Bispecific antibody compounds

Abstract

Disclosed are novel compounds comprising two antigen binding sites and a tetramerization site, wherein the antigen binding sites are selected from native binding sites. The novel compounds have improved manufacturability, stability, and solubility compared to similar compounds based on scFv binding sites, such that the disclosed compounds become significantly better candidates for active ingredients in pharmaceutical compositions.

Description

Technical Field

[0001] This specification includes a sequence listing submitted together with this application in computer-readable format. The sequence listing forms part of this disclosure and is incorporated herein by reference in its entirety.

[0002] The present invention relates to bispecific antibodies for radioimmunotherapy, and in particular, bispecific antibodies that are capable of dimerizing or tetramerizing and that comprise 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 Art

[0003] Pre-targeted radioimmunotherapy (PRIT) is a method suitable for treating solid cancers using bispecific antibodies that have an antigen-binding site capable of binding an antigen exposed on the surface of a tumor and another antigen-binding site capable of binding a radionuclide or a chelator that binds a radionuclide.

[0004] PRIT is carried out by first administering the bispecific antibody to a patient and when the bispecific antibody has bound to the tumor, administering a chelator with a bound radionuclide which will be bound by the bispecific antibody and thereby localized to the tumor. Delaying the administration of the chelator with the radionuclide until the bispecific antibody has been cleared from the circulation is beneficial to reduce the systemic exposure to radiation. Some protocols use an additional step of administering a scavenger between the administration of the bispecific antibody and the radionuclide in order to facilitate the clearance of the bispecific antibody.

[0005] Many different forms 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 that comprise an scFv capable of binding a tumor antigen, an scFv capable of binding DOTA chelated to a metal ion, and a tetramerization region. Depending on the concentration, these bispecific antibodies are capable of existing in monomeric form and multimeric form, meaning that they can be administered in multimeric form and when the administered antibody is diluted in the patient's bloodstream, it will convert to monomeric form, which facilitates clearance from the plasma via the kidney. Thus, the disclosed bispecific antibodies can be used without the need to administer a scavenger.

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

[0008] Protein engineering modification techniques have been used in the past to partially address such challenges, such as introducing stable disulfide bonds between the variable light chain and variable heavy chain of scFv, introducing one or more additional substitutions, or engineering the host cells that produce scFv (see Kang and Seong, Frontiers in Microbiology (2020), Volume 11, Article 1927).

[0009] Despite recent developments, there is still a need for bispecific antibodies for PRIT that have improved manufacturability compared to previously designed bispecific antibodies. SUMMARY OF THE INVENTION

[0010] The present invention relates to a compound comprising a first antigen-binding site capable of binding to a tumor antigen and a second antigen-binding site capable of binding to a chelator, wherein one of the first antigen-binding site and / or the second antigen-binding site is a VHH fragment and the other of the first antigen-binding site and / or the second antigen-binding site is selected from a Fab fragment and a VHH-binding fragment.

[0011] In some embodiments, the compound further comprises a tetramerization region and / or a third or subsequent antigen-binding site.

[0012] In another aspect, the present invention relates to a composition comprising the compound of the present invention.

[0013] In another aspect, the present invention relates to a method of treating and / or diagnosing cancer, comprising the steps of:

[0014] a. Administering to an individual in need of treatment and / or diagnosis a compound according to the present invention or a composition according to the present invention.

[0015] b. Administering a radionuclide bound to a chelator that can be bound by the second antigen-binding site.

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

[0017] The present invention also relates to a nucleic acid encoding the compound of the present invention, an expression vector comprising the nucleic acid of the present invention, a host cell comprising the nucleic acid of the present invention, and a method of producing the compound of the present invention.

[0018] Detailed Description

[0019] Definitions

[0020] Amino acid change: The term "amino acid change" is intended to mean a change of an amino acid found in an original amino acid sequence, where the change is selected from substitution, deletion, or insertion of an additional amino acid immediately following the amino acid in question.

[0021] Amino acid substitution: The term "amino acid substitution" is intended to mean the replacement of one amino acid by a different amino acid. In this specification, the term amino acid substitution with respect to a reference sequence is intended to mean that the amino acid sequence in question can be generated starting from the reference sequence and introducing the amino acid substitution, even if the sequence in question is actually generated by another method not involving the reference sequence.

[0022] Antibody: The term "antibody" is a term recognized in the art and is intended to include a molecule or an active fragment of a molecule that binds to an antigen. Except for heavy chain antibodies, a natural antibody is composed of two heavy chains each containing one variable region and three or more constant regions (CH1, CH2, and CH3), and two light chains each containing one variable region and one constant region (CL). One light chain is bound to one heavy chain by a disulfide bond located in the constant regions (CH1 and CL), and the two heavy chains are bound to each other by multiple disulfide bonds located in the constant region (CH2).

[0023] There are several isotypes of known natural antibodies, including IgA, IgD, IgE, IgG, such as IgG1, IgG2, IgG3, and IgM, and these isotypes differ mainly in the constant regions.

[0024] Fab fragment: The term "Fab" or "Fab fragment" is an antibody fragment composed of a first polypeptide containing a light chain variable region and a light chain constant region and a second polypeptide containing a heavy chain variable region and a heavy chain constant region, where the two polypeptides are linked by a disulfide bond located in the constant region. The Fab fragment can be provided by proteolytic cleavage of a natural antibody, or it can be prepared by expressing and combining the two polypeptides, for example using recombinant DNA techniques known in the art. The Fab fragment is capable of binding to the same antigen recognized by the intact antibody. The term "Fab" or "Fab fragment" encompasses 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 that have the same overall structure as a natural Fab fragment, but in which the sequence has been engineered by introducing one or more amino acid changes (such as substitutions, deletions, or insertions) in one or both amino acid chains.

[0025] Bispecific Antibody: A bispecific antibody is an antibody that can bind simultaneously to two targets with different structures. A bispecific antibody (BsAb) is a non-natural, engineered antibody that has at least one binding site that specifically binds to one antigen, such as a tumor antigen, and at least one other binding site that specifically binds to another antigen, such as a chelator that binds a radionuclide. A variety of bispecific fusion proteins can be generated using molecular engineering. In one form, the bispecific fusion protein is bivalent and consists of, for example, a VHH that has a single binding site for one antigen and a Fab fragment that has a single binding site for a second antigen. In another form, the bispecific fusion protein is tetravalent and consists of, for example, an IgG that has two binding sites for one antigen and two identical scFvs for a second antigen.

[0026] CDR: Complementary 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 composed of two heavy chains and two light chains has 6 CDR sequences, three in the light chain and three in the heavy chain.

[0027] DOTA: Dodecane tetraacetic acid (DOTA), also known as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, and has the formula (CH 2 CH 2 NCH 2 CO 2 H) 4 ,also known as C 16 H 28 N 4 O 8 ·xH 2 O.

[0028] Derivatives of DOTA: Intended to mean compounds that contain the DOTA ring system, which is linked to some other chemical groups or moieties and is capable of chelating metal ions. Examples of such compounds include benzyl-DOTA and the bispecific chelators disclosed in WO2019010299A (Proteus-DOTA) or WO2022005998. Additional DOTA derivatives are disclosed in WO2010099536 A1.

[0029] DOTAM: Is a chelator that contains a ring system capable of binding metal ions. It 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 N 6 O 6 ·2H 2 O.

[0030] Effective amount: As used herein, the term "effective amount" refers to an amount of a given compound or composition that is necessary or sufficient to effect the desired biological result. The effective amount of a given compound or composition according to the methods of the invention will be an amount that achieves the selected result, and such amount can be determined routinely by those skilled in the art without undue experimentation.

[0031] Plasma half-life: The "plasma half-life" of 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 in the plasma half-life is size, as the kidney is known to have a filtration function that retains molecules of approximately 70 kDa in size, while smaller molecules can be eliminated via the kidney. Additionally, some molecules can interact with receptors, which may affect the plasma half-life.

[0032] Prevention: 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 an individual as a result of administration of a prophylactic or therapeutic agent.

[0033] Radioisotopes: Examples of radioisotopes that can be used, for example, for diagnostic or therapeutic use by binding to an antibody or by using a chelating agent to bind to an antibody include, but are not limited to 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 F and α-emitting particles. Non-limiting examples of α-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.

[0034] Sequence alignment: Sequence alignment simply refers to any way of aligning two sequences one below the other. It is a way of arranging DNA, RNA, or protein sequences to identify regions of similarity between the sequences. There are different alignment algorithms, and they usually have a scoring function that assigns a numerical score indicating the goodness of the alignment to each alignment and tries to find the best alignment according to its scoring function.

[0035] 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 (%).

[0036] A variety of computational algorithms are available to those skilled in the art for generating sequence alignments and calculating sequence identity. As used herein, a sequence alignment refers to a pairwise alignment. Several algorithms perform this operation, including the sequence alignment program Clustal Omega [doi:10.1038 / msb.2011.75].

[0037] As used herein, a sequence alignment may refer to the following algorithms and parameters:

[0038] Algorithm: Clustal Omega (1.2.4), http: / / www.clustal.org / omega /

[0039] A heavy-chain antibody is an antibody that contains two heavy chains and lacks the two light chains that are normally present in natural antibodies. Heavy-chain antibodies occur naturally in members of the camelid family and further occur in cartilaginous fish (such as certain sharks).

[0040] Structurally, a heavy-chain antibody is composed of a single variable region that contains complementarity-determining regions (CDRs) and two to five constant regions (depending on its origin).

[0041] Heavy-chain antibodies derived from the camelidae family contain two constant regions, while heavy-chain antibodies derived from cartilaginous fish may have up to five constant regions.

[0042] A single-domain antibody (sdAb) or VHH fragment is a fragment of a heavy-chain antibody that contains the variable region of the heavy-chain antibody, i.e., the antigen-binding region, but lacks the constant region. A VHH fragment (also known as a nanobody) is a relatively small molecule with a molecular weight of less than 20 kDa. Throughout this specification and the claims, the terms single-domain antibody (sdAb), VHH fragment, VHH, VHH binder, or nanobody may be used interchangeably.

[0043] Treatment: As used herein, the terms "treatment", "treat", "treated", or "treating" refer to prevention and / or treatment, particularly where the goal is to prevent or slow down (mitigate) an undesired physiological change or condition, such as the progression of cancer. Beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, diminishment of disease extent, stabilization of the disease state (i.e., not getting worse), delay or slowing of disease progression, improvement or palliation of the disease state, and remission (partial or total), whether detectable or not. "Treatment" can also mean an extended survival period compared to the expected survival period without receiving treatment. Those in need of treatment include those who already have a condition or disorder, those who are predisposed to having a condition or disorder, or those in whom a condition or disorder in the body is to be prevented.

[0044] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" is intended to mean a composition for use as a medicament or medicine to be administered to a patient in need. The pharmaceutical composition comprises at least one active ingredient and at least one pharmaceutical grade ingredient, such as a solvent, diluent, salt, stabilizer, pH regulator, antioxidant, etc. The pharmaceutical composition is prepared from pharmaceutical grade ingredients (e.g., as described in the 10th Edition of the European Pharmacopoeia) using methods and techniques known in the field of pharmacy or medicine.

[0045] Compound

[0046] 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 antigen-binding site and the second antigen-binding site is a VHH fragment, and the other of the first binding site and the second binding site is a VHH fragment or a Fab fragment. One of the first binding site and the second binding site is capable of binding to a tumor antigen, and the other of the first binding site and the second binding site is capable of binding to a chelating agent or a chelating agent that binds to a metal ion.

[0047] In some embodiments, the compound of the present invention further comprises a tetramerization region.

[0048] In some embodiments, the compound of the present invention may even comprise a third or subsequent binding site in the form of a second or subsequent VHH fragment.

[0049] The compound 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, 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.

[0050] In embodiments where both the first and second antigen-binding sites are in the form of VHHs, the entire compound can be produced as a single polypeptide strand containing two binding sites and having or not having a tetramerization region.

[0051] 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 strands and several combinations are possible.

[0052] In this embodiment, the VHH fragment can be included in the same polypeptide strand as the light chain of the Fab, or in the same polypeptide chain as the heavy chain of the Fab, or on both the light and heavy chains. It is even contemplated in the present invention that one VHH fragment is included in the same strand as the light chain of the Fab, and a second VHH fragment, which can be the same as or different from the first VHH fragment, is included in the same strand as the heavy chain of the Fab. In the latter case, the compound can contain two binding specificities, one binding specificity provided by 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, one binding specificity provided by the Fab and two binding specificities provided by two different VHH fragments (such a structure is referred to herein as a heterodimeric molecule).

[0053] It is even contemplated to prepare molecules of the present invention in which one or both of the polypeptides of the molecule contain a VHH fragment in the N-terminal portion of the polypeptide and a second VHH fragment in the C-terminal portion of the polypeptide. In this way, it is possible to prepare molecules of the present invention containing two, three or even more specificities.

[0054] Similarly, if present, the tetramerization region can be in the same polypeptide strand as the light chain of the Fab or in the same polypeptide chain as the heavy chain of the Fab.

[0055] This gives rise to several possible combinations since the compounds of this embodiment can contain:

[0056] A polypeptide containing a VHH region, the light chain of the Fab and a tetramerization region; and a second polypeptide chain containing the heavy chain of the Fab;

[0057] A polypeptide containing a VHH region and the light chain of the Fab; and a second polypeptide chain containing the heavy chain of the Fab and a tetramerization region;

[0058] A polypeptide containing a VHH region, the heavy chain of the Fab and a tetramerization region; and a second polypeptide chain containing the light chain of the Fab;

[0059] A polypeptide containing a VHH region, the heavy chain of the Fab and a tetramerization region; and a second polypeptide chain containing the light chain of the Fab;

[0060] A polypeptide containing a VHH region, the heavy chain of the Fab and a tetramerization region; and a second polypeptide chain containing the light chain of the Fab;

[0061] A polypeptide comprising a VHH region and the heavy chain of a Fab; and a second polypeptide chain comprising the light chain of a Fab and a tetramerization region;

[0062] A polypeptide comprising a VHH region and the heavy chain of a Fab; and a second polypeptide chain comprising a VHH region, which VHH region may be the same VHH region or it may be a different VHH region as present in the first polypeptide, and the light chain of a Fab;

[0063] A polypeptide comprising a VHH region, the light chain of a Fab; and a second polypeptide chain comprising the heavy chain of a Fab;

[0064] A polypeptide comprising a VHH region, the heavy chain of a Fab; and a second polypeptide chain comprising the light chain of a Fab; or

[0065] A polypeptide comprising a VHH region and the heavy chain of a Fab; and a second polypeptide chain comprising a VHH region, which VHH region may be the same VHH region or it may be a different VHH region as present in the first polypeptide, and the light chain of a Fab and a tetramerization region.

[0066] A polypeptide comprising a VHH region, the heavy chain of a Fab and a tetramerization region;

[0067] and a second polypeptide chain comprising a VHH region, which VHH region may be the same VHH region or it may be a different VHH region as present in the first polypeptide,

[0068] and the light chain of a Fab.

[0069] Those skilled in the art will appreciate that even other combinations may be possible, especially for the compounds of the present invention that contain second or subsequent VHH fragment binding sites.

[0070] For the present invention, the order of the regions in the polypeptide is not decisive. Instead, it is contemplated that the VHH fragment can be placed in the N-terminal portion, C-terminal portion or internal portion of the polypeptide in which it is present. In addition, a polypeptide that forms one of the chains of the compounds of the present invention may even contain two VHH fragments, for example, one VHH fragment at each of the two ends of the polypeptide, with a Fab fragment between the two VHH fragments in one chain. Preferably, the tetramerization region is located at the C-terminal end of the polypeptide that contains this region.

[0071] A preferred embodiment of the present invention is a form in which the first antigen-binding site and the second antigen-binding site are in the form of VHH fragments, and the tetramerization region is located at the C-terminal end of the polypeptide. Such compounds are schematically shown in Figure 1B .

[0072] Another preferred embodiment of the invention is a form in which one of the first antigen-binding site and the second antigen-binding site is a VHH and the other antigen-binding site is in the form of a Fab fragment, wherein the tetramerization region is located at the C-terminal end of the first polypeptide or the second polypeptide. An example of a compound according to this embodiment is schematically shown in Figure 1A and Figure 1E .

[0073] Another preferred embodiment of the invention is a form in which one of the first antigen-binding site and the second antigen-binding site is a VHH and the other antigen-binding site is in the form of a Fab fragment, wherein the tetramerization region is located at the C-terminal end of the first polypeptide or the second polypeptide, and wherein there are two VHH fragments. The two VHH fragments can be identical, such as shown in Figure 1C and Figure 1F . In this case, due to the coordination of the binding sites of the two VHH fragments, the affinity of the binding of the VHH fragments is increased. Alternatively, the two VHH fragments can be different and capable of binding different tumor antigens, such as two different tumor antigens expressed on the surface of the same tumor, as shown in Figure 1D and Figure 1G .

[0074] In a preferred embodiment, the total molecular weight of the compound is below the renal clearance limit, such as below 70 kDa. However, the compound in the tetrameric form will have a size above the renal clearance limit, meaning that when the compound of the invention is administered to a patient, the compound in the tetrameric form will have a long plasma half-life and the compound in the monomeric form will have a short plasma half-life. In addition, due to the presence of four binding sites, the compound in the tetrameric form will have a higher affinity for tumors.

[0075] Tumor antigens can in principle be any antigen that is exposed on the tumor in an antibody-accessible manner. Many such tumor antigens are known in the art and the invention is not limited to any particular such tumor antigen.

[0076] Examples of tumor antigens include, but are not limited to: 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, connexin 4, PSMA, STEAP1, Trop-2, 5T4, AGS-16, αvβ6, CA19.9, CAIX, CD138, CD174, CD180, CD227, CD326, CD79a, CEACAM5, CRIPTO, DLL3, DS6, endothelin B receptor, FAP, GD2, GPA33, mesothelin, PMEL 17, SLC44A4, TENB2, TIM-1, CD98, endosialin / CD248 / TEM1, fibronectin extra domain B, LIV-1, mucin 1, p-cadherin, periostin, Fyn, SLTRK6, tenascin c, VEGFR2, BAFF, BAFFR, and PRLR.

[0077] Preferred tumor antigens include HER2, B7-H3, GD2, CD20, CD38, GPA33, CEA, EGFRvIII, and CD33.

[0078] Antigen-binding sites that are capable of binding to a tumor antigen or to a chelator or a chelator that binds to a metal ion can be provided by selecting sequences that form Fab sites or VHH fragments from isolated intact antibodies having the desired binding properties and expressing the sequences in suitable production cells using methods well known in the art. Alternatively, such antigen-binding sites that are capable of binding to a tumor antigen or to a chelator or a chelator that binds to a metal ion can be provided by screening a library of Fab or VHH fragments for binders having the desired binding properties. This is within the capabilities of those skilled in the art.

[0079] In one embodiment, the first antigen-binding site capable of binding to a tumor antigen is a Fab, which can be derived from a tumor antigen-binding antibody known in the art. "Derived from" means that the Fab is made from the corresponding fragment of the antibody from which it is derived, and optionally modified by one or more amino acid changes, and / or it can even be humanized using methods known in the art. Examples of antibodies from which the Fab used 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).

[0080] In one embodiment, the second antigen-binding site that binds to a chelator is a Fab, which can be derived from a chelator-binding antibody.

[0081] A preferred example of an antibody capable of binding to a chelator is an antibody that is capable of binding to DOTA, or a derivative of DOTA, such as disclosed in WO 2010 / 099536 incorporated by reference, or derived from one of these antigen-binding sites. "Derived from" means that the Fab is made from the corresponding fragment of the antibody from which it is derived, and optionally modified by one or more amino acid changes, and / or it can even be humanized using methods known in the art. Examples of antibodies from which the Fab used according to the present invention can be derived include the antibody named 2D12.5 (Corneillie et al., J. Am. Chem. Soc-125:15039-15048, 2003), and the corresponding antibody optionally containing one or more substitutions in the CDR sequences as disclosed in WO 2010 / 099536.

[0082] Another preferred example of an antibody capable of binding to a chelator is an antibody that is capable of binding to DOTAM, such as disclosed in WO2019201959 incorporated by reference, or derived from one of these antigen-binding sites.

[0083] In some preferred embodiments, the compounds of the present invention are further modified by selected changes intended to improve selected properties of the compounds (e.g., removing liabilities of the compounds) in order to obtain improved molecules.

[0084] One of the key attributes of a therapeutic monoclonal antibody or antibody-derived protein candidate is that it must have biochemical and biophysical properties that render it stable, soluble, and not prone to degradation or alteration during manufacture and storage. A wide variety of potential liabilities can exist in a therapeutic candidate that need to be corrected, including a tendency to aggregate, poor solubility, a tendency to fragment, deamidate, oxidize, cyclize, or otherwise chemically modify key residues, disulfide bond shuffling, glycosylation, and the like. During antibody discovery and development, it is crucial to use protein sequence analysis algorithms, antibody modeling, and engineering to optimize the antibody for stability, solubility, and other biophysical characteristics. If the antibody does not perform appropriately, two significant potential consequences are immunogenicity and lack of batch-to-batch comparability.

[0085] Post-translational modifications (PTMs) of antibodies can affect the affinity, stability, potency, and homogeneity of the antibody, and this will create complex processes in downstream development. The bioactivity and yield of multiple isoforms of the product may be affected. PTMs generally include deamidation, isomerization, oxidation, N-glycosylation, glycosylation, free thiol modification, pyro-Glutamate, O-glycosylation, C-terminal lysine removal, and the like.

[0086] To identify sequences with liabilities, standard rules applied to the primary sequence of an antibody provide predictions of residues to be modified or avoided. Several bioinformatics software packages include algorithms for providing this information. Not all PTMs can be confidently predicted by strict sequence rules and are only revealed during a more in-depth characterization of the antibody (e.g., O-glycans).

[0087] Ideally, the molecule should be selected from a small group of molecules. Such selection should only allow molecules that do not have predicted liabilities. However, in some cases, the favorite lead candidate does retain less significant liabilities. These issues can be mitigated by generating variant molecules of the amino acids in question. In antibodies and antibody-derived therapeutic agents, the most critical parts of the sequence are the CDRs that participate in target binding. Liabilities in these sequences can affect binding and are therefore generally of higher priority for change compared to framework liabilities.

[0088] Many such specific substitutions intended to remove the identified liabilities are known in the art, and identifying such residues and replacing such residues with suitable other residues is within the skill of the average practitioner.

[0089] The tetramerization region can be selected from any region capable of tetramerizing at high concentrations and dissociating into monomers at low concentrations. In this context, "high concentration" is intended to mean the concentration typically present in a pharmaceutical composition, such as in the range of 1 - 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 invention in tetrameric form, and after administration, the compound is diluted in plasma and will gradually dissociate into monomeric form.

[0090] Examples of such tetramerization regions include the p53, p63, p73, hnRNPC, SNAP - 23, StefinB, KCNQ4, and CBFA2T1 regions, which have the amino acid sequences disclosed in SEQ ID NOs: 1 - 8, and regions 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 with one of SEQ ID NOs: 1 - 8.

[0091] Preferred tetramerization regions are the p53 region having the sequence of SEQ ID NO: 1; and regions having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 97% sequence identity with SEQ ID NO: 1.

[0092] In another embodiment, the tetramerization region is a region 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, said changes being selected from substitutions, insertions, or deletions of a single amino acid residue.

[0093] A preferred tetramerization region according to the invention is a region comprising a sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity with amino acids 6 - 36 of SEQ ID NO: 1 and that is different from the sequence of SEQ ID NO: 1 having one or more substitutions, wherein the region retains the ability to dimerize or tetramerize.

[0094] Alternatively stated, a preferred tetramerization region according to the invention is a region comprising the sequence of amino acids 6 - 36 of SEQ ID NO: 1 or a sequence that differs from the sequence of amino acids 6 - 36 of SEQ ID NO: 1 by 1, 2, 3, 4, 5, or 6 changes, wherein the region maintains the ability to dimerize or tetramerize.

[0095] One of ordinary skill in the art can readily determine whether such a region with a given substitution maintains the ability to dimerize or tetramerize by simple routine experiments, or such information can be found in the literature, such as J. Gencel - Augusto and G - Lozano; Genes & Development 34:1128 - 1146, which is incorporated by reference.

[0096] A preferred tetrameric region according to the present invention is a region having an amino acid sequence that differs from the sequence of amino acids 6 - 36 of SEQ ID NO:1 by 1, 2, 3, 4, 5, or 6 substitutions selected from the following:

[0097] E6V, Q, K, G, D, or A;

[0098] Y7S, N, H, F, D, or C;

[0099] F8Y, V, S, L, I, or C;

[0100] T9S, P, N, or A;

[0101] L10V, I, or F;

[0102] Q11R, L, K, H, or E;

[0103] I12V, T, M, L, or F;

[0104] R13S, P, L, H, G, or C;

[0105] G14W, R, or A;

[0106] R15S, P, L, H, G, or C;

[0107] E16V, Q, K, G, D, or A;

[0108] F18Y, V, S, L, I, or C;

[0109] E19V, Q, K, G, D, or A;

[0110] M20V, T, R, L, K, or I;

[0111] F21L or I;

[0112] R22L or G;

[0113] E23V, Q, K, G, D, or A;

[0114] L24M;

[0115] N25S, I, or D;

[0116] E26V, Q, K, G, D, or A;

[0117] A27V, T, S, G or D;

[0118] L28W, V, M or F;

[0119] E29Q, G or D;

[0120] L30V, R, I, H or F;

[0121] K31T, R, Q, N, M or E;

[0122] D32Y, V, N, H, G or A;

[0123] A33V, T, S, P, G or D;

[0124] Q34R, L, K, H or E;

[0125] It uses the numbering of SEQ ID NO:1. All such substitutions are known in the art and are known not to eliminate the ability to form dimers and / or tetramers in the region.

[0126] The L24P substitution completely eliminates tetramers and should not be applied.

[0127] The p53 tetramerization region containing the sequence of amino acids 6 - 36 of SEQ ID NO:1 is a preferred SADA region.

[0128] The compounds of the present invention may further comprise one or more linkers that separate different parts of the separated compound, for example, separating the first binding site from the second binding site or separating the second binding site from the tetramerization region. The purpose of the linker is to separate different regions, allowing the different regions to fold and function without being hindered by other elements of the compound. The linker preferably consists of hydrophilic residues that do not form strong secondary structures and is usually rich in residues such as glycine, serine and / or threonine. A preferred linker is a linker composed of G and S residues (such as GGGGS (SEQ ID NO:9)), optionally repeated two or more times to obtain the desired length.

[0129] In a 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 region is p53. The Fab may preferably be derived from antibody 2D12.5, optionally having the C825 substitution disclosed in WO 2010 / 099536. An example of this embodiment is a compound comprising a first polypeptide and a second polypeptide, the first polypeptide comprising the sequence of SEQ ID NO:10 and the second polypeptide comprising the sequence of SEQ ID NO:11.

[0130] In another preferred embodiment, the first antigen-binding site is a VHH that binds CD38, the second antigen-binding site is a Fab that is capable of binding DOTA, and the tetramerization region is p53. The Fab may preferably be derived from antibody 2D12.5, optionally having the C825 substitution disclosed in WO 2010 / 099536.

[0131] An example of this embodiment is a compound comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the sequence of SEQ ID NO:12 and the second polypeptide comprises the sequence of SEQ ID NO:11; a compound comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the sequence of SEQ ID NO:12 and the second polypeptide comprises the sequence of SEQ ID NO:14; a compound comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the sequence of SEQ ID NO:12 and the second polypeptide comprises the sequence of SEQ ID NO:15; a compound comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the sequence of SEQ ID NO:23 and the second polypeptide comprises the sequence of SEQ ID NO:27; or a compound comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the sequence of SEQ ID NO:24 and the second polypeptide comprises the sequence of SEQ ID NO:25; a compound comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the sequence of SEQ ID NO:23 and the second polypeptide comprises the sequence of SEQ ID NO:25.

[0132] 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 is capable of binding DOTA. This embodiment does not include the tetramerization region p53.

[0133] An example of this embodiment is a compound comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the sequence of SEQ ID NO:12 and the second polypeptide comprises the sequence of SEQ ID NO:13.

[0134] In another preferred embodiment, the first antigen-binding site is a VHH that binds CD38, and the second antigen-binding site is a humanized Fab that is capable of binding DOTA, and the tetramerization region is p53.

[0135] An example of this embodiment is a compound comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the sequence of SEQ ID NO:28 and the second polypeptide comprises the sequence of SEQ ID NO:29.

[0136] In another preferred embodiment, the first antigen-binding site is a VHH that binds CD38, the second antigen-binding site is a Fab that is capable of binding DOTAM, and the tetramerization region is p53.

[0137] An example of this embodiment is a compound comprising a first polypeptide and a second polypeptide, the first polypeptide comprising the sequence of SEQ ID NO: 16 and the second polypeptide comprising the sequence of SEQ ID NO: 17.

[0138] 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 is capable of binding DOTAM. This embodiment does not include the tetramerization region p53.

[0139] An example of this embodiment is a compound comprising a first polypeptide and a second polypeptide, the first polypeptide comprising the sequence of SEQ ID NO: 16 and the second polypeptide comprising the sequence of SEQ ID NO: 18.

[0140] 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 that is capable of binding DOTA, and the tetramerization region is p53. The Fab may preferably be derived from antibody 2D12.5, optionally having the C825 substitution disclosed in WO 2010 / 099536.

[0141] An example of this embodiment is a compound comprising a first polypeptide and a second polypeptide, the first polypeptide comprising the sequence of SEQ ID NO: 19 and the second polypeptide comprising the sequence of SEQ ID NO: 27.

[0142] In another preferred embodiment, the first antigen-binding site is a VHH that binds CD33, the second antigen-binding site is a Fab that is capable of binding DOTA, and the tetramerization region is p53. The Fab may preferably be derived from antibody 2D12.5, optionally having the C825 substitution disclosed in WO 2010 / 099536.

[0143] An example of this embodiment is a compound comprising a first polypeptide and a second polypeptide, the first polypeptide comprising the sequence of SEQ ID NO: 20 and the second polypeptide comprising the sequence of SEQ ID NO: 27.

[0144] 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 that is capable of binding DOTA, and the tetramerization region is p53. The Fab may preferably be derived from antibody 2D12.5, optionally having the C825 substitution disclosed in WO 2010 / 099536.

[0145] An example of this embodiment is a compound comprising a first polypeptide and a second polypeptide, the first polypeptide comprising the sequence of SEQ ID NO: 21 and the second polypeptide comprising the sequence of SEQ ID NO: 27.

[0146] 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 region is p53. The Fab may preferably be derived from antibody 2D12.5, optionally having the C825 substitution disclosed in WO 2010 / 099536.

[0147] An example of this embodiment is a compound comprising a first polypeptide and a second polypeptide, the first polypeptide comprising the sequence of SEQ ID NO:22 and the second polypeptide comprising the sequence of SEQ ID NO:27.

[0148] 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 region is p53. The Fab may preferably be derived from antibody 2D12.5, optionally having the C825 substitution disclosed in WO 2010 / 099536.

[0149] An example of this embodiment is a compound comprising a first polypeptide and a second polypeptide, the first polypeptide comprising the sequence of SEQ ID NO:26 and the second polypeptide comprising the sequence of SEQ ID NO:27.

[0150] 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 region is p53. The third binding site is a VHH that binds CD276. The Fab may preferably be derived from antibody 2D12.5, optionally having the C825 substitution disclosed in WO 2010 / 099536.

[0151] An example of this embodiment is a compound comprising a first polypeptide and a second polypeptide, the first polypeptide comprising the sequence of SEQ ID NO:30 and the second polypeptide comprising the sequence of SEQ ID NO:31.

[0152] Another example of this embodiment is a compound comprising a first polypeptide and a second polypeptide, the first polypeptide comprising the sequence of SEQ ID NO:30 and the second polypeptide comprising the sequence of SEQ ID NO:32.

[0153] Use of the compound

[0154] The compounds of the present invention are suitable for immunotherapy, particularly suitable for pretargeted radioimmunotherapy (PRIT).

[0155] PRIT is used to treat cancer in a method in which a bispecific antibody is administered to a patient in need thereof, the bispecific antibody comprising a first binding site capable of binding to a tumor antigen and a second binding site capable of binding to a radionuclide, a chelator bound to the radionuclide, or a molecule linked to the chelator (e.g., a peptide bound to a chelator group), wherein the first binding site is capable of binding to a peptide moiety. After allowing the antibody to bind to the tumor and clearing unbound antibody from the plasma, a radionuclide or a chelator bound to the radionuclide is administered to the patient and bound by the bispecific antibody located at the tumor, the radionuclide or the chelator bound to the radionuclide being recognized by the bispecific antibody. Unbound radionuclide or chelator bound to the radionuclide will be rapidly cleared from the plasma via renal clearance.

[0156] Preferably, the bispecific antibody is cleared from the plasma prior to the administration of the radionuclide in order to protect other tissues from radiation. In some embodiments, a clearing agent is administered between the administration of the bispecific antibody and the administration of the radionuclide in order to improve clearance.

[0157] In one embodiment, a compound of the invention comprising a tetrameric region is preferably administered in tetrameric form and will bind to a target tumor antigen after administration. Due to the coordination between the four tumor antigen binding sites in the tetrameric form, the compound in tetrameric form will have a higher affinity for the tumor antigen, and thus it is foreseeable that the compound in tetrameric form will exhibit improved binding properties to the tumor due to the tetrameric form. The unbound compound in tetrameric form will rapidly disassemble into monomeric form due to the reduced concentration in the plasma and the monomer will be rapidly cleared from the plasma due to its size being below the renal clearance limit. These properties of the compounds of the invention mean that they effectively bind to the tumor antigen and rapidly clear unbound compounds from the plasma, thus achieving very high efficiency clearance even without the use of a clearing agent.

[0158] In another embodiment, a compound of the invention comprises two identical tumor binding sites. In this embodiment, the compound will have a higher affinity for the tumor antigen due to the coordination between the two tumor antigen binding sites as compared to a similar compound having the same binding sites but only one tumor binding site.

[0159] In another embodiment, the compounds of the invention comprise two different tumor-binding sites. In this embodiment, the compound will have improved binding properties for tumors expressing two tumor antigens due to the coordination between the two different tumor antigen-binding sites, as compared to a similar compound having only one of the two tumor-binding sites. An example of a tumor carrying two different tumor antigens is breast cancer, where the use of a compound according to this embodiment would be beneficial. In some breast cancers, there is overexpression of both B7H3 and HER2 on the cell surface. The cell line BT-474 derived from breast cancer can be used to experimentally address this situation.

[0160] Accordingly, in a preferred aspect, the invention relates to a method of treating or diagnosing cancer in a patient, comprising the steps of:

[0161] i. administering to an individual in need of such treatment or diagnosis a compound according to the invention, which is capable of binding to a tumor antigen and further capable of binding to a chelator having a bound radionuclide;

[0162] ii. after a holding period, administering to the individual a chelator that binds the radionuclide.

[0163] In the case where the compound comprises a tetrameric region, the compound will disassemble into monomers and unbound compound will be cleared from the plasma during the holding period.

[0164] The holding period can be selected in the range of 12 h to 7 days, such as 12 h, 18 h, 24 h, 36 h, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days.

[0165] Even though satisfactory high clearance can be achieved using the compounds of the invention, a step of administering a scavenger can also be included to improve clearance. However, in most cases, it is redundant and needs to be omitted in order to avoid further administration steps and inconvenience to the patient associated with such additional steps.

[0166] Similar treatment methods have been previously disclosed, for example, in WO 2018 / 204873, with the significant difference that the methods of the prior art use bispecific antibody constructs with antigen-binding sites in the scFv format.

[0167] It is known that there are some inherent difficulties in the scFv format, such as low productivity, and some heterogeneity is often observed in the product, which may be caused by the mispairing of disulfide bonds, and this problem may be exacerbated by the practice of frequently introducing stable disulfide bonds between the variable light chain and the variable heavy chain. In addition, many scFvs have low solubility and low stability is also often observed upon storage.

[0168] Thus, it is known that some scFvs require several rounds of amino acid alterations in order to alleviate such inherent difficulties. In contrast, the molecules of the present invention are dominated by native binding regions that do not give rise to such difficulties, or generally have fewer and less severe problems that need to be addressed as part of the development of a pharmaceutical product.

[0169] The compounds of the present invention are superior to the scFv-based conjugates disclosed in the art in terms of manufacturability and product stability, and offer at least the following benefits:

[0170] Higher expression levels,

[0171] Easier to purify, for example using robust manufacturing capture purification methods,

[0172] Higher solubility,

[0173] Wider formulation options,

[0174] Construction of native regions,

[0175] Improved stability, and

[0176] Less heterogeneity.

[0177] Thus, in many ways, the compounds of the present invention are "better molecules" that are more suitable for the preparation of pharmaceutical products compared to scFv-based compounds.

[0178] 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 tumor, and non-small cell lung cancer.

[0179] Those skilled in the art will appreciate that the chelating agent used in the therapeutic / diagnostic methods of the present invention can be any chelating agent that is recognized and can be bound by the second antigen-binding site.

[0180] In a preferred embodiment, the second antigen-binding site is capable of binding DOTA or a derivative of DOTA. In this embodiment, the chelating agent is DOTA or a derivative of DOTA that can be bound by the second antigen-binding site, such as a compound containing a DOTA ring system, for example the compounds disclosed in WO 2010 / 099536, WO2019 / 010299, and WO 2022 / 005998 (incorporated herein by reference).

[0181] The radionuclide can be any radionuclide that can be bound by a chelating agent (usually 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, 133 La, 177 Lu, 237 Np, 189m Os, 231 Pa, 203 Pb, 212 Pb, 210 Po, 211 Po, 212 Po, 214 Po, 215 Po, 216 Po, 218 Po, 195m Pt, 238 Pu, 239 Pu, 240 Pu, 244 Pu, 223 Ra, 224 Ra, 226 Ra, 82 Rb, 186 Re, 188 Re, 103m Rh,119 Sb, 75 Se, 89 Sr, 149 Tb, 151 Tb, 161 Tb, 99m Tc, 94m Tc, 227 Th, 228 Th, 229 Th, 230 Th, 232 Th, 201 Tl, 90 Y, 86 Y, 89 Zr.

[0182] In one embodiment, the method of the present invention is a method for treating cancer. In this embodiment, one of ordinary skill in the art will select a suitable radionuclide, such as a radionuclide that delivers high energy and preferably has limited penetration, so that only the targeted tissue is affected. The treating physician will be able to select a suitable radionuclide for treatment without the use of any inventive activity.

[0183] In one embodiment, the method of the present invention is a method for diagnosing cancer. In this embodiment, the method generally includes the subsequent step of detecting a radionuclide that binds to the compound of the present invention and is located on the surface of tumor cells. In this embodiment, one of ordinary skill in the art may select a radionuclide having a long penetration range and depositing low energy in surrounding tissues. Selecting a radionuclide suitable for diagnosis is within the skill of an ordinary practitioner. Detection can be carried out using well-known methods and equipment for detecting radionuclides, such as PET or SPECT scanners.

[0184] In some embodiments, the method of the invention includes a second and optional subsequent step of administering a chelator having a bound radionuclide. Such a second administration of a chelator having a bound radionuclide typically occurs 1 - 7 days after the first administration of a chelator having a bound radionuclide, and subsequent administrations of a chelator having a bound radionuclide typically occur 1 - 7 days after the previous administration of a chelator having a bound radionuclide. The radionuclide and / or chelator administered in the first, second, and optional subsequent administrations may be the same or they may be different radionuclides and / or chelators. For example, an α - emitter may be administered at the first administration and a β - emitter may be administered at the second and optional subsequent administrations. Or in another example, a radionuclide suitable for PET or SPECT scanning is administered at the first administration and a scan is performed to detect a tumor, and a radionuclide more suitable for eradicating tumor cells is administered at the second and optional subsequent administrations.

[0185] Nucleic acid sequences, etc.

[0186] The invention is also directed to nucleic acid sequences encoding the compounds of the invention. The nucleic acids can be provided by methods known in the art, for example starting with nucleic acid sequences of the individual elements encoding the compound and assembling and modifying the sequences using methods known in the art.

[0187] Alternatively, the nucleic acid sequence can be obtained by DNA synthesis, for example by designing the amino acid sequence of the desired compound, obtaining a suitable nucleic acid sequence encoding the desired amino acid sequence and synthesizing the sequence using methods known in the current art. This method has the advantage of easily adapting codon usage to the intended host cell and also providing the nucleic acid with suitable sequences required for expression in the intended host cell, such as promoters, RBS, Kozak sequences, terminators, polyadenylation sites, etc. Once the desired amino acid sequence has been designed, a suitable nucleic acid sequence encoding the desired amino acid sequence can be designed, all within the skill of the average practitioner.

[0188] Production of the molecule

[0189] The nucleic acid sequence encoding the compound 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.

[0190] In embodiments where the first antigen - binding site and the second antigen - binding site are VHH fragments, the complete nucleic acid sequence encoding the compound can be contained in a single expression construct for production as a single polypeptide chain.

[0191] In embodiments where one of the first antigen-binding site and the second antigen-binding site is a VHH and the other is a Fab fragment, those skilled in the art will appreciate that two expression constructs are required, one for each strand of the compound. The two expression constructs can be inserted and expressed in the same cell. The two chains can be expressed in a single cell by transfection with two vectors or by a vector having a bicistronic expression site.

[0192] Host cells suitable for use in accordance with the present invention can in principle be any host cell capable of expressing the polypeptides of the compound. Such host cells and expression systems suitable for specific polypeptides are known in the art, and the selection of an expression system suitable for a particular compound, including expression vectors and host cells, is within the skill of the average practitioner.

[0193] Examples of suitable host cells include bacterial cells such as Escherichia coli (E. coli), Bacillus sp. such as Bacillus licheniformis and Bacillus subtilis; fungal cells such as Saccharomyces cerevisiae, Pichia pastoris, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Penicillium chrysogenum; insect cells, mammalian cells such as HeLa cells, CHO cells, HEK cells.

[0194] Mammalian cells such as HeLa cells, CHO cells and HEK cells are preferred because it is well known that these cells can not only produce two polypeptide chains, but they can also properly combine the two chains and produce the complete molecule of the present invention.

[0195] To produce the compounds of the present invention, where one of the first antigen-binding site and the second antigen-binding site is a VHH and the other is a Fab fragment, it is expedient to use substantially the same techniques known in the art for producing recombinant antibodies to produce the compounds. Using this method, two nucleic acids encoding the two strands of the compound are provided with suitable expression signals and transfected into the same host cell. When the two nucleic acids are expressed and the two strands are formed, they will assemble in vivo and be secreted from the host cell as a single protein product, even though it is composed of two independent polypeptide strands and expressed as two independent polypeptide strands. The results show that this method is highly effective for producing the compounds of the present invention in high purity and yield.

[0196] After production, the compounds of the present invention are recovered from cell culture supernatants using methods known in the art, such as precipitation, affinity purification, and chromatography methods. For compounds of the present invention that comprise Fab fragments, recovery methods similar to those used for intact antibodies, such as Protein A or Protein L affinity purification, may be suitably used.

[0197] Composition

[0198] The present invention also relates to compositions comprising one or more compounds of the present invention.

[0199] In addition to the compounds of the present invention, the compositions may also comprise one or more of a diluent, salt, pH regulator, stabilizer, antioxidant, tonicity regulator, etc.

[0200] In a preferred embodiment, the composition is a pharmaceutical composition comprising only pharmaceutically acceptable ingredients, such as those disclosed in recognized pharmacopoeias, for example, as described in the European Pharmacopoeia 10 th Edition, using methods and techniques known in the pharmaceutical or pharmacy arts.

[0201] All references cited are incorporated by reference in their entirety.

[0202] The figures and examples are provided for purposes of illustration and not limitation of the present invention. Those skilled in the art will appreciate that aspects, embodiments, claims, and any items of the present invention may be combined.

[0203] Unless otherwise stated, all percentages are by 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 10. BRIEF DESCRIPTION OF THE DRAWINGS

[0204] Brief Description of the Drawings

[0205] Figure 1A . A schematic illustration showing a compound of the present invention, which comprises a VHH comprising a first binding site, which is linked to a Fab comprising a second binding site with an optional C-terminal p53 tetramerization region. In this example, the compound is composed of two polypeptide chains linked to each other by a disulfide bond located in the constant region of the Fab. One chain comprises the VHH binding fragment and the light or heavy chain of the Fab, and the other chain comprises the remaining chain (light or heavy chain) of the Fab and the optional p53 tetramerization region.

[0206] Figure 1B. A schematic illustration showing a compound of the present invention, the compound comprising a first VHH comprising a first binding site, which is linked to a second VHH comprising a second binding site having an optional C-terminal p53 tetramerization region. In this example, the compound consists of a single polypeptide chain comprising two binding sites and an optional tetramerization region.

[0207] Figure 1C . A schematic illustration showing a compound of the present invention, the compound comprising two VHHs each comprising a first binding site, which are linked to a Fab comprising a second binding site having an optional C-terminal p53 tetramerization region. In this example, the compound consists of two polypeptide chains linked to each other via a disulfide bond located in the constant region of the Fab. One chain comprises the first VHH binding fragment and the light or heavy chain of the Fab, and the other chain comprises a second VHH binding fragment identical to the first VHH and the remaining chain (light or heavy chain) of the Fab and an optional p53 tetramerization region.

[0208] Figure 1D . A schematic illustration showing a compound of the present invention, the compound comprising two different VHHs each comprising a binding site different from the other, which are linked to a Fab comprising a second binding site having an optional C-terminal p53 tetramerization region. In this example, the compound consists of two polypeptide chains linked to each other via a disulfide bond located in the constant region of the Fab. This compound is a trispecific YPRIT. One chain comprises the first VHH binding fragment and the light or heavy chain of the Fab, and the other chain comprises a second VHH binding fragment different from the first VHH and the remaining chain (light or heavy chain) of the Fab and an optional p53 tetramerization region.

[0209] Figure 1E . A schematic illustration showing a compound of the present invention, the compound comprising a VHH comprising a first binding site, which is linked to a Fab comprising a second binding site having an optional C-terminal p53 tetramerization region. In this example, the compound consists of two polypeptide chains linked to each other via a disulfide bond located in the constant region of the Fab. One chain comprises the light or heavy chain of the Fab and a C-terminal VHH binding fragment. The other chain comprises the remaining chain (light or heavy chain) of the Fab and an optional p53 tetramerization region.

[0210] Figure 1F.Schematic illustration of a compound of the invention, which compound comprises two identical VHHs each comprising a first binding site, which are linked to a Fab comprising a second binding site with an optional C-terminal p53 tetramerization region. In this example, the compound consists of two polypeptide chains linked to each other via a disulfide bond located in the constant region of the Fab. This compound is bivalent at the first antigen-binding site. One chain comprises 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 chain) of the Fab, a C-terminal second VHH identical to the first VHH fragment, and an optional p53 tetramerization region.

[0211] Figure 1G .Schematic illustration of a compound of the invention, which compound comprises two different VHHs each comprising a binding site different from the other, which are linked to a Fab comprising a second binding site with an optional C-terminal p53 tetramerization region. In this example, the compound consists of two polypeptide chains linked to each other via a disulfide bond located in the constant region of the Fab. This compound is trispecific YPRIT. One chain comprises 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 chain) of the Fab, a C-terminal second VHH different from the first VHH fragment, and an optional p53 tetramerization region.

[0212] Figure 2A .HPLC-SEC analysis of HER2-DOTA(fab)-p53. For other details, see Example 2.

[0213] Figure 2B .HPLC-SEC analysis of CD38-DOTA(fab)-p53. For other details, see Example 2.

[0214] Figure 3A .HPLC-SEC analysis of CD38-DOTA(fab)-p53_C-TERM-LC. For other details, see Example 3.

[0215] Figure 3B .HPLC-SEC analysis of CD33-DOTA(fab)-p53. For other details, see Example 3.

[0216] Figure 4 .HPLC-SEC analysis of CD38-DOTA(fab)-dp53-. For other details, see Example 4.

[0217] Figure 5 .Dynamic light scattering (DLS) results for CD38-DOTA(fab)-p53 and HER2-DOTA(fab)-p53. For other details, see Example 5.

[0218] Figure 6 . Nano differential scanning fluorimetry (DSF) results of CD38-DOTA(fab)-p53 and HER2-DOTA(fab)-p53. For other details, see Example 7.

[0219] Figure 7 and Figure 8 . Capillary isoelectric focusing (cIEF) profiles of the bispecific compounds CD38-DOTA(fab)-p53 and CD38-DOTA(fab)-dp53. For other details, see Example 12.

[0220] Figure 9 . Analysis of YMS9a, YMS9c and YMS9d on non-reducing SDS-PAGE. For other details, see Reference Example 1.

[0221] Figure 10 . Demonstration of the tetramerization of CD38-DOTA(fab)-p53 and HER2-DOTA(fab)-p53. For other details, see Example 13.

[0222] Figure 11A . Demonstration of the pk curve of CD38(bivalent)-DOTA(fab)-p53_C-terminus. For other details, see Example 14.

[0223] Figure 11B . Demonstration of the pk curve of CD38-DOTA(fab)-p53_C-terminus-HC. For other details, see Example 14.

[0224] Figure 11C . Demonstration of the pk curve of CD38-DOTA(fab)-p53_C-terminus-LC. For other details, see Example 14.

[0225] Figure 11D . Demonstration of the pk curve of CD38(bivalent)-DOTA(fab)-dp53. For other details, see Example 14.

[0226] Figure 11E . Demonstration of the pk curve of CD38(bivalent)-DOTA(Fab)-p53. For other details, see Example 14.

[0227] Figure 12A . Demonstration of the EC50 assay, which is a graphical representation of the binding titration curve of YPRIT molecules against the target expressed in cell lines for EGFR-DOTA(fab)-p53. For other details, see Example 15.

[0228] Figure 12B . Displays the EC50 assay, which is a graphical representation of the titration curve of the binding of YPRIT molecule to the target expressed in the cell line for CEA-DOTA(fab)-p53. For other details, see Example 15.

[0229] Figure 12C . Displays the EC50 assay, which is a graphical representation of the titration curve of the binding of YPRIT molecule to the target expressed in the cell line for CD33-DOTA(fab)-p53. For other details, see Example 15.

[0230] Figure 12D . Displays the EC50 assay, which is a graphical representation of the titration curve of the binding of YPRIT molecule to the target expressed in the cell line for CD276-DOTA(fab)-p53. For other details, see Example 15.

[0231] Figure 12E . Displays the EC50 assay, which is a graphical representation of the titration curve of the binding of YPRIT molecule to the target expressed in the cell line for CD38-DOTA(fab)-p53. For other details, see Example 15.

[0232] Figure 12F . Displays the EC50 assay, which is a graphical representation of the titration curve of the binding of YPRIT molecule to the target expressed in the cell line for CD38-DOTA(fab)-dp53. For other details, see Example 15.

[0233] Figure 12G . Displays the EC50 assay, which is a graphical representation of the titration curve of the binding of YPRIT molecule to the target expressed in the cell line for CD38(bivalent)-DOTA(Fab)-p53. For other details, see Example 15.

[0234] Figure 12H . Displays the EC50 assay, which is a graphical representation of the titration curve of the binding of YPRIT molecule to the target expressed in the cell line for CD38(bivalent)-DOTA(Fab)-dp53. For other details, see Example 15.

[0235] Figure 12I . Displays the EC50 assay, which is a graphical representation of the titration curve of the binding of YPRIT molecule to the target expressed in the cell line for HER2 / CD276_23F11-DOTA(humanized fab)-p53. For other details, see Example 15.

[0236] Figure 12J. Displays an EC50 determination, which is a graphical representation of the binding titration curve of HER2 / CD276_23F11-DOTA (humanized fab)-p53 to the YPRIT molecule expressed in the cell line. For other details, see Example 15.

[0237] Figure 13A . Displays the in vivo distribution of CD38-DOTA (Fab)-p53 by SPECT-based quantitative assessment. For other details, see Example 16.

[0238] Figure 13B . Displays the in vivo distribution of CD38-DOTA (humanized fab)-p53 by SPECT-based quantitative assessment. For other details, see Example 16.

[0239] Figure 14A Displays the biodistribution of CD38 (bivalent)-DOTA (Fab)-p53

[0240] Figure 14B Displays the biodistribution of CD38 (bivalent)-DOTA (fab)-dp53

[0241] Figure 14C Displays the biodistribution of CD38-DOTA (fab)-p53_C-terminal-LC

[0242] Figure 14D Displays the biodistribution of CD38-DOTA (fab)-p53_C-terminal-HC

[0243] Figure 14E Displays the biodistribution of CD38 (bivalent)-DOTA (fab)-p53_C-terminal

[0244] Figure 15 Displays YPRIT binding to Pb-TCMC-PEG4-biotin (a biotinylated variant of Pb-DOTAM / Pb-TCMC). Detailed implementation

[0245] Brief Description of the Sequences

[0246] SEQ ID NO:1: Displays the amino acid sequence containing the P53 tetramer region. The P53 tetramer region is composed of amino acids 6 to 36 of the sequence;

[0247] SEQ ID NO:2: Displays the amino acid sequence of the P63 tetramer region;

[0248] SEQ ID NO:3: Displays the amino acid sequence of the P73 tetramer region;

[0249] SEQ ID NO:4: Displays the amino acid sequence of the hnRNPC tetramerization region;

[0250] SEQ ID NO:5: Displays the amino acid sequence of the SNAP23 tetramerization region;

[0251] SEQ ID NO:6: Displays the amino acid sequence of the StefinB tetramerization region;

[0252] SEQ ID NO:7: Displays the amino acid sequence of the KCNQ4 tetramerization region;

[0253] SEQ ID NO:8: Displays the amino acid sequence of the CBFA2T1 tetramerization region;

[0254] SEQ ID NO:9: Displays the amino acid sequence of the G 4 S linker;

[0255] SEQ ID NO:10: Displays 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 VHH fragment that binds to HER2, 116-132 is the G4S linker, and 133-347 is the light chain of the Fab that binds to DOTA.

[0256] SEQ ID NO:11: Displays the amino acid sequence of the other polypeptide chain of the YPRIT compound disclosed in Example 1. Amino acids 1-222 are the heavy chain of the Fab that binds to DOTA, 223-232 is the G4S linker, 238-268 is the p53 tetramerization region, and 279-284 is the His tag.

[0257] The same polypeptide chain is used for the CD38-DOTA(Fab)-p53 compound.

[0258] SEQ ID NO:12: Displays 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 to CD38, 125-145 is the G4S linker, and 146-360 is the light chain of the Fab that binds to DOTA.

[0259] SEQ ID NO:13: Displays the amino acid sequence of the heavy chain of CD38-DOTA(fab)-dp53. Amino acids 1-222 are the heavy chain of the Fab that binds to DOTA, 225-230 is the His tag.

[0260] SEQ ID NO:14: Displays the amino acid sequence of the heavy chain of CD38 (bivalent)-DOTA(Fab)-p53. Amino acids 1-124 are the VHH fragment that binds to CD38, 125-145 is the G4S linker, 146-367 is the heavy chain of the Fab that binds to DOTA, 368-377 is the G4S linker, and 383-413 is the p53 tetramerization region.

[0261] SEQ ID NO:15: Displays the amino acid sequence of the heavy chain of CD38 (bivalent)-DOTA(fab)-dp53. Amino acids 1-124 are the VHH fragment that binds to CD38, 125-145 is the G4S linker, and 146-368 is the heavy chain of the Fab that binds to DOTA.

[0262] SEQ ID NO:16: Displays the amino acid sequence of the light chain of YPRIT_CD38_DOTAM_p53. Amino acids 1-124 are the VHH fragment that binds to CD38, 125-145 is the G4S linker, and 146-364 is the light chain of the Fab that binds to DOTAM.

[0263] SEQ ID NO:17: Displays the amino acid sequence of the heavy chain of CD38-DOTAM(fab)-p53. Amino acids 1-224 are the heavy chain of the Fab that binds to DOTAM, 225-234 is the G4S linker, and 240-270 is the p53 tetramerization region.

[0264] SEQ ID NO:18: Displays the amino acid sequence of the heavy chain of CD38-DOTAM(fab)-dp53. Amino acids 1-225 are the heavy chain of the Fab that binds to DOTAM.

[0265] SEQ ID NO:19: Displays the amino acid sequence of the light chain of BAFF-DOTA(fab)-p53. Amino acids 1-115 are the VHH fragment that binds to BAFF, 116-136 is the G4S linker, and 137-351 is the light chain of the Fab that binds to DOTA.

[0266] SEQ ID NO:20: Displays the amino acid sequence of the light chain of CD33-DOTA(fab)-p53. Amino acids 1-126 are the VHH fragment that binds to CD33(22), 127-147 is the G4S linker, and 148-362 is the light chain of the Fab that binds to DOTA.

[0267] SEQ ID NO:21: Displays the amino acid sequence of the light chain of EGFR-DOTA(fab)-p53. Amino acids 1-120 are the VHH fragment that binds to EGFR, 121-141 are the G4S linker, and 142-356 are the light chain of the Fab that binds to DOTA.

[0268] SEQ ID NO:22: Displays the amino acid sequence of the light chain of CEA-DOTA(fab)-p53. Amino acids 1-120 are the VHH fragment that binds to CEA, 121-141 are the G4S linker, and 142-356 are the light chain of the Fab that binds to DOTA.

[0269] SEQ ID NO:23: Displays the amino acid sequence of the light chain of monovalent CD38-DOTA(fab)-p53_C-terminal-LC. Amino acids 1-215 are the light chain of the Fab that binds to DOTA, 216-236 are the G4S linker, and 237-360 are the VHH fragment that binds to CD38.

[0270] SEQ ID NO:24: Displays the amino acid sequence of the light chain of monovalent CD38-DOTA(fab)-p53_C-terminal-HC.

[0271] SEQ ID NO:25: Displays the amino acid sequence of the heavy chain of monovalent CD38-DOTA(fab)-p53_C-terminal-HC. Amino acids 1-222 are the heavy chain of the Fab that binds to DOTA, 223-243 are the G4S linker, 244-367 are the VHH fragment that binds to CD38, 368-377 are the G4S linker, and 383-413 are p53.

[0272] SEQ ID NO:26: Displays the amino acid sequence of the light chain of CD276-DOTA(fab)-p53. Amino acids 1-117 are the VHH fragment that binds to CD276 (G8), 118-137 are the G4S linker, and 138-352 are the light chain of the Fab that binds to DOTA.

[0273] SEQ ID NO:27: Displays the amino acid sequence of the heavy chain of the YPRIT compound without the His tag (6xHis) disclosed in Example 1. Amino acids 1-222 are the heavy chain of the Fab that binds to DOTA, 223-232 are the G4S linker, and 238-268 are the p53 tetramerization region.

[0274] SEQ ID NO:28: Displays the amino acid sequence of the LC sequence of CD38-DOTA (humanized fab)-p53

[0275] SEQ ID NO:29: Amino acid sequence showing the HC sequence of CD38-DOTA (humanized fab)-p53

[0276] SEQ ID NO:30: Amino acid sequence showing the LC sequence of HER2-DOTA (humanized fab)-p53

[0277] SEQ ID NO:31: Amino acid sequence showing the HC sequence of HER2 / CD276_23F11-DOTA (humanized fab)-p53

[0278] SEQ ID NO:32: Amino acid sequence showing the HC sequence of HER2 / CD276_23A04-DOTA (humanized fab)-p53

[0279] Materials and Methods

[0280] Antibodies and Antibody Fragments:

[0281] Fab conjugated to DOTA : The Fab that binds DOTA 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), the substitutions identified during affinity maturation as disclosed in WO10099536 were introduced and its humanization was incorporated. The Fab that binds DOTA consists of: a light chain (DOTA-FAB-LC) having the amino acid sequence disclosed at amino acid positions 133-347 of SEQ ID NO:10; and a heavy chain (DOTA-FAB-HC) having the amino acid sequence disclosed at amino acid positions 1-222 of SEQ ID NO:11.

[0282] Fab conjugated to DOTAM : The Fab that binds DOTAM used in the examples is derived from the antibody that binds DOTAM disclosed in WO2019 / 201959. The Fab that binds DOTAM consists of: a light chain (DOTAM-FAB-LC) having the amino acid sequence disclosed at amino acid positions 146-364 of SEQID NO:16, and a heavy chain (DOTAM-FAB-HC) having the amino acid sequence of amino acids 1-224 of SEQID NO:17.

[0283] SPR analysis was performed on a Biocore 8K+ using a CFJB0944 CM5 sensor chip for immobilization and SPR operating buffer 1XHBS-EP+ pH 7.4 (Cytiva, catalog number BR100669) according to the manufacturer's instructions.

[0284] Example

[0285] Example 1. Production of Molecules of the Invention Comprising HER2-Binding Sites or CD38-Binding Sites

[0286] This example demonstrates the production of exemplary bispecific compounds having a first binding region that binds to a cellular target (e.g., a cell surface target), a second binding region that binds to a payload, and a tetramerization region. Specifically, this example describes the production of two exemplary bispecific antibody-based compounds, CD38-DOTA(Fab)-p53 and HER2-DOTA(Fab)-p53, which comprise a VHH linked via a G4S linker to a FAB DOTA binder having a C-terminal p53 tetramerization region and a His tag.

[0287] To this end, a HEK suspension cell line was transformed with an expression cassette encoding a first polypeptide (SEQ ID NO:12) and a second polypeptide (SEQ ID NO:11), where the first polypeptide consists of a VHH against CD38, (G4S) 4 and DOTA-FAB_LC, and the second polypeptide consists of DOTA-FAB_HC having a C-terminal p53 and His tag. (SEQ ID NO:11). This molecule was named CD38-DOTA(Fab)-p53.

[0288] The HEK transformants were cultured in expression medium and the product was captured from the medium using Protein L resin after centrifugation.

[0289] The same procedure was carried out to prepare the molecule HER2-DOTA(Fab)-p53 of the invention having a HER2 binding site, which has a first polypeptide (SEQ ID NO:10) comprising 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.

[0290] Example 2. Analysis of Exemplary Bispecific Compounds

[0291] The two compounds prepared in Example 1 were analyzed by HPLC-SEC, and the chromatograms are shown in Figure 2A and Figure 2B .

[0292] HPLC-SEC analysis showed that HER2-DOTA(Fab)-p53 and CD38-DOTA(Fab)-p53 each had only one peak, showing good symmetry without shoulders, indicating that the products were homogeneous and no compounds with different molecular weights or aggregates were visible.

[0293] Example 3. Production of Other Molecules of the Invention

[0294] Other molecules were prepared using the procedure described in Example 1 as listed in Table 1 below. Note that the two molecules prepared in Example 1 are included in the table with molecule numbers 1 and 2, and CD38-DOTA(fab)-dp53 from Example 4 is included with molecule number 15. The linker and His tag are not mentioned in the table, but the complete sequences can be seen in the sequence listing.

[0295] Table 1: Molecules generated by the present invention

[0296]

[0297] The compounds prepared in this example were analyzed by HPLC-SEC, and as examples of the resulting chromatograms, the chromatograms of YPRIT_CD38-DOTA(fab)-p53_C-terminal-LC (molecule 12) and CD33-DOTA(fab)-p53 (molecule 8) are shown in Figure 3A and Figure 3B .

[0298] HPLC-SEC analysis showed that all the analyzed molecules had only one peak, showing good symmetry without shoulders, indicating that the products were homogeneous and no compounds with different molecular weights or aggregates were visible.

[0299] Example 4. Production and Analysis of Exemplary Bispecific Compounds without a Tetramerization Region.

[0300] This example demonstrates the production of exemplary bispecific compounds having a first binding region that binds to a cellular target (such as a cell surface target) and a second binding region that binds to a payload. Specifically, this example describes the production of an exemplary bispecific antibody-based compound, CD38-DOTA(fab)-dp53, which comprises a VHH and a His tag linked via a G4S linker to FAB DOTA (molecule 15 of Example 3).

[0301] To this end, HEK suspension cell lines were transformed with expression components encoding a first polypeptide (SEQ ID NO:12) and a second polypeptide (SEQ ID NO:13), the first polypeptide consisting of a VHH against CD38, (G4S) 4 It consists of DOTA-FAB-LC, and this second polypeptide consists of DOTA-FAB-HC and a His tag. This molecule is named CD38-DOTA(fab)-dp53.

[0302] HEK transformants were cultured in an expression medium and the product was captured from the medium using Protein L resin after centrifugation.

[0303] The compound was analyzed by HPLC-SEC, and the chromatogram is shown in Figure 4 In.

[0304] HPLC-SEC analysis showed only one peak, presenting good symmetry without shoulders, indicating that the product is homogeneous and no compounds with different molecular weights or aggregates are visible. It was also observed that the retention time of the corresponding molecule with p53 was later, which is based on the monomer rather than the tetramer size of the product.

[0305] Example 5. Colloidal Stability I (DLS)

[0306] This example describes the colloidal stability of the exemplary bispecific compounds HER2-DOTA(Fab)-p53 and CD38-DOTA(Fab)-p53 (Molecules 1 and 2 of Example 3).

[0307] Dynamic light scattering (DLS) was used to determine the hydrodynamic diameter of CD38-YPRIT and HER2-YPRIT.

[0308] The results are shown in Figure 5 And Table 2 below.

[0309] Table 2: DLS Results:

[0310]

[0311] For HER2-DOTA(Fab)-p53, a radius of 7.37 nm was detected. The polydispersity was extremely low (PDI = 0.03), indicating a highly monodisperse sample.

[0312] For CD38-DOTA(Fab)-p53, a radius of 7.69 nm was detected. The polydispersity was extremely low (PDI = 0.05), indicating a highly monodisperse sample.

[0313] Therefore, both samples showed high colloidal stability.

[0314] Example 6. Colloidal Stability Ib (DLS)

[0315] The colloidal stability of the following 6 molecules prepared in Example 3 was described using dynamic light scattering (DLS) for determining the hydrodynamic diameter:

[0316] ● CD38 (bivalent)-DOTA(fab)-dp53 - Molecule 4 in Table 1

[0317] ● CD38-DOTAM(fab)-p53 - Molecule 5 in Table 1

[0318] ● CD38-DOTAM(fab)-dp53 - Molecule 6 in Table 1

[0319] ● CD38-DOTA(fab)-p53_C - terminal - LC - Molecule 12 in Table 1

[0320] ● CD38-DOTA(fab)-p53_C - terminal - HC - Molecule 13 in Table 1

[0321] ● CD38 (bivalent)-DOTA(fab)-p53_C - terminal - Molecule 14 in Table 1.

[0322] Table 3: DLS Results:

[0323]

[0324] The hydrodynamic radius of the variants was observed to be between 4.23 and 7.82 nm. For all six samples, PDI < 0.1 (0.00 to 0.07), indicating a highly monodisperse sample quality.

[0325] Therefore, all samples showed high colloidal stability and homogeneous sample quality. It was also evident that the constructs without the p53 region (Molecules 4 and 6) were smaller in size.

[0326] Example 7. Colloidal Stability II (nanoDSF)

[0327] This example describes the thermal stability of the exemplary bispecific compounds HER2 - DOTA(Fab)-p53 and CD38 - DOTA(Fab)-p53 (Molecules 2 and 1 in Table 1).

[0328] Nanoscale differential scanning fluorimetry (DSF) was used to determine the unfolding transitions and sample aggregation.

[0329] The results are shown in Figure 6 and Table 4 below.

[0330] Table 4: Nano DSF Results:

[0331]

[0332] As can be seen from Table 4 above and Figure 6 it is visible that for HER2 - DOTA(Fab)-p53, secondary unfolding transitions were observed at 63.55 °C and 76.09 °C. Protein unfolding (ratio T ON) starts at 55.34 °C. From 61.32 °C (turbidity T ON ) microscopic sample aggregation was observed. The scattering amplitude is high and the signal / noise ratio is excellent.

[0333] For CD38-DOTA(Fab)-p53, a secondary unfolding transition was observed at 62.72 °C and 75.37 °C. Protein unfolding (ratio T ON ) starts at 56.05 °C. From 53.57 °C (turbidity T ON ) microscopic sample aggregation was observed. The scattering amplitude is high and the signal / noise ratio is excellent.

[0334] Thus, the two proteins show unfolding profiles indicative of good colloidal stability.

[0335] Example 8. Colloidal Stability IIb (nanoDSF)

[0336] The colloidal stability of the following 6 molecules prepared in Example 3 was described using nano differential scanning fluorimetry (DSF) for determining unfolding transitions and sample aggregation:

[0337] ● CD38 (divalent)-DOTA(fab)-dp53 - Molecule 4 in Table 1

[0338] ● CD38-DOTAM(fab)-p53 - Molecule 5 in Table 1

[0339] ● CD38-DOTAM(fab)-dp53 - Molecule 6 in Table 1

[0340] ● CD38-DOTA(fab)-p53_C - terminal - LC - Molecule 12 in Table 1

[0341] ● CD38-DOTA(fab)-p53_C - terminal - HC - Molecule 13 in Table 1

[0342] ● CD38 (divalent)-DOTA(fab)-p53_C - terminal - Molecule 14 in Table 1.

[0343] Table 5: Nano DSF results:

[0344]

[0345] As can be seen from the above table, a major first unfolding transition was observed between approximately 68 °C (IP#1) and 69 °C. For CD38-DOTA(fab)-p53_C - terminal - HC, a second unfolding transition was observed.

[0346] Macroscopic sample aggregation was observed from 53 °C to 68 °C (turbidity TON). The scattering amplitude is high and the signal / noise ratio is excellent.

[0347] Thus, the protein shows unfolding profiles indicative of higher colloidal and thermal stability.

[0348] Example 9. SPR Data: In Vitro Binding to DOTA

[0349] This example demonstrates the binding characteristics of the exemplary bispecific compounds HER2-DOTA(Fab)-p53 and CCD38-DOTA(Fab)-p53 to Lu-DOTA.

[0350] Surface plasmon resonance was used to determine the association, dissociation, and equilibrium constants.

[0351] Table 6: SPR

[0352]

[0353]

[0354] It shows that the exemplary HER2-DOTA(Fab)-p53 and CD38-DOTA(Fab)-p53 bind effectively to Lu-DOTA in vitro.

[0355] Example 10. SPR Data, In Vitro Tumor Antigen Binding

[0356] This example demonstrates the binding characteristics of the exemplary bispecific compounds HER2-DOTA(Fab)-p53 and CD38-DOTA(Fab)-p53 to bio-huHER2 / Erb2 and bio-huCD38, respectively. In addition, it demonstrates the binding characteristics of the exemplary bispecific compound CD38-DOTA(fab)-dp53.

[0357] Surface plasmon resonance was used to determine the association, dissociation, and equilibrium constants.

[0358] Table 7: SPR

[0359]

[0360] It shows that the exemplary HER2-DOTA(Fab)-p53 and CD38-DOTA(Fab)-p53 bind effectively to their respective tumor targets in vitro. The values obtained for CD38-DOTA(fab)-dp53 indicate that the addition of p53 enhances binding by affinity for bio-huCD38.

[0361] Example 11. SPR Data, In Vitro Tumor Antigen Binding

[0362] The SPR analysis was performed essentially as outlined in Example 10.

[0363] In the first part of this example, the antibody was immobilized on the chip, and the antigen was dissolved in the SPR operating buffer flowing on the chip. The measurement values shown in Table 8 were thus obtained.

[0364] Table 8: SPR: Antibody in ligand setting

[0365]

[0366]

[0367] In the second part of this example, the antigen was immobilized on the chip, and the antibody was dissolved in the SPR operating buffer flowing on the chip. The measurement values shown in Table 9 were thus obtained.

[0368] Table 9: SPR: Antigen in ligand setting

[0369]

[0370] *The kd value could not be determined due to the limitations of the instrument. The KD value was estimated based on the obtained ka and the limit kd detection level (1.00E-07) of the instrument.

[0371] **The value was considered indicative due to the reasons described in *.

[0372] It is inferred from Table 8 and Table 9 that the exemplary molecules bind effectively in vitro to their corresponding tumor targets, which confirms that molecules against different tumor targets can be produced. In addition, it shows that molecules with different valences can be produced and both N-terminal and C-terminal VHH linkages can be produced.

[0373] Example 12. cIEF Heterogeneity

[0374] Capillary isoelectric focusing was applied for antibody charge heterogeneity analysis, in which molecules were separated based on the isoelectric point (pI).

[0375] Analysis was performed on the exemplary bispecific compounds CD38-DOTA(Fab)-p53 and CD38-DOTA(fab)-dp53 (Molecules 1 and 15 in Table 1).

[0376] As can be seen from Figure 7 CD38-DOTA(Fab)-p53 has a highly heterogeneous cIEF profile and the different main peaks are difficult to annotate.

[0377] As can be seen from Figure 8 CD38-DOTA(Fab)-dp53 has a cIEF profile with a main peak at 9.45 and two smaller acidic peaks at 9.25 and 8.99.

[0378] These data show that deletion of the tetramerization region results in a much more homogeneous charge population compared to YPRIT with a C-terminal P53 region.

[0379] Reference Example 1: CD38SADA

[0380] This example is included as reference and was originally disclosed in Example 4 of application PCT / DK2022 / 050280.

[0381] In this example, variants of bispecific antibodies were generated that were able to bind CD38 and DOTA.

[0382] Each variant consists of an anti-CD38 scFv, an anti-DOTA scFv, and a SADA region, and differs only in the number of interchain disulfide bonds within the scFv. More details can be found in PCT / DK2022 / 050280. The constructs in Table 10 were generated.

[0383] Table 10: Constructs generated

[0384]

[0385] Constructs were analyzed on non-reducing SDS-PAGE, see Figure 9 .

[0386] The results showed that YMS9a and YMS9c contained a large amount of multimers, while the amount of multimers in YMS9d was significantly reduced or absent.

[0387] The results also showed that YMS9a and YMS9c caused some heterogeneity in the monomer bands. The heterogeneity disappeared under reducing conditions.

[0388] Example 13 Tetramerization :

[0389] The sample molecules were dissolved in PBS at different concentrations and allowed to reach equilibrium by incubation for 180 minutes at 37° C. The samples were then analyzed for monomer and tetramer content using a Refeyn second generation mass spectrometer, where the results are expressed as the number of monomer molecules in the monomeric or tetrameric state, calculated as a percentage of the total number of available monomers.

[0390] The molecules used in this example are molecules 1 and 2 from Table 1 and the CD38-SADA molecule of Example 12, designated YMS9d.

[0391] The results can be seen in Figure 10 middle.

[0392] The results show that, depending on the concentration, the molecules of the invention are able to tetramerize and monomerize, and the distribution between monomers and tetramers is similar to that of the corresponding SADA molecules.

[0393] Example 14. Analysis of Mouse PK Samples :

[0394] Mouse PK samples were analyzed using ELISA to evaluate the level of CD38 molecules in the plasma of BALB / C mice.

[0395] 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 collected at different times after administration, and the concentration of the sample molecules was measured using ELISA.

[0396] Table 11_Tested CD38 variants:

[0397]

[0398] ELISA analysis was first tested and optimized with CD38-DOTA (humanized fab)-p53 and showed acceptable precision and accuracy in assay buffer and 1% BALB / C plasma matrix.

[0399] In three independent experiments for each variant, each of the five molecules was tested in 1% BALB / C plasma matrix. The results showed acceptable precision and accuracy for all five variants. The potency of the five variants was comparable to that of the CD38-DOTA (humanized fab)-p53 variant.

[0400] The PK curves are shown in Figure 11A - Figure 11E .

[0401] The results showed that the molecules were cleared rapidly at a rate similar to the clearance rate observed for CD38-SADA (data not shown). The rapid PK of the tested molecules confirmed that the molecules of the present invention are suitable for two-step PRIT, substantially using the two-step PRIT method disclosed in WO2018204873, without the need to administer a scavenger between the tumor-binding bispecific molecule and the chelator conjugated with the radionuclide.

[0402] Example 15. EC50 Determination of YPRIT Molecules by Binding Fluorescence-Activated Cell Sorting (FACS).

[0403] QC was performed on Daudi, THP-1, A-431, HEK293T, MKN-45, and Jurkat E6-1 cell lines for CD38, CD33, EGFR, CEA, and CD276 expression prior to using reference antibodies for EC50 determination by FACS.

[0404] Subsequently, the EC50 of the antibody samples was determined by titration binding FACS to Daudi cells (for CD38 binding assessment), to THP-1 cells (for CD33 binding assessment), to A-431 cells (for EGFR binding assessment), to MKN-45 cells (for CEACAMS binding assessment), and to HEK293T (for CD276 binding assessment). When applicable, the Jurkat E6-1 or HEK293T cell lines were used as negative controls.

[0405] As can be seen from Figure 12A - Figure 12D EGFR-DOTA(fab)-p53 and CD276-DOTA(fab)-p53 showed specific binding and did not bind to the control cell line Jurkat E6-1. CEA-DOTA(fab)-p53 showed specific binding to MKN-45 and did not bind to the control cell line HEK293T. CD33-DOTA(fab)-p53 showed background binding to both the Jurkat E6-1 cell line and the HEK293T cell line.

[0406] All CD38 YPRIT molecules showed fully titrated binding in Daudi cells within the tested concentration range and at a similar EC50 determined in a similar assay. For CD38, no background binding was observed on HEK293T cells. Figure 12E - Figure 12H Titration curves of CD38-DOTA(fab)-p53, CD38-DOTA(fab)-dp53, CD38(bivalent)-DOTA(Fab)-p53, and CD38(bivalent)-DOTA(Fab)-dp53 are shown respectively.

[0407] 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 from Figure 12I - Figure 12J as can be seen.

[0408] The obtained EC50 values of the YPRIT molecules are shown in Table 12.

[0409] Table 12: EC50 values of the YPRIT molecules.

[0410]

[0411] For CD33-DOTA(fab)-p53, we did see binding to cells above the background of HEK293T and Jurkat cells, but the concentration range did not allow determination of the EC50. These data demonstrate that we can generate tumor-binding YPRIT using VHH sequences targeting a broad range of sequences. Our data also demonstrate that some VHHs can provide non-specific binding to control cells. Since the VHH sequences in these examples were selected from published sources, it suggests that the selection from a small panel of candidate VHH sequences will be beneficial when engineering YPRIT for optimal clinical profiles.

[0412] Example 16 In Vivo Distribution :

[0413] For this example, BRGSF mice implanted with CD38-positive tumors were provided.

[0414] The sample molecule of the present invention was administered to the mice. After a delay time after administration of the molecule, 177 Lu-DOTA was administered to the mice as a single dose. The delay times for CD38-DOTA(humanized fab)-p53, CD38(bivalent)-DOTA(Fab)-p53, and CD38(bivalent)-DOTA(Fab)-dp53 were 24 hours, for CD38-DOTA(fab)-p53_C-terminal-HC and CD38-DOTA(Fab)-p53 were 48 hours, and for CD38-DOTA(fab)-p53_C-terminal-LC and CD38(bivalent)-DOTA(fab)-p53_C-terminal were 72 hours.

[0415] At 2, 24, and 48 hours after administration of 177Lu-DOTA, the mice were scanned in a SPECT scanner, and the tissue distribution of 177 Lu was determined by image analysis of the scanned images.

[0416] The results showed that the tested molecules of the present invention bound well to tumor antigens in vivo. In addition, the tumor uptake was higher than or similar to that of the reference CD38-SADA molecule (not shown).

[0417] Figure 13A - Figure 13B Shows the in vivo distribution evaluated by image analysis of the scanned images for CD38-DOTA(Fab)-p53 (molecule 1) and CD38-DOTA(humanized fab)-p53 (molecule 16).

[0418] Example 17

[0419] In subsequent studies, the animal groups received the following test compounds: CD38(bivalent)-DOTA(fab)-p53_C-terminal, CD38-DOTA(fab)-p53_C-terminal-HC, CD38-DOTA(fab)-p53_C-terminal-LC, CD38(bivalent)-DOTA(fab)-dp53 and CD38(bivalent)-DOTA(Fab)-p53. At 48 h after administration of 177Lu-DOTA, the animals were euthanized after SPECT scanning, and the following samples were collected: blood, bone, kidney, large intestine, liver, muscle, small intestine, spleen, stomach, tail and tumor. They were transferred to dry plastic tubes for radioactivity measurement. Figure 14A - Figure 14E Respectively show the tissue biodistributions of CD38(bivalent)-DOTA(Fab)-p53, CD38(bivalent)-DOTA(fab)-dp53, CD38-DOTA(fab)-p53_C-terminal-LC, CD38-DOTA(fab)-p53_C-terminal-HC and CD38(bivalent)-DOTA(fab)-p53_C-terminal.

[0420] The results show higher tumor-to-kidney ratios for CD38(bivalent)-DOTA(fab)-dp53 and CD38(bivalent)-DOTA(fab)-p53, especially CD38(bivalent)-DOTA(fab)-dp53.

[0421] In addition, bivalent CD38-YPRIT provides a better tumor-to-kidney ratio compared to the monovalent CD38-YPRIT with a C-terminal VHH geometry.

[0422] Example 18: In Vitro Binding to Pb-TCMC-PEG4-Biotin

[0423] This example demonstrates the binding characteristics of the YPRIT compound with Pb-TCMC-PEG4-biotin, which is a biotinylated variant of Pb-DOTAM.

[0424] Dishes with wells were coated overnight at 4 °C with SADA / YPRIT (1 μg / mL, 100 μL / well), then the dishes were washed 2 times with a dish washer, blocked for 1 h with PBST + 1% BSA at room temperature and washed 2 times.

[0425] Diluted Pb-TCMC-PEG4-biotin was added to the coated wells and incubated for 90 minutes at room temperature on a dish shaker (400 rpm), 100 μL per well, and the dishes were washed 5 times.

[0426] Add streptavidin-HRP (1:5,000, 100 μL / well) and incubate for 30 minutes at room temperature on a plate shaker (400 rpm), 100 μL per well, and wash the plate 5 times.

[0427] Add TMB substrate and incubate for 15 minutes at room temperature. 100 μL per well. Add stop solution, 100 μL per well, and read the plate at 450 nm by Synergy H1.

[0428] CD38-SADA (YMS9a, see Reference Example 1) was used as a negative control because it only binds Lu-DOTA, and a variant of YMS9a containing anti-Pb-DOTAM scFv instead of anti-DOTA scFv (sequence not shown) was used as a positive control because it uses the same anti-Pb-DOTAM sequence as the tested YPRIT.

[0429] From Figure 15 As can be seen, 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 the binding of YPRIT-Pb-DOTAM-p53 and YPRIT-Pb-DOTAM-dp53 were 1.1 and 1.2 ng / ml, respectively, indicating that the affinity of Pb-TCMC is in the same range.

Claims

1. A compound comprising: a. A first antigen-binding site capable of binding to a tumor antigen; and b. A second antigen-binding site capable of binding to a chelator; wherein the first antigen-binding site is selected from Fab fragments and VHH binding fragments; the second antigen-binding site is selected from Fab fragments and VHH binding fragments; and at least one of the first antigen-binding site and the second antigen-binding site is a VHH fragment.

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

3. The compound according to claim 1 or 2, which further comprises a tetramerization region.

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

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

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

7. The compound according to claim 5 or 6, wherein the third or subsequent binding site is identical to the first binding site.

8. The compound according to any one of claims 1 to 7, wherein the first binding site is capable of binding to 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, connexin 4, PSMA, STEAP1, Trop-2, 5T4, AGS-16, αvβ6, CA19.9, CAIX, CD138, CD174, CD180, CD227, CD326, CD79a, CEACAM5, CRIPTO, DLL3, DS6, endothelin B receptor, FAP, GD2, mesothelin, PMEL 17, SLC44A4, TENB2, TIM-1, CD98, endosialin / CD248 / TEM1, fibronectin extra domain B, LIV-1, mucin 1, p-cadherin, periostin, Fyn, SLTRK6, tenascin c, VEGFR2 or PRLR.

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

26.

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

13. The compound according to 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 133-347 of the sequence of SEQ ID NO:10 and amino acids 1-222 of SEQ ID NO:

11.

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

17.

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

17. The compound according to any one of the preceding claims, wherein the tetramerization region is a region having an amino acid sequence that differs from the sequence of amino acids 6-36 of SEQ ID NO:1 by 1, 2, 3, 4, 5, or 6 substitutions selected from the following: 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; It uses the numbering of SEQ ID NO:

1.

18. The compound according to claim 15 or 16, wherein the tetrameric region comprises the sequence of amino acids 6 - 36 of SEQ ID NO:

1.

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

20. The compound according to 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. The compound according to any one of claims 1 to 20, which is selected from the compounds comprising the following sequences: 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.

22. A composition comprising the compound according to any one of the preceding claims.

23. The composition according to claim 22, which is a pharmaceutical composition.

24. A method for treating and / or diagnosing cancer, comprising the following steps: 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 that binds to the chelator and is recognized by the compound.

25. The method according to claim 24, further comprising the steps of: iii. Scanning the patient to detect the location of the radioactivity.

26. The method according to 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 tumor, and non-small cell lung cancer.

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

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

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

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

31. The method according to claims 24 to 30, further comprising a second and optionally one or more subsequent administrations of a radionuclide that binds to a chelator.

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

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

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

35. A method of producing the compound of claims 1 to 21, comprising the steps of: i. Providing a host cell of claim 34, ii. Culturing the host cell under conditions that cause expression of the nucleic acid encoding the compound, and iii. Recovering the compound from the culture medium.

36. The method according to claim 35, wherein the compound is a compound according to claim 2a), the method comprising the steps of: 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 cell under conditions that cause expression of the two nucleic acids, and iii. Recovering the compound from the culture medium.

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

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