Preparations containing SADA complex

Abstract

Formulations are disclosed that include SADA complexes. The formulations provide sufficient shelf life without excessive degradation or multimerization of the SADA complexes. Use of the formulations to treat cancer is further disclosed.

Description

[Technical field]

[0001] This specification contains a Sequence Listing in computer readable format that has been submitted with this application, which forms part of this disclosure and is incorporated herein in its entirety.

[0002] The present invention relates to a composition comprising a SADA complex, said SADA complex being stabilized in a tetrameric form. Preferably, the composition is a pharmaceutical composition.

[0003] The present invention further relates to the treatment of cancer using the compositions of the present invention. [Background technology]

[0004] Protein drugs are typically formulated as aqueous preparations that contain ingredients that stabilize the protein to ensure adequate shelf life.

[0005] Self-assembling and disassembling (SADA) technology, first disclosed in WO 2018 / 204873, exploits the SADA domain, which has concentration-dependent assembly and disassembly properties. Complexes containing SADA domains typically exist in at least two distinct forms, a tetrameric form at high concentrations and a monomeric form at low concentrations.

[0006] The SADA conjugates may be designed such that the tetrameric form has a molecular weight well above the renal clearance limit and the monomeric form has a molecular weight below the renal clearance limit, meaning that the tetrameric form has a high plasma half-life and the monomeric form has a low plasma half-life. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2018 / 204873 Summary of the Invention [Problem to be solved by the invention]

[0008] Because the SADA complex is administered primarily in tetrameric form, its formulation poses a challenge, as the formulation should not only provide sufficient stability to the protein, but also ensure that the SADA complex remains in tetrameric form without excessive degradation to monomers or agglomeration to multimers. [Means for solving the problem]

[0009] Summary of the Invention The present disclosure provides a composition comprising a SADA domain as part of a SADA complex that allows for effective delivery of a payload to a desired target site while minimizing the risk of off-target interactions. For optimal delivery of a payload, it is desirable for the SADA complex in tetrameric form to be highly stable in the composition / solution. However, ensuring the stability of the composition is a challenge. The challenge is to ensure that the composition contains predominantly SADA complexes in a high order tetramerization state, that the SADA complexes remain in tetrameric form, while avoiding their multimerization, aggregation and precipitation, and loss of product.

[0010] It is desirable to administer the SADA complex in tetrameric form, since the tetrameric form, which has a size far above the renal clearance limit, remains in the blood circulation for a sufficient time to allow binding to the site of interest, while the monomeric form is quickly lost from the circulation because its size is below the renal clearance. Taken together, this provides a particularly desirable property of the SADA complex, which when administered in tetrameric form, remains in the circulation long enough to bind to the site of interest, and the complex that does not bind to the target gradually degrades into a monomer that is lost via the kidney. The present disclosure provides a composition that ensures the necessary stability of the SADA complex.

[0011] In a first aspect, the present invention provides an aqueous composition comprising: a. A SADA complex comprising a SADA domain and at least one additional domain in an amount of 5-50 g / L; b. Buffer system; c. one or more stabilizers; and d. one or more surfactants Including, The pH is in the range of 5-6, the SADA complex is predominantly in the tetrameric form, and the ionic strength is in the range of 5-150 mM. Concerning the composition.

[0012] Surprisingly, it is realized that the formulation is capable of stabilizing the SADA complex upon storage and furthermore is capable of maintaining the SADA complex predominantly in a tetrameric form.

[0013] The SADA conjugate preferably comprises a SADA domain and two binding sites, one capable of binding to a tumor antigen and the other binding site capable of binding to a chelating agent that complexes with a metal ion, which may be DOTA or a compound containing the DOTA ring system.

[0014] In a second aspect, the present invention relates to the use of a composition of the invention for treating or diagnosing cancer.

[0015] In a preferred embodiment, the present invention provides a method for producing a pharmaceutical composition comprising the steps of: a. administering a composition of the invention to a patient in need of treatment or diagnosis; and b. after a period of time, administering a DOTA compound conjugated to a radionuclide; The present invention relates to the use of the composition of the present invention in a method comprising the steps of:

[0016] In a third aspect, the present invention relates to a kit comprising the composition of the invention and preferably instructions for use, and / or DOTA conjugated to a radionuclide.

[0017] Further aspects are provided in the claims. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The present invention relates to the self-assembly and disassembly (SADA) technology described in the International Patent Application having the publication number WO 2018 / 204873, which is incorporated herein by reference. This technology is based on SADA domains, which are small polypeptides with concentration-dependent self-assembly and disassembly properties. Examples of SADA polypeptides are polypeptides that contain the tetramerization domains of p53, p63, p76, hnRNPC, SNAP-23, Stefin B, KCNQ4, CBFA2T1, and any other examples of such polypeptides provided in said International Patent Application without limitation.

[0019] According to the present specification, a SADA complex is intended to mean a polypeptide comprising a SADA domain and at least one additional domain.

[0020] SADA complex is self-assembled, forms multimer, especially tetramer, at high concentration, and breaks down into monomer at low concentration.This has the consequence that when SADA complex in tetramer form is administered to patient, it is diluted in plasma and gradually breaks down into monomer.When SADA complex is designed such that multimer form has a size above renal clearance limit and monomer has a size below renal clearance limit, multimer has a long plasma half-life, while monomer has a short plasma half-life.

[0021] For SADA complexes that contain a binding site that binds to tissue antigens, the SADA complexes rapidly bind to antigen targets and are localized to the target tissue, while unbound SADA complexes are rapidly degraded and removed from plasma by renal clearance.

[0022] Some embodiments of the invention are set forth in the claims.

[0023] According to one embodiment, the present invention provides an aqueous composition comprising: a. A SADA complex comprising a SADA domain and at least one additional domain in an amount of 5-50 g / L; b. Buffer system; c. one or more stabilizers; and d. one or more surfactants Including, The pH is in the range of 5 to 6, and the ionic strength is in the range of 5 to 150 mM. Concerning the composition.

[0024] Preferably, the SADA complex is predominantly in a multimeric / tetrameric form.

[0025] The formulation of the present invention has the advantage of ensuring the stability of the composition. The inventors have found that the formulation of the present invention ensures high stability of the SADA complex and can maintain the tetrameric form of the SADA complex during storage, further protecting the protein from proteolysis. Thus, the composition of the present invention provides a solution of tetrameric SADA complex that remains in tetrameric form, reducing proteolysis after and during storage. Thus, the formulation of the present invention provides SADA molecules with the desired high shelf life.

[0026] According to one embodiment, the present invention relates to a composition of the present invention, the ionic strength of which is in the range of 5 to 150 mM, 10 to 135 mM, 20 to 120 mM, or 25 to 100 mM.

[0027] According to one embodiment, the present invention relates to a composition of the present invention comprising an amount of SADA complex selected from the group consisting of 5-50 g / L, 6.25-45 g / L, 7.5-40 g / L, 9.75-35 g / L, 10-20 g / L, preferably 10-15 g / L.

[0028] According to one embodiment, the SADA complex comprises two binding sites and a SADA domain, a first binding site capable of binding to a target antigen and a second binding site capable of binding to a payload, such as a cytotoxic agent, a radionuclide, or a compound capable of binding to a payload.

[0029] In some embodiments, the first and / or second binding site is or comprises an antibody component, such as an antigen-binding fragment of an antibody, an scFv, or a nanobody. Preferably, the first and / or second binding site is an scFv.

[0030] In some embodiments, the first binding site is specific for a cell surface target, for example a tumor antigen.

[0031] According to one embodiment, the binding site specific for a tumor antigen is an anti-GD2, anti-CD20, anti-CD38, anti-Globo H, anti-GPA33, anti-PSMA, anti-polysialic acid, anti-Lewy, anti-LiCAM, anti-HER2, anti-B7H3, anti-CD33, anti-peptide / MHC, anti-glypican 3 or anti-GD3 binding domain.

[0032] Thus, the present invention relates to a composition of the invention, wherein the first binding site is capable of binding to a tumor antigen.

[0033] According to one embodiment, the invention relates to a composition according to the invention, wherein the first binding site is capable of binding to GD2, B7-H3, CD20, GPA33 or CD38.

[0034] GD2 is a disialoganglioside, and disialogangliosides may be considered tumor-associated antigens.

[0035] B7-H3, also known as CD276, is an immune checkpoint molecule and costimulatory / co-inhibitory immunoregulatory protein; B7-H3 may be considered a tumor-associated antigen.

[0036] CD20 is a membrane-embedded surface molecule and CD20 can be considered a tumor-associated antigen.

[0037] GPA33 is a glycoprotein and a cell surface antigen, and GPA33 can be considered a tumor-associated antigen.

[0038] CD38, also known as cyclic ADP ribose hydrolase, is a glycoprotein and CD38 may be considered a tumor-associated antigen.

[0039] According to one embodiment, the present invention relates to a method for the preparation of a polypeptide comprising the steps of: a. Contains the CDR sequences of SEQ ID NOs: 1 to 6, and b. has at least 90%, 95%, 96%, 97%, 98% or preferably at least 95% sequence identity to SEQ ID NO:7 The present invention relates to a composition of matter comprising a sequence.

[0040] According to one preferred embodiment, the first binding site is capable of binding to GD2 and comprises the sequence of SEQ ID NO:7.

[0041] According to one embodiment, the present invention relates to a method for the preparation of a polypeptide comprising the steps of: a. a sequence having at least 90%, 95%, 96%, 97%, 98% or preferably at least 95% sequence identity to SEQ ID NO:8, and b. A sequence having at least 90%, 95%, 96%, 97%, 98% or preferably at least 95% sequence identity to SEQ ID NO:9 The present invention relates to a composition of matter comprising:

[0042] CD38, also known as cyclic ADP ribose hydrolase, is a glycoprotein and CD38 may be considered a tumor-associated antigen.

[0043] According to one embodiment, the present invention relates to a method for the preparation of a polypeptide comprising the steps of: a. comprises the CDR sequences of SEQ ID NOs: 29-34, and b. has at least 90%, 95%, 96%, 97%, 98% or preferably at least 95% sequence identity to SEQ ID NO: 35; The present invention relates to a composition of matter comprising a sequence.

[0044] Preferably, the first binding site in this embodiment is capable of binding to CD38.

[0045] In a preferred embodiment, the first binding site is capable of binding to CD38 and comprises the sequence of SEQ ID NO:35.

[0046] According to another embodiment, the present invention relates to a method for the preparation of a polypeptide comprising the steps of: a. a sequence having at least 90%, 95%, 96%, 97%, 98% or preferably at least 95% sequence identity to SEQ ID NO: 36, and b. A sequence having at least 90%, 95%, 96%, 97%, 98% or preferably at least 95% sequence identity to SEQ ID NO: 37 The present invention relates to a composition of matter comprising:

[0047] According to one embodiment, the present invention relates to a composition of the invention, wherein the second binding site is capable of binding to a chelating agent.

[0048] In principle, any chelating agent may be used in the present invention, provided that the second binding moiety is capable of binding to said chelating agent.

[0049] According to one embodiment, the present invention relates to a compound in which the second binding moiety is capable of binding to DOTA or to a compound comprising a DOTA ring system, or where DOTA is capable of binding to a metal ion, e.g. 175 Lu 3+ Or 177 Lu 3+ When chelated with lutetium, such as lutetium ionomer, the present invention relates to compositions capable of binding to DOTA or to compounds containing the DOTA ring system.

[0050] DOTA (dodecanetetraacetic acid) is also known as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid and is C 16 H 28 It has the formula (CH2CH2NCH2CO2H)4, also known as N4O8·xH2O.

[0051] A compound comprising a DOTA ring system is intended herein to mean a compound comprising DOTA to which an additional group or moiety is attached. Examples of such compounds include benzyl DOTA and the bispecific chelators disclosed in WO 2019 / 010299, which is incorporated by reference.

[0052] According to one embodiment, the present invention relates to a method for the preparation of a medicament for the treatment of cancer, the medicament comprising the step of: a. Contains the CDR sequences of SEQ ID NOs: 23 to 28; b. comprises a polypeptide having at least 90%, 95%, 96%, 97%, 98% or preferably at least 95% sequence identity to SEQ ID NO: 35 The present invention relates to a composition.

[0053] Preferably, the second binding site in this embodiment is a DOTA-metal, i.e., a metal ion, such as lutetium, preferably Lu. 3+ It is possible to bind DOTA which chelates

[0054] According to this embodiment, the present invention provides a method for the preparation of a medicament for a medicament comprising the steps of: a. a sequence having at least 90%, 95%, 96%, 97%, 98% or preferably at least 95% sequence identity to SEQ ID NO: 10, and b. A sequence having at least 90%, 95%, 96%, 97%, 98% or preferably at least 95% sequence identity to SEQ ID NO: 11 The present invention relates to a composition of matter comprising:

[0055] According to one embodiment, the invention relates to a composition of the invention, wherein the SADA domain comprises a sequence as disclosed in SEQ ID NOs: 12 to 19 or a sequence which differs from one of the sequences of SEQ ID NOs: 12 to 19 by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions.

[0056] A p53 tetramerization domain comprising the sequence of SEQ ID NO: 12, more preferably comprising amino acids 6 to 36 of SEQ ID NO: 12, is a preferred SADA domain.

[0057] SADA complexes, according to the present invention, may contain additional elements, including, but not limited to, linkers separating different portions of the complex, antibody fragments that are separate from the binding site, such as constant regions, and antigen fragments that are capable of binding to and eliciting an effector or immune response.

[0058] According to one embodiment, the SADA conjugate comprises a linker.

[0059] A linker, sometimes known as a spacer, is a short amino acid sequence engineered to separate two domains in a single polypeptide, allowing the two domains to fold and function without steric hindrance from adjacent domains. Linkers are known in the art, and the present invention is not limited to any particular sequence of a linker. In general, the purpose of a linker is to connect and / or separate different elements of a complex, and typically consists mainly of small hydrophilic amino acids, such as glycine, serine, and threonine.

[0060] According to one embodiment, the invention relates to a composition according to the invention, wherein the SADA complex comprises a linker having a sequence selected from SEQ ID NO: 20 multiplied by an integer from 1 to 6.

[0061] Another suitable linker that may be used in the present invention is an IgG3 spacer domain, for example the IgG3 spacer domain disclosed in SEQ ID NO:21.

[0062] In some embodiments, the SADA complex consists of a SADA domain and two binding sites, e.g., scFv. In some embodiments, the SADA complex comprises an anti-GD2 scFv-anti-DOTA scFv-p53 tetramerization domain connected by a linker and / or spacer. In some embodiments, the SADA complex has the following structure: anti-GD2 light chain Fv-anti-GD2 heavy chain Fv-anti-DOTA heavy chain Fv-anti-DOTA light chain Fv-p53 tetramerization domain connected by a linker and / or spacer.

[0063] Examples of suitable SADA conjugates according to the present invention include a GD2-SADA conjugate comprising the amino acid sequence of SEQ ID NO:22, a CD38-SADA conjugate comprising the amino acid sequence of SEQ ID NO:38, a B7-H3-SADA conjugate comprising the amino acid sequence of SEQ ID NO:39, a CD20-SADA conjugate comprising the amino acid sequence of SEQ ID NO:40, and a GPA33-SADA conjugate comprising the amino acid sequence of SEQ ID NO:41.

[0064] According to the invention, the composition of the invention comprises a buffer system, for example an organic acid or an alkali metal salt thereof.

[0065] Preferably, the buffer is selected from acetate, citrate, histidine, citrate-histidine, acetate-histidine and succinate.

[0066] Preferred examples include acetate buffers containing acetic acid and sodium acetate.

[0067] Sodium acetate is also known as sodium acetate and has the formula CH3COONa.

[0068] According to one embodiment, the present invention relates to a composition of the present invention comprising a buffer in an amount selected from 5 to 30 mM, 10 to 25 mM, preferably in an amount of 20 mM.

[0069] According to one embodiment, the stabilizing agent is selected from polyols, in particular sugar alcohols and non-reducing sugars.

[0070] Preferred examples include sucrose, trehalose, sorbitol, glycerol and inositol.

[0071] The stabilizer maintains or extends the time while the active pharmaceutical ingredient maintains a desired property during storage.

[0072] According to one embodiment, the present invention relates to a composition according to the invention comprising a stabilizing agent, preferably sucrose, in an amount selected from the group consisting of 200-600 mM, 225-500 mM, 250-300 mM, preferably in an amount of 275 mM.

[0073] The present invention also relates to a composition wherein the surfactant is a non-ionic surfactant.

[0074] Non-ionic surfactants may comprise / consist of undissociated long chain polymers consisting of a hydrophilic head group and a hydrophobic tail.

[0075] According to one embodiment, the present invention relates to a composition wherein the surfactant is polyethylene glycol sorbitan monolaurate, poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) or polyethylene glycol sorbitan monooleate, polyoxyethylene sorbitan monooleate.

[0076] Polyethylene glycol sorbitan monolaurate, also known as polyoxyethylene sorbitan monolaurate, is known in the art. A preferred polyethylene glycol sorbitan monolaurate is commercially available as Polysorbate® 20 or TWEEN® 20.

[0077] Polyethylene glycol sorbitan monooleate, also known as polyoxyethylene sorbitan monooleate, is known in the art. A preferred polyethylene glycol sorbitan monooleate is commercially available as Polysorbate® 80 or TWEEN® 80.

[0078] Poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) are also known in the art. A preferred poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) is commercially available as Kolliphor® P188 or Poloxamer® 188.

[0079] Polyethylene glycol sorbitan monolaurate is a preferred surfactant for use in the present invention.

[0080] According to one embodiment, the present invention relates to a composition comprising a surfactant in an amount selected from 0.1-0.3 g / L, 0.15-0.25 g / L, 0.16-0.24 g / L, 0.17-0.23 g / L, 0.18-0.22 g / L, 0.19-0.21 g / L, preferably 0.20 g / L.

[0081] The composition of the present invention may have a pH selected from 5 to 6, 5.1 to 5.9, 5.2 to 5.8, 5.3 to 5.7, 5.4 to 5.6, preferably a pH of 5.5.

[0082] The compositions of the present invention may further comprise an antioxidant.

[0083] Antioxidants can be added to the composition to protect the contents from damage caused by oxidative stress. This can be beneficial for proteins that contain amino acids that are sensitive to oxidation, and can be particularly beneficial for proteins that contain amino acids that are sensitive to oxidation, where the amino acids are exposed on the surface of the protein. Examples of amino acids that are sensitive to oxidation include residues such as methionine and (free) cysteine.

[0084] A preferred antioxidant for use in the present invention is methionine.

[0085] According to one embodiment, the present invention relates to a composition comprising an antioxidant such as methionine in an amount selected from the group consisting of 5-15 mM, 6-14 mM, 7-13 mM, 8-12 mM, 9-11 mM, preferably in an amount of 10 mM.

[0086] The present invention further relates to compositions according to the invention which are salt-free or contain only low concentrations of salt, for example less than 50 mM.

[0087] The inventors appreciate that salts generally destabilize the SADA complex and therefore it is preferred to limit the amount of salts such as NaCl, KCl or similar salts in the present compositions.

[0088] A preferred composition of the present invention comprises a. SADA complex in an amount of 15 g / L; b. Sodium acetate in an amount of 20 mM; c. sucrose in an amount of 275 mM; d. Polysorbate 20 in an amount of 0.2 g / L; Including, At pH 5.5, the SADA complex is predominantly in a tetrameric form.

[0089] Optionally, the composition further comprises 10 mM methionine.

[0090] The term predominantly tetrameric form is intended in the present specification and claims to mean that the majority of the SADA complexes are in tetrameric form, e.g., at least 50% w / w; at least 60% w / w; at least 70% w / w; at least 80% w / w; at least 90% w / w; or at least 95% w / w is in tetrameric form.

[0091] According to one embodiment, the composition of the invention is a pharmaceutical composition.

[0092] The SADA conjugates of the present invention can be prepared using methods known in the art.

[0093] In a preferred embodiment, a nucleic acid encoding the desired amino acid sequence of the complex is provided. A construct containing the nucleic acid sequence with the necessary regulatory elements to direct expression in a selected host organism, e.g., promoter, signal sequence, ribosomal recognition site, Kozak sequence, enhancer, terminator, polyadenylation site, etc., is prepared and inserted into the selected host organism, and the selected host organism is grown under conditions conducive to expression of the SADA complex. Finally, the SADA complex is recovered from the growth broth using well-known separation and recovery techniques.

[0094] The formulations of the present invention may be prepared by dissolving the SADA conjugate and other ingredients in sterile water using methods known in the art.

[0095] The present invention further relates to the use of the compositions of the present invention for treating or diagnosing cancer.

[0096] According to one embodiment, the compositions of the invention may be used to treat or diagnose cancers expressing tumor antigens recognized by SADA conjugates, such as GD2, CD38, B7-H3, CD20 or GPA33.

[0097] The cancer may be selected from neuroblastoma, melanoma, sarcoma, brain tumor or carcinoma.

[0098] According to one embodiment, the invention relates to the use of the composition of the invention, wherein said cancer is selected from among osteosarcoma, liposarcoma, fibrosarcoma, malignant fibrous histiocytoma, leiomyosarcoma, spindle cell sarcoma, brain tumor, small cell lung cancer, retinoblastoma, HTLV-1-infected T-cell leukemia, and other tumors positive for GD2, CD38, B7-H3, CD20 or GPA33.

[0099] According to one embodiment, the present invention comprises: a. administering a composition of the invention to a patient in need of treatment or diagnosis; and b. after a period of time, administering a DOTA compound containing a radionuclide; The present invention relates to the use of a composition according to any of the preceding claims in a method comprising the steps of:

[0100] The duration of step b is typically selected from 48 hours to 72 hours, for example from 50 hours to 65 hours or from 55 hours to 60 hours. Preferably, the duration is selected to allow the majority of unbound SADA complexes to degrade and be removed from the bloodstream.

[0101] The method of the present invention may further comprise administering a removal agent after step a and before step b.

[0102] According to one embodiment, the invention relates to the use of the composition of the invention, wherein the radionuclide is selected among alpha, beta and positron emitting radionuclides.

[0103] According to one embodiment, the present invention relates to a radionuclide comprising: 211 At, 51 Cr, 57 Co, 58 Co, 67 Cu, 152 EU, 67 Ga, 111 In, 59 Fe, 212 Pb, 177 Lu, 223 Ra, 224 Ra, 186 Re, 188Re, 75 Se, 99m Tc, 227 Th, 89 Zr, 90 Y, 94m Tc, 64 Cu, 68 Ga, 66 Ga, 86 Y, 82 Rb, 110m In, 209 Bi, 211 Bi, 212 Bi, 213 Bi, 210 Po, 211 Po, 212 Po, 214 Po, 215 Po, 216 Po, 218 Po, 211 At, 215 At, 217 At, 218 At, 221 Fr, 223 Ra, 224 Ra, 226 Ra, 225 Ac, 227 Ac, 227 Th, 228 Th, 229 Th, 230 Th, 232 Th, 231 Pa, 233 U, 234 U, 235 U, 236 U, 238 U, 237 Np, 238 Pu, 239 Pu, 240 Pu, 244 Pu, 241 Am, 244 Cm, 245 Cm, 248 Cm, 249 Cf and 252 Cf.

[0104] Preferred examples of radionuclides for use in the present invention include: 177 Lu, 99 mTc,64 Cu, 90 Y and 89 Zr is an example.

[0105] According to one embodiment, the invention relates to a kit comprising a composition of the invention and a DOTA compound.

[0106] Typically the kit further comprises instructions for use or at least information to the user as to where to find the information.

[0107] According to one embodiment, the present invention relates to a kit of the present invention further comprising a radionuclide.

[0108] [Definition] Antibody fragment: An antibody fragment is a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of structure, an antibody fragment binds to the same antigen recognized by the intact antibody. For example, the 3F8 monoclonal antibody fragment binds to the epitope recognized by 3F8. The term "antibody fragment" also includes any synthetic or engineered protein that acts like an antibody by binding to a specific antigen to form a complex. For example, antibody fragments include isolated fragments consisting of variable regions, such as "Fv" fragments consisting of heavy and light chain variable regions, recombinant single chain polypeptide molecules in which light and heavy variable regions are connected by a peptide linker ("scFv protein"), and minimal recognition units consisting of amino acid residues that mimic the hypervariable region.

[0109] DOTA: DOTA (dodecanetetraacetic acid) is also known as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid. 16 H 28 It has the formula (CH2CH2NCH2CO2H)4, also known as N4O8·xH2O.

[0110] DOTA metal chelate: refers to DOTA having a complex bound to a metal ion.

[0111] Derivatives of DOTA: is intended to mean compounds that contain a DOTA ring system and are capable of chelating metal ions. Examples of such compounds include benzyl DOTA and the bispecific chelators disclosed in WO 2019 / 010299. Additional DOTA derivatives are disclosed in WO 2010 / 099536.

[0112] Radioisotopes: Examples of radioisotopes that can be conjugated to antibodies for diagnostic and therapeutic uses include: 211 At, 14 C. 51 Cr, 57 Co, 58 Co, 67 Cu, 152 EU, 67 Ga, 3 H, 111 In, 59 Fe, 177 Lu, 32 P, 223 Ra, 224 Ra, 186 Re, 188 Re, 75 Se, 35 S, 99m Tc, 227 Th, 89 Zr, 90 Y, 123 I, 124 I, 125 I, 131 I, 94m Tc, 64 Cu, 68 Ga, 66 Ga, 76 Br, 86 Y, 82 Rb, 110m In, 13 N, 11 C. 18 Non-limiting examples of alpha-emitting particles include: 209 Bi, 211 Bi, 212 Bi, 213 Bi, 212 Pb, 210 Po, 211 Po,212 Po, 214 Po, 215 Po, 216 Po, 218 Po, 211 At, 215 At, 217 At, 218 At, 218 Rn, 219 Rn, 220 Rn, 222 Rn, 226 Rn, 221 Fr, 223 Ra, 224 Ra, 226 Ra, 225 Ac, 227 Ac, 227 Th, 228 Th, 229 Th, 230 Th, 232 Th, 231 Pa, 233 U, 234 U, 235 U, 236 U, 238 U, 237 Np, 238 Pu, 239 Pu, 240 Pu, 244 Pu, 241 Am, 244 Cm, 245 Cm, 248 Cm, 249 Cf and 252 Cf are included.

[0113] Treatment: As used herein, the terms "treatment", "treat", "treated" or "treating" refer to prophylactic and / or therapeutic approaches, particularly where the progression of an undesirable physiological change or disorder in a subject, such as multiple sclerosis, should be prevented or slowed (alleviated). Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, reduction in the extent of disease, a stable (i.e., not worsening) state of disease, delay or slowing of disease progression, improvement or palliation of the disease state, and remission (whether partial or total). "Treatment" can also mean prolonged survival compared to expected survival in the absence of treatment. Those in need of treatment include those already with the condition or disorder, as well as those prone to having the condition or disorder, or those in whom the condition or disorder is to be prevented.

[0114] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" is intended to mean a composition for administering a drug or medicine to a patient in need thereof. Pharmaceutical compositions are prepared from pharmaceutical grade ingredients using methods and techniques known in the pharmaceutical or pharmacological field, for example as described in the European Pharmacopoeia, 10th edition.

[0115] Sequence identity: The term sequence identity is intended to mean a measure of the relatedness of two nucleic acid or amino acid sequences. Sequence identity is determined by aligning two sequences, finding the longest overlap, counting the number of matches in the overlap, and dividing the number of matches by the number of nucleotide or amino acid residues in the overlap to calculate the sequence identity. Sequence identity is typically expressed as a percentage (%).

[0116] A variety of computational algorithms are available to those skilled in the art for generating sequence alignments and calculating sequence identity. As used herein, sequence alignment refers to pairwise alignment. Several algorithms perform this, including the sequence alignment program Clustal Omega [doi:10.1038 / msb.2011.75].

[0117] As used herein, sequence alignment is performed using the following algorithm: Algorithm: Clustal Omega (1.2.4), (http: / / www.clustal.org / omega / ). [Brief description of the drawings]

[0118] [Figure 1] The results of two mass spectrometric measurements of the GD2-SADA construct at concentrations of 100 nM and 5 nM are shown. For further details, see Example 1. [Diagram 2] 1 shows the concentration-dependent distribution of monomers, dimers and tetramers of the GD2-SADA construct. For further details, see Example 1. [Diagram 3]

[0023] Figure 1 shows the design of Part A of the Phase I study of GD2-SADA. For further details, see Example 10. [Figure 4] Figure 1 shows the effect of tetramerization on the tumor killing effect of GD2-SADA conjugates. For further details, see Example 9. [Diagram 5]SPECT images of tumor-implanted IMR-32 athymic nude mice receiving GD2-SADA (>90% tetramer, 10 mg / kg, IV) (top panel), GD2 monomer (2.4 mg / kg, IV) (middle panel) and GD2 monomer (9.6 mg / kg) (bottom panel) followed by 177Lu-DOTA are shown. In each panel, the first mouse was scanned 2 hours after administration of 177Lu-DOTA, and subsequent mice were scanned 24, 48 and 120 hours after administration of 177Lu-DOTA, respectively. For further details, see Example 9. [Sequences] SEQ ID NOs: 1 to 6: CDR sequence of GD2-binding antibody 3F8 equivalent to the CDR sequence of GD2-scFv SEQ ID NO: 7: anti-GD2 scFv SEQ ID NO: 8: anti-GD2 VL SEQ ID NO: 9: anti-GD2 VH SEQ ID NO: 10: huC825 VL SEQ ID NO: 11: huC825 VH SEQ ID NOs: 12 to 19: SADA domain disclosed in WO 2018 / 204873 SEQ ID NO: 20: linker sequence SEQ ID NO: 21: IgG3 spacer sequence SEQ ID NO: 22: GD2-SADA complex SEQ ID NOs: 23 to 28: CDR sequence of C825 SEQ ID NOs: 29 to 34: CDR sequence of CD38-binding antibody SEQ ID NO: 35: anti-CD38 scFv SEQ ID NO: 36: anti-CD38 VL SEQ ID NO: 37: anti-CD38 VH SEQ ID NO: 38: CD38-SADA complex SEQ ID NO: 39: B7-H3-SADA complex SEQ ID NO: 40: CD20-SADA complex SEQ ID NO: 41: GPA33-SADA complex All cited references are incorporated by reference.

[0119] The accompanying drawings and examples are provided to illustrate, not to limit, the present invention. It is apparent to one skilled in the art that the aspects, embodiments, claims and any items of the present invention may be combined.

[0120] All percentages are on a weight / weight basis unless otherwise stated. All measurements are made under standard conditions (ambient temperature and pressure) unless otherwise stated. Test conditions comply with European Pharmacopoeia 10.0 unless otherwise stated.

[0121] [Methods and Materials] GD2-SADA complex: This complex comprises an anti-GD2 scFv domain, a humanized C825 domain and a P53 SADA domain, which is disclosed in WO 2018 / 204873 as SEQ ID NO: 31 and in the present patent application as SEQ ID NO: 22.

[0122] CD38-SADA complex: This complex comprises an anti-CD38 scFv domain, a humanized C825 domain and a P53 SADA domain. This complex is disclosed in (unpublished Danish patent application) DK 2021 / 70621 as SEQ ID NO: 41 and in this patent application as SEQ ID NO: 38.

[0123] B7-H3-SADA complex: This complex comprises an anti-B7-H3 scFv domain, a humanized C825 domain and a P53 SADA domain. This complex has the amino acid sequence shown in SEQ ID NO:39.

[0124] CD20-SADA complex: This complex comprises an anti-CD20 scFv domain, a humanized C825 domain and a P53 SADA domain. This complex is disclosed in (unpublished Danish patent application) DK 2021 / 70621 as SEQ ID NO: 42 and in this patent application as SEQ ID NO: 40.

[0125] GPA33-SADA complex: This complex comprises an anti-GPA33 scFv domain, a humanized C825 domain and a P53 SADA domain. This complex is disclosed in (unpublished Danish patent application) DK 2021 / 70621 as SEQ ID NO: 61 and in this patent application as SEQ ID NO: 41.

[0126] [Surfactants] · Polysorbate 20: TWEEN 20®, polyethylene glycol sorbitan monolaurate, polyoxyethylene sorbitan monolaurate. · Polysorbate 80: TWEEN 80®, polyethylene glycol sorbitan monolaurate, polyoxyethylene sorbitan monolaurate. Poloxamer 188: Poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol). · Kollifor: Poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol). EXAMPLES

[0127] Example 1: Effect of SADA concentration on the relative number of oligomeric forms measured by GD2-SADA In this example, a Refeyn mass photometer (Refeyn Ltd, Oxford UK) was used according to the manufacturer's instructions with the following settings: Measurement principle: Label-free / interference light scattering Mass range: 4kDa~5MDa Measurement accuracy: + / -2% Single measurement mass error + / -5% Resolution (FWHM) 25kDa@66kDa, 88kDa@660kDa Concentration range: 100pM to 100nM (particle concentration) Sensitivity: Protein <1ng Sample volume: 5-20μl Frame rate / standard setting) 1kHz (raw), 100Hz (integrated) Field of view: 3×10μm (@1kHz) to 10×10μm (@300Hz) ·Wavelength 525nm -Pixel size 21nm

[0128] The GD2-SADA construct was diluted in PBS and incubated for 60 min at 37° C. before size distribution was measured. The following dilutions were made: 150 nM, 100 nM, 50 nM, 10 nM, 5 nM, 1 nM and 100 pM and subjected to mass spectrometer using a Refeyn mass spectrometer.

[0129] An exemplary spectrogram is shown in Figure 1, with the top one being 100nM and the bottom one being 5nM. In this spectrogram, three peaks can be seen, representing monomeric, dimeric and tetrameric GD2-SADA, and at 100nM concentration, a broader peak can also be seen around 500kDa, representing more multimers of GD2-SADA. It can be further seen that at high concentrations (100nM), a high proportion of GD2-SADA constructs are present in tetrameric form, while at low concentrations, the majority are present in monomeric form.

[0130] FIG. 2 shows the concentration-dependent distribution of the GD2-SADA construct.

[0131] Example 2: Effect of buffer and pH GD2-SADA was prepared in 20 mM buffer at the pH shown in the table below. The stability of the construct was determined by nanoDSF, while Tm indicates the temperature at which 50% of the protein is unfolded, meaning that a higher temperature indicates a higher stability. Measurements were performed in quadruplicate. The following results were obtained:

[0132] [Table 1]

[0133] The results showed that acetate and histidine offered greater stability than citrate and succinate buffers. pH values ​​>5.5 were found to offer the best stability.

[0134] [Example 3] Effects of salt and sucrose GD2-SADA was prepared in 20 mM buffer at pH 5.5 and salt (sodium chloride) or sucrose added as indicated. The stability of the construct was determined by nanoDSF, while Tm indicates the temperature at which 50% of the protein is unfolded, meaning that higher temperatures indicate higher stability. Measurements were performed in quadruplicate. The following results were obtained:

[0135] [Table 2]

[0136] The results showed that sucrose stabilized the construct, whereas salt (sodium chloride) destabilized it.

[0137] Example 4: Effect of surfactants in accelerated aging studies An exemplary formulation was prepared that contained 20 mM acetate buffer and 275 mM sucrose, respectively, and further contained the following:

[0138] [Table 3]

[0139] The solutions were stored at 40° C. for 1 or 2 weeks, after which the purity was analyzed by size exclusion HPLC and the purity loss and main recovery of the main peak were calculated based on the recovery before incubation. The results are shown in Tables 4 and 5 below.

[0140] [Table 4]

[0141] [Table 5]

[0142] The results showed that surfactants improved the average recovery. In this example, better recoveries were obtained by using polysorbate 20 / 80 or high concentrations of poloxamer 188.

[0143] [Example 5] GD2-SADA formulation An exemplary formulation of GD2-SADA was made utilizing the findings of Examples 1 to 4. Additionally, methionine was added as an antioxidant to protect M199 and polysorbate from oxidation.

[0144] [Table 6]

[0145] [Example 6] Suitability of GD2-SADA formulation To demonstrate the suitability of the formulation for clinical administration, an in-use stability study was performed to demonstrate suitable product recovery and stability of the GD2-SADA formulation of Example 6 at room temperature over a 4-hour period, including the time of administration.

[0146] The study covered a range of concentrations from 0.05mg / mL to 10mg / mL. Dilutions of GD2-SADA were made in saline (NaCl 0.9%). The 50mL IV bag containing the GD2-SADA dilutions was connected to an infusion set and an infusion filter. Samples were taken by passing the GD2-SADA dilutions from the 50mL IV bag through the infusion set and filter.

[0147] Purity, potency, physicochemical and particle results were all within expected ranges and comparable at T=0 and T=4 hours.

[0148] [Table 7] TIFF2024546804000009.tif70168

[0149] Protein recoveries were 99 and 106% at the concentrations tested and were calculated as the percentage difference between the observed and expected theoretical concentrations.

[0150] To assess the potential effect of surface adhesion to the injection material under worst case conditions, the lowest dose concentration (0.05 mg / mL) was prepared by use of a precision pipette to minimize variance due to preparation procedures that would not typically affect the clinically administered dose, where the entire volume is injected. This experiment confirmed a protein recovery of 106%, indicating that GD2-SADA surface adhesion to the injection material is negligible.

[0151] In conclusion, the formulation used was evaluated to be stable within the tested concentration range of 0.05 mg / mL to 10 mg / mL, as well as during storage and handling for up to 4 hours at room temperature for GD2-SADA.

[0152] Example 7: Shelf-life supporting stability Stability results from the supportive shelf-life studies are summarized in Table 8 for the long-term stability study (5±3° C.) and Table 9 for the accelerated conditions (25±2° C.).

[0153] Long-term stability studies show that GD2-SADA is stable in the tetrameric form (>94%) for at least 9 months at 2-8° C. in a formulation containing 20 mM sodium acetate buffer, 275 mM sucrose, and 0.2 g / L polysorbate 20 at a pH of 5.5. In addition, accelerated stability data shows that GD2-SADA is stable in the tetrameric form (>94%) for at least 3 months at 25° C.

[0154] In addition, stability data indicates that GD2-SADA is stable in this formulation with respect to potency, purity and impurities.

[0155] [Table 8] TIFF2024546804000011.tif73162

[0156] [Table 9] TIFF2024546804000013.tif89162

[0157] Example 8: Thermal stability of additional SADA conjugates The sample solutions were centrifuged at 20,000×g for 1 h at 4° C. in a tabletop centrifuge. The supernatants were buffer exchanged into storage buffer (histidine and acetate with and without 150 mM NaCl) and the samples were further diluted to 10 μM. Sucrose was spiked in all conditions with the target sucrose concentration. Each sample was analyzed using nanoDSF (Protein Unfolding Temp. m The fluorescence intensity was measured in duplicate by 350 / 330 nm ratio for each antibody. Five molecules were included in this study. These molecules have the same DOTA binding and P53 sequences but different antigen binding sites, e.g., GD2, CD38, B7H3, CD20 and GPA33.

[0158] As shown in Tables 10 and 11, 150 mM NaCl has a negative effect on the thermal stability of all investigated SADA molecules, as indicated by the decreased Tm values ​​compared to the buffer group (acetate at pH 5.5 and histidine at pH 6.0). In addition, 275 mM sucrose has a positive effect on the thermal stability of all investigated SADA molecules, as indicated by the increased Tm values ​​compared to the buffer group (acetate at pH 5.5 and histidine at pH 6.0). This salt destabilizing and sugar stabilizing effect is universal in all investigated SADA molecules, despite the different antigen binding sites. Therefore, we conclude that the salt destabilizing and sugar stabilizing effects are mainly induced by DOTA binding and P53 moiety.

[0159] [Table 10]

[0160] [Table 11]

[0161] [Example 9] GD2-SADA tetramer has a better PK profile and therefore better tumor up-to-date information, as well as tumor killing effect.

[0162] To better evaluate the role of the tetramer, we compared the GD2-SADA drug candidate to a true monomeric form, designated (P53- / -)GD2-SADA, in which the entire SADA domain was eliminated, resulting in a final protein size of approximately 54 kDa.

[0163] GD2-SADA and (P53- / -)GD2-SADA were compared in terms of plasma pharmacokinetics and antitumor efficacy. In summary, the results demonstrate that GD2-SADA tetramers have altered plasma exposure profiles (Table 12) and improved therapeutic efficacy of GD2-SADA (Figure 4) compared with monomeric forms of GD2-SADA and (P53- / -)GD2-SADA. This was also supported by SPECT / CT imaging (Figure 5), which demonstrated substantially higher tumor binding and persistence of GD2-SADA compared with (P53- / -)GD2-SADA monomer. Therefore, it is important to keep GD2-SADA in tetrameric form by the composition claimed in the present invention.

[0164] [Table 12]

[0165] Example 10: Phase I study of GD2-SADA in patients with recurrent or refractory metastatic solid tumors known to express GD2, including small cell lung cancer, sarcoma, and melanoma 177 Lu-DOTA drug conjugates.

[0166] The exam is divided into three separate parts: A. Optimize the dose of a safe tumor-targeting protein component (GD2-SADA) and compare GD2-SADA with 177 GD2-SADA dose escalation to optimize the dosing interval between doses of Lu-DOTA.

[0167] B. To establish optimal and safe payload delivery in therapeutics 177 Dose escalation of Lu-DOTA.

[0168] C. Repeat dosing to assess cumulative toxicity signals and safety profile after repeat dosing and to determine the recommended Phase 2 dose.

[0169] The patient population consists of adult and adolescent patients with relapsed or refractory metastatic solid tumors known to express GD2, including small cell lung cancer (SCLC), sarcoma, and melanoma.

[0170] The purpose of the study is to establish a safe dosing schedule.

[0171] The study design for Part A can be seen in Figure 3 and Table 13 below.

[0172] [Table 13]

[0173] Cohort 1: Patients received an intravenous infusion of GD2-SADA on day 1, followed by 177 An intravenous infusion of the Lu-DOTA imaging dose is administered. On day 15, a repeat dose of GD2-SADA is administered, followed by day 20. 177 A therapeutic dose of Lu-DOTA is administered.

[0174] Cohort 2: Patients received an intravenous infusion of GD2-SADA on day 1, followed by 177 An intravenous infusion of the Lu-DOTA imaging dose is administered. On day 15, a repeat dose of GD2-SADA is administered, followed by day 17. 177 A therapeutic dose of Lu-DOTA is administered.

[0175] Cohorts 3-5: Patients were randomized to receive GD2-SADA on the same date as either Cohort 1 or Cohort 2, selected after analysis of data from the first two cohorts. 177 It is administered according to the dosing interval with Lu-DOTA.

[0176] In Part A dosimetry, including tumor absorbed dose and whole body, selected organ and blood dosimetry of absorbed dose will be assessed.

[0177] Part A consists of a 6-week DLT observation period and a follow-up observation period lasting up to 24 weeks after the first treatment.

[0178] GD2-SADA and 177 Assuming a 48-hour interval between Lu-DOTA is selected in Part A, patients will receive an intravenous infusion of GD2-SADA on day 1, followed by an intravenous infusion of Lu-DOTA on day 3. 177 An intravenous infusion of Lu-DOTA is administered. On day 15, a repeat dose of GD2-SADA is administered, followed by day 17. 177 A therapeutic dose of Lu-DOTA is administered.

[0179] Day 43 (GD2-SADA) and Day 45 ( 177 Lu-DOTA) is administered a second treatment cycle.

[0180] The study design for Part B can be seen in Table 14 below.

[0181] [Table 14]

[0182] Part B consists of a 6-week DLT observation period and a follow-up observation period lasting until 24 weeks after the first treatment.

[0183] The treatment schedule for Part C is GD2-SADA and 177 Assume that a 48 hour interval between Lu-DOTA is selected in Part A. Patients will receive an intravenous infusion of GD2-SADA on day 1, followed by an intravenous infusion of GD2-SADA on day 3. 177 An intravenous infusion of the Lu-DOTA imaging dose is administered.

[0184] On day 15, a repeat dose of GD2-SADA was administered, followed by day 17. 177 A therapeutic dose of Lu-DOTA is administered.

[0185] The first treatment cycle (imaging part, followed by treatment part) is planned to have a duration of 6 weeks, and the subsequent cycles (cycles 2 to 5) are planned to have a duration of 4 weeks (or a maximum delay of 8 weeks is allowed within this protocol in case of recovery from the toxicity of the radiation suffered), with GD2-SADA administered on day 1 and IVF on day 3 in each cycle. 177 Lu-DOTA is administered.

[0186] See Table 15 for treatment schedules.

[0187] [Table 15]

[0188] Part C consists of a 6-week treatment cycle (Cycle 1), followed by up to four treatment cycles (Cycles 2-5) of 4-week duration each, and a follow-up observation period lasting up to 52 weeks after the first treatment.

[0189] Premedication, including analgesics, is mandatory and will be empirically introduced with anti-GD2 IgG-based monoclonal antibodies. The dosing scheme can be seen in Table 16 below.

[0190] [Table 16]

[0191] [array]

[0192] [ka] TIFF2024546804000022.tif248165TIFF2024546804000023.tif252165TIFF2024546804000024.tif182164

Claims

1. 1. An aqueous composition comprising: a. a SADA complex comprising a SADA domain, a first binding site, and a second binding site in an amount of 5 to 50 g / L; b. a buffer system in an amount selected from 5-100 mM; c. one or more stabilizers in an amount selected from 200-350 mM; and d. one or more surfactants in an amount selected from the range of 0.1 to 0.3 g / L wherein the first binding site is capable of binding to GD2 and the SADA complex comprises the sequence of SEQ ID NO:

22.

2. The composition described in claim 1, containing an amount of SADA complex selected from the range of 10 to 30 g / L.

3. 2. The composition of claim 1, wherein the buffer system comprises an organic acid or an alkali metal salt thereof selected from acetate, citrate, histidine, citrate-histidine, acetate-histidine, and succinate.

4. The composition of claim 3 , wherein the buffer comprises sodium acetate.

5. The composition of claim 1 , wherein the one or more stabilizers are selected from the group consisting of polyols, sugar alcohols, and non-reducing sugars.

6. The composition of claim 5 , wherein the stabilizer is sucrose.

7. The composition of claim 1 , wherein the one or more surfactants are selected from nonionic surfactants.

8. 8. The composition of claim 7, wherein the one or more surfactants are selected from the group consisting of polyethylene glycol sorbitan monolaurate, poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), or polyethylene glycol sorbitan monooleate.

9. 9. The composition of claim 8, wherein the surfactant is polyethylene glycol sorbitan monolaurate.

10. 2. The composition of claim 1, wherein the pH is selected from the range of 5 to 6.

11. The composition described in claim 1, further comprising an antioxidant in an amount selected from 5 to 15 mM.

12. 12. The composition of claim 11, wherein the antioxidant is methionine.

13. 10. The composition of claim 1, which is free of NaCl or contains a low concentration of NaCl.

14. a. a SADA complex comprising or consisting of the amino acid sequence of SEQ ID NO: 22 in an amount of about 15 g / L; b. sodium acetate in an amount of about 20 mM; c. sucrose in an amount of about 275 mM; d. Polysorbate 20 in an amount of about 0.2 g / L; and e. 10 mM methionine; Including, 10. The composition of claim 1, wherein the pH is 5.

5.

15. A composition described in claim 1 for use in the treatment or diagnosis of cancer that expresses GD2.

16. The treatment or diagnosis a. administering the composition; and b. After a period of time, administering a DOTA compound containing a radionuclide; 16. The composition for use according to claim 15, comprising:

17. A composition for use as described in claim 16, wherein the treatment or diagnosis further comprises administering a remover after step a and before step b.

18. The radionuclide is １７７ Lu, ９９ mTc, ６４ Cu, ９０ Y and ８９ 17. The composition for use according to claim 16, wherein the metal is selected from the group consisting of Zr.

19. The radionuclide is １７７ Lu, ９９ mTc, ６４ Cu, ９０ Y and ８９ 18. The composition for use according to claim 17, wherein the metal is selected from the group consisting of Zr.

20. A kit comprising the composition of any one of claims 1 to 19.

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