CD38 binding protein comprising deimmunized Shiga toxin A subunit effector

Abstract

The present invention provides binding proteins ("CD38 binding proteins"), each of which comprises (1) a CD38 binding region for cell targeting and (2) a Shiga toxin A subunit effector polypeptide ("Shiga toxin effector polypeptide"). The Shiga toxin effector polypeptide component of the CD38 binding protein may comprise a combination of mutations relative to a wild-type Shiga toxin sequence, wherein the combination of mutations provides (1) deimmunization and / or (2) reduced protease sensitivity; wherein each Shiga toxin effector polypeptide retains one or more Shiga toxin functions, such as stimulation of cellular internalization, intracellular routing, catalytic activity and / or potent cytotoxicity. The CD38 binding protein may have one or more uses, such as selectively killing specific cell types expressing CD38; and more generally, for diagnosing and treating cancers and disorders involving CD38-expressing cells, for example, CD38-positive hematopoietic cancers such as multiple myeloma.

Description

[0001] I. Cross-reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 945,107, filed December 6, 2019, U.S. Provisional Application No. 62 / 945,106, filed December 6, 2019, and U.S. Provisional Application No. 62 / 795,633, filed January 23, 2019, each of which is incorporated herein by reference in its entirety.

[0003] II. Description of Text Files Submitted Electronically

[0004] The contents of the electronically submitted text file are incorporated herein by reference in their entirety: a computer-readable copy of the Sequence Listing (file name: MTEM_009_01WO_SeqList_ST25 UPDATED 23JAN 2020.txt, recorded on January 23, 2020, file size 238 kb).

[0005] III. Technical Field

[0006] The present invention relates to binding proteins (each a "CD38 binding protein"), each comprising (1) a CD38 binding region or domain and (2) a Shiga toxin A subunit effector polypeptide ("Shiga toxin effector polypeptide").

[0007] IV. Background Art

[0008] The following includes information that may be helpful in understanding the present invention. No admission is made that any of the information provided herein is prior art or relevant to the present invention, nor is an admission made that any publication or document specifically or implicitly referenced herein is prior art.

[0009] CD38 is highly expressed on malignant plasma cells in subjects with multiple myeloma. Multiple myeloma is a hematological malignancy of plasma cells that involves excessive proliferation of plasma cells, leading to end-organ damage. In multiple myeloma, malignant plasma cells can cause disease by producing an abnormal immunoglobulin called M-protein (also called myeloma protein or monoclonal protein), resulting in monoclonal gammopathy or abnormally high levels of M-protein in the plasma. Furthermore, malignant plasma cells accumulate in the bone marrow, crowding out healthy cells.

[0010] It is desirable to have CD38 binding proteins that can be used as therapeutic molecules to treat a variety of diseases (e.g., hematopoietic cancers) that can be treated by selectively killing CD38 expressing cells or selectively delivering beneficial agents to CD38 expressing cells. It is desirable to have cytotoxic, CD38 binding, cell targeting proteins that exhibit low antigenicity and / or immunogenicity, low off-target toxicity, and potent on-target cytotoxicity. For example, it is desirable to have a CD38 binding protein comprising a cytotoxic Shiga toxin A subunit derived component that has 1) reduced potential for undesirable antigenicity and / or immunogenicity (e.g., by mutation) and / or 2) reduced potential for nonspecific toxicity (e.g., by improved stability) while maintaining potent cytotoxicity to CD38 expressing cells. In addition, it is desirable to have therapeutic and / or diagnostic CD38 binding proteins that exhibit low antigenicity and / or immunogenicity, low off-target toxicity, high stability, and / or the ability to deliver cargo to target cells expressing CD38.

[0011] V. Summary of the Invention

[0012] As described herein, the present invention provides CD38 binding fusion proteins comprising at least one Shiga toxin A subunit effector polypeptide and at least one CD38 binding domain, and optionally a linker.

[0013] Thus, in some aspects, the invention provides a CD38 binding fusion protein comprising: a) a Shiga toxin A subunit effector polypeptide; b) a heavy chain variable domain (VH) comprising: 1) a vHCDR1 comprising the sequence of SEQ ID NO: 34; 2) a vHCDR2 comprising the sequence of SEQ ID NO: 35; and 3) a vHCDR3 comprising the sequence of SEQ ID NO: 36; and c) a light chain variable domain (VL) comprising: 1) a vLCDR1 comprising the sequence of SEQ ID NO: 31; 2) a vLCDR2 comprising the sequence of SEQ ID NO: 32; and 3) a vLCDR3 comprising the sequence of SEQ ID NO: 33.

[0014] In a further embodiment, the CD38 binding fusion protein comprises the sequence of SEQ ID NO:79.

[0015] In a further aspect, the CD38 binding fusion protein has a VL comprising the sequence of SEQ ID NO:43 and a VH comprising the sequence of SEQ ID NO:44.

[0016] In a further aspect, the CD38 binding fusion protein has a) a VL comprising the sequence of SEQ ID NO:43, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to SEQ ID NO:43, and b) a VH comprising the sequence of SEQ ID NO:44, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to SEQ ID NO:44.

[0017] In a further aspect, the CD38 binding fusion protein has a VL comprising the sequence of SEQ ID NO:41 and a VH comprising the sequence of SEQ ID NO:44.

[0018] In a further aspect, the CD38 binding fusion protein comprises a VL comprising the sequence of SEQ ID NO:41, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to SEQ ID NO:41, and a VH comprising the sequence of SEQ ID NO:44, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to SEQ ID NO:44.

[0019] In additional aspects, the CD38 binding fusion protein comprises: a) a Shiga toxin A subunit effector polypeptide; b) a heavy chain variable domain (VH) comprising: 1) vHCDR1 comprising the sequence of SEQ ID NO: 22; 2) vHCDR2 comprising the sequence of SEQ ID NO: 23; and 3) vHCDR3 comprising the sequence of SEQ ID NO: 24; and c) a light chain variable domain (VL) comprising: 1) vLCDR1 comprising the sequence of SEQ ID NO: 19; 2) vLCDR2 comprising the sequence of SEQ ID NO: 20; and 3) vLCDR3 comprising the sequence of SEQ ID NO: 21.

[0020] In a further aspect, the CD38 binding fusion protein has a VL comprising the sequence of SEQ ID NO:37 and a VH comprising the sequence of SEQ ID NO:38.

[0021] In additional aspects, the CD38 binding fusion protein comprises a VL comprising the sequence of SEQ ID NO: 37, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to SEQ ID NO: 37, and a VH comprising the sequence of SEQ ID NO: 37, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to SEQ ID NO: 37.

[0022] In a further aspect, the CD38 binding fusion protein comprises: a) a Shiga toxin A subunit effector polypeptide; b) a heavy chain variable domain (VH) comprising: 1) vHCDR1 comprising the sequence of SEQ ID NO:28; 2) vHCDR2 comprising the sequence of SEQ ID NO:29; and 3) vHCDR3 comprising the sequence of SEQ ID NO:30; and c) a light chain variable domain (VL) comprising: 1) vLCDR1 comprising the sequence of SEQ ID NO:25; 2) vLCDR2 comprising the sequence of SEQ ID NO:26; and 3) vLCDR3 comprising the sequence of SEQ ID NO:27.

[0023] In a further aspect, the CD38 binding fusion protein has a VL comprising the sequence of SEQ ID NO:39 and a VH comprising the sequence of SEQ ID NO:40.

[0024] In a further aspect, the CD38 binding fusion protein comprises a VL comprising the sequence of SEQ ID NO:39, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to SEQ ID NO:39, and a VH comprising the sequence of SEQ ID NO:40, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to SEQ ID NO:40.

[0025] In a further aspect, the CD38 binding fusion protein comprises a first linker between the Shiga toxin A subunit effector polypeptide and the CD38 binding domain. In some embodiments, the first linker is a protein linker and can be about 1 to about 40 amino acids. In some embodiments, the first linker comprises the sequence of SEQ ID NO: 70.

[0026] In additional aspects, the CD38 binding fusion protein has a first linker comprising the sequence of any one of SEQ ID NOs: 70-75, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to SEQ ID NOs: 70-75.

[0027] In a further aspect, the CD38 binding fusion protein comprises a second linker positioned between VH and VL. In some embodiments, the second linker comprises the sequence (Gly4Ser)n (SEQ ID NO: 195), wherein n is equal to 1, 2, 3, 4 or 5, and in some embodiments n=1.

[0028] In another aspect, the CD38 binding fusion protein comprises, from its N- to C-terminus, Shiga toxin A subunit effector polypeptide-first linker-VH-second linker-VL.

[0029] In a further aspect, the CD38 binding fusion protein comprises, from its N- to C-terminus, Shiga toxin A subunit effector polypeptide-first linker-VL-second linker-VH.

[0030] In a further aspect, the CD38 binding fusion protein has a Shiga toxin A subunit effector polypeptide comprising the sequence of SEQ ID NO:46.

[0031] In a further aspect, the CD38 binding fusion protein has a Shiga toxin A subunit effector polypeptide comprising the sequence of any one of SEQ ID NOs: 45-69, or a sequence having at least 95%, at least 96%, at least 97%, at least 95%, or at least 99% amino acid sequence identity to SEQ ID NOs: 45-69.

[0032] In a further aspect, the CD38 binding fusion protein of claim 1, wherein the Shiga toxin A subunit effector polypeptide comprises the sequence of SEQ ID NO:46, VL comprises the sequence of SEQ ID NO:43, and VH comprises the sequence of SEQ ID NO:44.

[0033] In a further aspect, the CD38 binding fusion protein has at least 95% amino acid sequence identity to the sequence of SEQ ID NO:79.

[0034] In other aspects, the CD38 binding fusion protein of the present invention is a homodimer. In some embodiments, the homodimer comprises two identical polypeptides, each polypeptide comprising the sequence of SEQ ID NO: 79. In some embodiments, the homodimer comprises two identical polypeptides, each polypeptide comprising a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to SEQ ID NO: 79. In some embodiments, at least about 90%, 95%, or 98% of the CD38 binding fusion protein is in the form of a homodimer.

[0035] In some aspects, the present invention provides a composition of a CD38 binding fusion protein of the present invention and a pharmaceutically acceptable carrier or excipient.

[0036] In a further aspect, the present invention provides nucleic acids encoding CD38-binding fusion proteins, expression vectors comprising the nucleic acids, and host cells comprising the nucleic acids and / or expression vectors described herein. Similarly, the present invention provides methods comprising culturing a host cell containing the nucleic acids and / or expression vectors under conditions that express the CD38-binding fusion protein, and recovering the protein. In some embodiments, the protein is purified by contacting the CD38-binding fusion protein with a bacterial protein L. In some cases, the protein L is isolated or derived from Peptostreptococcus magnus (P. magnus), which can be conjugated to a resin.

[0037] In additional aspects, the present invention provides a method of treating multiple myeloma comprising administering a CD38 binding fusion protein of the present invention to a subject in need thereof.

[0038] VI. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figures 1A-1B : Figure 1A and Figure 1B Schematic diagrams of illustrative CD38 targeting molecules are provided in , each molecule comprising at least one CD38 binding region and at least one Shiga toxin A subunit effector. Figure 1A In the present invention, the CD38 binding region is typically a scFv, which comprises anti-CD38 VH and VL domains separated by a linker, and the scFv is located at the N-terminus or C-terminus of the Shiga toxin component. In an exemplary CD38 targeting molecule, the CD38 binding region is a scFv, and the scFv is shown to participate in intermolecular variable domain exchange with an adjacent scFv ( Figure 1B , lower left). Figures 1A-1B The depiction of the illustrative molecules is intended to illustrate certain general arrangements of structural features of a limited set of embodiments of the present invention. It should be understood that these illustrative molecules are not intended to and should not be interpreted as fully defining any structural features and / or arrangement of components of the molecules of the present invention. Figures 1A-1B The relative size, position, or number of features shown in the schematic diagrams may be simplified. Figures 1A-1B The schematic diagrams in are not intended to accurately depict any information regarding the relative sizes of the molecular structures in any embodiment of the invention.

[0040] Figure 2 : Figure 2 Representative CD38-positive H929 human myeloma cells are shown for each of the 51 CD38-targeting molecules in the pool. 50 As shown in the figure, the CD38 targeting molecules can be in either orientation, i.e. VH-VL or VL-VH. The grey squares represent the additional members in the pool. The screening cut-off value of 1x103 pM is indicated by the vertical dashed line, where the CD 50 The value is more powerful than the cutoff value, and the CD on the right 50 None of the 52 tested molecules exhibited a CD of 20,000 pM or less in this assay. 50 Any molecule not included in Figure 2 Some of the most potent molecules tested were in the CD38-targeting molecule families #6 and #7.

[0041] Figures 3A-3C : Figures 3A-3C Representative cytotoxicity assays using different concentrations of CD38 targeting molecules #3 and #4 are shown. CD38 targeting reference molecule #1 and Shiga effector polypeptide without targeting domain were used as positive and negative controls, respectively. CTM #3 and CTM #4 showed cytotoxicity against CD38 positive H929 cells ( Figure 3A ) and MOLP-8 cells ( Figure 3B ) concentration-dependent cytotoxicity. By comparing the cytotoxic activity against CD38-positive H929 and MOLP-8 cells ( Figures 3A-3B ) and cytotoxic activity against CD38-negative U266 cells ( Figure 3C ) to study the specificity of cytotoxicity against cells expressing CD38. CTM#3 and CTM#4 did not show significant cytotoxic activity against U266 cells under the test conditions ( Figure 3C ).

[0042] Figures 4A-4B : Figures 4A-4B showed that using human cells ( Figure 4A ) or non-human primates ( Figure 4BRepresentative cytotoxicity assay of CD38 targeting molecules #1, #2, and #4 tested on BLCL-C162 cells. BLCL-C162 is a rhesus macaque cell line expressing rhesus macaque CD38. CD38 targeting reference molecule #1 and Shiga effector peptide without targeting domain were used as positive and negative controls, respectively. CTM#1, CTM#2, and CTM#4 showed cytotoxicity against CD38 positive MOLP-8 cells ( Figure 4A ) and CD38-positive BLCL-C162 cells ( Figure 4B Note that the sequence of CD38 is identical in rhesus and cynomolgus monkeys.

[0043] Figure 5 : Figure 5 The concentration-dependent protein synthesis inhibition activity of CD38-targeting molecule #4 in a cell-free in vitro protein translation assay is shown; protein synthesis inhibition is an activity of the Shiga toxins described herein. CD38-targeting reference molecule #1 and a Shiga effector polypeptide without a targeting domain served as positive controls. Compared to the positive control reference molecule, CTM#4 reduced luciferase protein synthesis, as measured by relative luminescence units (RLU), in a concentration-dependent manner.

[0044] Figures 6A-6B :exist Figures 6A-6B In the study, CD38 targeting molecules (CTM#2, CTM#3 and CTM#4) were combined with recombinant human CD38 protein ( Figure 6A ) or recombinant cynomolgus monkey CD38 protein ( Figure 6B The binding properties of the molecules CD38 targeting molecule #2 (SEQ ID NO: 77), CD38 targeting molecule #3 (SEQ ID NO: 78), and CD38 targeting molecule #4 (SEQ ID NO: 79) each bound to human and cynomolgus monkey CD38, while CD38 targeting reference molecule #1 (SEQ ID NO: 83) bound to human CD38 ( Figure 6A ) but not to cynomolgus monkey CD38 ( Figure 6B ).

[0045] Figure 7 : Figure 7 Shown is a 3D protein structure diagram of CD38 and the positions of key residues in CD38 bound by CD38 targeting molecule #4, which were determined by using human CD38 extracellular domain epitope mapping mutagenesis. Figure 7 In the figure, the key surface accessible contact residues F216 and L262 are shown in red, the key structural residues L124 and L230 are shown in purple, and the key structural residues C119 / C201 and C254 / C275 involved in the disulfide bond pair are shown in gray.

[0046] Figures 8A-8H : Figures 8A-8H The cytotoxic activity of various illustrative CD38 targeting molecules (CTM#1, CTM#2, CTM#4, CTM#7 and CTM of family #5) against cells expressing CD38 in the presence of daratumumab is shown. Human myeloma H929 cells ( Figure 8A )), human lymphoma ST486 cells ( Figure 8B ) and human multiple myeloma MOLP-8 cells ( Figure 8C ) measured the cytotoxic activity of 0.5 μg / mL CTM#1, CTM#7, or family#5 CD38-targeted molecules against CD38-positive cells pretreated with serially diluted daratumumab. CD38-positive human lymphoma ST486 cells ( Figure 8D ), human multiple myeloma MOLP-8 cells ( Figure 8E ) The cytotoxic activity of the administered dilution series of CTM#1, CTM#7, or CTM of family #5 against cells pretreated with 10 μg / mL daratumumab was measured. The cytotoxic activity of the administered dilution series of CTM#4 against cells expressing human CD38 exposed to 50 μg / mL daratumumab was measured using human myeloma H929 cells or human multiple myeloma MOLP-8 cells ( Figure 8F ). Figure 8G Representative CD4 staining of human myeloma MOLP-8 cells exposed to a concentration series of daratumumab and measured 48 hours after administration of CTM#4 is shown. 50 Value (nM). Figure 8H Shown are the cytotoxic activity and CD4 T cells of a dilution series of CTM#4 administered to human multiple myeloma MOLP-8 cells treated with a range of concentrations of daratumumab 48 hours after exposure to CTM#4. 50 Value (nM). Figure 8H CD of CTM#4 at specific daratumumab concentration (nM) 50 The values ​​(nM) are shown in the table below. The cytotoxic activity of CTM#4 was retained in the presence of daratumumab, with CD400 at a daratumumab concentration of approximately 10,000 nM. 50 The value varies moderately.

[0047] Figure 9 : Figure 9Shown are changes in body weight over time in BALB / c mice that were administered six doses of 1 mg / kg body weight (mg / kg) of CD38-targeted molecule #3 or CD38-targeted molecule #4 over 12 days. Because half of the mice receiving CTM#3 died before the last scheduled dose on day 12, dosing was stopped for the CTM#3 group before day 12.

[0048] Figure 10 : Figure 10 The amount of serum anti-drug immunoglobulin G is shown, which was measured using an in-solution ELISA assay suitable for serum samples taken from mice administered CTM#1, CTM#2, or CD38-targeted reference molecule #1 compared to a less deimmunized CD38-targeted control molecule (SEQ ID NO: 84). Each mouse was administered 0.25 mg / kg of the corresponding CD38-targeted molecule three times a week for two weeks, then again three times a week in weeks 4 and 5, for a total of 12 doses over a 5-week period. Mouse serum was collected at various time points during the dosing phase of the study and after the end of dosing.

[0049] Figures 11A-11D : Figures 11A-11D Figure 2 shows the growth of human tumors over time in an in vivo xenograft mouse model of human cancer. Mice were injected with human myeloma tumor cells (LP1 cells, Figure 11A ; H929 cells, Figure 11B ;MM1.S cells, Figure 11C ;MOLP-8 cells, Figure 11D ), and administered CD38 targeting molecule #4 or a negative control of vehicle alone. Blue represents the measurement value of the vehicle-only control; all other data are data for CTM #4 at different doses (e.g., 1.5, 3, 4.5, 5, 6, 9, 10, or 12 mg / kg) and administration schedules (e.g., once every two weeks, BIW; once a week, QW; or once every other week, Q2W).

[0050] Figure 12: Figure 12A Shown are the average bioluminescent signals in a disseminated xenograft mouse model of cancer treated with illustrative CD38-targeting molecules. Figure 12A Shown are the average bioluminescent signals in the MM1.S-luc disseminated multiple myeloma mouse model. Figure 12BShown are images of mice collected using bioluminescent imaging of luciferase activity in xenograft tumor cells: mice administered only the vehicle exhibited a strong signal throughout their bodies, while mice administered the CD38 targeting molecule #4 (CTM#4) appeared to have no detectable MM1.S-luc tumor cells by this method. Complete tumor elimination was observed in 10 of 10 mice. Figure 12C Shown are the average bioluminescent signals in the DDaudi-luc disseminated xenograft mouse model. Figure 12C Shown are images of mice collected using bioluminescent imaging of luciferase activity in xenograft tumor cells: mice administered vehicle alone exhibited strong signal widespread throughout their bodies, while mice administered CD38-targeted reference molecule #1 exhibited reduced signal on the relevant study days.

[0051] Figure 13 : Figure 13 The diagram shows the size and proportion of protein species present in a sample preparation of CTM#4 analyzed by size exclusion chromatography (SEC) under native conditions. For SEC analysis, the absorbance of ultraviolet (UV) light at a wavelength of 280 nanometers (nm) after passage through the SEC column was plotted in milliabsorbance units (mAU) against the elution time in minutes (min). Software was used to identify the individual peaks in the 280 nm trace and the elution time of each peak at the maximum absorbance of UV light at 280 nm. The peak centered at 18.81 minutes (#2) represents the non-covalent homodimeric form of CTM#4, which contains 1014 amino acid residues and has a theoretical molecular weight of approximately 110 kDa, while the peak centered at 17.38 minutes (#1) represents one or more higher molecular weight species, such as multimers and / or aggregates of CTM#4. The purity of a single peak can be calculated by dividing the area of ​​that peak by the total peak area of ​​the sample. The peak at approximately 25 minutes represents the expected signal from UV absorbance due to buffer elution.

[0052] Figures 14A-14B : Figure 14A is a schematic diagram of an illustrative CD38 targeting molecule, such as CTM#4. CTM#4 comprises a contiguous polypeptide comprising an engineered deimmunized (DI) Shiga toxin subunit A effector polypeptide (SLT-1A) and an anti-CD38 single-chain variable fragment (scFv) fused via a protein linker. Figure 14Bis a schematic diagram showing the potential mechanism of action of an illustrative CD38-targeting molecule, such as CTM#4, which involves specific binding to CD38-expressing cells via cell-surface CD38, internalization into these target cells; intracellular self-routing in a retrograde pathway from endosomes to the Golgi apparatus, then to the endoplasmic reticulum, and then to the cytosol; and once in the cytosol, irreversible enzymatic inactivation of ribosomes leading to target cell death. This hypothetical mechanism of action of CTM#4 is independent of the patient's immune status.

[0053] Figure 15 : Figure 15 CTM#4 was shown to kill patient-derived multiple myeloma cells in primary cell culture. Figure 15 Shown are the total multiple myeloma cell survival percentages after administration of a concentration series of CTM#4 (Conc) and the CD400 activity of CTM#4 against multiple myeloma cell samples. 50 Values ​​(nM) were obtained by bone marrow aspiration (BMA) from six different patients (010, 011, 012, 013, 014, and 015), including one sample from a daratumumab-resistant patient (#13). Figure 15 The middle column of the table reports the CD38 receptor density of MM cells in patient samples. 48 hours after CTM#4 administration, CD38 receptors were expressed on MM cells within a wide range of CD38 expression levels (e.g., 1.4 to 67.5 × 10 5 CD38 cell surface density of 100 CD38 molecules / cell), and potent cytotoxicity of CTM#4 against primary multiple myeloma cells was observed (e.g., CD 50 values ​​ranged from 3.5 to 8.5 nM).

[0054] Figure 16 : Figure 16 Shown are the results of a time course study of cell viability measured using the Real-Time Glo assay following administration of CTM#4 to human multiple myeloma cells in vitro. Figure 16 Shown are CTM#4 CD over time across different cell types from a multiple myeloma cell panel (ANBL-6, NCI-H929, RMPI-8226, and MOLP-8) and in a non-multiple myeloma control cell line (HCT116). 50 Value (ng / mL). Figure 16 In the table, CD is reported in the bottom two rows (CTM#4 or CD38TM4). 50 Value (pM). Figure 16 In the table, the expression level of cell surface CD38 is reported and the value is characterized as high or medium in the CD38 expression row.

[0055] Figure 17 : Figure 17 Shown is the time course of tumor volume after mice were inoculated with human multiple myeloma MM1.S cells to form disseminated xenografts and treated with CTM#4. Mice were treated with CTM#4 once a week (QW) or every other week (Q2W) and optionally co-treated with the current standard of care (SOC) for multiple myeloma.

[0056] Figures 18A-18B : Figures 18A-18B Shown is an ex vivo analysis of total natural killer (NK) cell counts in human whole blood (left panel) and cynomolgus monkey whole blood using flow cytometry following administration of a dilution series of CTM#4. The calculated CTM#4 concentration that eliminates half of the NK population in this assay is shown (CD 50 ).

[0057] Figure 19 : Figure 19 Table summarizing the dose-response curves and results for human multiple myeloma ANBL-6 cells administered CTM#4. ANBL-6 cells were incubated with CTM#4 continuously for 48 or 72 hours, or exposed to CTM#4 for 2 hours, washed, and maintained in CTM#4-free medium until the 48 or 72 hour time point. Figure 19 The CD4+ expression of CTM#4 on ANBL-6 cells under four test conditions is shown. 50 Values: 0.03 nM without washing and 0.16 nM with a 2-hour wash at the 48-hour time point; 0.01 nM without washing and 0.1 nM with a 2-hour wash at the 72-hour time point.

[0058] Figure 20 : Figure 20 Schematic diagram showing the design of the first phase of the CTM#4 monotherapy clinical study for patients classified as having relapsed and / or refractory multiple myeloma (RRMM). The study design includes a dose escalation phase (Part 1) and an expansion phase (Part 2). In both parts, patients are treated until progressive disease (PD), unacceptable toxicity, or withdrawal.

[0059] Figure 21 : Figure 21 Figure 2 is a schematic diagram illustrating the dosing regimen for the CTM#4 monotherapy clinical study. Dose escalation will begin at 50 μg / kg CTM#4 and then be continued based on the number of dose-limiting toxicities (DLTs) observed in cycle 1. Figure 21After evaluation of the 50 μg / kg dose, subsequent dose escalation and maximum tolerated dose (MTD) decisions will be informed by a Bayesian logistic regression model (BLRM) with overdose control, along with consideration of other available non-DLT safety, clinical efficacy, pharmacokinetic (PK), and pharmacodynamic (PD) data.

[0060] Figures 22A-22B : Figure 22A List the primary and secondary objectives of the CTM#4 monotherapy clinical study, and Figure 22B Its exploratory objectives are listed.

[0061] Figures 23A-23D : Figure 23A and Figure 23B Shown is the binding of CD38 binding proteins, including CD38 targeting reference molecule #1 (SEQ ID NO: 83), to CD38-expressing MOLP-8 cells in the presence of anti-CD38 antibodies daratumumab, HB-7, AT13-5, and OKT-10. Figure 23C Competitive binding of CD38 binding proteins, including CD38 targeting reference molecule #1 (SEQ ID NO: 83), to MOLP-8 cells expressing CD38 is shown. All signals were detected by flow cytometry. Figure 23D Table summarizing the binding of CD38 binding proteins (top column) in the presence of anti-CD38 antibodies daratumumab, HB-7, AT13-5, OKT-10, and anti-CD38 binding scFv (first column on the left). Figure 23A -D: CD38TM1 fusion protein refers to a CD38 binding fusion protein comprising the amino acid sequence of SEQ ID NO: 228; CD38TM2 fusion protein refers to a CD38 binding fusion protein comprising the sequence of SEQ ID NO: 229; CD38TM3 fusion protein refers to a CD38 binding fusion protein comprising the sequence of SEQ ID NO: 230.

[0062] Figure 24A and 24B The purification of CTM#3 and CTM#4 by IMAC column and Protein L (Pro-L) column are shown, respectively. The MW mark indicates the position of the molecular weight marker. The VH and VL of CD38TM3 do not bind to the standard monoclonal antibody purification resin (Protein A or Protein L) and are purified using the His-tag and IMAC column ( Figure 24A To facilitate purification, the CD38TM3 light chain, which is a lambda light chain, was modified by framework mutations to enable it to bind to protein L, allowing affinity purification without an additional tag. The resulting CD38TM4 is able to bind to protein L and can therefore be purified by protein L affinity.

[0063] Figures 25A-25D : Figures 25A-25D Shown are the sequences of various illustrative CD38 targeting moieties suitable for use as components of CD38 targeting molecules. Figure 25A The sequences of CD38 binding domain family #1 and CD38 binding protein #1 (CD38TM1) are provided. Figure 25B The sequences of CD38 binding domain family #2 and CD38 binding protein #2 (CD38TM2) are provided. Figure 25C The sequences of CD38 binding region family #3 and CD38 targeting moiety #3 (CD38TM3) are provided. Figure 25D The sequences of CD38 binding domain family #3 and CD38 targeting moiety #4 (CD38TM4) are provided. Figure 25D As shown, the first 21 amino acids of the CD38TM3 VL domain were substituted with the first 22 amino acids of the VL domain of CD38TR1, a kappa light chain, to derive the VL domain of CD38TM4. The CDR sequences are underlined.

[0064] Figure 26 : Figure 26 Shown is an X-ray structure illustration of a diabody formed by two identical scFvs, each containing VH-GGGGS-VL from N- to C-terminus, where the VH and VL are derived from CD38TM4. The VH of one scFv chain (chain A) is complexed with the VL of the other scFv chain (chain B) to form the CD38 binding domain.

[0065] VII. Specific Implementation Methods

[0066] The present invention provides various embodiments of CD38 binding proteins and compositions thereof, wherein each CD38 binding protein comprises (1) at least one Shiga toxin A subunit effector polypeptide derived from the A subunit of at least one member of the Shiga toxin family, and (2) at least one CD38 binding region capable of specifically binding to the extracellular portion of the CD38 molecule, optionally with a linker, as described below. For each CD38 binding protein of the invention, at least one binding region is heterologous to the Shiga toxin A subunit effector polypeptide, e.g., an immunoglobulin-type binding region.

[0067] A. Definition

[0068] These and other features, aspects and advantages of the present invention will be better understood through the following description, appended claims and drawings. The aforementioned elements of the present invention may be combined individually or removed freely to form other embodiments of the present invention, and there is no statement in the following that opposes such combination or removal.

[0069] In order to make the present invention more easily understood, some terms are defined below. Additional definitions can be found in the detailed description of the invention.

[0070] As used in the specification and the appended claims, the terms "a," "an," and "the" include singular and plural referents unless the context clearly dictates otherwise.

[0071] As used in the specification and the appended claims, the term "and / or" when referring to two substances A and B means at least one of A and B. As used in the specification and the appended claims, the term "and / or" when referring to more than two substances, such as A, B, and C, means at least one of A, B, or C, or at least one of any combination of A, B, or C (with single or multiple possibilities for each substance).

[0072] The term "amino acid residue" or "amino acid" includes reference to an amino acid incorporated into a protein, polypeptide, or peptide. The term "polypeptide" includes any polymer of amino acids or amino acid residues. The term "polypeptide sequence" refers to the series of amino acids or amino acid residues that physically comprise a polypeptide.

[0073] A "protein" is a macromolecule comprising one or more polypeptides or polypeptide "chains." For example, a protein can comprise one, two, three, four, five, six, seven, eight, nine, ten, or more polypeptides. In embodiments where a protein comprises more than one polypeptide, the polypeptides of the protein can be the same or different from one another. A protein can be a monomer or a multimer, e.g., a dimer, trimer, tetramer, etc.

[0074] A "peptide" is a small polypeptide having a size less than about 15 to 20 total amino acid residues. The term "amino acid sequence" refers to a series of amino acids or amino acid residues that physically comprise a peptide or polypeptide in terms of length. Sometimes, "residue" as used herein is intended to refer to a position in a protein and its associated amino acid identity. Unless otherwise indicated, the polypeptide and protein sequences disclosed herein are written from left to right, representing their order from amino terminus to carboxyl terminus.

[0075] The terms "amino acid," "amino acid residue," "amino acid sequence," or polypeptide sequence include naturally occurring amino acids (including L and D isomers) and, unless otherwise limited, also include known analogs of natural amino acids that can function in a manner similar to the naturally occurring amino acids, such as selenocysteine, pyrrolysine, N-formylmethionine, γ-carboxyglutamate, hydroxyprolinehypusine, pyroglutamic acid, and selenomethionine.

[0076] As used herein, "modification" refers to an amino acid substitution, insertion, and / or deletion in a polypeptide sequence, or to a change in a portion chemically linked to a protein. For example, a modification can be an altered carbohydrate or PEG structure attached to a protein. As used herein, "amino acid modification" refers to an amino acid substitution, insertion, and / or deletion in a polypeptide sequence. For clarity, unless otherwise indicated, amino acid modifications are always directed to amino acids encoded by DNA, e.g., the 20 amino acids for which codons exist in DNA and RNA.

[0077] "Amino acid replacement" or "replacement" herein refers to replacing an amino acid at a specific position in a parent polypeptide sequence with a different amino acid. In particular, in some embodiments, the replacement is for an amino acid that is not naturally occurring at a specific position, or an amino acid that is not naturally occurring in an organism or in any organism. For example, replacement N297A refers to a variant polypeptide, in this case an Fc variant, in which the asparagine at position 297 is replaced by alanine. For clarity, proteins that are engineered to change the nucleic acid coding sequence but do not change the starting amino acid (e.g., CGG (encoding arginine) is exchanged for CGA (still encoding arginine) to increase host organism expression levels) are not "amino acid replacements"; that is, although a new gene encoding the same protein is generated, if the protein has the same amino acid at its starting specific position, it is not an amino acid replacement.

[0078] The phrase "conservative substitutions" with respect to amino acid residues of a peptide, peptide region, polypeptide region, protein or molecule refers to changes in the amino acid composition of the peptide, peptide region, polypeptide region, protein or molecule that do not substantially alter the function and structure of the entire peptide, peptide region, polypeptide region, protein or molecule (see Creighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, New York (2nd ed., 1992))).

[0079] As used herein, "amino acid insertion" or "insertion" refers to the addition of an amino acid sequence at a specific position in a parent polypeptide sequence. For example, -233E or 233E indicates the insertion of glutamic acid after position 233 and before position 234. In addition, 233ADE or A233ADE indicates the insertion of AlaAspGlu after position 233 and before position 234.

[0080] As used herein, "amino acid deletion" or "deletion" refers to the removal of an amino acid sequence at a specific position in a parent polypeptide sequence. For example, E233- or E233#, E233(), or E233del indicates a deletion of glutamic acid at position 233. In addition, EDA233- or EDA233# indicates a deletion of the sequence GluAspAla starting at position 233.

[0081] As used herein, "protein" refers to at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides, and peptides. The peptidyl group comprises naturally occurring amino acids and a peptide bond, or a synthetic peptide mimetic structure, i.e., an "analog," such as a peptoid (see Simon et al., PNAS USA 89(20):9367(1992), incorporated by reference in its entirety). Amino acids can be naturally occurring or synthetic (e.g., amino acids not encoded by DNA); as will be understood by those skilled in the art. For example, for the purposes of the present invention, homophenylalanine, citrulline, ornithine, and norleucine are considered synthetic amino acids, and amino acids in both the D- and L- (R or S) configurations can be used. Variants of the present invention may include modifications including the use of synthetic amino acids incorporated using techniques developed, for example, by Schultz and colleagues, including but not limited to those described by Cropp & Shultz, 2004, Trends Genet. 20(12):625-30, Anderson et al., 2004, Proc Natl Acad Sci USA 101(2):7566-71, Zhang et al., 2003, 303(5656):371-3, and Chin et al., 2003, Science 301(5635):964-7, which are incorporated by reference in their entireties. In addition, the polypeptides may include synthetic derivatization of one or more side chains or termini, glycosylation, pegylation, cyclic permutations, cyclizations, linkers to other molecules, fusions to proteins or protein domains, and the addition of peptide tags or labels.

[0082] "Wild type or WT" herein refers to an amino acid sequence or nucleotide sequence found in nature, including allelic variations. A WT protein has an amino acid sequence or nucleotide sequence that has not been intentionally modified.

[0083] As used herein, "variant protein" or "protein variant" or "variant" refers to a protein that is different from a parent protein due to at least one amino acid modification. Protein variants can refer to the protein itself, a composition comprising the protein, or the amino acid sequence of the encoded protein. In some embodiments, the protein variant has at least one amino acid modification compared to the parent protein, for example, about one to about seventy amino acid modifications compared to the parent, and in some embodiments, about one to about five amino acid modifications. As described below, in some embodiments, the parent polypeptide (e.g., Fc parent polypeptide) is a human wild-type sequence, for example, from the Fc region of IgG1, IgG2, IgG3, or IgG4, although the human sequence with a variant can also serve as a "parent polypeptide." In some embodiments, the protein variant sequence herein has at least about 80% identity with the parent protein sequence, and in some embodiments has at least about 90% identity, at least about 95%, at least about 95%, or at least about 99% identity with the parent protein sequence. Variant protein can refer to the variant protein itself, a composition comprising the protein variant, or the DNA sequence encoding it.

[0084] As used herein, an "antigen binding domain" or "ABD" refers to a set of six complementarity determining regions (CDRs) that, when present as part of a polypeptide sequence, specifically bind to a target antigen as described herein. Thus, an "anti-CD38 antigen binding domain" binds to the CD38 antigen as outlined herein. As known in the art, these CDRs typically exist as a first set of variable heavy CDRs (vhCDRs or VHCDRs) and a second set of variable light CDRs (vlCDRs or VLCDRs), each comprising three CDRs: vhCDR1, vhCDR2, and vhCDR3 for the heavy chain, and vlCDR1, vlCDR2, and vlCDR3 for the light chain. As understood in the art, the CDRs are separated in each of the heavy and light chain variable regions by framework regions: for the light chain variable region these are (VL)FR1-vlCDR1-(VL)FR2-vlCDR2-(VL)FR3-vlCDR3-(VL)FR4, and for the heavy chain variable region they are (VH)FR1-vhCDR1-(VH)FR2-vhCDR2-(VH)FR3-vhCDR3-(VH)FR4.

[0085] The antigen-binding domains of the present invention can be embodied in a variety of formats, such as Fab, Fv, and scFv. In the "Fab" format, this set of six CDRs is contributed by two different polypeptide sequences, a heavy chain variable region (vh or VH; comprising vhCDR1, vhCDR2, and vhCDR3) and a light chain variable region (vl or VL; comprising vlCDR1, vlCDR2, and vlCDR3), wherein the C-terminus of VH is attached to the N-terminus of the heavy chain CH1 domain, and the C-terminus of VL is attached to the N-terminus of the constant light domain (thereby forming a light chain). The heavy chain variable region and the light chain variable region together form Fv, as described herein, which can be scFv or Fab. Therefore, in some cases, the six CDRs of the antigen-binding domain are contributed by VH and VL. In the scFv format, VH and VL are typically covalently attached to a single polypeptide sequence using a linker outlined herein, which can be (starting from the N-terminus) VH-linker-VL or VL-linker-VH.

[0086] As used herein, "Fab" or "Fab region" refers to a polypeptide comprising the VH, CH1, VL, and CL immunoglobulin domains. Fab can refer to this region in isolation, or in the context of a full-length antibody or antibody fragment.

[0087] As used herein, "Fv" or "Fv fragment" or "Fv region" refers to a polypeptide comprising the VL and VH domains of a single antibody. Those skilled in the art will appreciate that these consist of two domains, a variable heavy domain and a variable light domain.

[0088] "Single-chain Fv" or "scFv" herein refers to a variable heavy domain covalently linked to a variable light domain, typically using an scFv linker as described herein to form an scFv or scFv domain. The scFv domain can be in either direction from the N-terminus to the C-terminus (vh-linker-vl or vl-linker-vh). Typically, the linker is an scFv linker well known in the art and described above.

[0089] The term "antibody" is used in the broadest sense and includes, for example, complete immunoglobulins or antigen-binding portions of immunoglobulins or antigen-binding proteins associated with or derived from immunoglobulins. Complete antibody structural units typically comprise tetrameric proteins. Each tetramer is typically composed of two pairs of identical polypeptide chains, each pair having a "light" chain (typically having a molecular weight of about 25 kDa) and a "heavy" chain (typically having a molecular weight of about 50 to 70 kDa). Human immunoglobulin light chains can be classified as having either κ or λ light chains. In some embodiments, the present invention provides antibody structures comprising antigen-binding domains (e.g., antibody heavy and / or light chains) typically based on the IgG class, which has several subclasses, including but not limited to IgG1, IgG2, IgG3, and IgG4. In general, IgG1 has different allotypes, with polymorphisms at 356 (D or E), IgG2, and 358 (L or M). The sequences described herein use the 356D / 358M allotype, but other allotypes are also included herein. That is, any sequence comprising an IgG1 Fc domain encompassed herein may have a 356E / 358L alternative to a 356D / 358M allotype. IgG4 is used more frequently than IgG3.

[0090] It should be noted that IgG1 has different allotypes, with polymorphisms at 356 (D or E) and 358 (L or M). The sequences described herein use the 356D / 358M allotype, but other allotypes are also contemplated herein. That is, any sequence comprising an IgG1 Fc domain contemplated herein may have 356E / 358L instead of 356D / 358M allotypes.

[0091] Therefore, " isotype " as used herein refers to any subclass of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions. As used herein, " IgG subclass modification " or " isotype modification " refers to an amino acid modification in which one amino acid of an IgG isotype is converted into the corresponding amino acid in a different, aligned IgG isotype. For example, because IgG1 contains a tyrosine at EU position 296 and IgG2 contains a phenylalanine, the F296Y substitution in IgG2 is considered an IgG subclass modification. Similarly, because IgG1 has a proline at position 241 and IgG4 has a serine there, an IgG4 molecule with S241P is considered an IgG subclass modification. Note that subclass modification is considered herein as an amino acid substitution.

[0092] As used herein, "Fc" or "Fc region" or "Fc domain" refers to the polypeptide comprising the constant region of an antibody excluding the first constant region immunoglobulin domain (e.g., CHI) and, in some cases, part of the hinge. For IgG, the Fc domain comprises the immunoglobulin domains CH2 and CH3 (Cγ2 and Cγ3) and the lower hinge region between CHI (Cγ1) and CH2 (Cγ2). Although the boundaries of the Fc region can vary, the human IgG heavy chain Fc region is generally defined as including residues C226 or P230 to its carboxyl terminus, where numbering is according to the EU index as in Kabat. Thus, the "CH" domain in the context of IgG is as follows: "CH1" refers to positions 118-215 according to the EU index as in Kabat. "Hinge" refers to positions 216-230 according to the EU index as in Kabat. "CH2" refers to positions 231-340 according to the EU index as in Kabat, and "CH3" refers to positions 341-447 according to the EU index as in Kabat. Thus, an "Fc domain" includes a -CH2-CH3 domain, and optionally a hinge domain (hinge-CH2-CH3). In some embodiments, as described more fully below, the Fc region is subjected to amino acid modifications, e.g., to alter binding to one or more FcR receptors or to the FcRn receptor.

[0093] The "heavy constant region" herein refers to the CH1-hinge-CH2-CH3 portion of a human IgG antibody.

[0094] "Light constant region" refers to the CL domain of either kappa or lambda.

[0095] As used herein, "variable domain" refers to a region of an immunoglobulin comprising one or more Ig domains, which are substantially encoded by any of the Vκ (V.κ), Vλ (V.λ) and / or VH genes that constitute the kappa, lambda and heavy chain immunoglobulin genetic loci, respectively. Thus, a "variable heavy domain" comprises (VH)FR1-vhCDR1-(VH)FR2-vhCDR2-(VH)FR3-vhCDR3-(VH)FR4, and a "variable light domain" comprises (VL)FR1-vlCDR1-(VL)FR2-vlCDR2-(VL)FR3-vlCDR3-(VL)FR4.

[0096] The amino-terminal portion of each chain contains a variable region of about 100 to 110 or more amino acids that is primarily responsible for antigen recognition, commonly referred to in the art and herein as an "Fv domain" or "Fv region." In the variable region, each V domain of the heavy and light chains brings together three loops to form an antigen binding site. Each loop is called a complementarity determining region (hereinafter referred to as "CDR"), where the variation in amino acid sequence is most significant. "Variable" refers to the fact that certain segments of the variable region differ greatly in sequence between antibodies. The variability within the variable region is not evenly distributed. Instead, the V region consists of relatively unchanging stretches of 15-30 amino acids called framework regions (FRs), separated by shorter regions of great variation called "hypervariable regions," each of which is 9-15 amino acids or longer.

[0097] Each VH and VL is composed of three hypervariable regions ("complementarity determining regions," "CDRs") and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0098] The hypervariable region generally encompasses amino acid residues 24-34 (LCDR1; "L" for light chain), 50-56 (LCDR2), and 89-97 (LCDR3) from the light chain variable domain and from about 31-35B (HCDR1; "H" for heavy chain), 50-65 (HCDR2), and 95-102 (HCDR3) from the heavy chain variable domain; Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5th ed. Public Health Service, National Institutes of Health. of Health, Bethesda, Md. (1991) and / or those residues that form the hypervariable loops (e.g., residues 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3) in the light chain variable region, and 26-32 (HCDR1), 53-55 (HCDR2), and 96-101 (HCDR3) in the heavy chain variable region; Chothia and Lesk (1987) J. Mol. Biol. 196:901-917. Specific CDRs of the invention are described below.

[0099] As will be appreciated by those skilled in the art, the exact numbering and position of the CDRs may differ between different numbering systems. However, it will be understood that inventions of variable heavy and / or variable light chain sequences include inventions of the associated (intrinsic) CDRs. Thus, the invention of each variable heavy region is an invention of vhCDRs (e.g., vhCDR1, vhCDR2, and vhCDR3), and the invention of each variable light region is an invention of vlCDRs (e.g., vlCDR1, vlCDR2, and vlCDR3).

[0100] A useful comparison of CDR numbering is as follows (Table 4), see Lafranc et al., Dev. Comp. Immunol. 27(1):55-77 (2003):

[0101] Table 1. Antibody CDR nomenclature

[0102]

[0103] In this specification, the Kabat numbering system is generally used when referring to residues in the variable domain (approximately residues 1-107 in the light chain variable region and residues 1-113 in the heavy chain variable region), and the EU numbering system is used for the Fc region (e.g., Kabat et al., supra (1991)).

[0104] The present invention provides a large number of different CDR sets. In this case, a "full CDR set" comprises three variable light chain CDRs and three variable heavy chain CDRs, such as vlCDR1, vlCDR2, vlCDR3, vhCDR1, vhCDR2, and vhCDR3. These can be part of a larger variable light domain or a variable heavy domain, respectively. In addition, as more fully outlined herein, when heavy and light chains are used (e.g., when Fab is used), the variable heavy and variable light domains can be on separate polypeptide chains, or, in the case of scFv sequences, on a single polypeptide chain.

[0105] The CDRs contribute to the formation of the antigen binding site, or more specifically, epitope binding site, of an antibody.

[0106] An "epitope" refers to a determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, called a paratope. An epitope is a group of molecules, such as amino acids or sugar side chains, that typically have specific structural characteristics as well as specific charge characteristics. A single antigen may have more than one epitope. An epitope can include amino acid residues that are directly involved in binding (also called the immunodominant component of the epitope) and other amino acid residues that are not directly involved in binding, such as amino acid residues that are effectively blocked by the specific antigen-binding peptide; in other words, amino acid residues that are within the footprint of the specific antigen-binding peptide. Epitopes can be conformational or linear. Conformational epitopes are produced by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are produced by adjacent amino acid residues in a polypeptide chain. Conformational and non-conformational epitopes are distinguished in that, in the presence of denaturing solvents, the former does not bind but the latter does.

[0107] "Specific binding" or "specifically binds to" or "specifically for" a particular antigen or epitope refers to binding that is significantly different from non-specific interactions. Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule, which is typically a molecule of similar structure but without binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target.

[0108] As used herein, the term "Kassoc" or "Ka" is intended to refer to the association rate of a particular antibody-antigen interaction, while the term "Kdis" or "Kd" as used herein is intended to refer to the dissociation rate of a particular antibody-antigen interaction. D " is intended to refer to the dissociation constant, which is obtained from the ratio of KD to Ka (i.e., KD / Ka) and expressed as a molar concentration (M). The KD value of an antibody can be determined using methods well established in the art. In some embodiments, the KD value of an antibody is determined by D The method is to use surface plasmon resonance, for example by using a biosensor system such as In some embodiments, the K D is determined by biolayer interferometry. In some embodiments, K D is measured using flow cytometry with antigen-expressing cells. In some embodiments, K D The value is measured using immobilized antigen. In other embodiments, K D The K value is measured when the antibody (e.g., parent mouse antibody, chimeric antibody, or humanized antibody variant) is immobilized. In some embodiments, K D The value is measured in bivalent binding mode. In other embodiments, K D The values ​​are measured in monovalent binding mode. Specific binding to a particular antigen or epitope can be determined, for example, by the K of the antibody for the antigen or epitope.D is at least about 10 - 7 M, at least about 10 -8 M, at least about 10 -9 M, at least about 10 -10 M, at least about 10 -11 M, at least about 10 -12 M, at least about 10 -13 M, at least about 10 -14 Typically, the K of an antibody that specifically binds to an antigen is D is the K of the control molecule relative to the antigen or epitope D 20 times, 50 times, 100 times, 500 times, 1000 times, 5,000 times, 10,000 times or more of.

[0109] " Percent (%) amino acid sequence identity " with respect to a protein sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a specific (parent) sequence, after the sequences are aligned and, if necessary, gaps are introduced to obtain maximum percentage sequence identity, and any conservative substitutions are not considered part of the sequence identity. Alignment for determining percentage amino acid sequence identity can be achieved in various ways well known to those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine suitable parameters for measuring alignment, including any algorithm required for achieving maximum alignment over the full length of the compared sequences. A specific program is the ALIGN-2 program outlined in paragraphs

[0279] to

[0280] of U.S. Pre-Grant Publication No. 20160244525, which is incorporated herein by reference. Another approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics, 2:482-489 (1981). This algorithm can be applied to amino acid sequences by using a scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, MO Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, DC, USA and standardized by Gribskov, Nucl. Acids Res. 14 (6): 6745-6763 (1986).

[0110] The example that performs this algorithm to determine sequence identity percentage is provided by Genetics Computer Group (Madison, WI) in " BestFit " utility program.The default parameters of this method are described in Wisconsin Sequence Analysis Package Program Manual, the 8th edition (1995) (available from Genetics Computer Group, Madison, WI).Another method of setting up percentage identity in the context of the present invention is to use copyright by the MPSRCH program package all by Edinburgh University, this program package is developed by John F.Collins and Shane S.Sturrok and issued by IntelliGenetics, Inc. (Mountain View, CA).From this package, Smith-Waterman algorithm can be used, wherein default parameters are used for score table (for example, gap opening penalty is 12, gap extension penalty is 1, and gap is 6).From the data generated, " match " value reflects " sequence identity ".Other suitable programs for calculating percentage identity or similarity between sequences are well known in the art, for example, another kind of alignment program is BLAST, uses default parameters. For example, BLASTN and BLASTP can be used using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expectation = 10; matrix = BLOSUM62; description = 50 sequences; sort by = high score; database = non-redundant; GenBank + EMBL + DDBJ + PDB + GenBank CDS translation + Swiss protein + Spupdate + PIR. Detailed information on these programs can be found by typing http: / / in front of blast.ncbi.nlm.nih.gov / Blast.cgi.

[0111] The degree of identity between an amino acid sequence of the invention ("inventive sequence") and a parent amino acid sequence is calculated as the number of exact matches in the alignment of the two sequences divided by the length of the "inventive sequence" or the length of the parent sequence, whichever is shorter. The result is expressed as a percentage of identity.

[0112] The term "nucleic acid" includes any form of RNA or DNA molecules having more than one nucleotide, including single-stranded, double-stranded, oligonucleotide or polynucleotide. The term "nucleotide sequence" includes the order of nucleotides in an oligonucleotide or polynucleotide in the form of a single-stranded nucleic acid.

[0113] As used herein, the term "promoter" includes a DNA sequence that is operably linked to a nucleic acid sequence to be transcribed, such as a nucleic acid sequence encoding a desired molecule. A promoter is typically located upstream of a nucleic acid sequence to be transcribed and provides a site for specific binding of RNA polymerase and other transcription factors.

[0114] A "vector" is capable of transferring a gene sequence to a target cell. In general, "vector construct," "expression vector," and "gene transfer vector" refer to any nucleic acid construct capable of directing the expression of a gene of interest and capable of transferring a gene sequence to a target cell, either by genomic integration of all or a portion of the vector, or by transient or heritable maintenance of the vector as an extrachromosomal element. Thus, the term encompasses cloning and expression vehicles, as well as integrating vectors.

[0115] Term used herein " regulating element " comprises the nucleotide sequence of some aspect of controlling nucleic acid sequence expression.The example of regulating element illustratively comprises enhancer, internal ribosome entry site (IRES), intron, replication origin, polyadenylation signal (pA), promoter, enhancer, transcription termination sequence and upstream control structure domain, and it contributes to the duplication, transcription and / or transcriptional post-processing of nucleic acid sequence.In some cases, regulating element can also comprise cis-regulating DNA element and transposable element (TE).Those of ordinary skill in the art can select and use these and other regulating elements in expression construct when being no more than routine experiment.Expression construct can use genetic recombination method or use well-known method synthetic generation.

[0116] A "control element" or "control sequence" is a nucleotide sequence that participates in molecular interactions that contribute to the functional regulation of a polynucleotide, including replication, duplication, transcription, splicing, translation, or degradation of the polynucleotide. The regulation may affect the frequency, speed, or specificity of the process and may be enhancing or inhibitory in nature. Control elements known in the art include, for example, transcriptional regulatory sequences, such as promoters and enhancers. A promoter is a region of DNA that is capable of binding RNA polymerase under certain conditions and initiating transcription of a coding region that is typically located downstream (in the 3' direction) of the promoter.

[0117] For the purposes of the present invention, when referring to a polypeptide or polypeptide region, the phrase "derived from" means that the polypeptide or polypeptide region comprises the amino acid sequence originally found in the "parent" protein, and that it may now contain some amino acid residue additions, deletions, truncations, rearrangements, or other changes relative to the original polypeptide or polypeptide region, as long as some functions and structures of the "parent" molecule are substantially conserved. A skilled person will be able to identify the parent molecule from which the polypeptide or polypeptide region is derived using techniques known in the art, such as protein sequence alignment software.

[0118] For the purposes of the present invention, with respect to Shiga toxin polypeptide sequences or Shiga toxin-derived polypeptides, the term "wild type" generally refers to a naturally occurring Shiga toxin protein sequence found in a living species (e.g., a pathogenic bacterium), wherein the Shiga toxin protein sequence is one of the most frequently occurring variants. This is in contrast to infrequently occurring Shiga toxin protein sequences, which, while still naturally occurring, are found in less than 1% of the individual organisms of a species when a statistically robust number of naturally occurring individual organisms of that species are sampled that include at least one Shiga toxin protein variant. Clonal expansion of a natural isolate outside of its natural environment (regardless of whether the isolate is an organism or molecule containing biological sequence information) does not alter the natural requirements, as long as the clonal expansion does not introduce new sequence variants not present in the natural population of the species and / or does not alter the relative proportions of the sequence variants to one another.

[0119] As used herein, the terms "associated," "associated," "linked," or "linked" refer to the state in which two or more components of a molecule are bound, attached, connected, or otherwise coupled to form a single molecule or the act of associating two molecules with each other to form a single molecule by creating an association, connection, attachment, and / or any other connection between the two molecules. For example, the term "linked" may refer to the association of two or more components to form a single molecule through one or more atomic interactions, and wherein the atomic interactions may be covalent and / or non-covalent. Non-limiting examples of covalent associations between two components include peptide bonds and cysteine-cysteine ​​disulfide bonds. Non-limiting examples of non-covalent associations between two molecular components include ionic bonds and hydrogen bonds.

[0120] For purposes of the present invention, the term "linked" refers to the association of two or more molecular components through one or more atomic interactions to form a single molecule, and wherein the atomic interactions include at least one covalent bond. For purposes of the present invention, the term "linked" refers to the act of producing a linked molecule as described above.

[0121] For the purposes of the present invention, the term "fused" refers to two or more protein components that are associated by at least one covalent bond that is a peptide bond, regardless of whether the peptide bond involves the participation of a carbon atom of a carboxylic acid group or involves another carbon atom, such as a π-carbon, a π-carbon, a π-carbon, a π-carbon, etc. Non-limiting examples of two protein components that are fused together include, for example, an amino acid, a peptide, or a polypeptide fused to a polypeptide via a peptide bond such that the resulting molecule is a single continuous polypeptide. For the purposes of the present invention, the term "fused" refers to the act of producing a fusion molecule as described above (e.g., a fusion protein produced by recombinant fusion of genetic regions that produces a single protein molecule when translated).

[0122] The symbol "::" means that the polypeptide regions preceding and following it are physically linked together to form a continuous polypeptide.

[0123] As used herein, the term "expressed," "expressing," or "expresses" and its grammatical variants refer to the translation of a polynucleotide or nucleic acid into a protein. The expressed protein may be retained within the cell, become a component of the cell surface membrane, or be secreted into the extracellular space.

[0124] As used herein, a cell that expresses a significant amount of an extracellular target biomolecule on at least one cell surface is a "target positive cell" or "target+ cell" and is a cell that is physically coupled to a specific extracellular target biomolecule.

[0125] As used herein, the symbol "?" is shorthand for an immunoglobulin-type binding region that is capable of binding to the biomolecule following the symbol. The symbol "?" is used to refer to the functional characteristics of an immunoglobulin-type binding region, based on its ability to bind to the biomolecule following the symbol. The binding affinity is expressed as 10 -5 or lower dissociation constant (KD) description.

[0126] As used herein, the term "heavy chain variable (VH) domain" or "light chain variable (VL) domain" refers to any antibody VH or VL domain (e.g., a human VH or VL domain) and any derivative that retains at least the qualitative antigen binding ability of the corresponding native antibody (e.g., a humanized VH or VL domain derived from a native murine VH or VL domain). A VH or VL domain comprises a "framework" region interrupted by three CDRs or antigen-binding regions (ABRs). The framework region serves to arrange the CDRs or ABRs to specifically bind to an antigenic epitope. From amino terminus to carboxyl terminus, both VH and VL domains comprise the following framework (FR) and CDR regions or ABR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4; or, similarly, FR1, ABR1, FR2, ABR2, FR3, ABR3, and FR4. As used herein, the terms "HCDR1," "HCDR2," or "HCDR3" are used to refer to CDR1, 2, or 3, respectively, in a VH domain, and the terms "LCDR1," "LCDR2," and "LCDR3" are used to refer to CDR1, 2, or 3, respectively, in a VL domain. As used herein, the terms "HABR1," "HABR2," or "HABR3" are used to refer to ABR1, 2, or 3, respectively, in a VH domain, and the terms "LABR1," "LABR2," or "LABR3" are used to refer to CDR1, 2, or 3, respectively, in a VL domain. For camelid VHH fragments, cartilaginous fish IgNARs, VNAR fragments, some single-domain antibodies, and derivatives thereof, there is a single heavy chain variable domain comprising the same basic arrangement: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. As used herein, the terms "HCDR1," "HCDR2," or "HCDR3" may be used to refer to CDR1, 2, or 3, respectively, in a single heavy chain variable domain.

[0127] For the purposes of the present invention, the term "effector" refers to a molecule that provides a biological activity, such as cytotoxicity, biological signaling, enzyme catalysis, subcellular homing and / or leads to an allosteric effect and / or the recruitment of one or more factors.

[0128] For the purposes of the present invention, the phrases "Shiga toxin effector polypeptide," "Shiga toxin effector polypeptide region," and "Shiga toxin effector region" refer to a polypeptide or polypeptide region derived from at least one Shiga toxin A subunit of a member of the Shiga toxin family, wherein the polypeptide or polypeptide region is capable of exhibiting at least one Shiga toxin function. For example, SEQ ID NOs: 45-69 are derived from StxA and SLT-1A.

[0129] For the purposes of the present invention, Shiga toxin effector function is the biological activity conferred by the polypeptide region derived from the Shiga toxin A subunit. Non-limiting examples of Shiga toxin effector function include promoting cell entry; lipid membrane deformation; promoting cell internalization; stimulating clathrin-mediated endocytosis; sending the cell to various intracellular compartments, such as the Golgi apparatus, endoplasmic reticulum, and cytosol, by route; guiding the intracellular self-selection path of cargo; inhibiting ribosome function; inhibiting protein synthesis, catalytic activity, such as N-glycosidase activity and catalytic inhibition of ribosomes; reducing protein synthesis, inducing caspase activity, activating effector caspase, and achieving cell inhibition and cytotoxicity. Shiga toxin catalytic activity includes, for example, ribosome inactivation, protein synthesis inhibition, N-glycosidase activity, polynucleotide: adenosine glycosidase activity, RNA enzyme activity, and DNA enzyme activity. Shiga toxin is a ribosome inactivating protein (RIP). RIP can depurinate nucleic acids, polynucleosides, polynucleotides, rRNA, ssDNA, dsDNA, mRNA (and olyA), and viral nucleic acids (see, e.g., Barbieri L et al., Biochem J 286: 1-4 (1992); Barbieri L et al., Nature 372: 624 (1994); Ling J et al., FEBS Lett 345: 143-6 (1994); Barbieri L et al., Biochem J 319: 507-13 (1996); Roncuzzi L, Gasperi-Campani A, FEBS Lett 392: 16-20 (1996); Stirpe F et al., FEBS Lett 382: 309-12 (1996); Barbieri L et al., Nucleic Acids Res 25: 518-22 (1997); Wang P, Tumer J et al., Biochem J 319: 507-13 (1996); Roncuzzi L, Gasperi-Campani A, FEBS Lett 392: 16-20 (1996); Stirpe F et al., FEBS Lett 382: 309-12 (1996); Barbieri L et al., Nucleic Acids Res 25: 518-22 (1997); Wang P, Tumer J et al., Biochem J 319: 507-13 (1996). N, Nucleic Acids Res 27:1900-5 (1999); Barbieri L et al., Biochim Biophys Acta 1480:258-66 (2000); Barbieri L et al., J Biochem 128:883-9 (2000); Brigotti M et al., Toxicon 39:341-8 (2001); Brigotti M et al., FASEB J 16:365-72 (2002); Bagga S et al., J Biol Chem 278:4813-20 (2003); Picard D et al., J Biol Chem 280:20069-75 (2005)).Some RIPs have shown antiviral activity and superoxide dismutase activity (Erice A et al., Antimicrob Agents Chemother 37:835-8 (1993); Au T et al., FEBS Lett 471:169-72 (2000); Parikh B, Tumer N, Mini Rev Med Chem 4:523-43 (2004); Sharma N et al., Plant Physiol 134:171-81 (2004)). Shiga toxin catalytic activity has been observed both in vitro and in vivo. Non-limiting examples of Shiga toxin effector activity assays measure various activities, such as protein synthesis inhibitory activity, depurinating activity, cell growth inhibition, cytotoxicity, supercoiled DNA relaxation activity, and nuclease activity.

[0130] As used herein, retention of Shiga toxin effector function refers to the ability to exhibit a level of Shiga toxin functional activity compared to a wild-type Shiga toxin effector polypeptide control (e.g., Shiga toxin A1 fragment) or a CD38 binding protein comprising a wild-type Shiga toxin effector polypeptide (e.g., Shiga toxin A1 fragment) under the same conditions, as measured by a suitable quantitative assay with reproducibility. For ribosome inactivation or ribosome inhibition of Shiga toxin effector function, the retained Shiga toxin effector function exhibits an IC50 of 10,000 pM or less in an in vitro setting, for example, by using assays known to the skilled artisan and / or described herein. For cytotoxic Shiga toxin effector function in a target positive cell killing assay, the retained Shiga toxin effector function exhibits a CD50 of 1,000 nM or less. 50 , depending on the cell type and its expression of appropriate extracellular target biomolecules, as shown, for example, by using assays known to the skilled person and / or described herein.

[0131] For the purposes of the present invention, the term "equivalent" with respect to ribosome inhibition refers to an empirically measured level of ribosome inhibitory activity as measured by an appropriate quantitative assay with reproducibility that is reproducible under identical conditions to within 10% or less of the activity of a reference molecule (e.g., a second CD38 binding protein or a third CD38 binding protein).

[0132] For the purposes of the present invention, the term "equivalent" with respect to cytotoxicity refers to an empirically measured level of cytotoxicity as measured by an appropriate quantitative assay with reproducibility that is reproducible under identical conditions to within 10% or less of the activity of a reference molecule (e.g., a second CD38 binding protein or a third binding protein).

[0133] As used herein, the term "reduced" with respect to cytotoxicity refers to a molecule that exhibits a CD40 inhibitory effect under the same conditions. 50 is the CD exhibited by the reference molecule 50 between 10 and 100 times.

[0134] Inaccurate IC values ​​should not be considered when determining the level of Shiga toxin effector function activity. 50 and CD 50 For some samples, it may not be possible to obtain IC values ​​due to the inability to collect the data points required for accurate curve fitting. 50 or CD 50 For example, in theory, if no more than 50% ribosome inhibition or cell death occurs, respectively, in a concentration series for a given sample, then the IC 50 and CD 50 As described in the analysis of data from illustrative Shiga toxin effector function assays (such as the assays described in the Examples below), data that are insufficient to accurately fit the curves should not be considered representative of actual Shiga toxin effector function.

[0135] Failure to detect Shiga toxin effector function activity may be due to improper expression, polypeptide folding and / or protein stability, rather than lack of cellular entry, subcellular routing and / or enzymatic activity. Assays for Shiga toxin effector function may not require many polypeptides of the present invention to measure a large amount of Shiga toxin effector function activity. If the underlying cause of poor or absent effector function is determined empirically to be related to protein expression or stability, one skilled in the art may be able to compensate for such factors using protein chemistry and molecular engineering techniques known in the art so that functional Shiga toxin effector activity can be restored and measured. As an example, improper cell-based expression may be compensated by using different expression control sequences; and improper polypeptide folding and / or stability may benefit from stabilizing terminal sequences, or compensatory mutations in non-effector regions that stabilize the three-dimensional structure of the molecule.

[0136] Some Shiga toxin effector functions are not easily measured, such as subcellular routing functions. For example, there are no conventional quantitative assays to distinguish whether the cytotoxicity and / or failure to deliver heterologous epitopes of a Shiga toxin effector polypeptide is due to inappropriate subcellular routing, but when tests are available, the Shiga toxin effector polypeptide can be analyzed for any significant level of subcellular routing compared to an appropriate wild-type Shiga toxin effector polypeptide. However, if the Shiga toxin effector polypeptide component of a binding protein of the invention exhibits cytotoxicity comparable or equivalent to that of a wild-type Shiga toxin A subunit construct, then it is inferred that the level of subcellular routing activity is comparable or equivalent to that of the wild-type Shiga toxin A subunit construct, at least under the conditions tested.

[0137] As new assays for individual Shiga toxin functions become available, Shiga toxin effector polypeptides and / or binding proteins comprising Shiga toxin effector polypeptides may be assayed for any level of those Shiga toxin effector functions, such as those whose activity is within 1000-fold or 100-fold or less of the activity of the wild-type Shiga toxin effector polypeptide or which exhibit 3-fold to 30-fold or greater activity compared to a functional knockout Shiga toxin effector polypeptide.

[0138] Adequate subcellular routing can be inferred simply by observing the level of cytotoxic activity of a molecule in a cytotoxic assay, such as one based on T cell epitope presentation or on toxin effector function involving a cytosolic and / or endoplasmic reticulum localized target substrate.

[0139] As used herein, "significant" retention of Shiga toxin effector function means that the level of Shiga toxin functional activity is comparable to that of a wild-type Shiga toxin effector polypeptide control (e.g., a Shiga toxin A1 fragment) as measured by a suitable quantitative assay with reproducibility. For in vitro ribosome inhibition, the IC50 for significant Shiga toxin effector function is 300 pM or less, depending on the ribosome origin used in the assay (e.g., bacterial, archaeal, or eukaryotic (algal, fungal, plant, or animal) origin). This is in contrast to the IC50 of approximately 100,000 pM exhibited by the catalytically impaired SLT-1A1-251 double mutant (Y77S / E167D). 50 This is a significantly greater inhibition than that of the cytotoxicity in target-positive cell killing assays in laboratory cell culture. Significant Shiga toxin effector function demonstrated CD40 activity of 100 nM, 50 nM, 30 nM or less. 50, which depends on the target biomolecule of the binding domain and the cell type, in particular the expression of the cell type and / or cell surface presentation of the appropriate extracellular target biomolecule and / or extracellular epitope targeted by the molecule being evaluated. 50 This is significantly more cytotoxic to appropriate target-positive cell populations compared to 100-10,000 nM (100-10,000 nM, depending on the cell line).

[0140] For the purposes of the present invention and with respect to the Shiga toxin effector function of the molecules of the present invention, the term "reasonable activity" refers to exhibiting at least an intermediate level (e.g., within 11-fold to 1,000-fold) of Shiga toxin effector activity as defined herein relative to a molecule comprising native (or wild-type) Shiga toxin, wherein Shiga toxin effector activity is selected from the group consisting of: internalization efficiency, subcellular routing efficiency to the cytosol, epitope presentation for delivery by target cells, ribosome inhibition, and cytotoxicity. For cytotoxicity, reasonable levels of Shiga toxin effector activity include within 1,000-fold of wild-type Shiga toxin constructs, for example, when a wild-type Shiga toxin construct exhibits a CD of 0.5 nM. 50 When (e.g., a binding protein comprising a wild-type Shiga toxin A1 fragment), it exhibits a CD of 500 nM or less 50 .

[0141] For the purposes of the present invention and with respect to the cytotoxicity of the molecules of the invention, the term "optimal" means that the level of cytotoxicity mediated by the Shiga toxin catalytic domain is within 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold of the cytotoxicity of a molecule comprising a wild-type Shiga toxin A1 fragment (e.g., Shiga toxin A subunit or some truncated variant thereof) and / or native (or wild-type) Shiga toxin.

[0142] It should be noted that even if the cytotoxicity of a Shiga toxin effector polypeptide is reduced relative to a wild-type Shiga toxin A subunit or fragment thereof, in practice, applications using attenuated Shiga toxin effector polypeptides may be equally effective or more effective than using a wild-type Shiga toxin effector polypeptide, as the most potent variants may exhibit undesirable effects that are minimized or reduced in variants with reduced cytotoxic efficacy. Wild-type Shiga toxin is very potent, being able to kill an intoxicated cell after only 1 toxin molecule has reached the cytosol of the intoxicated cell, or perhaps after only 40 toxin molecules have been internalized into the intoxicated cell. Shiga toxin effector polypeptides having even significantly reduced Shiga toxin effector function (such as subcellular routing or cytotoxicity) compared to a wild-type Shiga toxin effector polypeptide may still be sufficiently potent for practical applications (such as applications involving targeted cell killing, cargo molecule delivery, and / or detection of specific cells and their subcellular compartments). Furthermore, some Shiga toxin effector polypeptides with reduced activity may be particularly useful for delivering molecular cargo (eg, additional exogenous material) to certain intracellular locations or subcellular compartments of CD38-positive target cells.

[0143] The term "selective cytotoxicity" with respect to the cytotoxic activity of a molecule refers to the relative level of cytotoxicity between a CD38 target-positive cell population (e.g., a targeted cell type) and a non-targeted bystander cell population (e.g., a CD38 target-negative cell type), which can be expressed as the half-maximal cytotoxic concentration (CD38) of the targeted cell type. 50 ) relative to the CD of non-targeted cell types 50 The ratio of the expression of the target cell to the expression of the target cell can be used to provide a measure of cytotoxic selectivity or an indication of the selectivity of killing of targeted cells relative to non-targeted cells.

[0144] The cell surface presentation and / or density of a given extracellular target biomolecule (or extracellular epitopes of a given target biomolecule) may affect the application for which some binding proteins are most suitable. Differences in the cell surface presentation and / or density of a given target biomolecule between cells may quantitatively and qualitatively alter the efficiency of cellular internalization and / or cytotoxic efficacy of a given binding protein. The cell surface presentation and / or density of a given target biomolecule may vary greatly between target biomolecule-positive cells or even on the same cell at different points in the cell cycle or cell differentiation. The total cell surface presentation of a given target biomolecule and / or some extracellular epitopes of a given target biomolecule on a particular cell or cell population can be determined using methods known to those of skill, such as methods involving fluorescence activated cell sorting (FACS) flow cytometry.

[0145] As used herein, with respect to a polypeptide region or feature within a polypeptide, the term "disruption" and its grammatical variants refer to a change in at least one amino acid within the region or constituting the disrupted feature. Amino acid changes include various mutations, such as deletions, inversions, insertions, or substitutions that alter the amino acid sequence of a polypeptide. Amino acid changes also include chemical changes, such as changing one or more atoms in an amino acid functional group or adding one or more atoms to an amino acid functional group.

[0146] As used herein, "deimmunization" refers to reduced antigenicity and / or immunogenicity potential compared to a reference molecule (e.g., a wild-type peptide region, polypeptide region, or polypeptide) after administration to a subject (e.g., a human subject). This includes reduced overall antigenicity and / or immunogenicity compared to a reference molecule despite the introduction of one or more de novo antigenicity and / or immunogenic epitopes. For some embodiments, "deimmunization" refers to a molecule that, after administration to a mammal, exhibits reduced antigenicity and / or immunogenicity compared to the "parent" molecule from which it is derived (e.g., a wild-type Shiga toxin A1 fragment or a binding protein comprising a wild-type Shiga toxin A1 fragment). In some embodiments, the deimmunized Shiga toxin effector polypeptide can exhibit a relative antigenicity that is reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or more compared to the reference "parent" molecule as measured by the same assay under the same conditions, wherein the assay is, for example, an assay known to a skilled artisan and / or described herein, such as a quantitative ELISA or Western blot analysis. In some embodiments, the deimmunized Shiga toxin effector polypeptide can exhibit a relative immunogenicity that is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 97%, about 99% or more less than that of a reference molecule as measured by the same assay under the same conditions, e.g., an assay known to those of skill and / or described herein, e.g., a quantitative measure of anti-molecule antibodies produced in a mammal at a given time point following parenteral administration of the molecule.

[0147] The relative immunogenicity of illustrative binding proteins is determined using measurements of in vivo antibody responses to the binding proteins following repeated parenteral administration over a period of time.

[0148] For the purposes of the present invention, the phrase "B-cell and / or CD4+ T-cell deimmunization" refers to a molecule that, upon administration to a mammal, has reduced antigenicity and / or immunogenicity potential with respect to B cell antigenicity or immunogenicity and / or CD4+ T cell antigenicity or immunogenicity. For some embodiments, "B cell deimmunization" refers to a molecule that, upon administration to a mammal, exhibits reduced B cell antigenicity and / or immunogenicity compared to the "parent" molecule from which it is derived (e.g., a wild-type Shiga toxin A1 fragment). For some embodiments, "CD4+ T cell deimmunization" refers to a molecule that, upon administration to a mammal, exhibits reduced CD4+ T cell antigenicity and / or immunogenicity compared to the "parent" molecule from which it is derived (e.g., a wild-type Shiga toxin A1 fragment).

[0149] The term "endogenous" with respect to a B cell epitope, CD4+ T cell epitope, B cell epitope region or CD4+ T cell epitope region in a Shiga toxin effector polypeptide refers to an epitope that is present in the wild-type Shiga toxin A subunit.

[0150] For the purposes of the present invention, the phrase "CD8+ T cell hyperimmunization" refers to a molecule having increased antigenicity and / or immunogenic potential with respect to CD8+ T cell antigenicity or immunogenicity when present within nucleated cells in a living subject (e.g., a human subject). Typically, CD8+ T cell immune molecules are capable of cellular internalization into the early endosomal compartment of nucleated cells, either due to inherent characteristics or as a component of the CD38 binding protein of the present invention.

[0151] For the purposes of the present invention, the term "heterologous" is, for example, as opposed to endogenous, meaning having a different origin, e.g., a heterologous Shigella A subunit polypeptide is not naturally found as part of any A subunit of a native Shiga toxin. The fusion proteins of the present invention comprise non-heterologous components, e.g., a Shiga toxin and an anti-CD38 antigen binding domain.

[0152] The term "embedded" and its grammatical variants with respect to the polypeptide component of the binding protein of the present invention refers to the internal replacement of one or more amino acids within a polypeptide region with different amino acids to produce a new polypeptide sequence that shares the same total number of amino acid residues as the starting polypeptide region. Thus, the term "embedded" does not include any external terminal fusions of any additional amino acids, peptides or polypeptide components to the starting polypeptide, nor does it include any additional internal insertions of any additional amino acid residues, but rather only includes the replacement of existing amino acids. Internal replacement can be achieved solely by amino acid residue replacement or by a series of replacements, deletions, insertions and / or inversions. If the insertion of one or more amino acids is used, an equal number of proximal amino acids must be deleted next to the insertion to produce the embedded peptide. This is in contrast to the use of the term "insertion" with respect to the polypeptide component of the binding protein of the present invention, which refers to the insertion of one or more amino acids into the interior of a polypeptide, resulting in a new polypeptide with an increased number of amino acid residues compared to the starting polypeptide.

[0153] The term "insertional" and its grammatical variants with respect to the polypeptide component of the binding protein of the present invention refers to the insertion of one or more amino acids into a polypeptide, resulting in a new polypeptide sequence having an increased number of amino acid residues compared to the starting polypeptide. With respect to the polypeptide component of the binding protein of the present invention, the phrases "partial insertion," "embedded and inserted," and their grammatical variants refer to situations where the length of the resulting polypeptide is increased, but the increase is less than the number of amino acid residues equivalent to the length of the entire inserted polypeptide. Whether the insertion is "pure" or "partial," it includes any of the previously described insertions, even if other polypeptide regions not adjacent to the insertion site within the polypeptide are deleted, resulting in a reduction in the overall length of the final polypeptide, because the final polypeptide still contains an internal insertion of one or more amino acids of the T cell epitope-peptide within the polypeptide region.

[0154] For the purposes of the present invention, the phrase "proximal to the amino terminus" with respect to the position of the Shiga toxin effector polypeptide region of a binding protein of the invention refers to a distance wherein at least one amino acid residue of the Shiga toxin effector polypeptide region is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more (e.g., up to 18-20) amino acid residues of the amino terminus of the binding protein, so long as the binding protein is capable of exhibiting an appropriate level of Shiga toxin effector function activity as described herein (e.g., a certain level of cytotoxic potency). Thus, for some embodiments of the present invention, any amino acid residue fused to the Shiga toxin effector polypeptide at the amino terminus should not reduce any Shiga toxin effector function (e.g., by sterically hindering structures near the amino terminus of the Shiga toxin effector polypeptide region) such that the functional activity of the Shiga toxin effector polypeptide is reduced below the appropriate activity level required herein.

[0155] For the purposes of the present invention, the phrase "closer to the amino terminus" with respect to the position of a Shiga toxin effector polypeptide region within a binding protein of the invention compared to another component (e.g., a cell targeting, binding region, molecular moiety and / or additional exogenous material) refers to the position of at least one amino acid residue at the amino terminus of the Shiga toxin effector polypeptide that is closer to the amino terminus of the linear polypeptide component of the binding protein of the invention than to the other reference component.

[0156] For the purposes of the present invention, the phrase "an active enzymatic domain derived from an A subunit of a member of the Shiga toxin family" refers to the ability to inhibit protein synthesis by catalyzing a ribosome inactivation mechanism. The enzymatic activity of naturally occurring Shiga toxins can be defined by the ability to inhibit protein translation, as determined using assays known to the skilled person, such as in vitro assays involving RNA translation in the absence of living cells or in vivo assays involving RNA translation in living cells. The potency of Shiga toxin enzymatic activity can be assessed directly by observing N-glycosidase activity against ribosomal RNA (rRNA), such as ribosomal nicking assays, and / or indirectly by observing inhibition of ribosome function and / or protein synthesis, using assays known to the skilled person and / or described herein.

[0157] For the purposes of the present invention, the term "Shiga toxin A1 fragment region" refers to a polypeptide region that essentially consists of a Shiga toxin A1 fragment and / or is derived from a Shiga toxin A1 fragment of Shiga toxin.

[0158] For the purposes of the present invention, the terms "terminus," "amino terminus," or "carboxyl terminus" with respect to a binding protein generally refer to the last amino acid residue of a polypeptide chain (e.g., a single, continuous polypeptide chain) of a binding protein. A binding protein may comprise more than one polypeptide or protein, and thus, a binding protein of the invention may comprise multiple amino termini and carboxyl termini. For example, the "amino terminus" of a binding protein may be defined by the first amino acid residue of a polypeptide chain representing the amino terminus of a polypeptide, which is generally characterized by a starting amino acid residue that does not have a peptide bond with any amino acid residue related to the primary amino group of the starting amino acid residue or to the equivalent nitrogen of the starting amino acid residue that is a member of the class of N-alkylated alpha amino acid residues. Similarly, the "carboxyl terminus" of a binding protein may be defined by the last amino acid residue of a polypeptide chain representing the carboxyl terminus of a polypeptide, which is generally characterized by a final amino acid residue that does not have any amino acid residue attached by a peptide bond to the alpha-carbon of its primary carboxyl group.

[0159] For the purposes of the present invention, the terms "terminal," "amino terminus," or "carboxyl terminus" with respect to a polypeptide region refer to the region boundary of that region, regardless of whether additional amino acid residues are connected by peptide bonds outside of that region. In other words, the terminus of a polypeptide region is independent of whether that region is fused to another peptide or polypeptide. For example, a fusion protein comprising two protein regions (e.g., a binding region comprising a peptide or polypeptide and a Shiga toxin effector polypeptide) may have a Shiga toxin effector polypeptide region in which the carboxyl terminus terminates at amino acid residue 251 of the Shiga toxin effector polypeptide region, despite the presence of a peptide bond from residue 251 to an amino acid residue at position 252 representing the beginning of another protein region (e.g., a binding region). In this example, the carboxyl terminus of the Shiga toxin effector polypeptide region refers to residue 251, which is not the terminus of the fusion protein, but rather represents an internal region boundary. Thus, with respect to polypeptide regions, the terms "terminal," "amino terminus," and "carboxyl terminus" are used to refer to the boundary of a polypeptide region, regardless of whether that boundary is a physical terminus or a position embedded within a larger polypeptide chain.

[0160] For the purposes of the present invention, the phrase "the carboxyl-terminal region of the Shiga toxin A1 fragment" refers to a region of a polypeptide derived from a naturally occurring Shiga toxin A1 fragment, which region begins with a hydrophobic residue (e.g., V236 of StxA-A1 and SLT-1A1, and V235 of SLT-2A1), is followed by hydrophobic residues, and terminates with a forfurin cleavage site that is conserved between Shiga toxin A1 fragment polypeptides and terminates at the junction between the A1 fragment and the A2 fragment in the native Shiga toxin A subunit. For the purposes of the present invention, the carboxyl-terminal region of the Shiga toxin A1 fragment includes a peptide region derived from the carboxyl terminus of a Shiga toxin A1 fragment polypeptide, e.g., comprises or consists essentially of the carboxyl terminus of the Shiga toxin A1 fragment. Non-limiting examples of peptide regions derived from the carboxyl terminus of the Shiga toxin A1 fragment include the naturally located amino acid residue sequences from position 236 to position 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, or 251 in Stx1A (SEQ ID NO: 2) or SLT-1A (SEQ ID NO: 1); and from position 235 to position 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, or 250 in SLT-2A (SEQ ID No: 3).

[0161] For the purposes of the present invention, the phrase "adjacent to the carboxyl terminus of the A1 fragment polypeptide" with respect to the linking molecule moiety and / or binding region means within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid residues from the last amino acid residue defining the Shiga toxin A1 fragment polypeptide.

[0162] For the purposes of the present invention, the phrase "sterically covering the carboxyl terminus of the A1 fragment-derived region" includes any molecular moiety (e.g., an immunoglobulin-type binding region) of 4.5 kDa or larger that is linked and / or fused to the amino acid residues in the carboxyl terminus of the A1 fragment-derived region, such as amino acid residues derived from amino acid residues naturally located at any of positions 236 to 251 in Stx1A (SEQ ID NO: 2) or SLT-1A (SEQ ID NO: 1), or 235 to 250 in SLT-2A (SEQ ID No: 3). For the purposes of the present invention, the phrase "sterically covering the carboxyl terminus of the A1 fragment-derived region" also includes any molecular moiety (e.g., an immunoglobulin-type binding region) of 4.5 kDa or larger that is linked and / or fused to the amino acid residues in the carboxyl terminus of the A1 fragment-derived region, such as the amino acid residue carboxyl terminus of the last amino acid in the A1 fragment-derived region and / or the Shiga toxin effector polypeptide. For the purposes of the present invention, the phrase "sterically covering the carboxyl terminus of the A1 fragment-derived region" also includes any molecular moiety (e.g., an immunoglobulin-type binding region) of 4.5 kDa or greater in size that physically prevents cellular recognition of the carboxyl terminus of the A1 fragment-derived region, such as recognition by the ERAD machinery of eukaryotic cells.

[0163] For the purposes of the present invention, a binding region (such as an immunoglobulin-type binding region) comprising a polypeptide comprising at least 40 amino acids and linked (e.g., fused) to the carboxyl terminus of a Shiga toxin effector polypeptide region comprising an A1 fragment-derived region is a portion of a molecule that "sterically covers the carboxyl terminus of the A1 fragment-derived region."

[0164] For the purposes of the present invention, a binding region (such as an immunoglobulin-type binding region) comprising a polypeptide comprising at least 40 amino acids and linked (e.g., fused) to the carboxyl terminus of a Shiga toxin effector polypeptide region comprising an A1 fragment-derived region is the portion of a molecule that "blocks the carboxyl terminus of the A1 fragment-derived region."

[0165] For the purposes of the present invention, the term "A1 fragment of a Shiga toxin family member" refers to the amino-terminal fragment remaining of the Shiga toxin A subunit after proteolysis by furin at the furin cleavage site, which is conserved between Shiga toxin A subunits and is located between the A1 fragment and the A2 fragment in the wild-type Shiga toxin A subunit.

[0166] For the purposes of the present invention, the phrase "furin cleavage motif at the carboxyl terminus of the A1 fragment region" refers to a specific furin cleavage motif that is conserved between Shiga toxin A subunits and bridges the junction between the A1 and A2 fragments in naturally occurring Shiga toxin A subunits.

[0167] For the purposes of the present invention, the phrase "a furin cleavage site proximal to the carboxyl terminus of the A1 fragment region" refers to any identifiable furin cleavage site having amino acid residues within a distance of less than 1, 2, 3, 4, 5, 6, 7, or more amino acid residues from the amino acid residue defining the last amino acid residue in the A1 fragment region or A1 fragment-derived region, including a furin cleavage motif located at the carboxyl terminus of the A1 fragment region or A1 fragment-derived region, such as at a position proximal to the attachment of the A1 fragment-derived region to another component of a molecule, such as a molecular portion of a binding protein of the present invention.

[0168] As used herein, the term "additional CD38-targeted therapeutic agent" refers to an additional therapeutic agent (e.g., molecule) that targets CD38 to produce a therapeutic effect or benefit. Such additional CD38-targeted therapeutic agent is complementary to the binding protein and does not directly compete with the binding protein in its CD38 targeting activity. The additional CD38-targeted therapeutic agent may comprise, consist essentially of, or consist of an anti-CD38 antibody or small molecule inhibitor that interferes with CD38 signaling. For example, the additional CD38-targeted therapeutic agent may comprise, consist essentially of, or consist of an anti-CD38 antibody therapy that binds to an antigenic determinant that does not overlap with the antigenic determinant bound by the binding protein, or that binds to the CD38 molecule in such a way that, upon binding, the additional CD38-targeted therapeutic agent does not prevent the binding of the binding protein of the invention to the CD38 molecule. For example, the additional CD38-targeted therapeutic agent may comprise, consist essentially of, or consist of an anti-CD38 monoclonal antibody therapy (such as daratumumab).

[0169] For purposes of the present invention, the phrase "disrupted furin cleavage motif" refers to (i) a specific furin cleavage motif as described in Section IB herein and (ii) a furin cleavage motif comprising a mutation and / or truncation that can confer reduced furin cleavage to a molecule compared to a reference molecule, e.g., furin cleavage can be reproducibly observed to be reduced by 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or less (including 100% in the absence of cleavage) compared to the furin cleavage of the reference molecule observed in the same assay under the same conditions. The furin cleavage percentage compared to a reference molecule can be expressed as the ratio of cleavage of the molecule of interest:uncleaved material divided by cleavage of the reference molecule:uncleaved material (e.g., see WO 2015 / 191764; WO 2016 / 196344). Non-limiting examples of suitable reference molecules include molecules comprising a wild-type Shiga toxin furin cleavage motif and / or furin cleavage site as described herein.

[0170] For purposes of the present invention, the phrase "resistant to furin cleavage" means that a molecule or a specific polypeptide region thereof can reproducibly exhibit less furin cleavage than: (i) the carboxyl terminus of the Shiga toxin A1 fragment in the wild-type Shiga toxin A subunit or (ii) the carboxyl terminus of a Shiga toxin A1 fragment-derived region of a construct in which the native furin cleavage site naturally located at the junction between the A1 and A2 fragments is not disrupted; as determined by any available means available to the skilled artisan, including by using the methods described herein.

[0171] For the purposes of the present invention, the phrase "an active enzymatic domain derived from the A subunit of a member of the Shiga toxin family" refers to a polypeptide structure that is capable of inhibiting protein synthesis via catalytic inactivation of the ribosome based on Shiga toxin enzymatic activity. The ability of a molecular structure to exhibit protein synthesis inhibitory activity and / or catalytic inactivation of the ribosome can be observed using various assays known to those skilled in the art, such as in vitro assays involving RNA translation assays in the absence of living cells or in vivo assays involving ribosomes in living cells. For example, using assays known to those skilled in the art, the enzymatic activity of a molecule based on Shiga toxin enzymatic activity can be directly assessed by observing N-glycosidase activity against ribosomal RNA (rRNA), such as a ribosomal nicking assay, and / or indirectly assessed by observing inhibition of ribosome function, RNA translation, and / or protein synthesis.

[0172] As used herein, with respect to Shiga toxin effector polypeptides, "combination" describes a Shiga toxin effector polypeptide comprising two or more subregions, wherein each subregion comprises at least one of the following, e.g., (1) disruption of an endogenous epitope or epitope region; (2) a disrupted furin cleavage motif at the carboxyl terminus of the A1 fragment region.

[0173] As used herein, "binding protein" is used interchangeably with "CD38 binding protein" or "CD38 binding molecule" or "CD38 binding fusion protein" with respect to the molecules of the invention. All of the above types of molecules include various "CD38 binding proteins." Generally, proteins of the invention are referred to herein as "CD38 binding fusion proteins," which comprise a CD38 binding domain (also referred to herein as an "anti-CD38 antigen binding domain" (ABD) and an optional linker, as more fully described herein) and a Shiga toxin A subunit effector polypeptide," with an optional linker, as more fully described herein.

[0174] B. Introduction

[0175] The present invention provides a class of binding proteins that bind to CD38 and comprise a Shiga toxin A subunit effector polypeptide (with an optional linker; referred to herein as a "CD38 binding fusion protein"). The CD38 binding fusion proteins of the present invention can be used, for example, (1) as molecules that inhibit the growth of CD38-expressing cells, (2) as cytotoxic molecules that kill CD38-expressing cells, (3) to selectively kill specific CD38-positive cell types among other cells, (4) as non-toxic delivery vehicles that deliver cargo molecules to the interior of CD38-expressing cells, (5) as diagnostic molecules for the diagnosis, prognosis, or characterization of diseases and conditions involving CD38-expressing cells, and (6) as therapeutic molecules for the treatment of various diseases, disorders, and conditions involving CD38-expressing cells, such as some blood cancers, including hematopoietic cancers (e.g., multiple myeloma), and CD38-positive cell growth disorders. In some embodiments, the CD38 binding proteins can be used to treat non-Hodgkin lymphoma (NHL), Burkitt lymphoma (BL), B chronic lymphocytic leukemia (B-CLL), B and T acute lymphoblastic leukemia (ALL), T cell lymphoma (TCL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin lymphoma (HL) and chronic myeloid leukemia (CML) in subjects in need thereof. In some embodiments, the CD38 binding proteins of the present invention can be used to treat relapsed or refractory multiple myeloma in subjects in need thereof. In some embodiments, the CD38 binding proteins of the present invention can be used to treat melanoma in subjects in need thereof.

[0176] Shiga toxin A subunit effector polypeptides provide a robust and powerful scaffold for engineering new binding proteins (see, for example, WO 2014 / 164680, WO 2014 / 164693, WO 2015 / 138435, WO 2015 / 138452, WO 2015 / 113005, WO 2015 / 113007, WO 2015 / 191764, WO 2016 / 196344, WO 2017 / 019623, WO 2018 / 106895 and WO 2018 / 140427). The association of CD38-binding immunoglobulin-derived fragments as cell-targeting moieties with Shiga toxin A subunit effector polypeptides enables the engineering of therapeutic and diagnostic molecules that target CD38-expressing cells.

[0177] C. General Structure of CD38 Binding Fusion Proteins

[0178] The CD38 binding fusion proteins of the present invention may comprise (1) a binding region capable of specifically binding to the extracellular portion of CD38 associated with the cell surface; and (2) a Shiga toxin effector polypeptide region comprising a Shiga toxin A subunit effector polypeptide (referred to herein as a "Shiga toxin effector polypeptide"). In some embodiments, the binding proteins of the present invention comprise two or more identical CD38 binding regions and two or more Shiga toxin effector polypeptide regions, whether identical or different. A non-limiting example of a CD38 binding fusion protein of the present invention is a Shiga toxin effector polypeptide fused to an immunoglobulin-type binding region comprising a single-chain variable fragment that specifically binds to the extracellular portion of CD38 when associated with the cell surface; or a homodimer of the foregoing, as described more fully below.

[0179] In some embodiments, the Shiga toxin A subunit effector polypeptide of the binding protein of the present invention combines structural elements that produce two or more properties in a single molecule, such as the ability to: 1) exhibit reduced antigenicity and / or immunogenicity compared to a variant of the molecule lacking the particular combination of elements, 2) exhibit reduced protease cleavage compared to a variant of the molecule lacking the particular combination of elements, 3) exhibit reduced non-specific toxicity to multicellular organisms compared to a variant of the molecule lacking the particular combination of elements, and / or 4) exhibit potent cytotoxicity.

[0180] In some embodiments, the CD38 binding fusion protein of the present invention is a monomer. In some embodiments, the CD38 binding fusion protein is a homodimer. In some embodiments, the CD38 binding fusion protein is a homodimer comprising two identical polypeptides. In some embodiments, the monomeric CD38 binding fusion protein comprises the sequence of SEQ ID NO: 233, or a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0181] 1.CD38 binding region

[0182] In some embodiments, the CD38-binding proteins of the present invention comprise a binding region (also referred to as a "binding domain" or "antigen binding domain (ABD)") comprising an immunoglobulin-type polypeptide (e.g., VH and VL), wherein the polypeptide is capable of exhibiting specific and high affinity binding to CD38 and / or CD38 on the cell surface of cells such as CD38-expressing cells or CD38-positive cells present in mammals (e.g., humans, non-human primates, mice, etc.).

[0183] The present invention provides multiple CD38 binding domains. Specific CDRs of the CD38 binding domains are described below. As described above, the exact numbering and position of the CDRs may vary between different numbering systems. However, it should be understood that the invention of the heavy chain variable region and / or light chain variable region includes the invention of the CDRs present therein. Thus, the invention of each heavy chain variable region is the invention of a vhCDR (e.g., vhCDR1, vhCDR2, and vhCDR3), and the invention of each light chain variable region is the invention of a vlCDR (e.g., vlCDR1, vlCDR2, and vlCDR3).

[0184] The present invention provides binding proteins comprising novel CD38 binding domains. Such antigen binding domains (ABDs) are capable of binding to human and cynomolgus monkey CD38 proteins. Figure 25A -D depicts four different binding proteins comprising four different anti-CD38 ABDs capable of binding to human and cynomolgus monkey CD38. In some embodiments, the heavy chain variable region and the light chain variable region are arranged, for example, on two different polypeptide chains in a Fab format, and in some embodiments, the heavy chain variable region and the light chain variable region are fused together to form an scFv as generally described herein. Alternatively, as shown in FIG1 and described in more detail below, the CD38 binding domains from two CD38 binding fusion protein monomers that have already dimerized are then associated with each other; that is, a VH from one monomeric polypeptide chain associates with a VL from the other monomeric polypeptide chain, or vice versa, such that two anti-CD38 binding domains are formed in a non-covalently associated homodimer, which also comprises two Shigella components.

[0185] Also included herein are anti-CD38 ABDs having amino acid modifications in one or more CDRs and / or framework regions. As described herein, in some embodiments, a set of 6 CDRs may have 0, 1, 2, 3, 4, 5, or 6 amino acid modifications (where amino acid substitutions are found to be particularly useful). The CDRs can have amino acid modifications (e.g., 1, 2, 3, 4, 5, or 6 amino acid modifications in a set of CDRs (in other words, the CDRs can be modified so long as the total number of changes in the set of 6 CDRs is less than 6 amino acid modifications; for example, there can be one change in vlCDR1, two changes in vhCDR2, no changes in vhCDR3, etc.). In some embodiments, each CDR has no more than a single amino acid substitution. In some embodiments, amino acid substitutions in vhCDR3 are avoided. In some cases, the binding affinity to one or both of human and cynomolgus CD38 can be increased, while in other embodiments, the binding affinity can be decreased. In these embodiments, binding to both human and cynomolgus CD38 is retained. Suitable assays for testing whether an anti-CD38 antigen binding domain contains mutations compared to the VH and VL sequences outlined herein are known in the art, such as Biacore assays or the binding assays outlined in Examples 1, 2, or 3.

[0186] In some embodiments, the anti-CD38 ABDs outlined herein may also have amino acid modifications (again, amino acid substitutions have been found to be particularly useful) in the framework regions of one or both of the variable heavy chain framework region and the variable light chain framework region, as long as the framework (excluding the CDRs) remains at least about 80%, about 85%, or about 90% identical to the human germline sequence. Thus, for example, the same CDRs as described herein can be combined with different framework sequences from human germline sequences, as long as the framework regions remain at least 80%, at least 85%, or at least 90% identical to the human germline sequence.

[0187] In another aspect, the present invention provides a CD38 binding domain comprising a variant of the heavy chain variable region and light chain variable region listed above. The heavy chain variable region can have at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the "VH" sequence herein, and / or contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid changes. The light chain variable region can be at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the "VL" sequences herein, and / or contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid changes. In these embodiments, the invention encompasses these variants, as long as the antigen binding domain still binds to human and cynomolgus monkey CD38. Suitable assays for testing whether an anti-CD38 antigen binding domain contains mutations compared to the VH and VL sequences outlined herein are known in the art, such as Biacore assays and those of Examples 1, 2 or 3.

[0188] In some embodiments, the CD38 binding domain is CD38TM1 ( Figure 25A ), and includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 109, and wherein the light chain variable region has an amino acid sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 113.

[0189] In some embodiments, the CD38 binding domain is CD38TM1 ( Figure 25A), and includes a vhCDR1 comprising SEQ ID NO: 110, a vhCDR2 comprising SEQ ID NO: 111, a vhCDR3 comprising SEQ ID NO: 112, a vlCDR1 comprising SEQ ID NO: 114, a vlCDR2 comprising SEQ ID NO: 115, and a vlCDR3 comprising SEQ ID NO: 116. In some embodiments, one or more of these six CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In embodiments, any single CDR comprises no more than 1 or 2 amino acid substitutions, and the modified anti-CD38 antigen binding domain retains binding to human CD38.

[0190] In some embodiments, the CD38 binding domain of CD38TM1 ( Figure 25A ) having a VH domain with no more than 1, 2, 3, 4 or 5 amino acid changes in SEQ ID NO: 109.

[0191] In some embodiments, the CD38 binding domain of CD38TM1 ( Figure 25A ) having a VL domain with no more than 1, 2, 3, 4 or 5 amino acid changes in SEQ ID NO: 113.

[0192] In some embodiments, the CD38 binding domain of CD38TM1 ( Figure 25A ) having a VH domain with no more than 1, 2, 3, 4 or 5 amino acid changes in SEQ ID NO: 109 and a VL domain with no more than 1, 2, 3, 4 or 5 amino acid changes in SEQ ID NO: 113.

[0193] In some embodiments, the CD38 binding domain is CD38TM1 ( Figure 25A ) and having a VH comprising SEQ ID NO: 109 and a VL comprising SEQ ID NO: 113.

[0194] In some embodiments, the CD38 binding domain is CD38TM2 ( Figure 25B), and includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 117, and wherein the light chain variable region has an amino acid sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 121.

[0195] In some embodiments, the CD38 binding domain is CD38TM2 (Figure 25b) and includes vhCDR1 comprising SEQ ID NO: 118, vhCDR2 comprising SEQ ID NO: 119, vhCDR3 comprising SEQ ID NO: 120, vlCDR1 comprising SEQ ID NO: 122, vlCDR2 comprising SEQ ID NO: 23, and vlCDR3 comprising SEQ ID NO: 124. In some embodiments, one or more of these six CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In some embodiments, a single CDR comprises no more than 1 or 2 amino acid substitutions, and the modified anti-CD38 antigen binding domain retains binding to human and / or cynomolgus monkey CD38.

[0196] In some embodiments, the CD38 binding domain of CD38TM2 ( Figure 25B ) having a VH domain with no more than 1, 2, 3, 4 or 5 amino acid changes in SEQ ID NO: 117.

[0197] In some embodiments, the CD38 binding domain of CD38TM1 ( Figure 25A ) having a VL domain with no more than 1, 2, 3, 4 or 5 amino acid changes in SEQ ID NO: 121.

[0198] In some embodiments, the CD38 binding domain of CD38TM2 ( Figure 25B ) having a VH domain with no more than 1, 2, 3, 4 or 5 amino acid changes in SEQ ID NO: 117 and a VL domain with no more than 1, 2, 3, 4 or 5 amino acid changes in SEQ ID NO: 121.

[0199] In some embodiments, the CD38 binding domain is CD38TM2 ( Figure 25B ) and having a VH comprising SEQ ID NO: 117 and a VL comprising SEQ ID NO: 121.

[0200] In some embodiments, the CD38 binding domain is CD38TM3 ( Figure 25C ), and includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 101, and wherein the light chain variable region has an amino acid sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 105.

[0201] In some embodiments, the CD38 binding domain is CD38TM3 ( Figure 25C ), and includes a vhCDR1 comprising SEQ ID NO: 102, a vhCDR2 comprising SEQ ID NO: 103, a vhCDR3 comprising SEQ ID NO: 104, a vlCDR1 comprising SEQ ID NO: 106, a vlCDR2 comprising SEQ ID NO: 7, and a vlCDR3 comprising SEQ ID NO: 108. In some embodiments, one or more of these six CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In embodiments, a single CDR comprises 1 or 2 amino acid substitutions, and the modified anti-CD38 antigen-binding domain retains binding to human and / or cynomolgus monkey CD38.

[0202] In some embodiments, the CD38 binding domain of CD38TM3 ( Figure 25C ) having a VH domain with no more than 1, 2, 3, 4 or 5 amino acid changes in SEQ ID NO: 101.

[0203] In some embodiments, the CD38 binding domain of CD38TM3 ( Figure 25C ) having a VL domain with no more than 1, 2, 3, 4 or 5 amino acid changes in SEQ ID NO: 105.

[0204] In some embodiments, the CD38 binding domain of CD38TM3 ( Figure 25C ) having a VH domain with no more than 1, 2, 3, 4 or 5 amino acid changes in SEQ ID NO: 101 and a VL domain with no more than 1, 2, 3, 4 or 5 amino acid changes in SEQ ID NO: 105.

[0205] In some embodiments, the CD38 binding domain is CD38TM3 ( Figure 25C ) and having a VH comprising SEQ ID NO: 101 and a VL comprising SEQ ID NO: 105.

[0206] In some embodiments, the CD38 binding domain of the present invention is CD38TM4 ( Figure 25D ), which includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 101, and wherein the light chain variable region has an amino acid sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 125.

[0207] In some embodiments, the CD38 binding domain is CD38TM4 ( Figure 25D ), and includes a vhCDR1 comprising SEQ ID NO: 102, a vhCDR2 comprising SEQ ID NO: 3, a vhCDR3 comprising SEQ ID NO: 104, a vlCDR1 comprising SEQ ID NO: 106, a vlCDR2 comprising SEQ ID NO: 107, and a vlCDR3 comprising SEQ ID NO: 108. In some embodiments, one or more of these six CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In embodiments, a single CDR comprises 1 or 2 amino acid substitutions, and the modified anti-CD38 antigen-binding domain retains binding to human and / or cynomolgus monkey CD38.

[0208] In some embodiments, the CD38 binding domain of CD38TM4 ( Figure 25D) having a VH domain with no more than 1, 2, 3, 4 or 5 amino acid changes in SEQ ID NO: 101.

[0209] In some embodiments, the CD38 binding domain of CD38TM1 ( Figure 25A ) having a VL domain with no more than 1, 2, 3, 4 or 5 amino acid changes in SEQ ID NO: 125.

[0210] In some embodiments, the CD38 binding domain of CD38TM4 ( Figure 25D ) has a VH domain with no more than 1, 2, 3, 4 or 5 amino acid changes in SEQ ID NO: 101 and a VL domain with no more than 1, 2, 3, 4 or 5 amino acid changes in SEQ ID NO: 125.

[0211] In some embodiments, the CD38 binding domain is CD38TM4 ( Figure 25D ) and having a VH comprising SEQ ID NO: 101 and a VL comprising SEQ ID NO: 125.

[0212] In addition to the sequence variants described herein for the heavy and light chain variable regions and / or CDRs of each CD38 binding domain, the framework regions of the heavy and / or light chain variable regions can also be altered. In some embodiments, changes are made in the framework regions that maintain at least 80%, 85%, 90%, or 95% identity to the framework region sequences described in Table 4, while leaving the six CDRs unchanged and maintaining binding to human and / or cynomolgus monkey CD38.

[0213] In some embodiments, changes are made in both the framework regions and the six CDRs while maintaining binding of the CD38 binding domain to human and / or cynomolgus CD38. In these embodiments, the CDRs can have amino acid modifications (e.g., 1, 2, 3, 4, or 5 amino acid modifications in a set of six CDRs, i.e., as long as the total number of changes in the set of six CDRs is less than 6 amino acid modifications, the CDRs can be modified, wherein any combination of CDRs are changed; e.g., one change can be made in vl CDR1, two changes can be made in vh CDR2, no changes can be made in vh CDR3, etc.).

[0214] a. Format of the CD38 binding domain

[0215] As shown in FIG1 and described herein, the CD38 binding domains of the present invention comprise VH and VL domains, which can be located on different polypeptide chains or on a single polypeptide chain. It should be noted that in some embodiments, the VH and VL can form an scFv, where, for example, the CD38 binding fusion protein is a monomer. In some cases, as described herein and shown in FIG1 , the VH and VL are located on a single polypeptide chain, but the linker between them is too short to allow intramolecular association, and thus forms a homodimer comprising two anti-CD38 binding regions and two Shigella components. In other embodiments, the linker between the VH and VL is long enough to allow scFv formation, and thus the protein fusion is a monomer.

[0216] In some embodiments, the composition comprises an scFv comprising a CD38 binding domain as described herein. scFvs bind to human and cynomolgus monkey CD38 and comprise a heavy chain variable region (VH) and a light chain variable region (VL) connected by an scFv linker. In some embodiments, the CD38 binding domain comprises VH-linker-VL from its N- to C-terminus. In some embodiments, the CD38 binding domain comprises VL-linker-VH from its N- to C-terminus.

[0217] 2. Shiga toxin effector polypeptide components

[0218] The binding protein of the present invention comprises at least one Shiga toxin effector polypeptide derived from a Shiga toxin A subunit. A Shiga toxin effector polypeptide is a polypeptide derived from a Shiga toxin A subunit member of the Shiga toxin family that is capable of exhibiting one or more Shiga toxin functions (see, e.g., Cheung M et al., Mol Cancer 9:28 (2010); WO 2014 / 164680, WO 2014 / 164693, WO 2015 / 138435, WO 2015 / 138452, WO 2015 / 113005, WO 2015 / 113007, WO 2015 / 191764, WO 2016 / 196344, WO 2017 / 019623, WO 2018 / 106895 and WO 2018 / 140427). Functions of Shiga toxins include, for example, increased cellular internalization, subcellular routing from endosomal compartments to the cytosol, avoidance of intracellular degradation, catalytic inactivation of ribosomes (preventing protein synthesis), and achieving cytostatic and / or cytotoxic effects.

[0219] The Shiga toxin family of protein toxins consists of various naturally occurring toxins that are structurally and functionally related, such as Shiga toxin, Shigella-like toxin 1, and Shigella-like toxin 2 (Johannes L, W, Nat Rev Microbiol 8:105-16 (2010)). The holotoxin members of the Shiga toxin family contain a targeting domain that selectively binds to specific glycosphingolipids present on the surface of some host cells and an enzymatic domain that can permanently inactivate ribosomes once inside the cell (Johannes L, W, Nat Rev Microbiol 8:105-16 (2010)). Members of the Shiga toxin family share the same overall structure and mechanism of action (Engedal N et al., Microbial Biotech 4:32-46 (2011)). For example, Stx, SLT-1, and SLT-2 show indistinguishable enzymatic activities in cell-free systems (Head S et al., J Biol Chem 266:3617-21 (1991); Tesh V et al., Infect Immun 61:3392-402 (1993); Brigotti M et al., Toxicon 35:1431–1437 (1997)).

[0220] The Shiga toxin family encompasses true Shiga toxin (Stx) isolated from Shigella dysenteriae serotype 1, Shiga-like toxin 1 variants (SLT1 or Stx1 or SLT-1 or Slt-1) isolated from enterohemorrhagic Escherichia coli serotypes, and Shiga-like toxin 2 variants (SLT2 or Stx2 or SLT-2) isolated from enterohemorrhagic Escherichia coli serotypes. SLT1 differs from Stx by only one amino acid residue, and both are referred to as Verocytotoxin or Verotoxin (VT) (O'Brien A, Curr Top Microbiol Immunol 180:65-94 (1992)). Although SLT1 and SLT2 variants are only about 53-60% similar to each other at the primary amino acid sequence level, they share the enzymatic activity and cytotoxicity mechanism common to members of the Shiga toxin family (Johannes L, W, Nat Rev Microbiol 8: 105-16 (2010)). More than 39 different Shiga toxins have been described, such as the defined subtypes Stx1a, Stx1c, Stx1d and Stx2a-g (Scheutz F et al., J Clin Microbiol 50: 2951-63 (2012)). Members of the Shiga toxin family are not naturally restricted to any bacterial species, as Shiga toxin-encoding genes can spread between bacterial species by horizontal gene transfer (Strauch E et al., Infect Immun 69: 7588-95 (2001); Bielaszewska M et al., Appl Environ Micrbiol 73: 3144-50 (2007); Zhaxybayeva O, Doolittle W, Curr Biol 21: R242-6 (2011)). As an example of interspecific transfer, Shiga toxins have been found in A. haemolyticus strains isolated from human subjects (Grotiuz G et al., J Clin Microbiol 44:3838-41 (2006)). Once Shiga toxin encoding polynucleotides enter new subspecies or species, it is speculated that the Shiga toxin amino acid sequence can produce slight sequence variations due to genetic drift and / or selection pressure while still maintaining the cytotoxic mechanism shared by the Shiga toxin family members (see Scheutz F et al., J Clin Microbiol 50:2951-63 (2012)).

[0221] In some embodiments of the CD38 binding proteins of the present invention, as described more fully below, the Shiga toxin A subunit effector polypeptide component comprises a combination of at least the following Shiga toxin effector polypeptide subregions: (1) a deimmunization subregion, and (2) a protease cleavage resistance subregion near the carboxyl terminus of the Shiga toxin A1 fragment region.

[0222] In some embodiments, the CD38 binding protein comprises a Shiga toxin A subunit effector polypeptide comprising the sequence of any one of SEQ ID NOs: 45-69, or a sequence having at least 95%, at least 96%, at least 97%, at least 95%, or at least 99% amino acid sequence identity thereto. In some embodiments, the Shiga toxin A subunit effector polypeptide comprises the sequence of SEQ ID NO: 46.

[0223] a. Shiga toxin components useful in certain embodiments

[0224] As described in the art, there are a variety of Shiga toxin components that can be used in combination with the CD38 binding region of the present invention as outlined below. In particular, the entire contents of embodiments #1 to #11 of U.S. Provisional Application No. 62 / 795,633, filed January 23, 2019, are specifically incorporated herein by reference.

[0225] (i) Wild-type Shiga toxin components

[0226] In some embodiments, wild-type Shiga toxin effector polypeptides such as those described in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 of Table 20 may be used (see, for example, WO 2014 / 164693, WO 2015 / 138435, WO 2015 / 138452, WO 2017 / 019623, the entire contents of which are incorporated herein by reference, particularly sequences and fragments of wild-type sequences, such as those cited in paragraph

[21] of WO 2017 / 019623).

[0227] (ii) Shiga toxin component with a disrupted furin cleavage motif

[0228] In some embodiments, the Shiga toxin effector polypeptide comprises a disrupted furin cleavage motif at the carboxyl terminus of the Shiga toxin A1 fragment region (see, e.g., WO 2015 / 191764, expressly incorporated herein by reference in its entirety, particularly paragraphs [8], [9], and the sequences of the disrupted motif and toxin polypeptides comprising the disrupted motif). A particularly useful embodiment in this regard is SEQ ID NO: 46.

[0229] The Shiga toxin A subunit of the Shiga toxin family members contains a conserved furin cleavage site at the carboxyl terminus of its A1 segment region that is important for Shiga toxin function. A skilled artisan can identify furin cleavage site motifs and furin cleavage sites using standard techniques and / or by using the information herein.

[0230] The Shiga toxin cytotoxicity model is that the intracellular proteolytic processing of the Shiga toxin A subunit by furin in intoxicated cells is crucial for: 1) the release of the Al fragment from the remainder of the Shiga holotoxin, 2) the escape of the Al fragment from the endoplasmic reticulum by exposing a hydrophobic domain in the carboxyl terminus of the Al fragment, and 3) the enzymatic activation of the Al fragment (see Johannes L, et al., 2001). W, Nat Rev Microbiol 8:105-16 (2010). Efficient release of the Shiga toxin A1 fragment from the endoplasmic reticulum of intoxicated cells, the A2 fragment, and the remaining components of the Shiga holotoxin are crucial for efficient intracellular routing to the cytosol, maximum enzyme activity, efficient ribosome inactivation, and achieving optimal cytotoxicity, i.e., comparable to wild-type Shiga toxin (e.g., see WO 2015 / 191764 and references therein).

[0231] During shiga toxin poisoning, the A subunit is proteolytically cleaved by furin at the carboxyl bond of a conserved arginine residue (e.g., the arginine residue at position 251 in StxA and SLT-1A and the arginine residue at position 250 in Stx2A and SLT-2A). Furin cleavage of the Shiga toxin A subunit occurs in endosomes and / or Golgi compartments. Furin is a special serine endoprotease that is expressed by numerous cell types in all human tissues examined and by most animal cells. Furin cleaves polypeptides comprising accessible motifs that are generally concentrated in the minimal binary consensus motif Rx-(R / K / x)-R (SEQ ID NO: 181). The A subunits of the Shiga toxin family members contain a conserved, surface-exposed, extended loop structure (e.g., 242-261 in StxA and SLT-1A, and 241-260 in SLT-2) with a conserved SR / YxxR (SEQ ID NO: 182) motif cleaved by furin. Furin-induced StxA cleavage requires a surface-exposed extended loop structure located at amino acid residues 242-261 in StxA, including features flanked by a minimal furin cleavage motif, RxxR (SEQ ID NO: 183).

[0232] The furin cleavage motif and furin cleavage site in Shiga toxin A subunit and Shiga toxin effector polypeptide can be identified by the skilled person using standard methods and / or by using the information herein. Furin cleaves the minimal consensus motif RxxR (SEQ ID NO: 183) (Schalken J et al., J Clin Invest 80: 1545-9 (1987); Bresnahan P et al., J Cell Biol 111: 2851-9 (1990); Hatsuzawa K et al., J Biol Chem 265: 22075-8 (1990); Wise R et al., Proc Natl Acad Sci USA 87: 9378-82 (1990); Molloy S et al., J Biol Chem 267: 16396-402 (1992)). In accordance with this, many furin inhibitors include peptides containing the motif RxxR (SEQ ID NO: 183). The example of a furin synthetic inhibitor is a molecule comprising the peptide RVKR (SEQ ID NO: 190) (Henrich S et al., Nat Struct Biol 10: 520-6 (2003)). Typically, peptides or proteins comprising a surface accessible dibasic amino acid motif (wherein having two positively charged amino acids, amino acids separated by two amino acid residues) can be predicted to be sensitive to furin cleavage, wherein cleavage occurs at the carboxyl bond of the last basic amino acid in the motif.

[0233] The consensus motif in the substrate cut by furin has been identified as having a certain degree of specificity. The furin cleavage site motif in the region comprising 20 continuous amino acid residues has been described, which can be labeled as P14 to P6 ' (Tian S et al., Int J Mol Sci 12:1060-5 (2011)) using the nomenclature set forth in S Schechter I, Berger, A, Biochem Biophys Res Commun 32:898-902 (1968). According to this nomenclature, the furin cleavage site is at the carboxyl bond of the amino acid residue designated as P1, and the amino acid residue of the furin cleavage motif is numbered as P2, P3, P4 etc. from this reference P1 residue towards amino-terminal. Utilize symbol P2 ', P3 ', P4 ' etc. to number amino acid residues in the motif from P1 reference residue towards carboxyl terminal. Using this nomenclature, the P6 to P2' region delineates the core substrate of the furin cleavage motif bound by the enzymatic domain of furin. The two flanking regions, P14 to P7 and P3' to P6', are typically enriched in polar amino acid residues to increase accessibility to the core furin cleavage site located between them.

[0234] The common motif of furin cleavage site is generally described by the consensus motif RxxR (SEQ ID NO:183) corresponding to P4-P3-P2-P1 (SEQ ID NO:192); wherein " R " represents an arginine residue (see Table 1 above), a dash "-" represents a peptide bond, and lowercase "x" represents any amino acid residue. However, other residues and positions may contribute to further defining the furin cleavage motif. A slightly more sophisticated common motif of furin cleavage site is generally reported as the consensus motif Rx-[K / R]-R (SEQ ID NO:191) (wherein slash " / " represents "or", and separates the alternative amino acid residues of the same position), which corresponds to P4-P3-P2-P1 (SEQ ID NO:192) because it is observed that furin has a strong preference for the cleavage substrates containing this motif.

[0235] The furin cleavage motif of furin is described in the present invention.Except the minimum furin cleavage site RxxR (SEQ ID NO:183), the larger furin cleavage motif with some amino acid residue preference in certain position has been described.By comparing various known furin substrates, some physicochemical properties of the amino acid residues in the furin cleavage site motif of 20 amino acid residues length have been characterized.The P6 of furin cleavage motif has been described the core furin cleavage site to P2 ' zone, and this site interacts physically with the enzymatic domain of furin.Two flanking regions P14 to P7 and P3 ' to P6 ' are normally hydrophilic, are rich in polar amino acid residues, to increase the surface accessibility of the core furin cleavage site positioned between them.

[0236] In some embodiments, the furin protease cleavage motif region of the present invention tends to include amino acid residues with positive charge and / or high isoelectric point from position P5 to P1. Particularly, the P1 position of the furin proteolytic position is usually occupied by arginine, but other positively charged amino acid residues may appear in this position. Positions P2 and P3 tend to be occupied by flexible amino acid residues, and particularly P2 tends to be occupied by arginine, lysine or sometimes by very small flexible amino acid residues such as glycine. The P4 position tends to be occupied by positively charged amino acid residues in the furin substrate. However, if the P4 position is occupied by an aliphatic amino acid residue, the lack of positively charged functional group can be compensated by the positively charged residues at P5 and / or P6 positions. Positions P1 ' and P2 ' are usually occupied by aliphatic and / or hydrophobic amino acid residues, and the P1 ' position is most often occupied by serine.

[0237] The two hydrophilic flanking regions tend to be occupied by polar, hydrophilic amino acid residues with small amino acid functional groups; however, in some validated furin substrates, the flanking regions do not contain any hydrophilic amino acid residues (see Tian S, Biochem Insights 2:9-20 (2009)).

[0238] The 20 amino acid furin cleavage motif and furin cleavage site found in the native Shiga toxin A subunit at the junction between the Shiga toxin A1 and A2 fragments are well characterized in certain Shiga toxins. For example, in StxA (SEQ ID NO: 2) and SLT-1A (SEQ ID NO: 1), the furin cleavage motif is naturally located at L238 to F257, while in SLT-2A (SEQ ID NO: 3), the furin cleavage motif is naturally located at V237 to Q256. Based on amino acid homology, experiments and / or furin cleavage assays described herein, one can identify furin cleavage motifs in other native Shiga toxin A subunits or Shiga toxin effector polypeptides, where the motifs are actual furin cleavage motifs or are predicted to generate the A1 and A2 fragments following furin cleavage of these molecules in eukaryotic cells.

[0239] In some embodiments, the Shiga toxin effector polypeptide comprises (1) a Shiga toxin A1 fragment-derived polypeptide having a carboxyl terminus and (2) a disrupted furin cleavage motif at the carboxyl terminus of the Shiga toxin A1 fragment-derived polypeptide. The carboxyl terminus of the Shiga toxin A1 fragment-derived polypeptide can be identified by a skilled artisan using techniques known in the art, such as using protein sequence alignment software to identify (i) a furin cleavage motif that is conserved with native Shiga toxins, (ii) a surface-exposed extended loop that is conserved with native Shiga toxins, and / or (iii) a stretch of predominantly hydrophobic amino acid residues (i.e., a hydrophobic "patch") that can be recognized by the ERAD system.

[0240] The protease cleavage-resistant Shiga toxin effector polypeptide of the binding protein of the present invention (1) may completely lack any furin cleavage motif at the carboxyl terminus of its Shiga toxin A1 fragment region and / or (2) contain a disrupted furin cleavage motif at the carboxyl terminus of its Shiga toxin A1 fragment region and / or a region derived from the carboxyl terminus of the Shiga toxin A1 fragment. Disruption of the furin cleavage motif includes various changes in the amino acid residues in the furin cleavage motif, such as post-translational modification, change of one or more atoms in the amino acid functional group, addition of one or more atoms to the amino acid functional group, association with a non-protein moiety and / or connection with amino acid residues, peptides, polypeptides to produce, for example, a branched protein structure.

[0241] Using the methods described herein, described in WO 2015 / 191764, and / or known to the skilled artisan, Shiga toxin effector polypeptides that are resistant to protease cleavage can be produced from natural or non-natural Shiga toxin effector polypeptides and / or Shiga toxin A subunit polypeptides, wherein the resulting molecules still retain one or more Shiga toxin A subunit functions.

[0242] For the purposes of the present invention, with respect to a furin cleavage site or furin cleavage motif, the term "disruption" or "disrupted" refers to an alteration (such as a mutation) of a native furin cleavage site and / or furin cleavage motif that results in reduced furin cleavage near the carboxyl terminus of the Shiga toxin A1 fragment region or an identifiable region derived therefrom, compared to furin cleavage of a wild-type Shiga toxin A subunit or a polypeptide derived from a wild-type Shiga toxin A subunit comprising only the wild-type polypeptide sequence. Alterations of amino acid residues in a furin cleavage motif include mutations in the furin cleavage motif, such as deletions, insertions, inversions, substitutions, and / or carboxyl terminal truncations of the furin cleavage motif, and post-translational modifications, such as as a result of glycosylation, albumination, and the like of functional groups that couple or link the molecule to an amino acid residue. In some embodiments, the present invention provides the method for the furin protease cleavage motif to be used for the furin protease cleavage.Because the furin protease cleavage motif comprises about 20 amino acid residues, in theory, the change, modification, sudden change, disappearance, insertion and / or brachymemma of any one or more amino acid residues relating to these 20 positions may cause reducing furin protease cleavage sensitivity (people such as Tian S, Sci Rep 2:261 (2012)).The destruction of furin protease cleavage site and / or furin protease cleavage motif may or may not increase the resistance to cutting of other proteases (for example, common trypsin and extracellular protease in mammalian vascular system).Technician can use technology known in the art to test the influence of given destruction on given protease cleavage sensitivity.

[0243] For purposes of the present invention, a "disrupted furin cleavage motif" is a furin cleavage motif comprising an alteration of one or more amino acid residues derived from a 20 amino acid residue region representing the conserved furin cleavage motif found in native Shiga toxin A subunit at the junction between the Shiga toxin Al fragment and the A2 fragment regions and positioned such that furin cleavage of the Shiga toxin A subunit produces the Al and A2 fragments; wherein the disrupted furin cleavage motif exhibits reduced furin cleavage in an experimentally reproducible manner as compared to a reference molecule comprising the wild-type Shiga toxin Al fragment region fused to a carboxy-terminal polypeptide of a size sufficient to enable monitoring of furin cleavage using appropriate assays known to the skilled artisan and / or described herein.

[0244] The example of the sudden change type that can destroy furin cleavage site and furin cleavage motif is amino acid residue deletion, insertion, brachymemma, inversion and / or displacement, comprises the displacement with non-standard amino acid and / or non-natural amino acid.In addition, furin cleavage site and furin cleavage motif can be destroyed by sudden change, and this sudden change comprises by adding the structure of covalent attachment to amino acid whose modification, this covalent attachment structure shields at least one amino acid in this site or motif, such as due to coupling and / or site-specific albuminization of pegylation, small molecule adjuvant.

[0245] If the furin cleavage motif has been disrupted by mutation and / or the presence of non-natural amino acid residues, some of the disrupted furin cleavage motifs may not be easily identifiable as being associated with any furin cleavage motif; however, the carboxyl terminus of the Shiga toxin A1 fragment-derived region will be identifiable and will define the position of the furin cleavage motif if the furin cleavage motif is not disrupted. For example, a disrupted furin cleavage motif may comprise fewer than 20 amino acid residues of the furin cleavage motif compared to the Shiga toxin A subunit and / or the Shiga toxin A1 fragment due to a truncation of the carboxyl terminus.

[0246] In some embodiments, the Shiga toxin effector polypeptide comprises (1) a Shiga toxin A1 fragment-derived polypeptide having a carboxyl terminus and (2) a disrupted furin cleavage motif at the carboxyl terminus of the Shiga toxin A1 fragment polypeptide region; wherein the Shiga toxin effector polypeptide (and any binding protein comprising it) is more resistant to furin cleavage than a reference molecule (e.g., a wild-type Shiga toxin polypeptide comprising the carboxyl terminus of the A1 fragment and / or a conserved furin cleavage motif between the A1 and A2 fragments). For example, a reduction in furin cleavage of a molecule compared to a reference molecule can be determined by using an in vitro furin cleavage assay as described in WO2015 / 191764, the assay being performed using identical conditions and then quantifying the band density of any fragments resulting from cleavage to quantitatively measure changes in furin cleavage.

[0247] In some embodiments, the Shiga toxin effector polypeptide is more resistant to furin cleavage in vitro and / or in vivo compared to wild-type Shiga toxin A subunit.

[0248] In general, the susceptibility of a binding protein of the invention to protease cleavage is tested by comparing the binding protein of the invention to the same molecule in which the Shiga toxin effector polypeptide rendered resistant to furin cleavage is replaced with a wild-type Shiga toxin effector polypeptide comprising a Shiga toxin A1 fragment. In some embodiments, the CD38 binding fusion protein of the invention comprising a disrupted furin cleavage motif exhibits at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or more reduction in in vitro furin cleavage compared to a reference molecule comprising a wild-type Shiga toxin A1 fragment fused at the carboxyl terminus to a peptide or polypeptide.

[0249] Several furin cleavage motifs have been described to destroy.For example, two conservative arginines in minimum RxxR motif (SEQ ID NO:183) are mutated to alanine to prevent the processing of furin and / or furin sample protease completely (see, for example, Duda A et al., J Virology 78:13865-70 (2004)).Because furin cleavage site motif comprises about 20 amino acid residues, therefore in theory, relate to arbitrary some sudden change in one or more these 20 amino acid residue positions and can eliminate furin cutting or reduce furin cutting efficiency (see, for example, Tian S et al., Sci Rep 2:261 (2012)).

[0250] In some embodiments, the CD38-binding fusion proteins of the present invention comprise a Shiga toxin effector polypeptide derived from at least one A subunit of a member of the Shiga toxin family, wherein the Shiga toxin effector polypeptide comprises a disruption in one or more amino acids of a conserved, highly accessible, protease-sensitive loop derived from the Shiga toxin A subunit. For example, in StxA and SLT-1A, this highly accessible, protease-sensitive loop is naturally located at amino acid residues 242 to 261, while in SLT-2A, this conserved loop is naturally located at amino acid residues 241 to 260. Based on polypeptide sequence homology, one skilled in the art can identify this conserved, highly accessible loop structure in other Shiga toxin A subunits. Certain mutations in amino acid residues within this loop can reduce the accessibility of certain amino acid residues within the loop to proteolytic cleavage, and this may reduce furin cleavage sensitivity.

[0251] In some embodiments, the molecules of the invention comprise a Shiga toxin effector polypeptide comprising a disrupted furin cleavage motif comprising mutations in a conserved surface-exposed protease-sensitive loop in the Shiga toxin A subunit. In some embodiments, the molecules of the invention comprise a Shiga toxin effector polypeptide comprising a disrupted furin cleavage motif comprising mutations in the protease-sensitive loop of the Shiga toxin A subunit that reduce the surface accessibility of certain amino acid residues in the loop, resulting in reduced susceptibility to furin cleavage.

[0252] In some embodiments, the disrupted furin cleavage motif of the Shiga toxin effector polypeptide comprises a disruption in the presence, position, or functional group of one or both of the consensus amino acid residues P1 and P4 (such as the amino acid residues in positions 1 and 4 of the minimal furin cleavage motif R / YxxR (SEQ ID NO: 180). For example, mutating one or both of the two arginine residues in the minimal furin consensus site RxxR (SEQ ID NO: 183) to alanine will disrupt the furin cleavage motif and prevent furin cleavage at that site. Similarly, replacing one or both of the arginine residues in the minimal furin cleavage motif RxxR (SEQ ID NO: 183) with any non-conserved amino acid residue known to those skilled in the art will reduce the furin cleavage sensitivity of the motif. In particular, substitution of an arginine amino acid residue with any non-basic amino acid residue lacking a positive charge (e.g., A, G, P, S, T, D, E, Q, N, C, I, L, M, V, F, W, and Y) will result in a disrupted furin cleavage motif.

[0253] In some embodiments, the disrupted furin cleavage motif of the Shiga toxin effector polypeptide comprises a disruption in the interval between the consensus amino acid residues P4 and P1 (in terms of the number of intervening amino acid residues being other than two), and, thus, changing P4 and / or P1 to a different position and eliminating the P4 and / or P1 designation. For example, deletions within the furin cleavage motif of the minimal furin cleavage site or the core furin cleavage motif will reduce the furin cleavage sensitivity of the furin cleavage motif.

[0254] In some embodiments, the disrupted furin cleavage motif comprises one or more amino acid residue replacements compared to the wild-type Shiga toxin A subunit. In some embodiments, the disrupted furin cleavage motif comprises one or more amino acid residue replacements within the minimal furin cleavage site R / YxxR (SEQ ID NO: 180), such as, for example, for StxA and SLT-1A derived Shiga toxin effector polypeptides, the naturally located amino acid residue R248 is replaced by any non-positively charged amino acid residue and / or R251 is replaced by any non-positively charged amino acid residue; and, for SLT-2A derived Shiga toxin effector polypeptides, the naturally located amino acid residue Y247 is replaced by any non-positively charged amino acid residue and / or R250 is replaced by any non-positively charged amino acid residue.

[0255] In some embodiments, the disrupted furin cleavage motif comprises an undisrupted, minimal furin cleavage site R / YxxR (SEQ ID NO: 180), but comprises disrupted flanking regions, such as amino acid residue replacements in one or more amino acid residues naturally located in, for example, 241-247 and / or 252-259 of the furin cleavage motif flanking regions. In some embodiments, the disrupted furin cleavage motif comprises replacements of one or more amino acid residues located in the P1-P6 region of the furin cleavage motif; mutation of P1' to bulky amino acids, such as R, W, Y, F, and H; and mutation of P2' to polar and hydrophilic amino acid residues; and substitution of one or more amino acid residues located in the P1'-P6' region of the furin cleavage motif to one or more bulky hydrophobic amino acid residues.

[0256] In some embodiments, the disruption of the furin cleavage motif comprises a deletion, insertion, inversion and / or mutation of at least one amino acid residue within the furin cleavage motif. In some embodiments, the protease cleavage resistant Shiga toxin effector polypeptide comprises a disruption of the following amino acid sequence: the amino acid sequence is naturally located at amino acids 249-251 of the A subunit of Shiga-like toxin 1 (SEQ ID NO: 1) or Shiga toxin (SEQ ID NO: 2) or amino acids 247-250 of the A subunit of Shiga-like toxin 2 (SEQ ID NO: 3) or at an equivalent position in a conserved Shiga toxin effector polypeptide and / or a non-natural Shiga toxin effector polypeptide sequence. In some embodiments, the protease cleavage resistant Shiga toxin effector polypeptide comprises a disruption comprising a deletion of at least one amino acid within the furin cleavage motif. In some embodiments, the protease cleavage resistant Shiga toxin effector polypeptide comprises a disruption comprising an insertion of at least one amino acid within the protease cleavage motif region. In some embodiments, the protease cleavage-resistant Shiga toxin effector polypeptide comprises a disruption comprising an amino acid inversion, wherein at least one of the inverted amino acids is within the protease motif region. In some embodiments, the protease cleavage-resistant Shiga toxin effector polypeptide comprises a disruption comprising a mutation, such as an amino acid substitution with a non-standard amino acid or an amino acid with a chemically modified side chain.

[0257] In some embodiments of the CD38-binding fusion proteins of the present invention, the disrupted furin cleavage motif comprises a deletion of nine, ten, eleven or more carboxyl-terminal amino acid residues within the furin cleavage motif. In these embodiments, the disrupted furin cleavage motif will not comprise a furin cleavage site or a minimal furin cleavage motif. In other words, some embodiments lack a furin cleavage site at the carboxyl terminus of the A1 segment region.

[0258] In some embodiments, the disrupted furin cleavage motif comprises amino acid residue deletions and amino acid residue substitutions compared to the wild-type Shiga toxin A subunit. In some embodiments, the disrupted furin cleavage motif comprises one or more amino acid residue deletions and substitutions within the minimal furin cleavage site R / YxxR (SEQ ID NO: 180), such as, for example, for StxA and SLT-1A derived Shiga toxin effector polypeptides, the naturally located amino acid residue R248 is replaced by any non-positively charged amino acid residue and / or R251 is replaced by any non-positively charged amino acid residue; and, for SLT-2A derived Shiga toxin effector polypeptides, the naturally located amino acid residue Y247 is replaced by any non-positively charged amino acid residue and / or R250 is replaced by any non-positively charged amino acid residue.

[0259] In some embodiments, the disrupted furin cleavage motif comprises amino acid residue deletions and amino acid residue substitutions and carboxyl-terminal truncation compared to the wild-type Shiga toxin A subunit. In some embodiments, the disrupted furin cleavage motif comprises one or more amino acid residue deletions and substitutions within the minimal furin cleavage site R / YxxR (SEQ ID NO: 180), such as, for example, for StxA and SLT-1A derived Shiga toxin effector polypeptides, the naturally located amino acid residue R248 is replaced by any non-positively charged amino acid residue and / or R251 is replaced by any non-positively charged amino acid residue; and, for SLT-2A derived Shiga toxin effector polypeptides, the naturally located amino acid residue Y247 is replaced by any non-positively charged amino acid residue and / or R250 is replaced by any non-positively charged amino acid residue.

[0260] In some embodiments, the disrupted furin cleavage motif comprises a minimal furin cleavage site R / YxxR (SEQ ID NO:180) and carboxyl-terminal truncations, e.g., for StxA and SLT-1A derived Shiga toxin effector polypeptides, truncations ending at native amino acid positions 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291 or greater, and, where appropriate, including substitutions of the naturally-positioned amino acids with any non-positively charged amino acid residue. residues R248 and / or R251; and for the SLT-2A derived Shiga toxin effector polypeptide, the truncations terminate at native amino acid positions 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 27 0, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291 or greater and, where appropriate, comprises naturally positioned amino acid residues Y247 and / or R250 substituted with any non-positively charged amino acid residue.

[0261] In some embodiments, the disrupted furin cleavage motif comprises the insertion of one or more amino acid residues compared to the wild-type Shiga toxin A subunit, as long as the inserted amino residues do not create a new furin cleavage site. In some embodiments, the insertion of one or more amino acid residues destroys the natural spacing between arginine residues in the minimal furin cleavage site R / YxxR (SEQ ID NO: 180), such as StxA and SLT-1A derived polypeptides comprising the insertion of one or more amino acid residues at 249 or 250 and thus between R248 and R251; or SLT-2A derived polypeptides comprising the insertion of one or more amino acid residues at 248 or 249 and thus between Y247 and R250.

[0262] In some embodiments, the disrupted furin cleavage motif comprises both an amino acid residue insertion and a carboxyl terminal truncation compared to the wild-type Shiga toxin A subunit. In some embodiments, the disrupted furin cleavage motif comprises both an amino acid residue insertion and an amino acid residue substitution compared to the wild-type Shiga toxin A subunit. In some embodiments, the disrupted furin cleavage motif comprises both an amino acid residue insertion and an amino acid residue deletion compared to the wild-type Shiga toxin A subunit.

[0263] In some embodiments, the disrupted furin cleavage motif comprises an amino acid residue deletion, an amino acid residue insertion, and an amino acid residue substitution compared to a wild-type Shiga toxin A subunit.

[0264] In some embodiments, the disrupted furin cleavage motif comprises amino acid residue deletions, insertions, substitutions, and carboxyl-terminal truncations compared to wild-type Shiga toxin A subunit.

[0265] In some embodiments, the Shiga toxin effector polypeptide comprising a disrupted furin cleavage motif is fused directly to a portion of a molecule comprising amino acids, peptides, and / or polypeptides via a peptide bond, wherein the fusion structure involves a single continuous polypeptide. In these fusion embodiments, the amino acid sequence following the disrupted furin cleavage motif should not create a new furin cleavage site at the fusion junction.

[0266] Any of the above-described protease cleavage-resistant, Shiga toxin effector polypeptide subregions and / or disrupted furin cleavage motifs may be used alone or in combination with each of the individual embodiments of the invention, including the methods of the invention.

[0267] In some embodiments, the Shiga toxin effector polypeptide may comprise a disrupted furin cleavage motif and / or furin cleavage site at the carboxyl terminus of the Shiga toxin A1 fragment-derived region. In some embodiments, the Shiga toxin effector polypeptide does not comprise any known compensatory structures that provide for furin cleavage near the carboxyl terminus of the Shiga toxin A1 fragment-derived region. Non-limiting examples of disrupted furin cleavage motifs and furin cleavage sites suitable for use in the present invention are described in WO 2015 / 191764.

[0268] The destruction of some furin cleavage motifs is indicated in this article by the specific amino acid position of the natural shiga toxin A subunit provided in the reference sequence table, it should be noted that the naturally occurring shiga toxin A subunit includes a precursor form of the signal sequence containing about 22 amino acids in its amino terminal, and the signal sequence is removed to produce a mature shiga toxin A subunit and can be identified by a technician. In addition, the destruction of some furin cleavage motifs comprising sudden change is indicated in this article by following: with reference to the specific amino acid (for example R represents an arginine residue) (for example R251 represents the arginine residue at position 251 from the amino terminal) that is naturally present in a specific position in the natural shiga toxin A subunit, afterwards for the amino acid (for example R251A represents the amino acid replacement of arginine with amino acid residue 251 at alanine from the amino terminal) that replaces this residue in the specific sudden change in question.

[0269] In some embodiments, the Shiga toxin effector polypeptide comprises a disrupted furin cleavage motif at the carboxyl terminus of the Shiga toxin A1 fragment-derived region, and such embodiments are referred to herein as "furin cleavage-resistant" or "protease cleavage-resistant" Shiga toxin effector polypeptides to describe their properties relative to wild-type Shiga toxin A subunit and / or wild-type Shiga toxin A1 fragment fusion protein.

[0270] In some embodiments, the protease cleavage-resistant Shiga toxin effector polypeptide consists essentially of a truncated Shiga toxin A subunit having two or more mutations.

[0271] In some embodiments, the protease cleavage-resistant Shiga toxin effector polypeptide comprises a disrupted furin cleavage motif comprising a replacement of one or two arginine residues in the minimal furin cleavage site consensus motif with an amino acid residue of A, G, or H (relative to a wild-type Shiga toxin polypeptide). In some embodiments, the protease cleavage-resistant Shiga toxin effector polypeptide comprises a disruption comprising an amino acid replacement within the furin cleavage motif region, wherein the replacement occurs at a naturally located amino acid selected from the group consisting of amino acid 247 of SEQ ID NO: 3, amino acid 248 of SEQ ID NO: 1 or SEQ ID NO: 2, amino acid 250 of SEQ ID NO: 3, amino acid 251 of SEQ ID NO: 1 or SEQ ID NO: 2, or an equivalent position in a conserved Shiga toxin effector polypeptide and / or a non-natural Shiga toxin effector polypeptide sequence. In some embodiments, the replacement is for any non-conserved amino acid and occurs at a naturally located amino acid residue position. In some embodiments, the mutation comprises an amino acid replacement selected from the group consisting of R247A, R248A, R250A, R251A, or an equivalent position in a conserved Shiga toxin effector polypeptide and / or a non-native Shiga toxin effector polypeptide sequence.

[0272] In some embodiments, the protease cleavage-resistant Shiga toxin effector polypeptide comprises a disrupted furin cleavage motif comprising a deletion mutation. In some embodiments, the disrupted furin cleavage motif comprises a mutation that is a deletion of the region naturally located at 247-252 in StxA (SEQ ID NO: 2) and SLT-1A (SEQ ID NO: 3) or the region naturally located at 246-251 in SLT-2A (SEQ ID NO: 3); a deletion of the region naturally located at 244-246 in StxA (SEQ ID NO: 2) and SLT-1A (SEQ ID NO: 3) or the region naturally located at 243-245 in SLT-2A (SEQ ID NO: 3); or a deletion of the region naturally located at 253-259 in StxA (SEQ ID NO: 2) and SLT-1A (SEQ ID NO: 3) or the region naturally located at 252-258 in SLT-2A (SEQ ID No: 3).

[0273] In some embodiments, the protease cleavage-resistant Shiga toxin effector polypeptide comprises a disrupted furin cleavage motif comprising a carboxyl-terminal truncation mutation compared to a wild-type Shiga toxin A subunit, the truncation resulting in the deletion of one or more amino acid residues in the furin cleavage motif. In some embodiments, the disrupted furin cleavage motif comprises a carboxy-terminal truncation that deletes one or more amino acid residues within the minimal cleavage site Y / RxxR (SEQ ID NO: 180), such as for StxA and SLT-1A derived Shiga toxin effector polypeptides, the truncation ends at native amino acid residue positions 250, 249, 248, 247, 246, 245, 244, 243, 242, 241, 240 or less; and for SLT-2A derived Shiga toxin effector polypeptides, the truncation ends at native amino acid residue positions 249, 248, 247, 246, 245, 244, 243, 242, 241 or less. Where possible, some embodiments include a disrupted furin cleavage motif comprising a combination of any of the above mutations.

[0274] In some embodiments, the disrupted furin cleavage motif comprises a mutation that is a partial carboxyl-terminal truncation of the furin cleavage motif; however, some CD38-binding fusion proteins of the invention do not comprise a disrupted furin cleavage motif that is a complete carboxyl-terminal truncation of the entire 20 amino acid residue furin cleavage motif. For example, some Shiga toxin effector polypeptides comprise a disrupted furin cleavage motif that comprises a partial carboxyl-terminal truncation of the Shiga toxin A1 fragment region up to native position 240 in StxA (SEQ ID NO: 2) or SLT-1A (SEQ ID NO: 1), but no carboxyl-terminal truncation at position 239 or less. Similarly, some Shiga toxin effector polypeptides comprise a disrupted furin cleavage motif that comprises a partial carboxyl-terminal truncation of the Shiga toxin A1 fragment region up to native position 239 in SLT-2A (SEQ ID NO: 3), but no carboxyl-terminal truncation at position 238 or less. In the largest carboxy-terminal truncation of the furin cleavage-resistant Shiga toxin effector polypeptide, mutations comprising a disrupted furin cleavage motif (positions P14 and P13 of the furin cleavage motif) are still present.

[0275] In some other embodiments, the disrupted furin cleavage motif comprises a mutation that is a complete or partial carboxyl terminal truncation of the furin cleavage motif. For example, certain Shiga toxin effector polypeptides comprise a disrupted furin cleavage motif comprising a complete carboxyl terminal truncation of the Shiga toxin A1 fragment region up to native position 236 in StxA (SEQ ID NO: 2) or SLT-1A (SEQ ID NO: 1) or up to 235 in SLT-2A (SEQ ID NO: 3). For example, certain Shiga toxin effector polypeptides with reduced cytotoxicity comprise a disrupted furin cleavage motif comprising a complete carboxyl terminal truncation of the Shiga toxin A1 fragment region other than native position 240 in StxA (SEQ ID NO: 2) or SLT-1A (SEQ ID NO: 1) or 239 in SLT-2A (SEQ ID NO: 3). Shiga toxin effector polypeptides with reduced cytotoxicity can be used to deliver exogenous materials to cells and / or specific subcellular compartments.

[0276] In some embodiments, the disrupted furin cleavage motif comprises a minimal furin cleavage site R / YxxR (SEQ ID NO:180) and carboxyl-terminal truncations, e.g., for StxA and SLT-1A derived Shiga toxin effector polypeptides, truncations ending at native amino acid residue positions 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291 or greater, and, where appropriate, comprising replacement of the naturally located amino acid residue with any non-positively charged amino acid residue. amino acid residues R248 and / or R251; and for the SLT-2A derived Shiga toxin effector polypeptide, the truncations terminate at native amino acid residue positions 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291 or greater, and, where appropriate, comprises naturally positioned amino acid residues Y247 and / or R250 substituted with any non-positively charged amino acid residue. In some embodiments, the truncated Shiga toxin effector polypeptide comprising a disrupted furin cleavage motif further comprises a furin cleavage motif (amino acid residues at positions P9, P8, and / or P7) to maintain optimal cytotoxicity.

[0277] In some embodiments, the disrupted furin cleavage motif comprises a mutation comprising one or more internal amino acid residue deletions compared to the wild-type Shiga toxin A subunit. In some embodiments, the disrupted furin cleavage motif comprises a mutation comprising one or more amino acid residue deletions within the minimal furin cleavage site R / YxxR (SEQ ID NO: 180). For example, StxA and SLT-1A derived Shiga toxin effector polypeptides comprise internal deletions of naturally located amino acid residues R248 and / or R251, which can be combined with deletions of surrounding residues such as 249, 250, 247, 252, etc.; and SLT-2A derived Shiga toxin effector polypeptides comprise internal deletions of naturally located amino acid residues Y247 and / or R250, which can be combined with deletions of surrounding residues such as 248, 249, 246, 251, etc. In some embodiments, the disrupted furin cleavage motif comprises a deletion mutation of four consecutive amino acid residues that deletes the minimal furin cleavage site R / YxxR (SEQ ID NO: 180), such as StxA and SLT-1A derived Shiga toxin effector polypeptides lacking R248-R251 and SLT-2A derived Shiga toxin effector polypeptides lacking Y247-R250. In some embodiments, the disrupted furin cleavage motif comprises a mutation with one or more amino acid residue deletions in the amino acid residues flanking the core furin cleavage motif, such as deletions of amino acids 244-247 and / or amino acids 252-255 in SLT-1A or StxA. In some embodiments, the disrupted furin cleavage motif comprises a mutation that comprises an internal deletion of the entire surface-exposed protease cleavage-sensitive loop compared to the wild-type Shiga toxin A subunit, such as a deletion of naturally located amino acid residues 241-262 for StxA and SLT-1A derived Shiga toxin effector polypeptides; and a deletion of naturally located amino acid residues 240-261 for SLT-2A derived Shiga toxin effector polypeptides.

[0278] In some embodiments, the disrupted furin cleavage motif comprises a mutation that deletes an internal amino acid residue in the furin cleavage motif and a mutation that truncates the carboxyl terminus compared to the wild-type Shiga toxin A subunit. In some embodiments, the disrupted furin cleavage motif comprises a mutation that deletes an amino acid residue in the minimal furin cleavage site R / YxxR (SEQ ID NO: 180) and a mutation that truncates the carboxyl terminus compared to the wild-type Shiga toxin A subunit. For example, a Shiga toxin effector polypeptide resistant to protease cleavage may comprise a disrupted furin cleavage motif comprising a mutation that deletes amino acid residues 248-249 and / or 250-251 naturally located in a truncated StxA or SLT-1A polypeptide still having amino acid residues 247 and / or 252, or a mutation that deletes amino acid residues 247-248 and / or 249-250 in a truncated SLT-2A still having amino acid residues 246 and / or 251. In some embodiments, the disrupted furin cleavage motif comprises a mutation that makes the minimal furin cleavage site R / YxxR (SEQ ID NO:180), and carboxy-terminal truncations, e.g., for StxA and SLT-1A derived Shiga toxin effector polypeptides, the truncations terminate at native amino acid residue positions 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291 or greater and lacks R248-R251; and for an SLT-2A derived Shiga toxin effector polypeptide, the truncation ends at native amino acid residue position 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291 or greater and lacks Y247-R250.

[0279] In some embodiments, the disrupted furin cleavage motif comprises one or more mutations relative to the wild-type Shiga toxin A subunit, the one or more mutations altering at least one amino acid residue naturally located in the region of amino acids 248-251 of the Shiga toxin A subunit (SEQ ID NOs: 1-2 and 4-6) or amino acids 247-250 of the A subunit of Shiga-like toxin 2 (SEQ ID NOs: 3 and 7-18), or an equivalent region of the Shiga toxin A subunit or a derivative thereof. In some embodiments, the disrupted furin cleavage motif comprises one or more mutations relative to the wild-type Shiga toxin A subunit in the minimal furin cleavage site of the furin cleavage motif. In some embodiments, the minimal furin cleavage site is represented by the consensus amino acid sequence R / YxxR (SEQ ID NO: 180) and / or RxxR (SEQ ID NO: 183).

[0280] (iii) Shiga toxin components with a carboxyl-terminal endoplasmic reticulum retention / recycling signal motif

[0281] In some embodiments, the Shiga toxin effector polypeptide comprises a carboxy-terminal endoplasmic reticulum retention / recycling signal motif of a KDEL family member, such as described in paragraph

[52] of WO2018 / 140427 and paragraphs

[28] ,

[107] to

[113] of WO2015 / 138435, all of which are expressly incorporated by reference.

[0282] (iv) Deimmunized Shiga toxin A subunit effector polypeptide

[0283] In some embodiments, the Shiga toxin effector polypeptide of the binding protein is deimmunized, for example, compared to wild-type Shiga toxin, wild-type Shiga toxin polypeptide, and / or Shiga toxin effector polypeptide comprising only wild-type polypeptide sequence. Shiga toxin effector polypeptides and / or Shiga toxin A subunit polypeptides, whether naturally occurring or not, can be deimmunized by methods described herein, described in WO2015 / 113005, WO2015 / 113007, WO2016 / 196344, and WO2018 / 140427, and / or known to those skilled in the art, wherein the resulting molecule retains one or more Shiga toxin A subunit functions. The deimmunized Shiga toxin effector polypeptide may comprise the destruction of at least one putative endogenous epitope region to reduce the antigenicity and / or immunogenic potential of the polypeptide after administration of the Shiga toxin effector polypeptide to a subject (such as a human subject).

[0284] In some embodiments, the Shiga toxin effector polypeptide comprises a disruption of an endogenous epitope or epitope region, such as a B cell and / or CD4+ T cell epitope. In some embodiments, the Shiga toxin effector polypeptide comprises a disruption of at least one endogenous epitope region described herein, wherein the disruption reduces the antigenicity and / or immunogenic potential of the Shiga toxin effector polypeptide following administration of the polypeptide to a subject, and wherein the Shiga toxin effector polypeptide is capable of exhibiting one or more Shiga toxin A subunit functions, such as a significant level of Shiga toxin cytotoxicity.

[0285] As used herein, the term "disrupted" or "disruption" with respect to an epitope region refers to a deletion of at least one amino acid residue in the epitope region; an inversion of two or more amino acid residues, wherein at least one of the inverted amino acid residues is within the epitope region; at least one amino acid is inserted into the epitope region; and the substitution of at least one amino acid residue in the epitope region. Disruption of the epitope region by mutation includes amino acid substitution with non-standard amino acids and / or non-natural amino acids. Alternatively, the epitope region can be disrupted by mutation, comprising modification of the amino acid by adding a covalently linked chemical structure that masks at least one amino acid in the epitope region, see, for example, pegylation (see Zhang C et al., BioDrugs 26:209-15 (2012), small molecule adjuvants (Flower D, Expert Opin Drug Discov 7:807-17 (2012), and site-specific albuminization (Lim S et al., J Control Release 207-93 (2015)).

[0286] Some epitope regions and disruptions are indicated herein by reference to the specific amino acid positions of the natural Shiga toxin A subunits provided in the sequence table, it being noted that naturally occurring Shiga toxin A subunits may include a precursor form containing a signal sequence of about 22 amino acids at their amino termini, which is removed to produce mature Shiga toxin A subunits and can be identified by a technician. In addition, some epitope region disruptions are indicated herein by reference to specific amino acids naturally present at specific positions within natural Shiga toxin A subunits (e.g., S represents a serine residue) (e.g., S33 represents a serine residue at position 33 from the amino terminus), followed by the amino acid that replaces the residue in the specific mutation in question (e.g., S33I represents an amino acid replacement of serine with isoleucine at amino acid residue 33 from the amino terminus).

[0287] In some embodiments, the deimmunized Shiga toxin effector polypeptide comprises a disruption of at least one epitope region provided herein. In some embodiments, the deimmunized Shiga toxin effector polypeptide comprises a disruption of at least one epitope region described in WO2015 / 113005 or WO2015 / 113007.

[0288] In some embodiments, the deimmunized Shiga toxin effector polypeptide comprises a full-length Shiga toxin A subunit (e.g., SLT-1A (SEQ ID NO: 1), StxA (SEQ ID NO: 2), or SLT-2A (SEQ ID NO: 3)) containing at least one amino acid sequence disruption, wherein the amino acid sequence is selected from the group of naturally located amino acids consisting of: amino acids 1-15 of SEQ ID NO: 1 or SEQ ID NO: 2; amino acids 3-14 of SEQ ID NO: 3; amino acids 26-37 of SEQ ID NO: 3; amino acids 27-37 of SEQ ID NO: 1 or SEQ ID NO: 2; amino acids 39-48 of SEQ ID NO: 1 or SEQ ID NO: 2; amino acids 42-48 of SEQ ID NO: 3; amino acids 53-66 of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; amino acids 94-115 of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; SEQ ID NO: 1 or SEQ ID NO: 2; amino acids 141-153 of SEQ ID NO: 2; amino acids 140-156 of SEQ ID NO: 3; amino acids 179-190 of SEQ ID NO: 1 or SEQ ID NO: 2; amino acids 179-191 of SEQ ID NO: 3; amino acid 204 of SEQ ID NO: 3; amino acid 205 of SEQ ID NO: 1 or SEQ ID NO: 2; amino acids 210-218 of SEQ ID NO: 3; amino acids 240-258 of SEQ ID NO: 3; amino acids 243-257 of SEQ ID NO: 1 or SEQ ID NO: 2; amino acids 254-268 of SEQ ID NO: 1 or SEQ ID NO: 2; amino acids 262-278 of SEQ ID NO: 3; amino acids 281-297 of SEQ ID NO: 3; and amino acids 206 of SEQ ID NO: 1 or SEQ ID NO: 2. Amino acids 285-293 of ID NO: 2, or an equivalent position in a Shiga toxin A subunit polypeptide, a conserved Shiga toxin effector polypeptide subregion, and / or a non-native Shiga toxin effector polypeptide sequence.

[0289] In some embodiments, the Shiga toxin effector polypeptide comprises a truncated Shiga toxin A subunit. The truncation of the Shiga toxin A subunit may result in the deletion of the entire epitope region without affecting the Shiga toxin effector function. The smallest Shiga toxin A subunit fragment that exhibits significant enzymatic activity is a polypeptide consisting of residues 75-247 of StxA (Al-Jaufy A et al., Infect Immun 62:956-60 (1994)). Truncating the carboxyl terminus of SLT-1A, StxA or SLT-2A to amino acids 1–251 can remove two predicted B cell epitope regions, two predicted CD4 positive (CD4+) T cell epitopes and predicted discontinuous B cell epitopes. Truncating the amino terminus of SLT-1A, StxA or SLT-2A to 75-293 removes at least three predicted B cell epitope regions and three predicted CD4+ T cell epitopes. Truncation of both the amino and carboxyl termini of SLT-1A, StxA, or SLT-2A to 75-251 deleted at least five predicted B-cell epitope regions; four putative CD4+ T-cell epitopes; and one predicted discontinuous B-cell epitope.

[0290] In some embodiments, the Shiga toxin effector polypeptides of the present invention comprise a full-length or truncated Shiga toxin A subunit having at least one mutation, such as a deletion, insertion, inversion, or substitution in a B or T cell epitope region. In some embodiments, the polypeptide comprises a disruption comprising a deletion of at least one amino acid within the epitope region. In some embodiments, the polypeptide comprises a disruption comprising an insertion of at least one amino acid within the epitope region. In some embodiments, the polypeptide comprises a disruption comprising an inversion of an amino acid, wherein at least one inverted amino acid is within the epitope region. In some embodiments, the polypeptide comprises a disruption comprising a mutation, such as a substitution of an amino acid with a non-standard amino acid or an amino acid with a chemically modified side chain.

[0291] In some embodiments, the Shiga toxin effector polypeptide of the invention comprises or consists essentially of a full-length or truncated Shiga toxin A subunit having one or more mutations compared to the native sequence, which subunit comprises at least one amino acid substitution selected from the group consisting of: A, G, V, L, I, P, C, M, F, S, D, N, Q, H, and K. In some embodiments, the polypeptide comprises a full-length or truncated Shiga toxin A subunit having a single mutation compared to the native sequence, wherein the substitution is selected from the group consisting of: D to A, D to G, D to V, D to L, D to I, D to F, D to S, D to Q, E to A, E to G, E to V, E to L, E to I, E to F, E to S, E to Q, E to N, E to D, E to M, E to R, G to A, H to A, H to G, H to V, H to L, H to I, H to F, H to M, K to A, K to G, K to V, K to L, K to I, K to M, K to H, L to is replaced by A, L is replaced by G, N is replaced by A, N is replaced by G, N is replaced by V, N is replaced by L, N is replaced by I, N is replaced by F, P is replaced by A, P is replaced by G, P is replaced by F, R is replaced by A, R is replaced by G, R is replaced by V, R is replaced by L, R is replaced by I, R is replaced by F, R is replaced by M, R is replaced by Q, R is replaced by S, R is replaced by K, R is replaced by H, S is replaced by A, S is replaced by G, S is replaced by V, S is replaced by L, S is replaced by I, S is replaced by F, S is replaced by M, T is replaced by A, T is replaced by G, T is replaced by V, T is replaced by L, T is replaced by I, T is replaced by F, T is replaced by M, T is replaced by S, Y is replaced by A, Y is replaced by G, Y is replaced by V, Y is replaced by L, Y is replaced by I, Y is replaced by F, and Y is replaced by M.

[0292] In some embodiments, the Shiga toxin effector polypeptide of the invention comprises, or consists essentially of, a full-length or truncated Shiga toxin A subunit having one or more mutations compared to the native amino acid residue sequence, the subunit comprising at least one amino acid substitution within the immunogenic residue and / or epitope region, wherein at least one substitution occurs in a naturally located amino acid group selected from the group consisting of: 1 of SEQ ID NO: 1 or SEQ ID NO: 2; 4 of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; 8 of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; 9 of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; 11 of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; 33 of SEQ ID NO: 1 or SEQ ID NO: 2; 43 of SEQ ID NO: 1 or SEQ ID NO: 2; 44 of SEQ ID NO: 1 or SEQ ID NO: 2; 45 of SEQ ID NO: 1 or SEQ ID NO: 2; 46 of SEQ ID NO: 1 or SEQ ID NO: 2; NO: 47 of SEQ ID NO: 1 or SEQ ID NO: 2; 48 of SEQ ID NO: 1 or SEQ ID NO: 3; 49 of SEQ ID NO: 1 or SEQ ID NO: 2; 50 of SEQ ID NO: 1 or SEQ ID NO: 2; 51 of SEQ ID NO: 1 or SEQ ID NO: 2; 53 of SEQ ID NO: 1 or SEQ ID NO: 2; 54 of SEQ ID NO: 1 or SEQ ID NO: 2; 55 of SEQ ID NO: 1 or SEQ ID NO: 2; 56 of SEQ ID NO: 1 or SEQ ID NO: 2; 57 of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; 58 of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; 59 of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; 60 of SEQ ID NO: 1 or SEQ ID NO: 2; 61 of SEQ ID NO: 1 or SEQ ID NO: 2; 62 of SEQ ID NO: 1 or SEQ ID NO: 2; 84 of SEQ ID NO: 1 or SEQ ID NO: 2; 88 of SEQ ID NO: 1 or SEQ ID NO: 2; 94 of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3;SEQ ID NO: 1 or SEQ ID NO: 96; SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3; SEQ ID NO: 104; SEQ ID NO: 105; SEQ ID NO: 1 or SEQ ID NO: 2; SEQ ID NO: 107; SEQ ID NO: 1 or SEQ ID NO: 2; SEQ ID NO: 108; SEQ ID NO: 109; SEQ ID NO: 110; SEQ ID NO: 1 or SEQ ID NO: 2; SEQ ID NO: 111; SEQ ID NO: 112; SEQ ID NO: 141; SEQ ID NO: 1 or SEQ ID NO: 2; SEQ ID NO: 147; SEQ ID NO: 1 or SEQ ID NO: 2; SEQ ID NO: 154; SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; SEQ ID NO: 179; SEQ ID NO: 1 or SEQ ID NO: 2 NO:2; 180 of SEQ ID NO:1 or SEQ ID NO:2; 181 of SEQ ID NO:1 or SEQ ID NO:2; 183 of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3; 184 of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3; 185 of SEQ ID NO:1 or SEQ ID NO:2; 186 of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3; 187 of SEQ ID NO:1 or SEQ ID NO:2; 188 of SEQ ID NO:1 or SEQ ID NO:2; 189 of SEQ ID NO:1 or SEQ ID NO:2; 198 of SEQ ID NO:2; 204 of SEQ ID NO:1 or SEQ ID NO:2; 205 of SEQ ID NO:1 or SEQ ID NO:2; 241 of SEQ ID NO:3; 242 of SEQ ID NO:1 or SEQ ID NO:2; 247 of SEQ ID NO:1 or SEQ ID NO:2; 247 of SEQ ID NO: 3; 248 of SEQ ID NO: 1 or SEQ ID NO: 2; 250 of SEQ ID NO: 3; 251 of SEQ ID NO: 1 or SEQ ID NO: 2; 264 of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3;265 of SEQ ID NO:1 or SEQ ID NO:2; and 286 of SEQ ID NO:1 or SEQ ID NO:2.

[0293] In some embodiments, the Shiga toxin effector polypeptides of the invention comprise or consist essentially of a full-length or truncated Shiga toxin A subunit having at least one substitution within an immunogenic residue and / or epitope region, wherein at least one amino acid substitution is with a non-conservative amino acid relative to a native amino acid positioned at one of the following native positions (e.g., see Table 3 below): 1 of SEQ ID NO: 1 or SEQ ID NO: 2; 4 of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; 8 of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; 9 of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; 11 of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; 33 of SEQ ID NO: 1 or SEQ ID NO: 2; 43 of SEQ ID NO: 1 or SEQ ID NO: 2; 44 of SEQ ID NO: 1 or SEQ ID NO: 2; 45 of SEQ ID NO: 1 or SEQ ID NO: 2; NO:2; 46 of SEQ ID NO:1 or SEQ ID NO:2; 47 of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3; 48 of SEQ ID NO:1 or SEQ ID NO:2; 49 of SEQ ID NO:1 or SEQ ID NO:2; 50 of SEQ ID NO:1 or SEQ ID NO:2; 51 of SEQ ID NO:1 or SEQ ID NO:2; 53 of SEQ ID NO:1 or SEQ ID NO:2; 54 of SEQ ID NO:1 or SEQ ID NO:2; 55 of SEQ ID NO:1 or SEQ ID NO:2; 56 of SEQ ID NO:1 or SEQ ID NO:2; 57 of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3; 58 of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3; 59 of SEQ ID NO:1 or SEQ ID NO:2; 60 of SEQ ID NO:1 or SEQ ID NO:2; 61 of SEQ ID NO:1 or SEQ ID NO:2; 62 of SEQ ID NO: 2; 84 of SEQ ID NO: 1 or SEQ ID NO: 2; 88 of SEQ ID NO: 1 or SEQ ID NO: 2; 94 of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3;SEQ ID NO: 1 or SEQ ID NO: 96; SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3; SEQ ID NO: 104; SEQ ID NO: 105; SEQ ID NO: 1 or SEQ ID NO: 2; SEQ ID NO: 107; SEQ ID NO: 1 or SEQ ID NO: 2; SEQ ID NO: 108; SEQ ID NO: 109; SEQ ID NO: 1 or SEQ ID NO: 2; SEQ ID NO: 110; SEQ ID NO: 1 or SEQ ID NO: 2; SEQ ID NO: 111; SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; SEQ ID NO: 141; SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; SEQ ID NO: 147; SEQ ID NO: 1 or SEQ ID NO: 2; SEQ ID NO: 154; SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; SEQ ID NO: 179; SEQ ID NO: 1 or SEQ ID NO: 2 NO:2; 180 of SEQ ID NO:1 or SEQ ID NO:2; 181 of SEQ ID NO:1 or SEQ ID NO:2; 183 of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3; 184 of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3; 185 of SEQ ID NO:1 or SEQ ID NO:2; 186 of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3; 187 of SEQ ID NO:1 or SEQ ID NO:2; 188 of SEQ ID NO:1 or SEQ ID NO:2; 189 of SEQ ID NO:1 or SEQ ID NO:2; 198 of SEQ ID NO:2; 204 of SEQ ID NO:1 or SEQ ID NO:2; 205 of SEQ ID NO:1 or SEQ ID NO:2; 241 of SEQ ID NO:3; 242 of SEQ ID NO:1 or SEQ ID NO:2; 247 of SEQ ID NO:1 or SEQ ID NO:2; 247 of SEQ ID NO: 3; 248 of SEQ ID NO: 1 or SEQ ID NO: 2; 250 of SEQ ID NO: 3; 251 of SEQ ID NO: 1 or SEQ ID NO: 2; 264 of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3;265 of SEQ ID NO:1 or SEQ ID NO:2; and 286 of SEQ ID NO:1 or SEQ ID NO:2.

[0294] In some embodiments, the Shiga toxin effector polypeptide of the invention comprises or consists essentially of a full-length or truncated Shiga toxin A subunit having at least one amino acid substitution selected from the group consisting of: K1 substituted with A, G, V, L, I, F, M, or H; T4 substituted with A, G, V, L, I, F, M, or S; D6 substituted with A, G, V, L, I, F, S, Q, or R; S8 substituted with A, G, V, I, L, F, or M; T8 substituted with A, G, V, I, L, F, or M; T9 substituted with A, G, V, I, L, F, M, or S; S9 substituted with A, G, V, L, I, F, or M; K11 substituted with A, G, V, L, I, F, M, or H; T12 substituted with A, G, V, I, L, F , M, S or K; S12 is substituted with A, G, V, I, L, F or M; S33 is substituted with A, G, V, L, I, F, M or C; S43 is substituted with A, G, V, L, I, F or M; G44 is substituted with A or L; S45 is substituted with A, G, V, L, I, F or M; T45 is substituted with A, G, V, L, I, F or M; G46 is substituted with A or P; D47 is substituted with A, G, V, L, I, F, S, M or Q; N48 is substituted with A, G, V, L, M or F; L49 is substituted with A, V, C or G; Y49 is substituted with A, G, V, L, I, F, M or T; F50 is substituted with A, G, V, L, I or T; A51 is substituted with V; D53 is substituted with A, G, V, L, I, F , S or Q; V54 is substituted with A, G, I or L; R55 is substituted with A, G, V, L, I, F, M, Q, S, K or H; G56 is substituted with A or P; I57 is substituted with A, G, V or M; L57 is substituted with A, V, C, G, M or F; D58 is substituted with A, G, V, L, I, F, S or Q; P59 is substituted with A, G or F; E60 is substituted with A, G, V, L, I, F, S, Q, N, D, M, T or R; E61 is substituted with A, G, V, L, I, F, S, Q, N, D, M or R; G62 is substituted with A; R84 is substituted with A, G, V, L, I, F, M, Q, S, K or H; V88 is substituted with A or G; I88 is substituted with A, V, C or G; D94 is substituted with is A, G, V, L, I, F, S or Q; S96 is substituted with A, G, V, I, L, F or M; T104 is substituted with A, G, V, L, I, F, M or N; A105 is substituted with L; T107 is substituted with A, G, V, L, I, F, M or P; S107 is substituted with A, G, V, L, I, F, M or P; L108 is substituted with A, V, C or G; S109 is substituted with A, G, V, I, L, F or M; T109 is substituted with A, G, V, I, L, F, M or S; G110 is substituted with A; S112 is substituted with A, G, V, L, I, F or M; D111 is substituted with A, G, V, L, I, F, S, Q or T; S112 is substituted with A, G, V, L, I, F or M;D141 is substituted with A, G, V, L, I, F, S or Q; G147 is substituted with A; V154 is substituted with A or G; R179 is substituted with A, G, V, L, I, F, M, Q, S, K or H; T180 is substituted with A, G, V, L, I, F, M or S; T181 is substituted with A, G, V, L, I, F, M or S; D183 is substituted with A, G, V, L, I, F, S or Q; D184 is substituted with is A, G, V, L, I, F, S or Q; L185 is replaced by A, G, V or C; S186 is replaced by A, G, V, I, L, F or M; G187 is replaced by A; R188 is replaced by A, G, V, L, I, F, M, Q, S, K or H; S189 is replaced by A, G, V, I, L, F and M; D197 is replaced by A, G, V, L, I, F, S or Q; D198 is replaced by A, G, V, L, I, F, S or Q; R204 is replaced by A, G, V, L, I, F, M, Q, S, K or H; R205 is replaced by A, G, V, L, I, F, M, Q, S, K or H; C242 is replaced by A, G or V; R247 is replaced by A, G, V, L, I, F, M, Q, S, K or H; S247 is replaced by A, G, V, I, L, F or M; Y247 is replaced by A, G, V, L, I , F or M; R248 is substituted with A, G, V, L, I, F, M, Q, S, K or H; R250 is substituted with A, G, V, L, I, F, M, Q, S, K or H; R251 is substituted with A, G, V, L, I, F, M, Q, S, K or H; D264 is substituted with A, G, V, L, I, F, S or Q; G264 is substituted with A; and T286 is substituted with A, G, V, L, I, F, M or S. ;

[0295] In some embodiments, the Shiga toxin effector polypeptide of the invention comprises or consists essentially of a full-length or truncated Shiga toxin A subunit having at least one of the following amino acid substitutions: K1A, K1M, T4I, D6R, S8I, T8V, T9I, S9I, K11A, K11H, T12K, S33I, S33C, S43N, G44L, S45V, S45I, T45 V,T45I,G46P,D47M,D47G,N48V,N48F,L49A,F50T,A51V,D53A,D53N,D53G,V54L,V54I, R55A,R55V,R55L,G56P,I57F,I57M,D58A,D58V,D58F,P59A,P59F,E60I,E60T,E60R,E61 A,E61V,E61L,G62A,R84A,V88A,D94A,S96I,T104N,A105L,T107P,L108M,S109V,T109V ,G110A,D111T,S112V,D141A,G147A,V154A,R179A,T180G,T181I,D183A,D183G,D184A, D184A, D184F, L185V, L185D, S186A, S186F, G187A, G187T, R188A, R188L, S189A, D198A, R204A, R205A, C242S, S247I, Y247A, R248A, R250A, R251A or D264A, G264A, T286A and / or T286I. These epitope-disrupting substitutions can be combined to form deimmunized Shiga toxin effector polypeptides having multiple substitutions per epitope region and / or multiple epitope regions disrupted while still retaining Shiga toxin effector function. For example, where possible, the naturally located K1A, K1M, T4I, D6R, S8I, T8V, T9I, S9I, K11A, K11H, T12K, S33I, S33C, S43N, G44L, S45V, S45I, T45V, T45I, G46P, D47M, D47G, N48V, N48F, L49A, F50T, A51V, D53A, D53N, D53G, V54L, V54I, R55 A,R55V,R55L,G56P,I57F,I57M,D58A,D58V,D58F,P59A,P59F,E60I,E60T,E60R,E61A,E61V,E61L,G62A ,R84A,V88A,D94A,S96I,T104N,A105L,T107P,L108M,S109V,T109V,G110A,D111T,S112V,D141A,G147A,V154A, R179A, T180G, T181I, D183A, D183G, D184A, D184A, D184F, L185V, L185D, S186A, S186F, G187A, G187T, R188A, R188L, S189A, D198A, R204A, R205A, C242S, S247I, Y247A, R248A, R250A, R251A or D264A, G264A, T286A and / or T286 The replacement of I with naturally located residues K1A, K1M, T4I, D6R, S8I, T8V, T9I, S9I, K11A, K11H, T12K, S33I, S33C, S43N, G44L, S45V, S45I, T45V, T45I, G46P, D47M, D47G, N48V, N48F, L49A, F50T, A51V, D53A, D53N, D53G, V54L, V54I, R55A, R55V, R55L, G56P, I5 7F,I57M,D58A,D58V,D58F,P59A,P59F,E60I,E60T,E60R,E61A,E61V,E61L,G62A,R84A,V88A,D94A,S96I,T104N ,A105L,T107P,L108M,S109V,T109V,G110A,D111T,S112V,D141A,G147A,V154A,R179A,T180G,T181I,D183A,D1 83G, D184A, D184A, D184F, L185V, L185D, S186A, S186F, G187A, G187T, R188A, R188L, S189A, D198A, R204A, R205A, C242S, S247I, Y247A, R248A, R250A, R251A or D264A, G264A, T286A and / or T286I are combined to generate the deimmunized Shiga toxin effector polypeptides of the present invention.

[0296] Any of the deimmunized Shiga toxin effector polypeptide subregions and / or epitope disrupting mutations described herein may be used alone or in combination, and further in combination with each individual embodiment of the CD38 binding proteins of the invention, including their use in the methods of the invention.

[0297] In some embodiments, the deimmunized Shiga toxin effector polypeptide of the CD38 binding protein of the present invention can consist essentially of a truncated Shiga toxin A subunit having two or more mutations. Truncation of the Shiga toxin A subunit can result in the deletion of an entire epitope and / or epitope region, a B cell epitope, a CD4+ T cell epitope, and / or a furin cleavage site without affecting Shiga toxin effector function, such as catalytic activity and cytotoxicity. Truncation of the carboxyl terminus of SLT-1A, StxA, or SLT-2A to amino acids 1–251 removes two predicted B cell epitope regions, two predicted CD4 positive (CD4+) T cell epitopes, and a predicted discontinuous B cell epitope. Truncation of the amino terminus of SLT-1A, StxA, or SLT-2A to amino acids 75–293 removes at least three predicted B cell epitope regions and three predicted CD4+ T cell epitopes. Truncation of both the amino and carboxyl termini of SLT-1A, StxA, or SLT-2A to 75-251 deleted at least five predicted B-cell epitope regions; four putative CD4+ T-cell epitopes; and one predicted discontinuous B-cell epitope.

[0298] In some embodiments, the deimmunized Shiga toxin effector polypeptide may comprise or consist essentially of a full-length or truncated Shiga toxin A subunit having at least one mutation (relative to the wild-type Shiga toxin polypeptide) in a provided endogenous B cell and / or CD4+ T cell epitope region, such as a deletion, insertion, inversion, or substitution. In some embodiments, the Shiga toxin effector polypeptide comprises a disruption comprising a mutation (relative to the wild-type Shiga toxin polypeptide) comprising a deletion of at least one amino acid residue within the endogenous B cell and / or CD4+ T cell epitope region. In some embodiments, the Shiga toxin effector polypeptide comprises a disruption, wherein the disruption comprises an insertion of at least one amino acid residue within the endogenous B cell and / or CD4+ T cell epitope region. In some embodiments, the Shiga toxin effector polypeptide comprises a disruption comprising an amino acid residue inversion, wherein at least one inverted amino acid residue is within the endogenous B cell and / or CD4+ T cell epitope region. In some embodiments, the Shiga toxin effector polypeptide comprises a disruption comprising a mutation (relative to a wild-type Shiga toxin polypeptide), such as an amino acid substitution, an amino acid substitution with a non-standard amino acid, and / or an amino acid residue with a chemically modified side chain. Non-limiting examples of deimmunized Shiga toxin effector subregions suitable for use as described herein are described in WO2015 / 113005, WO2015 / 113007, WO2015 / 191764, WO2016 / 196344, and WO / 187204.

[0299] In other embodiments, the deimmunized Shiga toxin effector polypeptide comprises a truncated Shiga toxin A subunit that is shorter than the full-length Shiga toxin A subunit, wherein at least one amino acid residue is disrupted in the region of a naturally localized B cell and / or CD4+ T cell epitope.

[0300] To generate deimmunized Shiga toxin effector polypeptides, in principle, the epitope can be destroyed by modifying any amino acid residue in the epitope region provided in various ways, such as deletions, insertions, inversions, rearrangements, substitutions, and chemical modifications of side chains relative to the wild-type Shiga toxin polypeptide. However, modifying certain amino acid residues and using certain amino acid modifications are more likely to successfully reduce antigenicity and / or immunogenicity while maintaining a certain level of Shiga toxin effector function. For example, terminal truncations and internal amino acid substitutions are preferred because these types of modifications maintain the overall spacing of amino acid residues in the Shiga toxin effector polypeptide and are therefore more likely to maintain the structure and function of the Shiga toxin effector polypeptide.

[0301] In some embodiments of the invention, the deimmunized Shiga toxin effector polypeptide comprises or consists essentially of amino acids 75 to 251 of SLT-1A (SEQ ID NO: 1), StxA (SEQ ID NO: 2) and / or SLT-2A (SEQ ID NO: 3), wherein at least one amino acid residue is disrupted in the naturally located epitope region. In some other embodiments, the deimmunized Shiga toxin effector polypeptide comprises or consists essentially of amino acids 1 to 241 of SLT-1A (SEQ ID NO: 1), StxA (SEQ ID NO: 2) and / or SLT-2A (SEQ ID NO: 3), wherein at least one amino acid residue is disrupted in the naturally located epitope region. An embodiment is a deimmunized Shiga toxin effector polypeptide comprising or consists essentially of amino acids 1 to 251 of SLT-1A (SEQ ID NO: 1), StxA (SEQ ID NO: 2) and / or SLT-2A (SEQ ID NO: 3), wherein at least one amino acid residue is disrupted in the naturally located epitope region provided. An embodiment is a Shiga toxin effector polypeptide comprising amino acids 1 to 261 of SLT-1A (SEQ ID NO: 1), StxA (SEQ ID NO: 2) and / or SLT-2A (SEQ ID NO: 3), wherein at least one amino acid residue is disrupted in a naturally located epitope region.

[0302] There are many different internal amino acid substitutions that can be used to generate deimmunized Shiga toxin effector polypeptides of the present invention. Of the possible replacement amino acids used within the epitope region, the following replacement amino acid residues are predicted to be most likely to reduce the antigenicity and / or immunogenicity of the epitope: G, D, E, S, T, R, K, and H. With the exception of glycine, these amino acid residues can all be classified as polar and / or charged residues. Of the possible replacement amino acids, the following amino acids A, G, V, L, I, P, C, M, F, S, D, N, Q, H, and K are predicted to be most likely to reduce antigenicity and / or immunogenicity while retaining a significant level of Shiga toxin effector function, depending on the amino acid replaced. Generally, substitutions should change polar and / or charged amino acid residues to non-polar and uncharged residues (see, e.g., WO2015 / 113007). In addition, reducing the overall size and / or length of the R-functional side chains of amino acid residues may be beneficial for epitope disruption (see, e.g., WO2015 / 113007). However, despite this versatility in substitutions that are most likely to confer epitope disruption, since the goal is to retain significant Shiga toxin effector function, the substituting amino acid is more likely to retain Shiga toxin effector function if it is similar to the amino acid it replaces, such as a similarly sized non-polar and / or uncharged residue replacing a polar and / or charged residue.

[0303] In WO2015 / 113007, a number of mutations were empirically tested for their effects on Shiga toxin effector function of various Shiga toxin effector polypeptides and binding proteins. Table 2 summarizes the results described in WO2015 / 113007 and WO2016 / 196344, where amino acid substitutions (alone or in combination with one or more other substitutions) did not prevent the display of potent levels of Shiga toxin effector function. Table 2 uses the epitope region numbering scheme described in WO2016 / 196344.

[0304] Table 2 Amino acid substitutions in Shiga toxin effector polypeptides

[0305]

[0306]

[0307]

[0308] Based on empirical evidence in WO2015 / 113007 and WO2016 / 196344, certain amino acid positions in the Shiga toxin A subunit are predicted to be resistant to epitope disruption while still retaining significant Shiga toxin effector function. For example, the following natural positions tolerate amino acid substitutions, alone or in combination, while retaining Shiga toxin effector function, such as cytotoxicity: amino acid 1 of SEQ ID NO: 1 or SEQ ID NO: 2; 4 of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; 8 of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; 9 of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; 11 of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; 33 of SEQ ID NO: 1 or SEQ ID NO: 2; 43 of SEQ ID NO: 1 or SEQ ID NO: 2; 44 of SEQ ID NO: 1 or SEQ ID NO: 2; 45 of SEQ ID NO: 1 or SEQ ID NO: 2; 46 of SEQ ID NO: 1 or SEQ ID NO: 2; 47 of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; 48 of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; NO:2; SEQ ID NO: 49; SEQ ID NO: 1 or SEQ ID NO: 2; SEQ ID NO: 50; SEQ ID NO: 1 or SEQ ID NO: 2; SEQ ID NO: 51; SEQ ID NO: 1 or SEQ ID NO: 2; SEQ ID NO: 53; SEQ ID NO: 1 or SEQ ID NO: 2; SEQ ID NO: 1 or SEQ ID NO: 2; SEQ ID NO: 54; SEQ ID NO: 1 or SEQ ID NO: 2; SEQ ID NO: 55; SEQ ID NO: 1 or SEQ ID NO: 2; SEQ ID NO: 56; SEQ ID NO: 1 or SEQ ID NO: 2; SEQ ID NO: 57; SEQ ID NO: 1 or SEQ ID NO: 2; SEQ ID NO: 1 or SEQ ID NO: 3; SEQ ID NO: 58; SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; SEQ ID NO: 60; SEQ ID NO: 1 or SEQ ID NO: 2; SEQ ID NO: 1 or SEQ ID NO: 2; SEQ ID NO: 1 or SEQ ID NO: 2; SEQ ID NO: 1 or SEQ ID NO: 2; SEQ ID NO: 1 or SEQ ID NO: 2; SEQ ID NO: 1 or SEQ ID NO: 2; SEQ ID NO: 1 or SEQ ID NO: 2; SEQ ID NO: 1 or SEQ ID NO: 2; SEQ ID NO: 1 or SEQ ID NO: 2 NO:3 of 94;SEQ ID NO: 1 or SEQ ID NO: 96; SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3; SEQ ID NO: 104; SEQ ID NO: 105; SEQ ID NO: 1 or SEQ ID NO: 2; SEQ ID NO: 107; SEQ ID NO: 1 or SEQ ID NO: 2; SEQ ID NO: 108; SEQ ID NO: 109; SEQ ID NO: 110; SEQ ID NO: 1 or SEQ ID NO: 2; SEQ ID NO: 111; SEQ ID NO: 112; SEQ ID NO: 141; SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; SEQ ID NO: 147; SEQ ID NO: 1 or SEQ ID NO: 2; SEQ ID NO: 154; SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; SEQ ID NO: 179; SEQ ID NO: 1 or SEQ ID NO: 2 NO:2; 180 of SEQ ID NO:1 or SEQ ID NO:2; 181 of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3; 183 of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3; 184 of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3; 185 of SEQ ID NO:1 or SEQ ID NO:2; 186 of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3; 187 of SEQ ID NO:1 or SEQ ID NO:2; 188 of SEQ ID NO:1 or SEQ ID NO:2; 189 of SEQ ID NO:1 or SEQ ID NO:2; 198 of SEQ ID NO:3; 204 of SEQ ID NO:1 or SEQ ID NO:2; 205 of SEQ ID NO:1 or SEQ ID NO:2; 241 of SEQ ID NO:3; 242 of SEQ ID NO:1 or SEQ ID NO:2; 247 of SEQ ID NO:1 or SEQ ID NO:2; 247 of SEQ ID NO: 3; 248 of SEQ ID NO: 1 or SEQ ID NO: 2; 250 of SEQ ID NO: 3; 251 of SEQ ID NO: 1 or SEQ ID NO: 2; 264 of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; 265 of SEQ ID NO: 1 or SEQ ID NO: 2;and 286 of SEQ ID NO: 1 or SEQ ID NO: 2. ;

[0309] Empirical data in WO2015 / 113007 and WO2016 / 196344 indicate other epitope-disrupting substitutions and combinations of epitope-disrupting substitutions that can reduce the antigenicity and / or immunogenicity of Shiga toxin effector polypeptides while retaining the ability of Shiga toxin effector polypeptides to exhibit significant Shiga toxin effector function, such as novel combinations of truncations and positions that tolerate substitutions mentioned above and novel substitutions at the same positions or conserved positions in related Shiga toxin A subunits.

[0310] It is anticipated that other amino acid substitutions of amino acid residues in the conserved functional groups tested herein may reduce antigenicity and / or immunogenicity while retaining significant Shiga toxin effector function. For example, other substitutions known to those skilled in the art similar to any of the following substitutions may disrupt endogenous epitopes while maintaining at least one Shiga toxin effector function: K1A, K1M, T4I, D6R, S8I, T8V, T9I, S9I, K11A, K11H, T12K, S33I, S33C, S43N, G44L, S45V, S45I, T45V, T45I, G46 P,D47M,D47G,N48V,N48F,L49A,F50T,A51V,D53A,D53N,D53G,V54L,V54I,R55A,R55V ,R55L,G56P,I57F,I57M,D58A,D58V,D58F,P59A,P59F,E60I,E60T,E60R,E61A,E61V, E61L,G62A,R84A,V88A,D94A,S96I,T104N,A105L,T107P,L108M,S109V,T109V,G110A ,D111T,S112V,D141A,G147A,V154A,R179A,T180G,T181I,D183A,D183G,D184A,D184 A, D184F, L185V, L185D, S186A, S186F, G187A, G187T, R188A, R188L, S189A, D198A, R204A, R205A, C242S, S247I, Y247A, R248A, R250A, R251A, or D264A, G264A, T286A and / or T286I. In particular, amino acid substitutions for conserved amino acid residues similar to those of the following may have the same or similar effects: K1A, K1M, T4I, S8I, T8V, T9I, S9I, K11A, K11H, S33I, S33C, S43N, G44L, S45V, S45I, T45V, T45I, G46P, D47M, N48V, N48F, L49A, A51V, D53A, D53N, V54L, V54I, R55A, R55V, R55L ,G56P,I57F,I57M,D58A,D58V,D58F,P59A,E60I,E60T,E61A,E61V,E61L,G62A,R84A,V88A,D94A,S96I,T104N ,T107P,L108M,S109V,T109V,G110A,D111T,S112V,D141A,G147A,V154A,R179A,T180G,T181I,D183A,D183G,D184A, D184F, L185V, S186A, S186F, G187A, R188A, R188L, S189A, D198A, R204A, R205A, C242S, S247I, Y247A, R248A, R250A, R251A, D264A, G264A, T286A, and T286I. In certain embodiments, the Shiga toxin effector polypeptides of the invention may comprise conservative amino acid substitutions similar to those tested empirically, for example, K1 substituted with G, V, L, I, F, and H; T4 substituted with A, G, V, L, F, M, and S; S8 substituted with A, G, V, L, F, and M; T9 substituted with A, G, L, F, M, and S; S9 substituted with A, G, L, I, F, and M; K11 substituted with G, V, L, I, F, and M; S33 substituted with A, G, V, L, F, and M; S43 substituted with A, G, V, L, I, F, and M; S45 substituted with A, G, L, F, and M; T45 substituted with A, G, L, F and M; D47 is replaced by A, V, L, I, F, S and Q; N48 is replaced by A, G, L and M; L49 is replaced by G; Y49 is replaced by A; D53 is replaced by V, L, I, F, S and Q; R55 is replaced by G, I, F, M, Q, S, K and H; D58 is replaced by G, L, I, S and Q; P59 is replaced by G; E60 is replaced by A, G, V, L, F, S, Q, N, D and M; E61 is replaced by G, I, F, S, Q, N, D, M and R; R84 is replaced by G, V, L, I, F, M, Q, S, K and H; V88 is replaced by G; I88 is replaced by G; D94 is replaced by G, V, L, I, F, S and Q; S96 substituted with A, G, V, L, F and M; T107 substituted with A, G, V, L, I, F, M and S; S107 substituted with A, G, V, L, I, F and M; S109 substituted with A, G, I, L, F and M; T109 substituted with A, G, I, L, F, M and S; S112 substituted with A, G, L, I, F and M; D141 substituted with V, L, I, F, S and Q; V154 substituted with G; R179 substituted with G, V, L, I, F, M, Q, S, K and H; T180 substituted with A, V, L, I, F, M and S; T181 substituted with A , G, V, L, F, M and S; D183 is replaced by V, L, I, F, S and Q; D184 is replaced by G, V, L, I, S and Q; S186 is replaced by G, V, I, L and M; R188 is replaced by G, V, I, F, M, Q, S, K and H; S189 is replaced by G, V, I, L, F and M; D197 is replaced by V, L, I, F, S and Q; D198 is replaced by A, V, L, I, F, S and Q; R204 is replaced by G, V, L, I, F, M, Q, S, K and H; R205 is replaced by G, V, L, I, F, M, Q, S, K and H; S247 is replaced by A, G,V, I, L, F and M; Y247 is substituted with A, G, V, L, I, F and M; R248 is substituted with G, V, L, I, F, M, Q, S, K and H; R250 is substituted with G, V, L, I, F, M, Q, S, K and H; R251 is substituted with G, V, L, I, F, M, Q, S, K and H; D264 is substituted with A, G, V, L, I, F, S and Q; and T286 is substituted with A, G, V, L, I, F, M and S.

[0311] Similarly, amino acid substitutions that remove charge, polarity, and / or reduce side chain length can disrupt the epitope while retaining at least one Shiga toxin effector function. In some embodiments, the Shiga toxin effector polypeptides of the invention may comprise one or more epitopes disrupted by substitutions that result in removal of side chain charge, removal of polarity, and / or reduction of side chain length, such as substitutions of the amino acid residues at the following positions: 1 of SEQ ID NO: 1 or SEQ ID NO: 2; 4 of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; 6 of SEQ ID NO: 1 or SEQ ID NO: 2; 8 of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; 9 of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; 11 of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; 12 of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; 33 of SEQ ID NO: 1 or SEQ ID NO: 2; or 4 of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. NO:2; 43 of SEQ ID NO:1 or SEQ ID NO:2; 44 of SEQ ID NO:1 or SEQ ID NO:2; 45 of SEQ ID NO:1 or SEQ ID NO:2; 46 of SEQ ID NO:1 or SEQ ID NO:2; 47 of SEQ ID NO:1 or SEQ ID NO:2; 48 of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3; 49 of SEQ ID NO:1 or SEQ ID NO:2; 50 of SEQ ID NO:1 or SEQ ID NO:2; 51 of SEQ ID NO:1 or SEQ ID NO:2; 53 of SEQ ID NO:1 or SEQ ID NO:2; 54 of SEQ ID NO:1 or SEQ ID NO:2; 55 of SEQ ID NO:1 or SEQ ID NO:2; 56 of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3; 57 of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3; 58 of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3; 59 of SEQ ID NO:2 or SEQ ID NO:3; 60 of SEQ ID NO:1 or SEQ ID NO:2; 61 of SEQ ID NO:1 or SEQ ID NO:2;SEQ ID NO: 1 or SEQ ID NO: 62; SEQ ID NO: 1 or SEQ ID NO: 2; SEQ ID NO: 1 or SEQ ID NO: 2; SEQ ID NO: 1 or SEQ ID NO: 3; SEQ ID NO: 1 or SEQ ID NO: 62; SEQ ID NO: 1 or SEQ ID NO: 2; SEQ ID NO: 1 or SEQ ID NO: 2; SEQ ID NO: 1 or SEQ ID NO: 2; SEQ ID NO: 1 or SEQ ID NO: 3 ...2; SEQ ID NO: 1 or SEQ ID NO: 3; SEQ ID NO: 1 or SEQ ID NO: 2; SEQ ID NO: 1 or SEQ ID NO: NO:1 or SEQ ID NO:2; 141 of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3; 147 of SEQ ID NO:1 or SEQ ID NO:2; 154 of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3; 179 of SEQ ID NO:1 or SEQ ID NO:2; 180 of SEQ ID NO:1 or SEQ ID NO:2; 181 of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3; 183 of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3; 184 of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3; 185 of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:2; 186 of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3; 187 of SEQ ID NO:1 or SEQ ID NO:2; 188 of SEQ ID NO:1 or SEQ ID NO:2; 189 of SEQ ID NO:1 or SEQ ID NO:2; 197 of SEQ ID NO: 3; 198 of SEQ ID NO: 1 or SEQ ID NO: 2; 204 of SEQ ID NO: 3; 205 of SEQ ID NO: 1 or SEQ ID NO: 2; 241 of SEQ ID NO: 3; 242 of SEQ ID NO: 1 or SEQ ID NO: 2;247 of SEQ ID NO:1 or SEQ ID NO:2; 247 of SEQ ID NO:3; 248 of SEQ ID NO:1 or SEQ ID NO:2; 250 of SEQ ID NO:3; 251 of SEQ ID NO:1 or SEQ ID NO:2; 264 of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3; 265 of SEQ ID NO:1 or SEQ ID NO:2; and 286 of SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the Shiga toxin effector polypeptide may comprise one or more of the following amino acid substitutions: K1 is substituted with A, G, V, L, I, F, M, or H; T4 is substituted with A, G, V, L, I, F, M, or S; D6 is substituted with A, G, V, L, I, F, S, or Q; S8 is substituted with A, G, V, I, L, F, or M; T8 is substituted with A, G, V, I, L, F, M, or S; T9 is substituted with A, G, V, I, L, F, M, or S; S9 is substituted with A, G, V, L, I, F, or M; K11 is substituted with A, G, V, L, I, F, M or H; T12 is substituted with A, G, V, I, L, F, M or S; S33 is substituted with A, G, V, L, I, F or M; S43 is substituted with A, G, V, L, I, F or M; G44 is substituted with A or L; S45 is substituted with A, G, V, L, I, F or M; T45 is substituted with A, G, V, L, I, F or M; G46 is substituted with A or P; D47 is substituted with A, G, V, L, I, F, S or Q; N48 is substituted with A, G, V, L or M; L49 is substituted with A or G; F50; A51 is substituted with V; D53 is substituted with A, G, V, L , I, F, S or Q; V54 is replaced by A, G or L; R55 is replaced by A, G, V, L, I, F, M, Q, S, K or H; G56 is replaced by A or P; I57 is replaced by A, G, M or F; L57 is replaced by A, G, M or F; D58 is replaced by A, G, V, L, I, F, S or Q; P59 is replaced by A, G or F; E60 is replaced by A, G, V, L, I, F, S, Q, N, D, M or R; E61 is replaced by A, G, V, L, I, F, S, Q, N, D, M or R; G62 is replaced by A; D94 is replaced by A, G, V, L, I, F, S or Q; R84 is substituted with A, G, V, L, I, F, M, Q, S, K or H; V88 is substituted with A or G; I88 is substituted with A, G or V; D94; S96 is substituted with A, G, V, I, L, F or M; T104 is substituted with A, G, V, I, L, F, M or S; A105 is substituted with L; T107 is substituted with A, G, V, I, L, F, M or S; S107 is substituted with A, G, V, L, I, F or M; L108 is substituted with A, G or M; S109 is substituted with A, G, V, I, L, F or M;T109 is substituted with A, G, V, I, L, F, M or S; G110 is substituted with A; D111 is substituted with A, G, V, L, I, F, S or Q; S112 is substituted with A, G, V, L, I, F or M; D141 is substituted with A, G, V, L, I, F, S or Q; G147 is substituted with A; V154 is substituted with A or G; R179 is substituted with A, G, V, L, I, F, M, Q, S, K or H; T180 is substituted with A, G, V, L, I, F, M or S; T181 is substituted with A, G, V, L, I, F, M or S; D183 is substituted with A, G, V, L, I, F, S or Q; D184 is substituted with A, G, V, L, I, F, S or Q; L185 is substituted with A, G or V; S186 is substituted with A, G, V, I, L, F or M; G187 is substituted with A; R188 is substituted with A, G, V, L, I, F, M, Q, S, K or H; S189 is substituted with A, G, V, I , L, F or M; D197 is replaced by A, G, V, L, I, F, S or Q; D198 is replaced by A, G, V, L, I, F, S or Q; R204 is replaced by A, G, V, L, I, F, M, Q, S, K or H; R205 is replaced by A, G, V, L, I, F, M, Q, S, K or H; C242 is replaced by A, G, V or S; S247 is replaced by A, G, V, I, L, F or M; Y247 is replaced by A, G, V, L, I , F or M; R248 is replaced by A, G, V, L, I, F, M, Q, S, K or H; R250 is replaced by A, G, V, L, I, F, M, Q, S, K or H; R251 is replaced by A, G, V, L, I, F, M, Q, S, K or H; C262 is replaced by A, G, V or S; D264 is replaced by A, G, V, L, I, F, S or Q; G264 is replaced by A; or T286 is replaced by A, G, V, L, I, F, M or S.;

[0312] In addition, any amino acid substitution in one epitope region of a Shiga toxin effector polypeptide that disrupts an epitope while retaining significant Shiga toxin effector function can be combined with any other amino acid substitution in the same or different epitope that disrupts an epitope while retaining significant Shiga toxin effector function to form a deimmunized Shiga toxin effector polypeptide in which multiple epitope regions are disrupted while still retaining significant levels of Shiga toxin effector function. In some embodiments, the Shiga toxin effector polypeptide of the present invention may comprise a combination of two or more of the above substitutions and / or a combination of substitutions described in WO2015 / 113007, WO2016 / 196344, and / or WO2018 / 140427.

[0313] Based on the work described in WO2015 / 113007, WO2016 / 196344, and WO2018 / 140427, it is predicted that certain amino acid regions within the Shiga toxin A subunit are tolerant to epitope disruption while still retaining significant Shiga toxin effector function. For example, the epitope regions naturally located at 1–15, 39–48, 53–66, 55–66, 94–115, 180–190, 179–190, and 243-257 are tolerant to multiple amino acid substitution combinations without compromising Shiga toxin enzymatic activity or cytotoxicity.

[0314] (v) Deimmunized Shiga toxin with a disrupted furin cleavage motif

[0315] The combined Shiga toxin effector polypeptide comprises two or more subregions (i.e., non-overlapping subregions), wherein each subregion comprises at least one of: (1) a disruption of an endogenous epitope or epitope region, and (2) a disrupted furin cleavage motif at the carboxyl terminus of the A1 fragment-derived region.

[0316] Some embodiments of the combination Shiga toxin effector polypeptides comprise both: (1) disruption of an endogenous epitope or epitope region, and (2) a disrupted furin cleavage motif at the carboxyl terminus of the A1 fragment-derived region. It is predicted that any of the individual deimmunized Shiga toxin effector subregions described in WO 2015 / 113007, WO 2016 / 196344, and WO 2018 / 140427 (e.g., see Table 2 above) can generally be combined with any Shiga toxin effector subregion described herein, described in WO 2015 / 191764, and / or known in the art that comprises a disrupted furin cleavage motif to generate Shiga toxin effector polypeptides for use as binding protein components.

[0317] In combination, Shiga toxin effector polypeptides can combine features of their respective subregions, such as disruption of the furin cleavage motif, disruption of one or more individual epitopes, and / or heterologous molecular cargo, and these combinations sometimes result in Shiga toxin effector polypeptides whose immunogenicity is synergistically reduced compared to the sum of the partially deimmunized subregions of the Shiga toxin effector polypeptides.

[0318] Deimmunized Shiga toxin effector polypeptides that do not exhibit cytotoxicity or reduced cytotoxicity at certain concentrations, for example, Shiga toxin effector polypeptides comprising Y77S, R179A and / or E167D or truncated at the carboxyl terminus beyond native position 240, can still be used as deimmunized Shiga toxin effector polypeptides for delivering exogenous substances into cells.

[0319] In some embodiments, the Shiga toxin effector polypeptide comprises: (i) a furin cleavage motif disrupted at the carboxyl terminus of its Shiga toxin A1 fragment region (see, e.g., WO2015 / 191764, the entire contents of which are hereby expressly incorporated by reference, particularly paragraphs [8], [9] and the sequences of the disrupted motif and toxin polypeptides comprising the disrupted motif) and (ii) is deimmunized; see, e.g., WO2018 / 140427, paragraphs

[80] et seq., "Embodiment Set #5," and paragraphs

[81] and

[82] , which are expressly incorporated herein by reference.

[0320] b. Other structural variations

[0321] The skilled artisan will recognize that the Shiga toxin effector polypeptides and binding proteins of the present invention, and the polynucleotides encoding any of the foregoing, can be mutated without diminishing their biological activity, for example by maintaining the overall structure and function of the Shiga toxin effector polypeptide, such as in conjunction with one or more 1) destruction of endogenous epitopes that reduce antigenicity and / or immunogenicity potential and / or 2) destruction of furin cleavage motifs that reduce proteolytic cleavage. For example, some modifications can enhance expression, enhance purification, improve pharmacokinetic properties, and / or improve immunogenicity. Such modifications are well known to the skilled artisan and include, for example, the addition of a methionine at the amino terminus to provide a start site, the placement of additional amino acids at either end to create a restriction site or stop codon for convenient localization, and the fusion of a biochemical affinity tag to either end to provide convenient detection and / or purification. A common modification to improve the immunogenicity of polypeptides produced using microbial systems (e.g., prokaryotic cells) is to remove the initial methionine residue (which may be formylated during production in, for example, a bacterial host system) after the polypeptide is produced, because, for example, the presence of N-formylmethionine (fMet) can induce an undesirable immune response in a subject (e.g., a human subject).

[0322] Also provided herein are additional amino acid residues, such as epitope tags or other part sequences, at the amino and / or carboxyl termini of the protein components of the associated proteins of the present invention or the associated proteins of the present invention. Additional amino acid residues can be used for various purposes, including, for example, promoting cloning, promoting expression, post-translational modification, promoting synthesis, purification, promoting detection and administering. Non-limiting examples of epitope tags and parts are chitin-binding protein domains, enteropeptidase cleavage sites, Xa factor cleavage sites, FIASH tags, FLAG tags, green fluorescent protein (GFP), glutathione-S-transferase moieties, HA tags, maltose-binding protein domains, myc tags, polyhistidine tags, ReAsH tags, strep tags, strep tag II, TEV protease sites, thioredoxin domains, thrombin cleavage sites and V5 epitope tags.

[0323] In some of the above embodiments, the polypeptide sequence of the Shiga toxin effector polypeptide and / or binding protein of the present invention is varied by introducing one or more conservative amino acid substitutions into the polypeptide region, as long as all desired structural features are still present and the Shiga toxin effector polypeptide is able to exhibit any desired function alone or as a component of a binding protein. As used herein, the term "conservative substitution" means that one or more amino acids are replaced by another biologically similar amino acid residue. Examples include replacement of amino acid residues with similar characteristics, such as small amino acids, acidic amino acids, polar amino acids, basic amino acids, hydrophobic amino acids, and aromatic amino acids (see, e.g., Table 3). An example of a conservative substitution with a residue not typically found in endogenous mammalian peptides and proteins is a conservative substitution of an arginine or lysine residue with, for example, ornithine, canavanine, aminoethylcysteine, or another basic amino acid. For further information on phenotypically silent substitutions in peptides and proteins, see, e.g., Bowie J et al., Science 247: 1306-10 (1990).

[0324] In some embodiments, the Shiga toxin effector polypeptides and binding proteins of the present invention may comprise a functional fragment or variant of a polypeptide region of the present invention as described herein having up to 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions compared to the polypeptide sequence described herein, so long as it comprises (1) a disrupted furin cleavage motif at the carboxyl terminus of the Shiga toxin A1 fragment-derived region and (2) at least one disrupted amino acid in the endogenous B cell and / or CD4+ T cell epitope region, wherein the disrupted amino acid does not overlap with the disrupted furin cleavage motif. Variants of the Shiga toxin effector polypeptides and binding proteins of the present invention are within the scope of the present invention because variants of the polypeptides described herein are altered by changing one or more amino acid residues or deleting or inserting one or more amino acid residues (such as within the binding region or Shiga toxin effector polypeptide region) to achieve desired properties (such as altered cytotoxicity, altered cytostatic effect, altered immunogenicity, and / or altered serum half-life). The Shiga toxin effector polypeptide and CD38 binding protein of the present invention may further contain or not contain a signal sequence.

[0325] Thus, in some embodiments, the Shiga toxin effector polypeptide comprises or consists essentially of an amino acid sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% overall sequence identity to a native Shiga toxin A subunit or a fragment thereof, such as a Shiga toxin A subunit, such as SLT-1A (SEQ ID NO: 1), StxA (SEQ ID NO: 2) and / or SLT-2A (SEQ ID NO: 3), wherein the Shiga toxin effector polypeptide has one or more activities associated with a native SLT-1A subunit, e.g., target-mediated internalization, catalytic activity and / or cytotoxic activity.

[0326] In some embodiments, the Shiga toxin effector polypeptide has one or more amino acid residues that have been mutated, inserted, or deleted to increase the enzymatic activity of the Shiga toxin effector polypeptide. In some embodiments, the Shiga toxin effector polypeptide has one or more amino acid residues that have been mutated or deleted to reduce or eliminate the catalytic and / or cytotoxic activity of the Shiga toxin effector polypeptide. For example, the catalytic and / or cytotoxic activity of the A subunit of a member of the Shiga toxin family may be reduced or eliminated by mutation or truncation.

[0327] The cytotoxicity of the A subunits of Shiga toxin family members can be altered, reduced or eliminated by mutation and / or truncation. The positions marked as tyrosine-77, glutamate-167, arginine-170, tyrosine-114, and tryptophan-203 have been shown to be important for the catalytic activity of Stx, Stx1, and Stx2 (Hovde C et al., Proc Natl Acad Sci USA 85:2568-72 (1988); Deresiewicz R et al., Biochemistry 31:3272-80 (1992); Deresiewicz R et al., Mol Gen Genet 241:467-73 (1993); Ohmura M et al., Microb Pathog 15:169-76 (1993); Cao C et al., Microbiol Immunol 38:441-7 (1994); Suhan M, Hovde C, Infect Immun 66:5252-9 (1998)). Mutation of both glutamate-167 and arginine-170 abolished the enzymatic activity of Slt-I A1 in a cell-free ribosome inactivation assay (LaPointe P et al., J Biol Chem 280:23310-18 (2005)). In another approach using de novo expression of Slt-I A1 in the endoplasmic reticulum, mutation of both glutamate-167 and arginine-170 abolished the cytotoxicity of the Slt-I A1 fragment at this expression level (LaPointe P et al., J Biol Chem 280:23310-18 (2005)). Truncation assays showed that a StxA fragment from residues 75 to 268 still retained significant enzymatic activity in vitro (Haddad J et al., J Bacteriol 175:4970-8 (1993)). A truncated fragment of Slt-I A1 containing residues 1-239 exhibited significant in vitro enzymatic activity and cytotoxicity upon de novo expression in the cytosol (LaPointe P et al., J Biol Chem 280:23310-18 (2005)). Expression of the Slt-I A1 fragment truncated to residues 1-239 in the endoplasmic reticulum was not cytotoxic because it was unable to retrotranslocate to the cytosol (LaPointe P et al., J Biol Chem 280:23310-18 (2005)).

[0328] The most critical residues for enzymatic activity and / or cytotoxicity in the Shiga toxin A subunit are located at the following residue positions: asparagine-75, tyrosine-77, tyrosine-114, glutamate-167, arginine-170, arginine-176, and tryptophan 203 (Di R et al., Toxicon 57:525-39 (2011)). In particular, the Stx2A double mutant construct containing glutamate E167 to lysine and arginine 176 to lysine mutations is completely inactive; whereas many single mutations in Stx1 and Stx2 show a 10-fold reduction in cytotoxicity. In addition, truncating Stx1A to 1-239 or 1-240 reduces its cytotoxicity, and similarly, truncating Stx2A to conserved hydrophobic residues reduces its cytotoxicity. The most critical residues in the Shiga toxin A subunit for binding to eukaryotic ribosomes and / or eukaryotic ribosome inhibition have been mapped to the following residue positions: arginine-172, arginine-176, arginine-179, arginine-188, tyrosine-189, valine-191 and leucine 233, etc. (McCluskey A et al., PLoS One 7:e31191 (2012). However, certain modifications may increase the Shiga toxin functional activity exhibited by the Shiga toxin effector polypeptide. For example, mutation of residue position alanine 231 in Stx1A to glutamic acid increased the in vitro enzymatic activity of Stx1A (Suhan M, Hovde C, Infect Immun 66:5252-9 (1998)).

[0329] In some embodiments, the Shiga toxin effector polypeptide derived from SLT-1A (SEQ ID NO: 1) or StxA (SEQ ID NO: 2) has one or more mutated amino acid residues, the mutations comprising substitution of asparagine at position 75, tyrosine at position 77, tyrosine at position 114, glutamic acid at position 167, arginine at position 170, arginine at position 176, and / or substitution of tryptophan at position 203. Based on the prior art, one skilled in the art will recognize examples of such substitutions, such as substitution of asparagine at position 75 to alanine, substitution of tyrosine at position 77 to serine, substitution of tyrosine at position 114 to serine, substitution of glutamic acid at position 167 to glutamic acid, substitution of arginine at position 170 to alanine, substitution of arginine at position 176 to lysine, substitution of tryptophan at position 203 to alanine, and / or substitution of alanine at position 231 to glutamic acid. Other mutations that increase or decrease Shiga toxin enzymatic activity and / or cytotoxicity are within the scope of the invention and can be determined using the well-known techniques and assays disclosed herein.

[0330] 3. Linkers connecting components and / or their subcomponents

[0331] The individual CD38 binding regions, Shiga toxin effector polypeptides and / or components of the binding protein may be suitably linked to one another via one or more linkers well known in the art and / or described herein. H ), light chain variable region (V L ), CDR and / or ABR regions can be suitably linked to each other via one or more linkers known in the art and / or described herein. Protein components of the present invention, such as multi-chain binding regions, can be suitably linked to each other or to other polypeptide components via one or more linkers known in the art. Peptide components (e.g., KDEL family endoplasmic reticulum retention / recycling signal motifs) can be suitably linked to another component of the present invention via one or more linkers well known in the art, such as protein linkers.

[0332] " Linker " herein refers to the domain linker that two protein domains are connected together, for example, for scFv and / or other proteins and protein fusion structures. Generally, there are many suitable linkers that can be used, including traditional peptide bonds, which are produced by recombinant technology, allowing to reorganize the two domains with sufficient length and flexibility to allow each domain to retain its biological function. The linker peptide can mainly include the following amino acid residues: Gly, Ser, Ala or Thr. The length of the linker peptide should be enough to connect the two molecules so that they present the correct conformation relative to each other, thereby maintaining the required activity. In some embodiments, the length of the linker is about 1 to about 50 amino acids. In some embodiments, the length of the linker is about 1 to about 30 amino acids. In one embodiment, a linker with a length of 1 to 20 amino acids can be used, and in some embodiments, about 5 to about 10 amino acids or 8 to 15 amino acids can be used. Useful linkers include glycine-serine polymers, including, for example, (GS)n (SEQ ID NO: 193), (GSGGS)n (SEQ ID NO: 194), (GGGGS)n (SEQ ID NO: 195), and (GGGS)n (SEQ ID NO: 196), where n is an integer of at least 1 (and typically 3 to 4), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. Alternatively, a variety of non-protein polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol, can be used as linkers. Other linker sequences can include any sequence of the CL / CH1 domain of any length, but not all residues of the CL / CH1 domain; for example, the first 5-12 amino acid residues of the CL / CH1 domain. The linker can also be derived from an immunoglobulin light chain, such as C or C. In one embodiment, the present invention relates to a scFv linker or a scFv heavy chain immunoglobulin. The scFv linker can be derived from an immunoglobulin heavy chain of any isotype, including, for example, C1, C2, C3, C4, C1, C2, C, C and C. The linker sequence can also be derived from other proteins, such as Ig-like proteins (for example, TCR, FcR, KIR), hinge region derived sequences and other native sequences from other proteins. Although any suitable linker can be used, many embodiments use glycine-serine polymers, including, for example, (GS) n (SEQ ID NO: 193), (GSGGS) n (SEQ ID NO: 194), (GGGGS) n (SEQ ID NO: 195) and (GGGS) n (SEQ ID NO: 196), wherein n is an integer of at least 1 (and typically 2 to 3 to 4 to 5). " scFv linker " typically includes these glycine-serine polymers.

[0333] Suitable linkers are generally those that allow each polypeptide component of the invention to fold into a three-dimensional structure that closely resembles the polypeptide component produced individually without any linker or other component. Suitable linkers include single amino acids, peptides, polypeptides, and linkers lacking any of the foregoing, such as various non-proteinaceous carbon chains, whether branched or cyclic.

[0334] Suitable linkers can be non-proteinaceous, for example, chemical linkers. Various non-proteinaceous linkers known in the art can be used to connect the cell targeting binding region to the Shiga toxin effector polypeptide component of the binding protein of the present invention, such as linkers commonly used to conjugate immunoglobulin polypeptides to heterologous polypeptides. For example, the polypeptide regions can be connected using their amino acid residues and functional side chains of carbohydrate moieties, such as carboxyl, amine, sulfhydryl, carboxylic acid, carbonyl, hydroxyl and / or cyclic groups. For example, disulfide bonds and thioether bonds can be used to connect two or more polypeptides. In addition, non-natural amino acid residues can be used with other functional side chains such as keto groups. Examples of non-protein chemical linkers include, but are not limited to, N-succinimidyl (4-iodoacetyl)-aminobenzoate, S-(N-succinimidyl)thioacetate (SATA), N-succinimidyl-oxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (SMPT), N-succinimidyl 4-(2-pyridyldithio)-pentanoate (SPP), succinimidyl 4-(N-maleimidomethyl)cyclohexanecarboxylate (SMCC or MCC), sulfosuccinimidyl (4-iodoacetyl)-aminobenzoate, 4-succinimidyl-oxycarbonyl-α-(2-pyridyldithio)toluene, sulfosuccinimidyl-6-(α-methyl-α-(pyridyldithio)-toluamido)hexanoate, N-succinimidyl-3-(2-pyridyldithio)-pentanoate. pyridyldisulfide)-propionate (SPDP), succinimidyl 6(3(-(-2-pyridyldisulfide)-propionamido)hexanoate, sulfosuccinimidyl 6(3(-(-2-pyridyldisulfide)-propionamido)hexanoate, maleimidocaproyl (MC), maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB), 3-maleimidocaproyl Aminobenzoic acid N-hydroxysuccinimide ester (MBS), α-alkyl derivatives, sulfoNHS-ATMBA (sulfosuccinimidyl N-[3-(acetylthio)-3-methylbutyryl-β-alanine]), sulfodichlorophenol, 2-iminothiophene, 3-(2-pyridyldithio)-propionylhydrazide, Ellman's reagent, dichlorotriazinic acid, and S-(2-thiopyridyl)-L-cysteine.

[0335] Protein linkers can be selected for incorporation into the recombinant fusion binding proteins of the present invention. For the recombinant fusion cell targeting proteins of the present invention, the linker typically comprises from about 1 to about 50 amino acid residues. In some embodiments, the linker may comprise from about 5 to 30 amino acid residues. Typically, protein linkers comprise a majority of amino acid residues with polar, uncharged and / or charged residues, such as threonine, proline, glutamine, glycine, and alanine. Non-limiting examples of protein linkers include alanine-serine-glycine-glycine-proline-glutamate (ASGGPE, SEQ ID NO: 197), valine-methionine (VM), alanine-methionine (AM), AM (G 2至4 S) x AM, wherein G is glycine, S is serine, and x is an integer from 1 to 10 (SEQ ID NO: 198).

[0336] Protein joints can be selected based on required characteristics. Technicians can select protein joints according to specific characteristics, such as, compared with the activity of the same domain itself, optimize the folding, stability, expression, solubility, pharmacokinetic properties, pharmacodynamic properties and / or one or more of the activity of the fusion domain under the background of the fusion construct of the fusion molecule. For example, protein joints can be selected based on flexibility, rigidity and / or cleavability. Technicians can use databases and joint design software tools when selecting joints. In some cases, certain joints can be selected to optimize expression. Certain joints can be selected to promote the intermolecular interaction between the same polypeptide or protein to form homomultimers, such as homodimers.

[0337] Flexible protein linkers are generally greater than 12 amino acid residues in length and are rich in small non-polar amino acid residues, polar amino acid residues and / or hydrophilic amino acid residues, such as glycine, serine and threonine. Flexible protein linkers can be selected to increase the spatial separation between components and / or allow intramolecular interactions between components. For example, various "GS" linkers are known to the skilled artisan and are composed of multiple glycine and / or one or more serines, sometimes in repeating units, such as (G x S) n (SEQ ID NO: 199), (S x G) n (SEQ ID NO:200), (GGGGS) n (SEQ ID NO: 195) and (G) n(SEQ ID NO: 201), wherein x is 1 to 6, and n is 1 to 30. Non-limiting examples of flexible protein linkers include GKSSGSGSESKS (SEQ ID NO: 202), EGKSSGSGSESKEF (SEQ ID NO: 203), GSTSGSGKSSEGKG (SEQ ID NO: 204), GSTSGSGKSSEGSGSTKG (SEQ ID NO: 205), GSTSGSGKPGSGEGSTKG (SEQ ID NO: 206), SRSSG (SEQ ID NO: 207), and SGSSC (SEQ ID NO: 208).

[0338] Rigid protein linkers are typically rigid α-helical structures and are rich in proline residues and / or one or more strategically placed prolines. Rigid linkers can be selected to prevent intramolecular interactions between the linked components.

[0339] In some embodiments of the CD38 binding proteins of the invention, a linker comprising one or more protease-sensitive sites may be used to provide for cleavage by proteases present in target cells. In some embodiments of the binding proteins of the invention, a non-cleavable linker may be used to reduce undesirable toxicity following administration to a vertebrate organism.

[0340] In some embodiments of the CD38 binding proteins of the invention, the CD38 binding region is linked to the Shiga toxin effector polypeptide using any number of means known to those skilled in the art, including covalent and non-covalent linkages.

[0341] In some embodiments of the CD38 binding proteins of the present invention, the molecule comprises a binding region having a heavy chain variable (V H ) domain and light chain variable (V L ) domain. There are many linkers known in the art that are suitable for this purpose, such as the 15-residue (Gly4Ser)3 peptide (SEQ ID NO: 183). Suitable scFv linkers that can be used to form non-covalent homodimer structures include GGGS (SEQ ID NO: 185), GGGGS (SEQ ID NO: 186), GGGGSGGG (SEQ ID NO: 187), GGSGGGG (SEQ ID NO: 188), GSTSGGGSGGGSGGGGSS (SEQ ID NO: 189), and GSTSGSGKPGSSEGSTKG (SEQ ID NO: 190).

[0342] Suitable methods for linking the components of the CD38 binding protein can be any method currently known in the art for achieving this purpose, so long as the linkage does not substantially interfere with the binding ability of the binding region, cellular internalization of the Shiga toxin effector polypeptide component, and / or the desired Shiga toxin effector function as measured by appropriate assays, including the assays described herein, as appropriate.

[0343] Components of the binding protein, such as the Shiga toxin A subunit effector polypeptide and / or the immunoglobulin-type CD38 binding region, can be engineered to provide suitable attachment moieties for linking additional components (e.g., additional exogenous materials) (see WO2018 / 106895).

[0344] In some embodiments, the CD38 binding protein comprises a Shiga toxin A subunit effector polypeptide, a linker, and a CD38 binding domain, or consists thereof. The linker can be a protein linker and can comprise from about 1 to about 50 amino acid residues. In some embodiments, the protein linker consists of 1 to 50 amino acid residues. In some embodiments, the linker comprises the sequence of any one of SEQ ID NOs: 70-75, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to SEQ ID NOs: 70-75, or consists thereof. In some embodiments, the linker comprises the sequence of SEQ ID NO: 70, or consists thereof.

[0345] D. For the purposes of the CD38 binding proteins of the present invention, the specific order or orientation of the following components is not fixed: Unless otherwise specified, the Shiga toxin effector polypeptide, binding region, and any optional linker can be varied relative to each other or the binding protein as a whole (e.g., see Figure 1). The components of the binding proteins of the present invention can be arranged in any order, provided that the desired activity of the binding region and the Shiga toxin effector polypeptide is not abolished. In some embodiments, the CD38 binding protein comprises, from its N- to C-terminus, Shiga toxin A subunit effector polypeptide-first linker-VH-second linker-VL. In some embodiments, the CD38 binding protein comprises, from its N- to C-terminus, Shiga toxin A subunit effector polypeptide-first linker-VL-second linker-VH. Useful embodiments of CD-38 binding fusion proteins

[0346] As will be appreciated by those skilled in the art, the CD38 binding fusion proteins of the present invention may be in a variety of forms, as generally depicted in FIG1 .

[0347] In some embodiments, as Figure 13As shown, a wild-type Shiga toxin effector polypeptide component (e.g., as shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3) can be linked to any one of the CD38 targeting moieties (also referred to herein as CD38 binding regions or domains) selected from CD38TM1, CD38TM2, CD38TM3, and CD38TM4 using a linker.

[0348] In certain embodiments, such as Figure 13 As shown, the Shiga toxin effector polypeptide component is SLT-1A-D1-1 (SEQ ID NO: 47), which is linked to a CD38 binding domain selected from CD38TM1, CD38TM2, CD38TM3, and CD38TM4.

[0349] In certain embodiments, such as Figure 13 As shown, the Shiga toxin effector polypeptide component is SLT-1A-D1 (SEQ ID NO: 45), which is linked to a CD38 binding domain selected from CD38TM1, CD38TM2, CD38TM3, and CD38TM4.

[0350] In certain embodiments, such as Figure 13 As shown, the Shiga toxin effector polypeptide component is SLT-1A-D1-2 (SEQ ID NO: 47), which is linked to a CD38 binding domain selected from CD38TM1, CD38TM2, CD38TM3, and CD38TM4.

[0351] In certain embodiments, such as Figure 13 As shown, the Shiga toxin effector polypeptide component is SLT-1A-D1-3 (SEQ ID NO: 48), which is linked to a CD38 binding domain selected from CD38TM1, CD38TM2, CD38TM3, and CD38TM4.

[0352] In certain embodiments, such as Figure 13 As shown, the Shiga toxin effector polypeptide component is SLT-1A-D1-4 (SEQ ID NO: 49), which is linked to a CD38 binding domain selected from CD38TM1, CD38TM2, CD38TM3, and CD38TM4.

[0353] In certain embodiments, such as Figure 13 As shown, the Shiga toxin effector polypeptide component is SLT-1A-combo variant 1 (SEQ ID NO: 55) linked to a CD38 binding domain selected from CD38TM1, CD38TM2, CD38TM3, and CD38TM4.

[0354] In certain embodiments, such as Figure 13 As shown, the Shiga toxin effector polypeptide component is SLT-1A-combo variant 2 (SEQ ID NO: 56) linked to a CD38 binding domain selected from CD38TM1, CD38TM2, CD38TM3, and CD38TM4.

[0355] In certain embodiments, such as Figure 13 As shown, the Shiga toxin effector polypeptide component is SLT-1A-combo variant 3 (SEQ ID NO: 57) linked to a CD38 binding domain selected from CD38TM1, CD38TM2, CD38TM3, and CD38TM4.

[0356] In certain embodiments, such as Figure 13 As shown, the Shiga toxin effector polypeptide component is SLT-1A-combo variant 4 (SEQ ID NO: 58) linked to a CD38 binding domain selected from CD38TM1, CD38TM2, CD38TM3, and CD38TM4.

[0357] In certain embodiments, such as Figure 13 As shown, the Shiga toxin effector polypeptide component is SLT-1A-combo variant 5 (SEQ ID NO: 59) linked to a CD38 binding domain selected from CD38TM1, CD38TM2, CD38TM3, and CD38TM4.

[0358] In certain embodiments, such as Figure 13 As shown, the Shiga toxin effector polypeptide component is SLT-1A-combo variant 6 (SEQ ID NO: 60) linked to a CD38 binding domain selected from CD38TM1, CD38TM2, CD38TM3, and CD38TM4.

[0359] In certain embodiments, such as Figure 13 As shown, the Shiga toxin effector polypeptide component is SLT-1A-combo variant 7 (SEQ ID NO: 61) linked to a CD38 binding domain selected from CD38TM1, CD38TM2, CD38TM3, and CD38TM4.

[0360] In certain embodiments, such as Figure 13As shown, the Shiga toxin effector polypeptide component is SLT-1A-combo variant 8 (SEQ ID NO: 62) linked to a CD38 binding domain selected from CD38TM1, CD38TM2, CD38TM3, and CD38TM4.

[0361] In certain embodiments, such as Figure 13 As shown, the Shiga toxin effector polypeptide component is SLT-1A-combo variant 9 (SEQ ID NO: 63) linked to a CD38 binding domain selected from CD38TM1, CD38TM2, CD38TM3, and CD38TM4.

[0362] In certain embodiments, such as Figure 13 As shown, the Shiga toxin effector polypeptide component is SLT-1A-combo variant 10 (SEQ ID NO: 64) linked to a CD38 binding domain selected from CD38TM1, CD38TM2, CD38TM3, and CD38TM4.

[0363] In certain embodiments, such as Figure 13 As shown, the Shiga toxin effector polypeptide component is SLT-1A-combo variant 11 (SEQ ID NO: 65) linked to a CD38 binding domain selected from CD38TM1, CD38TM2, CD38TM3, and CD38TM4.

[0364] In certain embodiments, such as Figure 13 As shown, the Shiga toxin effector polypeptide component is SLT-1A-combo variant 12 (SEQ ID NO: 66) linked to a CD38 binding domain selected from CD38TM1, CD38TM2, CD38TM3, and CD38TM4.

[0365] In certain embodiments, such as Figure 13 As shown, the Shiga toxin effector polypeptide component is SLT-1A-combo variant 13 (SEQ ID NO: 67) linked to a CD38 binding domain selected from CD38TM1, CD38TM2, CD38TM3, and CD38TM4.

[0366] In certain embodiments, such as Figure 13 As shown, the Shiga toxin effector polypeptide component is SLT-1A-combo variant 14 (SEQ ID NO: 68) linked to a CD38 binding domain selected from CD38TM1, CD38TM2, CD38TM3, and CD38TM4.

[0367] In certain embodiments, such as Figure 13 As shown, the Shiga toxin effector polypeptide component is SLT-1A-combo variant 15 (SEQ ID NO: 69) linked to a CD38 binding domain selected from CD38TM1, CD38TM2, CD38TM3, and CD38TM4.

[0368] The following embodiments describe in more detail certain structures of illustrative CD38 binding proteins, including CD38 binding fusion proteins, that target cells physically coupled to CD38 at the cell surface, including cells expressing CD38.

[0369] As understood in the art, the CD38 binding fusion protein of the present invention comprises at least a Shiga toxin A subunit effector polypeptide and a CD38 binding domain, and, if necessary, an appropriate linker. The CD38 binding domain typically comprises a VH and a VL, which, when properly associated, will bind to human CD38.

[0370] Particularly useful CD38 binding proteins of the present invention include, but are not limited to, CD38 binding protein #1 (SEQ ID NO:76), CD38 binding protein #2 (SEQ ID NO:77), CD38 binding protein #3 (SEQ ID NO:78), CD38 binding protein #4 (SEQ ID NO:79), CD38 binding protein #5 (SEQ ID NO:80), CD38 binding protein #6 (SEQ ID NO:81) and CD38 binding protein #7 (SEQ ID NO:82).

[0371] In a particularly useful embodiment, the CD38 binding protein of the invention comprises, from N-terminus to C-terminus, a Shiga toxin A subunit effector polypeptide-linker-VH-linker-VL. In this embodiment, the Shiga toxin polypeptide has SEQ ID NO: 46, and the VH and VL are selected from the following pairs: SEQ ID NO: 101 and 105; SEQ ID NO: 101 and 125; SEQ ID NO: 109 and 113; and SEQ ID NO: 117 and 121.

[0372] In a particularly useful embodiment, the CD38 binding protein of the invention comprises, from N-terminus to C-terminus, a Shiga toxin A subunit effector polypeptide-linker-VH-linker-VL. In this embodiment, the Shiga toxin polypeptide has SEQ ID NO: 46, and the VH and VL are selected from the following pairs: SEQ ID NO: 101 and 105; SEQ ID NO: 101 and 125; SEQ ID NO: 109 and 113; and SEQ ID NO: 117 and 121.

[0373] In some embodiments, the CD38 binding fusion protein comprises a Shiga toxin A subunit effector polypeptide and a CD38 binding domain from the N-terminus to the C-terminus or from the C-terminus to the N-terminus. The CD38 binding fusion protein may further comprise a first linker connecting the Shiga toxin A subunit effector polypeptide and the CD38 binding domain.

[0374] In some embodiments, the CD38 binding fusion protein comprises, from N-terminus to C-terminus or from C-terminus to N-terminus, a Shiga toxin A subunit effector polypeptide, a heavy chain variable domain (VH), and a light chain variable domain (VL).

[0375] In some embodiments, the CD38 binding fusion protein comprises, from N-terminus to C-terminus or from C-terminus to N-terminus, a Shiga toxin A subunit effector polypeptide, a first linker, VH, and VL.

[0376] In some embodiments, the CD38 binding fusion protein comprises, from N-terminus to C-terminus or from C-terminus to N-terminus, a Shiga toxin A subunit effector polypeptide, a first linker, a VH, a second linker, and a VL.

[0377] In some embodiments, the CD38 binding fusion protein comprises, from N-terminus to C-terminus or from C-terminus to N-terminus, a Shiga toxin A subunit effector polypeptide, a VL, and a VH.

[0378] In some embodiments, the CD38 binding fusion protein comprises, from N-terminus to C-terminus or from C-terminus to N-terminus, a Shiga toxin A subunit effector polypeptide, a first linker, VL, and VH.

[0379] In some embodiments, the CD38 binding fusion protein comprises, from N-terminus to C-terminus or from C-terminus to N-terminus, a Shiga toxin A subunit effector polypeptide, a first linker, a VL, a second linker, and a VH.

[0380] In some embodiments, the CD38 binding fusion protein comprises (i) a Shiga toxin A subunit effector polypeptide; (ii) a VH comprising a vHCDR1 comprising the sequence of SEQ ID NO: 34, a vHCDR2 comprising the sequence of SEQ ID NO: 35, and a vHCDR3 comprising the sequence of SEQ ID NO: 36; and (iii) a VL comprising a vLCDR1 comprising the sequence of SEQ ID NO: 31, a vLCDR2 comprising the sequence of SEQ ID NO: 32, and a vLCDR3 comprising the sequence of SEQ ID NO: 33. In some embodiments, the CD38 binding fusion protein further comprises a first linker connecting (i) the Shiga toxin A subunit effector polypeptide and (ii) VH or (iii) VL. In some embodiments, the CD38 binding protein further comprises a second linker connecting (ii) VH and (iii) VL. In some embodiments, the first linker comprises the sequence of SEQ ID NO: 70. In some embodiments, the second linker comprises the sequence of SEQ ID NO: 75.

[0381] In some embodiments, the VL comprises the sequence of SEQ ID NO: 43, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity thereto. In some embodiments, the VH comprises the sequence of SEQ ID NO: 44, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity thereto. In some embodiments, the VL comprises the sequence of SEQ ID NO: 43, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity thereto, and the VH comprises the sequence of SEQ ID NO: 44, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity thereto. In some embodiments, the VH comprises the sequence of SEQ ID NO: 43, and the VL comprises the sequence of SEQ ID NO: 44.

[0382] In some embodiments, the VL comprises the sequence of SEQ ID NO: 41, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity thereto. In some embodiments, the VH comprises the sequence of SEQ ID NO: 44, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity thereto. In some embodiments, the VL comprises the sequence of SEQ ID NO: 41, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity thereto, or consists thereof, and the VH comprises the sequence of SEQ ID NO: 44, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity thereto. In some embodiments, the VL comprises the sequence of SEQ ID NO: 41, and the VH comprises or consists of the sequence of SEQ ID NO: 44.

[0383] In some embodiments, the CD38 binding fusion protein comprises (i) a Shiga toxin A subunit effector polypeptide; (ii) a VH comprising a vHCDR1 comprising the sequence of SEQ ID NO: 22, a vHCDR2 comprising the sequence of SEQ ID NO: 23, and a vHCDR3 comprising the sequence of SEQ ID NO: 24; and (iii) a VL comprising a vLCDR1 comprising the sequence of SEQ ID NO: 19, a vLCDR2 comprising the sequence of SEQ ID NO: 20, and a vLCDR3 comprising the sequence of SEQ ID NO: 21. In some embodiments, the VL comprises or consists of the sequence of SEQ ID NO: 37, and the VH comprises the sequence of SEQ ID NO: 38. In some embodiments, the VL comprises the sequence of SEQ ID NO: 37, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity thereto, and the VH comprises the sequence of SEQ ID NO: 37, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity thereto. In some embodiments, the CD38 binding fusion protein further comprises a first linker connecting (i) the Shiga toxin subunit effector polypeptide and (ii) the VH or (iii) the VL. In some embodiments, the CD38 binding fusion protein further comprises a second linker connecting (ii) the VH and (iii) the VL.

[0384] In some embodiments, the CD38 binding fusion protein comprises (i) a CD38 binding protein comprising (i) a Shiga toxin A subunit effector polypeptide; (ii) a VH comprising a vHCDR1 comprising the sequence of SEQ ID NO: 28, a vHCDR2 comprising the sequence of SEQ ID NO: 29, and a vHCDR3 comprising the sequence of SEQ ID NO: 30; and (iii) a VL comprising a vLCDR1 comprising the sequence of SEQ ID NO: 25, a vLCDR2 comprising the sequence of SEQ ID NO: 26, and a vLCDR3 comprising the sequence of SEQ ID NO: 27. In some embodiments, the VL comprises the sequence of SEQ ID NO: 39, and the VH comprises the sequence of SEQ ID NO: 40. In some embodiments, the VL comprises the sequence of SEQ ID NO: 39, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity thereto, and the VH comprises the sequence of SEQ ID NO: 40, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity thereto. In some embodiments, the CD38 binding fusion protein further comprises a first linker connecting (i) the Shiga toxin subunit effector polypeptide and (ii) the VH or (iii) the VL. In some embodiments, the CD38 binding protein further comprises a second linker connecting (ii) the VH and (iii) the VL.

[0385] In some embodiments, the CD38 binding fusion protein of the present invention comprises a polypeptide set forth in any one of SEQ ID NOs: 76-82 and 228-232. Optionally, the CD38 binding fusion protein comprises an amino-terminal methionine residue. In some embodiments, the CD38 binding fusion protein has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to any one of SEQ ID NOs: 79-82. In some embodiments, the CD38 binding fusion protein comprises the sequence of SEQ ID NO: 79. In some embodiments, the CD38 binding fusion protein comprises two identical polypeptides, each polypeptide comprising a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to SEQ ID NO: 79. In some embodiments, the CD38 binding fusion protein comprises two identical polypeptides, each polypeptide comprising the sequence of SEQ ID NO: 79. In some embodiments, the CD38 binding fusion protein consists of two identical polypeptides, each polypeptide consisting of the sequence of SEQ ID NO: 79.

[0386] General functions of CD38-binding fusion proteins

[0387] The CD38 binding proteins of the present invention can be used in a variety of applications involving, for example, cell killing; cell growth inhibition; intracellular cargo delivery; biological information collection; immune response stimulation and / or remediation of health conditions. In some embodiments, the CD38 binding proteins of the present invention target CD38 (i.e., are molecules that target CD38). The CD38 binding proteins of the present invention can be used as therapeutic and / or diagnostic molecules, for example, as cell-targeted cytotoxic therapeutic molecules; cell-targeted non-toxic delivery vehicles; and / or cell-targeted diagnostic molecules; for example, in applications involving in vivo targeting of specific CD38-expressing cell types for diagnosis or treatment of a variety of diseases (including cancers and immune disorders associated with CD38 expression).

[0388] In some embodiments, the CD38 binding proteins of the present invention are capable of binding to the extracellular portion of the CD38 molecule associated with the cell surface of a specific cell type and are capable of entering these cells. Once internalized within the targeted cell type, some embodiments of the binding proteins of the present invention are capable of routing enzymatically active, cytotoxic Shiga toxin effector polypeptide fragments to the cytosol of the target cell and ultimately killing the cell. Alternatively, non-toxic or reduced toxicity variants of the binding proteins of the present invention can be used to deliver additional exogenous materials to CD38-positive target cells. The system is modular in that any number of different Shiga toxin effector polypeptides can be associated with the CD38 binding region to generate variants of the binding proteins of the present invention with different functional characteristics, for example, deimmunized effectors for applications involving administration of the CD38 binding proteins to subjects (such as human subjects) and effectors with reduced sensitivity to protease cleavage, particularly to improve in vivo stability.

[0389] E. Killing of CD38-positive cells via Shiga toxin A subunit cytotoxicity

[0390] Some embodiments of the Shiga toxin effector polypeptides and binding proteins of the present invention are cytotoxic. Some embodiments of the CD38 binding proteins of the present invention are cytotoxic solely due to the presence of one or more Shiga toxin effector polypeptide components. The A subunits of members of the Shiga toxin family each contain a polypeptide region with enzymatic activity that, once in the cytosol of a eukaryotic cell, is capable of killing the eukaryotic cell. Because members of the Shiga toxin family are suitable for killing eukaryotic cells, molecules derived from Shiga toxins (e.g., CD38 binding proteins comprising some embodiments of Shiga toxin effector polypeptides) can exhibit potent cell-killing activity.

[0391] For some embodiments of the binding proteins of the present invention, upon contact with a cell (e.g., a CD38-positive cell) physically coupled to CD38 bound by the binding region of the binding protein, the binding protein is capable of causing cell death. 50 A value of less than 5, less than 2.5, less than 1, less than 0.5, or less than 0.25 nM is significantly more potent than a non-targeted wild-type Shiga toxin effector polypeptide (e.g., SEQ ID NOs: 1-18). For example, a CD38 binding protein comprising, consisting essentially of, or consisting of any one of SEQ ID NOs: 76, 78-81, and 228-232 potently kills CD38 expressing cells, including cells expressing attenuated levels of CD38 (see Examples, Tables 7 to 9, and Figure 3 below), for example, characterized by a CD38 binding protein of about 0.1 to about 0.2 nM. 50 Those that are worth it.

[0392] For some embodiments, the CD of the binding protein 50 Values ​​of less than 50, less than 30, less than 20, less than 15, less than 10, less than 5, less than 1, less than 0.5, less than 0.4, less than 0.3, less than 0.2, less than 0.1 ng / mL. For example, a CD38 binding protein comprising, consisting essentially of, or consisting of any one of SEQ ID NOs: 76, 78-81, and 228-232 potently kills CD38 expressing cells, including cells expressing attenuated levels of CD38 (see Examples, Tables 7 to 9, and Figure 3 below), for example, characterized by a CD38 binding protein of about 0.05 or 0.1 ng / mL. 50 Those that are worth it.

[0393] Cell killing can be achieved using the molecules of the invention under different conditions of target cells, such as target cells manipulated ex vivo, target cells cultured in vitro, target cells within a tissue sample cultured in vitro, or target cells in an in vivo environment (e.g., within a multicellular organism).

[0394] In some embodiments, the Shiga toxin effector polypeptides and binding proteins of the present invention comprise (1) a deimmunized Shiga toxin effector subregion, (2) a protease cleavage resistance region near the carboxyl terminus of the Shiga toxin A1 fragment-derived region, (3) a carboxyl terminal endoplasmic reticulum retention / recycling signal motif; and / or (4) a heterologous molecular cargo; however, for some embodiments, these structural modifications do not significantly alter the potency of Shiga toxin cytotoxicity compared to a reference molecule comprising a wild-type Shiga toxin A subunit polypeptide (e.g., a wild-type Shiga toxin A1 fragment). Thus, the deimmunized, protease cleavage-resistant, and / or molecular cargo-carrying Shiga toxin effector polypeptides and CD38 binding proteins of the present invention can maintain potent cytotoxicity while providing one or more other functions or properties.

[0395] The binding protein component can be selected from the prior art or produced using conventional methods known to the skilled person (see, for example, WO 2014 / 164680, WO 2014 / 164693, WO 2015 / 138435, WO 2015 / 138452, WO 2015 / 113005, WO 2015 / 113007, WO 2015 / 191764, WO 2016 / 196344, PCT / US2017 / 065074 and WO 2018 / 106895). For example, deimmunized and furin cleavage-resistant Shiga toxin effector polypeptides have been previously described in WO 2015 / 113007, WO 2015 / 191764 and WO 2016 / 196344. Shiga toxin effector polypeptides and Shiga toxin effector backbones comprising site-specific conjugation sites have been previously described in WO2018 / 106895.

[0396] F. Selective cytotoxicity between cell types

[0397] Some CD38 binding proteins of the present invention are used to selectively kill specific target cells in the presence of non-targeted bystander cells. By targeting the Shiga toxin effector polypeptide to specific CD38-positive cells via the cell-targeting binding region, the binding proteins of the present invention can exhibit cell-type-specific, restricted cell-killing activity, thereby resulting in exclusive or selective killing of selected cell types in the presence of non-targeted cells. In addition, cell-targeted delivery of the cytotoxic Shiga toxin effector polypeptide region using the CD38 binding proteins of the present invention allows for the restricted, exclusive, or selective delivery of the cytotoxic component to CD38-positive target cells in the presence of non-targeted cells.

[0398] For some embodiments, the binding proteins of the present invention are cytotoxic at certain concentrations. In some embodiments, when the binding proteins of the present invention are applied to a mixture of cell types, the cytotoxic binding proteins are capable of selectively killing those cells that are physically coupled to the extracellular CD38 bound by the binding region, compared to cell types that are not physically coupled to any extracellular CD38. For some embodiments, the cytotoxic binding proteins of the present invention are capable of selectively or preferentially causing the death of a specific cell type in a mixture of two or more different cell types. This enables targeting cytotoxic activity to a specific cell type with a high priority (such as a 3-fold cytotoxic effect) relative to a "bystander" cell type that does not express the target biomolecule. Alternatively, if the target biomolecule is expressed in sufficiently low amounts and / or is physically coupled to a cell type that is not targeted in low amounts, the expression of the target biomolecule in the binding region may not be limited to one cell type. This enables targeted cell killing of a specific type of cell to have a high priority, such as a 3-fold cytotoxic effect, relative to a "bystander" cell type that does not express a significant amount of the target biomolecule or is not physically coupled to a significant amount of the target biomolecule.

[0399] For some embodiments, when the cytotoxic binding protein is administered to two different cell type populations, the cytotoxic binding protein is capable of causing cell death (e.g., by a half maximal cytotoxic concentration (CPC)) with respect to the target cell population. 50 ), wherein the target cell population members express the extracellular target biomolecule of the cytotoxic binding protein binding region, and the dose that causes cell death is greater than the CD40 dose of the same cytotoxic binding protein against the cell population whose members do not express the extracellular target biomolecule of the cytotoxic binding protein binding region. 50 The dose is at least three times lower.

[0400] For some embodiments, the binding protein of the invention exhibits at least three times greater cytotoxic activity against a population of cell types physically coupled to the extracellular CD38 bound by the binding region than against a population of cell types not physically coupled to any extracellular CD38 bound by the binding region. According to the invention, selective cytotoxicity can be quantified as the ratio (a) of cytotoxicity against a population of cells of a specific cell type physically coupled to the extracellular CD38 bound by the binding region to (b) cytotoxicity against a population of cells of a cell type not physically coupled to any extracellular CD38 bound by the binding region (a / b). In some embodiments, the cytotoxicity ratio indicates that the selective cytotoxicity of a cell or cell type population physically coupled to the target biomolecule of the binding region is at least 3-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 75-fold, at least 100-fold, at least 250-fold, at least 500-fold, at least 750-fold, or at least 1000-fold greater than that of a cell or cell type population not physically coupled to the target biomolecule of the binding region.

[0401] This preferential cell-killing function allows certain cytotoxic binding proteins of the invention to kill target cells under a variety of conditions and in the presence of non-target bystander cells, such as mixtures of cell types manipulated ex vivo, cultured tissues in vitro with a mixture of multiple cell types, or in vivo in the presence of multiple cell types (e.g., in situ or in a natural location within a multicellular organism).

[0402] G. Production, Manufacturing, and Purification of Shiga Toxin Effector Peptides and CD38 Binding Proteins

[0403] The Shiga toxin effector polypeptides and CD38 binding proteins disclosed herein can be produced using techniques well known to those skilled in the art. For example, the Shiga toxin effector polypeptides and CD38 binding proteins of the present invention can be produced by standard synthetic methods, by using a recombinant expression system, or by any other suitable method. Thus, the Shiga toxin effector polypeptides and binding proteins of the present invention can be synthesized in a variety of ways, including, for example, methods comprising: (1) synthesizing the polypeptide or polypeptide component of the binding protein stepwise or by fragment assembly using standard solid phase or liquid phase methods, and isolating and purifying the final polypeptide compound product; (2) expressing a polynucleotide encoding a protein or protein component of the binding protein of the present invention in a host cell and recovering the expression product from the host cell or host cell culture; or (3) expressing a polynucleotide encoding a polypeptide or polypeptide component of the binding protein of the present invention in vitro without cells and recovering the expression product; or obtaining fragments of the protein component by any combination of methods (1), (2), or (3), followed by joining (e.g., connecting) the peptide or polypeptide fragments to obtain the polypeptide component, and recovering the polypeptide component.

[0404] In some embodiments, the CD38 binding proteins of the present invention or the protein components of the binding proteins of the present invention can be synthesized by solid-phase or liquid-phase peptide synthesis. The polypeptides and binding proteins of the present invention can be suitably manufactured by standard synthetic methods. Thus, peptides can be synthesized, for example, by a method comprising synthesizing the peptide stepwise or by fragment assembly by standard solid-phase or liquid-phase methods, and isolating and purifying the final peptide product. In this context, reference is made to WO 1998 / 011125 or, in particular, Fields G et al., Principles and Practice of Solid-Phase Peptide Synthesis (Synthetic Peptides, Grant G, ed., Oxford University Press, UK, 2nd edition, 2002) and the synthetic examples therein.

[0405] The Shiga toxin effector polypeptides and CD38 binding proteins of the present invention can be prepared (produced and purified) using recombinant techniques well known in the art. In general, methods for preparing proteins by culturing host cells transformed or transfected with vectors comprising encoding polynucleotides and purifying or recovering proteins from cell cultures are described in, for example, Sambrook J et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, NY, US, 1989); Dieffenbach C et al., PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press, NY, US, 1995). Any suitable host cell can be used to produce the polypeptides and / or binding proteins of the present invention. The host cell can be a cell stably or transiently transfected, transformed, transduced or infected with one or more expression vectors driving the expression of the polypeptides of the present invention. In addition, the Shiga toxin effector polypeptides and / or binding proteins of the present invention can be produced by modifying the polynucleotides encoding the polypeptides of the present invention or the binding proteins, which results in changing one or more amino acids or deleting or inserting one or more amino acids to obtain desired properties, such as changed cytotoxicity, changed cell growth inhibition effect and / or changed serum half-life.

[0406] A variety of expression systems can be selected to produce the polypeptides or CD38 binding proteins of the present invention. For example, host organisms for expressing the binding proteins of the present invention include prokaryotes, such as Escherichia coli and Bacillus subtilis; eukaryotic cells, such as yeast and filamentous fungi (e.g., Saccharomyces cerevisiae, Pichia pastoris, Aspergillus awamori, and Kluyveromyces lactis); algae (e.g., Chlamydomonas reinhardtii); insect cell lines; mammalian cells (e.g., CHO cells); plant cell lines; and eukaryotic organisms, such as transgenic plants (e.g., Arabidopsis thaliana and Nicotiana benthamiana).

[0407] Thus, the present invention also provides methods for producing the Shiga toxin effector polypeptides and / or binding proteins of the invention according to the above methods and using: a polynucleotide encoding part or all of a polypeptide of the invention or a protein component of a binding protein of the invention, an expression vector comprising at least one polynucleotide of the invention capable of encoding part or all of a polypeptide of the invention or a binding protein of the invention when introduced into a host cell, and / or a host cell comprising a polynucleotide or expression vector of the invention.

[0408] In some embodiments, nucleic acids encoding CD38 binding proteins are prepared for use in methods for preparing CD38 binding proteins of the present invention. In some embodiments, methods for preparing CD38 binding proteins of the present invention comprise contacting host cells with nucleic acids encoding CD38 binding proteins. CD38 binding proteins can be produced by culturing host cells under conditions that express CD38 binding proteins and recovering the protein. Culture conditions for producing recombinant proteins using various host cells are known to those skilled in the art. For example, in some embodiments, host cells can be maintained in a culture medium at 95°C and a 5% CO2 atmosphere for a period of time sufficient to express the protein.

[0409] When using recombinant technology to express protein in host cell or cell-free system, advantageously desired protein is separated (or purified) from other components such as host cell factors, to obtain preparation with high purity or substantially homogeneous.Purification can be completed by method well known in the art, such as centrifugal technique, extraction technique, chromatography and fractionation technique (such as by gel filtration to carry out size separation, by ion exchange column to carry out charge separation, hydrophobic interaction chromatography, reversed phase chromatography, chromatography, chromatofocusing and protein A agarose chromatography on silicon dioxide or cation exchange resin (such as DEAE etc.) to remove pollutants) and precipitation technique (such as ethanol precipitation or ammonium sulfate precipitation).Any amount of biochemical purification techniques can be used to increase the purity of polypeptide of the present invention and / or conjugated protein.In some embodiments, polypeptide of the present invention and conjugated protein can optionally be purified with homomultimer form (such as comprising two or more polypeptide of the present invention or conjugated protein molecular complex).

[0410] In embodiments, provided herein is a method for preparing a CD38 binding protein as described herein, the method comprising culturing a host cell as described herein under conditions expressing a CD38 binding protein, and recovering the protein. In some embodiments, the method comprises contacting the CD38 binding protein with protein L. In some embodiments, protein L is Peptostreptococcus magnus protein L. In some embodiments, protein L is recombinantly produced. In some embodiments, protein L fragments or variants can be used, wherein the fragments or variants maintain the binding of protein L to a kappa light chain variable domain (e.g., human VκI, VκIII, and VκIV subtypes). In some embodiments, protein L is conjugated to a resin. In some embodiments, protein L is a matrix. Examples of protein L purification of antibody fragments are described in Royce, BioProcess International 2015; 13:6.

[0411] In the following examples, non-limiting examples of methods for producing exemplary Shiga toxin effector polypeptides and CD38 binding proteins of the invention are described, as well as specific but non-limiting aspects of the production methods.

[0412] Production or manufacture of pharmaceutical and / or diagnostic compositions comprising CD38 binding fusion proteins

[0413] Provided herein are compositions comprising the CD38 binding proteins of the present invention. In some embodiments, the CD38 binding proteins can be in the form of dimers (e.g., homodimers). In some embodiments, the CD38 binding proteins are members of a CD38 binding protein population, wherein at least about 50%, at least about 55%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% of the CD38 binding proteins of the population are in the form of homodimers. In some embodiments, at least about 90% of the CD38 binding proteins of the population are in the form of homodimers. In some embodiments, at least about 95% of the CD38 binding proteins of the population are in the form of homodimers.

[0414] In some embodiments, the CD38 binding protein is in the form of a dimer (e.g., a homodimer). In some embodiments, the CD38 binding protein is a member of a population of CD38 binding proteins, wherein the population is substantially free of monomers and multimers higher than dimers (e.g., trimers, tetramers, etc.). In some embodiments, the population that is "substantially free" of monomers and multimers higher than dimers (e.g., trimers, tetramers, etc.) has a ratio of dimers to monomers or multimers higher than dimers of no more than about 2:1 to about 9:1. In some embodiments, the ratio is about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, or about 9:1.

[0415] Any pharmaceutically acceptable salt or solvate of the Shiga toxin effector polypeptide and CD38 binding protein of the invention is within the scope of the invention.

[0416] In the context of the present invention, the term "solvate" refers to a complex with a defined stoichiometry formed between a solute (e.g., a protein compound according to the invention or a pharmaceutically acceptable salt thereof) and a solvent. When the solvent in question is water, such solvates are often referred to as hydrates.

[0417] The polypeptides and proteins of the present invention, or salts thereof, can be formulated into pharmaceutical compositions for storage or administration, which typically comprise an effective amount of a molecule of the present invention, or a salt thereof, in a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" includes any standard pharmaceutical carrier. Pharmaceutically acceptable carriers for therapeutic molecules are well known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences (Mack Publishing Co. (A. Gennaro, ed., 1985). As used herein, "pharmaceutically acceptable carrier" includes any and all physiologically acceptable (i.e., compatible) solvents, dispersion media, coatings, antimicrobial agents, isotonic agents, and absorption delaying agents, etc.

[0418] VIII. Polynucleotides, Expression Vectors, and Host Cells

[0419] In addition to the polypeptide of the present invention and associated proteins, the polynucleotides encoding the polypeptide components of the present invention and associated proteins or their functional parts are also included within the scope of the present invention.Term "polynucleotide" is equivalent to term "nucleic acid", which each includes one or more of the following: the polymer of deoxyribonucleic acid (DNA), the polymer of ribonucleic acid (RNA), analogs and derivatives thereof, fragments and homologues of these DNA or RNA generated using nucleotide analogs.The polynucleotide of the present invention can be single-stranded, double-stranded or triple-stranded.This type of polynucleotide is particularly disclosed to include all polynucleotides that can encode exemplary proteins, for example, considering the known wobble (wobble) that can be tolerated at the third position of the RNA codon, different RNA codons encode identical amino acids (see Stothard P, Biotechniques 28:1102-4 (2000)).

[0420] In one aspect, the present invention provides polynucleotides encoding Shiga toxin effector polypeptides and / or binding proteins of the present invention, or fragments or derivatives thereof. The polynucleotides may include, for example, nucleic acid sequences encoding polypeptides having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more identity to a polypeptide comprising one of the amino acid sequences of a polypeptide or binding protein of the present invention. The present invention also includes polynucleotides comprising a nucleotide sequence that hybridizes under stringent conditions to a polynucleotide encoding a Shiga toxin effector polypeptide component and / or binding protein of the present invention, or a fragment or derivative thereof, or an antisense or complementary sequence to any such sequence.

[0421] Derivatives or analogs of the molecules of the invention (e.g., CD38 binding proteins) particularly include polynucleotide (or polypeptide) molecules having regions of substantial homology to the polynucleotide (or binding protein of the invention), for example, at least about 45%, about 50%, about 70%, about 80%, about 95%, about 98%, or even about 99% identity (e.g., 80%-99% identity) relative to a polynucleotide (or polypeptide) sequence of the same size or when compared to an aligned sequence (wherein the alignment is performed by a computer homology program known in the art). An illustrative program is the GAP program (Wisconsin Sequence Analysis Package, Version 8 for UNIX, Genetics Computer Group, University Research Park, Madison, WI, US) using default settings, which uses the algorithm of Smith T, Waterman M, Adv Appl Math 2:482-9 (1981). Also included are polynucleotides capable of hybridizing under stringent conditions to a sequence complementary to a sequence encoding a binding protein of the invention (see, e.g., Ausubel F et al., Current Protocols in Molecular Biology (John Wiley & Sons, New York, NY, US, 1993) and below). Stringent conditions are known to those skilled in the art and can be found, for example, in Current Protocols in Molecular Biology (John Wiley & Sons, NY, US, Ch. Sec. 6.3.1-6.3.6 (1989)).

[0422] The present invention further provides expression vectors comprising polynucleotides within the scope of the present invention. Polynucleotides encoding the Shiga toxin effector polypeptide components and / or binding proteins of the present invention can be inserted into known vectors, including bacterial plasmids, viral vectors, and phage vectors, using materials and methods well known in the art to produce expression vectors. Such expression vectors will include the necessary polynucleotides to support production of the Shiga toxin effector polypeptides and / or binding proteins of the present invention in any selected host cell or cell-free expression system (e.g., pTxb1 and pIVEX2.3). Expression vectors comprising specific polynucleotides for use with a particular type of host cell or cell-free expression system are well known to those of ordinary skill in the art, can be determined using routine experimentation, and / or can be purchased.

[0423] As used herein, the term "expression vector" refers to a linear or circular polynucleotide comprising one or more expression units. The term "expression unit" refers to a polynucleotide fragment that encodes a polypeptide of interest and is capable of providing expression of a nucleic acid segment in a host cell. An expression unit typically comprises a transcription promoter, an open reading frame encoding a polypeptide of interest, and a transcription terminator, all of which are in an operable configuration. An expression vector comprises one or more expression units. Thus, in the context of the present invention, an expression vector encoding a Shiga toxin effector polypeptide and / or binding protein of the present invention comprising a single polypeptide chain includes at least an expression unit for the single polypeptide chain, while an expression vector comprising, for example, two or more polypeptide chains (e.g., one chain comprising V L domain and the second chain comprises a V linked to a toxin effector polypeptide H The multi-chain binding protein of the present invention may be expressed in a manner that is specific to the protein (e.g., a domain) and includes at least two expression units, one for each of the two polypeptide chains of the protein. To express the multi-chain binding protein of the present invention, the expression units for each polypeptide chain may be contained on separate expression vectors (e.g., expression may be achieved using a single host cell into which the expression vectors for each polypeptide chain have been introduced).

[0424] Expression vectors capable of directing transient or stable expression of polypeptides and proteins are well known in the art. Expression vectors typically include, but are not limited to, one or more of the following: a heterologous signal sequence or peptide, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence, each of which is well known in the art. Optional regulatory sequences, integration sequences, and useful markers that can be used are known in the art.

[0425] The term "host cell" refers to a cell capable of supporting the replication or expression of an expression vector. The host cell can be a prokaryotic cell, such as E. coli, or a eukaryotic cell (e.g., yeast, insect, amphibian, bird, or mammalian cell). The generation and isolation of host cell lines comprising a polynucleotide of the present invention or capable of producing a polypeptide and / or binding protein of the present invention can be accomplished using standard techniques known in the art.

[0426] Shiga toxin effector polypeptides and / or proteins within the scope of the present invention may be variants or derivatives of the polypeptides and molecules described herein, which are produced by modifying the polynucleotide encoding the polypeptide and / or protein component of the binding protein by changing one or more amino acids or deleting or inserting one or more amino acids to make it better suited to achieve desired properties, such as better expression in a host cell.

[0427] IX. Molecules Immobilized on a Solid Substrate

[0428] Some embodiments of the present invention include molecules of the present invention (such as associated proteins of the present invention, fusion proteins or polynucleotides) or any effector fragments thereof that are fixed on solid substrates.Solid substrates disclosed herein include but are not limited to microbeads, nanoparticles, polymers, matrix materials, microarrays, microtiter plates or any solid surface known in the art (see, for example, US 7,771,955).According to these embodiments, the molecule of the present invention can be covalently or non-covalently attached to solid substrates using technology known to technicians, such as beads, particles or plates (see, for example, Jung Y et al., Analyst 133:697-701 (2008)). The immobilized molecules of the present invention can be used in screening applications using techniques known in the art (see, for example, Bradbury A et al., Nat Biotechnol 29:245-54 (2011); Sutton C, Br J Pharmacol 166:457-75 (2012); Diamante L et al., Protein Eng Des Sel 26:713-24 (2013); Houlihan G et al., J Immunol Methods 405:47-56 (2014)).

[0429] Non-limiting examples of solid substrates to which the CD38 binding fusion proteins of the invention can be immobilized include microbeads, nanoparticles, polymers, nanopolymers, nanotubes, magnetic beads, paramagnetic beads, superparamagnetic beads, streptavidin-coated beads, reverse-phase magnetic beads, carboxyl-terminated beads, hydrazine-terminated beads, silica (sodium silica) beads and iminodiacetic acid (IDA) modified beads, aldehyde-modified beads, epoxy-activated beads, diaminodipropylamine (DADPA) modified beads (containing primary amine surface groups), biodegradable polymer beads, polystyrene substrates, amino-polystyrene particles, carboxyl-terminated beads, and polystyrene-modified beads. -polystyrene particles, epoxy-polystyrene particles, dimethylamino-polystyrene particles, hydroxy-polystyrene particles, colored particles, flow cytometer particles, sulfonate-polystyrene particles, nitrocellulose surfaces, reinforced nitrocellulose membranes, nylon membranes, glass surfaces, activated glass surfaces, activated quartz surfaces, polyvinylidene fluoride (PVDF) membranes, polyacrylamide-based substrates, polyvinyl chloride substrates, polymethyl methacrylate substrates, poly(dimethylsiloxane) substrates, and photopolymers containing photoreactive species capable of forming covalent bonds (such as nitrenes, carbenes, and ketyl radicals). Other examples of solid substrates to which the CD38-binding fusion proteins of the present invention can be immobilized are commonly used in molecular display systems, such as cell surfaces, bacteriophages, and viral particles.

[0430] X. Delivery Devices and Kits

[0431] In some embodiments, the present invention relates to a device comprising one or more compositions of matter of the present invention, such as pharmaceutical compositions or diagnostic compositions, for delivery to a subject in need thereof. Thus, a delivery device comprising one or more compositions of the present invention can be used to administer the compositions of matter of the present invention to a subject by various delivery methods including intravenous, subcutaneous, intramuscular, or intraperitoneal injection; or by other suitable means recognized by those skilled in the art.

[0432] Also within the scope of the present invention are kits comprising at least one composition of matter of the present invention and optional packaging and instructions for use. The kits can be used for drug administration and / or diagnostic information collection. The kits of the present invention may optionally comprise at least one additional reagent (e.g., a standard, a marker, etc.). The kits typically include a label indicating the intended use of the kit contents. The kits may further include reagents and other tools for detecting cell types (e.g., tumor cells) in a sample or subject, or for diagnosing whether a subject belongs to a group that responds to a treatment strategy utilizing a compound, composition, or related method (e.g., a method described herein).

[0433] XI. Methods of Using CD38 Binding Proteins and / or Pharmaceutical and / or Diagnostic Compositions Thereof

[0434] In general, the present invention aims to provide pharmacologically active agents and compositions comprising the same that can be used to prevent and / or treat diseases or conditions associated with overexpression of CD38, such as certain hematopoietic cancers, such as multiple myeloma, and other conditions associated with uncontrolled growth of CD38-positive cells. Accordingly, the present invention provides methods for using the polypeptides, CD38-binding proteins, and pharmaceutical compositions of the present invention for targeted killing of CD38-expressing cells, for delivering additional exogenous materials to the CD38-targeted cells, for labeling the interior of targeted cells, and for collecting diagnostic information.

[0435] In particular, the present invention aims to provide such pharmacologically active agents, compositions and / or methods that have certain advantages over agents, compositions and / or methods currently known in the art. Accordingly, the present invention provides methods of using Shiga toxin effector polypeptides and binding proteins having specific protein sequences and pharmaceutical compositions thereof. For example, any of the amino acid sequences described herein may be used in particular as a component of a binding protein for use in the following methods or in any method known to the skilled person using a binding protein, such as the various methods described in WO 2014 / 164680, WO 2014 / 164693, WO 2015 / 138435, WO 2015 / 138452, WO 2015 / 113005, WO 2015 / 113007, WO 2015 / 191764, US20150259428, WO 2016 / 196344, WO 2017 / 019623, WO 2018 / 106895 and WO 2018 / 140427.

[0436] The present invention provides methods for inhibiting the growth of CD38-positive cells or CD38-positive cell populations, comprising contacting the cells or cell populations in vitro, ex vivo, or in vivo with a CD38 binding protein comprising a cytotoxic Shiga toxin effector polypeptide, or a pharmaceutical composition comprising a CD38 cell-binding protein of the present invention. The present invention also provides methods fo...

Claims

1. A CD38 binding fusion protein, the sequence of which is shown in SEQ ID NO:

79.

2. A CD38 binding fusion protein, wherein the protein is a homodimer composed of two identical polypeptides, each polypeptide consisting of the sequence of SEQ ID NO:

79.

3. A nucleic acid encoding the CD38 binding fusion protein according to claim 1 or 2. An expression vector comprising the nucleic acid according to claim 3 . A host cell comprising the expression vector according to claim 4 .

6. A composition comprising (i) the CD38 binding fusion protein of claim 1 or 2 and (ii) a pharmaceutically acceptable carrier.

7. A composition comprising (i) the CD38 binding fusion protein of claim 1 or 2 and (ii) a pharmaceutically acceptable excipient.

8. The composition of claim 6 or 7, wherein at least 90% of the CD38 binding fusion protein is in the form of a homodimer.

9. The composition of claim 8, wherein at least 95% of the CD38 binding fusion protein is in the form of a homodimer.

10. A method for preparing the CD38 binding fusion protein according to claim 1 or 2, comprising culturing the host cell according to claim 5 under conditions for expressing the CD38 binding fusion protein, and recovering the protein.

11. The method for purifying a CD38-binding fusion protein according to claim 1 or 2, comprising contacting the CD38-binding fusion protein with bacterial protein L.

12. The method of claim 11, wherein the protein L is isolated or derived from Peptostreptococcus magnus.

13. The method of claim 11 or 12, wherein the protein L is conjugated to a resin.

14. Use of the CD38 binding fusion protein of claim 1 or 2 or the composition of any one of claims 6 to 9 in the preparation of a medicament for treating multiple myeloma in a subject in need thereof.

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