Methods of Treatment with Asparaginase

Abstract

2 Methods of Treatment with Asparaginase Abstract The invention relates to methods of treating diseases with L-asparaginase. Methods of Treatment with Asparaginase Abstract The invention relates to methods of treating diseases with L-asparaginase. 2 20 24 27 82 86 11 D ec 2 02 4 2 0 2 4 A b s t r a c t 2 0 2 4 2 7 8 2 8 6 1 1 D e c 2

Description

The conjugates s of the invention can be used in the treatment of a disease treatable by depletion of asparagine and / or glutamine. For example, the conjugate is useful in the treatment or the manufacture of a medicament for use in the treatment of acute lymphoblastic Leukemia (ALL) in both adults and children, as well as other conditions where asparagine and / or glutamine depletion is expected to have a useful effect. Such conditions include, but are not limited to the following: malignancies, or cancers, including but not limited to hematologic malignancies, lymphoma, large cell immunoblastic lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, NK lymphoma, Hodgkin's disease, acute myelocytic Leukemia, acute promyelocytic Leukemia, acute myelomonocytic Leukemia, acute monocytic Leukemia, acute T-cell Leukemia, acute myeloid Leukemia (AML), biphenotypic B-cell myelomonocytic Leukemia, chronic lymphocytic Leukemia, lymphosarcoma, reticulosarcoma, and melanosarcoma. In some embodiments, the disease may be acute myeloid leukemia or diffuse large B-cell lymphoma. Malignancies or cancers, include but not limited to, renal cell carcinoma, renal cell adenocarcinoma, glioblastoma including glioblastoma multiforma and glioblastoma astrocytoma, medulloblastoma, rhabdomyosarcoma, malignant melanoma, epidermoid carcinoma, 2024278286   11 Dec 2024 squamous cell carcinoma, lung carcinoma including large cell lung carcinoma and small cell lung carcinoma, endometrial carcinoma, ovarian adenocarcinoma, ovarian tetratocarcinoma, cervical adenocarcinoma, breast carcinoma, breast adenocarcinoma, breast ductal carcinoma, pancreatic adenocarcinoma, pancreatic ductal carcinoma, colon carcinoma, colon adenocarcinoma, colorectal adenocarcinoma, bladder transitional cell carcinoma, bladder papilloma, prostate carcinoma, osteosarcoma, epitheloid carcinoma of the bone, prostate carcinoma, and thyroid cancer. Representative non-malignant hematologic diseases which respond to asparagine and / or glutamine depletion include immune system-mediated Blood diseases, e.g., infectious diseases such as those caused by HIV infection (i.e., AIDS). Non-hematologic diseases associated with asparagine and / or glutamine dependence include autoimmune diseases, for example rheumatoid arthritis, systemic lupus erythematosus (SLE), collagen vascular diseases, etc. Other autoimmune diseases include osteo-arthritis, Issac's syndrome, psoriasis, insulin dependent diabetes mellitus, multiple sclerosis, sclerosing panencephalitis, rheumatic fever, inflammatory bowel disease (e.g., ulcerative colitis and Crohn's disease), primary billiary cirrhosis, chronic active hepatitis, glomerulonephritis, myasthenia gravis, pemphigus vulgaris, and Graves' disease. Cells suspected of causing disease can be tested for asparagine and / or glutamine dependence in any suitable in vitro or in vivo assay, e.g., an in vitro assay wherein the growth medium lacks asparagine and / or glutamine. Thus, in one aspect, the invention is directed to a method of treating a disease treatable in a patient, the method comprising administering to the patient an effective amount of a conjugate of the invention. In another aspect, the conjugate of the invention is co-administered with another active pharmaceutical ingredient. In some embodiments, the conjugate of the invention is co-administered with Oncaspar®, daunorubicin, cytarabine, Vyxeos®, ABT-737, Venetoclax, dactolisib, bortezomib, carfilzomib, vincristine, prednisolone, everolimus, and / or CB-839. In a specific embodiment, the disease is ALL. In a particular embodiment, the conjugate used in the treatment of a disease treatable by asparagine and / or glutamine depletion comprises an L-asparaginase from Erwinia species, more specifically Erwinia chrysanthemi, and more specifically, the L-asparaginase comprising the sequence of SEQ. ID NO: 1 as described herein. In one embodiment, treatment with a conjugate of the invention will be administered as a first line therapy. In another embodiment, treatment with a conjugate of the invention will be administered as a second line therapy in patients, particularly patients with ALL, where objective signs of allergy or hypersensitivity, including "silent hypersensitivity," have developed to other asparaginase preparations, in particular, the native Escherichia-coli-demed L-asparaginase or its PEGylated variant (pegaspargase). 2024278286   11 Dec 2024 Non-limiting examples of objective signs of allergy or hypersensitivity include testing "antibody positive" for an asparaginase enzyme. In a specific embodiment, the conjugate of the invention is used in second line therapy after treatment with pegaspargase. In a more specific embodiment, the conjugate used in second line therapy comprises an L-asparaginase from Erwinia species, more specifically Erwinia chrysanthemi, and more specifically, the L-asparaginase comprising the sequence of SEQ. ID NO: 1. In a more specific embodiment, the conjugate further comprises PEG (e.g., mPEG) having a molecular weight of less than or equal to about 5000 Da, more specifically about 5000 Da. In an even more specific embodiment, at least about 40% to about 100% of accessible amino groups (e.g., lysine residues and / or the N-terminus) are PEGylated, more particularly about 40-55% or 100%. In another aspect, the invention is directed to a method for treating acute lymphoblastic leukemia comprising administering to a patient in need of the treatment a therapeutically effective amount of a conjugate of the invention. In another aspect, the invention is directed to a method for treating acute myeloid leukemia comprising co-administering to a patient in need of the treatment a therapeutically effective amount of a conjugate of the invention in combination with daunorubicin, cytarabine, Vyxeos®, ABT-737, venetoclax, dactolisib, bortexomib, and / or carfilzomib. In another aspect, the invention is directed to a method for treating acute myeloid leukemia comprising coadministering to a patient in need of the treatment a therapeutically effective amount of a conjugate of the invention in combination with venetoclax. In another aspect, the invention is directed to a method for treating diffuse large B-cell lymphoma comprising co-administering to a patient in need of the treatment a therapeutically effective amount of a conjugate of the invention in combination with ABT-737, venetoclax, carfilzomib, vincristine, and / or prednisolone. In another aspect, the invention is directed to a method for treating diffuse large B-cell lymphoma comprising co-administering to a patient in need of the treatment a therapeutically effective amount of a conjugate of the invention in combination with vincristine. In another aspect, the conjugate described herein will be administered at a dose ranging from about 1500 IU / m2 to about 15,000 IU / m2, typically about 10,000 to about 15,000 IU / m2 (about 20-30 mg protein / m2), at a schedule ranging from about twice a week to about once a month, typically once per week or once every other week, as a single agent (e.g., monotherapy) or as part of a combination of chemotherapy drugs, including, but not limited to glucocorticoids, corticosteroids, anticancer compounds or other agents, including, but not limited to methotrexate, dexamethasone, prednisone, prednisolone, vincristine, cyclophosphamide, and anthracycline. As an example, patients with ALL will be 2024278286   11 Dec 2024 administered the conjugate of the invention as a component of multi-agent chemotherapy during chemotherapy phases including induction, consolidation or intensification, and maintenance. In a specific example, the conjugate is not administered with an asparagine synthetase inhibitor (e.g., such as set forth in U.S. Patent No. 9,920,311 which is herein incorporated by reference in its entirety). In another specific example, the conjugate is not administered with an asparagine synthetase inhibitor, but is administered with other chemotherapy drugs. The conjugate can be administered before, after, or simultaneously with other compounds as part of a multi-agent chemotherapy regimen. In a specific embodiment, the method comprises administering a conjugate of the invention at an amount of about 1 U / kg to about 25 U / kg (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 U / kg) or an equivalent amount thereof 20 (e.g., on a protein content basis). In a more specific embodiment, the conjugate is administered at an amount selected from the group consisting of about 5, about 10, and about 25 U / kg. In another specific embodiment, the conjugate is administered at a dose ranging from about 1,000 IU / m2 to about 20,000 IU / m2 (e.g., 1,000 IU / m2, 2,000 IU / m2, 3,000 IU / m2, 4,000 IU / m2, 5,000 IU / m2, 6,000 IU / m2, 7,000 IU / m2, 8,000 IU / m2, 9,000 IU / m2, 10,000 IU / m2, 11,000 IU / m2, 12,000 IU / m2, 13,000 IU / m2, 14,000 IU / m2, 15,000 IU / m2, 16,000 IU / m2, 17,000 IU / m2, 18,000 IU / m2, 19,000 IU / m2, or 20,000 IU / m2). In another specific embodiment, the conjugate is administered at a dose that depletes L-asparagine and / or glutamine to undetectable levels using methods and apparatus known in the art for a period of about 3 days to about 10 days (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 days) for a single dose. In another embodiment, the method comprises administering a conjugate of the invention that elicits a lower immunogenic response in a patient compared to an unconjugated L-asparaginase. In another embodiment, the method comprises administering a conjugate of the invention that has a longer in vivo circulating half-life after a single dose compared to the unconjugated L-asparaginase. In one embodiment, the method comprises administering a conjugate that has a longer t1 / 2 than pegaspargase administered at an equivalent protein dose. In a specific embodiment, the method comprises administering a conjugate that has a t1 / 2 of at least about 50, 52, 54, 56, 58, 59, 60, 61, 62, 63, 64, or 65 hours at a dose of about 50 pg / kg (protein content basis). In another specific embodiment, the method comprises administering a conjugate that has a t1 / 2 of at least about 30, 32, 34, 36, 37, 37, 39, or 40 hours at a dose of about 10 pg / kg (protein content basis). In another specific embodiment, the method comprises administering a conjugate that has a t1 / 2 at least about 100 to about 200 hours at a dose ranging from about 10,000 to about 15,000 IU / IU / m2 (about 20-30 mg protein / IU / m2). In one 2024278286   11 Dec 2024 embodiment, the method comprises administering a conjugate that has a mean AUC that is at least about 2, 3, 4 or 5 times greater than pegaspargase at an equivalent protein dose. The incidence of relapse in ALL patients following treatment with L-asparaginase remains high, with approximately 10-25% of pediatric ALL patients having early relapse (e.g. some during maintenance phase at 30-36 month post-induction) (Avramis (2005) Clin. Pharmacokinet. 44, 367-393). If a patient treated with E. coli-demed L-asparaginase has a relapse, subsequent treatment with E. coli preparations could lead to a "vaccination" effect, whereby the E. coli preparation has increased immunogenicity during the subsequent administrations. In one embodiment, the conjugate of the invention may be used in a method of treating patients with relapsed ALL who were previously treated with other asparaginase preparations, in particular those who were previously treated with E. coli-demed asparaginases. In some embodiments, the uses and methods of treatment of the invention comprise administering an L-asparaginase conjugate having properties or combinations of properties described herein above (e.g., in the section entitled L-asparaginase PEG conjugates or PASylated L-asparaginase) or herein below. Compositions, Formulations, and Routes of Administration The invention also includes a pharmaceutical composition comprising a conjugate of the invention. In a specific embodiment, the pharmaceutical composition is contained in a vial as a lyophilized powder to be reconstituted with a solvent, such as currently available native L-asparaginases, whatever the bacterial source used for its production (Kidrolase®, Elspar®, Erwinase®). In another embodiment, the pharmaceutical composition may further comprises a "ready to use" solution, such as pegaspargase (Oncaspar®) enabling, further to an appropriate handling, an administration through, e.g., intramuscular, intravenous (infusion and / or bolus), intra-cerebro-ventricular (icv), subcutaneous routes. In additional embodiments, the pharmaceutical composition may comprise the conjugate of the invention in combination with Oncaspar®, daunorubicin, cytarabine, ABT-737, Venetoclax, dactolisib, bortezomib, carfilzomib, vincristine, prednisolone, everolimus, and / or CB-839. Conjugates of the invention, including compositions comprising conjugates of the invention (e.g., a pharmaceutical composition) can be administered to a patient using standard techniques. Techniques and formulations generally may be found in Remington's Pharmaceutical Sciences (2013) 22nd ed., Mack Publishing herein incorporated by reference. Suitable dosage forms, in part, depend upon the use or the route of entry, for example, oral, 2024278286   11 Dec 2024 transdermal, transmucosal, or by injection (parenteral). Such dosage forms should allow the therapeutic agent to reach a target cell or otherwise have the desired therapeutic effect. For example, pharmaceutical compositions injected into the Blood stream preferably are soluble. Conjugates and / or pharmaceutical compositions according to the invention can be formulated as pharmaceutically acceptable salts and complexes thereof. Pharmaceutically acceptable salts are nontoxic salts present in the amounts and concentrations at which they are administered. The preparation of such salts can facilitate pharmaceutical use by altering the physical characteristics of the compound without preventing it from exerting its physiological effect. Useful alterations in physical properties include lowering the melting point to facilitate transmucosal administration and increasing solubility to facilitate administering higher concentrations of the drug. The pharmaceutically acceptable salt of an asparaginase may be present as a complex, as those in the art will appreciate. Pharmaceutically acceptable salts include acid addition salts such as those containing sulfate, hydrochloride, fumarate, maleate, phosphate, sulfamate, acetate, citrate, lactate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate, and quinate. Pharmaceutically acceptable salts can be obtained from acids, including hydrochloric acid, maleic acid, sulfuric acid, phosphoric acid, sulfamic acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid, fumaric acid, and quinic acid. Pharmaceutically acceptable salts also include basic addition salts such as those containing benzathine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine, procaine, aluminum, calcium, lithium, magnesium, potassium, sodium, ammonium, alkylamine, and zinc, when acidic functional groups, such as carboxylic acid or phenol are present. For example, see Remington's Pharmaceutical Sciences, supra. Such salts can be prepared using the appropriate corresponding bases. Pharmaceutically acceptable carriers and / or excipients can also be incorporated into a pharmaceutical composition according to the invention to facilitate administration of the particular asparaginase. Examples of carriers suitable for use in the practice of the invention include calcium carbonate, calcium phosphate, various sugars such as lactose, glucose, or sucrose, or types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, and physiologically compatible solvents. Examples of physiologically compatible solvents include sterile solutions of water for injection (WFI), saline solution and dextrose. Pharmaceutical compositions according to the invention can be administered by different 2024278286   11 Dec 2024 routes, including intravenous, intraperitoneal, subcutaneous, intramuscular, oral, topical (transdermal), or transmucosal administration. For systemic administration, oral administration is preferred. For oral administration, for example, the compounds can be formulated into conventional oral dosage forms such as capsules, tablets, and liquid preparations such as syrups, elixirs, and concentrated drops. Alternatively, injection (parenteral administration) may be used, e.g., intramuscular, intravenous, intraperitoneal, and subcutaneous injection. For injection, pharmaceutical compositions are formulated in liquid solutions, preferably in physiologically compatible buffers or solutions, such as saline solution, Hank's solution, or Ringer's solution. In addition, the compounds may be formulated in solid form and redissolved or suspended immediately prior to use. For example, lyophilized forms of the conjugate can be produced. In a specific embodiment, the conjugate is administered intramuscularly. In another specific embodiment, the conjugate is administered intravenously. Systemic administration can also be accomplished by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are well known in the art, and include, for example, for transmucosal administration, bile salts, and fusidic acid derivatives. In addition, detergents may be used to facilitate permeation. Transmucosal administration, for example, may be through nasal sprays, inhalers (for pulmonary delivery), rectal suppositories, or vaginal suppositories. For topical administration, compounds can be formulated into ointments, salves, gels, or creams, as is well known in the art. The amounts of the conjugate to be delivered will depend on many factors, for example, the IC50, EC5o, the biological half-life of the compound, the age, size, weight, and physical condition of the patient, and the disease or disorder to be treated. The importance of these and other factors to be considered are well known to those of ordinary skill in the art. Generally, the amount of the conjugate to be administered will range from about 10 International Units per square meter of the surface area of the patient's body (IU / m2) to 50,000 IU / m2, with a dosage range of about 1,000 IU / m2 to about 15,000 IU / m2 being preferred, and a range of about 6,000 IU / m2 to about 15,000 IU / m2 being more preferred, and a range of about 10,000 to about 15,000 IU / m2 (about 20-30 mg protein / m2) being particularly preferred to treat a malignant hematologic disease, e.g., Leukemia. Typically, these dosages arc administered via intramuscular or intravenous injection at an interval of about 3 times weekly to about once per month, typically once per week or once every other week during the course of therapy. Of course, other dosages and / or treatment regimens may be employed, as determined by the attending 2024278286   11 Dec 2024 physician. This invention is further illustrated by the following additional examples that should not be construed as limiting. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made to the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. EXAMPLES The subject matter of U.S. Patent No. 9,920,311 is herein incorporated by reference including the Examples disclosing methods of producing and testing PEGylated Asparaginase. The mPEG-r-crisantaspase conjugate used in the following examples was prepared as set forth in U.S. Patent No. 9,920,311. Example 1 mPEG-r-crisantaspase conjugate (Pegcrisantaspase) was tested against various cell lines as shown below in two stages. Cell preparation. All cell lines have been licensed from the American Type Culture Collection (ATCC) Manassas, Virginia (US). Master and Working Cell banks (MCB and WCB) were prepared by subculturing in ATCC-recommended media and freezing according to ATCC recommended protocols (www.atcc.org). Compound preparation. Test compounds were prepared as stock solutions in DMSO or aqueous buffers as appropriate and serially diluted to obtain a dilution series. Cell proliferation assay. Cell proliferation was assessed using a commercially available luminescence assay using ATP as the endpoint. Controls. t=0 signal. On a parallel plate, 45 pl cells were dispensed and incubated in a humidified atmosphere of 5% CO2 at 37°C. After 24 hours 5 pl DMSO-containing Hepes buffer and 25 pl ATPIite IStep™ solution were mixed, and luminescence measured after 10 minute incubation (= luminescencet=0). Reference compound. The IC$o of the reference compound doxorubicin is measured on a separate plate. The IC$o is trended. If the IC$o is out of specification (0.32 - 3.16 times deviating from historic average) the assay is invalidated. 2024278286   11 Dec 2024 Cell growth control. The cellular doubling times of all cell lines are calculated from the t=0 hours and t=end growth signals of the untreated cells. If the doubling time is out of specification (0.5 - 2.0 times deviating from historic average) the assay is invalidated. Maximum signals. For each cell line, the maximum luminescence was recorded after incubation until t=end without compound in the presence of 0.4% DMSO (=luminescenceUntreated,t=end). Drug sensitivity. The 10log IC50 differences between the "modified and "wild type' groups of cell lines were analyzed in three ways. First, for the eighteen most frequent genetic changes, drug sensitivities of individual cell lines were visualized in waterfall plots. Secondly, a larger subset of the most commonly occurring and best known cancer genes (38 in total) was analyzed with type II Anova analysis in the statistical program R. The results are displayed in a volcano plot. Thirdly, the complete set of 114 cancer genes was analyzed by a two-sided homoscedastic t-test in R. The p-values from Anova and t-test were subjected to a Benjamini-Hochberg multiple testing correction, and only genetic associations with a false discovery rate less than 20% are considered significant. The type II Anova analysis on 38 cancer genes is a different test than the homoscedastic t-test on 114 cancer genes, meaning that the significance of the associations may differ. For more information on Oncolines™ methods see www.ntrc.nl / services / oncolinestm. ICso were calculated by non-linear regression using IDBS XLfit. The percentage growth after incubation until t=end (%-growth) was calculated as follows: 100% x (luminescence^^ I luminescenceUntreate<i,t=en<i)- This was fitted to the 10log compound concentration (cone) by a 4-parameter logistics curve: %-growth = bottom + (top - bottom) I (1+ io(|oe|C5°where hiii is the Hillcoefficient, and bottom and top the asymptotic minimum and maximum cell growth that the compound allows in that assay. 2024278286 11 Dec 2024 Pegcrisantaspase in Oncolines™ Cell line name ATCC ref D isease ICM(IU / mL) Max effect (%) Glgjliu / mq LDm (HJ / mL) 769-P CRL-1933 Renal cell adenocarcinoma 0.036 5 : 0.029 0.195 786 -O CRL-1932 Rena I cell adenocarcinoma 0 032 98 0.028 0.345 A-172 CRL-1620 GI io b la stoma 0.054 99 0.051 0.201 A-204 HTB-S2 Rha bdomyosa rcoma 0 078 31 0.064 0.581 A3 75 CRL-1619 Malignant melanoma 0.099 5- 0.095 >10 A3 88 CRL-7905 Epidermoid carcinoma 0 105 79 0.032 >10 A-427 HTB-53 Lung careinoma 0 043 93 0.037 0 201 A-498 HTB-44 Re na I ca rein o ma 0.051 98 0.041 0.243 A-549 CCL-185 Lung carcinoma 0.025 3C 0.024 >10 A-704 HTB-45 Renal cell adenocarcinoma 0 072 e 3 0.047 0.310 ACHN CRL-1611 Renal cell adenocarcinoma 0.035 8 3 0.032 >10 AN 3 CA HTB-111 Endometrium adenocarcrioma 0.070 34 0.059 0.187 AsPC-1 CRL-1682 Pancreas ductal carcinoma 0 060 67 0.064 >10 AU-56 5 CRL-2351 Breast adenocarcinoma 0.083 34 0.892 >10 ET-20 HTB-1 9 Breast carcinoma 0.044 74 0.033 >10 BT-549 HTB-122 Breast ductal carcinoma 0 084 93 0.073 0.765 BxPC-3 CRL-1687 Pa ncrea s a den oca rcinoma 0 104 92 0.063 0.592 C-33 A HTB-31 Cervix carcinoma 0.059 87 0.061 >10 CAL 27 CRL-2095 Squamous cell carcinoma, tongue 0 050 s: 0.050 >10 CCRF-CEM CCL-119 Acute lymph o b la stic Ie u kem s, T ly mp h o bla st 0.00042 43 0.0015 0.058 * COLO 20 5 CCL-222 Colorectal adenocarcinoma 0.080 98 0.069 0.308 COLO 829 CRL-1974 Maligna nt mela noma 0.139 86 0.111 1 .679 Daoy HTB-186 Medulloblastoma, central nervous system 0.054 95 0.050 0.220 DLD-1 CCL-221 Colon adenocarcinoma 0.098 3 5 0.093 >10 DoTc2 4510 CRL-7920 Cervix carcinoma 0.168 78 0.149 >10 DU 145 HTB-81 Prostate carcinoma 0.061 8 7 0.060 >10 FaDu HTB-43 Sq u a mou s ce II ca rein o ma, Ph a rynx 0.045 8 3 0.049 >10 HCT 116 CCL-247 Colon carcinoma 0.025 99 0.024 0 133 H CT-1 5 CCL-225 Colon adenocarcinoma 0 068 95 0.067 >10 Hs 57ST HTB-126 Breast carcinoma 0.163 86 0.142 >10 JB2 HTB-1 Transitional cell carcinoma. urina ry bladder 0.00024 21 0.035 >10 • Jurkat E6.1 TIB-152 Leukemia, T lymphocyte 0.045 99 0.039 0.112 K-562 CCL-243 Chronic myelogenous leukemia (CML) 0 069 76 0.033 >10 KU812 CRL-2099 Chronic myelogenous leukemia: (CML) 0.189 77 0.166 >10 2024278286 11 Dec 2024 LNCaP FGC CRL-1740 Prostate cardnoma 0 098 26 0 180 >10 LoVo CCL-229 Colorectal adenocarcinoma 0 048 77 0 051 >10 LS 174T CL-188 Colorectal adenocarcinoma 0 079 85 0 080 >10 MCF7 HTB-22 Breast adenocarcinoma 0 100 66 0 080 >10 Me Wo HTB-65 Malignant melanoma 0 059 77 0 054 >10 MG-63 CRL-1427 O«eo»arcom» 0 075 92 0 070 >10 MIA PaCa-2 CRL-1420 Pancreas ductal cardnoma 0 118 96 0 111 0 868 MOLT-4 CRL-1582 Acute lymphoblastic leukemia 0.00047 88 0 00045 0 020 • NCI-H460 HTB-177 Large cell lung carcinoma 0 058 98 0 055 0 435 NCI-H82 HTB-175 Small ceil lung caronoma 0 082 50 0 407 >10 OVCAR-3 HTB-161 Ovary adenocarcinoma 0 050 72 0 040 >10 PA-1 CRL-1572 Ovary tera to carcinoma 0 0031 57 0 010 >10 RKO CRL-2577 Colon carcinoma 0 070 98 0 063 0 950 RPMI-7951 HTB-66 Malignant melanoma 0 048 92 0 044 >10 RT4 HTB-2 Bladder papilloma 0 066 85 0 050 0 232 SHP-77 CRL-2195 Small cell lung cardnoma 0 073 56 0 094 >10 SJCRH30 CRL-2061 Rhabdomyosarcoma 0 101 89 0 089 >10 SK-N-AS CRL-2137 Neuroblastoma 0 315 76 0 270 >10 SK-N-FI CRL-2142 Neuroblastoma 0 126 31 0 447 >10 SNU-C2B CCL-250 Colorectal cardnoma 0 137 77 0 110 >10 SR CRL-2262 Large cel immunoblastic lymphoma 0 053 100 0 047 0.134 SUP-T1 CRL-1942 T-CeU lymphoblastic lymphoma 0 098 85 0.091 >10 SW48 CCL-231 Co Io rectal adenocarcinoma 0 058 97 0 042 0431 SW480 CCL-228 Colorectal adenocarcinoma 0 150 94 0 126 0.741 SW620 CCL-227 Colorectal adenocarcinoma 0 172 97 0 148 2 294 SW948 CCL-237 Colorectal adenocarcinoma 0.064 84 0 060 >10 T24 HTB-4 Bladder transtonal cell carcmoma 0 040 96 0.035 0 406 T98G CRL-1690 Glioblastoma multiforma, brain 0 058 91 0 053 >10 TT CRL-1803 Thyroid carcinoma 1 936 27 3.798 >10 $ U-2 OS HTB-96 Osteosarcoma 0 042 97 0 033 0 288 U-87 MG HTB-14 Glioblastoma: astrocytoma 0 068 89 0 061 >10 VA-ES-BJ CRL-2138 Epith el io »d carcinoma, bone 0 073 85 0 065 >10 $ max effect < 20%, curve invalidated * biphasic curve less potent most potent 2024278286   11 Dec 2024 NCI60 parameters. The LDso, the concentration at which 50% of cells die, is the concentration where luminescence^ end = ½ x luminescencei = oh- The Glso, the concentration of 50% growth inhibition, is the concentration where cell growth is half maximum. This is concentration associated with the signal: ((luminescenceUntreated,t=end - luminescence's) / 2) + luminescence^. Curve fitting. Curves calculated automatically by the software were adjusted manually according to the following protocol: The curve bottom was fixed at 0% when the calculated curve had a bottom below zero. The hill was fixed on -6 when the software calculated a lower value. Curves were invalidated when the F-test value for fitting quality was >1.5 or when the compound was inactive (<20% maximal effect), in which cases curves were removed from the graphs. When a curve had a biphasic character, it was fitted on the most potent ICso- Incidentally, when technical failures were likely, concentration points were knocked out. This is always shown in the dose-response graphs. The maximal effect (Max effect) was calculated as 100% (signal of untreated cells) minus the curve bottom when the dose-response curve was completely determined for more than 85%. A dose-response curve is considered 100% complete when the data points at the highest concentrations reach the curve bottom. If the completeness was smaller than 85%, Max effect was calculated as 100% minus the average of the lowest signal. In cases where the bottom of the curve was locked on 0%, the maximal effect was always calculated as 100% minus the growth inhibition at the highest concentration. Volcano plot. The volcano plot in Figure 8 shows how genetic transformations in 38 important genes are statistically associated with shifts in compound sensitivity (as measured by 10loglC50). The p-value (y-axis in the volcano plot) indicates the confidence level for genetic association of mutations in a particular gene with a IC50 shift. The factor of the IC50 shift is indicated on the x-axis. The areas of the circles are proportional to the number of mutants in the cell panel (each mutation is present at least three times). To compute significance, p-values are subjected to a Benjamin-Hochberg multiple testing correction, and only genetic associations with a <20% false discovery rate are colored grey. The relevant cutoff p-value (0.059) is indicated by a horizontal line. If there are no significant associations, no grey circles and horizontal line are drawn. Results of the T-test. For 98 validated cancer driver genes, of which mutations also occur in patients, it was tested if presence of 'wild type' and 'mutant' variants of the gene in cell lines, is associated with a significant IC50S shift of the investigated compound. The column 'ICso shift' indicates the 10loglCso difference. A negative ICso shift indicates that the compound is more potent in cell lines that carry the 'mutant' gene. The column 'p-value' indicates the result of a two-sided t-test. To compute 2024278286 11 Dec 2024 significance, p-values were subjected to a Benjamin-Hochberg multiple testing correction, and only genetic associations with a <20% false discovery rate are highlighted (column 'adj. p-value'). If there are no significant associations, there are no grey cells in the table below. Gere IQnsttft rndua ad. Plaice IQa sffit IQi shift    pAdte ad-P-vd KRAS -1.31 227ECB 1.77EO6 t.HE3 024 041 092 FMA 012 068 092 F7HJ -070 7.55666 296&03 GATA3 030 041 092 fF2 013 058 0=2 AR-GAP35 -0.81 0C2 049 M14 •018 041 092 MrtXD 014 069 092 MH1 -0.66 001 049 SMARCS 029 0.42 092 EZKB 014 069 092 O-D4 -0.74 Q01 049 AELdw 029 042 092 ZHO 009 070 0=2 CMEA -029 001 0.49 RK3R1 023 0.46 092 TSC1 O10 071 092 CCKDt ■072 001 049 MSA 025 049 092 BCM O10 072 0=2 ASL1 -0.43 006 051 RFC 014 051 092 SETC2 010 074 092 S^Z -0.61 oca 066 CASF6 017 052 092 O10 075 092 HEAP1 -0.52 010 066 RK3CA 012 052 092 RATI 007 077 0=2 CIK42C 099 010 066 M0F2K1 023 0.52 092 Q-DB 009 079 0=2 ACUR1B -0.50 010 066 017 051 092 CUD 009 079 092 -0.32 013 092 1P53 009 051 092 B3FR 009 081 0=2 FEW -0.31 020 092 rm 018 0.57 092 FB3R1 008 082 092 n&E -029 024 092 CLK1 020 0.58 092 m 006 082 0=2 FO-2F2 043 024 092 an 017 0.59 092 SIKH -008 083 0=2 FFM1D -0.37 024 092 RASA1 019 060 092 -007 081 092 twi -0.36 025 092 ARCE 017 060 092 AWC 007 085 0=2 ILFAiPS -028 ■028 092 SWDl 013 0.60 022 B1W 006 085 092 AR DIA -021 033 092 AR DIB 015 060 092 CIW31 -006 086 0=2 CRT -025 033 092 CH 015 0.63 092 RBI 006 087 0=2 TCHKi 029 035 092 EFCA2 013 063 092 rccra 001 088 092 AFC 021 037 092 MrC 012 0.63 092 aa -004 089 0=2 JAK1 -023 038 092 S3Q 017 0.61 092 KRAS -002 090 092 Svf 022 039 0=2 S=300 ■oro 066 092 LFP1B -003 093 091 FHT 021 039 092 RCA 015 0.68 092 MIKU 001 097 097 The special volcano plot of Figure 9 relates compound sensitivity (as measured by 10loglC5o) to the presence of cancer hotspot mutations. This provides increased focus on clinically relevant cancer driver mutations in comparison to the previous analyses. The hotspot mutations were derived from statistical analyses of the recurrence patterns of mutations and copy number alterations in patients through separate studies. Axes and statistical analyses are identical to the volcano plot of Figure 8. The cutoff p-level for significance is 0.32. Example 3: Synergistic activity of Pegcrisantaspase and Oncaspar®. Effect2o For determination of the effect of the compound on the activity of other anti-cancer agents in SynergyScreen™ experiments, a low, fixed concentration is used, corresponding to the concentration at which cell growth is inhibited by 20%. This concentration is determined using the dose-response curves of the single compounds. The concentration is the value on the x-as, corresponding to 80% viability of untreated at the y-axis. 2024278286   11 Dec 2024 Oncaspar in Oncolines Cell line ATCC ref. Disease Effect2o (lU / mL) KG-1 CCL-246 Acute myelogenous leukemia (AML) 0.0001 HL-60 CCL-240 Acute promyelocytic leukemia 0.0003 THP-1 TIB-202 Acute monocytic leukemia 0.31 DB CRL-2289 Large cell lymphoma, B lymphoblast 0.47 HT CRL-2260 Diffuse mixed lymphoma, B lymphoblast 0.28 RL CRL-2261 Non-Hodgkin's lymphoma, B lymphoblast 0.37 MO LT-4 CRL-1582 Acute lymphoblastic leukemia (ALL) 0.00019 U-87-MG HTB-14 Glioblastoma, brain 0.4 HT-1080 CCL-121 Fibrosarcoma 0.00025 MV-4-11 CRL-9591 biphenotypic B myelomonocytic leukemia 0.26 Pegcrisantaspase in Oncolines Cell line ATCC ref. Disease Effect2o (lU / mL) KG-1 CCL-246 Acute myelogenous leukemia (AML) 0.00018 HL-60 CCL-240 Acute promyelocytic leukemia 0.00028 THP-1 TIB-202 Acute monocytic leukemia 0.012 DB CRL-2289 Large cell lymphoma, B lymphoblast 0.059 HT CRL-2260 Diffuse mixed lymphoma, B lymphoblast 0.033 RL CRL-2261 Non-Hodgkin's lymphoma, B lymphoblast 0.037 MO LT-4 CRL-1582 Acute lymphoblastic leukemia (ALL) 0.00019 U-87-MG HTB-14 Glioblastoma, brain 0.057 HT-1080 CCL-121 Fibrosarcoma 0.013 MV-4-11 CRL-9591 biphenotypic B myelomonocytic leukemia 0.0088 mPEG-r-crisantaspase conjugate (Pegcrisantaspase; see first table below) or Oncaspar® (see second table below) were tested with other agents that are typically used in the standard of care (SOC) for AML or DLBCL. There was an increased effect in AML when used with daunorubicin, cytarabine, ABT-737, Venetoclax, dactolisib, bortezomib, and carfilzomib. Additionally, there was an increased effect in 2024278286 11 Dec 2024 DLBCLwhen used with vincristine, prednisolone, ABT-737, venetoclax, everolimus, dactolisib, bortezomib, carfilzomib, and CB-839. See the table below. Grey shading indicates synergistic activity. Light grey shading indicates one experiment and dark grey indicates two experiments. AML DLBCL ALL ICHI mutant refers nee KG-1 HL-GO THP-1 DB HT RL MO LT-4 HI-1060 MV-4-11 dautKnubiriti daunorubicin daunorubicin daunorubicin daunorubicin cytarabine cytarabine cytarabine cytarabine cytarabine doxorubicin doxorubicin dcxctwkbi vincristine vincristine vincristine prednisolone predttEotone pedtEofon ABT- 737 ABT-737 ABT-737 ABT-737 ABT-73 7 ABT- 73 7 ABT-737 venetodax venetoclax venetoclax venetadax venetodax venetoclax venetoclax e^urolimiE everolimus e va c Emm everolimus everolimus everolinus everolimus. dactolisib dactolisib dactolisib dactolisib dactolisib dactolisib dactolisib bortezimib bortezomib bortezMnib bortezmiib bortezjmib bortezitnib bortezomib carabiMnib cairilzomib caralzciuib carulzimib caiulzcrnib caralzimib carilzomib CB-839 CB-839 CB-839 CB-839 CB-839 CB-839 CB-839 iwsidenib ivoridenib ivosidenib ivosidenib Bay- Bay Bay Bay ibrutinib ibrutinib ibrutinib methotrexate AML DLBCL ALL Glioblastoma IDH1 mutant reference KG-1 HL-60 THP-1 DB HT RL MO LT-4 U-87-MG HT-1080 MV-4-11 daunorubicin daunorubicin daunombich daunorubicin daunorubicin cytarabine cytarabine cytarabine cytarabine cytarabiie doxorubicin doxonibicii doxorubicin vincristine vincristine vincristine prednisolone prednisolone prednisolone temozolomide ABT-737 ABT-737 ABT-737 ABT-737 ABT-73 7 .ABT-73 7 .ABT-737 venetoclax venetoclax venetoclax venetoclax venetoclax venetoclax venetoclax everolimus everolimus everolimus everolimus everolimus everolimus everolimus dactolisib dactolisib dactolisib dactolisib dactolisib dactolisib dactolisib bortezomb boitezomb bortezomib bortezomib bortezomib bortezomib bortezomib carfilzomib carfilzomib carfilzomib carfilzomib carfilzomib carfilzomib carfilzomib CB-839 CB-839 CB-839 CB-839 CB-839 CB-839 CB-839 hosidenb ivosidenib ivosidenib ivosidenib Bay-1436032 Bay-1436032 Bay-1436032 Bay-1436032 ibrutinib ibnitiub ibrutinib methotrexate Example 3: mPEG-r-crisantaspase conjugates (Pegcrisantaspases) were tested in vivo with cytarabine and daunorubicin. Groups of 5 mice each were given mPEG-r-crisantaspase (PegC) as a single agent (5 & 50 IU / kg) and given in combination with SOC agent cytarabine (50 mg / kg once a day for 5 days followed by 2 days rest for 2 cycles) and daunorubicin (1 mg / kg administered weekly for 2 weeks). These doses were well tolerated. See Figure 1. Group 1 is PBS control, Group 3 is PegC, Group 11 is Daunorubicin 2024278286   11 Dec 2024 plus PegC and Group 13 is Daunorubicin. The approximate 10% decrease in mean relative body weight was due to daunorubicin. Example 4: The present example was conducted in a manner similar to Example 1 but mPEG-r-crisantaspase conjugates (Pegcrisantaspases) were tested in combination with other compounds. Figure 2 shows that Pegcrisantaspase potentiates the effect of cytarbine, venetoclax, and ABT-737, indicating synergy. Example 5: mPEG-r-crisantaspase conjugates (Pegcrisantaspases) was tested in combination with ABT-737 against HL-60 cell line. Plate preparation. The stocks of the mixtures and single agents were diluted in DMSO or 0.9% sodium chloride to generate a 7-points dose-response series. After further 31.6 times dilution in 20 mM sterile Hepes buffer pH 7.4 (reference compounds) or medium (pegcrisantaspase), 5 pl of pegcrisantaspase solution, and 5 pl of reference compound was added to 40 pl pre-plated cells in duplicate in a 384-well assay plate. The final DMSO concentration during incubation was 0.4% in all wells. Final assay concentrations range, for the single agents, between 10 and 0.01 times their ICso (10 and 0.01 ICso equivalents). Cell proliferation assay. A cell assay stock was thawed and diluted in appropriate medium and dispensed in a 384-well plate, depending on the cell line used, at a concentration of 800 - 3200 cells per well in 45 pl medium: i.e., DB: 800 cells per well; RL: 1000 cells per well; MV-4-11: 1600 cells per well; KG-1, HL-60 and HT 3200 cells per well. For each used cell line the cell density was optimized previously. The margins of the plate were filled with phosphate-buffered saline. Plated cells were incubated in a humidified atmosphere of 5% CO2 at 37 9C. After 24 hours, 5 pl of pegcrisantaspase solution, and 5 pl of reference compound was added, and plates were further incubated for another 72 hours. After 72 hours, plates were cooled in 30 minutes to room temperature and 25 pl of ATPIite IStep™ (PerkinElmer) solution was added to each well, and subsequently shaken for 2 minutes. After 5 minutes of incubation in the dark at room temperature, the luminescence was recorded on an Envision multimode reader (PerkinElmer). Controls: t = 0 signal. On a parallel plate, 40 pl cells were dispensed in quadruplicate and incubated in a humidified atmosphere of 5 % CO2 at 37 9C. After 24 hours, plates were cooled to room temperature in 30 minutes. 5 pl DMSO-containing Hepes buffer, 5 pl 0.9% sodium chloride-containing 2024278286   11 Dec 2024 medium and 25 pl ATPIite IStep™ solution were added and subsequently mixed for 2 minutes. Luminescence was measured after 10 minute incubation (= luminescence^) in the dark. Cell growth control. The cellular doubling times of all cell lines are calculated from the t = 0 hours and t = end growth signals of the untreated cells. If the doubling time is out of specification (0.5 -2.0 times deviating from historic average) the assay is invalidated. Maximum signals. On each 384-well plate, the maximum luminescence was recorded after incubation for 72 hours without compound in the presence of 0.4% DMSO. All equivalent wells (usually 14) were averaged. This average is defined as: / um / nescenceuntreated,t=72h- Dose response curves. Accurate single agent IC50s are needed for combination analysis. For each single agent its dose-response signal was fitted by a 4-parameter logistics curve using XL-fit 5 (IDBS software): luminescence = bottom + (top-bottom) / (1+ io{'oglC50~'og[cpdn'hlll]) [cpd] is the compound concentration tested, hill is the Hill-coefficient. Bottom and top are the asymptotic minimum and maximum of the curve. Combination Index (Cl) determination. The Cl is one of the most widely used quantitative indications of synergy. The Cl evaluates the concentrations needed to achieve a fixed-effect. A Cl of below 1 indicates synergy. A Cl of less than 0.3 indicates strong synergy. For example, a Cl of 0.1 indicates that the combination needs a ten-fold lower concentration than expected from the single agent data, to achieve the same effect level. For instance, when a potent and less potent compound with a Cl of 0.1 are combined, the effective concentration of the potent compound is improved tenfold by the less potent compound. Cl is defined for a certain percentage cell viability (V), which is the signal related to a nonexposed control: V = 100 % x / um / nescencetreated,t=72h / / um / nescenceuntreated,t=72h- The concentrations of the two compounds cpdl and cpd2 needed to reach a certain percentage cell viability Vin combination are then compared to the concentrations needed as single agents: Cl (ioo-v;= [cpdl]v / IC(ioo-v;,cpdi + [cpd2]v / IC(ioo-v / cpd2 For example, [cpdljso signifies the concentration of cpdl in a mixture that gives 50% viability. ICso,cPdi would signify the IC50 of cpdl alone. The Cl is labelled by %-effect, to follow conventions, so CI75 signifies the Cl at 25 % viability Curve shift analysis. This analysis provides a visual confirmation of synergy1. The concentrations of the mixtures of compounds 1 and 2 (cpdl and cpd2), and the single agents, were expressed in terms of IC50 equivalents (in 'units' of IC50): 2024278286   11 Dec 2024 [mix] = [cpdl] I IC5o,cpdi + [cpd2] I IC50,cpd2 The dose-response signal was fitted by a 4-parameter logistics curve using XL-fit 5 (IDBS software) luminescence = bottom + (top-bottom) / (1+ io^x-'0slmixJyhl11^ Here hill is the Hill-coefficient and X the inflection point of the curve. Bottom and top are the asymptotic minimum and maximum of the curve. Because [mix] is expressed in terms of IC50 equivalents, the curves of the single agents will overlap and their inflection point will lie at a value of 1. The IC50 values that are used in the calculations, are those determined in parallel for the single agents. In mixtures where synergy is absent, curves will overlap those of the single agents. In mixtures where there is synergy, curves will shift leftward towards lower IC50 equivalents: the mixture appears more potent than expected on basis of the individual constituents. This is a good indicator of synergy. Isobolograms. An isobologram is a dose-oriented plot which reveals whether drug combinations are synergistic. It is defined at a certain effect level, which is usually 75 %. If the single agent curves do not achieve this efficacy level, the isobologram level is set at 50 %, 30 %, 25% or 20%. If single agents do not reach the 20% effect, no isobologram is drawn. On the axis, the calculated doses of the single compounds are plotted that give the pre-set growth effect. Both points are connected with a straight line (additivity line). For the drug combinations, it is calculated which dilutions give the pre-set growth effect and the concentrations of the individual components at this point are plotted in the isobologram. In case of an additive drug effect, the drug combination will lie close to the additivity line. In case of synergy or antagonism, the points will lie under or above the line, respectively. Experiments with inactive agents. In certain agreed cases, synergy experiments are performed in the presence of 'inactive' agents, which are compounds that do not give a dose-response curve as single agents, at the concentrations tested. The experiments are executed as described above except that the 'inactive' agent is added in a fixed concentration to each well of the experiment. Because the single 'inactive' agent shows no effect, its contribution to Cl is insignificant. Cl values are then based on the response of the active agents. Curve shift of the mixture is determined compared to the other, active agent. No isobologram is calculated. The dose-response curves with single agents is depicted in Figure 3. ABT-737 has an IC50 of 835 nM and a maximal effect at 67% while pegcrisantaspase had an IC50 of 0.15 nM and a maximal effect at 88%. Curve shift analysis: The x-axes of the single agent curves (grey and dark grey) and the mixture curves (red, orange and pink) were translated to an IC50 equivalent, based on the IC50S of the single agents, and are compared to the dose-response curves of the mixture as shown in Figure 4. 2024278286   11 Dec 2024 For dose-response curves on the mixtures on an IC50 basis, all curves were superimposed and shifts recorded. A leftward shit of the mixtures curves compared to the single agent curves (grey and dark grey) indicates synergy, a rightward shift indicates antagonism (see Figure 5 and tables below). IC50 Shifts of Mixtures Compared To Single Agents 7^? e in Hl-Wce ^S ABT-737 1 2 1 pegcrisantaspase 1 1 2 Alog IC50 -0.41 -0.66 -0.47 Factor IC50 shift 2.56 4.61 2.95 $4 os ABT-737 1 5 1 pegcrisantaspase 1 1 5 Alog IC50 -0.41 -0.66 -0.29 Factor IC50 shift 2.56 4.55 1.95 AST-737 * pe COO'WOUfW ABT-737 1 10 1 pegcrisantaspase 1 1 10 Alog IC50 -0.41 -0.59 -0.25 Factor IC50 shift 2.56 3.86 1.77 Results using the combination of pegcrisantaspase and ABT-737 are shown below. Cl values calculated from the mixture data, ED75 corresponds to 25% viability. A representative value is the average Cl value for the three mixtures at 50% viability, which is indicated in the summary. ■Bl ABT-737 + pegcrisantaspase 1 :1 in HL-60 0.26 0.14 0.18 ABT-737 + pegcrisantaspase 2 :1 in HL-60 0.22 0.09 0.16 ABT-737 + pegcrisantaspase 1 : 2 in HL-60 0.29 0.22 0.27 average 0.26 0.15 0.20 standard deviation 0.04 0.06 0.06 2024278286   11 Dec 2024 IMIII Mb® ABT-737 + pegcrisantaspase 1 : 1 in HL-60 0.26 0.14 0.18 ABT-737 + pegcrisantaspase 5 : 1 in HL-60 0.18 0.04 0.09 ABT-737 + pegcrisantaspase 1 : 5 in HL-60 0.44 0.31 0.27 average 0.29 0.17 0.18 standard deviation 0.14 0.14 0.09 ABT-737 + pegcrisantaspase 1 :1 in HL-60 0.26 0.14 0.18 ABT-737 + pegcrisantaspase 10 :1 in HL-60 0.21 0.03 0.64 ABT-737 + pegcrisantaspase 1 :10 in HL-60 0.54 0.39 0.32 average 0.33 0.19 0.38 standard deviation 0.18 0.19 0.24 The combination data were used to generated isobolograms as shown in Figure 6. An isobologram is a dose-oriented plot that reveals whether drug combinations are synergistic. In case of synergy, combination points lie under the straight additivity line. The concentration of pegcrisantaspase is shown in lU / mL. The additivity line (dark grey) indicates concentration combinations that would give theoretical additivity. Drug combinations are plotted as the red, pink and orange dots, in summary, strong synergy between pegcrisantaspase and ABT-737 in HL-60 cell line was found as shown below. Compound ICso (lU / mL) Therapeutic ICso(nM) Cell line Average Cl value 1:1, 1:2 and 2:1* Average Cl value 1:1, 1:5 and 5:1* Average Cl value 1:1, 1:10 and 10:1* Curve shift pegcrisantaspase 0.070 ABT-747 1213 HL-60 0.26 0.29 0.33 yes * Average Combination index of the mixtures at ED50; Ci ~ 1.0: no synergy; Ci < 1.0: synergy; Ci < 0.3: strong synergy; Cl > 1.5: antagonistic ND: not determined, tested compound had an efficacy <20% NA: not applicable 2024278286 11 Dec 2024 Example 6: The present example was conducted in a manner similar to Example 5 but the synergy with additional anti-cancer agents in different cell types were tested as shown below. AML DLBCL Cell line anti-cancer agent conclusion Cell line anti-cancer agent conclusion KG-1 daunorubicin synergy DB ABT-737 synergy KG-1 cytarabine synergy DB venetoclax synergy KG-1 ABT-737 strong synergy DB carfilzomib synergy KG-1 venetoclax strong synergy DB prednisolone no synergy KG-1 dactolisib strong synergy DB vincristine strong synergy KG-1 bortezomib synergy HT carfilzomib no synergy / antagonism KG-1 carfilzomib synergy HT vincristine synergy HL-60 daunorubicin antagonism HT ABT-737 synergy HL-60 cytarabine synergy HT venetoclax no synergy HL-60 ABT-737 strong synergy RL ABT-737 synergy HL-60 venetoclax strong synergy RL venetoclax synergy HL-60 dactolisib synergy RL carfilzomib synergy HL-60 bortezomib antagonism RL prednisolone synergy HL-60 carfilzomib antagonism RL vincristine synergy MV-4- 11 daunorubicin MV-4- 11 cytarabine MV-4- 11 ABT-737 MV-4- 11 venetoclax MV-4- 11 dactolisib MV-4- 11 bortezomib MV-4- 11 carfilzomib Example 7: The present example was conducted in a manner similar to Example 1 but mPEG-r-crisantaspase conjugates were tested for activity against CNS cell lines, including for example, 2024278286 11 Dec 2024 glioblastoma, medulloblastoma, glioblastoma multiforma and glioblastoma astrocytoma. Results are displayed in Figure 7. Additional experiments using different cell lines were performed, and the results are displayed in Figure 10. Example 8: mPEG-r-crisantaspase conjugates (Pegcrisantaspases) in combination of additional compounds were tested against AML (acute myeloid leukemia) and DLBCL (diffuse large B-cell lymphoma) cell lines in accordance with the methods described in Example 1. Results are shown below. KG-1, HL-60 and MV4-11 are AML cell lines, and DB, HT and RL are DLBCL cell lines. The combination data with pegcrisantaspase and venetoclax showed strong synergy in the AML cell lines. 2024278286 11 Dec 2024 Pegcrisantaspase combined with reference compounds in AML and DLBCL cell lines Cl 50 Heatmap per cell line 10:1 5:1 2:1 1:1 1:2 1:5 LIO daunorubidn 093 086 084 0.77 0.94 078 067 cytarabine 109 111 104 111 0.90 064 >2.03 ABT-737 116 086 072 0.54 0.24 013 009 KG-1 venetodax <0.10 <0.10 <0.10 <0.10 <0.10 <0.10 <0.10 dactolisib 078 082 103 0.60 0.74 063 044 bortezomib 090 102 079 0.57 1.95 na na carfilzomib 089 102 ICO 0.94 0.95 089 072 10:1 5:1 2:1 1:1 1:2 1:5 110 HL-60 daunorubicin cytarabine ABT-737 venetodax dactolisib bortezomib carfilzomib 116 125 174 1.74 1.52 137 118 078 069 077 0.83 1.12 098 091 021 018 022 026 0.29 044 054 004 007 014 021 0.31 042 062 081 0.80 085 077 0.87 104 098 135 128 197 187 1.42 136 120 119 132 116 1.35 1.10 142 126 10:1 5:1 2:1 1:1 1:2 1:5 LIO MV-4-11 daunorubidn cytarabine ABT-737 venetcdax dactolisib bortezomib carfilzomib 090 103 111 1.20 113 113 096 053 074 076 0.81 0.93 082 081 068 075 090 0.71 0.89 0.88 096 044 059 060 0.65 0.80 090 093 074 072 099 0.75 1.QB 120 119 115 114 128 1.23 1.10 094 089 066 087 077 0.90 0.95 1(5 106 10:1 5:1 2:1 1:1 1:2 1:5 LIO ABT-737 090 081 083 0.75 076 058 049 venetodax 097 096 094 0.98 099 127 099 DB carfilzomib 080 084 102 0.99 0.96 120 102 prednisolone 069 085 0.99 1.07 106 108 vincristine 055 039 050 0.45 0.41 0.44 051 101 5:1 2:1 1:1 1:2 1:5 LIO HT carfilzomib vincristine ABT-737 venetodax 097 108 128 1.40 1.31 126 085 077 095 160 1.00 1.00 125 098 na na na 0.83 na na na na na na 0.93 na na na 10:1 5:1 2:1 1:1 1:2 1:5 LIO RL ABT-737 venetodax carfilzomib prednisolone vincristine 124 144 121 1.18 1.09 115 132 104 126 090 1.21 1.02 090 097 088 099 088 0.90 0.95 133 113 022 052 038 0.73 0.92 082 087 085 109 115 1.07 1.34 135 107 10:1 5:1 2:1 KG-1 daunorubidn cytarabine ABT-737 venetodax dactolisib bortezomib carfilzomib 093 086 084 109 111 104 116 086 072 <0.10 <0.10 <0.10 078 082 103 090 102 079 OS 102 100 HL-60 daunorubidn cytarabine ABT-737 venetodax dactolisib bortezomib carfilzomib 116 125 174 078 069 077 021 018 022 004 007 014 081 080 085 135 128 197 119 132 116 MV-4-11 daunorubidn cytarabine ABT-737 venetodax dactolisib bortezomib carfilzomib 090 103 111 053 074 076 068 075 090 044 059 060 074 072 099 115 114 128 066 087 077 DB ABT-737 venetodax carfilzomib prednisolone vincristine 090 081 083 097 096 094 080 084 102 069 085 >203 055 039 050 HT carfilzomib vincristine ABT-737 venetodax 097 108 128 077 095 160 na na na na na na RL ABT-737 venetodax carfilzomib prednisolone vincristine 124 144 121 104 126 090 088 099 088 022 052 038 085 109 115 2024278286 11 Dec 2024 Example 9 Pasylated conjugates of crisantaspases were tested against pegylated (PEG-crisantaspase) and non-pegylated (Erwinase) versions of crisantaspases along with E. coli L-asparaginase (Oncaspar) in multiple cell lines in accordance with the methods described in Example 1. PA-20 and PA-40 are pasylated crisantaspase conjugates produced in Corynebacterium or Pseudomonas expression systems and PA-200 is a pasylated fusion protein produced in Pseudomonas expression system. The PA-20, PA-40, PA-200 and PA-400 constructs are SEQ ID NO: 2, 3, 6 and 7. Results are shown below. CCRF-CEM, MOLT-4 and RS4:11 are all AML cell lines, Jurkat E6-1 is an acute T-cell leukemia cell line, HL-60 is an acute promyelocytic leukemia cell line, MV4-11 is a biphenotypic B-cell myelomonocytic leukemia cell line, THP-1 is an AML cell line, RL is a non-Hodgin's lymphoma cell line, and H9 is a lymphoma cell line. Erwinase in Oncolines™ Cell line name ATCC ref Disease IC^ (lU / mL) Max effect (%) GIsoOU / mL) LDM (lU / mL) CCRF-CEM CCL-119 Acute lymphoblastic leukemia (ALL) 0.0028 47 0.0045 > 0.1 ■ HL-60 CCL-240 Acute promyelocytic leukemia 0.0019 40 0.0038 > 0.1 * Jurkat E6-1 TIB-152 Acute T cell leukemia 0.084 99 0.065 0.21 MOLT-4 CRL-1582 Acute lymphoblastic leukemia (ALL) 0.0020 86 0.0020 > 0.1 * RL CRL-2261 Non-Hodgkin’s lymphoma, B lymphoblast 0.069 89 0.069 > 10 RS4; 11 CRL-1873 Acute lymphoblastic leukemia (ALL) 0.0020 90 0.0018 0.0042 THP-1 TIB-202 Acute monocytic leukemia (AML) 0.0022 24 > 0.0316 > 0.1 • MV-4-11 CRL-9591 biphenotypic B myelomonocytic leukemia 0.12 94 0.090 0.53 H9 HTB-176 lymphoma 0.069 95 0.057 0.21 # F-test value >1.5, curve invalidated                                                       |||| • biphasic curve                                                                 less potent                                            most potent 2024278286 11 Dec 2024 Oncaspar in Oncolines1M Ceti lint name ATCC ret Disease IC^CUmL) Max effect (%) Gl^U / mU LD«(IU / mL) CCRF-CEM CCL-119 Acute lymphoblastic leukemia (ALL) 0.00W7 49 0 00098 > O1 HL-60 CCL-240 Acute promyelocyte leukoma 0-00028 38 0 00035 > 0-1 JurkalBB-1 TIB-152 Acute T col leukemia 0.66 97 0-56 11 MOLT-4 CRL-15S2 Acute lymphoblastic leukemia (AH) 003025 ST 0 00026 > 01 RL CRL-2261 Non-Hodgkin's lymphoma. B lymphoblast 049 Si 0.52 > 10 R$4: 11 CRL-1873 Acute lymphoblastic leukemia (ALL) 0.00033 89 0 00031 0 00054 THP«1 TIB-202 Acute monocytic leukemia (AML) 0.00039 21 > 01 > 01 MV-4-11 CRL9591 biphenotypic B myc io monocytic leukemia 0 71 89 0.61 > 10 H9 HTB-176 lymphoma 046 91 0.39 16 # F-teSt value >1.5, Curve Invalidated • bi phasic curve less potent most polent PEG-crisantaspase in Oncolines,M Cell line name ATCC ref Disease tCM {IU / mL) Max effect (%) GtwflU / mLI UDw(IU / mL) CCRFCEM CCL’119 Acute lymphoblastic leukemia (ALL) 0,00075 40 0.091 > 00316 • HL-60 CCL-240 Acute promyekjeyte leukemia 0026 89 0017 017 Jurkat E6-1 TIB-152 Acute T cel leukem«a 0.036 99 0030 0.089 MOLT-4 CRL-1582 Acute lymphoblastic leukemia (ALL) 0 00033 77 0,00034 > 00316 • RL CRL-2261 Non-Hbdgkin’s lymphoma. 0 lymphoblast 0,026 87 0.026 > 10 RS4; 11 CRL-18T3 Acute lymphoblastic leukemia (ALL) 000015 88 O00040 000009 THP-1 TIB-202 Acute monocytic leukemia (AML) 00032 39 0012 > 01 MV-4-11 CRL9591 biphanotypic B myolo monocytic loukerha 0068 94 0.049 0.18 H9 HTB-176 lymphoma 0,027 96 0.022 0.15 W F-test value >15, curve invalidated • biphasic curve less potent I I most potent 2024278286 11 Dec 2024 PA-20 Corynebacterium in Oncolines’M Cell line name ATCC ref Disease IC„(IU / mL) Max effect (%) GIM (lU / mL) LD„(IU / mL) CCRF-CEM CCL-119 Acute lymphoblasts leukemia (ALL) 0.00067 36 0010 > 01 • HL-60 CCL-240 Acute promyelocyte leukemia 00050 42 0034 > 01 • Jurkat E6-1 TIB-152 Acute T cel leukemia 00M 99 0044 0 129 MOLT-4 CRL-1582 Acute lymphoblastic leukemia (ALL) 000079 64 O00061 0.032 • RL CRL-2261 Non-Hodgkin's lymphoma. B lymphoblast 0.019 89 0018 > 10 RS4; 11 CRL-1873 Acute lymphoblastic leukemia (ALL) 0.00077 87 000071 00013 THP-1 TIB-202 Acute monocytic leukemia (AML) O0015 33 > 00316 > 01 • MV-4-11 CRL-9591 biphenotypic B myclomonocytic leukoma 0.062 94 0.053 018 H9 HTB-176 lymphoma 0.041 97 0032 0.14 • Ftcst value >L5, curve invalidated • biphasic curve less potent most potent PA-40 Corynebacterium in Oncolines™ Cell line name ATCC ref Disease IC„(IU(mL) Max effect (%) GI»(IU / mL) LDw(IU / mL) CCRF-CEM CCL-119 Acute lymphoblastic leukemia (ALL) 0 00078 34 > 001 > O1 • HL-60 CCL-240 Acute promyelocytic leukemia 0037 83 0 025 0.15 Jurkat E6-1 TIB-152 Acute T cel leukemia 0.036 99 0 028 0096 MOLT-4 CRL-1582 Acute lymphoblasts leukemia (ALL) 0.00061 80 0 00063 > 0.316 • RL CRL-2261 Non-Hodgkin's lymphoma. B lymphoblast 0028 74 0.030 > 10 • RS4; 11 CRL-1873 Acute lymphoblastc leukoma (ALL) 0.00068 88 000064 0.001 THP-1 TIB-202 Acute monocyte leukemia (AML) 0 039 75 0.030 > 10 MV-4-11 CRL-9591 biphenotypic B myelomonocytic leukemia 0.045 93 0034 0.24 H9 HTB-176 lymphoma 0.020 93 0016 0066 « F-test value >L5, curve invalidated                                                   |          |                  | • biphasic curve                                                                  less potent                                             most potent PA-20 Pseudomonas in Oncolines™ Cell line name ATCC ref Disease ICM (lU / mL) Max effect (%) GIM(IU / mL) LDm (lU / mL) CCRF-CEM CCL-119 Acute lymphoblastic leukemia (ALL) 0.00083 39 0.0014 > 0.1 * HL-60 CCL-240 Acute promyelocytic leukemia 0.00087 30 0.0011 > 0.1 • Jurkat E6-1 TIB-152 Acute T cell leukemia 0.061 99 0.052 0.15 MOLT-4 CRL-1582 Acute lymphoblastic leukemia (ALL) 0.00083 82 0.00085 > 0.0316 * RL CRL-2261 Non-Hodgkin's lymphoma. B lymphoblast 0.056 o7 0.056 > 10 RS4; 11 CRL-1873 Acute lymphoblastic leukemia (ALL) 0.00075 87 0.00070 0.0013 THP-1 TIB-202 Acute monocytic leukemia (AML) 0.056 75 0.042 > 10 MV-4-11 CRL-9591 biphenotypic B myelomonocytic leukemia 0.062 92 0.048 0.25 H9 HTB-176 lymphoma 0.039 95 0.029 0.16 U F-test value >L5, curve invalidated * biphasic curve less potent most potent 2024278286 11 Dec 2024 PA-40 Pseudomonas in Oncolines,M Call line name ATCC ref Disease ICM (tU / mL) Max effect (%) Glw(IU?mL) LDw(IU / mL) CCRF-CEM CCL-119 Acute lymphoblastic leukemia (AIL) 000086 43 0 0014 > 0.10 ♦ HL-60 CCL-240 Acute promyelocytic leukemia 0 00025 34 0.00042 > 0.0316 • Jurkat E6-1 TIB-152 Acute T cell leukemia 0.037 99 0.028 0.14 MOLT-4 CRL-1582 Acute lymphoblastic leukemia (ALL) 000043 83 0 00046 > 0.0316 • RL CRL-2261 Non-Hodgkin's lymphoma. B lymphoblast 0028 69 0 031 > 10 • RS4; 11 CRL-1873 Acute lymphoblastic leukemia (ALL) 000067 88 0.00063 0.0012 THP-1 TIB-202 Acute monocytic leukemia (AML) 0028 72 0.021 > 10 MV-4-11 CRL9591 biphenotypic B myelomonocytic leukemia 0.054 94 a(M6 0.23 H9 HTB-176 lymphoma 0026 94 0.021 0.14 W F-test value >15. curve invalidated • biphasic curve less potent most potent PA-200 Pseudomonas in Oncolines™ Cell line name ATCC ref Disease ICM(iU / mL) Max effect (%) Gl^jlU / mL) LDw(IU / mL) CCRF-CEM CCL-119 Acute lymphoblastic leukemia (ALL) 0 0028 41 0.0067 > 0.10 * HL-60 CCL-240 Acute promyelocytic leukemia 0.0026 0.0035 > 0.10 • Jurkat E6-1 TIB-152 Acute T cell leukemia 0.14 99 012 0.36 MOLT-4 CRL-1582 Acute tymphoblastc leukemia (ALL) 0.0026 81 0.0327 > a io * RL CRL-2261 Non-Hodgkin's lymphoma. B lymphoblast 0 098 .-. 0.10 > 0.10 RS4; 11 CRL-1873 Acute lymphoblastic leukemia (ALL) 0.0025 87 0.0023 0.0041 THP-1 TlB-202 Acute monocytic leukemia (AML) an 71 0.064 > a io MV-4-11 CRL-9591 biphenotypic B myefomonocytic leukemia 0.16 94 0.13 0.76 H9 HTB-176 lymphoma 0089 94 0.076 0.30 if F-tcst value >15, curve invalidated * biphasic curve less potent most potent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. A method of treating cancer in a patient comprising administering to the patient an effectiveamount of a conjugate comprising:(a) an Erwinia L-asparaginase protein having an amino acid sequence comprising SEQ ID NO:1, linked to (b) a polyethylene glycol (PEG) molecule having a molecular weight less than or equal to about 5000 Da, wherein the cancer is a solid tumor selected from the group consisting of renal cell carcinoma, renal cell adenocarcinoma, glioblastoma including glioblastoma multiforma and glioblastoma astrocytoma, medulloblastoma, rhabdomyosarcoma, malignant melanoma, epidermoid carcinoma, squamous cell carcinoma, lung carcinoma including large cell lung carcinoma and small cell lung carcinoma, endometrial carcinoma, ovarian adenocarcinoma, ovarian tetratocarcinoma, cervical adenocarcinoma, breast carcinoma, breast adenocarcinoma, breast ductal carcinoma, pancreatic adenocarcinoma, pancreatic ductal carcinoma, colon carcinoma, colon adenocarcinoma, colorectal adenocarcinoma, bladder transitional cell carcinoma, bladder papilloma, prostate carcinoma, osteosarcoma, epitheloid carcinoma of the bone, prostate carcinoma, and thyroid cancer, andwherein the cancer comprises cells carrying a mutation in a gene selected from NRAS, PTEN, CDKN2A, ERBB2, or any combination thereof.

2. The method of claim 1, wherein the L-asparaginase has an amino acid sequence consisting ofSEQ ID NO: 1.

3. The method of claim 1 or 2, wherein the PEG has a molecular weight of about 5000 Da, 4000,Da, 3000 Da, 2500 Da, or 2000 Da.

4. The method of any one of claims 1-3, wherein the conjugate:(a) has an in vitro activity of at least 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% as compared to the L-asparaginase when not conjugated to PEG;(b) has an L-asparagine depletion activity at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times more potent than the L-asparaginase when not conjugated to PEG; and / or(c) depletes plasma L-asparagine levels to an undetectable level for at least about 12, 24, 48, 96, 108, or 120 hours.2024278286   15 May 20265.     The method of any one of claims 1-4, wherein the conjugate has:(a) a longer in vivo circulating half-life compared to the L-asparaginase when not conjugated to PEG;(b) a longer tV than pegaspargase administered at an equivalent protein dose;(c) a tV of at least about 58 to about 65 hours at a dose of about 50 ^g / kg on a protein content basis, and a tV of at least about 34 to about 40 hours at a dose of about 10 ^g / kg on a protein content basis, following iv administration in mice; and / or(d) a tV of at least about 100 to about 200 hours at a dose ranging from about 10,000 to about 15,000 IU / m2 (about 20-30 mg protein / m2).

6. The method of any one of claims 1-5, wherein the conjugate:(a) has a greater area under the curve (AUC) compared to the L-asparaginase when not conjugated to PEG;(b) the conjugate has a mean AUC that is at least about 3 times greater than pegaspargase at an equivalent protein dose.

7. The method of any one of claims 1-6, wherein the PEG is covalently linked to one or more aminogroups of the L-asparaginase, optionally wherein the PEG is covalently linked to the one or more amino groups by an amide bond.

8. The method of any one of claims 1-7, wherein the PEG is covalently linked to at least from about40% to about 100% of the accessible amino groups or at least from about 40% to about 90% of total amino groups.

9. The method of any one of claims 1-8, wherein the conjugate has the formula:Asp-[NH-CO-(CH2)x-CO-NH-PEG]nwherein Asp is the L-asparaginase, NH is one or more of the NH groups of the lysine residues and / or the N-terminus of the Asp, PEG is a polyethylene glycol moiety, n is a number that represents at least about 40% to about 100% of the accessible amino groups in the Asp, and x is an integer ranging from about 1 to about 8.2024278286   15 May 202610.    The method of any one of claims 1-9, wherein the PEG is monomethoxy-polyethylene glycol(mPEG).

11. The method of any one of claims 1-10, wherein the conjugate is administered at an amount of about 5 U / kg body weight to about 50 U / kg body weight.

12. The method of any one of claims 1-11, wherein the conjugate is administered at a dose ranging from about 10,000 to about 15,000 IU / m2.

13. The method of any one of claims 1-12, wherein the administration is intravenous or intramuscular and is once per week, twice per week, or three times per week.

14. The method of any one of claims 1-13, wherein the conjugate is administered as monotherapy.

15. The method of any one of claims 1-13, wherein the conjugate is administered as part of acombination therapy.

16. The method of claim 15, wherein the conjugate is administered as part of a combination therapy with pegaspargase, daunorubicin, cytarabine, daunorubicine and cytarabine, ABT-737, Venetoclax, dactolisib, bortezomib, carfilzomib, vincristine, prednisolone, everolimus, and / or CB-839.

17. The method of any one of claims 1-16, wherein the patient receiving treatment has had:(a) a previous hypersensitivity to an E. coli asparaginase or PEGylated form thereof or to an Erwinia asparaginase; and / or(b) a cancer relapse, in particular a relapse that occurs after treatment with an E. coli asparaginase or PEGylated form thereof.

18. Use of a conjugate comprising (i) an Erwinia L-asparaginase and (ii) polyethylene glycol (PEG) in the manufacture of a medicament for treating cancer,wherein the PEG has a molecular weight less than or equal to about 5000 Da,wherein the L-asparaginase has an amino acid sequence comprising SEQ ID NO:1,wherein the cancer is a solid tumor selected from the group consisting of renal cell carcinoma, renal cell adenocarcinoma, glioblastoma including glioblastoma multiforma and glioblastoma astrocytoma, medulloblastoma, rhabdomyosarcoma, malignant melanoma, epidermoid carcinoma, squamous cell carcinoma, lung carcinoma including large cell lung carcinoma and small cell lung2024278286   15 May 2026carcinoma, endometrial carcinoma, ovarian adenocarcinoma, ovarian tetratocarcinoma, cervical adenocarcinoma, breast carcinoma, breast adenocarcinoma, breast ductal carcinoma, pancreatic adenocarcinoma, pancreatic ductal carcinoma, colon carcinoma, colon adenocarcinoma, colorectal adenocarcinoma, bladder transitional cell carcinoma, bladder papilloma, prostate carcinoma, osteosarcoma, epitheloid carcinoma of the bone, prostate carcinoma, and thyroid cancer, andwherein the cancer comprises cells carrying a mutation in a gene selected from NRAS, PTEN, CDKN2A, ERBB2, or any combination thereof.

19. Use of a conjugate comprising (i) an Erwinia L-asparaginase and (ii) polyethylene glycol (PEG) in the manufacture of a medicament for treating a colon cancer in a patient,wherein the PEG has a molecular weight less than or equal to about 5000 Da,wherein the L-asparaginase has an amino acid sequence comprising SEQ ID NO:1, and wherein the colon cancer comprises cells carrying a mutation in a gene selected from NRAS, PTEN, CDKN2A, ERBB2, or any combination thereof.

20. A method of treating a colon cancer in a patient comprising administering to the patient aneffective amount of a conjugate comprising:(a) an Erwinia L-asparaginase protein having an amino acid sequence comprising SEQ ID NO:1, linked to (b) a polyethylene glycol (PEG) molecule having a molecular weight less than or equal to about 5000 Da, wherein the colon cancer comprises cells carrying a mutation in a gene selected from NRAS,PTEN, CDKN2A, ERBB2, or any combination thereof.

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