Dual-specific antibody compounds

Abstract

Novel compounds are disclosed that contain two antigen-binding sites and a tetramerization site, where the antigen-binding sites are selected from naturally occurring binding sites. The novel compounds have improved manufacturability, stability, and solubility compared to similar compounds based on scFv binding sites, making the disclosed compounds much better candidates for active ingredients in pharmaceutical compositions.

Description

[Technical Field]

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

[0002] The present invention relates to bispecific antibodies for use in radioimmunotherapy, in particular bispecific antibodies that can dimerize or tetramerize and that contain two antigen-binding sites, one capable of binding a tumor antigen and one capable of binding a chelator molecule with or without a radionuclide. [Background technology]

[0003] Pretargeted radioimmunotherapy (PRIT) is a useful method for treating solid tumors using bispecific antibodies that have an antigen-binding site capable of binding an antigen exposed on the surface of the tumor and an additional antigen-binding site capable of binding a radionuclide or a chelator to which a radionuclide is attached.

[0004] PRIT is performed by first administering a bispecific antibody to a patient. Once the bispecific antibody binds to the tumor, a chelating agent with an attached radionuclide is administered, which is bound by the bispecific antibody and thereby localized to the tumor. To reduce systemic exposure to radiation, it is beneficial to delay administration of the radionuclide-containing chelating agent until the bispecific antibody has been cleared from the circulation. Some protocols use an additional step of administering a clearing agent between administering the bispecific antibody and the radionuclide to facilitate clearance of the bispecific antibody.

[0005] Many different formats of bispecific antibodies have been used for PRIT, some of which rely on the use of single-chain variable fragments (scFv).

[0006] WO 2018 / 204873 discloses bispecific antibodies for PRIT, each comprising an scFv capable of binding a tumor antigen, an scFv capable of binding DOTA chelated to a metal ion, and a tetramerization domain. These bispecific antibodies have the ability to exist in monomeric and multimeric forms depending on the concentration. These bispecific antibodies can be administered in multimeric form, and when the administered antibody is diluted in the patient's bloodstream, it is converted to a monomeric form, which facilitates its removal from plasma via the kidney. Therefore, the disclosed bispecific antibodies can be used without the need for administering a removal agent.

[0007] scFv is a synthetic binding site containing variable fragments of antibody light and heavy chains, and this format has been widely used in many applications. However, experience has shown that there are many challenges associated with the scFv format, such as low production yield, low stability, and low solubility.

[0008] These challenges have previously been partially addressed using protein engineering techniques, such as introducing a stabilizing disulfide bond between the light and heavy variable chains of the scFv, introducing one or more additional substitutions, or engineering the host cell that produces the scFv (see Kang and Seong in Frontiers in Microbiology (2020) vol 11, Article 1927). [Prior art documents] [Patent documents]

[0009] [Patent Document 1] International Publication No. 2018 / 204873 [Non-patent literature]

[0010] [Non-Patent Document 1] Kang and Seong in Frontiers in Microbiology(2020)vol 11,Article 1927 Summary of the Invention [Problem to be solved by the invention]

[0011] Despite recent advances, there remains a need for bispecific antibodies for PRIT that have improved manufacturability compared to previously designed bispecific antibodies. [Means for solving the problem]

[0012] The present invention relates to compounds comprising a first antigen-binding site capable of binding a tumor antigen and a second antigen-binding site capable of binding a chelating agent, wherein one of the first and / or second antigen-binding site is a VHH fragment, and the other of the first and / or second antigen-binding site is selected from a Fab fragment and a VHH binding fragment.

[0013] In some embodiments, the compound further comprises a tetramerization domain and / or a third or subsequent antigen binding site.

[0014] In another aspect, the present invention relates to compositions comprising the compounds of the present invention.

[0015] In another aspect, the present invention provides a method of treating and / or diagnosing cancer, comprising: a. Administering a compound according to the invention or a composition according to the invention to a human in need of treatment and / or diagnosis. b. administering a radionuclide conjugated to a chelator capable of being bound by the second antigen binding site The present invention relates to a method, including:

[0016] When the method is for diagnosing cancer, the method may further comprise the step of detecting radioactivity, such as using a scanning step.

[0017] The invention also relates to nucleic acids encoding the compounds of the invention, expression vectors comprising nucleic acids of the invention, host cells comprising nucleic acids of the invention, and methods of making the compounds of the invention. DETAILED DESCRIPTION OF THE INVENTION

[0018] [Definition] Amino acid change: The term "amino acid change" is intended to mean a change in one amino acid found in the original amino acid sequence, the change being selected from among a substitution, deletion or insertion of an additional amino acid immediately after that amino acid.

[0019] Amino acid substitution: The term "amino acid substitution" is intended to mean the replacement of one amino acid with a different amino acid. As used herein, the term amino acid substitution(s) relative to a reference sequence is intended to mean that an amino acid sequence can be generated starting from the reference sequence and introducing said amino acid substitution(s), even if the amino acid sequence was actually generated by another process not involving the reference sequence.

[0020] Antibody: The term "antibody" is an art-recognized term and is intended to include molecules or active fragments of molecules that bind to an antigen. Naturally occurring antibodies, excluding heavy chain antibodies, are composed of two heavy chains, each containing one variable domain and three or more constant domains (CH1, CH2, and CH3), and two light chains, each containing one variable domain and one constant domain (CL). One light chain is linked to one heavy chain by disulfide bonds located in the constant domains (CH1 and CL), and the two heavy chains are linked to each other by several disulfide bonds located in the constant domain (CH2).

[0021] Several isotypes of natural antibodies are known, including IgA, IgD, IgE, IgG such as IgG1, IgG2, IgG3, and IgM, and these isotypes differ mainly in the constant domains.

[0022] Fab fragment: The term "Fab" or "Fab fragment" refers to an antibody fragment consisting of a first polypeptide containing a light chain variable domain and a light chain constant domain and a second polypeptide containing a heavy chain variable domain and a heavy chain constant domain, the two polypeptides being connected via a disulfide bond located in the constant region. Fab fragments can be provided by proteolytic cleavage of a natural antibody, or can be produced by expressing and combining two polypeptides, for example, using recombinant DNA techniques known in the art. Fab fragments are capable of binding to the same antigen recognized by an intact antibody. The term "Fab" or "Fab fragment" encompasses both natural Fab fragments, i.e., fragments having the same sequence as found in a natural antibody, and synthetic or engineered Fab fragments; i.e., fragments having the same overall structure as a natural Fab fragment, but in which the sequence has been engineered in one or both amino acid chains by introducing one or more amino acid changes, such as substitutions, deletions, or insertions.

[0023] Bispecific antibodies: Bispecific antibodies are antibodies that can simultaneously bind two targets of different structures. Bispecific antibodies (BsAbs) are non-naturally engineered antibodies that have at least one binding site that specifically binds one antigen, e.g., a tumor antigen, and at least one other binding site that specifically binds another antigen, e.g., a chelator that binds a radionuclide. Various bispecific fusion proteins can be generated using molecular engineering. In one form, the bispecific fusion protein is bivalent, e.g., consisting of a VHH with a single binding site for one antigen and a Fab fragment with a single binding site for a second antigen. In another form, the bispecific fusion protein is tetravalent, e.g., consisting of an IgG with two binding sites for one antigen and two identical scFvs for a second antigen.

[0024] CDR: Complementarity-determining region (CDR) is a part of the variable region of an antibody and is crucial for the binding specificity of the antibody. A typical antibody consisting of two heavy chains and two light chains has six CDR sequences, three in the light chain and three in the heavy chain.

[0025] DOTA: DOTA (dodecanetetraacetic acid), also known as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, is a C 16 H 28 It has the formula (CH2CH2NCH2CO2H)4, also known as N4O8·xH2O.

[0026] Derivatives of DOTA: is intended to mean compounds comprising a DOTA ring system to which some other chemical group or moiety is attached and capable of chelating metal ions. Examples of such compounds include benzyl-DOTA and the bispecific chelators disclosed in WO2019010299 (Proteus-DOTA) or WO2022005998. Further DOTA derivatives are disclosed in WO2010099536.

[0027] DOTAM: A chelating agent containing a ring system capable of binding metal ions. DOTAM has the systematic name 1,4,7,10-tetraazacyclododecane-1,7-bis(acetate)-4,10-bis(acetamide) and has the formula C 16 H 30 It has N6O6·2H2O.

[0028] Effective amount: As used herein, the term "effective amount" refers to the amount of a given compound or composition necessary or sufficient to realize a desired biological effect. An effective amount of a given compound or composition in the methods of the invention is the amount that achieves this selected result, and such an amount can be determined as a matter of routine by one of ordinary skill in the art without necessitating undue experimentation.

[0029] Plasma half-life: The term "plasma half-life" for a given compound is the time required for the plasma concentration of the given compound to decrease by 50%. The plasma half-life depends on various factors and properties of the given compound. An important factor for plasma half-life is size, since it is known that the kidney has a filtering function that retains molecules with a size of about 70 kDa, while smaller molecules can be eliminated through the kidney. Furthermore, some molecules may interact with receptors that can affect the plasma half-life.

[0030] Prevent: As used herein, the terms "prevent," "preventing," and "prevention" refer to the prevention of the recurrence or onset of one or more symptoms of a disorder in a subject as a result of the administration of a prophylactic or therapeutic agent.

[0031] Radioisotopes: Examples of radioisotopes that can be attached to antibodies for diagnostic or therapeutic use, for example, by conjugation or use of a chelating agent, include: 211 At, 14 C. 51 Cr, 57 Co, 58 Co, 67 Cu, 165 Dy 152 EU, 67 Ga, 3 H, 111 In, 59 Fe, 133 La, 177Lu, 32 P, 223 Ra, 224 Ra, 186 Re, 188 Re, 75 Se, 89 Sr 149 Tb, 151 Tb, 161 Tb 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, 221 Fr, 223 Ra, 224 Ra, 226 Ra, 225 Ac, 227 Ac, 227 Th, 228 Th, 229 Th, 230 Th, 232 Th, 231 Pa, 237 Np, 238 Pu, 239 Pu, 240 Pu, 244 Pu, 241 Am, 244 Cm, 245 Cm, 248 Cm, 249 Cf and 252 Cf is an example.

[0032] Sequence alignment: Sequence alignment simply refers to any method of lining up two sequences so that one sequence is underneath the other. Sequence alignment is a method of arranging DNA, RNA, or protein sequences to identify areas of similarity between the sequences. Various alignment algorithms exist, and they usually have a scoring function that assigns a numerical score to every alignment indicating how good the alignment is and attempts to find the best alignment according to the scoring function.

[0033] 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 the two sequences, finding the longest overlap, counting the number of matches in the overlap, and calculating the sequence identity by dividing the number of matches by the number of nucleotides or amino acid residues in the overlap. Sequence identity is typically expressed as a percentage (%).

[0034] A variety of algorithms are available to those skilled in the art for generating sequence alignment and calculating sequence identity.As used herein, sequence alignment refers to pairwise alignment.Some algorithms perform this, including the sequence alignment program Clustal Omega [doi:10.1038 / msb.2011.75].

[0035] As used herein, sequence alignment may refer to the following algorithms and parameters. Algorithm: Clustal Omega (1.2.4), http: / / www.clustal.org / omega /

[0036] Heavy chain antibodies are antibodies that contain two heavy chains and lack the two light chains normally found in naturally occurring antibodies. Heavy chain antibodies are found naturally in members of the camelid order, as well as in cartilaginous fish such as some sharks.

[0037] Structurally, heavy chain antibodies consist of a single variable domain containing the complementarity determining regions (CDRs) and two to five constant domains, depending on the origin of the heavy chain antibody.

[0038] Heavy chain antibodies from camelids contain two constant regions, whereas heavy chain antibodies from cartilaginous fish can have up to five constant domains.

[0039] Single domain antibodies (sdAbs) or VHH fragments are fragments of heavy chain antibodies that contain the variable domains of the heavy chain antibodies, i.e., the antigen-binding domains, but lack the constant domains. VHH fragments, also called nanobodies, are fairly small molecules with a molecular weight of less than 20 kDa. The terms single domain antibody (sdAb), VHH fragment, VHH, VHH binder or nanobody may be used interchangeably throughout this specification and claims.

[0040] Treatment: As used herein, the terms "treatment," "treat," "treated," or "treating" refer to prophylaxis and / or therapy, particularly where the purpose is to prevent or slow (alleviate) an undesirable physiological change or disorder, such as the progression of cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, lessening of the extent of disease, stabilized (i.e., not worsening) disease, slowing or slowing of disease progression, improvement or palliation of the disease state, and remission (whether partial or complete), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in whom the condition or disorder is to be prevented.

[0041] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" is intended to mean a composition intended for administration as a drug or medicine to a patient in need thereof. Pharmaceutical compositions comprise at least one active ingredient and at least one pharmaceutical grade ingredient, such as a solvent, diluent, salt, stabilizer, pH adjuster, antioxidant, etc. Pharmaceutical compositions are prepared from pharmaceutical grade ingredients using methods and techniques known in the art of pharmacy or compounding, for example, as described in the European Pharmacopoeia, 10th Edition.

[0042] compound The present invention relates to a compound comprising a first antigen-binding site and a second antigen-binding site, wherein at least one of the first and second antigen-binding sites is a VHH fragment and the other of the first and second binding sites is a VHH fragment or a Fab fragment, one of the first and second binding sites is capable of binding a tumor antigen, and the other of the first and second binding sites is capable of binding a chelator or a chelator that binds a metal ion.

[0043] In some embodiments, the compounds of the invention further comprise a tetramerization domain.

[0044] In some embodiments, the compounds of the invention may even comprise a third or subsequent binding site in the form of a second or subsequent VHH fragment.

[0045] The compounds of the present invention may have a structure in which the first binding site is a Fab fragment and the second binding site is a VHH fragment, or the first binding site is a VHH fragment and the second binding fragment is a Fab, or the first binding fragment is a VHH fragment and the second binding fragment is a VHH fragment.

[0046] In embodiments in which both the first and second antigen-binding sites are in the form of a VHH, the entire compound comprises two binding sites and can be produced as a single polypeptide chain, either with or without a tetramerization domain.

[0047] In embodiments where one of the antigen binding sites is in the form of a Fab and the other binding site is in the form of a VHH, the compound generally consists of two polypeptide chains, with several combinations possible.

[0048] In this embodiment, the VHH fragment may be contained in the same polypeptide chain as the light chain of the Fab, or in the same polypeptide chain as the heavy chain of the Fab, or in both the light and heavy chains. It is even encompassed by the present invention that one VHH fragment is contained in the same chain as the light chain of the Fab and a second VHH fragment, which may be identical to or different from the first VHH fragment, is contained in the same chain as the heavy chain of the Fab. In the latter situation, the compound may comprise two binding specificities, i.e., one binding specificity provided through the Fab and one binding specificity provided by two identical VHH fragments (such a structure is referred to herein as a homodimeric molecule), or three different binding specificities, i.e., one binding specificity provided through the Fab and two binding specificities provided by two different VHH fragments (such a structure is referred to herein as a heterodimeric molecule).

[0049] It is even contemplated to prepare molecules of the invention in which one or both of the polypeptides of the molecule comprises one VHH fragment located in the N-terminal portion of the polypeptide and a second VHH fragment located in the C-terminal portion of the polypeptide. In this way, it is possible to prepare molecules of the invention that comprise two, three or even more specificities.

[0050] Similarly, if present, the tetramerization domain may be located on the same polypeptide chain as the light chain of the Fab or on the same polypeptide chain as the heavy chain of the Fab.

[0051] The compound of this embodiment is one polypeptide chain comprising a VHH domain, a light chain of a Fab, and a tetramerization domain; and a second polypeptide chain comprising a heavy chain of a Fab; one polypeptide chain comprising a VHH domain and a light chain of a Fab; and a second polypeptide chain comprising a heavy chain of a Fab and a tetramerization domain; one polypeptide chain comprising a VHH domain, a heavy chain of a Fab, and a tetramerization domain; and a second polypeptide chain comprising a light chain of a Fab; one polypeptide chain comprising a VHH domain and a heavy chain of a Fab; and a second polypeptide chain comprising a light chain of a Fab and a tetramerization domain; one polypeptide comprising a VHH domain and a heavy chain of a Fab; and a second polypeptide chain comprising a VHH domain, which may be the same VHH domain as present in the first polypeptide or may be a different VHH domain, and a light chain of a Fab; one polypeptide chain comprising a VHH domain and a light chain of a Fab; and a second polypeptide chain comprising a heavy chain of a Fab; one polypeptide chain comprising a VHH domain and a heavy chain of a Fab; and a second polypeptide chain comprising a light chain of a Fab; or one polypeptide comprising a VHH domain and a heavy chain of a Fab; and a second polypeptide chain comprising a VHH domain, which may be the same VHH domain as present in the first polypeptide or may be a different VHH domain, a light chain of a Fab, and a tetramerization domain; one polypeptide comprising a VHH domain, a heavy chain of a Fab, and a tetramerization domain; and a second polypeptide chain comprising a VHH domain and a light chain of a Fab, which may be the same VHH domain as present in the first polypeptide or may be a different VHH domain. This results in several possible combinations, since the

[0052] Those skilled in the art will appreciate that even further combinations may be possible, especially for compounds of the invention that comprise second or subsequent VHH fragment binding sites.

[0053] The order of domains in a polypeptide is not critical to the present invention. Rather, it is contemplated that the VHH fragment may be located at the N-terminal portion, the C-terminal portion, or an internal portion of the polypeptide in which the VHH fragment is located. Furthermore, a polypeptide forming one of the chains of a compound of the present invention may even contain two VHH fragments, for example, one chain of Fab fragments between two VHH fragments, and one VHH fragment at each end of the polypeptide. Preferably, the tetramerization domain is located at the C-terminus of the polypeptide containing this domain.

[0054] In one preferred embodiment of the present invention, the first and second antigen-binding sites are in the form of VHH fragments and the tetramerization domain is located at the C-terminus of the polypeptide. Such a compound is shown schematically in Figure 1B.

[0055] Another preferred embodiment of the present invention is a format in which one of the first and second antigen-binding sites is a VHH, the other antigen-binding site is in the form of a Fab fragment, and the tetramerization domain is located at the C-terminus of the first or second polypeptide. Examples of compounds according to this embodiment are shown schematically in Figures 1A and 1E.

[0056] Another preferred embodiment of the present invention is a format in which two VHH fragments are present, one of the first and second antigen-binding sites being a VHH and the other antigen-binding site being in the form of a Fab fragment, with the tetramerization domain located at the C-terminus of the first or second polypeptide. The two VHH fragments may be identical, as shown in Figures 1C and 1F, in which case the avidity of the binding of the VHH fragments is increased due to the coordination of the two VHH fragment-binding sites. Alternatively, the two VHH fragments may be different and both may bind different tumor antigens, such as two different tumor antigens expressed on the surface of the same tumor, as shown in Figures 1D and 1G.

[0057] In one preferred embodiment, the total molecular weight of compound is less than the renal clearance limit, such as less than 70 kDa.However, the size of tetrameric compound exceeds the renal clearance limit, which means that when the compound of the present invention is administered to a patient, the tetrameric compound has a long plasma half-life, while the monomeric compound has a short plasma half-life.In addition, the tetrameric compound has a higher affinity to tumors due to the existence of four binding sites.

[0058] A tumor antigen can in principle be any antigen exposed on a tumor so as to be accessible to antibodies. Many such tumor antigens are known in the art, and the present invention is not limited to any particular such tumor antigen.

[0059] Examples of tumor antigens include HER2, B7-H3, CA6, CD138, CD20, CD19, CD22, CD27L, CD30, CD33, CD37, CD38, CD47, CD56, CD66e, CD70, CD74, CD79b, EGFR, CEA, EGFRvIII, FRα, GCC, GPNMB, mesothelin, MUC16, NaPi2b, nectin 4, PSMA, STEAP1, Trop-2, 5T4, AGS-16, αvβ6, CA19.9, CAIX, CD138, CD174, CD180, CD227, These include, but are not limited to, CD326, CD79a, CEACAM5, CRIPTO, DLL3, DS6, endothelin B receptor, FAP, GD2, GPA33, mesothelin, PMEL17, SLC44A4, TENB2, TIM-1, CD98, endosialin / CD248 / TEM1, fibronectin extra domain B, LIV-1, mucin 1, p-cadherin, peritosin, Fyn, SLTRK6, tenascin c, VEGFR2, BAFF, BAFFR, or PRLR.

[0060] Preferred tumor antigens include HER2, B7-H3, GD2, CD20, CD38, GPA33, CEA, EGFRvIII, and CD33.

[0061] Antigen-binding sites capable of binding tumor antigens or capable of binding chelators or chelators that bind metal ions can be provided by selecting sequences that form Fab or VHH fragments from isolated intact antibodies with the desired binding properties and expressing the sequences in suitable production cells using methods well known in the art. Alternatively, such antigen-binding sites capable of binding tumor antigens or capable of binding chelators or chelators that bind metal ions can be provided by screening Fab or VHH fragment libraries for binders with the desired binding properties. This is all within the capabilities of one skilled in the art.

[0062] In one embodiment, the first antigen-binding site capable of binding a tumor antigen is a Fab, and the Fab can be derived from a tumor antigen-binding antibody known in the art. By "derived" it is meant that the Fab is made from the respective fragment of the antibody from which it is derived, optionally modified by one or more amino acid changes, and / or the Fab can even be humanized using methods known in the art. Examples of antibodies from which Fabs for use in the present invention can be derived include the anti-B7H3 antibody 8H9 (Modak et al (2001) Cancer. Res. 61:4048-56), the anti-GD2 antibody 3F8 (Cheung et al (1998) J. Clin. Oncol. 3052-3060), the anti-CD38 antibody daratumumab (Lee (2006) Mol. Med. 12:317-23) or AT13 / 5 (Ellis et al. (1995) J. Immunol 155:925-37).

[0063] In one embodiment, the second antigen-binding site that binds the chelator is a Fab, which can be derived from a chelator-conjugated antibody.

[0064] A preferred example of an antibody capable of binding a chelator is an antibody capable of binding DOTA or a DOTA derivative, or is derived from one of these antigen-binding sites, as disclosed, for example, in WO 2010 / 099536, which is incorporated by reference. By "derived from," we mean that the Fab is made from the respective fragment of the antibody from which the Fab is derived, and optionally modified by one or more amino acid changes, and / or the Fab may even be humanized using methods known in the art. An example of an antibody from which a Fab for use in accordance with the present invention may be derived is the antibody designated 2D12.5 (Corneillie et al., J. Am. Chem. Soc-125:15039-15048, 2003), optionally containing one or more substitutions in the CDR sequence, as disclosed in WO 2010 / 099536.

[0065] Another preferred example of an antibody capable of binding a chelator is an antibody capable of binding DOTAM, or derived from one of these antigen binding sites, as disclosed, for example, in WO2019201959, which is incorporated by reference.

[0066] In some preferred embodiments, the compounds of the present invention are further modified with selected alterations intended to improve selected properties of the compound, e.g., to eliminate disadvantages of the compound, to obtain an improved molecule.

[0067] One of the key attributes of a therapeutic monoclonal antibody or antibody-derived protein candidate is that it must possess biochemical and biophysical properties that make it stable, soluble, and resistant to degradation or modification during manufacturing and storage. A wide range of potential drawbacks may exist in a therapeutic candidate, including a tendency toward strong aggregation, poor solubility, a tendency to fragment, deamidation, oxidation, cyclization, or other chemical modifications of key residues, disulfide bond shuffling, glycosylation, and the like, which require correction. During antibody discovery and development, it is crucial to use protein sequence analysis algorithms, antibody modeling, and engineering to optimize antibodies for stability, solubility, and other biophysical characteristics. Two significant potential consequences of an antibody not behaving properly are immunogenicity and a lack of batch-to-batch comparability.

[0068] Post-translational modifications (PTMs) of antibodies can potentially affect antibody affinity, stability, potency, and homogeneity, leading to complex downstream development processes. The biological activity and production of multiple product isoforms can potentially be affected. PTMs typically include deamidation, isomerization, oxidation, N-glycosylation, glycation, free thiol modification, pyroglutamic acid, O-glycosylation, and C-terminal lysine removal.

[0069] To identify disadvantageous sequences, standard rules applied to the primary antibody sequence provide predictions of residues to modify or avoid. Several bioinformatics software packages include algorithms to provide this information. Not all PTMs can be confidently predicted by strict sequence rules and are only revealed during deeper characterization of the antibody (e.g., O-glycans).

[0070] Ideally, molecules should be selected from a group of molecules. Such selection should allow only molecules without predicted disadvantages to move forward. However, in some cases, preferred lead candidates may have some minor disadvantages. These can be alleviated by generating variant molecules of the amino acids in question. In antibodies and antibody-derived therapeutics, the most important parts of the sequence are the CDRs involved in target binding. These sequence disadvantages are likely to affect binding, and therefore often have a higher priority for modification than framework disadvantages.

[0071] Many such specific substitutions intended to eliminate identified disadvantages are known in the art, and it is within the skill of the average practitioner to identify such residues and replace them with suitable others.

[0072] The tetramerization domain can be selected from any domain that has the ability to tetramerize at high concentrations and dissociate into monomers at low concentrations. In this context, "high concentration" is intended to mean concentrations typically found in pharmaceutical compositions, such as in the range of 1 to 50 mg / L, and "low concentration" is intended to mean the concentration of the compound in plasma after administration of a dose of the compound, such as less than 50 μg / L. These properties allow for the administration of the compounds of the present invention in tetrameric form, which gradually dissociates into monomeric form after administration as the compound is diluted in plasma.

[0073] Examples of such tetramerization domains include the p53, p63, p73, hnRNPC, SNAP-23, StefinB, KCNQ4 and CBFA2T1 domains having the amino acid sequences disclosed in SEQ ID NOs: 1-8, and domains having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity or at least 98% sequence identity to one of SEQ ID NOs: 1-8.

[0074] Preferred tetramerization domains are p53 domains having the sequence of SEQ ID NO:1 and domains having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 97% sequence identity to SEQ ID NO:1.

[0075] In another embodiment, the tetramerization domain is a domain comprising the sequence of SEQ ID NO: 1 or a sequence that differs from this sequence by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 changes selected from among a substitution, insertion or deletion of a single amino acid residue.

[0076] Preferred tetramerization domains according to the present invention are those that have at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity to amino acids 6 to 36 of SEQ ID NO: 1, and that comprise a sequence that differs from the sequence of SEQ ID NO: 1 by one or more substitutions, wherein the domain maintains the ability to dimerize or tetramerize.

[0077] In other words, a preferred tetramerization domain according to the present invention is a domain comprising the sequence of amino acids 6 to 36 of SEQ ID NO: 1 or a sequence that differs from amino acids 6 to 36 of SEQ ID NO: 1 by 1, 2, 3, 4, 5 or 6 changes, which domain maintains the ability to dimerize or tetramerize.

[0078] Those skilled in the art can readily determine by simple routine experimentation whether such domains with a given substitution maintain the ability to dimerize or tetramerize, or can find such information in the literature, e.g., J. Gencel-Augusto and G. Lozano; Genes & Development 34:1128-1146, incorporated by reference.

[0079] Preferred tetramerization domains according to the present invention have the following substitutions, using the numbering of SEQ ID NO: 1: E6V, Q, K, G, D or A; Y7S, N, H, F, D or C; F8Y, V, S, L, I or C; T9S, P, N or A; L10V, I or F; Q11R, L, K, H or E; I12V, T, M, L or F; R13S, P, L, H, G or C; G14W, R or A; R15S, P, L, H, G or C; E16V, Q, K, G, D or A; F18Y, V, S, L, I or C; E19V, Q, K, G, D or A; M20V, T, R, L, K or I; F21L or I; R22L or G; E23V, Q, K, G, D or A; L24M; N25S, I or D; E26V, Q, K, G, D or A; A27V, T, S, G or D; L28W, V, M or F; E29Q, G or D; L30V, R, I, H or F; K31T, R, Q, N, M or E; D32Y, V, N, H, G or A; A33V, T, S, P, G or D; Q34R, L, K, H or E A domain having an amino acid sequence that differs from the sequence of amino acids 6 to 36 of SEQ ID NO: 1 by one, two, three, four, five, or six substitutions selected from the following: All of these substitutions are known in the art and are known not to abolish the ability of the domain to form dimers and / or tetramers.

[0080] The L24P substitution completely abolishes tetramerization and should not be applied.

[0081] The p53 tetramerization domain comprising the sequence of amino acids 6 to 36 of SEQ ID NO: 1 is a preferred SADA domain.

[0082] The compounds of the present invention may further comprise one or more linkers separating different parts of the compound, for example, separating the first binding site from the second binding site, or separating the second binding site from the tetramerization domain. The purpose of the linker is to separate the different domains so that they can fold and function without interference from other elements of the compound. The linker is preferably composed of hydrophilic residues that do not form strong secondary structures, and is typically rich in residues such as glycine, serine, and / or threonine. A preferred linker is one composed of G and S residues, such as GGGGS (SEQ ID NO: 9), optionally repeated two or more times to obtain the desired length.

[0083] In one preferred embodiment, the first antigen-binding site is a VHH that binds HER2, the second antigen-binding site is a Fab that can bind DOTA, and the tetramerization domain is p53. The Fab can be conveniently derived from antibody 2D12.5, optionally with a C825 substitution, as disclosed in WO 2010 / 099536. An example of this embodiment is a compound comprising a first polypeptide comprising the sequence of SEQ ID NO: 10 and a second polypeptide comprising the sequence of SEQ ID NO: 11.

[0084] In another preferred embodiment, the first antigen-binding site is a VHH that binds CD38, the second antigen-binding site is a Fab that can bind DOTA, and the tetramerization domain is p53. The Fab can be conveniently derived from antibody 2D12.5, optionally with the C825 substitution, disclosed in WO 2010 / 099536.

[0085] Examples of this embodiment are a compound comprising a first polypeptide comprising the sequence of SEQ ID NO: 12 and a second polypeptide comprising the sequence of SEQ ID NO: 11; a compound comprising a first polypeptide comprising the sequence of SEQ ID NO: 12 and a second polypeptide comprising the sequence of SEQ ID NO: 14; a compound comprising a first polypeptide comprising the sequence of SEQ ID NO: 12 and a second polypeptide comprising the sequence of SEQ ID NO: 15; a compound comprising a first polypeptide comprising the sequence of SEQ ID NO: 23 and a second polypeptide comprising the sequence of SEQ ID NO: 27; or a compound comprising a first polypeptide comprising the sequence of SEQ ID NO: 24 and a second polypeptide comprising the sequence of SEQ ID NO: 25; a compound comprising a first polypeptide comprising the sequence of SEQ ID NO: 23 and a second polypeptide comprising the sequence of SEQ ID NO: 25.

[0086] In another preferred embodiment, the first antigen-binding site is a VHH that binds CD38 and the second antigen-binding site is a Fab that can bind DOTA. This embodiment does not include the tetramerization domain p53.

[0087] An example of this embodiment is a compound comprising a first polypeptide comprising the sequence of SEQ ID NO:12 and a second polypeptide comprising the sequence of SEQ ID NO:13.

[0088] In another preferred embodiment, the first antigen-binding site is a VHH that binds CD38, the second antigen-binding site is a humanized Fab capable of binding DOTA, and the tetramerization domain is p53.

[0089] An example of this embodiment is a compound comprising a first polypeptide comprising the sequence of SEQ ID NO:28 and a second polypeptide comprising the sequence of SEQ ID NO:29.

[0090] In another preferred embodiment, the first antigen-binding site is a VHH that binds CD38, the second antigen-binding site is a Fab capable of binding DOTAM, and the tetramerization domain is p53.

[0091] An example of this embodiment is a compound comprising a first polypeptide comprising the sequence of SEQ ID NO:16 and a second polypeptide comprising the sequence of SEQ ID NO:17.

[0092] In another preferred embodiment, the first antigen-binding site is a VHH that binds CD38 and the second antigen-binding site is a Fab that can bind DOTAM. This embodiment does not include the tetramerization domain p53.

[0093] An example of this embodiment is a compound comprising a first polypeptide comprising the sequence of SEQ ID NO:16 and a second polypeptide comprising the sequence of SEQ ID NO:18.

[0094] In another preferred embodiment, the first antigen-binding site is a VHH that binds BAFF (B cell activating factor, (TNF)), the second antigen-binding site is a Fab capable of binding DOTA, and the tetramerization domain is p53. The Fab may conveniently be derived from antibody 2D12.5, optionally with a C825 substitution, as disclosed in WO 2010 / 099536.

[0095] An example of this embodiment is a compound comprising a first polypeptide comprising the sequence of SEQ ID NO:19 and a second polypeptide comprising the sequence of SEQ ID NO:27.

[0096] In another preferred embodiment, the first antigen-binding site is a VHH that binds CD33, the second antigen-binding site is a Fab that can bind DOTA, and the tetramerization domain is p53. The Fab can be conveniently derived from antibody 2D12.5, optionally with a C825 substitution, as disclosed in WO 2010 / 099536.

[0097] An example of this embodiment is a compound comprising a first polypeptide comprising the sequence of SEQ ID NO:20 and a second polypeptide comprising the sequence of SEQ ID NO:27.

[0098] In another preferred embodiment, the first antigen-binding site is a VHH that binds EGF-R variant 3 (EGFRvIII), the second antigen-binding site is a Fab capable of binding DOTA, and the tetramerization domain is p53. The Fab may conveniently be derived from antibody 2D12.5, optionally with a C825 substitution, as disclosed in WO 2010 / 099536.

[0099] An example of this embodiment is a compound comprising a first polypeptide comprising the sequence of SEQ ID NO:21 and a second polypeptide comprising the sequence of SEQ ID NO:27.

[0100] In another preferred embodiment, the first antigen-binding site is a VHH that binds CEA, the second antigen-binding site is a Fab that can bind DOTA, and the tetramerization domain is p53. The Fab can be conveniently derived from antibody 2D12.5, optionally with a C825 substitution, as disclosed in WO 2010 / 099536.

[0101] An example of this embodiment is a compound comprising a first polypeptide comprising the sequence of SEQ ID NO:22 and a second polypeptide comprising the sequence of SEQ ID NO:27.

[0102] In another preferred embodiment, the first antigen-binding site is a VHH that binds CD276, the second antigen-binding site is a Fab that can bind DOTA, and the tetramerization domain is p53. The Fab may conveniently be derived from antibody 2D12.5, optionally with a C825 substitution, as disclosed in WO 2010 / 099536.

[0103] An example of this embodiment is a compound comprising a first polypeptide comprising the sequence of SEQ ID NO:26 and a second polypeptide comprising the sequence of SEQ ID NO:27.

[0104] In another preferred embodiment, the first antigen-binding site is a VHH that binds HER2, the second antigen-binding site is a humanized Fab that can bind DOTA, and the tetramerization domain is p53. The third binding site is a VHH that binds CD276. The Fab can be conveniently derived from antibody 2D12.5, optionally with a C825 substitution, as disclosed in WO2010 / 099536.

[0105] An example of this embodiment is a compound comprising a first polypeptide comprising the sequence of SEQ ID NO:30 and a second polypeptide comprising the sequence of SEQ ID NO:31.

[0106] Another example of this embodiment is a compound comprising a first polypeptide comprising the sequence of SEQ ID NO:30 and a second polypeptide comprising the sequence of SEQ ID NO:32.

[0107] Use of the compound The compounds of the present invention are useful in immunotherapy, particularly pre-targeted radioimmunotherapy (PRIT).

[0108] PRIT is used to treat cancer in a method in which a bispecific antibody comprising a first binding site capable of binding a tumor antigen and a second binding site capable of binding a radionuclide, a radionuclide-binding chelator, or a molecule linked to a chelator, e.g., a peptide bound to a chelator group, is administered to a patient in need of treatment; if the second binding site is capable of binding a peptide bound to a chelator group, the first binding site can bind the peptide moiety. After binding the antibody to the tumor and removing unbound antibody from plasma, the radionuclide or radionuclide-binding chelator recognized by the bispecific antibody is administered to the patient and bound by the bispecific antibody localized in the tumor. Unbound radionuclide or radionuclide-binding chelator is rapidly removed from plasma via renal clearance.

[0109] To protect other tissues from radiation, the bispecific antibody is preferably cleared from plasma before the radionuclide is administered, hi some embodiments, a clearing agent is administered between administration of the bispecific antibody and the radionuclide to improve clearance.

[0110] In one embodiment, the compounds of the present invention that contain tetramerization domains are preferably administered in tetrameric form and bind to target tumor antigens after administration.Tetrameric compounds have higher avidity for tumor antigens due to the cooperation between the four tumor antigen binding sites of the tetrameric form, so it can be expected that the tetrameric form of the compounds will exhibit improved binding properties to tumors due to the tetrameric format.Due to the reduced concentration in plasma, unbound compounds in tetrameric form will quickly disintegrate into monomeric form, and the monomer will be rapidly removed from plasma due to its size below the renal clearance limit.These properties of the compounds of the present invention mean that the compounds of the present invention can efficiently bind to tumor antigens, and unbound compounds will be rapidly removed from plasma, thereby achieving highly efficient removal without the need for a removal agent.

[0111] In another embodiment, the compound of the present invention contains two identical tumor-binding sites, and in this embodiment, the compound has a higher avidity for a tumor antigen due to cooperation between the two tumor antigen-binding sites compared to a similar compound containing the same binding site but with only one tumor-binding site.

[0112] In another embodiment, the compound of the present invention comprises two different tumor-binding sites. In this embodiment, the compound has improved binding properties against tumors expressing both tumor antigens due to the cooperation between the two different tumor antigen-binding sites, compared to a similar compound with only one of the two tumor-binding sites. An example of a tumor with two different tumor antigens for which the use of a compound according to this embodiment is beneficial is breast cancer. Some breast cancers have overexpression of both B7H3 and HER2 on the cell surface. To address this experimentally, the breast cancer-derived cell line BT-474 can be used.

[0113] Accordingly, in one preferred embodiment, the present invention provides a method of treating or diagnosing cancer in a patient, comprising: i. administering a compound according to the present invention, which is capable of binding a tumor antigen and further capable of binding a chelator having an attached radionuclide, to a subject in need of such treatment or diagnosis; ii. After the incubation period, administering to the subject a chelating agent that binds the radionuclide. The present invention relates to a method comprising:

[0114] If the compound contains a tetramerization domain, the compound will degrade into monomers and unbound compound will be removed from the plasma during the standing period.

[0115] The leaving period can be selected within the range of 12 hours to 7 days, for example, 12 hours, 18 hours, 24 hours, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days.

[0116] Although satisfactory and efficient removal can be obtained using the compounds of the present invention, it is possible to include a step of administering a removal agent to improve clearance. However, in most situations, the step of administering a removal agent is redundant and is preferably omitted to avoid an additional administration step and the inconvenience to the patient involved in such an additional step.

[0117] Similar treatment methods have been previously disclosed, for example in WO 2018 / 204873, with the important difference that the prior art methods use bispecific antibody constructs based on antigen-binding sites in scFv format.

[0118] The scFv format has several inherent drawbacks, such as low productivity, and heterogeneity is often observed in the product, likely due to disulfide bond mismatching, which can be exacerbated by the frequently used practice of introducing a stabilizing disulfide bond between the variable light and heavy chains. Furthermore, many scFvs have low solubility, and poor stability during storage is also often observed.

[0119] As a result, some scFvs are known to require several amino acid changes to alleviate these inherent difficulties. In contrast, the molecules of the present invention are based on naturally occurring binding domains that do not present such difficulties, or generally present a lesser number and severity of issues that need to be addressed as part of the development of a pharmaceutical product.

[0120] The compounds of the present invention are superior to scFv-based conjugates disclosed in the art in terms of manufacturability and product stability, and offer at least the following advantages: Higher expression levels, Easier purification, for example, using robust production capture purification methods; Higher solubility, A wide range of formulation options, Built on natural domains Improved stability, and Lower heterogeneity to provide.

[0121] Thus, in many respects, the compounds of the present invention are "better molecules" that are more suitable for the preparation of pharmaceuticals compared to scFv-based compounds.

[0122] In one embodiment, the cancer is selected from osteosarcoma, neuroblastoma, liposarcoma, fibrosarcoma, carcinoma, malignant fibrous histiocytoma, leiomyosarcoma, spindle cell sarcoma, brain tumor, small cell lung cancer, retinoblastoma, HTLV-1 infected T-cell leukemia, breast cancer, colon cancer, prostate cancer, T-cell and B-cell lymphoma, glioblastoma multiforme, malignant glioma, head and neck cancer, solid tumors, and non-small cell lung cancer.

[0123] Those skilled in the art will appreciate that the chelating agent used in the treatment / diagnosis methods of the present invention can be any chelating agent that can be recognized and bound by the second antigen-binding site.

[0124] In one preferred embodiment, the second antigen-binding site is capable of binding DOTA or a derivative of DOTA. In this embodiment, the chelator is a compound comprising the DOTA ring system to which the second antigen-binding site can be bound, such as a derivative of DOTA, such as the compounds disclosed in WO 2010 / 099536, WO 2019 / 010299 and WO 2022 / 005998 (incorporated herein by reference).

[0125] The radionuclide can be any radionuclide that can be bound by a chelating agent, typically a radionuclide cation. Examples of suitable radionuclides include: 225 Ac, 227 Ac, 241 Am, 211 At, 215 At, 217 At, 218 At, 209 Bi, 211 Bi, 212 Bi, 213 Bi, 249 Cf, 252 Cf, 244 Cm, 245 Cm, 248 Cm, 57 Co, 58 Co, 51 Cr, 64 Cu,67 Cu, 152 Dy, 165 Dy, 152 Eu, 59 Fe, 221 Fr, 67 Ga, 68 Ga, 66 Ga, 161 Ho, 110m In, 111 In, 192 Ir,<00001​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​In one embodiment, the method of the present invention is a method for treating cancer. In this embodiment, a person skilled in the art will select an appropriate radionuclide, for example, a radionuclide that delivers high energy, preferably one with limited penetration so that only the targeted tissue is affected. The treating physician will be able to select an appropriate radionuclide for treatment without having to perform any inventive activity.

[0127] In one embodiment, the method of the present invention is a method for diagnosing cancer. In this embodiment, the method typically includes a subsequent step of detecting a radionuclide bound to a compound of the present invention and localized on the surface of tumor cells. In this embodiment, a person skilled in the art can select a radionuclide that has a long penetration range and deposits low energy in surrounding tissue. Selecting an appropriate radionuclide for diagnosis is within the skill of an average practitioner. Detection can be performed using well-known methods and devices for detecting radionuclides, such as PET or SPECT scanners.

[0128] In some embodiments, the methods of the present invention include a second and optional subsequent step of administering a chelating agent having a radionuclide bound thereto. Such a second administration of a chelating agent having a radionuclide bound thereto typically occurs 1-7 days after the first administration of a chelating agent having a radionuclide bound thereto, and a subsequent administration of a chelating agent having a radionuclide bound thereto typically occurs 1-7 days after the previous administration of a chelating agent having a radionuclide bound thereto. The radionuclide and / or chelating agent administered in the first, second, and optional subsequent administrations may be the same or may be different radionuclides and / or chelating agents. For example, an alpha-emitter may be administered in the first administration, and a beta-emitter may be administered in the second and optional subsequent administrations. Or in another example, a radionuclide suitable for PET or SPECT scanning is administered in a first dose, a scan is performed to detect tumor(s), and a radionuclide more suitable for eradicating tumor cells is administered in a second and optionally subsequent doses.

[0129] Nucleic acid sequences, etc. The present invention also relates to nucleic acid sequences encoding the compounds of the present invention. Nucleic acids can be provided by methods known in the art, for example, by starting from nucleic acid sequences encoding the separate elements of the compound and assembling and modifying the sequences using methods known in the art.

[0130] Alternatively, nucleic acid sequences can be obtained by DNA synthesis, for example, by designing an amino acid sequence for the intended compound, deriving an appropriate nucleic acid sequence encoding the intended amino acid sequence, and synthesizing the sequence using methods known in the prior art. This method has the advantage that it is easy to adapt the codon usage to the intended host cell, and it is also easy to provide the nucleic acid with the appropriate sequences required for expression in the intended host cell, such as a promoter, RBS, Kozak sequence, terminator, polyadenylation site, etc. Once the intended amino acid sequence is designed, all of this is within the skill of an average practitioner to design an appropriate nucleic acid sequence encoding the intended amino acid sequence.

[0131] Molecular Creation Nucleic acid sequences encoding the compounds of the invention can be inserted into an expression construct, such as an expression vector, transformed into a selected host cell, and expressed, resulting in the formation of the compound.

[0132] In embodiments in which the first and second antigen binding sites are VHH fragments, the complete nucleic acid sequences encoding the compounds may be contained in a single expression construct for production as a single polypeptide chain.

[0133] In embodiments in which one of the first and second antigen-binding sites is a VHH and the other is a Fab fragment, one skilled in the art will understand that two expression constructs are required, one for each chain of the compound. The two expression constructs can be inserted into and expressed in the same cell. The two chains can be expressed in a single cell either by transfection with two vectors or by a single vector with a bicistronic expression site.

[0134] Host cells suitable for use according to the present invention can in principle be any host cell capable of expressing the polypeptide of the compound. Such host cells and expression systems suitable for particular polypeptides are known in the art, and it is within the skill of the average practitioner to select an appropriate expression system, including an expression vector and a host cell, for a particular compound.

[0135] Examples of suitable host cells include bacterial cells such as E. coli, Bacillus sp., such as B. licheniformis and B. subtilis; fungal cells such as Saccharomyces cerevisiae, Pichia pastoris, Aspergillus niger, A. oryzae, Trichoderma reesei, and Penicillium chrysogenum; and mammalian cells such as insect cells, HeLa cells, CHO cells, and HEK cells.

[0136] Mammalian cells such as HeLa, CHO and HEK cells are preferred, as these cells are well known to be capable not only of producing two polypeptide chains but also of correctly assembling the two chains to produce the complete molecule of the invention.

[0137] To produce a compound of the present invention in which one of the first and second antigen-binding sites is a VHH and the other is a Fab fragment, it is advantageous to produce the compound using techniques essentially similar to those known in the art for producing recombinant antibodies. Using this approach, two nucleic acids encoding the two chains of the compound are provided with appropriate expression signals and transformed into the same host cell. Once the two nucleic acids are expressed and the two chains are formed, the two chains assemble in vivo and consist of two separate polypeptide chains that are secreted from the host cell as a single protein product, despite being expressed as two separate polypeptide chains. This method has been found to be highly effective in producing the compounds of the present invention with high purity and yield.

[0138] After production, the compounds of the invention are recovered from the cell culture supernatant using methods known in the art, such as precipitation, affinity purification, and chromatographic methods. For compounds of the invention comprising Fab fragments, the same recovery methods used for whole antibodies, such as Protein A or Protein L affinity purification, can be conveniently used.

[0139] composition The present invention also relates to compositions comprising one or more compounds of the present invention.

[0140] The compositions may contain, in addition to a compound of the invention, one or more of a diluent, salt, pH adjuster, stabilizer, antioxidant, tonicity adjuster, and the like.

[0141] In a preferred embodiment, the composition is a pharmaceutical composition containing only pharmaceutically acceptable ingredients, such as ingredients disclosed in a recognized pharmacopeia, e.g., as set forth in the European Pharmacopoeia, 10th Edition, using methods and techniques known in the art of pharmacy or compounding.

[0142] All cited references are incorporated by reference.

[0143] The accompanying figures and examples are provided to illustrate the invention, not to limit it. It will be apparent to one skilled in the art that the aspects, embodiments, claims and optional clauses of the invention may be combined.

[0144] All percentages are weight / weight unless otherwise stated. All measurements are made under standard conditions (ambient temperature and pressure) unless otherwise stated. Test conditions are in accordance with the European Pharmacopoeia 10th Edition unless otherwise stated. [Brief explanation of the drawings]

[0145] [Figure 1A] Schematic representation of a compound of the invention comprising a VHH containing a first binding site linked to a Fab containing a second binding site, with an optional C-terminal p53 tetramerization domain. In this example, the compound consists of two polypeptide chains attached to each other via disulfide bonds located in the constant region of the Fab. One chain contains a VHH-binding fragment and either the light or heavy chain of the Fab, while the other chain contains the remaining chain (light or heavy) of the Fab and, optionally, the p53 tetramerization domain. [Figure 1B] 1 shows a schematic diagram of a compound of the invention comprising a first VHH comprising a first binding site linked to a second VHH comprising a second binding site, with an optional C-terminal p53 tetramerization domain. In this example, the compound consists of a single polypeptide chain comprising two binding sites and an optional tetramerization domain. [Figure 1C] Schematic diagram of a compound of the present invention comprising two VHHs containing a first binding site linked to a Fab containing a second binding site, with an optional C-terminal p53 tetramerization domain. In this example, the compound consists of two polypeptide chains attached to each other via disulfide bonds located in the constant region of the Fab. One chain contains a first VHH-binding fragment and either the light or heavy chain of the Fab, while the other chain contains a second VHH-binding fragment identical to the first VHH, the remaining chain (light or heavy) of the Fab, and optionally a p53 tetramerization domain. [Figure 1D]Schematic diagram of a compound of the present invention comprising two different VHHs, each containing a binding site distinct from the other, linked to a Fab containing a second binding site, together with an optional C-terminal p53 tetramerization domain. In this example, the compound consists of two polypeptide chains attached to each other via disulfide bonds located in the constant region of the Fab. This compound is a trispecific YPRIT. One chain comprises a first VHH-binding fragment and either the light or heavy chain of the Fab, while the other chain comprises a second VHH-binding fragment distinct from the first VHH, the remaining chain (light or heavy) of the Fab, and optionally a p53 tetramerization domain. [Figure 1E] Schematic representation of a compound of the invention comprising a VHH containing a first binding site linked to a Fab containing a second binding site, together with an optional C-terminal p53 tetramerization domain. In this example, the compound consists of two polypeptide chains attached to each other via disulfide bonds located in the constant region of the Fab. One chain contains either the light or heavy chain of the Fab and a C-terminal VHH-binding fragment. The other chain contains the remaining chain (light or heavy) of the Fab and, optionally, a p53 tetramerization domain. [Figure 1F] Schematic diagram of a compound of the present invention comprising two identical VHHs containing a first binding site linked to a Fab containing a second binding site, together with an optional C-terminal p53 tetramerization domain. In this example, the compound consists of two polypeptide chains attached to each other via disulfide bonds located in the constant region of the Fab. The compound is bivalent with respect to the first antigen-binding site. One chain comprises either the light or heavy chain of the Fab and a C-terminal first VHH-binding fragment. The other chain comprises the remaining chain (light or heavy) of the Fab, a C-terminal second VHH identical to the first VHH fragment, and optionally a p53 tetramerization domain. [Figure 1G]Schematic diagram of a compound of the present invention comprising two different VHHs, each containing a binding site distinct from the other, linked to a Fab containing a second binding site, along with an optional C-terminal p53 tetramerization domain. In this example, the compound consists of two polypeptide chains attached to each other via disulfide bonds located in the constant region of the Fab. This compound is a trispecific YPRIT. One chain comprises either the light or heavy chain of the Fab and a first VHH-binding fragment at the C-terminus. The other chain comprises the remaining chain (light or heavy) of the Fab, a second VHH at the C-terminus distinct from the first VHH fragment, and optionally a p53 tetramerization domain. [Figure 2A] HPLC-SEC analysis of HER2-DOTA(fab)-p53. For further details, see Example 2. [Figure 2B] HPLC-SEC analysis of CD38-DOTA(fab)-p53. For further details, see Example 2. [Figure 3A] HPLC-SEC analysis of CD38-DOTA(fab)-p53_C-term-LC. For further details, see Example 3. [Figure 3B] HPLC-SEC analysis of CD33-DOTA(fab)-p53. For further details, see Example 3. [Figure 4] HPLC-SEC analysis of CD38-DOTA(fab)-dp53-See Example 4 for further details. [Figure 5] Dynamic light scattering (DLS) results for CD38-DOTA(fab)-p53 and HER2-DOTA(fab)-p53. For further details, see Example 5. [Figure 6] Nanodifferential scanning fluorimetry (DSF) results for CD38-DOTA(fab)-p53 and HER2-DOTA(fab)-p53. For further details, see Example 7. [Figure 7]Capillary isoelectric focusing (cIEF) profiles of bispecific compounds CD38-DOTA(fab)-p53 and CD38-DOTA(fab)-dp53. For further details, see Example 12. [Figure 8] Capillary isoelectric focusing (cIEF) profiles of bispecific compounds CD38-DOTA(fab)-p53 and CD38-DOTA(fab)-dp53. For further details, see Example 12. [Figure 9] YMS9a, YMS9c and YMS9d analyzed on non-reducing SDS-PAGE. For further details, see Reference Example 1. [Figure 10]

[0023] Figure 1 shows tetramerization of CD38-DOTA(fab)-p53 and HER2-DOTA(fab)-p53. For further details, see Example 13. [Figure 11A] Figure 1 shows the pk curves for CD38(bivalent)-DOTA(fab)-p53_C-term. For further details, see Example 14. [Figure 11B] Figure 1 shows the pk curve for CD38-DOTA(fab)-p53_Cterm-HC. For further details, see Example 14. [Figure 11C] Figure 1 shows the pk curve for CD38-DOTA(fab)-p53_Cterm-LC. For further details, see Example 14. [Figure 11D] Figure 1 shows the pk curve for CD38(bivalent)-DOTA(fab)-dp53. For further details, see Example 14. [Figure 11E] Figure 1 shows the pk curve for CD38(bivalent)-DOTA(Fab)-p53. For further details, see Example 14. [Figure 12A] EC50 determination is shown for EGFR-DOTA(fab)-p53 as a graphical representation of YPRIT molecule binding titration curve to target expressed in cell lines. For further details, see Example 15. [Figure 12B]EC50 determination is shown for CEA-DOTA(fab)-p53 as a graphical representation of YPRIT molecule binding titration curve to target expressed in cell lines. For further details, see Example 15. [Figure 12C] EC50 determination is shown for CD33-DOTA(fab)-p53 as a graphical representation of the YPRIT molecule binding titration curve to the target expressed in the cell line. For further details, see Example 15. [Figure 12D] EC50 determination is shown for CD276-DOTA(fab)-p53 as a graphical representation of YPRIT molecule binding titration curve to target expressed in cell lines. For further details, see Example 15. [Figure 12E] EC50 determination is shown for CD38-DOTA(fab)-p53 as a graphical representation of YPRIT molecule binding titration curve to target expressed in cell lines. For further details, see Example 15. [Figure 12F] EC50 determination is shown for CD38-DOTA(fab)-dp53 as a graphical representation of YPRIT molecule binding titration curve to target expressed in cell lines. For further details, see Example 15. [Figure 12G] EC50 determination is shown for CD38(bivalent)-DOTA(Fab)-p53 as a graphical representation of the YPRIT molecule binding titration curve to the target expressed in the cell line. For further details, see Example 15. [Figure 12H] EC50 determination is shown for CD38(bivalent)-DOTA(Fab)-dp53 as a graphical representation of the YPRIT molecule binding titration curve to the target expressed in the cell line. For further details, see Example 15. [Figure 12I] Figure 1 shows the EC50 determination for HER2 / CD276_23F11-DOTA(humanized fab)-p53 as a graphical representation of the YPRIT molecule binding titration curve to the target expressed in cell lines. For further details, see Example 15. [Figure 12J] Figure 1 shows the EC50 determination for HER2 / CD276_23F11-DOTA(humanized fab)-p53 as a graphical representation of the YPRIT molecule binding titration curve to the target expressed in cell lines. For further details, see Example 15. [Figure 13A]

[0033] Figure 1 shows the in vivo distribution of CD38-DOTA(Fab)-p53 as assessed by SPECT-based quantification. For further details, see Example 16. [Figure 13B]

[0033] Figure 1 shows the in vivo distribution of CD38-DOTA(humanized fab)-p53 as assessed by SPECT-based quantification. For further details, see Example 16. [Figure 14A] 1 shows the biodistribution of CD38(bivalent)-DOTA(Fab)-p53. [Figure 14B] 1 shows the biodistribution of CD38(bivalent)-DOTA(fab)-dp53. [Figure 14C] 1 shows the biodistribution of CD38-DOTA(fab)-p53_C-terminus-LC. [Figure 14D] The biodistribution of CD38-DOTA(fab)-p53_C-terminus-HC is shown. [Figure 14E] The biodistribution of CD38(bivalent)-DOTA(fab)-p53_C-terminus is shown. [Figure 15] YPRIT binding to Pb-TCMC-PEG4-biotin (a biotinylated variant of Pb-DOTAM / Pb-TCMC) is shown.

[0146] [Brief description of the sequence] SEQ ID NO: 1 shows an amino acid sequence containing the p53 tetramerization domain. The p53 tetramerization domain consists of amino acids 6 to 36 of this sequence.

[0147] SEQ ID NO: 2: Shows the amino acid sequence of the P63 tetramerization domain.

[0148] SEQ ID NO: 3: Shows the amino acid sequence of the P73 tetramerization domain.

[0149] SEQ ID NO: 4: Shows the amino acid sequence of the hnRNPC tetramerization domain.

[0150] SEQ ID NO: 5: Shows the amino acid sequence of the SNAP23 tetramerization domain.

[0151] SEQ ID NO: 6: Shows the amino acid sequence of the StefinB tetramerization domain.

[0152] SEQ ID NO: 7: Shows the amino acid sequence of the KCNQ4 tetramerization domain.

[0153] SEQ ID NO: 8: Shows the amino acid sequence of the CBFA2T1 tetramerization domain.

[0154] SEQ ID NO: 9: Shows the amino acid sequence of the G4S linker.

[0155] SEQ ID NO: 10: Shows the amino acid sequence of one polypeptide chain of the HER2-DOTA(Fab)-p53 compound disclosed in Example 1. Amino acids 1-115 are the HER2-binding VHH fragment, 116-132 are the G4S linker, and 133-347 are the light chain of the DOTA-binding Fab.

[0156] SEQ ID NO: 11 shows the amino acid sequence of another polypeptide chain of the YPRIT compound disclosed in Example 1. Amino acids 1-222 are the heavy chain of the DOTA-binding Fab, 223-232 are a G4S linker, 238-268 are the p53 tetramerization domain, and 279-284 are a His tag.

[0157] The same polypeptide chain was used for the CD38-DOTA(Fab)-p53 compound.

[0158] SEQ ID NO: 12: shows the amino acid sequence of one of the polypeptide chains of the CD38-DOTA(Fab)-p53 compound disclosed in Example 1. Amino acids 1-124 are the VHH fragment that binds CD38, 125-145 are a G4S linker, and 146-360 are the light chain of the DOTA-binding Fab.

[0159] SEQ ID NO: 13 shows the amino acid sequence of the heavy chain of CD38-DOTA(fab)-dp53. Amino acids 1 to 222 are the heavy chain of the DOTA-binding Fab, and 225 to 230 are the His tag.

[0160] SEQ ID NO: 14 shows the amino acid sequence of the heavy chain of CD38(bivalent)-DOTA(Fab)-p53: amino acids 1-124 are the CD38-binding VHH fragment, 125-145 are a G4S linker, 146-367 are the heavy chain of the Fab that binds DOTA, 368-377 are a G4S linker, and 383-413 are the p53 tetramerization domain.

[0161] SEQ ID NO: 15 shows the amino acid sequence of the heavy chain for CD38(bivalent)-DOTA(fab)-dp53. Amino acids 1 to 124 are the VHH fragment that binds CD38, 125 to 145 are a G4S linker, and 146 to 368 are the heavy chain of the DOTA-binding Fab.

[0162] SEQ ID NO: 16 shows the amino acid sequence of the light chain of YPRIT_CD38_DOTAM_p53. Amino acids 1 to 124 are a VHH fragment that binds CD38, 125 to 145 are a G4S linker, and 146 to 364 are the light chain of the DOTAM-binding Fab.

[0163] SEQ ID NO: 17 shows the amino acid sequence of the heavy chain for CD38-DOTAM(fab)-p53. Amino acids 1-224 are the heavy chain of the DOTAM-binding Fab, 225-234 are a G4S linker, and 240-270 are the p53 tetramerization domain.

[0164] SEQ ID NO: 18 shows the amino acid sequence of the heavy chain for CD38-DOTAM(fab)-dp53. Amino acids 1 to 225 are the heavy chain of the DOTAM-binding Fab.

[0165] SEQ ID NO: 19 shows the amino acid sequence of the light chain of BAFF-DOTA(fab)-p53. Amino acids 1 to 115 are the VHH fragment that binds BAFF, 116 to 136 are the G4S linker, and 137 to 351 are the light chain of the DOTA-binding Fab.

[0166] SEQ ID NO: 20 shows the amino acid sequence of the light chain of CD33-DOTA(fab)-p53. Amino acids 1-126 are the VHH fragment that binds CD33(22), 127-147 are the G4S linker, and 148-362 are the light chain of the DOTA-binding Fab.

[0167] SEQ ID NO: 21 shows the amino acid sequence of the light chain of EGFR-DOTA(fab)-p53. Amino acids 1 to 120 are the VHH fragment that binds EGFR, 121 to 141 are the G4S linker, and 142 to 356 are the light chain of the DOTA-binding Fab.

[0168] SEQ ID NO: 22 shows the amino acid sequence of the light chain of CEA-DOTA(fab)-p53. Amino acids 1 to 120 are the VHH fragment that binds CEA, 121 to 141 are the G4S linker, and 142 to 356 are the light chain of the DOTA-binding Fab.

[0169] SEQ ID NO: 23: Amino acid sequence of the light chain of monovalent CD38-DOTA(fab)-p53_Cterm-LC. Amino acids 1-215 are the light chain of the DOTA-binding Fab, 216-236 are a G4S linker, and 237-360 are a VHH fragment that binds CD38.

[0170] SEQ ID NO: 24: Shows the amino acid sequence of the light chain for monovalent CD38-DOTA(fab)-p53_Cterm-HC.

[0171] SEQ ID NO: 25 shows the amino acid sequence of the heavy chain of monovalent CD38-DOTA(fab)-p53_Cterm-HC: amino acids 1-222 are the heavy chain of the DOTA-binding Fab, 223-243 are a G4S linker, 244-367 are a VHH fragment that binds CD38, 368-377 are a G4S linker, and 383-413 are p53.

[0172] SEQ ID NO: 26 shows the amino acid sequence of the light chain of CD276-DOTA(fab)-p53. Amino acids 1 to 117 are the VHH fragment that binds CD276(G8), 118 to 137 are the G4S linker, and 138 to 352 are the light chain of the DOTA-binding Fab.

[0173] SEQ ID NO: 27 shows the amino acid sequence of the heavy chain for the YPRIT compound disclosed in Example 1 without the His tag (6xHis). Amino acids 1-222 are the heavy chain of the DOTA-binding Fab, 223-232 are the G4S linker, and 238-268 are the p53 tetramerization domain.

[0174] SEQ ID NO: 28: Amino acid sequence of the LC sequence of CD38-DOTA(humanized fab)-p53.

[0175] SEQ ID NO: 29: Amino acid sequence of the HC sequence of CD38-DOTA (humanized fab)-p53.

[0176] SEQ ID NO: 30: Shows the amino acid sequence of the LC sequence of HER2-DOTA(humanized fab)-p53.

[0177] SEQ ID NO: 31: Shows the amino acid sequence of the HC sequence of HER2 / CD276_23F11-DOTA(humanized fab)-p53.

[0178] SEQ ID NO: 32: Shows the amino acid sequence of the HC sequence of HER2 / CD276_23A04-DOTA(humanized fab)-p53.

[0179] Materials and Methods [Antibodies and antibody fragments] [DOTA-binding Fab] The DOTA-binding Fab used in the examples is derived from the 2D12 antibody (Corneillie et al. J. Am. Chem. Soc. 125:15039-15048 (2003)). In one example (CD38-DOTA(humanized fab)-p53), humanization was performed by introducing substitutions identified during the affinity maturation process disclosed in WO 10099536. The DOTA-binding Fab consists of a light chain (DOTA-FAB-LC) having the amino acid sequence disclosed as amino acids 133 to 347 of SEQ ID NO: 10 and a heavy chain (DOTA-FAB-HC) having the amino acid sequence disclosed as amino acids 1 to 222 of SEQ ID NO: 11.

[0180] [DOTAM-binding Fab] The DOTAM-binding Fab used in the examples is derived from the DOTAM-binding antibody disclosed in WO 2019 / 201959. The DOTAM-binding Fab consists of a light chain (DOTAM-FAB-LC) having the amino acid sequence disclosed as amino acids 146 to 364 of SEQ ID NO: 16 and a heavy chain (DOTAM-FAB-HC) having the amino acid sequence disclosed as amino acids 1 to 224 of SEQ ID NO: 17.

[0181] SPR analysis was performed on a Biocore 8K+ using a CFJB0944 CM5 sensor chip for immobilization and SPR running buffer 1X HBS-EP + pH 7.4 (Cytiva, catalog number BR100669) according to the manufacturer's instructions. [Example]

[0182] Example 1: Preparation of molecules of the present invention containing a HER2-binding site or a CD38-binding site This example demonstrates the generation of exemplary bispecific compounds having a first binding domain that binds a cellular target (e.g., a cell surface target), a second binding domain that binds a payload, and a tetramerization domain. Specifically, this example describes the generation of two exemplary bispecific antibody-based compounds, CD38-DOTA(Fab)-p53 and HER2-DOTA(Fab)-p53, which comprise a VHH with a C-terminal p53 tetramerization domain and a His tag, linked to a Fab DOTA binder by a G4S linker.

[0183] To do so, a HEK suspension cell line was transformed with an expression cassette encoding a first polypeptide (SEQ ID NO: 12) consisting of a VHH against CD38, (G4S)4, and DOTA-FAB_LC, and a second polypeptide (SEQ ID NO: 11) consisting of DOTA-FAB_HC with a C-terminal p53 and His tag (SEQ ID NO: 11). This molecule was designated CD38-DOTA(Fab)-p53.

[0184] HEK transformants were grown in expression medium, and protein L resin was used to capture the product from the medium after centrifugation.

[0185] The same procedure was followed to generate a molecule of the invention containing a HER2 binding site - HER2-DOTA(Fab)-p53 -, which has a first polypeptide (SEQ ID NO: 10) containing a VHH against HER2, (G4S)4 and DOTA-FAB_LC, and the same second polypeptide (SEQ ID NO: 11) as used in CD38-DOTA(Fab)-p53.

[0186] Example 2: Analysis of exemplary bispecific compounds The two compounds prepared in Example 1 were analyzed by HPLC-SEC, and the chromatograms are shown in Figures 2A and 2B.

[0187] HPLC-SEC analysis showed only one peak with a clean, symmetric appearance without shoulders for both HER2-DOTA(Fab)-p53 and CD38-DOTA(Fab)-p53, respectively, indicating that the products were homogeneous and no compounds with different molecular weights or strong aggregates could be observed.

[0188] Example 3: Preparation of additional molecules of the invention The procedure described in Example 1 was used to make additional molecules, which are listed in Table 1 below. Note that the two molecules prepared in Example 1 are included in the table as molecule numbers 1 and 2, and CD38-DOTA(fab)-dp53 from Example 4 is included as molecule number 15. The linker and His tag are not mentioned in the table, but the complete sequences can be found in the sequence listing.

[0189] [Table 1] TIFF2025540920000003.tif244164TIFF2025540920000004.tif176162

[0190] The compounds prepared in this example were analyzed by HPLC-SEC. Examples of the chromatograms obtained for YPRIT_CD38-DOTA(fab)-p53_C-terminus-LC (molecule 12) and CD33-DOTA(fab)-p53 (molecule 8) are shown in Figures 3A and 3B.

[0191] HPLC-SEC analysis showed only one peak with a clean, symmetric appearance without shoulders for all analyzed molecules, indicating that the products were homogeneous and no compounds with different molecular weights or strong aggregates could be observed.

[0192] Example 4: Generation and analysis of exemplary bispecific compounds without a tetramerization domain. This example demonstrates the generation of an exemplary bispecific compound having a first binding domain that binds a cellular target (e.g., a cell surface target) and a second binding domain that binds a payload. Specifically, this example describes the generation of an exemplary bispecific antibody-based compound, CD38-DOTA(fab)-dp53, which comprises a VHH (molecule 15 in Example 3) linked to FAB DOTA by a G4S linker and a His tag.

[0193] To do so, we transformed HEK suspension cells with expression cassettes encoding a first polypeptide (SEQ ID NO: 12) consisting of a VHH against CD38, (G4S)4, DOTA-FAB-LC, and a second polypeptide (SEQ ID NO: 13) consisting of DOTA-FAB-HC and a His tag. This molecule was designated CD38-DOTA(fab)-dp53.

[0194] HEK transformants were grown in expression medium, and protein L resin was used to capture the product from the medium after centrifugation.

[0195] The compound was analyzed by HPLC-SEC and the chromatogram is shown in FIG.

[0196] HPLC-SEC analysis showed only one peak with a clean, symmetric appearance without a shoulder, indicating that the product was homogeneous and no compounds with different molecular weights or strong aggregates were observed. A later retention time was also observed, which corresponded to the size of the monomeric product and not to the size of the corresponding p53 tetrameric molecule.

[0197] Example 5: Colloid Stability I (DLS) This example describes the colloidal stability of exemplary bispecific compounds HER2-DOTA(Fab)-p53 and CD38-DOTA(Fab)-p53 (molecules 1 and 2 in Example 3).

[0198] Dynamic light scattering (DLS) was used to determine the hydrodynamic diameter of CD38-YPRIT and HER2-YPRIT.

[0199] The results are shown in Figure 5 and Table 2 below.

[0200] [Table 2]

[0201] For HER2-DOTA(Fab)-p53, a radius of 7.37 nm was detected, with very low polydispersity (PDI=0.03), indicating a highly monodisperse sample.

[0202] For CD38-DOTA(Fab)-p53, a radius of 7.69 nm was detected, with very low polydispersity (PDI=0.05), indicating a highly monodisperse sample.

[0203] Thus, all samples exhibited high colloidal stability.

[0204] Example 6: Colloid Stability Ib (DLS) Dynamic light scattering (DLS) was used to determine the hydrodynamic diameter to describe the colloidal stability of the following six molecules prepared in Example 3: CD38(bivalent)-DOTA(fab)-dp53-molecule 4 in Table 1 CD38-DOTAM(fab)-p53-molecule 5 in Table 1 CD38-DOTAM(fab)-dp53-molecule 6 in Table 1 CD38-DOTA(fab)-p53_C-terminus-LC-molecule 12 in Table 1 CD38-DOTA(fab)-p53_C-term-HC-molecule 13 in Table 1 CD38(bivalent)-DOTA(fab)-p53_C-terminus - molecule 14 in Table 1.

[0205] [Table 3]

[0206] Hydrodynamic radii ranging from 4.23 to 7.82 nm were observed for these variants. PDIs were <0.1 (0.00–0.07) for all six samples, indicating highly monodisperse sample quality.

[0207] Thus, all samples showed high colloidal stability and uniform sample quality. The smaller size of the constructs without the p53 domain (molecules 4 and 6) is also evident.

[0208] Example 7: Colloidal Stability II (nano DSF) This example describes the thermal stability of exemplary bispecific compounds HER2-DOTA(Fab)-p53 and CD38-DOTA(Fab)-p53 (molecule 2 and molecule 1 in Table 1).

[0209] Nanodifferential scanning fluorimetry (DSF) was used to determine the unfolding transition and sample aggregates.

[0210] The results are shown in Figure 6 and Table 4 below.

[0211] [Table 4]

[0212] As can be seen from Table 4 above and Figure 6, two unfolding transitions were observed for HER2-DOTA(Fab)-p53 at 63.55°C and 76.09°C. Protein unfolding (ratio T オン ) began at 55.34°C. Microscopic aggregates were observed from 61.32°C (turbidity T オン ) The scattering amplitude was high and the signal-to-noise ratio was very good.

[0213] For CD38-DOTA(Fab)-p53, two unfolding transitions were observed at 62.72 °C and 75.37 °C. Protein unfolding (rate T オン) began at 56.05°C. Microscopic aggregates were observed at 53.57°C (turbidity T オン ) The scattering amplitude was high and the signal-to-noise ratio was very good.

[0214] Thus, both proteins exhibited unfolding profiles indicative of good colloidal stability.

[0215] [Example 8: Colloidal stability IIb (nanoDSF)] The colloidal stability of the following six molecules prepared in Example 3 was described using nanodifferential scanning fluorimetry (DSF) to determine the unfolding transition and sample aggregates. CD38(bivalent)-DOTA(fab)-dp53-molecule 4 in Table 1 CD38-DOTAM(fab)-p53-molecule 5 in Table 1 CD38-DOTAM(fab)-dp53-molecule 6 in Table 1 CD38-DOTA(fab)-p53_C-terminus-LC-molecule 12 in Table 1 CD38-DOTA(fab)-p53_C-term-HC-molecule 13 in Table 1 CD38(bivalent)-DOTA(fab)-p53_C-terminus - molecule 14 in Table 1.

[0216] [Table 5]

[0217] As can be seen from the table above, one major unfolding transition was observed at approximately 68 °C (IP#1) to 69 °C. For CD38-DOTA(fab)-p53_C-term-HC, a second unfolding transition was observed.

[0218] Macroscopic aggregates were observed between 53°C and 68°C (turbidity turned on). The scattering amplitude was high and the signal-to-noise ratio was very good.

[0219] Thus, these proteins exhibited unfolding profiles indicative of high colloidal and thermal stability.

[0220] Example 9: SPR data: in vitro DOTA binding This example describes the binding properties of exemplary bispecific compounds HER2-DOTA(Fab)-p53 and C CD38-DOTA(Fab)-p53 to Lu-DOTA.

[0221] Surface plasmon resonance was used to determine the association, dissociation and equilibrium constants.

[0222] [Table 6]

[0223] Exemplary HER2-DOTA(Fab)-p53 and CD38-DOTA(Fab)-p53 were demonstrated to effectively bind Lu-DOTA in vitro.

[0224] Example 10: SPR data, in vitro tumor antigen binding This example describes the binding properties of exemplary bispecific compounds HER2-DOTA(Fab)-p53 and CD38-DOTA(Fab)-p53 to bio-huHER2 / Erb2 and bio-huCD38, respectively. Additionally, this example describes the binding properties of exemplary bispecific compound CD38-DOTA(fab)-dp53.

[0225] Surface plasmon resonance was used to determine the association, dissociation and equilibrium constants.

[0226] [Table 7]

[0227] We demonstrate that exemplary HER2-DOTA(Fab)-p53 and CD38-DOTA(Fab)-p53 effectively bind to their respective tumor targets in vitro. The values ​​obtained for CD38-DOTA(fab)-dp53 suggest that the addition of p53 enhances binding by avidity to bio-huCD38.

[0228] Example 11: SPR data, in vitro tumor antigen binding SPR analysis was performed essentially as outlined in Example 10.

[0229] In the first part of this example, the antibody was immobilized on a chip and the antigen dissolved in SPR running buffer was flowed over the chip, which gave the measurements shown in Table 8.

[0230] [Table 8]

[0231] In the second part of this example, the antigen was immobilized on a chip and the antibody dissolved in SPR running buffer was flowed over the chip, which gave the measurements shown in Table 9.

[0232] [Table 9]

[0233] From Tables 8 and 9, it can be concluded that the exemplary molecules effectively bound to their respective tumor targets in vitro, confirming that molecules can be generated against different tumor targets. Furthermore, it demonstrated that molecules with different valencies can be generated, as well as that both N- and C-terminal VHH binding can be generated.

[0234] Example 12: cIEF heterogeneity For antibody charge heterogeneity analysis, capillary isoelectric focusing was applied to separate molecules based on their isoelectric points (pI).

[0235] Analysis was performed on exemplary bispecific compounds CD38-DOTA(Fab)-p53 and CD38-DOTA(fab)-dp53, molecule 1 and molecule 15 in Table 1.

[0236] As is evident from Figure 7, CD38-DOTA(Fab)-p53 has a very heterogeneous cIEF profile, and it is difficult to note a clear major peak.

[0237] As can be seen from Figure 8, CD38-DOTA(Fab)-dp53 has a cIEF profile with a major peak at 9.45 and two minor acidic peaks at 9.25 and 8.99.

[0238] These data indicate that deleting the tetramerization domain results in a much more homogeneous charge population compared to YPRIT with the C-terminal P53 domain.

[0239] [Reference example 1: CD38 SADA] This example is incorporated by reference and is originally disclosed in Example 4 of application PCT / DK2022 / 050280.

[0240] In this example, bispecific antibody variants capable of binding CD38 and DOTA were generated.

[0241] Each variant consists of an anti-CD38 scFv, an anti-DOTA scFv, and a SADA domain, and differs only in the number of interchain disulfide bonds within the scFv. Further details can be found in PCT / DK2022 / 050280. The constructs in Table 10 were generated.

[0242] [Table 10]

[0243] These constructs were analyzed by non-reducing SDS-PAGE, see FIG.

[0244] The results show that YMS9a and YMS9c contained significant amounts of multimers, whereas in YMS9d the amount of multimers was significantly reduced or absent.

[0245] The results also showed that YMS9a and YMS9c produced some heterogeneity in the monomer band, which disappeared under reducing conditions.

[0246] Example 13: Tetramerization Sample molecules were dissolved in PBS at different concentrations and allowed to reach equilibrium by incubation for 180 min at 37° C. Samples were then analyzed for their monomer and tetramer content using a Refeyn Two Mass Photometer, and results were expressed as the number of monomeric molecules in either the monomeric or tetrameric state, calculated as a percentage of the total number of available monomers.

[0247] The molecules used in this example were molecule 1 and molecule 2 from Table 1 and the CD38-SADA molecule from Example 12, designated YMS9d.

[0248] The results can be seen in Figure 10.

[0249] The results showed that the molecules of the present invention can tetramerize and, in a concentration-dependent manner, can monomerize, and the distribution between monomers and tetramers is similar to that of the corresponding SADA molecules.

[0250] Example 14: Analysis of mouse PK samples ELISA was applied for the analysis of mouse PK samples to evaluate the levels of CD38 molecules in BALB / C mouse plasma.

[0251] To determine the clearance of the molecules of the present invention, the sample molecules of the present invention were administered to BALB / C mice, blood samples were taken at different time points after administration, and the concentration of the sample molecules was determined using ELISA.

[0252] [Table 11]

[0253] The ELISA assay was initially tested and optimized with CD38-DOTA(humanized fab)-p53 and showed acceptable precision and accuracy in both assay buffer and 1% BALB / C plasma matrix.

[0254] Each of the five molecules was tested in a 1% BALB / C plasma matrix, with three separate experiments for each variant. Results showed acceptable precision and accuracy for all five variants. The performance of the five variants was comparable to that of the CD38-DOTA (humanized fab)-p53 variant.

[0255] The pk curves are shown in Figures 11A to 11E.

[0256] Results showed rapid clearance of these molecules at rates similar to those observed for CD38-SADA (data not shown). The fast PK of the tested molecules confirms that the molecules of the invention are suitable for two-step PRIT, essentially using the two-step PRIT method disclosed in WO2018204873, without the need to administer a clearing agent between the administration of the tumor-binding bispecific molecule and the chelating agent that binds the radionuclide.

[0257] Example 15: EC50 determination of YPRIT molecules by coupled fluorescence-activated cell sorting (FACS). Reference antibodies were used to QC Daudi, THP-1, A-431, HEK293T, MKN-45 and Jurkat E6-1 cell lines for CD38, CD33, EGFR, CEA and CD276 expression prior to use in EC50 determinations by FACS.

[0258] The EC50 values ​​of antibody samples were then determined by titration binding FACS on Daudi cells (for CD38 binding), THP-1 cells (for CD33 binding), A-431 cells (for EGFR binding), MKN-45 cells (for CEACAMS binding), and HEK293T cells (for CD276 binding). Where appropriate, Jurkat E6-1 or HEK293T cell lines were used as negative controls.

[0259] As can be seen from Figures 12A-12D, EGFR-DOTA(fab)-p53 and CD276-DOTA(fab)-p53 showed specific binding but not to the control cell line Jurkat E6-1. CEA-DOTA(fab)-p53 showed specific binding to MKN-45 but not to the control cell line HEK293T. CD33-DOTA(fab)-p53 showed background binding to Jurkat E6-1 and HEK293T cell lines.

[0260] All CD38 YPRIT molecules showed full titration and similarly determined EC50 binding on Daudi cells across the range of concentrations tested. For CD38, no background binding was observed on HEK293T cells. Figures 12E-12H show titration curves for CD38-DOTA(fab)-p53, CD38-DOTA(fab)-dp53, CD38(bivalent)-DOTA(Fab)-p53, and CD38(bivalent)-DOTA(Fab)-dp53, respectively.

[0261] The trivalent molecules HER2 / CD276_23F11-DOTA(humanized fab)-p53 and HER2 / CD276_23A04-DOTA(humanized fab)-p53 showed specific binding and did not bind to the control cell line Jurkat E6-1, as can be seen in Figures 12I-12J.

[0262] The resulting EC50 values ​​for the YPRIT molecule can be seen in Table 12.

[0263] [Table 12]

[0264] For CD33-DOTA(fab)-p53, binding to HEK293T and Jurkat cells above background is observed, but the concentration range does not allow for the determination of an EC50. These data demonstrate that tumor-binding YPRITS can be generated using VHH sequences targeting a broad panel of sequences. Our data also indicate that some VHHs may provide nonspecific binding to control cells. Because the VHH sequences in these examples were selected from published sources, this suggests that selecting from a panel of candidate VHH sequences is beneficial when designing YPRITS with optimal clinical profiles.

[0265] Example 16: In vivo distribution In this example, BRGSF mice bearing transplanted CD38-positive tumors were provided.

[0266] The sample molecules of the present invention were administered to mice. After a delay period following administration of the molecules, 177Mice were given a single dose of Lu-DOTA with a lag time of 24 hours for CD38-DOTA(humanized fab)-p53, CD38(bivalent)-DOTA(Fab)-p53, and CD38(bivalent)-DOTA(Fab)-dp53, 48 hours for CD38-DOTA(fab)-p53_C-term-HC and CD38-DOTA(Fab)-p53, and 72 hours for CD38-DOTA(fab)-p53_C-term-LC and CD38(bivalent)-DOTA(fab)-p53_C-term.

[0267] At 2, 24, and 48 hours after administration of 177Lu-DOTA, the mice were scanned with a SPECT scanner, and image analysis of the scanned images revealed 177 The tissue distribution of Lu was determined.

[0268] The results showed that the tested molecules of the present invention bound well to tumor antigens in vivo. Furthermore, tumor uptake was higher or similar to that of the reference CD38-SADA molecule (not shown).

[0269] Figures 13A-B show the in vivo distribution assessed using image analysis of scanned images for CD38-DOTA(Fab)-p53 (molecule 1) and CD38-DOTA(humanized fab)-p53 (molecule 16).

[0270] [Example 17] In subsequent studies, groups of animals were given the following test compounds: CD38(bivalent)-DOTA(fab)-p53_C-term, CD38-DOTA(fab)-p53_C-term-HC, CD38-DOTA(fab)-p53_C-term-LC, CD38(bivalent)-DOTA(fab)-dp53, and CD38(bivalent)-DOTA(Fab)-p53. Forty-eight hours after Lu-DOTA administration, the animals were euthanized after SPECT scanning, and the following samples were collected: blood, bone, kidney, colon, liver, muscle, small intestine, spleen, stomach, tail, and tumor. They were transferred to dry plastic tubes for radioactivity content measurement. Figures 14A to 14E show the tissue biodistribution of CD38(bivalent)-DOTA(Fab)-p53, CD38(bivalent)-DOTA(fab)-dp53, CD38-DOTA(fab)-p53_C-term-LC, CD38-DOTA(fab)-p53_C-term-HC, and CD38(bivalent)-DOTA(fab)-p53_C-term, respectively.

[0271] The results showed higher tumor-to-kidney ratios for CD38(bivalent)-DOTA(fab)-dp53 and CD38(bivalent)-DOTA(fab)-p53, especially for CD38(bivalent)-DOTA(fab)-dp53.

[0272] Furthermore, bivalent CD38-YPRITS provided a better tumor-to-kidney ratio than monovalent CD38-YPRITS with a C-terminal VHH geometry.

[0273] Example 18: In vitro binding to Pb-TCMC-PEG4-biotin This example describes the binding properties of YPRIT compounds to Pb-TCMC-PEG4-biotin, a biotinylated variant of Pb-DOTAM.

[0274] Plates with wells were coated with SADA / YPRIT (1 μg / mL, 100 μL per well) overnight at 4°C, after which the plates were washed twice with a plate washer and blocked with PBST+1% BSA for 1 h at room temperature and washed twice.

[0275] The diluted Pb-TCMC-PEG4-biotin was added to the coated wells and incubated at room temperature for 90 minutes on a plate shaker (400 rpm), 100 μL per well, and the plate was washed five times.

[0276] Streptavidin-HRP (1:5,000, 100 μL per well) was added and incubated at room temperature for 30 minutes on a plate shaker (400 rpm), 100 μL per well, and the plate was washed five times.

[0277] TMB substrate was added and incubated for 15 minutes at room temperature, 100 μL per well. 100 μL stop solution was added per well and the plate was read at 450 nm using a Synergy H1.

[0278] CD38-SADA (YMS9a, see Reference Example 1) was used as a negative control since it only binds Lu-DOTA, and a variant of YMS9a (sequence not shown) containing anti-Pb-DOTAM scFv instead of anti-DOTA scFv was used as a positive control since it uses the same anti-Pb-DOTAM sequence as the test YPRITS.

[0279] It is clear from Figure 15 that the positive controls, YPRIT-Pb-DOTAM-p53 and YPRIT-Pb-DOTAM-dp53, all bind Pb-TCMC-PEG4-biotin in a concentration-dependent manner. The EC50 values ​​for YPRIT-Pb-DOTAM-p53 and YPRIT-Pb-DOTAM-dp53 were 1.1 and 1.2 ng / ml, respectively, indicating that their affinities for Pb-TCMC are in the same range.

Claims

1. a. a first antigen-binding site capable of binding a tumor antigen, and b. A second antigen-binding site capable of binding a chelator Including, the first antigen-binding site is selected from a Fab fragment and a VHH-binding fragment, and the second antigen-binding site is selected from a Fab fragment and a VHH-binding fragment; and At least one of the first and second antigen-binding sites is a VHH fragment. compound.

2. conditions: a) one of the first and second antigen-binding sites is a Fab fragment and the other antigen-binding site is a VHH; or b) the first and second antigen-binding sites are VHHs 2. The compound according to claim 1, according to one of the following:

3. The compound of claim 1 or 2, further comprising a tetramerization domain.

4. The compound of any one of claims 1 to 3, further comprising a third or subsequent binding site.

5. 5. The compound of claim 4, wherein the third or subsequent binding site is a tumor antigen binding site.

6. The compound of claim 5, wherein the third or subsequent tumor-binding site is formed by a second or subsequent VHH fragment.

7. 7. The compound of claim 5 or 6, wherein the third or subsequent binding site(s) is / are identical to the first binding site.

8. The first binding site is selected from the group consisting of HER2, B7-H3, CA6, CD138, CD20, CD19, CD22, CD27L, CD30, CD33, CD37, CD38, CD47, CD56, CD66e, CD70, CD74, CD79b, BAFF, BAFFR, EGFR, EGFRvIII, FRα, GCC, GPNMB, mesothelin, MUC16, NaPi2b, nectin4, PSMA, STEAP1, Trop-2, 5T4, AGS-16, αvβ6, CA19.9, CAIX, CD138, CD174, CD180, C The compound of any one of claims 1 to 7, which is capable of binding to D227, CD326, CD79a, CEACAM5, CRIPTO, DLL3, DS6, endothelin B receptor, FAP, GD2, mesothelin, PMEL17, SLC44A4, TENB2, TIM-1, CD98, endosialin / CD248 / TEM1, fibronectin extra domain B, LIV-1, mucin 1, p-cadherin, peritosin, Fyn, SLTRK6, tenascin c, VEGFR2, or PRLR.

9. The compound according to any one of claims 1 to 8, wherein the first antigen-binding site is a Fab derived from one of 8H9 and 3F8.

10. The compound according to any one of claims 1 to 8, wherein the first antigen-binding site is a VHH fragment.

11. 11. The compound of claim 10, wherein the first antigen-binding site comprises a sequence selected from among amino acids 1 to 115 of SEQ ID NO: 10, amino acids 1 to 124 of SEQ ID NO: 12, amino acids 1 to 124 of SEQ ID NO: 16, amino acids 1 to 115 of SEQ ID NO: 19, amino acids 1 to 126 of SEQ ID NO: 20, amino acids 1 to 120 of SEQ ID NO: 21, amino acids 1 to 120 of SEQ ID NO: 22, amino acids 237 to 360 of SEQ ID NO: 23, amino acids 244 to 367 of SEQ ID NO: 25, or amino acids 1 to 117 of SEQ ID NO:

26.

12. The compound according to any one of claims 1 to 11, wherein the second antigen-binding site is capable of binding DOTA, DOTAM, a DOTA derivative that binds a metal ion, or a DOTAM derivative that binds a metal ion.

13. The compound of claim 12, wherein the second antigen-binding site is a Fab derived from 2D12.

5.

14. The compound according to claims 12 to 13, wherein the second antigen binding site comprises amino acids 133 to 347 of the sequence SEQ ID NO: 10 and amino acids 1 to 222 of SEQ ID NO:

11.

15. 13. The compound of claim 12, wherein the second antigen-binding site comprises amino acids 146 to 364 of SEQ ID NO: 16 and amino acids 1 to 224 of SEQ ID NO:

17.

16. 16. The compound according to any one of claims 1 to 15, wherein the tetramerization domain is selected from among p53, p63, p73, hnRNPC, SNAP-23, StefinB, KCNQ4, CBFA2T1 domains, and domains having at least 80% sequence identity with one of these domains.

17. The tetramerization domain contains the following substitutions, using the numbering of SEQ ID NO: 1: E6V, Q, K, G, D or A; Y7S, N, H, F, D or C; F8Y, V, S, L, I or C; T9S, P, N or A; L10V, I or F; Q11R, L, K, H or E; I12V, T, M, L or F; R13S, P, L, H, G or C; G14W, R or A; R15S, P, L, H, G or C; E16V, Q, K, G, D or A; F18Y, V, S, L, I or C; E19V, Q, K, G, D or A; M20V, T, R, L, K or I; F21L or I; R22L or G; E23V, Q, K, G, D or A; L24M; N25S, I or D; E26V, Q, K, G, D or A; A27V, T, S, G or D; L28W, V, M or F; E29Q, G or D; L30V, R, I, H or F; K31T, R, Q, N, M or E; D32Y, V, N, H, G or A; A33V, T, S, P, G or D; Q34R, L, K, H or E 17. The compound of any one of claims 1 to 16, wherein the domain has an amino acid sequence that differs from the sequence of amino acids 6 to 36 of SEQ ID NO: 1 by 1, 2, 3, 4, 5, or 6 substitutions selected from:

18. The compound of claim 15 or 16, wherein the tetramerization domain comprises the sequence of amino acids 6 to 36 of SEQ ID NO:

1.

19. The compound of any one of claims 1 to 18, wherein the first binding site is capable of binding HER2 and the second binding site is capable of binding DOTA.

20. The compound of any one of claims 1 to 18, wherein the first binding site is capable of binding CD38 and the second binding site is capable of binding DOTA.

21. a. SEQ ID NO: 12 and SEQ ID NO: 11; b. SEQ ID NO: 10 and SEQ ID NO: 11 c. SEQ ID NO: 12 and SEQ ID NO: 14; d. SEQ ID NO: 12 and SEQ ID NO: 15; e. SEQ ID NO: 16 and SEQ ID NO: 17; f. SEQ ID NO: 16 and SEQ ID NO: 18; g. SEQ ID NO: 19 and SEQ ID NO: 27; h. SEQ ID NO:20 and SEQ ID NO:27; i. SEQ ID NO:21 and SEQ ID NO:27; j. SEQ ID NO:22 and SEQ ID NO:27; k. SEQ ID NO: 26 and SEQ ID NO: 27 l. SEQ ID NO:23 and SEQ ID NO:27; m. SEQ ID NO:24 and SEQ ID NO:25; n. SEQ ID NO: 23 and SEQ ID NO: 25; o. SEQ ID NO: 12 and SEQ ID NO: 13; p. SEQ ID NO:28 and SEQ ID NO:29; q. SEQ ID NO: 30 and SEQ ID NO: 31; and r. SEQ ID NO: 30 and SEQ ID NO: 32 The compound according to any one of claims 1 to 20, selected from compounds comprising the sequence:

22. A composition comprising a compound according to any one of claims 1 to 21.

23. 23. The composition of claim 22, which is a pharmaceutical composition.

24. 1. A method of treating and / or diagnosing cancer, comprising: i. administering to a person in need of treatment and / or diagnosis a compound according to claims 1 to 22 or a composition according to claim 23 or 24; ii. Administering a radionuclide bound to a chelator that is recognized by said compound. A method comprising:

25. iii. Scanning the patient to detect the location of the radioactivity.

25. The method of claim 24, further comprising:

26. 26. The method of claim 24 or 25, wherein the cancer is selected from osteosarcoma, neuroblastoma, liposarcoma, fibrosarcoma, carcinoma, malignant fibrous histiocytoma, leiomyosarcoma, spindle cell sarcoma, brain tumor, small cell lung cancer, retinoblastoma, HTLV-1 infected T-cell leukemia, breast cancer, colon cancer, prostate cancer, T-cell and B-cell lymphoma, glioblastoma multiforme, malignant glioma, head and neck cancer, solid tumors, and non-small cell lung cancer.

27. The method of claims 24-25, wherein the cancer is breast cancer and the first binding site is capable of binding HER2.

28. 28. The method of claim 27, wherein the compound is a compound according to claim 19.

29. The method of claims 24 to 26, wherein the cancer is a CD38-positive cancer and the first binding site is capable of binding CD38.

30. 30. The method of claim 29, wherein the compound is a compound according to claim 20.

31. The method of claims 24-30, further comprising a second administration and optionally one or more subsequent administrations of a radionuclide bound to a chelating agent.

32. A nucleic acid encoding a compound according to claims 1 to 21.

33. 33. An expression vector comprising the nucleic acid of claim 32.

34. 34. A host cell comprising the nucleic acid of claim 33.

35. A method for making a compound according to claims 1 to 21, comprising the steps of: i. providing a host cell according to claim 34; ii. culturing the host cell under conditions that result in expression of a nucleic acid encoding the compound; and iii. Recovering the compound from the culture broth. A method comprising:

36. The compound is a compound according to claim 2 a), i. providing a host cell comprising a first nucleic acid encoding a first polypeptide chain of the compound and a second nucleic acid encoding a second polypeptide chain of the compound; ii. culturing the host cells under conditions that result in expression of the two nucleic acids; and iii. Recovering the compound from the culture broth.

36. The method of claim 35, comprising:

37. 37. The method of claim 35 or 36, wherein the host cell is a HEK cell line.

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