorubicin), doxorubicin liposomal, dihydroxyanthracindione, mithramycin, actinomycin D, aldesleukin, allutamine, biaomycin, capecitabine, carboplain, chlorabusin, cyclarabine, daclinomycin, floxuridhe, lauprolide acetate, levamisole, lomusline, mercaptopurino, mesna, mitolanc, pegaspergase, pentoslatin, picamycin, riuxlmab, campath- 1, straplozocin, tretinoin, VEGF antisense oligonucleotide, vindesine, and vinorelbine. Compositions comprising one or more cancer therapeutics (e.g., FLAG, CHOP) are also contemplated by the present invention. FLAG comprises fludarabine, cytosine arabinoside (Ara-C) and G-CSF. CHOP comprises cyclophosphamide, vincristine, doxorubicin, and prednisone. For a full listing of cancer therapeutics known in the art, see, e.g., the latest editions of The Merck Index and the Physician's Desk Reference. Likewise, the immunoconjugate of the invention may be used in conjunction with radiation therapy or other known cancer therapeutic modalities.
[0063] An immunoconjugate of the present invention can be administered with a cancer therapeutic modality such as an antibody drug conjugate (ADC). An ADC comprises a
monoclonal antibody or antibody fragment, a cytotoxic payload or drug and a stable, chemical linker with labile bonds connecting the payload to the antibody. ADCs approved by the FDA and routinely used in the treatment of various cancers include gemtuzumab ozogamicin, ibritumomab tiuxetan, tositumomab, ado-trastuzumab emtansine, and Brentuximab Vedotin.
[0064] An immunoconjugate of the present disclosure can be administered with a cancer therapeutic modality such as immune checkpoint inhibitors. By checkpoint inhibitor it is meant that the compound inhibits one or more proteins in a number of inhibitory pathways that usually serve to modulate an immune response. The pathways are co-opted by tumors to evade the immune system and proliferate. Proteins in the checkpoint signaling pathways include for example, PD-1, PD-L1, PD-L2, TIM3, LAG3 and CTLA-4. Checkpoint inhibitors are known in the art. For example, the checkpoint inhibitor can be a small molecule. A "small molecule" as used herein, is meant to refer to a composition that has a molecular weight in the range of less than about 5 kD to 50 kD, for example less than about 4 kD, less than about 3.5 kD, less than about 3 kD, less than about 2.5 kD, less than about 2 kD, less than about 1.5 kD, less than about 1 kD, less than 750 daltons, less than 500 daltons, less than about 450 daltons, less than about 400 daltons, less than about 350 daltons, less than 300 daltons, less than 250 daltons, less than about 200 daltons, less than about 150 daltons, less than about 100 daltons. Small molecules can be, e.g., nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic or inorganic molecules. Alternatively, the checkpoint inhibitor is an antibody or antibody fragment thereof. For example, the antibody or antibody fragment thereof is specific to a protein in a checkpoint signaling pathway, such as PD-1, PD-L1, PD-L2, LAG3, TIM3 or CTLA-4.
[0065] In another embodiment, methods of treating cancer comprising administering an immunoconjugate in combination with a regimen of radiation therapy are provided. The therapy may also comprise surgery and/or chemotherapy. For example, the immunoconjugate may be administered in combination with radiation therapy and cisplatin, fluo-rouracil, carboplatin, and/or paclitaxel. Treatment with the immunoconjugate may allow use according to lower doses of radiation and/or less frequent radiation treatments, which may for example, reduce the incidence of severe sore throat that impedes swallowing function potentially resulting in undesired weight loss or dehydration.
[0066] In another embodiment, methods of treating cancer comprising administering an immunoconjugate in combination with an immunotherapeutic which includes, without limitation, rituxan, rituximab, campath-1, gemtuzumab, and trastuzumab. In another
embodiment, an immunoconjugate is administered in combination with one or more anti- angiogenic agents which include, without limitation, angiostatin, thalidomide, kringle 5, endostatin, Serpin (Serine Protease Inhibitor), anti-thrombin, 29 kDa N-terminal and a 40 kDa C-terminal proteolytic fragments of fibronectin, 16 kDa proteolytic fragment of prolactin, 7.8 kDa proteolytic fragment of platelet factor-4, a 13 amino acid peptide corresponding to a fragment of platelet factor-4 (Maione et al, 1990, Cancer Res. 51 : 2077- 2083), a 14-amino acid peptide corresponding to a fragment of collagen I (Tolma et al, 1993, J. Cell Biol. 122: 497-511), a 19 amino acid peptide corresponding to a fragment of Thrombospondin I (Tolsma et al, 1993, J. Cell Biol. 122: 497-511), a 20-amino acid peptide corresponding to a fragment of SPARC (Sage et al., 1995, J. Cell. Biochem. 57: 1329-1334), and a variant thereof, including a pharmaceutically acceptable salt thereof.
[0067] In yet another embodiment, an immunoconjugate is administered in combination with a cancer vaccine including, without limitation, autologous cells or tissues, non-autologous cells or tissues, carcinoembryonic antigen, alpha-fetoprotein, human chorionic gonadotropin, BCG live vaccine, melanocyte lineage proteins, and mutated, tumor- specific antigens.
[0068] In yet another embodiment, an immunoconjugate is administered in association with hormonal therapy. Hormonal therapeutics include, without limitation, a hormonal agonist, hormonal antagonist (e.g., flutamide, tamoxifen, leuprolide acetate), and steroid (e.g., dexamethasone, retinoid, betamethasone, Cortisol, cortisone, prednisone, dehydrotestosterone, glucocorticoid, mineralocorticoid, estrogen, testosterone, progestin).
[0069] In yet another embodiment, an immunoconjugate is administered in association with a gene therapy program to treat or prevent cancer.
[0070] Combination therapy with an immunoconjugate may sensitize the cancer or tumor to administration of an additional cancer therapeutic. Accordingly, the present invention contemplates combination therapies for treating, and/or preventing recurrence of cancer comprising administering an effective amount of an immunoconjugate prior to, subsequently, or concurrently with a reduced dose of a cancer therapeutic. For example, initial treatment with an immunoconjugate may increase the sensitivity of a cancer or tumor to subsequent challenge with a dose of cancer therapeutic. This dose is near, or below, the low range of standard dosages when the cancer therapeutic is administered alone, or in the absence of an immunoconjugate. When concurrently administered, the immunoconjugate may be administered separately from the cancer therapeutic, and optionally, via a different mode of administration. The cancer therapeutic may be selected from, for example, a steroid;
a biologic anti-autoimmune drug such as a monoclonal antibody, a fusion protein, or an anti- cytokine; a non-biologic anti-autoimmune drug; an immunosuppressant; an antibiotic; and anti-viral agent; a cytokine; or an agent otherwise capable of acting as an immune-modulator. In some embodiments, the cancer therapeutic is an immune checkpoint inhibitor, such as a a therapeutic that inhibits signalling from the PD-l/PD-Ll pathway, or CTLA4 pathway. For example, the cancer therapeutic may be an antibody or fragment thereof that is specific for PD-1, PD-L1, or CTLA4. For example, the cancer therapeutic may be selected from, without limitation, pembrolizumab, pidilizumab, nivolumab, ipilimumab, or tremelimumab.
[0071] Pharmaceutical compositions for combination therapy may also include, without limitation, antibiotics (e.g., dactinomycin, bleomycin, mithramycin, anthramycin), asparaginase, Bacillus and Guerin, procaine, tetracaine, lidocaine, propranolol, anti-mitotic agents, abrin, ricinA, Pseudomonas exotoxin, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, antihistaminic agents, anti-nausea agents, etc.
[0072] Clinical outcomes of cancer treatments using an immunoconjugate of the invention are readily discernible by one of skill in the relevant art, such as a physician. For example, standard medical tests to measure clinical markers of cancer may be strong indicators of the treatment's efficacy. Such tests may include, without limitation, physical examination, performance scales, disease markers, 12-lead ECG, tumor measurements, tissue biopsy, cytoscopy, cytology, longest diameter of tumor calculations, radiography, digital imaging of the tumor, vital signs, weight, recordation of adverse events, assessment of infectious episodes, assessment of concomitant medications, pain assessment, blood or serum chemistry, urinalysis, CT scan, and pharmacokinetic analysis. Furthermore, synergistic effects of a combination therapy comprising the immunoconjugate and another cancer therapeutic may be determined by comparative studies with patients undergoing monotherapy.
[0073] The effective dose of immunotoxin to be administered during a cycle varies according to the mode of administration. Direct administration (e.g., intratumoral injection) requires much smaller total body doses of immunoconjugate as compared to systemic, intravenous administration of the immunoconjugate. It will be evident to the skilled artisan that local administration can result in lower body doses, and in those circumstances, and resulting low circulating plasma level of immunoconjugate would be expected and desired.
[0074] Moreover, the effective dose of a specific immunoconjugate construct may depend on additional factors, including the type of cancer, the size of the tumor in the case of
HNSCC, the stage of the cancer, the immunoconjugate's toxicity to the patient, the specificity of targeting to cancer cells, as well as the age, weight, and health of the patient.
[0075] In one embodiment, the effective dose by direct administration of immunoconjugate may range from about 10 to 3000, 20 to 900, 30 to 800, 40 to 700, 50 to 600, 60 to 500, 70 to 400, 80 to 300, 90 to 200, or 100 to 150 micrograms/tumor/day. In other embodiments, the dose may range from approximately 10 to 20, 21 to 40, 41 to 80, 81 to 100, 101 to 130, 131 to 150, 151 to 200, 201 to 280, 281 to 350, 351 to 500, 501 to 1000, 1001 to 2000, or 2001 to 3000 micrograms/tumor/day. In specific embodiments, the dose may be at least approximately 20, 40, 80, 130, 200, 280, 400, 500, 750, 1000, 2000, or 3000 mi crograms/tumor/ day .
[0076] In another embodiment, the effective dose of immunoconjugate may range from about 100 to 5000, 200 to 4000, 300 to 3000, 400 to 2000, 500 to 1000, 600 to 900, or 700 to 1500 micrograms/tumor/month. In other embodiments, the dose may range from approximately 100 to 199, 200 to 399, 400 to 649, 650 to 999, 1000 to 1799, 1800 to 2499, 2500 to 3499, 3500 to 4999, 5000 to 7499, 7500 to 10000, or 10001 to 20000 micrograms/tumor/month. In specific embodiments, the dose may be at least approximately 100, 200, 400, 650, 1000, 1400, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 7500, 10000, or 20000 micrograms/tumor/month.
[0077] In another embodiment, the effective dose of immunoconjugate results in an intratumoral concentration of at least approximately 5, 10, 20, 30, 40, 50, 60, 75, 100, 125, 150, 100, 200, 300, 400, or 500 micrograms/cm3 of the immunoconjugate. In other embodiments, the resulting intratumoral concentration of immunoconjugate is approximately 5 to 500, 10 to 400, 15 to 300, 20 to 200, 25 to 100, 30 to 90, 35 to 80, 40 to 70, 45 to 60, or 50 to 55 micrograms/cm3. In other embodiments, the resulting intratumoral concentration of immunoconjugate is approximately 10 to 15, 16 to 20, 21 to 25, 26 to 30, 31 to 35, 36 to 40, 41 to 45, 46 to 50, 51 to 55, 56 to 60, 61 to 65, 66 to 70, 71 to 75, 76 to 80, 81 to 85, 86 to 90, 91 to 95, 96 to 100, or 100 to 200 micrograms/cm3.
[0078] In another embodiment, the effective dose of immunoconjugate results in a plasma concentration of less than approximately 0.1, 1, 2.5, 5, 7.5, 10, 15, 20, 30, 40, or 50 micrograms/liter. In other embodiments, the resulting circulating concentration of immunoconjugate is approximately 0.1 to 50, 1 to 40, 2.5 to 30, 5 to 20, or 7.5 to 10 micrograms/liter. In other embodiments, the resulting circulating concentration of immunoconjugate is approximately 0.1 to 1, 1.1 to 2.4, 2.5 to 5, 5.1 to 7.4, 7.5 to 10, 11 to 15, 16 to 20, 21 to 30, 31 to 40, or 41 to 50 micrograms/liter.
[0079] In a particular non-limiting embodiment, the effective dose of the immunoconjugate is between about 100 and 3000 micrograms/tumor/month, for example approximately 100, 200, 300, 400, 750, or 1000 micrograms/tumor/month, wherein the patient is administered a single dose per day. The single dose is administered approximately every month for approximately 1, 2, 3, 4, 5, or 6 consecutive months. After this cycle, a subsequent cycle may begin approximately 1, 2, 4, 6, or 12 months later. The treatment regime may include 1, 2, 3, 4, 5, or 6 cycles, each cycle being spaced apart by approximately 1, 2, 4, 6, or 12 months.
[0080] In a particular non-limiting embodiment, the effective dose of the immunoconjugate is between about 20 and 1240 micrograms/tumor/day, for example approximately 20, 40, 80, 130, 200, or 280 micrograms/tumor/day or approximately 100, 200, 330, 500, 700, 930, 1240 micrograms/tumor/day, wherein the patient is administered a single dose per day. The single dose is administered approximately every day (one or more days may optionally be skipped) for approximately 1, 2, 3, 4, 5, 6 or 7 consecutive days. After this cycle, a subsequent cycle may begin approximately 1, 2, 3, 4, 5, or 6 weeks later. The treatment regime may include 1, 2, 3, 4, 5, or 6 cycles, each cycle being spaced apart by approximately 1, 2, 3, 4, 5, or 6 weeks.
[0081] The injection volume preferably is at least an effective amount, which is appropriate to the type and/or location of the tumor. The maximum injection volume in a single dose may be between about 25% and 75% of tumor volume, for example approximately one-quarter, one-third, or three-quarters of the estimated target tumor volume. In a specific, non-limiting embodiment, the maximum injection volume in a single dose is approximately 30% of the tumor volume.
[0082] In another embodiment, the immunotoxin is administered intratumourally at a total dose per cycle equivalent to, or below the maximum tolerated dose established in a safety trial but the dosage is standardized in relation to the tumor volume. For example, subjects will receive between 1 microgram per cm3 and 500 microgram per cm3 tumor or a dose sufficient to reach about between 14 picomole and 7 nanomole per cm3 tumor tissue. The dose will be administered in a volume not exceeding about 20-50% of the tumor volume. The immunotoxin will be diluted in a suitable salt solution. For example, for a tumor of estimated volume of 3 cm3, a target dose of 14 picomoles (1 microgram per cm3), and a maximum injection relative volume of about ½ of the tumor, 3 microgram of immunotoxin will be diluted into about 1 ml of diluent.
[0083] In another particular embodiment, the effective dose of the immunoconjugate is between about 20 and 300 micrograms/tumor/day, for example approximately 20, 40, 80, 130, 200, or 280 micrograms/tumor/day, wherein the patient is administered a single dose per day. The maximum injection volume in a single dose may be between about 25% and 75% of tumor volume, for example approximately one-quarter, one-third, or three-quarters of the estimated target tumor volume. The single dose is administered every other day for approximately 5, 7, 9, 11 , 13, 15, 17, 19, 21, 23, 25, 27, 29, or 31 consecutive days. After this cycle, a subsequent cycle may begin approximately 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 weeks later. The treatment regime may include 1, 2, 3, 4, 5, or 6 cycles, each cycle being spaced apart by approximately 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 weeks.
[0084] Moreover, the effective dose of a specific immunoconjugate construct may depend on additional factors, including the type of cancer, the size of the tumor, the stage of the cancer, the immunoconjugate's toxicity to the patient, the specificity of targeting to cancer cells, as well as the age, weight, and health of the patient.
[0085] The immunoconjugates described herein may be administered to the patient via any suitable route. The immunoconjugates may be administered by injection into the vascular system or by injection into an organ. Administration routes include parenteral, intravascular, intravenous inj ection, topical, oral, buccal, or ocular routes, or intravaginally, by inhalation, by depot injections, or by implants. Parenteral administration includes subcutaneous, intramuscular, intraperitoneal, intracavity, intrathecal, intratumoral, transdermal and intravenous injection. In some embodiments, the immunoconjugates are administered intravenously as a bolus or by continuous infusion over a period of time. In other embodiments, the immunoconjugates may be administered directly to the cancer site.
[0086] In accordance with one aspect of the present invention, the immunoconjugate and/or other anticancer agent is delivered to the patient by direct administration. Accordingly, the immunoconjugate and/or other anticancer agent may be administered, without limitation, by one or more direct injections into the tumor, by continuous or discontinuous perfusion into the tumor, by introduction of a reservoir of the immunoconjugate, by introduction of a slow-release apparatus into the tumor, by introduction of a slow-release formulation into the tumor, and/or by direct application onto the tumor. By the mode of administration into the tumor, introduction of the immunoconjugate and/or other anticancer agent to the area of the tumor, or into a blood vessel or lymphatic vessel that substantially directly flows into the area of the tumor, is also contemplated. In each case, the pharmaceutical composition is administered in at least an amount sufficient to achieve the
endpoint, and if necessary, comprises a pharmaceutically acceptable carrier. It is contemplated that the immunoconjugates may be administered intratumorally, whereas any other anticancer agent may be delivered to the patient by other modes of administration (e.g., intravenously). Additionally, where multiple anticancer agents are intended to be delivered to a patient, the immunoconjugate and one or more of the other anticancer agent may be delivered intratumorally, whereas other anticancer agents may be delivered by other modes of administration (e.g., intravenously and orally).
[0087] In some embodiments, a composition may be an immunoconjugate described herein and a pharmaceutically acceptable excipient, carrier, buffer or stabilizer. For example, in some embodiments, the composition comprises an immunoconjugate in a formulation comprising 20 mM sodium phosphate and 0.1 % Polysorbate 80. An immunoconjugate according to the invention may be comprised in a pharmaceutical composition or medicament. Pharmaceutical compositions adapted for direct administration include, without limitation, lyophilized powders or aqueous or non-aqueous sterile injectable solutions or suspensions, which may further contain antioxidants, buffers, bacteriostats and solutes that render the compositions substantially isotonic with the blood of an intended recipient. Other components that may be present in such compositions include water, alcohols, polyols, glycerin and vegetable oils, for example. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets. Immunoconjugate may be supplied, for example but not by way of limitation, as a lyophilized powder which is reconstituted with sterile water or saline prior to administration to the patient.
[0088] Pharmaceutical compositions of the invention may comprise a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include essentially chemically inert and nontoxic compositions that do not interfere with the effectiveness of the biological activity of the pharmaceutical composition. Examples of suitable pharmaceutical carriers include, but are not limited to, water, saline solutions, glycerol solutions, ethanol, N-(l(2,3-dioleyloxy) propyl)N,N,N-trimethylammonium chloride (DOTMA), diolesylphosphotidyl-ethanolamine (DOPE), and liposomes. Such compositions should contain a therapeutically effective amount of the compound, together with a suitable amount of carrier so as to provide the form for direct administration to the patient.
[0089] In another embodiment, a pharmaceutical composition comprises an immunoconjugate and one or more additional anticancer agent, optionally in a pharmaceutically acceptable carrier.
[0090] The composition may be in the form of a pharmaceutically acceptable salt which includes, without limitation, those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylarnino ethanol, histidine, procaine, etc.
[0091] In some embodiments, the pharmaceutical carrier may include, without limitation, binders, coating, disintegrants, fillers, diluents, flavors, colors, lubricants, glidants, preservatives, sorbents, sweeteners, conjugated linoleic acid (CLA), gelatin, beeswax, purified water, glycerol, any type of oil, including, without limitation, fish oil or soybean oil, or the like. Pharmaceutical compositions of the immunoconjugates also can comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as, e.g., polyethylene glycols.
Methods of Production
[0092] A person skilled in the art will appreciate that the novel nucleic acid sequences of the present application can be used in a number of recombinant methods.
[0093] In some embodiments, the sequences, vectors, and constructs of the present invention are not codon optimized. An exemplary non-codon optimized immunoconjugate sequence is provided in SEQ ID NO: 1. Codon optimization methods are known in the art and can be found, for example, in US2008/0194511, and US2007/0292918, both of which are incorporated herein for all purposes.
[0094] Accordingly, the nucleic acid sequences of the present disclosure may be incorporated in a known manner into an appropriate expression vector which ensures good expression of the proteins encoded thereof. Possible expression vectors include, but are not limited to, cosmids, plasmids, or modified viruses (e.g. replication defective retroviruses, adenoviruses and adeno-associated viruses), so long as the vector is compatible with the host cell used. The expression vectors are "suitable for transformation of a host cell", which means that the expression vectors contain a nucleic acid molecule of the present application and regulatory sequences selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid molecule. Operatively linked is intended to mean that the nucleic acid is linked to regulatory sequences in a manner which allows expression of the
nucleic acid. In some embodiments, the expression vector is a plasmid. In further embodiments, the plasmid is a pING-RBS, pING3302, or pSJFI plasmid.
[0095] The present application therefore contemplates a recombinant expression vector of the present application containing a nucleic acid molecule of the present application, or a fragment thereof, and the necessary regulatory sequences for the transcription and translation of the inserted protein sequence.
[0096] Suitable regulatory sequences may be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes (For example, see the regulatory sequences described in (Goeddel, 1990), Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). Selection of appropriate regulatory sequences is dependent on the host cell chosen as discussed below, and may be readily accomplished by one of ordinary skill in the art. Examples of such regulatory sequences include: a transcriptional promoter and enhancer or RNA polymerase binding sequence, a ribosomal binding sequence, including a translation initiation signal. Additionally, depending on the host cell chosen and the vector employed, other sequences, such as an origin of replication, additional DNA restriction sites, enhancers, and sequences conferring inducibility of transcription may be incorporated into the expression vector.
[0097] The recombinant expression vectors of the present application may also contain a selectable marker gene which facilitates the selection of host cells transformed or transfected with a recombinant molecule of the present application. Examples of selectable marker genes are genes encoding a protein such as G418 and hygromycin which confer resistance to certain drugs, (3-galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or an immunoglobulin or portion thereof such as the Fc portion of an immunoglobulin preferably IgG Transcription of the selectable marker gene is monitored by changes in the concentration of the selectable marker protein such as β-galactosidase, chloramphenicol acetyltransferase, or firefly luciferase. If the selectable marker gene encodes a protein conferring antibiotic resistance such as neomycin resistance transformant cells can be selected with G418. Cells that have incorporated the selectable marker gene will survive, while the other cells die. This makes it possible to visualize and assay for expression of recombinant expression vectors of the present application and in particular to determine the effect of a mutation on expression and phenotype. It will be appreciated that selectable markers can be introduced on a separate vector from the nucleic acid of interest.
[0098] Recombinant expression vectors can be introduced into host cells to produce a transformed host cell. The terms "transformed with", "transfected with", "transformation" and
"transfection" are intended to encompass introduction of nucleic acid (e.g. a vector) into a cell by one of many possible techniques known in the art. The term "transformed host cell" as used herein is intended to also include cells capable of glycosylation that have been transformed with a recombinant expression vector of the present application. Prokaryotic cells can be transformed with nucleic acid by, for example, electroporation or calcium- chloride mediated transformation. For example, nucleic acid can be introduced into mammalian cells via conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran mediated transfection, lipofectin, electroporation or microinjection. Suitable methods for transforming and transfecting host cells can be found in (Sambrook et al, 2001) (Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, 2001), and other laboratory textbooks.
[0099] Suitable host cells include a wide variety of eukaryotic host cells and prokaryotic cells. For example, the proteins of the present application may be expressed in bacterial cells, yeast cells or mammalian cells. In certain embodiments, the immunoconjugates of the present disclosure may be expressed in prokaryotic cells, such as Escherichia coli (Zhang et al, 2004), Science 303(5656): 371-3). Other suitable host cells include yeast cells or mammalian cells, and can be found in (Goeddel, 1990), Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1991).
[00100] Accordingly, the present disclosure provides a recombinant expression vector comprising one or more of the novel nucleic acid sequences as well as methods and uses of the expression vectors in the preparation of recombinant proteins. Further, the disclosure provides a host cell comprising one or more of the novel nucleic acid sequences or expression vectors comprising one or more of the novel nucleic acid sequences.
[00101] In one aspect, the present disclosure provides methods for purifying an immunoconjugate comprising an EpCAM antibody fragment and a toxin. As used herein, the term "purification" refers to steps taken in order to reduce the amounts of foreign elements, such as biological macromolecules (e.g., DNA or protein) that are not the protein of interest and that may be present in a sample of the protein. The purification methods provided herein include one or more chromatographic steps in order to purify the immunoconjugate proteins provided herein from a biological source.
[00102] Chromatographic materials and methods are known in the art and described, for example, in Chromatography: 6th Edition (E. Heftmann, 2004), incorporated herein by reference in its entirety. For example, chromatographic materials useful in the present
disclosure include, but are not limited to, cation-exchange, ani on-exchange (e.g., Q- Sepharose), affinity, hydrophobic interaction, mixed mode (e.g., hydroxy apatite or Capto MMC, Capto MMC Impres and Nuvia C prime), reversed phase, size exclusion, metal affinity (e.g., Ni2+ column) and adsorption materials. The invention also contemplates many support medium, including agarose, cellulose, silica, and poly(stryrene-divinylbenzene) (PSDVB). In addition, multiple chromatographic methods can be used including conventional chromatography, HPLC (High Performance Liquid Chromatography or High pressure Liquid Chromatography), or perfusion chromatography. One skilled in the art will also appreciate that the size of the column (i.e., diameter and length) will depend on several factors such as the volume of material to be loaded, the concentration of protein to be purified, and the desired resolution or purity. In some embodiments, centrifugation and/or filtration and/or chromatography steps are used to clarify and/or concentrate the protein. One embodiment of the invention involves clarification by precipitation and/or centrifugation and/or filtration (e.g., microfiltration, diafiltration). In some embodiments, precipitation is carried out with carefully selected concentrations of the chemical agents as this reduces co-precipitation of contaminating proteins. Precipitated proteins may be separated from soluble materials by filtration or by centrifugation.
[00103] The primary mechanism of a Capto MMC column, which is a mixed mode column, is thought to be as a weak cation exchanger. For Capto MMC columns and other cation exchange columns, it is known in the art that the protein to be captured has to be at a lower pH relative to the pi of the molecule. In one aspect, the His tagged immunoconjugate molecules disclosed herein precipitate when the pH is dropped below the pi and therefore the same was expected for the non-his tagged version. Thus, the Capto MMC column (as a positively charged cationic exchanger) would not be expected to be capable of capturing the non-His tagged immunoconjugate at a pH above its pi value based on electrostatic charge. Surprisingly, however, the non-His tagged immunoconjugates were successfully captured by the Capto MMC column despite the fact that the protein applied had a higher pH than pi. Without wishing to be bound by theory, the column may exhibit hydrophobic interactions that lead to the successful capture of the non-His tagged immunoconjugates.
[00104] When a His tagged protein is purified, a hydroxyapatite column removes host cell proteins only, because the Nickel column removes the product-related impurities. However, in the context of a non-His tagged protein such as the immunoconjugates disclosed herein, a significant amount of disulfide bonded, product-related impurities must be removed by the hydroxyapatite column. Thus, in some embodiments, the present disclosure provides
methods for purifying a non-His tagged protein wherein the method comprises the use of a hydroxy apatite column, and wherein the hydroxyapatite column removes both host cell proteins and product-related impurities. In further embodiments, the flow through/wash is collected following contact with the hydroxyapatite column. In addition, impurities are generally removed as a final step in a purification process of a protein by ultrafiltration. However, the present inventors have found that surprisingly, in the context of the non-His tagged proteins provided herein, ultrafiltration did not successfully remove the small molecular weight impurities. However, a size exclusion step, which is not generally used in commercial processes due to the added cost and manufacturing time as the final purification step, successfully removed the impurities present non-His tagged proteins disclosed herein.
Definitions
[00105] As used herein, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to an "immunoconjugate" is a reference to one or more immunoconjugates and equivalents thereof known to those skilled in the art, and so forth.
[00106] As used herein, the term "about" means plus or minus 10% of the numerical value of the number with which it is being used. Therefore, about 50% means in the range of 45%-55%.
[00107] As used herein, the term "subject" or "patient" refers to any member of the subphylum cordata, including, without limitation, humans and other primates, including non- human primates such as chimpanzees and other apes and monkey species. The terms "mammals" and "animals" are included in this definition. In particular embodiments, the term "subject" as used herein refers to a human that has been diagnosed with cancer.
[00108] As used herein, the term "binding protein" includes antibodies and fragments thereof. As used herein, the term "antibody" is intended to include monoclonal antibodies, polyclonal antibodies, and chimeric antibodies. The antibody may be from recombinant sources and/or produced in transgenic animals. The term "antibody fragment" as used herein is intended to include without limitations Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, and multimers thereof, multispecific antibody fragments and Domain Antibodies. Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, dsFv, ds-
scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques.
[00109] As used herein the term "His tag" refers to a stretch of histidine amino acid residues included in a protein sequence. In some embodiments, the His tag is used for protein purification, for example using a Ni2+ column. While there is no limit on how many histidine residues make up a His tag, there should be as many as is required to effectively bind to, for example, a Ni2+ column. In some embodiments, the His tag comprises at least 2 histidine residues. In some embodiments, the His tag comprises at least 3, or at least 4, or at least 5 or at least 6 or at least 7 or at least 8 or at least 9 or at least 10 histidine residues. In particular embodiments, the His tag comprises 6 histidine residues and is also termed a "Hexahistitine tag" or a "HexaHis tag."
[00110] As used herein, the phrase "humanized antibody or antibody fragment" means that the antibody or fragment comprises human framework regions. The humanization of antibodies from non-human species has been well described in the literature. See for example EP-B1 0 239400 and Carter & Merchant 1997 (Curr Opin Biotechnol 8, 449-454, 1997).
[00111] As used herein, the term "immunoconjugate" comprises (1) a binding protein attached to (2) a toxin. The terms "immunotoxin" and "immunoconjugate" are used interchangeably herein.
[00112] As used herein, the phrase "treating cancer" refers to inhibition of cancer cell replication, inhibition of cancer spread (metastasis), inhibition of tumor growth, reduction of cancer cell number or tumor growth, decrease in the malignant grade of a cancer (e.g., increased differentiation), or improved cancer-related symptoms. As used herein, the phrase "preventing cancer" refers to prevention of cancer occurrence. In certain instances, the preventative treatment reduces the recurrence of the cancer. In other instances, preventative treatment decreases the risk of a patient from developing a cancer, or inhibits progression of a pre-cancerous state (e.g. a colon polyp) to actual malignancy.
[00113] As used herein, the term "anticancer agents" or "cancer therapeutics" refers to compounds or treatments that are effective in treating or preventing cancer including, without limitation, chemical agents, other immunotherapeutics, cancer vaccines, anti-angiogenic compounds, certain cytokines, certain hormones, gene therapy, radiotherapy, surgery, and dietary therapy.
[00114] As used herein, the term "effective amount" or "therapeutically effective amount" means an amount effective, at dosages and for periods of time necessary to achieve the desired result. Effective amounts of an immunoconjugate may vary according to factors
such as the disease state, age, sex, weight of the animal. Dosage regime may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
[00115] As used herein, the phrase "the immunoconjugate is administered directly to the cancer site" refers to direct or substantially direct introduction including, without limitation, single or multiple injections of the immunoconjugate directly into the tumor or peritumorally, continuous or discontinuous perfusion into the tumor or peritumorally, introduction of a reservoir into the tumor or peritumorally, introduction of a slow-release apparatus into the tumor or peritumorally, introduction of a slow-release formulation into the tumor or peritumorally, direct application onto the tumor, direct injection into an artery that substantially directly feeds the area of the tumor, direct injection into a lymphatic vessel that substantially drains into the area of the tumor, direct or substantially direct introduction in a substantially enclosed cavity (e.g., pleural cavity) or lumen (e.g., intravesicular). "Peritumoral" is a term that describes a region, within about 10 cm, preferably within 5 cm, more preferably within 1 cm, of what is regarded as the tumor boundary, such as, but not limited to, a palpable tumor border. "Direct administration" in the context of prevention of occurrence or prevention of recurrence is defined as administration directly into a site at risk for development or recurrence of a cancer. In one embodiment, direct administration is by systemic delivery.
[00116] As used herein, the phrase "ligand that binds to a protein on the cancer cell" includes any molecule that can selectively target the immunoconjugate to the cancer cell by binding to a protein on the cancer cells. The targeted protein on the cancer cell is preferably a tumor associated antigen that is expressed at higher levels on the cancer cell as compared to normal cells.
[00117] As used herein, the term "linker" or "peptide linker" refers to a short peptide sequence that occurs between protein domains. In one embodiment, linkers are composed of flexible residues like glycine and serine so that the adjacent protein domains are free to move relative to one another. Longer linkers are used when it is necessary to ensure that two adjacent domains do not sterically interfere with one another. In another embodiment, linkers are rigid and function to prohibit unwanted interactions between discrete protein domains. Fusion proteins or polypeptides can use linkers to connect the regions that do not naturally occur together in nature. In a particular embodiment, a furin protease sensitive peptide linker connects, links, joins or fuses a toxin to a binding protein that recognizes one or more tumor
associated antigens on the surface of cancer cells. A "furin protease sensitive peptide linker", "furin protease sensitive linker" or "furin linker" comprises a furin cleavage site that is recognized and cleaved by furin, an enzyme which belongs to the subtilisin-like proprotein convertase family. The members of this family are proprotein convertases that process latent precursor proteins into their biologically active products. This encoded protein is a calcium- dependent serine endoprotease that can efficiently cleave precursor proteins at their paired basic amino acid processing sites. In one embodiment, a furin protease sensitive peptide linker fuses a binding protein portion to a toxin portion in an immunoconjugate. The toxin is cleaved from the binding protein of the immunoconjugate by a furin enzyme once the immunoconjugate is internalized in a cancer cell, allowing the free toxin to exert its cytotoxic effect.
[00118] As used herein, the terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acid residues of any length. The polymer may be linear or branched, it may comprise modified amino acids or amino acid analogs, and it may be interrupted by chemical moieties other than amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling or bioactive component.
[00119] As used herein, the term "polynucleotide" is a polymeric form of nucleotides of any length, which contain deoxyribonucleotides, ribonucleotides, and analogs in any combination analogs. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The term "polynucleotide" includes double-, single-stranded, and triple-helical molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double stranded form of either the DNA, RNA or hybrid molecules.
[00120] The following are non-limiting examples of polynucleotides: a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracyl, other sugars and linking groups such as fluororibose and thioate, and nucleotide branches. The sequence of nucleotides may be interrupted by non-nucleotide components. A
polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications included in this definition are caps, substitution of one or more of the naturally occurring nucleotides with an analog, and introduction of means for attaching the polynucleotide to proteins, metal ions, labeling components, other polynucleotides, or a solid support.
[00121] As used herein, the term "recombinant" polynucleotide means a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which either does not occur in nature or is linked to another polynucleotide in a nonnatural arrangement.
[00122] As used herein, the phrase "pharmaceutically acceptable" refers to general clinical use and/or approval by a regulatory agency of the Federal or state government, listing in the United States Pharmacopoeia, or general acceptance by those skilled in the relevant art.
[00123] As used herein, "physiologic conditions" for antibody binding reflect but do not necessarily exactly duplicate the conditions in which a human epidermal growth factor receptor 2-binding polypeptide would encounter a human epidermal growth factor receptor 2 molecule in vivo. Binding under physiologic conditions should be reasonably predictive that binding in vivo will occur.
[00124] A "signal peptide" or "leader sequence" is a short amino acid sequence that directs a newly synthesized protein through a cellular membrane, usually the endoplasmic reticulum in eukaryotic cells, and either the inner membrane or both inner and outer membranes of bacteria. Signal peptides are typically at the N-terminal portion of a polypeptide and are typically removed enzymatically between biosynthesis and secretion of the polypeptide from the cell. The signal peptide is not present in the secreted protein, only during protein production. "Signal peptide" and "leader sequence" are used interchangeably herein.
[00125] As used herein, a "cell line" or "cell culture" denotes bacterial, plant, insect or higher eukaryotic cells grown or maintained in vitro. The descendants of a cell may not be completely identical (either morphologically, genotypically, or phenotypically) to the parent cell. A monoclonal antibody may be produced by a hybridoma or other cell. Methods of making hybridomas, both murine and human, are known in the art.
[00126] As used herein, the term "host cell" denotes a prokaryotic or eukaryotic cell that has been genetically altered, or is capable of being genetically altered by administration of an exogenous polynucleotide, such as a recombinant plasmid or vector. When referring to genetically altered cells, the term refers both to the originally altered cell, and to the progeny thereof. In some embodiments, the host cell is a bacterial cell. In further embodiments, the
host cell is an E. coli cell, such as E. coli El 04 or E. coli TGI cells. As used herein, the term "vector" refers to a recombinant DNA or RNA plasmid or virus that comprises a heterologous polynucleotide to be delivered into a target cell, either in vitro or in vivo. The heterologous polynucleotide may comprise a sequence of interest for purposes of therapy, and may optionally be in the form of an expression cassette. As used herein, a vector need not be capable of replication in the ultimate target cell or subject. The term includes cloning vectors for the replication of a polynucleotide, and expression vectors for translation of a polynucleotide encoding sequence. Also included are viral vectors, which comprise a polynucleotide encapsidated or enveloped in a viral particle.
[00127] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entireties for all purposes. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as, an acknowledgment or any form of suggestion that they constitute valid prior art in any country in the world.
[00128] All publications referenced herein are incorporated by reference in their entireties for all purposes. Compositions and methods encompassed in the present disclosure will be further described with respect to the following examples; however, the scope of the invention is not to be limited thereby.
EXAMPLE 1; EXPRESSION AND PURIFICATION OF VB4-847
[00129] VB4-845 is a recombinant fusion protein comprising an anti-Epithelial Cell Adhesion Molecule (EpCAM) single-chain antibody genetically fused with a truncated form of Pseudomonas exotoxin A (252-6Ο8) (ETA(252-608)) and having His tags at the N- and C- terminus of the amino acid sequence. VB4-847 is a novel recombinant fusion protein consisting of an EpCAM single-chain antibody fused with ETA(252-608) without any His tags. A study was conducted to compare the expression from different constructs of VB4-847 (non-His) to VB4-845 (His) in order to determine the methods for clinical production of VB4-847.
[00130] In the case of the codon optimized sequence, soluble protein expression was substantially reduced upon removal of the His tags. Surprisingly, deletion of the His tags did not have as dramatic of an impact on expression levels from the VB4-847 wild type sequence. Expression of VB4-847 using a pING plasmid containing a modified RBS was also evaluated, however very little difference in expression levels was observed compared to
expression from the pING3302 plasmid. In addition to soluble expression, VB4-847 was also successfully expressed in the periplasm of E. coli using another expression system.
[00131] Biological testing of purified VB4-847 demonstrated that the potency of the molecule is not affected by removal of the His tags. Furthermore, based on the yields obtained in a non-optimized fermentation and purification run, the VB4-847/pING-RBS clone was demonstrated to be a feasible option for producing drug product at the clinical scale.
Experimental Methods
VB4-847-CODA in pING3302 plasmid
[00132] The His tag at the N terminus of the VL of VB4-845-CODA was removed by
SOE PCR and the fragment containing EcoRl-¥e\B- L-Vu-BbvCl was cloned into the pCR 2.1 vector and transformed into 10F E. coli cells for sequencing. The pCR2.1 plasmid containing the correct insert was digested with EcoKl-BbvCl and ligated with BbvCl-EcoRl digested VB4-847 diabody/pING3302 consisting of the pING3302 plasmid and the BbvCl- VH-PE-A¾OI sequence of VB4-847 which does not contain a C-terminal His tag on PE. Chemically competent 10F E. coli cells were transformed with the ligation reaction and a transformed colony grown for plasmid extraction. Plasmid with the 2kb insert was then used to transform E. coli E104 and selected colonies grown for small-scale expression.
VB4-847-CODA in pING-RBS plasmid
[00133] The RBS sequence between the EcoKl site and PelB leader of VB4-847-
CODA was removed by SOE PCR. The fragment containing EcoRI-CATA-Met-PelB-VL- Yu-BbvCl was cloned into the pCR 2.1 vector and transformed into 10F E. coli cells for sequencing. The pCR2.1 plasmid containing the correct insert was digested with EcoKl- BbvCl and ligated along with the BbvCl-N n-VE-Xhol fragment of VB4-847-CODA into pING/RBS plasmid pre-digested with EcoR\-Xho\. Chemically competent 10F E. coli cells were transformed with the ligation reaction and a transformed colony grown for plasmid extraction. Plasmid with the 2kb insert was then used to transform E. coli E104 and selected colonies grown for small-scale expression.
VB4-847 in pING/RBS plasmid
[00134] The RBS sequence between the EcoBI site and PelB leader of VB4-847 was removed by SOE PCR. The fragment containing £coRI-CATA-Met-PelB-VL-VH-<SwaI was cloned into the pCR 2.1 vector and transformed into 10F E. coli cells for sequencing. The His tag at the C-terminus of PE was removed by PCR mutagenesis and the fragment containing Narl-PE-XhoI was cloned into the pCR 2.1 vector and transformed into 1 OF E.
coli cells for sequencing . The pCR2.1 plasmids containing the correct inserts were digested with EcoRl-Smal and Narl-Xhol for the 5' and 3' fragments, respectively, and ligated with the Smal-Vn-PE-Narl fragment into the pING3302 plasmid pre-digested with EcoRl-Xhol. Chemically competent 10F E. coli cells were transformed with the ligation reaction and a transformed colony grown for plasmid extraction. Plasmid with the 2kb insert was then used to subclone the complete VB4-847 fragment as an EcoRl-Xhol fragment into the pING-RBS plasmid pre-digested with the same enzymes. Chemically competent 10F E. coli cells were transformed with the ligation reaction and a transformed colony grown for plasmid extraction. Plasmid with the 2kb insert was then used to transform E. coli E104 and selected colonies grown for small-scale expression.
Small-scale soluble expression in E104 cells
[00135] Transformed El 04 cells containing VB4-847 and VB4-847-CODA expression plasmids were inoculated into 5 mL 2-YT containing 25 μg/mL tetracycline and incubated at 37°C with constant shaking at 225 rpm. After 16 hours of incubation, 300 μΕ overnight seed culture was inoculated into 30 mL TB (1% inoculum), and incubated at 37°C with constant shaking at 225 rpm until an OD6oo of 2.0 was attained. The culture was induced with 150 μΕ of L-Arabinose (0.1% final), and incubated at 25°C with constant shaking at 225 rpm. At 16 hours post-induction, the culture supernatant was collected for Western blot analysis.
VB4-847 Molecular Engineering and Periplasmic Expression- VB 4-847 -CODA in pSJFl plasmid
[00136] The RBS sequence between the EcoKl site and PelB leader of VB4-847-
CODA was removed by PCR mutagenesis using a 5' primer containing the EcoRl site followed by the initiation methionine codon and PelB sequence and a 3' primer in the VH region. The fragment containing i?coRI-Met-PelB-VL-VH- ¾vCI was cloned into the pCR 2.1 vector and transformed into 10F E. coli cells for sequencing. A Hindlll site was introduced between 3' end of PE and the Xhol site by PCR mutagenesis and the fragment containing ApaLl-VE-Hindlll-Xhol was cloned into the pCR 2.1 vector and transformed into 10F E. coli cells for sequencing. The pCR2.1 plasmids containing the correct inserts were digested with EcoRl-BbvCl and ApaLl-Xhol for the 5' and 3' fragments, respectively, and ligated with the BbvCl-ApaLl VH-PE fragment into the pING3302 plasmid pre-digested with EcoRl-Xhol. Chemically competent 10F E. coli cells were transformed with the ligation reaction and a transformed colony grown for plasmid extraction. Plasmid with the 2kb insert was then used to subclone the complete VB4-847-CODA fragment as an EcoRl-Hindlll
fragment into the pSJFI plasmid pre-digested with the same enzymes. Chemically competent 10F E. coli cells were transformed with the ligation reaction and a transformed colony grown for plasmid extraction. Plasmid with the 2kb insert was then used to transform E. coli TGI and selected colonies grown for small-scale expression.
Small-scale periplasmic expression in TGI cells
[00137] Transformed TGI cells containing VB4-847-CODA expressed from the pSJFI plasmid were inoculated into 5 mL 2-YT containing 100 μg/mL kanamycin and incubated at 37°C with constant shaking at 225 rpm. After 16 hours of incubation, 600 overnight seed culture was inoculated into 30 mL TB (2% inoculum), and incubated at 28°C with constant shaking at 225 rpm for 16 hours. The culture was induced with 30 of 100 mM IPTG (0.1 mM final) and incubated at 28°C with constant shaking at 225 rpm for 10 hours. The cells were pelleted by centrifugation and extraction buffer (3 M NaCl, 2 mM EDTA, 1 mM Tris- HC1 pH 8.0) added at a ratio of 1 mL of buffer per gram of cell pellet. Following a 30 minute incubation with mixing, the pellet was harvested and the supernatant transferred to a new tube. Shock buffer was added to the pellet at a ratio of 4 mL per original gram of cell pellet and incubated with mixing for 30 minutes. The supernatant was then harvested and combined with the extraction buffer supernatant to yield the periplasmic extract. The volume was corrected to adjust to the original culture volume for analysis by Western blot.
Western blot analysis
[00138] The level of expression was estimated by Western blot analysis. Briefly, 16 μΐ. of induced culture periplasmic extract or supernatant and 4 μΐ. LDS sample buffer were loaded onto a NuPAGE 10% Bis-Tris gel. The gel was then transferred to a nitrocellulose membrane at 40V for 1 hour. After blocking and washing the membrane, VB4-847 protein was detected using a rabbit anti-PE (1/5000) followed by a goat anti-rabbit antibody (1/1000) coupled to HRP. The membrane was developed using DAB and the level of expression of the VB4-847 proteins were compared to control VB4-845 as appropriate.
Fermentation and purification
[00139] E. coli E104 cells transformed with VB4-847/pING-RBS were cultivated in glycerol minimal media (GMM) + 25 μg/mL tetracycline in a 2 L Erlenmeyer flask in a shaker incubator (26°C, 200 rpm). When the optical density (OD6oo) of the culture was between 2.0-2.5, a volume of 150 mL was used to seed ~ 15 L of GMM in a 20 L Bioreactor. At an OD6oo of 50, the cells were induced with the addition of L-arabinose for VB4-847
expression for ~ 40 hours. The supernatant was then collected by centrifugation using a disk stacked continuous centrifuge and further clarified by microfiltration. The inventors surprisingly found that a Capto MMC column was not sufficient to capture all of the protein from the supernatant. Therefore, after concentration and diafiltration against 20 mM sodium phosphate pH 7.0, the supernatant containing the VB4-847 protein was applied onto a Q- sepharose column that was equilibrated and washed with 100 mM NaCl in 20 mM sodium phosphate buffer pH 7.5. The VB4-847 was then eluted with 300 mM NaCl in 20 mM sodium phosphate buffer pH 8.0. The eluate was then buffered exchanged by ultrafiltration against 20 mM sodium phosphate buffer pH 7.0 and applied to a Capto MMC column previously equilibrated with 20 mM sodium phosphate, pH 7.0. After sample loading, the column was washed with a 20 mM sodium phosphate, 100 mM NaCl pH 7.0 buffer until UV280 baselined. In this study, the Capto MMC column should not have successfully captured the protein because it generally requires that the protein to be captured has a lower pH than pi. However, the VB4-847 molecule precipitates when the pH falls below the pi. Surprisingly, the Capto MMC column was able to capture the VB4-847 protein despite the higher pH than pi. Bound VB4-847 was eluted with 20 mM sodium phosphate, 400 mM NaCl pH 7.0. The fractions containing the product were pooled, diluted to obtain a final concentration of 5 mM sodium phosphate, 200 mM NaCl, pH 7.1 and applied onto a ceramic hydroxy apatite (CHT) column previously equilibrated with 5 mM sodium phosphate, 200 mM NaCl pH 7.1 buffer. After washing to UV280 baseline with equilibration buffer, bound VB4-847 was eluted with 10 mM sodium phosphate, 200 mM NaCl pH 7.1 buffer. Based on the purity of the CHT flow through, wash and eluate assessed by SE-HPLC and SDS-PAGE gel, The CHT flow through and wash was pooled based purity and was then diluted 2-fold to reduce the salt concentration and pH adjusted to 8.0 for application on a Q-sepharose high performance column. The column was equilibrated with 20 mM sodium phosphate, 100 mM NaCl pH 8.0. After sample loading, the column was washed to UV280 baseline with equilibration buffer and bound VB4-847 was eluted in 20 mM sodium phosphate, 200 mM NaCl, pH 8.0. The peak fractions from the Q-sepharose column contained an approximately 2% impurity in the form of a small molecular weight fragment. The present inventors found that ultrafiltration did not remove this impurity; therefore, a size exclusion column was used at this stage to remove the impurity as expected. The peak fractions were concentrated and applied onto a size-exclusion column (Sephacryl S200) that was equilibrated in 150 mM NaCl in 20mM sodium phosphate pH 7.5 buffer. The VB4-847 peak fractions from the
Sephacryl S200 column was then concentrated and formulated to 5 mg/mL in 20 mM sodum phosphate buffer pH 8.0 containing 0.1 % Polysorbate 80.
MTS assay
[00140] Cal-27 and A-375 cells were used to assess the potency of VB4-847. The cytotoxicity was measured by MTS assay (Promega) following a 3 day treatment. Briefly, cells were seeded at 5000 cells per well in a 96 well plate and allowed to adhere at 37°C for 2-3 hours. Subsequently, indicated drugs were added to the cells over a range of concentrations. After 3 days, the cell viability was determined and the IC50 was interpolated from the resulting plot.
Results
VB4-847 Molecular Engineering and Soluble Expression
[00141] In order to evaluate the feasibility of expressing an EpCAM immunoconjugate without the His tags, an insert using the codon optimized nucleotide sequence (CODA) in the pING3302 plasmid was engineered and expressed in El 04 cells. As seen in Figure 2, the removal of the His tags negatively affected expression at the shake flask level. Compared with VB4-845-CODA-His expression, VB4-847-CODA soluble protein levels were dramatically lower.
[00142] As such, the expression of VB4-847 was then evaluated in E104 cells using the pING-RBS plasmid. The pING-RBS plasmid is a modification of the pING3302 plasmid in which the nucleotides between the end of the arabinose promoter and the RBS of the PelB leader sequence are deleted. The use of the pING/RBS plasmid has previously been shown to increase expression of both VB4-845 and an immunoconjugate comprising an EpCAM scFv and a de-immunized bouganin toxin (VB6-845). As seen in Figure 3, expression of VB4- 847-CODA from the pING-RBS plasmid did not rescue the drop in expression due to deletion of the His tags.
[00143] Although the use of the codon optimized nucleotide sequence has been previously shown to increase expression of VB4-845-His, the expression of VB4-847 using the non-optimized nucleotide sequence was evaluated for expression from the pING-RBS plasmid. Surprisingly, the construct with the original wild type sequence expressed higher levels of soluble protein than VB4-847-CODA at the shake flask level (Figure 4). The use of the pING-RBS plasmid however did not further increase VB4-847 expression above the levels observed with the pING3302 plasmid, as was the case with VB4-845-His.
VB 4-847 Molecular Engineering and Periplasmic Expression
[00144] To explore the possibility of expressing VB4-847 into the periplasmic space, an alternative IPTG-inducible system was used with TGI E. coli cells. The VB4-847-CODA insert was cloned into the pSJFl plasmid, placing VB4-847 expression under the control of an IPTG-inducible promoter. As seen in Figure 5, upon IPTG induction, the TGI cells expressed VB4-847 that could be extracted from the periplasmic space using osmotic shock. The expression levels were superior to the soluble material obtained with the E104 cells from the pING3302 plasmid.
VB 4-847 fermentation and biological testing
[00145] The VB4-847/pING-RBS clone in E104 was fermented at the 15 L scale and purification performed as described above. The cytotoxicity of purified VB4-847 was then tested against the EpCAM positive cell line, Cal-27 and the EpCAM negative A-375 cell line. As seen in Figure 6A, VB4-847 was highly potent against Cal-27 cells with an average IC50 of 0.385 pM. This was similar to the control VB4-845-His which showed an average IC50 of 0.405 pM. As expected, neither VB4-847 or VB4-845-His displayed any potency against the antigen negative A-375 cells (Figure 6B).
[00146] Thus, the results of the study showed that a non-codon optimized VB4-847 construct (i.e., an EpCAM-specific immunoconjugate that does not include His tags) could be efficiently expressed as well as exhibit highly potent cytotoxicity against EpCAM positive cell ines.
Discussion
[00147] This study provides the engineering and expression of VB4-847, a new
EpCAM-specific immunoconjugate. The effect of modifying the RBS in the pING plasmid has been mixed in past experiences, depending on the insert being expressed. In the case of the codon-optimized VB6-845 (VB6-845-CODA), the modified RBS increased expression, whereas VB4-845-CODA-His expression was negatively affected when the modified RBS was used. However, in the case of the wild type nucleotide sequence, VB4-845-His expression is greatly increased by the modified RBS. In this study, a comparison between VB4-845 (which contains His tags) and VB4-847 (no His tags) was conducted. Relative to the codon-optimized VB4-845, a decrease in expression was observed when the His tags were removed from the VB4-847-CODA sequence, and expression of VB4-847-CODA from
the pING-RBS plasmid did not restore expression levels. However, surprisingly, expression of the wild type VB4-847 construct from the pING-RBS plasmid was much higher than expression of the codon-optimized construct. Moreover, the product obtained from the pING- RBS expressed, non codon-optimized VB4-847 was highly potent against EpCAM positive cells.
[00148] In addition to soluble expression, VB4-847 was also amenable to periplasmic expression in E. coli. Although the amount of VB4-847-CODA extracted from the periplasm following induction was reasonable, periplasmic extraction is not the ideal process for large scale drug production and the use of a soluble expression system would be preferable.
[00149] Together, the results of the study showed that VB4-847 can be successfully produced and purified.
Claims
1. An immunoconjugate comprising an amino acid sequence according to SEQ ID NO: 2 or SEQ ID NO: 21.
2. The immunoconjugate of claim 1, wherein the amino acid sequence consists of SEQ ID NO: 2 or SEQ ID NO: 21.
3. The immunoconjugate of claim 1, wherein the amino acid sequence is encoded by a non codon-optimized nucleic acid sequence.
4. The immunoconjugate of claim 1, wherein the immunoconjugate exhibits cytotoxic activity against EpCAM-expressing cells.
5. A nucleic acid molecule encoding the immunioconjguate of claim 1.
6. An expression vector comprising a nucleic acid molecule according to claim 5.
7. A host cell comprising the expression vector of claim 7.
8. A method for treating cancer, the method comprising administering an
immunoconjugate of claim 1 to a subject in need thereof.
9. The method of claim 8, wherein the cancer is head and neck cancer.
10. The method of claim 9, wherein the head and neck cancer is squamous cell carcinoma of the head and neck (HNSCC).
11. The method of claim 8, wherein the composition is administered directly to the cancer site.
12. The method of claim 8, wherein the composition is administered intratumorally, intravesicularly, or peritumorally.
13. The method of claim 8, wherein the composition is administered systemically.
14. The method of claim 13, wherein the systemic administration is intravenous administration.
15. The method of claim 8, further comprising administering to the subject one or more additional therapeutic agent for simultaneous, separate or sequential treatment or prevention of cancer.
16. The method of claim 15, wherein the additional therapeutic agent is an immune checkpoint inhibitor, a chemotherapeutic drug, or a radiotherapeutic drug.
17. The method of claim 8, wherein the method reduces the size of one or more tumors present in the subj ect.
18. The method of claim 17, further comprising surgically removing one or more tumors from the subject following administration of the composition.
19. A kit for treating or preventing cancer comprising an effective amount of the composition according to claim 5 and directions for the use thereof to treat the cancer.
20 The kit of claim 19, further comprising reagents for detection of HPV in a sample from a subject suspected of having cancer.
21. A method for producing a protein, the method comprising
(i) inserting the nucleic acid molecule of claim 1 in a plasmid,
(ii) expressing the protein in a cell culture comprising E. coli cells, and
(iii) obtaining the protein from the cell culture supernatant.
22. The method of claim 21 , wherein the E. coli cells are E. coli E104 or TGI cells.
23. The method of claim 21 , wherein the plasmid is selected from the group consisting of pING-RBS, pING3302, and pSJFI.
24. The method of claim 21 , wherein the method further comprises purifying the protein.
25. The method of claim 24, wherein the protein is purified using a purification process comprising contacting the protein with at least one chromatographic material.
26. The method of claim 25, wherein the at least one chromatographic material is selected from hydroxy apatite and Capto MMC.
27. The method of claim 24, wherein impurities are removed from the protein using a size exclusion chromatographic step.
28. The method of claim 27, wherein the method further comprises applying the protein to a Q-sepharose column prior to applying the protein to the Capto MMC column.
29. The method of claim 27, wherein the method comprises the following steps:
(i) inserting a nucleic acid molecule encoding the amino acid sequence of claim 1 in a plasmid,
(ii) expressing the protein in a cell culture comprising E. coli cells,
(iii) inducing expression of the protein encoded by the nucleic acid sequence
(iv) obtaining the protein from the cell culture supernatant by centrifugation,
(v) clarifying the supernatant by microfiltration;
(vi) concentrating and diafiltrating the supernatant containing the protein,
(vii) contacting and eluting the protein on a Q-sepharose column,
(viii) contacting the protein with a Capto MMC column,
(ix) eluting the protein from the Capto MMC column,
(x) contacting the eluted protein with a hydroxy apatite column,
(xi) eluting the protein from the hydroxy apatite column,
(xii) adjusting the pH of the eluate to about 8.0,
(xiii) contacting the eluted protein with a Q-sepharose high performance column,
(xiv) eluting the protein from the Q-sepharose high performance column, and
(xv) contacting the protein on a size exclusion column.
30. The method of claim 29, wherein the nucleic acid sequence is according to SEQ ID NO: 1.
31. An immunoconjugate comprising (i) an antigen binding domain comprising a light chain CDR1 having at least 90% identity to SEQ ID NO: 5, a light chain CDR2 having at least 90% identity to SEQ ID NO: 6, a light chain CDR3 having at least 90% identity to SEQ ID NO: 7, a heavy chain CDR1 having at least 90% identity to SEQ ID NO: 12, a heavy chain CDR2 having at least 90% identity to SEQ ID NO: 13, and a heavy chain CDR3 having at least 90% identity to SEQ ID NO: 14; and (ii) a toxin;
wherein the amino acid sequence of the immunoconjugate does not comprise a His tag.
32. The immunoconjugate of claim 31, wherein the antigen binding domain comprises a light chain variable region comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 4 and a heavy chain variable region comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 11.
33. The immunoconjugate of claim 31, wherein the antigen binding domain is an scFv.
34. The immunoconjugate of claim 33, wherein the scFv comprises an amino acid seuqence having at least 90% identity to SEQ ID NO: 19.
35. The immunoconjugate of claim 31, wherein the toxin is ETA(252-608).
36. The immunoconjugate of claim 31, wherein the toxin comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 18.
37. The immunoconjugate of claim 29, wherein the antigen binding domain is conjugated to the toxin via a linker.
38. The immunoconjugate of claim 37, wherein the linker comprises a sequence having at least 90% identity to SEQ ID NO: 16.
39. An immunoconjugate comprising (i) an antigen binding domain comprising a light chain CDRl having at least 90% identity to SEQ ID NO: 5, a light chain CDR2 having at least 90% identity to SEQ ID NO: 6, a light chain CDR3 having at least 90% identity to SEQ ID NO: 7, a heavy chain CDRl having at least 90% identity to SEQ ID NO: 12, a heavy chain CDR2 having at least 90% identity to SEQ ID NO: 13, and a heavy chain CDR3 having at least 90% identity to SEQ ID NO: 14; and (ii) a toxin;
wherein the amino acid sequence of the immunoconjugate comprises a single His tag.
40. The immunoconjugate of claim 39, wherein the antigen binding domain comprises a light chain variable region comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 4 and a heavy chain variable region comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 11.
41. The immunoconjugate of claim 39, wherein the antigen binding domain is an scFv.
42. The immunoconjugate of claim 41, wherein the scFv comprises an amino acid seuqence having at least 90% identity to SEQ ID NO: 19.
43. The immunoconjugate of claim 39, wherein the toxin is ETA(252-608).
44. The immunoconjugate of claim 39, wherein the toxin comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 18.
45. The immunoconjugate of claim 39, wherein the antigen binding domain is conjugated to the toxin via a linker.
46. The immunoconjugate of claim 45, wherein the linker comprises a sequence having at least 90% identity to SEQ ID NO: 16.
47. The immunoconjugate of claim 39, wherein the His tag is an N-terminal His tag.
48. The immunoconjugate of claim 47, wherein the N-terminal His tag comprises 6 histidine residues.
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US9822182B2 (en) | 2013-10-02 | 2017-11-21 | Viventia Bio Inc. | Anti-EPCAM antibodies and methods of use |
US10576163B2 (en) | 2015-03-12 | 2020-03-03 | Viventia Bio Inc. | Methods of treatment for EpCAM positive bladder cancer |
US10583198B2 (en) | 2015-03-12 | 2020-03-10 | Viventia Bio Inc. | Dosing strategies for targeting EPCAM positive bladder cancer |
US10858429B2 (en) | 2003-04-30 | 2020-12-08 | University Of Zurich | Methods for treating cancer using an immunotoxin |
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WO2011116387A1 (en) * | 2010-03-19 | 2011-09-22 | Tetragenetics, Inc. | Production of aglycosylated monoclonal antibodies in ciliates |
SG11201508419PA (en) * | 2013-04-12 | 2015-11-27 | Viventia Bio Inc | Compositions and methods for detection and treatment of hepatocellular carcinoma |
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US10858429B2 (en) | 2003-04-30 | 2020-12-08 | University Of Zurich | Methods for treating cancer using an immunotoxin |
US9822182B2 (en) | 2013-10-02 | 2017-11-21 | Viventia Bio Inc. | Anti-EPCAM antibodies and methods of use |
US10576163B2 (en) | 2015-03-12 | 2020-03-03 | Viventia Bio Inc. | Methods of treatment for EpCAM positive bladder cancer |
US10583198B2 (en) | 2015-03-12 | 2020-03-10 | Viventia Bio Inc. | Dosing strategies for targeting EPCAM positive bladder cancer |
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