Patent Grant
11208634
This application contains a Sequence Listing electronically submitted via EFS-Web to the United States Patent and Trademark Office as an ASCII text file entitled “2016-09-30-SequenceListing_ST25.txt” having a size of 58 kilobytes and created on Sep. 30, 2016. The information contained in the Sequence Listing is incorporated by reference herein.
This disclosure describes, in one aspect, an engineered polypeptide that includes a diphtheria toxin (DT) domain and a targeting domain. The DT domain includes the DT catalytic site and at least one amino acid substitution that decreases induction of anti-toxin antibodies compared to wild-type diphtheria toxin. The targeting domain includes a targeting moiety that selectively binds to a target.
In some embodiments, the targeting domain selectively binds to a component of a tumor cell. In some embodiments, the polypeptide can include two or more targeting domains and/or two or more targeting moieties
In some embodiments, the DT domain includes at least three amino acid substitutions compared to the amino acid sequence of DT390.
In another aspect, this disclosure describes a method of killing a cell. Generally, the method includes contacting the cell to be killed with any embodiment of the polypeptide summarized above under conditions that allow the cell to internalize the polypeptide; and allowing the polypeptide to kill the cell. In various embodiments, the cell may be in vitro or in vivo.
In another aspect, this discloser describes a method of treating a subject having a tumor. Generally, the method includes administering to the subject any embodiment of the polypeptide summarized above in which the polypeptide includes a targeting domain selectively binds to a target present on cells of the tumor.
In another aspect, this discloser describes a method of deimmunizing a diphtheria toxin polypeptide. Generally, the method includes identifying hydrophilic amino acid residues positioned at the surface of the native diphtheria toxin polypeptide, constructing a modified diphtheria toxin polypeptide comprising a substitution of one of the identified hydrophilic amino acid residues, screening the modified diphtheria toxin polypeptide for biological function of the native diphtheria toxin polypeptide, and screening the modified diphtheria toxin polypeptide for induction of antibodies.
The above summary of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.
This disclosure describes compounds, compositions, and methods that involve deimmunized targeted toxins. The use of biological drugs can be limited because they can be recognized as foreign by the human immune system. This can be particularly true of bacterial proteins such as, for example, diphtheria toxin (DT). Diphtheria toxin has promise as a targeted toxin, but even federal drug administration (FDA) approved DT-based drugs such as denileukin diftitox (ONTAK, Eisai Co., Ltd., Tokyo, Japan) can elicit anti-toxin responses after multiple treatments. Furthermore, those who receive diphtheria, tetanus, and pertussis (DPT) immunization at an early age have a greater likelihood of generating an anti-toxin response on multiple treatment with DT-based drugs. DT targeted toxins (TT) have nevertheless proven effective in eliciting anti-cancer responses in many different animal models and in patients. Thus, pursuit of a deimmunized form of the toxin has been considered a desirable, but unattainable goal.
Diphtheria toxin is a 535 amino acid protein (mw 58.3 kDa), having two functional domains. The C-terminal B domain binds most eukaryotic cells. The B domain may be removed to form DT390 and/or replaced with ligands to form a DT targeted toxin. The N-terminal A domain includes the catalytic region that ADP-ribosylates elongation factor 2 (EF-2).
DTEGF13 is a bispecific ligand directed toxin (BLT) that includes truncated DT (DT390) and two ligands. The ligands include IL-13 and epidermal growth factor (EGF). The ligands bind to unrelated receptors. EGF binds to epidermal growth factor receptor (EGFR), a transmembrane signaling protein from the erbB family. EGFR is overexpressed on a range of carcinomas including carcinomas of the prostate, pancreatic, breast, and/or lung. IL-13 is a pleiotropic lymphokine and its receptor is overexpressed on tumors, B cells, and monocytes. DTEGF13 binds to its target via the EGF and IL-13 ligands, and once internalized it is toxic to target cells. In vivo, DTEGF13 is effective locally and systemically against cancers including but not limited to prostate, glioblastoma, and pancreatic carcinomas in local and systemic xenograft models. Thus, DTEGF13 generates an anti-diphtheria toxin response when injected in vivo and provides a model for deimmunization.
Hydrophilic amino acids having surface positions, as derived from an x-ray crystallographic model, away from the NAD-ribosylating catalytic site were selected for point mutation to create a deimmunized DT molecule of the invention. The mutants were sequentially screened for those that underwent screening for minimal activity loss. Candidate mutants with at least seven mutations were tested for their ability to generate anti-toxin IgG antibody when given multiple injections in animal models. Despite multiple immunizations, the deimmunized DTEGF13 (dDTEFG13; SEQ ID NO:8) showed reduced anti-toxin induction when compared to the non-mutated parental form.
Based on the molecular model derived from the x-ray crystallographic structure, twenty amino acid residues located in prominent surface positions were identified. A series of eight mutants were generated, each having three point mutations. Thus, the entire series probed 24 point mutations. Alanine and serine substitution were employed. The mutants were screened for activity loss using a standard, highly reproducible proliferation inhibition assay measuring thymidine uptake.
To determine whether DTEGF13 had been successfully deimmunized, groups of immunocompetent BALB/c mice (H-2d) were immunized weekly with 0.25 μg of either mutated dDTEGF13, or non-mutated parental DTEGF13. Animals were immunized intraperitoneally over a period of 82 days. Serum samples were obtained weekly and analyzed using ELISA to detect anti-dDTEGF 13 IgG. The results of the immunization experiment are summarized in
To determine if neutralizing antibodies were present in the sera from immunized mice, sera from the mice in
To test the ability of dDTEGF13 to inhibit tumor growth in vivo, PC-3 cells were injected into the flank of nude mice. Once the tumors were established and palpable, mice were treated with multiple intratumoral injections. dDTEGF13 was studied in a mouse model because the human EGF and IL-13 (of DTEGF13) reacts with mouse EGFR and IL13R, respectively. The upper panel of
This disclosure therefore describes, in one aspect, a deimmunized DT targeted toxin polypeptide that may be used in molecular drug development. Generally, the polypeptide includes a deimmunized diphtheria toxin domain that includes the DT catalytic site and at least one targeting domain selected to selectively bind to a target cell. In some cases, the targeting domain can include a domain that selectively binds to a tumor cell target. As used herein, the term “deimmunized” refers to a polypeptide that has been modified to reduce the extent to which the polypeptide induces an immune response in the subject to which it is administered as compared with a molecule that has not been deimmunized. In some cases, an antibody response to the DT targeted toxin molecule is measured. In one embodiment, antibody induction can be characterized in terms of antibody production induced in an immunocompetent mouse strain. Also, as used herein, the term “selectively” refers to having a differential affinity—e.g., greater than a general non-selective affinity—to any degree toward a particular desired target. An exemplary targeting domain can include a sufficient portion of a ligand or binding moiety, such as, for example, EGF, IL-13, anti-CD19, and/or anti-CD22, to selectively bind to its receptor that is expressed by a target cell. The resulting polypeptide exhibits measurable diphtheria toxin toxicity with reduced induction of anti-toxin antibodies in immunocompetent mice compared to wild-type diphtheria toxin. The polypeptide may be useful as a drug effective against, for example, neoplastic conditions such as pancreatic cancer, breast cancer, and glioma.
In some embodiments, a deimmunized DT molecule can include at least one amino acid substitution compared to DT390 (or full length DT). Thus, a dDT molecule can have at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least or 24 mutations relative to DT390 or full length DT.
Thus, in some embodiments, the dDT molecule can include a substitution of at least three of amino acid residues 15, 100, 104, 125, 173, 185, 227, 242, 245, 292, 318, or 385. In one embodiment, the dDT molecule can include amino acid substitutions of K125, R173, and Q245. In another embodiment, the dDT molecule can include amino acid substitutions K125S, R173A, and Q245S. In another embodiment, the dDT molecule can include amino acid substitutions K104, K385, and E292. In another embodiment, the dDT molecule can include amino acid substitutions K104S, K385G, and E292S. In another embodiment, the dDT molecule can include amino acid substitutions E100, Q184, and K227. In another embodiment, the dDT molecule can include amino acid substitutions E100S, Q184S, and K227S. In another embodiment, the dDT molecule can include amino acid substitutions E15S, D318, and K242S.
In other embodiments, the dDT molecule can include at least five amino acids substitutions. Thus, in one embodiment, the dDT molecule can include amino acid substitutions K125, R173, Q245, K385, and E292. In another embodiment, the dDT molecule can include amino acid substitutions K125S, R173A, Q245S, K385G, and E292S.
In still other embodiments, the dDT molecule can include at least seven amino acid substitutions. Thus, in one embodiment, the dDT molecule can include amino acid substitutions K125, R173, Q245, K385G, E292, Q184, and K227. In another embodiment, the dDT molecule can include amino acid substitutions K125S, R173A, Q245S, K385G, E292S, Q184S, and K227S. In another embodiment, the dDT molecule can include amino acid substitutions K125S, R173A, Q245S, K385G, E292S, Q184S, and K227S.
While the dDT molecule is described using substitutions of serine, alanine, and/or glycine, these are exemplary molecules and one of skill in the art would be able to readily determine if other amino acids would provide a dDT molecule as described herein. For example, alanine, serine, and glycine may be used interchangeably to create dDT molecules that can be screened for activity and reduced immunogenicity, as described in the Examples herein.
While described above in the context of an exemplary embodiment in which the targeting domain can include a sufficient portion of EGF, IL-13, anti-CD19, and/or anti-CD22 to selectively bind to a desired target, the targeting domain can include any suitable targeting moiety. A “targeting moiety” refers to that portion of a compound (e.g., a ligand) that possesses target-selective affinity (e.g., toward a receptor for the ligand) that may be employed in a targeting domain. The targeting moiety may be, or be derived from, a ligand, an antibody, a cytokine, or an interleukin. In some cases, the targeting moiety may be, or be derived from, an agent that recognizes at least a portion of a tumor-specific marker such as, for example, a ligand that binds to a receptor that is, to some extent, specifically expressed by a target cell. In other cases, the targeting moiety may be an antibody or be derived from an antibody (e.g., at least enough of the immunospecific portion of an antibody—e.g., enough of a light chain—to provide some degree of immunospecificity).
In various embodiments, a targeting domain can include more than one targeting moiety. For example, an exemplary embodiment referred to herein as dDTEGF13 includes a first and second targeting moiety. The first targeting moiety includes a sufficient portion of EGF to selectively bind to EGFR, while the second targeting moiety includes a sufficient portion of IL-13 to selectively bind to IL-13 receptor. Likewise, an exemplary embodiment referred to herein as dDT2219 also includes a first and second targeting moiety. The first targeting moiety includes a sufficient portion of an anti-CD19 scFv to selectively bind to CD-19, while the second targeting moiety includes a sufficient portion of an anti-CD22 scFv to selectively bind to CD-22.
Thus, in various embodiments, the targeting domain can include a polypeptide that selectively binds to, for example, EGFR, HER2/neu, EpCAM, CD19, CD20, CD22, CD30, CD52, CD33, ROR-1, UPAR, VEGFR, CEA, UPA, LIV-1, SGN-CD70A, CD70, IL-3 alphA receptor, IL-4R, CD133, ROR1, mesothelin, TRAIL, CD38, CD45, CD74, or CD23.
The dDT2219 molecule is an engineered variant of DT2219 in which the diphtheria toxin domain of the molecule is replaced with a deimmunized DT domain. DT2219 is a recombinant fusion protein that contains the catalytic and translocation enhancing domain of diphtheria toxin (DT390) fused with bispecific single chain variable fragments (scFV) of antibodies targeting human CD19 and CD22 cell surface receptors. The protein is engineered so that the native binding region of DT is replaced by the more avidly bound scFV. After binding, CD19 and CD22 readily internalize to promote toxin entry into the cytosol, inhibition of protein synthesis, and/or subsequent apoptotic cell death.
CD19, a 95 kDa membrane glycoprotein, is ubiquitously present on the surface of all stages of B lymphocyte development and is also expressed on most B-cell mature lymphoma cells and leukemia cells. CD22 is 135-kDa glycoprotein expressed on B lineage lymphoid precursors, including precursor B acute lymphoblastic leukemia, and often is co-expressed with CD19 on mature B cell malignancies. DT mediates potent cell-cycle independent cell death and therefore can be particularly effective as an alternative therapy for chemotherapy refractory malignancies.
Construction of the dDT2219 molecule is shown in
In order to show activity and kinetics of dDT2219 compared with the parental DT2219 form, proliferation assays we performed with the CD19+/CD22+ Burkitt lymphoma cell lines Daudi and Raji. Therefore, H3-Thymidine uptake by Daudi cells was evaluated after exposure to the respective drug (
For verification of the specificity of dDT2219 (DT2219 and BIC were used as controls), a proliferation assay was performed with HPB-MLT cells (T-cell leukemia cell line that expresses CD3 but not CD19 or CD22) and dDT2219, DT2219, and BIC3 in labeled increasing concentrations. No effect after dDT2219 and DT2219 exposure was visible. Only BIC3 induced apoptosis (
Binding and blocking characteristics were compared using flow cytometry. FITC-labeled dDT2219 (
In order to verify the binding capability in a clinical context, samples obtained from CLL patients were used to compare respective binding of dDT2219 and DT2219. Samples were exposed to increasing concentrations (no drug, 1 nM, 10 nM, 20 nM, 50 nM, or 100 nM) of FITC-labeled dDT2219 or DT2219. After gating on the CLL population, a dose-dependent increase in binding was seen for both constructs (
To evaluate whether neutralizing antibodies develop in the sera of immunized mice after repetitive exposure to dDT2219, BALB/c mice were divided into two groups of seven animals. Both groups were immunized simultaneously with either dDT2219 or DT2219 (control group). Both groups were treated with an equal concentration of dDT2219 or DT2219. In in all evaluated days, sera of the dDT2219 group showed a significantly lower (p<0.05) antibody induction, seen in an ELISA detecting α-DT390 (
In order to specify whether detected antibodies indeed neutralize dDT2219 or DT2219, a neutralization assay was performed using Raji targets and sera of both animal groups of day 160. A significantly lower amount of neutralizing antibodies (p<0.05) was found in the dDT2219 group (
Twenty-five patients with mature or precursor B-cell lymphoid malignancies expressing CD19 and/or CD22 were enrolled in a study to assess the safety of DT2219. All patients received a single course of DT2219. The most common adverse events (AE) including, weight gain, low albumin, transaminitis and fevers were transient grade 1-2 and occurred in patients in higher dose cohorts (≥40 μg/kg/day). Two subjects experienced DLT at dose levels 40 μg/kg and 60 μg/kg. Neutralizing antibodies were observed in 30% of patients after four courses of treatment and in all patients treated with at least 40 μg/kg (Bachanova et al., 2015. Clin Cancer Res 21:1267-1272).
Approaches to limit immunogenicity of DT2219 have been investigated, but none has proven to be a practical method for decreasing immune reactions to DT2219. Various approaches have included the use of polyethylene glycol (PEG) polymers or RNases, each of which can be conjugated to bioactive drugs, or co-administration with a B-cell depletive agents (e.g., rituximab).
Thus, this disclosure describes a deimmunized DT2219, dDT2219, as an exemplary targeted toxin with therapeutic activity. dDT2219 showed efficacy against B-cell malignancies, including two different Burkitt-lymphoma cell lines. No toxic effect for cell lines not expressing CD22 or CD19 was documented. The dDT2219 construct showed the same binding capacity to CLL as its parental form and a significantly lower induction of neutralizing antibodies in immunocompetent BALB/c mice. Since DT targeted toxin polypeptides and, in particular, DT2219 have provided promising results in induction of anti-cancer responses in vitro as well as in vivo, the deimmunized dDT2219 can provide anti-tumor therapy independent from limitation of anti-drug antibodies after multiple administrations and of vaccination status in a patient's records.
As noted above, the deimmunized targeted toxin can include a targeting domain that selectively binds to targets other than CD19 and/or CD22. Additional exemplary deimmunized bispecific targeted toxins have been produce and tested in an identical manner to dDT2219, as shown in Table 2.
dDTEpCAMe23 simultaneously targets EpCAM Epithelial cell adhesion molecule, a transmembrane glycoprotein mediating Ca2+-independent homotypic cell—cell adhesion in epithelia and e23 or HER2, a member of the human epidermal growth factor receptor (HER/EGFR/ERBB) family. dDTEpCAM133 simultaneously targets EpCAM and CD133, known as an established cancer stem cell marker. CD133, also known as prominin-1, is a glycoprotein that in humans is encoded by the PROM1 gene and a member of pentaspan transmembrane glycoproteins (5-transmembrane, 5-TM), which specifically localize to cellular protrusions. dDTEGFATF simultaneously targets EGFR and the amino terminal fragment (ATF) of urokinase (uPA). dDTROR1ATF simultaneously targets ATF and ROR1. ROR1 is a receptor tyrosine kinase modulating neurite growth. It is a type I membrane protein belonging to the ROR subfamily of cell surface receptors and is currently under investigation for its role in the metastasis of cancer cells.
In another aspect, this disclosure describes a method of killing a cell. Generally, the method includes contacting the cell with the deimmunized DT targeted toxin polypeptide described above, allowing the cell to internalize the deimmunized DT targeted toxin polypeptide, and allowing the deimmunized DT targeted toxin polypeptide to kill the cell. The method may be performed in vivo or in vitro.
In another aspect, this disclosure describes methods of treating a subject having a tumor. Generally, the method includes administering to the subject a deimmunized diphtheria toxin (dDT) targeted toxin compound in an amount effective to ameliorate at least one symptom or clinical sign of the tumor. “Treat” or variations thereof refer to reducing, limiting progression, ameliorating, or resolving, to any extent, the symptoms or signs related to a condition. As used herein, “ameliorate” refers to any reduction in the extent, severity, frequency, and/or likelihood of a symptom or clinical sign characteristic of a particular condition; “symptom” refers to any subjective evidence of disease or of a patient's condition; and “sign” or “clinical sign” refers to an objective physical finding relating to a particular condition capable of being found by one other than the patient.
A “treatment” may be therapeutic or prophylactic. “Therapeutic” and variations thereof refer to a treatment that ameliorates one or more existing symptoms or clinical signs associated with a condition. “Prophylactic” and variations thereof refer to a treatment that limits, to any extent, the development and/or appearance of a symptom or clinical sign of a condition. Generally, a “therapeutic” treatment is initiated after the condition manifests in a subject, while “prophylactic” treatment is initiated before a condition manifests in a subject. Thus, in certain embodiments, the method can involve prophylactic treatment of a subject at risk of developing a condition. “At risk” refers to a subject that may or may not actually possess the described risk. Thus, for example, a subject “at risk” for developing a specified condition is a subject that possesses one or more indicia of increased risk of having, or developing, the specified condition compared to individuals who lack the one or more indicia, regardless of the whether the subject manifests any symptom or clinical sign of having or developing the condition. Exemplary indicia of a condition can include, for example, genetic predisposition, ancestry, age, sex, geographical location, lifestyle, or medical history. Treatment may also be continued after symptoms have resolved, for example to prevent or delay their recurrence.
The dDT targeted toxin compound can be any embodiment of the dDT targeted toxin compound described above having a targeting domain that selectively binds to a target present on cells of the tumor.
In some cases, the tumor may be surgically resected or reduced through chemical (e.g., chemotherapeutic agents) and/or radiation therapy. In such embodiments, the dDT may be administered to the subject prior to, simultaneously with, or after the tumor is resected or reduced. Thus, in some embodiments, the dDT may be administered to the subject prior to, simultaneously with, or after a chemotherapeutic agent is administered to the subject.
The tumor may be a solid tumor or may be a liquid tumor. Accordingly, the dDT may be used to treat various forms of cancer including, for example, prostate cancer, lung cancer, colon cancer, rectum cancer, urinary bladder cancer, melanoma, kidney cancer, renal cancer, oral cavity cancer, pharynx cancer, pancreatic cancer, uterine cancer, thyroid cancer, skin cancer, head and neck cancer, cervical cancer, ovarian cancer, hematopoietic cancers and/or lymphatic cancers.
A “subject” may be any animal subject such as, for example, a mammalian subject (e.g., dog, cat, horse, cow, sheep, goat, monkey, primate, human, etc.). In some embodiments, the subject may be human, including both male and female subjects, and including neonatal, infant, juvenile, adolescent, adult and geriatric subjects.
Thus, in some embodiments, a targeting domain that includes a sufficient portion of epidermal growth factor (EGF) to selectively bind to epidermal growth factor receptor (EGFR) may be used to target a dDT targeted toxin compound to a tumor cell that expresses EGRF such as, for example, carcinomas of the prostate, pancreatic, breast, and/or lung.
In other embodiments, a targeting domain that includes an anti-CD19 and/or anti-CD 22 scFv may be used to target a dDT targeted toxin compound to a tumor cell that expressed CD19 and/or CD22 such as, for example, B cell malignancies such as non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), or Burkitt lymphoma.
A dDT targeted toxin described herein may be formulated with a pharmaceutically acceptable carrier. As used herein, “carrier” includes any solvent, dispersion medium, vehicle, coating, diluent, antibacterial, and/or antifungal agent, isotonic agent, absorption delaying agent, buffer, carrier solution, suspension, colloid, and the like. The use of such media and/or agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions. As used herein, “pharmaceutically acceptable” refers to a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with a dDT targeted toxin without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
A dDT targeted toxin may therefore be formulated into a pharmaceutical composition. The pharmaceutical composition may be formulated in a variety of forms adapted to a preferred route of administration. Thus, a composition can be administered via known routes including, for example, oral, parenteral (e.g., intradermal, transcutaneous, subcutaneous, intramuscular, intravenous, intraperitoneal, etc.), or topical (e.g., intranasal, intrapulmonary, intramammary, intravaginal, intrauterine, intradermal, transcutaneous, rectally, etc.). A pharmaceutical composition can be administered to a mucosal surface, such as by administration to, for example, the nasal or respiratory mucosa (e.g., by spray or aerosol). A composition also can be administered via a sustained or delayed release.
Thus, a dDT targeted toxin may be provided in any suitable form including but not limited to a solution, a suspension, an emulsion, a spray, an aerosol, or any form of mixture. The composition may be delivered in formulation with any pharmaceutically acceptable excipient, carrier, or vehicle. For example, the formulation may be delivered in a conventional topical dosage form such as, for example, a cream, an ointment, an aerosol formulation, a non-aerosol spray, a gel, a lotion, and the like. The formulation may further include one or more additives including such as, for example, an adjuvant, a skin penetration enhancer, a colorant, a fragrance, a flavoring, a moisturizer, a thickener, and the like.
A formulation may be conveniently presented in unit dosage form and may be prepared by methods well known in the art of pharmacy. Methods of preparing a composition with a pharmaceutically acceptable carrier include the step of bringing a dDT targeted toxin into association with a carrier that constitutes one or more accessory ingredients. In general, a formulation may be prepared by uniformly and/or intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulations.
The amount of dDT targeted toxin administered can vary depending on various factors including, but not limited to, the specific dDT targeted toxin being used, the weight, physical condition, and/or age of the subject, and/or the route of administration. Thus, the absolute weight of dDT targeted toxin included in a given unit dosage form can vary widely, and depends upon factors such as the species, age, weight and physical condition of the subject, and/or the method of administration. Accordingly, it is not practical to set forth generally the amount that constitutes an amount of dDT targeted toxin effective for all possible applications. Those of ordinary skill in the art, however, can readily determine the appropriate amount with due consideration of such factors.
In some embodiments, the method can include administering sufficient dDT targeted toxin to provide a dose of, for example, from about 100 ng/kg to about 50 mg/kg to the subject, although in some embodiments the methods may be performed by administering dDT targeted toxin in a dose outside this range. In some of these embodiments, the method includes administering sufficient dDT targeted toxin to provide a dose of from about 10 μg/kg to about 5 mg/kg to the subject, for example, a dose of from about 100 μg/kg to about 1 mg/kg.
Alternatively, the dose may be calculated using actual body weight obtained just prior to the beginning of a treatment course. For the dosages calculated in this way, body surface area (m2) is calculated prior to the beginning of the treatment course using the Dubois method: m2=(wt kg0.425×height cm0.725)×0.007184.
In some embodiments, the method can include administering sufficient dDT targeted toxin to provide a dose of, for example, from about 0.01 mg/m2 to about 10 mg/m2.
In some embodiments, a dDT targeted toxin compound may be administered, for example, from a single dose to multiple doses per week, although in some embodiments the method can be performed by administering a dDT targeted toxin at a frequency outside this range. In certain embodiments, a dDT targeted toxin may be administered from about once per month to about five times per week.
In yet another aspect, this disclosure describes a method of deimmunizing a toxin. Generally, the method involves targeting hydrophilic amino acid residues located at surface positions of the toxin polypeptides that can be involved in generating anti-B cell responses, constructing a modified polypeptide that includes a substitution of one of the identified hydrophilic amino acid residues, screening the modified polypeptide for biological function of the native polypeptide, and screening the modified polypeptide for induction of antibodies. As used herein, “biological function,” “biological activity,” and variations thereof refer to a native biological function of the polypeptide. In some cases, however, the biological function (or biological activity, etc.) can refer to a particular function that is relevant to a therapeutic use of the polypeptide such as, for example, cytokine induction, cellular signaling, and the like.
The deimmunizing method was much less time consuming and more cost-effective than, for example, epitope mapping techniques. Using the known x-ray crystallographic structure of DT390, 24 potential amino acids (R, K, D, E, and Q) located in prominent positions on the molecular surface were selected in order to introduce point mutations at the selected sites. The DTEGF13 parental construct was mutated simultaneously with three site-specific PCR primers to generate triple mutants. Those that did not exhibit activity loss were further mutated, finally resulting in two constructs each with seven point mutations, but with minimal activity loss. Mutating DTEGF13 and expressing the product allows one to effectively screen the mutants for activity in vitro anti-carcinoma assays compared to the non-muted parental form.
The structure and mechanism of diphtheria toxin are well established. The point mutations were selected to be located well away from the catalytic active site. Moreover, in order to address whether point mutation removed T cell or B cell epitopes, two different strains of mice with different MHC haplotypes (H-2b and H-2d) were immunized. MHC molecules are supposed to load different regions of peptide fragments for presentation as T cell epitopes and T and B epitopes are not necessarily linked. The mutated drug (dDTEGF13; SEQ ID NO:8) was equally effective in reducing anti-toxin responses in both strains, indicating that B cell epitopes rather than T cell epitopes have been eliminated from DT.
In some cases, therefore, the dDT targeted toxin compound can exhibit at least 5% of the biological activity of the unmodified native diphtheria toxin such as, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% of the biological activity of the unmodified native diphtheria toxin.
In some cases, the dDT targeted toxin compound can exhibit no more than 50% of the anti-toxin antibody induction of the unmodified native diphtheria toxin such as, for example, no more than 45%, no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 19%, no more than 18%, no more than 17%, no more than 16%, no more than 15%, no more than 14%, no more than 13%, no more than 12%, no more than 11%, no more than 10%, no more than 9%, no more than 8%, no more than 7%, no more than 6%, no more than 5%, no more than 4%, no more than 3%, no more than 2%, or no more than 1% of the anti-toxin antibody induction of the unmodified native diphtheria toxin.
In the preceding description and following claims, the term “and/or” means one or all of the listed elements or a combination of any two or more of the listed elements; the terms “comprises,” “comprising,” and variations thereof are to be construed as open ended—i.e., additional elements or steps are optional and may or may not be present; unless otherwise specified, “a,” “an,” “the,” and “at least one” are used interchangeably and mean one or more than one; and the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).
In the preceding description, particular embodiments may be described in isolation for clarity. Unless otherwise expressly specified that the features of a particular embodiment are incompatible with the features of another embodiment, certain embodiments can include a combination of compatible features described herein in connection with one or more embodiments.
For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.
The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.
Construction of dDTEGF13
The DTEGF13 coding region (SEQ ID NO:13) was originally synthesized using assembly PCR. In its final configuration, the coding region (from 5′ end to 3′ end) includes an NcoI restriction site, an ATG initiation codon, the first 389 amino acids of the DT molecule (DT390), the seven amino acid linker EASGGPE (SEQ ID NO:4), the coding regions for human EGF and IL-13 linked by a 20 amino acid segment of human muscle aldolase (hma), and an XhoI restriction site (
To create a deimmunized drug, DTEGF13 was mutated using the QuickChange Site-Directed Mutagenesis Kit (Stratagene. La Jolla, Calif.) and site specific mutations were confirmed by DNA sequencing.
Isolation of Inclusion Bodies, Refolding and Purification
These procedures were previously described (Stish et al., 2007, Clin Cancer Res 13:6486-6493). Plasmids were transformed into E. coli strain BL21(DE3) (Novagen, Madison, Wis.). Following overnight culture, bacteria were grown in Luria broth. Gene expression was induced with the addition of IPTG (FischerBiotech, Fair Lawn, N.J.). Two hours after induction, bacteria were harvested by centrifugation. Cell pellets were suspended and homogenized. Following sonication and centrifugation, the pellets were extracted and washed. Inclusion bodies were dissolved and protein refolded. Refolded proteins were purified by fast protein liquid chromatography ion exchange chromatography (Q SEPHAROSE Fast Flow, Sigma-Aldrich, St. Louis, Mo.) using a continuous gradient.
Antibodies and Cells
Anti-Ly5.2, a rat IgG2a from clone A20-1.7 was generously provided by Dr. Uli Hammerling, Sloan Kettering Cancer Research Center, New York, N.Y. Anti-Ly5.2 was used as a control since it recognized mouse CD45.1, a hematopoietic cell surface marker not expressed on human cells.
Human Cell Lines
The human prostate cancer cell line PC-3 (ATCC CRL-14435; Kaighn et al., 1979, Invest Urol 17:16-23), human colorectal cell line HT-29 (ATCC HTB-38; Fogh et al., 1977, J Natl Cancer Inst 59:221-226), the human pancreatic carcinoma MiaPaCa-2 (ATCC CRL-1420; Yunis et al. 1977, Int J Cancer 19:128-35), and the Burkitt's Lymphoma cell line Daudi (ATCC CCL-213; Klein et al., 1968, Cancer Res 28:1300-1310) were obtained from American Type Culture Collection (ATCC, Manassas, Va.). Cells were maintained in RPMI-1640 media (Cambrex, East Rutherford, N.J.) supplemented with 10% fetal bovine serum, 2 mmol/L L-glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin. All carcinoma cells were grown as monolayers and Daudi cells in suspension using culture flasks. Cell cultures were incubated in a humidified 37° C. atmosphere containing 5% CO2. When adherent cells were 80-90% confluent, they were passaged using trypsin-EDTA for detachment. Only cells with viability >95%, as determined by TRYPAN blue exclusion, were used for experiments.
Proliferation Assay
To determine the effect of drug on tumor cells, cells (2×104) were plated in a 96-well flat-bottom plate in RPMI supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin. BLT in varying concentrations was added to triplicate wells containing cells (Tsai et al., 2011, J Neurooncol 103:255-266). The plates were incubated at 37° C., 5% CO2 for 72 hours. Cells were then incubated with one μCi [methyl-3H]-thymidine (GE Healthcare, UK) per well for eight hours and harvested onto glass fiber filters, washed, dried and counted for ten minutes in a standard scintillation counter. Data were analyzed using Prism 4 (GraphPad Software, Inc., San Diego, Calif.) and were presented as “percent control response” calculated by dividing the counts per minute (cpm) of untreated cells by the cpm of the immunotoxin-treated cells (×100).
Blocking studies were conducted to test specificity. Briefly, 1 nM, 10 nM, 100 nM, or 1000 nM EGF13 devoid of toxin were added to media containing 0.001 nM, 0.01 nM, 0.1 nM, 1 nM, 10 nM, or 100 nM DTEGF13 (Vallera et al., 2010, Mol Cancer Ther 9:1872-1883). Resulting mixtures were added to wells containing tumor cells and proliferation was measured by 3H-thymidine uptake as described. Data were presented as percent control response.
To detect neutralizing antibodies, 90% serum from immunized mice was added to cells treated with a known inhibitory concentration of DTEGF13 (1000 ng/ml). Proliferation assays were then carried out as described above.
Detection of Serum IgG Anti-Toxin Content Using ELISA Assay
The assay to detect IgG anti-toxin antibodies was previously reported (Vallera et al., 2009, Leuk Res 33:1233-1242). Briefly, immunocompetent normal BALB/c mice (NCI) were immunized with weekly injections of 0.25 μg non-mutated 2219KDEL or mutated 2219KDEL 7 mut. After five injections, serum was collected four days after the final injection. A standard ELISA assay was used in which recombinant DT390 was adhered to the plate. Test serum from the immunized mice was then added followed by the detection antibody, anti-mouse IgG peroxidase (Sigma-Aldrich, St. Louis, Mo.). Plates were developed with o-phenylenediamine dihydrochloride (Pierce Biotechnology, Rockford, Ill.) for 15 minutes at room temperature. The reaction was stopped with the addition of 2.5 M H2SO4. Absorbance was read at 490 nm and the final concentration was determined from a standard curve using highly purified anti-DT390. All samples and standards were tested in triplicate.
In Vivo Efficacy Studies
Male nu/nu mice were purchased from the National Cancer Institute, Frederick Cancer Research and Development Center, Animal Production Area and housed in an Association for Assessment and Accreditation of Laboratory Animal Care-accredited specific pathogen-free facility under the care of the Department of Research Animal Resources, University of Minnesota. Animal research protocols were approved by the University of Minnesota Institutional Animal Care and Use Committee. All animals were housed in microisolator cages to minimize the potential of contaminating virus transmission.
Our flank tumor model was previously reported (Stish et al., 2007, Clin Canc Res 13:6486-6493). This route of administration was chosen because the major point of this experiment was not to mimic a clinical protocol, but was to determine if deimmunized DTEGF13 was efficacious. Direct intratumoral administration circumvents the major problems of systemic administration. These include inefficient distribution to tumor site related to the distance traveled and unfavorable tumor vasculature dynamics (high interstitial pressures) (Jain R K 1989, J Natl. Cancer Inst. 81:570-576). Consequently, intratumoral therapy guarantees more consistent and reliable delivery of recombinant toxin fusion proteins to the targeted site with limited systemic exposure, which maximizes efficacy.
For induction of tumor, mice were injected in the left flank with 4×106 PC-3 cells suspended in 100 μL of a 1:1 RPMI/Matri-Gel mixture. Once palpable tumors had formed (day 15), mice were divided into groups and treated with multiple injections of DTEGF13 from day 15 to day 25. All drugs were administered by intratumoral injection using 3/10 cc syringes with 29 gauge needles. All treatments were given in a 100-μL volume of sterile PBS. Tumor size was measured using a digital caliper, and volume was determined as a product of length, width, and height.
Construction of dDT2219
The dDT2219 coding region was synthesized using assembly PCR. The fully assembled coding region (from 5′ end to 3′ end) includes an NcoI restriction site, an ATG initiation codon, the first 390 amino acids of the mutated and deimmunized DT molecule (DT390), the seven amino acid linker EASGGPE (SEQ ID NO:4), the VL and VH regions of an anti-CD22 scFv, a GGGGS (SEQ ID NO:5) linker, the VL and VH regions of an anti-CD19 scFv, and a XhoI restriction site. The VL and VH gene of each scFv were joined by a linker (GSTSGSGKPGSGEGSTKG; SEQ ID NO:6) that was designated as aggregation reduced linker (ARL). The final 1755 bp NcoI/XhoI coding region was spliced into the pET21d expression vector under control of an isopropyl-b-D-thiogalactopyranoside (IPTG) inducible T7 promoter. DNA analysis was used to verify that the gene was in correct sequence (Biomedical Genomics Center, University of Minnesota, MN). To create a deimmunized drug, DT2219 was mutated using the QUICKCHANGE site-directed mutagenesis kit (Stratagene Corp., La Jolla Calif.) and site specific mutations were confirmed by DNA sequencing. The following amino acids were changed to deimmunize DT390: amino acid (aa) 125 changed from K to S; aa 173 R to A; aa 184 Q to S; aa 227 K to S; aa 245 Q to S; aa 292 E to S; and aa 385 K to G.
Inclusion Body Isolation, Refolding and Purification
Escherichia coli strain BL21 (DE3) (plasmid transfected) (Novagen, Inc., Madison, Wis., USA) was used for protein expression. Bacteria were cultured overnight in 800 ml Luria broth, containing 50 mg/ml carbenicillin. Expression was induced via addition of IPTG after the culture reached an optical density (OD) 600 of 0.65. Two hours after induction, grown bacteria were harvested and centrifuged. The cell pellet was homogenized in a solution buffer (50 mM tris, 50 mM NaCl, and 5 mM EDTA pH 8.0) and then sonicated and again centrifuged. Extraction of the pellet was performed using 0.3% sodium deoxycholate, 5% TRITON X-100, 10% glycerin, 50 mmol/L Tris, 50 mmol/L NaCl, 5 mmol/L EDTA (pH 8.0) and washed. Purification was described previously (Schmohl et al., 2015. Targeted Oncology 11(3):353-361; Schmohl et al., 2016. Mol Ther 24(7):1312-1322). Homogenized inclusion bodies were dissolved and proteins refolded. Purification was performed using an ion exchange chromatography (Q SEPHAROSE Fast Flow, Sigma-Aldrich, St. Louis, Mo.) with a continuous gradient.
Tissue Cultures
The following cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, Va.): Burkitt lymphoma cell line Daudi (ATCC CCL-213; Klein et al., 1968, Cancer Res 28:1300-1310) and Raji (ATCC CCL-86; Pulvertaft J V, 1964. Lancet 1:238-240), and human promyelocytic leukemia cell line HL-60 (ATCC CCL-240; Gallagher et al., 1979. Blood 54:713-733). T-cell leukemia HPB-MLT (Morikawa et al., 1978. Int J Cancer 21(2):166-170) was obtained from the German Collection of Microorganisms and Cell Cultures (Accession No. ACC 483). All cell lines were grown in suspension as previously described (Klein et al., 1968, Cancer Res 28:1300-1310). Cells were maintained in RPMI 1640 (supplemented with 10% fetal serum).
Proliferation Assay/Blocking Assay
In order to evaluate drug efficacy, (2×104) target cells were plated into a 96-well round bottom plate and suspended in RPMI supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin. DT2219, dDT2219, or BIC3 (bivalent scFv construct consisting of the catalytic and translocation domains of diphtheria toxin and two anti-CD3 scFv; Vallera et al., 2005. Leuk Res 29:331-341) was added in different concentrations. Assays were performed in triplicates. For time-dependent assays, cells were incubated for 12 hours, 24 hours, or 36 hours. For regular proliferation assays, 72 hours of incubation was performed at 37° C., 5% CO2. Cells were then incubated with one μCi [methyl-3H]-thymidine (GE Healthcare, UK) per well for eight hours and harvested onto glass fiber filters and washed. After a drying step, filters were counted for ten minutes in a standard scintillation counter. Data were analyzed using Prism 4 (GraphPad Software, Inc., La Jolla, Calif.) and were presented as “percent control response” calculated by dividing the counts per minute (CPM) of untreated cells by the CPM of the immunotoxin-treated cells (×100).
In order to assess specificity, blocking studies were performed. 100 nM, 200 nM, or 500 nM of 2219, the bispecific anti-CD19 and anti-CD22 scFv construct devoid of DT, was added to the respective amounts of either DT2219 or dDT2219 and incubated with target cells for 37° C. at 5% CO2. After incubation 3H-thymidine uptake was measured as described above.
Patient Samples and Binding Assay
Twelve patient samples from CLL patients were collected at the University of Minnesota. Samples were cryoconserved and stored according to standard procedures until use. For evaluation of binding, 4×105 peripheral blood nuclear cells (PBMCs) of the respective samples were washed and incubated in 4° C. with no, 1 nM, 10 nM, 20 nM, 50 nM, or 100 nM of fluorescein isothiocyate (FITC)-labeled constructs: dDT2219, anti-EpCAM scFv (negative control), or DT2219. After 30 minutes of incubation at 4° C., cells were washed in 1×PBS and stained with APC Cy 7 conjugated anti-CD20 monoclonal antibody (mAb) (BioLegend, San Diego, Calif.), Pacific blue conjugated anti-CD5 mAb (BioLegend, San Diego, Calif.), or PE-CF592 conjugated anti-CD3 mAb (BD Biosciences, San Jose, Calif.) as previously described (Hayes, et al., 2010. Leuk Res 34:809-815). After incubation for 15 minutes, (4° C.), cells were washed with 1×PBS and fluorescence intensity was evaluated by FACS analysis using a LSRII flow cytometer (BD Biosciences, San Jose, Calif.).
Detecting Anti-Toxin Antibodies in Mice
IgG anti-toxin antibodies were detected as previously described (Schmohl et al., 2015. Toxins 7:4067-4082). Briefly, immunocompetent normal BALB/c mice (NCI) were intraperitoneally immunized with non-mutated DT2219 (n=7 mice) or mutated DT2219 (dDT2219) (n=7 mice) (experiment registration number DAV529). Mice were immunized weekly for 12 weeks with 0.25 μg protein, followed by two weekly immunizations with 0.5 μg protein weekly, rested for six weeks, followed by weekly injections of 1 μg protein for three weeks. The experiment ended after 160 days. Blood was drawn on days 21, 35, 49, 63, 77, 99, and 160. A standard ELISA assay was used in which recombinant DT390 was adhered to the plate. Test serum from the immunized mice was then added followed by the detection antibody and anti-mouse IgG peroxidase (Sigma-Aldrich, St. Louis, Mo.). Plates were developed with o-phenylenediamine dihydrochloride (Thermo Fisher Scientific, Inc., Waltham, Mass.) for 15 minutes at room temperature. The reaction was stopped by adding 2.5 M H2504. Absorbance was read at 490 nm and the final concentration was determined from a standard curve using a mouse monoclonal antibody to diphtheria toxin ([11D9], Abcam Inc., Cambridge, Mass.). All samples and standards were tested in triplicates.
Detecting Neutralizing Antibodies in Mice
To detect neutralizing antibodies, 90% serum from immunized mice was added to cells, treated with a known inhibitory concentration of DT2219 and dDT2219 (1000 ng/ml). Proliferation assays were then carried out as described above. For detection of serum IgG anti-toxin content an ELISA Assay was used.
Statistical Analyses
Data are presented as mean+/−standard deviation. For evaluation of differences between the groups Student's t-test or one-way-ANOVA was used. Analysis and presentation of data was done with Graphpad prism 5 (GraphPad Software Inc., La Jolla, Calif., USA).
The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference in their entirety. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.
Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.
All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.
This application is the § 371 U.S. National Stage of International Application No. PCT/US2016/054823, filed Sep. 30, 2016, which claims priority to U.S. Provisional Application No. 62/236,568, filed Oct. 2, 2015, each of which is incorporated by reference herein by reference in its entirety.
This invention was made with government support under CA036725 awarded by National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2016/054823 | 9/30/2016 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2017/059270 | 4/6/2017 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 20090010966 | Davis | Jan 2009 | A1 |
| 20120121614 | Vallera | May 2012 | A1 |
| 20160340394 | Poma | Nov 2016 | A1 |
| Number | Date | Country |
|---|---|---|
| WO 2016187585 | Nov 2016 | WO |
| Entry |
|---|
| Lazar et al. Mol. Cellular Biol. 8: 1247-1252, 1988. |
| Cleton-Jansen et al. Mol. Gen. Genet. 229: 206-212, 1991. |
| International Patent Application No. PCT/US2016/054823, filed Sep. 30, 2016, International Search Report / Written Opinion dated Dec. 15, 2016; 14 pages. |
| International Patent Application No. PCT/US2016/054823, filed Sep. 30, 2016, International Preliminary Report on Patentability dated Apr. 12, 2018; 8 pages. |
| American Type Culture Collection, “ATCC CCL-86,” organism: Homo sapiens; [online]; Manassas, VA [retrieved on Feb. 5, 2019] from the Internet. Retrieved from the Internet: https://www.google.com/search?client=firefox-b-1-d&q=ATCC+CCL-86. pgs. |
| American Type Culture Collection, “ATTC No. CCL-213,” organism: Homo sapiens; [online]; Manassas, VA [retrieved on Feb. 5, 2019] from the Internet. Retrieved from the Internet: https://www.atcc.org/products/all/CCL-213.aspx; 3 pgs. |
| American Type Culture Collection, “ATTC No. CRL-1420,” organism: Homo sapiens; [online]; Manassas, VA [retrieved on Feb. 5, 2019] from the Internet. Retrieved from the Internet: https://www.atcc.org/products/all/CRL-1420.aspx; 3 pgs. |
| American Type Culture Collection, “ATTC No. HTB-38,” organism: Homo sapiens; [online]; Manassas, VA [retrieved on Feb. 5, 2019] from the Internet. Retrieved from the Internet: https://www.atcc.org/products/all/HTB-38.aspx; 3 pgs. |
| American Type Culture Collection, “ATTC No. CRL-1435,” organism: Homo sapiens; [online]; Manassas, VA [retrieved on Feb. 5, 2019] from the Internet. Retrieved from the Internet: https://www.atcc.org/products/all/CRL-1435.aspx#documentation; 3 pgs. |
| American Type Culture Collection, “ATTC No. CCL-240,” organism: Homo sapiens; [online]; Manassas, VA [retrieved on Feb. 5, 2019] from the Internet. Retrieved from the Internet: https://www.atcc.org/products/all/CCL-240.aspx#documentation; 3 pgs. |
| Bachanova, “Phase I Study of a Bispecific Ligand-Directed Toxin Targeting CD22 and CD19 (DT2219) for Refractory B-cell Malignancies” 2015 Clin Cancer Res., 21:1267-1272. |
| Barker, “Sequence Analysis Primer” Review, Jan. 1993 Biophys J., 64:292. |
| Berman, “The Protein Data Bank” 2000, Nucleic Acids Res., 28:235-242. |
| Brons, “Hierarchic T-cell help to non-linked B-cell epitopes” Nov. 1996 Scandinavian journal of immunology, 44:478-84. |
| Collier, “Diphtheria toxin: mode of action and structure” 1975 Bacteriological reviews, 39:54-85. |
| DSMZ German Collection of Microorganisms and Cell Cultures (Accession No. ACC 483). Retrieved from the Internet: https://www.dsmz.de/catalogues/details/culture/ACC-483.html; Feb. 5, 2019. 2 pgs. |
| Eisenberg, “The helical hydrophobic moment: a measure of the amphiphilicity of a helix” Oct. 1982 Nature, 299:371-4. |
| Fogh, “One hundred and twenty-seven cultured human tumor cell lines producing tumors in nude mice” 1977 Journal of the National Cancer Institute, 59:221-6. |
| Frankel, “DAB389IL2 (ONTAK) fusion protein therapy of chronic lymphocytic leukaemia” 2003 Expert opinion on biological therapy, 3:179-86. |
| Frankel, “Anti-CD3 recombinant diphtheria immunotoxin therapy of cutaneous T cell lymphoma” 2009 Current drug targets, 10:104-9. |
| Gallagher, “Characterization of the continuous, differentiating myeloid cell line (HL-60) from a patient with acute promyelocytic leukemia” 1979 Blood, 54:713-733. |
| Hayes, “Isolation of malignant B cells from patients with chronic lymphocytic leukemia (CLL) for analysis of cell proliferation: validation of a simplified method suitable for multi-center clinical studies” 2010 Leuk Res., 34:809-815. |
| He, “Effects of mutation at the D-JH junction on affinity, specificity, and idiotypy of anti-progesterone antibody DB3” Sep. 2006 Protein science: a publication of the Protein Society, 15:2141-8. |
| Hopp, “Prediction of protein antigenic determinants from amino acid sequences” Jun. 1981 Proceedings of the National Academy of Sciences of the United States of America, 78:3824-8. |
| Jain, “Delivery of novel therapeutic agents in tumors: physiological barriers and strategies” 1989 J Natl. Cancer Inst., 81:570-57. |
| Jameson, “The antigenic index: a novel algorithm for predicting antigenic determinants” 1988 Computer applications in the biosciences: CABIOS, 4:181-6. |
| Jemmerson, “Multiple overlapping epitopes in the three antigenic regions of horse cytochrome c1” Jan. 1987 J Immunol., 138:213-9. |
| Kaighn, “Establishment and characterization of a human prostatic carcinoma cell line (PC-3)” 1979 Investigative urology, 17:16-23. |
| Klein, “Surface IgM-kappa specificity on a Burkitt lymphoma cell in vivo and in derived culture lines” Jul. 1968 Cancer research, 28:1300-10. |
| Kreitman,“Immunotoxins in the treatment of hematologic malignancies” Jan. 2006 Current drug targets, 7:1301-11. |
| Lambotte, “Primary structure of diphtheria toxin fragment B: structural similarities with lipid-binding domains” Dec. 1980 The Journal of cell biology, 87:837-40. |
| Lin, “Expression cloning of human EGF receptor complementary DNA: gene amplification and three related messenger RNA products in A431 cells” 1984 Science, 224:843-8. |
| Morikawa, “Two E-rosette-forming lymphoid cell lines” Feb. 1978 Int J Cancer 21(2): 166-170. |
| Newman, “Patterns of antibody specificity during the BALB/c immune response to hen eggwhite lysozyme” Nov. 1992 J Immunol., 149:3260-72. |
| Oh, “A Deimmunized Bispecific Ligand-Directed Toxin That Shows an Impressive Anti-Pancreatic Cancer Effect in a Systemic Nude Mouse Orthotopic Model” 2012 Pancreas, 41(5):789-96. |
| Oh, “Intracranial elimination of human glioblastoma brain tumors in nude rats using the bispecific ligand-directed toxin, DTEGF13 and convection enhanced delivery” Dec. 2009 J Neurooncol., 95(3):331-342. |
| Oh, “A novel reduced immunogenicity bispecific targeted toxin simultaneously recognizing human epidermal growth factor and interleukin-4 receptors in a mouse model of metastatic breast carcinoma” 2009 Clinical cancer research: an official journal of the American Association for Cancer Research, 15:6137-47. |
| Onda, “Characterization of the B cell epitopes associated with a truncated form of Pseudomonas exotoxin (PE38) used to make immunotoxins for the treatment of cancer patients” 2006 J Immunol., 177:8822-34. |
| Onda, “Recombinant immunotoxin against B-cell malignancies with no immunogenicity in mice by removal of B-cell epitopes” Apr. 2011 Proc Natl Acad Sci USA, 108(14):5742-7. |
| Pastan, “Recombinant toxins as novel therapeutic agents” 1992 Annual review of biochemistry, 61:331-54. |
| Pulvertaft, “Cytology of Burkitt's Tumour (African Lymphoma)” 1964 Lancet, 1:238-240. |
| Rosenberg, “Intersite helper function of T cells specific for a protein epitope that is not recognized by antibodies” 1997 Immunological investigations, 26:473-89. |
| Schmohl, “Heterodimeric bispecific single chain variable fragments (scFv) killer engagers (BiKEs) enhance NK-cell activity against CD 133+ colorectal cancer cells” 2015 Targeted Oncology, 11(3):353-361. |
| Schmohl, “Mutagenic Deimmunization of Diphtheria Toxin for Use in Biologic Drug Development” Oct. 2015 Toxins (Basel):7(10): 4067-4082. |
| Schmohl, “Enhanced ADCC and NK Cell Activation of an Anticarcinoma Bispecific Antibody by Genetic Insertion of a Modified IL-15 Cross-linker” 2016 Mol Ther., 24(7): 1312-1322. |
| Stish, “A bispecific recombinant cytotoxin (DTEGF13) targeting human interleukin-13 and epidermal growth factor receptors in a mouse xenograft model of prostate cancer” Nov. 2007 Clinical cancer research: an official journal of the American Association for Cancer Research, 13:6486-93. |
| Stish, “Design and modification of EGF4KDEL 7Mut, a novel bispecific ligand-directed toxin, with decreased immunogenicity and potent anti-mesothelioma activity” Oct. 2009 British journal of cancer, 101:1114-23. |
| Stish, “Anti-glioblastoma effect of a recombinant bispecific cytotoxin cotargeting human IL-13 and EGF receptors in a mouse xenograft model” Dec. 2008 Journal of neuro-oncology, 87:51-61. |
| Tamilvanan, “Clinical concerns of immunogenicity produced at cellular levels by biopharmaceuticals following their parenteral administration into human body” Aug. 2010 Journal of drug targeting, 18:489-98. |
| Tsai, “A novel bispecific ligand-directed toxin designed to simultaneously target EGFR on human glioblastoma cells and uPAR on tumor neovasculature” Jun. 2011 Journal of neuro-oncology, 103:255-66. |
| Tsuneoka, “Evidence for involvement of furin in cleavage and activation of diphtheria toxin” Dec. 1993 The Journal of biological chemistry, 268:26461-5. |
| Vallera, “Genetic alteration of a bispecific ligand-directed toxin targeting human CD19 and CD22 receptors resulting in improved efficacy against systemic B cell malignancy” 2009 Leukemia research, 33:1233-42. |
| Vallera, “Bioengineering a unique deimmunized bispecific targeted toxin that simultaneously recognizes human CD22 and CD19 receptors in a mouse model of B-cell metastases” 2010 Molecular cancer therapeutics, 9:1872-83. |
| Vallera, “Genetically designing a more potent antipancreatic cancer agent by simultaneously co-targeting human IL13 and EGF receptors in a mouse xenograft model” May 2008 Gut, 57:634-41. |
| Vallera, “Molecular modification of a recombinant, bivalent anti-human CD3 immunotoxin (Bic3) results in reduced in vivo toxicity in mice” 2005 Leukemia research, 29:331-41. |
| Yunis, “Human pancreatic carcinoma (MIA PaCa-2) in continuous culture: sensitivity to asparaginase” 1977 International journal of cancer Journal international du cancer, 19:128-35. |
| Zurawski, “Interleukin 13, an interleukin 4-like cytokine that acts on monocytes and B cells, but not on T cells” 1994 Immunology today, 15:19-26. |
| Number | Date | Country | |
|---|---|---|---|
| 20180273920 A1 | Sep 2018 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62236568 | Oct 2015 | US |
