HK1263031A1 - Deimmunized therapeutic compositions and methods - Google Patents

Description

Therapeutic compositions and methods for deimmunization

Cross Reference to Related Applications

This application claims priority to U.S. provisional patent application No.62/236,568, filed on day 2/10/2015, which is incorporated herein by reference.

Government funding

The invention was made with government support from R01CA36725 awarded by the National Institutes of Health. The government has certain rights in this invention.

Sequence listing

This application contains the sequence listing as an ASCII text file, entitled "2016-09-30-sequence listing _ st25. txt", filed electronically via EFS-Web to the united states patent and trademark office, having a size of 58 kilobytes, generated on 9/30/2016. The information contained in this sequence listing is incorporated herein by reference.

Summary of The Invention

In one aspect, the disclosure describes engineered polypeptides comprising a Diphtheria Toxin (DT) domain and a targeting domain. The DT domain includes a DT catalytic site and at least one amino acid substitution that reduces induction by an antitoxin antibody 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 may 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 DT 390.

In another aspect, the present disclosure describes a method of killing a cell. Generally, the method comprises contacting the cell to be killed with any of the embodiments of the polypeptides summarized above under conditions that allow the cell to internalize (internalise) the polypeptide; and allowing the polypeptide to kill the cell. In various embodiments, the cell can be in vitro or in vivo.

In another aspect, the disclosure describes a method of treating a subject having a tumor. In general, the method comprises administering to the subject any of the embodiments of the polypeptides summarized above, wherein the polypeptide comprises a targeting domain that selectively binds to a target present on the tumor cell.

In another aspect, the disclosure describes methods of deimmunizing diphtheria toxin polypeptides. Generally, the methods include identifying hydrophilic amino acid residues at the surface of a native diphtheria toxin polypeptide, constructing a modified diphtheria toxin polypeptide comprising replacing one of the identified hydrophilic amino acid residues, screening the modified diphtheria toxin polypeptide for biological function of the native diphtheria toxin polypeptide, and antibody-induced screening the modified diphtheria toxin polypeptide.

The above summary of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. The following description more particularly exemplifies illustrative embodiments. In several places in the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each case, the list given serves only as a representative group and should not be interpreted as an exclusive list.

Brief description of the drawings

FIG. 1 construction of plasmid containing dDTEGF13 gene. (A) pET expression vector containing the coding sequence of dDTEGF13 (SEQ ID NO: 13). By downloading the Protein Data Bank (RCSB PDB; Berman et al, 2000, Nucleic acids sRs 28:235-242) diphtheria toxin x-ray crystallography Structure (DT; PBD ID:1MDT) to the PyMOL 3D molecular model program: (R) ((R))LLC, New York, NY) to generate PyMol sphere patterns. Shown are a frontal view of the protein and a 180 ° flip view of the molecule. (B) The amino acids (catalytic sites) involved in ADP ribosylation are darkened. (C) Amino acids mutated for deimmunization are darkened. (D) SDS-PAGE gel analysis was performed to confirm size and purity and stained with Coomassie Brilliant blue. Lane 1-molecular weight standard, lane 2-unreduced dDTEGF13, lane 3-reduced dDTEGF 13. The gel was stained with coomassie brilliant blue. (E) Also shown is an HPLC trace of the purified drug showing mainly a single peak obtained from a TSK3000 size exclusion column. Collecting only the single peak to obtain>95% purity.

Figure 2. three-stage strategy for generating and screening various DTEGF13 mutants to achieve deimmunization. In stage 1, 8 triple mutants (triple mutants) were synthesized and purified. 4 of these showed less than log of activity loss in the in vitro screening assay compared to the non-mutated parental control. In stage 2, the mutations are combined to generate five mutants (quintuplet mutants) that still have less than a loss of logarithmic activity compared to the parent. In stage 3, the seven mutant (septuplemutant) still had less than a log loss of activity. Glycine, alanine and serine substitutions were used.

Figure 3 in vitro activity of dtegf 13. (A) Bispecific DTEGF13 and its monospecific counterpart were tested and their reactivity against MiaPaCa-2 cells was compared. By analysis after 72 hours incubation with the targeting toxin³H-thymidine uptake was used for proliferation assays. Data are reported as percent control response. Each data point represents the mean of three replicate measurements ± s.d. The activity of de-immunized DTEGF13 and the non-mutated parent DTEGF13 against (B) HT-29 colon and (C) PC-3 prostate cancer cells was tested and compared in a thymidine uptake assay. (D) A blocking assay was performed in which the MiaPaca-2 pancreatic cancer cell line was incubated with an inhibitory dose of DTEGF13 and then blocked with increasing concentrations of the non-toxin EGF13 ligand. Thymidine uptake was then measured.

Figure 4 in vitro activity of dtegf13 against Raji cells. Bispecific dtegf13 and its monospecific counterpart were tested for their reactivity against Raji B cells. By analysis after 72 hours incubation with the targeting toxin³H-thymidine uptake was used for proliferation assays. Data are reported as percent control response.

Figure 5 immunogenicity of dtegf 13. The immune response of de-immunized DTEGF13 and the unmutated parent drug was determined by measuring anti-DT 390 serum IgG of weekly samples of mice immunized with 0.25 μ g DTEGF13 (n-5) or DTEGF13 (n-5). The measurements were done using indirect ELISA, and quantitation of the antibodies was determined using a standard curve generated with highly purified high titer anti-DT antibodies. (A) Response in BALB/c mice; (B) response in C57BL/6 mice.

FIG. 6 neutralizing antibodies. Sera were collected from mice immunized by multiple injections of (a) parental DTEGF13 or (B) DTEGF13 on day 56. Sera from individual mice were incubated with cells treated with known inhibitory concentrations of DTEGF13 to test for neutralization. Proliferation assays were performed by measuring tritiated thymidine uptake after 72 hours. The IgG antitoxin serum concentrations measured for each serum at day 56 are shown. There is a correlation between serum levels and the presence of neutralizing antibodies.

FIG. 7 Effect of treatment of established PC-3 flank tumors with dDTEGF 13. (A) Nude mice loaded with PC-3 flank tumors were treated intratumorally with ddegf13, control DT2219ARL, or not. Tumors were treated with 12 injections of dtegf 13. The mean tumor volume for each treatment group is shown. (B) Individual tumor volumes were shown for the irrelevant control BLT treated group (DT2219ARL) and untreated mice. The growth of each tumor over time is plotted.

FIG. 8, dDT2219(SEQ ID NO:3) in vitro activity. dDT2219 was tested in a trypan blue viability assay, in which different cell lines, CD22+ CD19+ Daudi, were seeded onto plates, followed by the addition of 10nM drug to the plates. Viability was determined daily. Dead cells take up trypan blue dye, while live cells do not. Cells were counted visually using a hemocytometer. No cells survived dDT2219 treatment after 3 days. Cells treated with Bic3 were minimally affected.

FIG. 9 comparison of exemplary de-immunized DT amino acid sequences (Sequence _ 1; SEQ ID NO:1) with non-de-immunized DT amino acid sequences (Sequence _ 2; SEQ ID NO: 2). Boxes show amino acid substitutions at exemplary sites.

Construction and purification of FIG. 10, dDT 2219. (A) The dDT2219 coding region has an NcoI restriction site, the first 390 amino acids of the deimmunized DT molecule (dDT390), splicing to anti-CD 22scFv, splicing to anti-CD 19scFv, and splicing to XhoI restriction sites. (B) The absorbance curve of dDT2219 is shown. The arrow marks the product of interest. (C) Size comparisons were made by SDS-PAGE gels and Coomassie blue staining. dDT2219, DT2219, molecular weight standards [ MW standards ], anti-CD 19scFv spliced to anti-CD 22scFv without DT2219 ], and diphtheria toxin [ DT ] alone. dDT2219 is 97.5kD in size. (D) Purity exceeded 90% after purification on a size exclusion column (product markers between C3 and C6 fractions).

Activity and kinetics of dDT2219 in fig. 11. Daudi cells were used to evaluate the kinetics and activity of (a) dDT2219, (B) DT2219, and (C) BIC3 (monospecific anti-CD 3scFv conjugated with DT). Comparisons were made after incubation for 12, 24 or 36 hour periods in increasing concentrations of drug as labeled. Although slightly enhanced activity was observed in DT2219 at higher concentrations (1nM, 10nM, 100nM) compared to dDT2219, no effect was seen with negative control BIC3 (Daudi cells expressing CD19 and CD22, but not CD 3). Direct comparisons were performed in (D) with different Burkitt (Burkitt) lymphoma cell lines (Raji cells). Despite the recorded efficacy of dDT2219 and DT2219, no effect was seen with BIC3 (Daudi cells expressing CD19 and CD22, but not CD 3).

FIG. 12 specificity of target cell depletion. To assess the specificity of dDT2219, (DT2219 and BIC3 are controls), proliferation assays were performed. (A) After exposure of each drug to HPB-MLT cells (CD3 expressing T cell leukemia cell line), only BIC3 was seen to inhibit proliferation. (B) When HL-60 cells (CD 3)^-、CD22^-、CD19^-Acute early childhoodMyeloid leukemia cell lines) had no detectable inhibition of proliferation when exposed to each drug.

FIG. 13 evaluation of specific binding. Daudi cells were exposed to labeled concentrations of FITC label dDT2219(a) or DT2219 (B). Fluorescence showed < 85% binding for dDT2219-FITC and 75% binding for DT 2219-FITC. In addition, Daudi cells were re-exposed to increasing concentrations of dDT2219-FITC and DT2219-FITC containing 100nM, 200nM or 500nM of the same unlabeled drug. Both FITC-labeled drugs showed reduced binding due to specific blocking of the unlabeled construct.

FIG. 14. binding in CLL. CLL samples from patients were exposed to FITC markers dDT2219, DT2219 and anti EpCAM scFv (control). (A) The results after exposure to increasing concentrations of various drugs, such as markers, are shown in (B) and (C). (D) CLL samples from patients were exposed to FITC label dDT2219 or DT 2219. Direct comparison of binding characteristics with 100nM of each construct showed statistically non-differential binding, 93 ± 3.5% and 88 ± 4.4% (p ═ 0.25), (n ═ 12) for the dDT2219 and DT2219 groups, respectively.

figure 15 neutralizing antibodies 14 BALB/C immunocompetent mice were divided into two groups of 7 mice each, two groups were immunized intraperitoneally with dDT2219 (experimental group) or DT2219 (control group) and blood was taken on days 21, 35, 49, 63, 77, 99, 160, serum was evaluated by performing ELISA for detection of α -DT390 antibodies (a) dDT2219 showed significantly lower concentrations of α -DT390 antibodies on all evaluation days (B) neutralization assay was performed using Raji target to evaluate the functionality of the detected antibodies dDT2219 group showed significantly lower amounts of functional neutralizing antibodies (C, D)6 of 7 mice (mice 2 to 7) showed high titers of functional neutralizing antibodies in the group immunized with DT2219, while 2 of 7 mice (mice 3 and 7) showed functional neutralizing antibodies in the group treated with dDT 2219.

Detailed Description

The present disclosure describes compounds, compositions, and methods relating to deimmunized (deimmunized) targeted toxins. The use of biological drugs may be limited because they are recognized as foreign by the human immune system. This is particularly true for bacterial proteins such as Diphtheria Toxin (DT). Diphtheria toxin has promise as a targeted toxin, but even DT-based drugs such as the denileukin bifittox, ONTAK, Eisai co., ltd., Tokyo, Japan, approved by the Federal Drug Administration (FDA) can elicit antitoxin responses after multiple treatments. Furthermore, those who received diphtheria, tetanus and pertussis (DPT) immunizations within hours are more likely to develop antitoxin responses to multiple treatments with DT-based drugs. However DT-Targeting Toxins (TT) have proven effective in eliciting anti-cancer responses in a number of different animal models and patients. Thus, the search for deimmunized forms of toxins has been considered a desirable, but unmet goal.

Diphtheria toxin is a 535 amino acid protein (mw 58.3kDa) with two functional domains. The C-terminal B domain binds to most eukaryotic cells. This B domain may be removed to form DT390 and/or replaced with a ligand to form a DT targeting toxin. The N-terminal A domain includes a catalytic region, the ADP-ribosylation of which extends factor 2 (EF-2).

DTEGF13 is a dual specific ligand directed toxin (BLT) that includes a truncated DT (DT390) and two ligands. These ligands include IL-13 and Epidermal Growth Factor (EGF). These 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 cancers, including prostate, pancreatic, breast and/or lung cancers. IL-13 is a pleiotropic lymphokine, the receptor of which is overexpressed on tumors, B cells and monocytes. DTEGF13 binds to its target via EGF and IL-13 ligands and once internalized is toxic to the target cell. In vivo, DTEGF13 is effective against cancers, including but not limited to prostate, glioblastoma, and pancreatic cancers, both locally and systemically in xenograft models. Thus, DTEGF13 produced an anti-diphtheria toxin response when injected in vivo and provided a model for deimmunization.

Hydrophilic amino acids having surface positions (e.g., derived from x-ray crystallography models) remote from the NAD-ribosylation catalytic site are selected for point mutation to generate the deimmunized DT molecules of the invention. Those mutants that underwent the minimal loss of activity screening were screened sequentially. Candidate mutants with at least 7 mutations were tested for their ability to produce anti-toxin IgG antibodies when injected multiple times in animal models. Despite multiple immunizations, deimmunized DTEGF13(dDTEFG 13; SEQ ID NO:8) showed reduced antitoxin induction compared to the unmutated parent form (parentalform).

Figure 1A shows the dedegf 13 plasmid construct and PyMol globular model of the x-ray crystallographic structure in the frontal and inverted (180 °) position. The amino acids within the active site are shown in figure 1B. Figure 1C shows 7 mutant amino acids used for deimmunization, darkened so that their surface positions on the molecule can be easily observed. An SDS-PAGE gel analysis of dDTEGF13 is shown in FIG. 1D, with a purity of greater than 95% as determined by Coomassie Brilliant blue. The molecular weight size estimated from the molecular weight standards was 68.9 kDa.

Based on molecular models derived from x-ray crystallographic structure, 20 amino acid residues located at prominent surface positions were identified. A series of 8 mutants were generated, each mutant having 3 point mutations. Thus, the entire system detects 24 point mutations. Glycine and serine substitutions were used. Mutant activity loss was screened using a standard, highly reproducible proliferation inhibition assay that measures thymidine uptake.

Figure 2 shows those mutants that exhibited minimal loss of activity and were subsequently combined with other mutants (stage 1). Point mutations that exhibited less than log loss mutants were combined onto the same molecule until DTEGF13 was obtained, having 7 mutations located in different regions of the molecule and having less than log activity reduction in an in vitro proliferation assay (stage 3). This mutant, shown in figure 1A, was then tested for its ability to generate an antitoxin response in immunocompetent mice.

FIG. 3A shows that the dual specific ligand-directed toxin DTEFG13 is very potent against EGF⁺IL13⁺IC of cell line MDA-MB-23 breast cancer cell line₅₀At 0.019nM, had greater activity than its monospecific counterpart in an in vitro proliferation screening assay. In addition, FIG. 3B shows that dDTEGF13 has similar activity against HT-29 human colon cancer cell line compared to the unmutated parental control. FIG. 3C shows that the same is true when dDTEGF13 and the unmutated parent are tested against PC-3 prostate cancer. Figure 3D shows that the EGF13 ligand portion of the DTEGF13 molecule is mediated by its selective binding to the target cell, since DTEGF13 is an EGF13 ligand that can block the toxin of MiaPaCA-2 cells without needles. FIG. 4 shows that dDTEGF13 does not inhibit EGFR-IL13R control B cell line Daudi, but a positive control anti-B cell BLT called DT2219ARL (Vallera et al, 2009, LeukRes 33:1233-1242) is inhibited. These data indicate that activity is mediated by selective binding of ligands.

To determine whether DTEGF13 had been successfully de-immunized, groups of immunocompetent BALB/c mice (H-2)^d) Immunization was weekly with 0.25 μ g of mutated DTEGF13 or unmutated parent DTEGF 13. Animals were immunized intraperitoneally over a 82 day period. Serum samples were obtained weekly and analyzed by ELISA for detection of anti-dtegf 13 IgG. The results of the immunization experiments are summarized in fig. 5A, which shows the statistical difference in the antitoxin response between the group of mice immunized with DTEGF13MC2 and the parental control. After 12 immunizations (day 82), the dtegf13MC2 group showed the smallest antibody response, whereas the parental group had an average anti-DT 390 response of more than 1,500 μ g/ml. To determine whether the antitoxin serum levels could be reduced in another mouse strain presenting a differential antigen, a mouse strain with a different H-2 haplotype (H-2)^b) The same experiment was repeated with C57BL/6 mice. The results were substantially consistent (fig. 5B).

To determine whether neutralizing antibodies were present in the sera from immunized mice, sera from the mice in fig. 5A were added to 1000ng/ml dtegf13 at a known inhibitory concentration. Treated cells were then tested for uptake of tritiated thymidine in a proliferation assay. Figure 6A shows the percent control response of treated MiaPaCa-2 cells, and the response of cells subsequently treated with day 56 sera from three different mice immunized multiple times with the parent DTEGF 13. High IgG serum antitoxin levels (2,429, 1,272, and 579. mu.g/ml) in these mice were associated with high neutralizing activity. In contrast, fig. 6B shows the response of cells treated with day 56 sera from three different mice immunized multiple times with dtegf 13. Low serum antitoxin levels are associated with a lack of neutralizing activity. These data indicate that there are no neutralizing antibodies present in the serum at day 56 of mice immunized with dtegf 13.

To test the ability of dtegf13 to inhibit tumor growth in vivo, PC-3 cells were injected into the flank of nude mice. After tumor establishment and palpability, mice were treated with multiple intratumoral injections. dDTEGF13 was studied in a mouse model because human EGF (of DTEGF 13) and IL-13 reacted with mouse EGFR and IL13R, respectively. Figure 7A shows mean tumor volume data from the first experiment in which groups of mice were injected without treatment or with dtegf13 treatment on days 0, 1, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77 (0.25 μ g/injection). Figure 7B shows tumor growth of each control mouse. Control mice were either untreated or treated with an unrelated immunotoxin control DT2219 ARL. Multiple ddegf13 injections were effective to prevent tumor growth until the end of the experiment at day 45, compared to negative controls.

Thus, in one aspect, the disclosure describes deimmunized DT-targeted toxin polypeptides that can be used in molecular drug development. Typically, the polypeptide includes a deimmunized diphtheria toxin domain including a 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 is modified to reduce the extent to which the polypeptide induces an immune response in a subject to which it is administered, as compared to a molecule that has not been deimmunized. In some cases, antibody responses to the DT-targeted toxic molecule were measured. In one embodiment, antibody induction can be characterized in terms of antibody production induced in immunocompetent mouse strains. In addition, as used herein, the term "selectively" refers to any degree of differential affinity for a particular desired target, e.g., greater than the usual non-selective affinity. Exemplary targeting domains can include a sufficient portion (affinity) of a ligand or binding moiety (e.g., EGF, IL-13, anti-CD 19, and/or anti-CD 22) to selectively bind its receptor expressed by a target cell. The resulting polypeptides exhibit measurable diphtheria toxin toxicity, as well as reduced induction of anti-toxin antibodies in immunocompetent mice compared to wild-type diphtheria toxin. The polypeptides can be used, for example, as medicaments effective against neoplastic conditions such as pancreatic cancer, breast cancer and glioma.

In some embodiments, the deimmunized DT molecules can include at least one amino acid substitution compared to DT390 (or full length DT). Thus, the dDT molecule can have at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, 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, or at least 24 mutations relative to DT390 or full length DT.

Thus, in some embodiments, the dDT molecule can include a substitution of at least 3 of amino acid residues 15, 100, 104, 125, 173, 185, 227, 242, 245, 292, 318, or 385. In one embodiment, the dDT molecule may include amino acid substitutions for K125, R173, and Q245. In another embodiment, the dDT molecule may include amino acid substitutions K125S, R173A, and Q245S. In another embodiment, the dDT molecule may include amino acid substitutions K104, K385, and E292. In another embodiment, the dDT molecule may include amino acid substitutions K104S, K385G, and E292S. In another embodiment, the dDT molecule may include amino acid substitutions E100, Q184, and K227. In another embodiment, the dDT molecule may include amino acid substitutions E100S, Q184S, and K227S. In another embodiment, the dDT molecule may include amino acid substitutions E15S, D318, and K242S.

In other embodiments, the dDT molecule can include at least 5 amino acid 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 may include amino acid substitutions K125S, R173A, Q245S, K385G, and E292S.

In other embodiments, the dDT molecule can include at least 7 amino acid substitutions. Thus, in one embodiment, the dDT molecule may include amino acid substitutions K125, R173, Q245, K385G, E292, Q184, and K227. In another embodiment, the dDT molecule may include amino acid substitutions K125S, R173A, Q245S, K385G, E292S, Q184S, and K227S. In another embodiment, the dDT molecule may include amino acid substitutions K125S, R173A, Q245S, K385G, E292S, Q184S, and K227S.

Although the dDT molecule is described using serine, alanine, and/or glycine substitutions, these are exemplary molecules and one of skill in the art will be able to readily determine whether other amino acids will provide the dDT molecules described herein. For example, alanine, serine, and glycine can be used interchangeably to produce dDT molecules that can be screened for activity and reduced immunogenicity as described in the examples herein.

Although described above in the context of exemplary embodiments (where the targeting domain may include sufficient portions of EGF, IL-13, anti-CD 19, and/or anti-CD 22 to selectively bind to a desired target), the targeting domain may include any suitable targeting moiety. A "targeting moiety" refers to a portion of a compound (e.g., a ligand) that can be used in a targeting domain that has a target-selective affinity (e.g., for a receptor for the ligand). 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, e.g., a ligand that binds to a receptor that is expressed with some degree of specificity by the target cell. In other cases, the targeting moiety may be an antibody or derived from an antibody (e.g., at least sufficient antibody immunospecific moieties, such as sufficient light chains, to provide some degree of immunospecificity).

In various embodiments, a targeting domain can include more than one targeting moiety. For example, the exemplary embodiment referred to herein as dtegf13 includes first and second targeting moieties. The first targeting moiety includes a sufficient portion of EGF to selectively bind EGFR, and the second targeting moiety includes a sufficient portion of IL-13 to selectively bind IL-13 receptor. Likewise, the exemplary embodiment referred to herein as dDT2219 also includes first and second targeting moieties. The first targeting moiety comprises a sufficient portion of the anti-CD 19scFv to selectively bind CD-19, while the second targeting moiety comprises a sufficient portion of the anti-CD 22scFv to selectively bind 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 α receptor, IL-4R, CD133, ROR1, mesothelin (mesothelin), TRAIL, CD38, CD45, CD74, or CD 23.

dDT2219 the molecule is an engineered variant of DT2219 in which the diphtheria toxin domain of the molecule is replaced by a deimmunized DT domain. DT2219 is a recombinant fusion protein containing a diphtheria toxin catalytic and translocation enhancing domain (DT390) fused to an antibody bispecific single chain variable fragment (scFV) targeting human CD19 and CD22 cell surface receptors. The protein was engineered such that the initial binding region of DT was replaced by scFv binding more aggressively. Upon binding, CD19 and CD22 readily internalize to promote entry of the toxin into the cytoplasm, inhibit protein synthesis, and/or subsequent apoptotic cell death.

CD19 is a 95kDa membrane glycoprotein that is widely present on the surface of all stages of B lymphocyte development and is also expressed on most B cell mature lymphocytes and leukemia cells. CD22 is a 135kDa glycoprotein expressed on B lineage lymphoid precursors (including precursor B acute lymphoid leukemia), and is commonly co-expressed with CD19 on mature B cell malignancies. DT mediates potent cell cycle independent cell death and is therefore particularly effective as an alternative therapy to chemotherapy-refractory malignancies.

dDT2219 construction of the 2219 molecule is shown in FIG. 10A. V encoding deimmunized DT (dDT390), anti-CD 22_HAnd V_LRegion, and V of anti-CD 19scFv_HAnd V_LThe polynucleotides of the regions are assembled as indicated. The active fragment was ligated via EASGGPE (SEQ ID NO:4) and ARL linker (SEQ ID NO:6) to form dDT 2219. The absorbance trace of dDT2219 eluted from the FFQ ion exchange column as the first stage in the purification of the drug using the three-step elution protocol is shown in fig. 10B. The first peak eluted from the column represents the product of interest. SDS-PAGE gels (FIG. 10C) and Coomassie blue staining showed purity after ion exchange and size exclusion column purification (FIG. 10D). The product is more than 90% pure, with a size of about 97.5 kD.

To show the activity and kinetics of dDT2219 compared to the parent DT2219 form, CD19 was used⁺/CD22⁺Proliferation assays were performed with Burkitt (Burkitt) lymphoma cell lines Daudi and Raji. Thus, Daudi cells were evaluated for H at 12 hours, 24 hours, and 36 hours after exposure to various drugs (FIG. 11A dDT2219, FIG. 11B DT2219) and increasing concentrations (0.001nM, 0.01nM, 0.1nM, 1nM, 10nM, and 100nM)³Uptake of thymidine. At higher concentrations (1-100nM), a slightly increased percent activity was seen in the DT2219 group after 24 and 36 hours, whereas there was no significant inhibition of proliferation at all time points in the control group exposed to BIC3 (Daudi cells did not express CD3) (fig. 11C). An additional burkitt lymphoma cell line Raji was used to confirm reproducibility of binding. IC of dDT2219 was estimated in a parallel proliferation assay with dDT2219, DT2219 and BIC3 incubation for 72 hours₅₀IC of 1.03, DT2219₅₀Is 0.23. BIC3 was also ineffective (fig. 11D).

To demonstrate the specificity of dDT2219 (DT2219 and BIC were used as controls), proliferation assays were performed with HPB-MLT cells (T cell lymphoma cell lines expressing CD3, but not expressing CD19 or CD22) and labeled increasing concentrations of dDT2219, DT2219 and BIC 3. There was no visible effect after dDT2219 and DT2219 exposure. Only BIC3 induced apoptosis (fig. 12A). Post-treatment inhibition was not seen in the CD22, CD19, and CD3 negative acute promyelocytic leukemia cell line HL-60 (fig. 12B).

Flow cytometry was used to compare binding and blocking characteristics. FITC-labeled dDT2219 (FIG. 13A) and DT2219 (FIG. 13B) were used at increasing concentrations (1nM, 5nM, 10nM, 50nM, 100nM, 200nM or 500nM) with Daudi cells. For dDT2219, increasing binding capacity was seen up to > 85%, for DT2219 > 75%. The same cells were treated with the same dose of dDT2219 or DT2219 and also exposed to 100nM, 200nM or 500nM of unlabeled dDT2219 or DT 2219. The deimmunized and parent unlabeled drug showed sufficient binding and sustained and dose-dependent blocking of the FITC-labeled drug as seen in the decrease in fluorescence intensity.

To demonstrate binding capacity in a clinical setting, samples obtained from CLL patients were used to compare dDT2219 and DT2219 for binding, respectively. Samples were exposed to increasing concentrations of FITC-labeled dDT2219 or DT2219 (no drug, 1nM, 10nM, 20nM, 50nM or 100 nM). After gating (gating) on the CLL population, a dose-dependent increase in binding of the two constructs was seen (fig. 14A and 14B). FITC-labeled anti-EpCAM scFv was used as a negative control (fig. 14C) and showed no binding. In a direct comparison between dDT2219 and DT2219, each patient sample was exposed to 100nM of dDT2219 or DT 2219. The assessed fluorescence (representing binding) seen in all 12 CLL samples was 93% and 88% with no significant difference between the two groups (p ═ 0.25) (fig. 14D), suggesting the same binding characteristics.

to evaluate whether neutralizing antibodies appeared in the sera of immunized mice after repeated exposure to dDT2219, BALB/c mice were divided into two groups of 7 animals each, two groups were immunized simultaneously with dDT2219 or DT2219 (control group), two groups were treated with equal concentrations of dDT2219 or DT2219, on all days of evaluation, sera of dDT2219 group showed significantly lower antibody induction (p <0.05) (fig. 15A) even after 4 boosts of 1 μ g of each drug immunization at the end of the experiment, dDT2219 group also showed significantly lower antibody induction, as seen in ELISA to detect α -DT 390.

To specify whether the antibodies tested did neutralize dDT2219 or DT2219, neutralization assays were performed using Raji target and sera from two animal groups on day 160. Significantly lower amounts of neutralizing antibodies were found in dDT2219 group (fig. 15B) (p <0.05) compared to DT2219 vaccinated control group. High titers of neutralizing antibodies were seen in 6 of 7 mice after DT2219 immunization (fig. 15C), while only 2 of 7 mice of dDT2219 group showed neutralizing antibodies (fig. 15D) (table 1).

Table 1: animal experiments, neutralizing antibodies

Abbreviations: DT, diphtheria toxin; dDT, deimmunizing diphtheria toxin; m, mouse; SD, standard deviation

25 patients with mature or precursor B-cell lymphoid malignancies (expressing CD19 and/or CD22) were involved in the study to assess DT2219 safety. All patients received a single course of DT 2219. The most common Adverse Events (AE) include weight gain, low albumin, elevated transaminases and fever, which are short-lived on a scale of 1-2 and occur in patients in the higher dose cohort (. gtoreq.40. mu.g/kg/day). Both subjects experienced DLT at dose levels of 40. mu.g/kg and 60. mu.g/kg. Neutralizing antibodies were observed in 30% of patients after 4 courses of treatment and in all patients treated with at least 40 μ g/kg (Bachanova et al 2015.Clin Cancer Res 21: 1267-1272).

Approaches to limiting the immunogenicity of DT2219 have been investigated, but there has not been a practical method demonstrated to reduce the immune response to DT 2219. Various routes have included the use of polyethylene glycol (PEG) polymers or ribonucleases (rnases), each of which can be conjugated to a biologically active drug, or co-administered with B-cell depleting agents (e.g., rituximab).

Thus, the present disclosure describes deimmunized DT2219, i.e., dDT2219, as an exemplary targeted toxin with therapeutic activity. dDT2219 shows efficacy against B cell malignancies, including two distinct sets of Burkitt's (Burkitt) lymphoma cell lines. Cell lines that do not express CD22 or CD19 are described as non-toxic (secreted). dDT2219 construct showed the same binding capacity to CLL as its parent form, and significantly lower induction of neutralizing antibodies in immunocompetent BALB/c mice. Since DT-targeted toxin polypeptides, in particular DT2219, have provided promising results in terms of inducing anti-cancer responses in vitro and in vivo, this deimmunization dDT2219 can provide anti-tumor therapy regardless of the limitations of anti-drug antibodies after multiple administrations, regardless of the vaccination status in the patient record.

As described above, the deimmunized targeting toxins may include a targeting domain that selectively binds to a target other than CD19 and/or CD 22. Additional exemplary deimmunized bispecific targeting toxins have been generated and tested in the same manner as dDT2219, as shown in table 2.

TABLE 2 additional exemplary deimmunized DT390 conjugates

Construct	IC50*	Immunogenicity	Efficacy #	SEQ ID NO:
					dDTEpCAMe23	<10nM	Whether or not	Is that	9
dDTEpCAM133	<17nM	Whether or not	Is that	10
					dDTEGFATF	<10nM	Whether or not	Is that	11
dDTROR1ATF	<19nM	Whether or not	Is that	12

Concentration of drug that inhibits 50% of drug activity as measured in an in vitro tritium-traced thymidine uptake proliferation assay or a tritium-traced leucine uptake protein synthesis assay.

Immunogenicity studies as described herein.

# efficacy as measured in the efficacy xenomodel (efficacy xenomodel) described herein.

dDTEpCAMe23 targets both EpCAM epithelial cell adhesion molecules (transmembrane glycoproteins mediating intraepithelial homocellular-cell adhesion independent of Ca2 +) and e23 or HER2 (members of the human epidermal growth factor receptor (HER/EGFR/ERBB) family). dDTEpCAM133 stimulates the targets EpCAM and CD133, which are known as established cancer stem cell markers. CD133, also known as prominin-1, is a glycoprotein encoded in humans by the PROM1 gene and is a member of the five transmembrane glycoprotein (5-transmembrane, 5-TM) that specifically localizes to the cell process (cellularistrution). ddtmegftf targets both EGFR and the amino-terminal fragment (ATF) of urokinase (uPA). dDTROR1ATF targets both ATF and ROR 1. ROR1 is a receptor tyrosine kinase that regulates neurite outgrowth. It is a type I membrane protein belonging to the ROR subfamily of cell surface receptors, and its role in cancer cell metastasis is currently under investigation.

In another aspect, the present disclosure describes a method of killing a cell. In general, the methods comprise contacting a cell with a deimmunized DT-targeting toxin polypeptide described above, allowing the cell to internalize the deimmunized DT-targeting toxin polypeptide and allowing the deimmunized DT-targeting toxin polypeptide to kill the cell. The method may be performed in vivo or in vitro.

In another aspect, the disclosure describes a method of treating a subject having a tumor. Generally, the method comprises administering to the subject a deimmunized diphtheria toxin (dDT) targeted toxin compound in an amount effective to alleviate at least one symptom or clinical sign (clinical sign) of the tumor. "treat" or variants thereof refers to any degree of attenuation, limiting progression, remission, or regression of symptoms or signs associated with the condition (condition). As used herein, "remission" refers to any reduction in the degree, severity, frequency, and/or likelihood of symptoms or clinical signs characteristic of a particular condition; "symptom (symptom)" refers to any subjective evidence of disease or patient condition; and "sign" or "clinical sign" refers to an objective physical finding related to a particular condition that can be found by someone other than the patient.

The "treatment" may be therapeutic or prophylactic. "therapeutic" and variations thereof refer to treatment that alleviates one or more symptoms or clinical signs associated with a condition. "prophylactic" and variations thereof refer to treatments that limit, to any extent, the occurrence and/or emergence of symptoms or clinical signs of a condition. Typically, a "therapeutic" treatment is initiated after the condition is manifested in the subject, while a "prophylactic" treatment is initiated before the condition is manifested in the subject. Thus, in some embodiments, the method may involve prophylactic treatment of a subject at risk of developing the condition. Risk (at risk) "means that the subject may or may not actually have the described risk. Thus, for example, a subject at risk of developing a particular condition is a subject having one or more signs of increased risk of having or developing a particular condition (indicia) as compared to an individual lacking the one or more signs, regardless of whether the subject exhibits any symptoms or clinical signs of having or developing the condition. Exemplary indications of a condition may include, for example, genetic predisposition, ancestry, age, gender, geographic location, lifestyle, or medical history. Treatment may also be continued after the symptoms have resolved, e.g., to prevent or delay their recurrence.

The dDT targeted toxin compound can be any of the embodiments of dDT targeted toxin compounds described above having a targeting domain that selectively binds to a target present on a tumor cell.

In some cases, the tumor may be surgically resected or attenuated by chemical (e.g., chemotherapeutic agents) and/or radiation therapy. In these embodiments, the dDT can be administered to the subject prior to, concurrently with, or after resection or attenuation of the tumor. Thus, in some embodiments, the dDT can be administered to a subject before, concurrently with, or after a chemotherapeutic agent is administered to the subject.

The tumor may be a solid tumor or may be a liquid tumor. Thus, the dDT can be used to treat various forms of cancer, including, for example, prostate cancer, lung cancer, colon cancer, rectal cancer, bladder cancer, melanoma, kidney cancer (kidneycancer), kidney cancer (renal cancer), oral cancer, pharyngeal cancer, pancreatic cancer, uterine cancer, thyroid cancer, skin cancer, head and neck cancer, cervical cancer, ovarian cancer, hematopoietic cancers, and/or lymphoid cancers.

A "subject" can be any animal subject, such as a mammalian subject (e.g., dog, cat, horse, cow, sheep, goat, monkey, primate, human, etc.). In some embodiments, the subject may be a human, including 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 dDT the targeted toxin compound to EGRF-expressing tumor cells, such as prostate, pancreatic, breast and/or lung cancer.

In other embodiments, targeting domains including anti-CD 19 and/or anti-CD 22scFv can be used to target dDT targeted toxin compounds to tumor cells expressing CD19 and/or CD22, e.g., B cell malignancies, such as non-hodgkin's lymphoma (NHL), Acute Lymphocytic Leukemia (ALL), Chronic Lymphocytic Leukemia (CLL), or Burkitt's lymphoma (Burkitt lymphoma).

The dDT targeting toxins described herein can 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 pharmaceutically active substances is well known in the art. It is contemplated that the use in therapeutic compositions will be with the exception that any conventional vehicle or agent is incompatible with the active ingredient. Supplementary active ingredients may also be incorporated into the composition. As used herein, "pharmaceutically acceptable" refers to a substance that is not biologically or otherwise undesirable, i.e., the substance can be administered to an individual with the 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.

dDT the targeted toxin may thus be formulated as a pharmaceutical composition. The pharmaceutical composition may be formulated in various forms suitable for the preferred route of administration. Thus, the compositions can be administered by known routes, including, for example, oral, parenteral (e.g., intradermal, transdermal, subcutaneous, intramuscular, intravenous, intraperitoneal, and the like), or topical (e.g., intranasal, intrapulmonary, intramammary, intraapproach, intrauterine, intradermal, transdermal, rectal administration, and the like). The pharmaceutical composition may be administered to a mucosal surface, for example, by administration to, for example, the nasal or respiratory mucosa (e.g., by a spray or aerosol). The compositions may also be administered by sustained or delayed release.

Thus, the dDT targeting toxin may be provided in any suitable form, including but not limited to a solution, suspension, emulsion, spray, aerosol, or any form of mixture. The composition can be delivered in a formulation with any pharmaceutically acceptable excipient, carrier or vehicle. For example, the formulation may be delivered in conventional topical dosage forms, such as creams, ointments, aerosol formulations, non-aerosol sprays, gels, lotions and the like. The formulation may also include one or more additives, for example, adjuvants, skin penetration enhancers, colorants, fragrances, flavors, humectants, thickeners, and the like.

The formulations may conveniently be presented in unit dosage form and may be prepared by methods well known in the art of pharmacy. A method of preparing a composition in a pharmaceutically acceptable carrier includes the step of bringing dDT the targeted toxin into association with the carrier which constitutes one or more accessory ingredients. In general, the formulations can be prepared by uniformly and/or intimately bringing into association the active compound with liquid carriers, finely divided solid carriers or both, and shaping the product into the desired formulation.

The amount of dDT targeted toxin administered can vary depending on a variety of factors, including, but not limited to, the particular dDT targeted toxin used, the weight, the physical condition, and/or the 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 on factors such as the species, age, weight, and physical condition of the subject, as well as the method of administration. Accordingly, it is not practical to universally state that the amount constituting the amount of dDT-targeted toxin effective for all possible applications is. However, suitable amounts can be readily determined by one of ordinary skill in the art by appropriate consideration of these factors.

In some embodiments, the method can include administering enough dDT targeted toxin to provide a dose to the subject, for example, from about 100ng/kg to about 50mg/kg, although in some embodiments the method can be practiced by targeting the toxin at a dose dDT that is outside of this range. In some of these embodiments, the method comprises administering enough dDT targeted toxin to provide a dose from about 10 μ g/kg to about 5mg/kg, for example a dose from about 100 μ g/kg to about 1mg/kg, to the subject.

Alternatively, the dose may be calculated using the actual body weight obtained before the start of the treatment process. For the doses calculated in this way, the body surface area (m) was calculated using the Dubois method before the start of the treatment process²)：m²＝(wt kg^0.425X height cm^0.725)×0.007184。

In some embodiments, the method comprises administering sufficient dDT targeting toxin to provide, for example, from about 0.01mg/m²To about 10mg/m²The dosage of (a).

In some embodiments, the dDT targeted toxin compound may be administered, for example, from a single dose per week to multiple doses, although in some embodiments the method may be practiced by administering dDT targeted toxin at a frequency outside of this range. In certain embodiments, the dDT targeted toxin may be administered about once a month to about 5 times a week.

In another aspect, the disclosure describes methods of toxin deimmunization. In general, the methods involve targeting hydrophilic amino acid residues at surface positions of the toxin polypeptide that can be involved in generating an anti-B cell response, constructing a modified polypeptide comprising 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 antibody induction. As used herein, "biological function," "biological activity," and variations thereof, refers to the native biological function of the polypeptide. However, in some instances, the biological function (or biological activity, etc.) may refer to a particular function associated with the therapeutic use of the polypeptide, such as cytokine induction, cell signaling, and the like.

This deimmunization method is for example much less time consuming and more cost effective than 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 surface of the molecule were selected to introduce point mutations at selected sites. Triple mutants (triplets) were generated by simultaneously mutating the DTEGF13 parent construct with three site-specific PCR primers. For those further mutations that did not show loss of activity, two constructs each with 7 point mutations but with minimal loss of activity were finally obtained. Mutation of DTEGF13 and expression of the product allowed for efficient screening of mutant activity in an in vitro anti-cancer assay compared to the unmutated parent form.

The structure and mechanism of diphtheria toxin is well defined. Point mutations are selected to be distant from the catalytically active site. In addition, to investigate whether point mutations removed T-cell or B-cell epitopes, two different strains of mice with different MHC haplotypes (H-2B and H-2d) were immunized. The MHC molecule should load different regions of the peptide fragment for presentation as T cell epitopes and the T and B epitopes are not necessarily linked. The mutant drug (dDTEGF 13; SEQ ID NO:8) was equally effective in reducing the antitoxin response in both lines, indicating that B cell epitopes, but not T cell epitopes, had been eliminated from DT.

In some cases, the dDT targeting toxin compound can exhibit at least 5% of the biological activity of the unmodified native diphtheria toxin, e.g., 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 may exhibit no more than 50% of the antitoxin antibody induction of unmodified native diphtheria toxin, e.g., 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 antitoxin antibody induction of unmodified.

In the foregoing description and the appended claims, the term "and/or" refers to 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, as they appear in the specification and claims, are intended to be open-ended, i.e., additional elements or limitations are optional and may or may not be present; unless otherwise indicated, "a," "an," "the," and "atleastone" are used interchangeably to refer to one or more than one; the recitation of numerical ranges by endpoints includes 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 foregoing description, certain embodiments have been described in isolation for the sake of clarity. Unless expressly stated otherwise that the features of a particular embodiment are incompatible with the features of another embodiment, certain embodiments may include a combination of compatible features described herein in connection with one or more embodiments.

For any of the methods disclosed herein that include separate steps, the steps may be performed in any order that is practicable. Optionally, any combination of two or more steps may be performed simultaneously.

The invention is illustrated by the following examples. It is understood that the specific examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as described herein.

Examples

Example 1

Construction of dDTEGF13

The coding region of DTEGF13 (SEQ ID NO:13) was initially synthesized using assembly PCR (assembly PCR). In its final configuration, the coding region (from 5 'end to 3' end) includes the Nco1 restriction site, the ATG start codon, the first 389 amino acids of the DT molecule (DT390), the 7 amino acid linker EASGGPE, the coding regions for human EGF and IL-13 joined by the 20 amino acid segment of human muscle aldolase, and the XhoI restriction site (FIG. 1A). The final 1755bp NcoI/XhoI target gene was spliced into pET21D expression vector under the control of isopropyl-b-D-thiogalactopyranoside (IPTG) inducible T7 promoter. DNA analysis was used to confirm that the genes were in the correct order (biological Genomics Center, University of Minnesota). DTCD3CD3 was synthesized as a control by fusing two duplicate scFv recognizing human CD3 epsilon to DT390 (Vallera et al, 2005, Leuk Res 29: 331-341).

To generate the deimmunized drug, DTEGF13 was mutated using the QuickChange site-directed mutagenesis kit (stratagene.la Jolla, CA) and the site-specific mutations were confirmed by DNA sequencing.

Inclusion bodies (inclusion bodies) isolation, refolding and purification

These operations were previously described (Stish et al 2007, Clin Cancer Res 13: 6486-6493). The plasmid was transformed into the e.coli (e. coli) strain BL21(DE3) (Novagen, Madison, WI). After overnight culture, the bacteria were grown in Luria broth. Gene expression was induced by addition of IPTG (Fischer Biotech, Fair Lawn, NJ). After 2 hours of induction, the bacteria were collected by centrifugation. Cell pellet (Cell pellet) was resuspended and homogenized. After sonication and centrifugation, the cell pellet was extracted and washed. The inclusion bodies were solubilized and the protein refolded. The refolded protein was purified by flash 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, rat IgG2a from clone A20-1.7, generously provided by Dr. Uli Hammerling, Sloan Kettering Cancer Research Center, New York, NY. Anti-ly5.2 was used as a control as it recognized mouse CD45.1, a hematopoietic cell surface marker that is not expressed on human cells.

Human cell line

The human prostate Cancer cell line PC-3(ATCC CRL-14435; Kaighn et al, 1979, Invest Urol 17:16-23), the human colorectal cell line HT-29(ATCC HTB-38; Fogh et al, 1977, J Natl Cancer Inst 59:221-226), the human pancreatic Cancer 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 the American type culture Collection (ATCC, Rockville MD). Cells were maintained in RPMI-1640 medium (Cambrex, East Rutherford, NJ) supplemented with 10% fetal bovine serum, 2mmol/L L glutamine, 100 units/mL penicillin, and 100. mu.g/mL streptomycin. Use the culture flask to let allCancer cells all grew as monolayers, while Daudi cells grew in suspension. Incubation of cell cultures in humidified, 5% CO₂At 37 deg.c in air. When adherent cells were 80-90% confluent, they were passaged using trypsin-EDTA dissociation. Cell viability Only>95% (as determined by trypan blue exclusion) was used for the experiment.

Proliferation assay

To determine the effect of the drug on tumor cells, cells (2X 10)⁴) Inoculation was in RPMI supplemented with 10% fetal bovine serum, 2mmol/L L glutamine, 100 units/mL penicillin and 100. mu.g/mL streptomycin in 96-well flat-bottom plates. Various concentrations of BLT were added to three replicate wells containing cells (Tsai et al, 2011, J neuroncol 103: 255-266). The plates were incubated at 37 ℃ with 5% CO₂The mixture was incubated for 72 hours. Followed by 1. mu. Ci [ methyl-³H]Thymidine (GE Healthcare, UK) incubates cells for 8 hours, collects the cells on glass fiber filters, washes, dries, and counts in a standard scintillation counter for 10 minutes. Data were analyzed using Prism 4(GraphPad Software, inc., San Diego, CA) and are expressed as a "percent control response" calculated by dividing the counts per minute (cpm) of untreated cells by the cpm (x 100) of immunotoxin-treated cells.

Blocking studies were performed to test specificity. Briefly, toxin-free 1nM, 10nM, 100nM or 1000nMEGF13 was added to a medium containing 0.001nM, 0.01nM, 0.1nM, 1nM, 10nM or 100nM DTEGF13 (Vallera et al, 2010, Mol Cancer Ther 9: 1872-1883). The resulting mixture was added to the wells containing tumor cells and passed as described³Proliferation was measured by H-thymidine uptake. Data are expressed as percent control response.

To detect neutralizing antibodies, 90% serum from immunized mice was added to cells treated with known inhibitory concentrations of DTEGF13(1000 ng/ml). Proliferation assays were then performed as described above.

Detection of serum IgG antitoxin content by ELISA assay

Assays for the detection of IgG antitoxin antibodies have been previously reported (Vallera et al, 2009, Leuk Res 33: 1233-1242). Briefly, immunocompetent normal BALB/c mice (NCI) were immunized with 0.25 μ g of either non-mutated 2219KDEL or mutated 2219KDEL 7mut injected weekly. After 5 injections, serum was collected 4 days after the last injection. Standard ELISA assays were used, in which recombinant DT390 was attached to the plate. Test sera from immunized mice were then added, followed by the addition of detection antibody, anti-mouse IgG peroxidase (Sigma-Aldrich, st. The plates were developed with o-phenylenediamine hydrochloride (Pierce Biotechnology, Rockford, IL) for 15 minutes at room temperature. By adding 2.5M H₂SO₄The reaction was terminated. The absorbance at 490nm was read and the final concentration determined from a standard curve using highly purified anti-DT 390. All samples and standards were tested in triplicate.

In vivo efficacy studies

Male nu/nu mice were purchased from the National Cancer Institute, Fredick Cancer research Development Center, Animal Production Area, and raised in specific pathogen-free locations approved by the Association for Association and registration of Laboratory Animal Care, and managed by the Department of research on Animal Resources, Minnesota. The Animal study protocol was approved by the University of Minnesota laboratory Animal Care and use Committee (University of Minnesota Institutional Animal Care and use Committee). All animals were housed in micro-isolation cages to minimize potential transmission of contaminating viruses.

Our flank tumor model has been previously reported (Stish et al, 2007, Clin Canc Res 13: 6486-6493). This route of administration was chosen because the gist of the experiment was not to mimic a clinical protocol, but rather to determine whether it was effective to de-immunize DTEGF 13. Direct intratumoral administration circumvents the major problem of systemic administration. These include the inefficient distribution to the tumor site related to distance traveled and unfavorable tumor vessel dynamics (high interstitial pressure) (Jain RK 1989, Jnat. cancer Inst.81: 570-576). Thus, intratumoral treatment ensures more consistent and reliable delivery of recombinant toxin fusion proteins to the targeted site with limited body exposure, which maximizes efficacy.

To induce tumors, mice were injected in the left flank with 4X 10 suspended in 100. mu.L of 1:1 RPMI/matrigel (matrix-Gel)⁶And (3) PC-3 cells. Once palpable tumors had formed (day 15), mice were divided into two groups and treated with multiple injections of DTEGF13 on days 15 to 25. All drugs were administered by intratumoral injection using a 3/10cc syringe with a 29 gauge needle. All treatments were given in sterile PBS in a volume of 100- μ L. Tumor size was measured with digital calipers and volume was determined as the product of length, width and height.

Example 2

dDT2219 construction

dDT2219 the 2219 coding region was synthesized using assembly PCR. The fully assembled coding region (from 5 'end to 3' end) includes the NcoI restriction site, the ATG start codon, the first 390 amino acids of the mutated and de-immunized DT molecule (DT390), the 7 amino acid linker ASGGPE (SEQ ID NO:4), the V against CD22scFv_LAnd V_HRegion, GGGGS (SEQ ID NO:5) linker, V against CD19scFv_LAnd V_HRegions and XhoI restriction sites. V per scFv_LAnd V_HThe genes were ligated via a linker (GSTSGSGKPGSGEGSTKG; SEQ ID NO:6) designated Aggregation Reduced Linker (ARL). The final 1755bp NcoI/XhoI coding region was spliced into pET21D expression vector under the control of isopropyl-b-D-thiogalactopyranoside (IPTG) inducible T7 promoter. DNA analysis was used to confirm that the genes were in the correct order (biological Genomics Center, University of Minnesota, MN). To generate the deimmunized drug, DT2219 was mutated using the quickkhange site-directed mutagenesis kit (Stratagene corp., La Jolla CA) and the site-specific mutations were confirmed by DNA sequencing. The following amino acids were changed to allow DT390 de-immunization: amino acid (aa)125 changed from K to S; aa 173R to A; aa 184Q to S; aa 227K to S; aa245Q to S; aa 292E to S; and aa385K to G.

Inclusion body isolation, refolding and purification

Coli strain BL21(DE3) (plasmid transformed) (Novagen, inc., Madison, WI, USA) was used for protein expression. The bacteria were cultured overnight in 800ml Luria broth containing 50mg/ml carbenicillin. Expression was induced by addition of IPTG after the culture reached an Optical Density (OD)600 of 0.65. After 2 hours of induction, the growing bacteria were harvested and centrifuged. The cell pellet was homogenized in buffer solution (50mM tris,50mM NaCl, and 5mM EDTA pH 8.0), followed by sonication and re-centrifugation. The precipitate was extracted with 0.3% sodium deoxycholate, 5% Triton X-100, 10% glycerol, 50mmol/L Tris,50 mmol/L NaCl, 5mmol/L EDTA (pH 8.0), and washed. Purification was previously described (Schmohl et al 2015.Targeted Oncology 11(3): 353-361; Schmohl et al 2016.MolTher24(7): 1312-1322). The homogenized inclusion bodies are solubilized and the protein refolded. Purification was performed using a continuous gradient of ion exchange chromatography (Q sepharose Fast Flow, Sigma-Aldrich, St. Louis, Mo.).

Tissue culture

The following cell lines were obtained from the American type culture Collection (ATCC, Rockville MD): burkitt lymphoma cell lines Daudi (ATCC CCL-213; Klein et al, 1968, Cancer Res 28:1300-1310) and Raji (ATCCCL-86; Pulveraft JV,1964.Lancet 1:238-240.), and the 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 number ACC 483). All cell lines were grown in suspension as described previously (Klein et al, 1968, Cancer Res 28: 1300-1310). Cells were maintained in RPMI 1640 (supplemented with 10% fetal serum).

Proliferation assay/blocking assay

To evaluate the efficacy of the drug, (2X 10)⁴) Target cells were seeded in 96-well round bottom plates and suspended in RPMI supplemented with 10% fetal bovine serum, 2mmol/L L glutamine, 100 units/mL penicillin, 100. mu.g/mL streptomycin. DT2219, dDT2219 or BIC3 (bivalent scFv constructs consisting of the catalytic and translocation domains of diphtheria toxin and two anti-CD 3 scFv; Vallera et al, 2005.Leuk Res 29:331-341) were added at different concentrations. The assay was performed in triplicate. For time-dependent assays, cells were incubated for 12 hours, 24 hours, or 36 hours. For conventional proliferation assays, at 37 ℃ and 5% CO₂The incubation was carried out for 72 hours. Followed by 1. mu. Ci of [ methyl-3H ] per well]Thymidine (GE Healthcare, UK) incubates cells for 8 hours, and the cells are collected on glass fiber filters and washed. After the drying step, the filters were counted in a standard scintillation counter for 10 minutes. Data were analyzed using Prism 4(GraphPad Software, inc., La Jolla, California) and expressed as the "percent control response" calculated by dividing the Counts Per Minute (CPM) of untreated cells by the CPM of immunotoxin-treated cells (x 100).

To assess specificity, blocking studies were performed. 100nM, 200nM or 500nM 2219 (DT-free bispecific anti-CD 19 and anti-CD 22scFv constructs) was added to each amount of DT2219 or dDT2219 and incubated with target cells at 37 ℃ and 5% CO₂The following incubation was performed. After incubation, measurements were made as described above³H-thymidine uptake.

Patient samples and binding assays

12 patient samples from CLL patients were collected at the university of minnesota. The samples were stored frozen and stored according to standard procedures until use. To evaluate binding, each sample was washed 4X 10⁵Peripheral Blood Mononuclear Cells (PBMC) and allowed to react at 4 ℃ with a Fluorescein Isothiocyanate (FITC) labeled construct at 1nM, 10nM, 20nM, 50nM or 100 nM: dDT2219, anti-EpCAM scFv (negative control) or DT 2219. After incubation at 4 ℃ for 30 min, cells were washed in 1 × PBS and spiked with APC Cy 7-conjugated anti-CD 20 monoclonal antibody (mAb) (BioLegend, San Diego, Calif.), Pacific blueConjugated anti-CD 5 mAbs (BioLegent, San Diego, Calif.) or PE-CF592 conjugated anti-CD 3 mAbs (BDbiosciences, San Jose, Calif.) were stained as described previously (Hayes et al, 2010.Leuk Res 34: 809-815). After 15 minutes of incubation, cells were washed with 1X PBS and fluorescence intensity was evaluated by FACS analysis using a LSRII flow cytometer (BD Biosciences, San Jose, CA).

Detection of antitoxin antibodies in mice

IgG antitoxin antibodies were detected as described previously (Schmohl et al, 2015.Toxins 7: 4067-4082). Briefly, immunocompetent normal BALB/c mice (NCI) (experimental accession number DAV529) were immunized intraperitoneally with either non-mutated DT2219(n ═ 7 mice) or mutated DT2219(dDT2219) (n ═ 7 mice). Mice were immunized with 0.25 μ g protein weekly for 12 weeks, followed by twice weekly immunization with 0.5 μ g protein weekly, stopped for 6 weeks, and then 1 μ g protein injection weekly for 3 weeks. The experiment was ended after 160 days. Blood was drawn on days 21, 35, 49, 63, 77, 99 and 160. Standard ELISA assays were used, in which recombinant DT390 was attached to the plate. Test sera from immunized mice were then added, followed by detection antibody, and anti-mouse IgG peroxidase (Sigma-Aldrich, st. Plates were developed with o-phenylenediamine hydrochloride (Thermo Fisher Scientific, inc., Waltham, MA) for 15 minutes at room temperature. By adding 2.5M H₂SO₄The reaction was terminated. Absorbance at 490nm was read using a mouse monoclonal antibody against diphtheria toxin ([11D 9)]Standard curve of Abcam inc., Cambridge, MA) to determine the final concentration. All samples and standards were tested in triplicate.

Detection of neutralizing antibodies in mice

To detect neutralizing antibodies, 90% serum from immunized mice was added to cells treated with known inhibitory concentrations of DT2219 and dDT2219(1000 ng/ml). Proliferation assays were then performed as described above. For the detection of serum IgG antitoxin content, ELISA assay was used.

Statistical analysis

Data are presented as mean +/-standard deviation. To evaluate differences between groups, Student's t test or one-way ANOVA analysis was used. Analysis and data presentation was done with Graphpad prism 5(Graphpad Software inc., La Jolla, CA, USA).

The complete disclosures of all patents, patent applications, and publications mentioned herein, as well as electronically supplied material (e.g., including nucleotide sequence submissions in, for example, GenBank and RefSeq, such as SwissProt, PIR, PRF, amino acid sequence submissions in PDB, and translations from annotated coding regions in GenBank and RefSeq) are incorporated by reference in their entirety. The disclosure of the present application applies if there is any inconsistency between the disclosure of the present application and the disclosure of any document incorporated herein by reference. 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, as variations obvious to those skilled in the art are intended to be included within the invention defined by the claims.

Unless otherwise indicated, all numbers expressing quantities of ingredients, 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 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 application of 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 measurements.

Unless otherwise noted, all headings are for the convenience of the reader and should not be used to limit the meaning of the text following the heading.

Sequence Listing free text

SEQ ID NO:3(dDT2219)

SEQ ID NO:4

EASGGPE

SEQ ID NO:5

GGGGS

SEQ ID NO:6

GSTSGSGKPG SGEGSTKGSEQ ID NO:7(dDT2219ARL)

SEQ ID NO:8(dDTEGF13)

SEQ ID NO:9(dDTEpCAMe23)

SEQ ID NO:10(dDTEpCAM133)

SEQ ID NO:11(dDTEGFATF)

SEQ ID NO:12(dDTROR1ATF)

13(dDTEGF13 coding sequence)

Claims (27)

1. A polypeptide, comprising:

a Diphtheria Toxin (DT) domain comprising:

a DT catalytic site; and

(ii) a reduction in at least one amino acid substitution induced by an antitoxin antibody as compared to wild-type diphtheria toxin; and

at least one targeting domain that selectively binds to a target.

2. The polypeptide of claim 1, wherein the polypeptide exhibits measurable diphtheria toxin toxicity.

3. The polypeptide of claim 1 or 2, wherein the targeting domain selectively binds to a component of a tumor cell.

4. The polypeptide of any preceding claim, wherein the targeting domain comprises an Epidermal Growth Factor (EGF) polypeptide that selectively binds to Epidermal Growth Factor Receptor (EGFR).

5. The polypeptide of any one of claims 1 to 3, wherein the targeting domain comprises an IL-13 polypeptide that selectively binds to an IL-13 receptor.

6. The polypeptide of any one of claims 1 to 3, wherein the targeting domain comprises a polypeptide that selectively binds CD 22.

7. The polypeptide of any one of claims 1 to 3, wherein the targeting domain comprises a polypeptide that selectively binds CD 19.

8. The polypeptide of any preceding claim, wherein the DT domain comprises DT 390.

9. The polypeptide of any preceding claim, wherein the polypeptide comprises at least two targeting domains.

10. The polypeptide of claim 9, wherein the first targeting domain comprises an IL-13 polypeptide and the second targeting domain comprises an EGF polypeptide.

11. The polypeptide of claim 9, wherein the first targeting domain comprises a polypeptide that selectively binds CD19 and the second targeting domain comprises a polypeptide that selectively binds CD 22.

12. The polypeptide of any preceding claim, wherein the DT domain comprises a variant of DT390 comprising at least 3 amino acid substitutions.

13. The polypeptide of claim 12, wherein the at least 3 amino acid substitutions comprise substitutions of amino acid residue 104, amino acid residue 385, and amino acid residue 292.

14. The polypeptide of claim 13, wherein the at least 3 amino acid substitutions comprise K104S, K385G, and E292S.

15. The polypeptide of claim 12, wherein the variant of DT390 comprises at least 7 amino acid substitutions.

16. The polypeptide of claim 15, wherein the at least 7 substitutions comprise substitutions of amino acid residue 125, amino acid residue 173, amino acid residue 245, amino acid residue 385, amino acid residue 292, amino acid residue 184, and amino acid residue 227.

17. The polypeptide of claim 16, wherein the at least 7 substitutions comprise K125S, R173A, Q245S, K385G, E292S, Q184S, and K227S.

18. A method of killing a cell comprising:

contacting a cell to be killed with a polypeptide of any preceding claim under conditions that allow the cell to internalize the polypeptide; and

allowing the polypeptide to kill the cell.

19. The method of claim 18, wherein the cell is in vitro.

20. The method of claim 18, wherein the cell is in vivo.

21. A method of treating a subject having a tumor, the method comprising:

administering to the subject the polypeptide of any one of claims 1 to 17, wherein the at least one targeting domain selectively binds to a target present on a cell of the tumor.

22. The method of claim 21, wherein the target comprises EGFR.

23. The method of claim 21, wherein said target comprises Her2, CD19, CD133, ROR1, CD20, CD22, CD33, CD52, EpCAM, CEA, UPA, or VEGFR.

24. The method of claim 21, wherein the polypeptide is administered prior to, concurrently with, or after administration of the chemotherapeutic agent.

25. A method of deimmunizing a diphtheria toxin polypeptide, the method comprising:

identifying hydrophilic amino acid residues 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 said modified diphtheria toxin polypeptide for biological function of said native diphtheria toxin polypeptide; and

the modified diphtheria toxin polypeptides are screened for antibody induction.

26. The method of claim 25, wherein the modified polypeptide is constructed to include two or more substitutions of the identified hydrophilic amino acid residues.

27. The method of claim 25 or 26, wherein the modified polypeptide exhibits at least 10% of the biological activity of the native polypeptide.