Tumor binding molecules and the use thereof for the diagnosis and treatment of cancer

Abstract

Disclosed are new bispecific antibodies comprising a VHH fragment and a Fab. The bispecific antibodies are particularly well suited for PRIT, where they have a rapid plasma clearance, a high tumor binding and a favorable tumor to kidney ratio.

Description

[0001] 10335 / PC

[0002] 26 November 2025

[0003] TUMOR BINDING MOLECULES AND THE USE THEREOF FOR THE DIAGNOSIS AND TREATMENT OF CANCER

[0004] The present specification comprises a sequence listing in computer readable format, submitted together with this application. The sequence listing forms part of the disclosure and is incorporated in the specification in its entirety.

[0005] The present invention relates to tumor binding antibodies and the use thereof for the diagnosis and / or treatment of cancer. In particular, the invention relates to pre-targeted Radioimmune methods for diagnosis and / or treatment of cancers that express the tumor antigen in high amounts.

[0006] Technical Background

[0007] Pretargeted Radioimmunotheray (PRIT) using a bispecific antibody comprising a binding site capable of binding a tumor antigen and a second binding site binding DOTA chelating a radionuclide has in recent years been exploited using different designs of bispecific antibodies.

[0008] An important part of such bispecific antibody is the tumor antigen binding sites.

[0009] B7H3 (CD276) is a member of the B7 ligand family and represents an attractive target for antibody-based immunotherapy. B7H3 has limited expression at low level in normal tissues, with a predominantly inhibitory role in adaptive immunity, suppressing T-cell activation and proliferation.

[0010] B7H3 is overexpressed on differentiated malignant cells and cancer initiating cells, with limited heterogeneity, and high frequency in many different cancer types. It inhibits tumor antigen-specific immune responses leading to a pro-tumorigenic effect, and also has non- immunological pro-tumorigenic functions, such as promoting migration and invasion, angiogenesis, chemoresistance and endothelial-to-mesenchymal transition as well as affecting tumor cell metabolism. 10335 / PC

[0011] 26 November 2025

[0012] Many antibody-based strategies utilizing distinct effector mechanisms to target B7-H3- expressing cancer cells have been developed. These strategies have demonstrated potent anti-tumor activity and acceptable safety profiles in preclinical models.

[0013] B7H3 exists in two isoforms, 2lgB7H3 and 4lgB7H3, determined by its extracellular domain that comprises one or two pairs of immunoglobulin variable (IgV)-like and immunoglobulin constant (IgC)-like domains respectively.

[0014] 4lg-B7H3 (Uniprot Q5ZPR3) is expressed at high to moderate levels on many solid tumors (e.g., colon, renal, lung, cervical, prostate, glioma), and importantly, expressed at very low to undetectable levels in normal tissues, making this antigen ideal for a targeted radio- theranostic.

[0015] Summary of the invention

[0016] According to an aspect the invention relates to a bispecific antibody comprising a first polypeptide comprising a first VHH capable of binding B7H3 and a light chain of a DOTA binding Fab, and a second polypeptide comprising a second VHH capable of binding B7H3 and a heavy chain of the DOTA binding Fab.

[0017] The bispecific antibodies of the invention have been shown to have a fast clearance from plasma after administration. This has the effect of minimizing exposure of healthy tissue to radiation during diagnosing / treatment, and also allows shorter interval between dosing of the bispecific antibody and the radioactivity, leading to improved patient compliance and shorter treatment periods.

[0018] Further, because of the fast plasma clearance of the bispecific antibody of the invention it may be possible to use same interval between dosing of bispecific antibody and dosing of chelator binding radionuclide, as typically used for bispecific antibodies of the prior art, such as the bispecific antibodies disclosed in WO 2018 / 204873, and thereby obtain an even higher clearing resulting in even less unintended exposure of healthy tissues such as the kidneys.

[0019] The bispecific antibodies provide a very high tumor uptake and retention resulting in a patient being administered the bispecific antibody will have a higher fraction of antibodies 10335 / PC

[0020] 26 November 2025 bound to the tumor tissue compared to prior art antibodies used in PRIT. This means that the tumor tissue will be exposed to an even higher radiation level compared with prior art antibodies used in PRIT, which will improve the efficacy in eradicating tumor cells.

[0021] The bispecific antibodies of the invention have the advantage of a high tumor to kidney ratio, thus providing a higher exposure to the tumor compared to the exposure of the kidney. This means that the risk of kidney damage is smaller compared to prior art PRIT methods.

[0022] According to another aspect the invention relates to new VHHs capable of binding B7H3.

[0023] According to another aspect, the invention relates to a nucleic acid encoding the bispecific antibody, an expression vector or a host cell comprising said nucleic acid, and a method for producing said antibody using said host cell.

[0024] According to another aspect, the invention relates to a composition comprising the bispecific antibody the invention.

[0025] The bispecific antibody of the invention or the composition of the invention for use in a method of treatment or diagnosis of cancer.

[0026] According to another aspect, the invention relates to a method for diagnosis or treatment of cancer comprising the steps of i. Administering the bispecific antibody comprising a first VHH capable of binding a tumor antigen and a light chain of a DOTA binding Fab, and a second polypeptide comprising a second VHH capable of binding a tumor antigen and a heavy chain of the DOTA binding Fab. ii. After an interval administering a chelator comprising a DOTA structure binding a radionuclide

[0027] If used for diagnosis preferably the method may further comprise a step of detecting the radioactivity.

[0028] Detailed Disclosure

[0029] Molecules 10335 / PC

[0030] 26 November 2025

[0031] The invention relates to a bispecific antibody comprising: a. a first polypeptide comprising a first VHH capable of binding a tumor antigen and a light chain of a DOTA binding Fab, and b. a second polypeptide comprising a second VHH capable of binding the same tumor antigen and a heavy chain of the DOTA binding Fab.

[0032] The first VHH may be identical to the second VHH or it may be different from the second VHH.

[0033] The tumor antigen may be selected among all tumor antigens, i.e. antigens that are highly expressed on tumor cells but not or only slightly expressed on the surface of other tissues. Examples of tumor antigens that may be selected according to the invention includes: B7H3, CD38, HER2, PSMA, GD2, GD3 ect.

[0034] A preferred tumor antigen is the B7H3 antigen.

[0035] Preferably, the invention relates to a bispecific antibody comprising: a. a first polypeptide comprising a first VHH capable of binding B7H3 and a light chain of a DOTA binding Fab, and b. a second polypeptide comprising a second VHH capable of binding B7H3 and a heavy chain of the DOTA binding Fab.

[0036] The term VHH is intended to mean a variable domain of a heavy chain antibody. VHH may also be known as "nanobodies". Heavy chain antibodies are a class of antibodies characterized in that they consist of only a heavy chain. Heavy chain antibodies are found in members of the camelid family and further in some cartilaginous fish, in particular in some sharks.

[0037] The VHH for use according to the invention may be any VHH having a high affinity to B7H3. The affinity is preferably in the nM or sub-nM range or higher, measured using SPR technology.

[0038] The VHH can be found by screening of a VHH library e.g. derived from a camelid organism. 10335 / PC

[0039] 26 November 2025

[0040] Preferred examples of VHHs for use according to the invention include Seq. ID. No. 1-8; and sequences with an identity of at least 90 %, 95 %, 96%, 97%, 98% or 99% to one of said sequences, preferably with same CDR sequences as identified for one of SEQ ID NO: 1-8 as shown in figure 1.

[0041] The term Fab is used to describe a fragment of an antibody comprising a variable heavy sequence (VH) and a variable light sequence (VL), said sequences are connected by a disulfide bond. The term Fab is well known in the art, and the skilled person will therefore fully understand the meaning of this term.

[0042] In a preferred embodiment the Fab binding a chelator is a Fab binding DOTA, more preferably such as a Fab derived from 2D12.5 antibody disclosed in Corneillie et al, J. Am. Chem. Soc. 125:15039-15048, 2003), optionally comprising one or more substitutions in the CDR sequences as disclosed in WQ2010 / 099536.

[0043] The expression a Fab derived from 2D12.5 should in this specification be understood as a synthetic Fab that has been designed based on the sequences of the 2D12.5 antibody by a number of amino acid substitutions.

[0044] Examples of Fabs derived from 2D12.5 includes humanized versions of the 2D12.5 Fab, generated by replacing some or all of the murine sequences of the 2D12.5 Fab with corresponding sequences derived from human antibodies. Humanization of antibodies is well known in the art and is typically made by aligning the sequences of the light and the heavy chain of the murine antibody being the starting point; with human light or heavy chain sequences, respectively, identifying the human sequences with the highest identity to the murine sequences and replacing the murine sequences in the constant / framework regions of the antibody with the corresponding sequences of the identified closest human sequences taking due case to maintain the CDR sequences of the murine antibody intact. Additionally amino acids substitutions may be made in order to achieve a certain effect of the new construct, such as substitutions increasing the binding affinity, removing glycosylation sites etc. All this is known in the art.

[0045] Preferred examples of a Fab derived from 2D12.5 include a Fab consisting of a light chain, comprising the sequence of amino acids no 146-360 of SEQ ID NO: 9; and a heavy chain comprising the sequence of amino acid no 146 to 368 of SEQ ID NO: 10; and a Fab consisting 10335 / PC

[0046] 26 November 2025 of a light chain, comprising the sequence of amino acids no 146-360 of SEQ ID NO:25; and a heavy chain comprising the sequence of amino acid no 146 to 368 of SEQ ID NO: 26.

[0047] The compound of the invention may further comprise one or more linkers separating the different part of the compound, e.g., separating the VHH from the Fab. The purpose of the linker is to separate the different domains allowing them to fold and function without steric hindrance from other domains of the compound. The linker is preferably composed of hydrophilic residues that do not generate strong secondary structures, and is typically rich in residues such as glycine, serine and / or threonine. A preferred linker is a linker composed of G and S residues.

[0048] The invention is not limited to any specific order of the component. The VHH fragment can be located in the C-terminal or N-terminal end of the VH or VL of the Fab respectively.

[0049] The bispecific antibody of the invention is monovalent with respect to the chelator binding Fab, and monovalent, bivalent, trivalent or tetravalent with respect to the B7H3 binding VHH.

[0050] Preferably the molecule is bivalent for the VHH and monovalent for the Fab.

[0051] In one embodiment the first VHH is identical to the second VHH.

[0052] Preferred bispecific antibodies of the invention consist of a first polypeptide containing a B7H3 binding VHH fragment, a G4S linker and the light chain of a DOTA binding Fab fragment derived from the 2D12.5 antibody (Corneille et al. J. Am. ChemSoc. 125:15039-15048), comprising the CDR substitutions of the C825 scFv disclosed in WO 2010 / 099536; and a second polypeptide containing a B7H3 binding VHH fragment, a G4S linker and the heavy chain of a DOTA binding Fab fragment derived from the 2D12.5 antibody (Corneille et al. J. Am. ChemSoc. 125:15039-15048), comprising the CDR substitutions of the C825 scFv disclosed in WO 2010 / 099536.

[0053] In one preferred embodiment, the bispecific antibody of the invention is a humanized bispecific antibody.

[0054] Technologies for humanization of the VHHs are known in the art, and the invention is not limited to a specific method for humanization of VHHs. In a preferred embodiment the VHH is compared to human VH3 in order to identify CDR sequences by comparison and identify 10335 / PC

[0055] 26 November 2025 framework regions. Based on the alignment, sequences of the isolated VHHs can be substituted to the corresponding amino acid of the human sequence.

[0056] Technologies for humanizing Fab fragments are also known in the art, and the invention is not limited to any specific method for humanizing the Fab fragment. Briefly, the amino acid sequences of the original VL and VH chains of the Fab fragment, e.g. a Fab derived from mouse, is aligned with corresponding human sequences for VL and VH sequences. Human sequences with the highest identity to the original sequences are identified, preferably sequences with the highest identity to the original framework sequences; and substitutions of the original sequences outside the CDR sequences, that increases the identity to the human sequence, are made, thereby generating a new sequence that has higher identity to the human sequence.

[0057] Examples of bispecific antibodies of the invention include: a bispecific antibody comprising a first polypeptide having a sequence of SEQ. ID: No. 9 and a second polypeptide comprising the sequence of SEQ. ID: NO. 10 a bispecific antibody comprising a first polypeptide having a sequence of SEQ. ID: No. 11 and a second polypeptide comprising the sequence of SEQ. ID: NO. 12 a bispecific antibody comprising a first polypeptide having a sequence of SEQ. ID: No. 13 and a second polypeptide comprising the sequence of SEQ. ID: NO. 14 a bispecific antibody comprising a first polypeptide having a sequence of SEQ. ID: No. 15 and a second polypeptide comprising the sequence of SEQ. ID: NO. 16 a bispecific antibody comprising a first polypeptide having a sequence of SEQ. ID: No. 17 and a second polypeptide comprising the sequence of SEQ. ID: NO. 18 a bispecific antibody comprising a first polypeptide having a sequence of SEQ. ID: No. 19 and a second polypeptide comprising the sequence of SEQ. ID: NO. 20 a bispecific antibody comprising a first polypeptide having a sequence of SEQ. ID: No. 21 and a second polypeptide comprising the sequence of SEQ. ID: NO. 22 10335 / PC

[0058] 26 November 2025 a bispecific antibody comprising a first polypeptide having a sequence of SEQ. ID: No. 23 and a second polypeptide comprising the sequence of SEQ. ID: NO. 24

[0059] Examples of humanized bispecific antibodies of the invention include: a bispecific antibody comprising a first polypeptide having a sequence of SEQ. ID: No. 25 and a second polypeptide comprising the sequence of SEQ. ID: NO. 26 a bispecific antibody comprising a first polypeptide having a sequence of SEQ. ID: No. 27 and a second polypeptide comprising the sequence of SEQ. ID: NO. 28 a bispecific antibody comprising a first polypeptide having a sequence of SEQ. ID: No. 29 and a second polypeptide comprising the sequence of SEQ. ID: NO. 30 a bispecific antibody comprising a first polypeptide having a sequence of SEQ. ID: No. 31 and a second polypeptide comprising the sequence of SEQ. ID: NO. 32 a bispecific antibody comprising a first polypeptide having a sequence of SEQ. ID: No. 33 and a second polypeptide comprising the sequence of SEQ. ID: NO. 34 a bispecific antibody comprising a first polypeptide having a sequence of SEQ. ID: No. 35 and a second polypeptide comprising the sequence of SEQ. ID: NO. 36 a bispecific antibody comprising a first polypeptide having a sequence of SEQ. ID: No. 37 and a second polypeptide comprising the sequence of SEQ. ID: NO. 38 a bispecific antibody comprising a first polypeptide having a sequence of SEQ. ID: No. 39 and a second polypeptide comprising the sequence of SEQ. ID: NO. 40.

[0060] Nucleic acid sequences etc.

[0061] The invention also relates to nucleic acid sequences encoding the compound of the invention. The nucleic acids may be provided by methods known in the art, e.g., starting from nucleic acid sequences encoding the separate elements of the compound, assembling and modifying the sequences using methods known in the art.

[0062] Alternatively, the nucleic acids sequences may be obtained by DNA synthesis, for example, by designing the amino acid sequences for the intended compound, deriving a suitable nucleic acid sequence encoding the intended amino acid sequence and synthesizing the 10335 / PC

[0063] 26 November 2025 sequence using methods known in the state of the art. This method has the benefit that it is easy to adapt the codon usage to the intended host cell and also to provide the nucleic acid with suitable sequences required for expression in the intended host cell, such as promoters, RBS, Kozak sequence, terminator, polyadenylation site etc. This is all within the skills of the average practitioner to design a suitable nucleic acid sequence encoding the intended amino acid sequence once the intended amino acid sequence has been designed.

[0064] Production of molecules

[0065] The nucleic acid sequences encoding the compound of the invention may be inserted into an expression construct, such as an expression vector, transformed into a selected host cell and expressed, leading to formation of the compound.

[0066] The skilled person will appreciate that two expression constructs are necessary, one for each strand of the compound. The two expression constructs may be inserted and expressed in the same cell. The two chains can be expressed in one cell either by transfection with two vectors or by one vector having bicistronic expression sites.

[0067] A suitable host cell for use according to the invention may in principle be any host cell capable of expressing the polypeptides of the compound. Such host cells and expression systems suitable for particular polypeptides are known in the art and selecting a suitable expression system for a particular compound, including expression vectors and host cells, are within the skills of the average practitioner.

[0068] Examples of suitable host cells include bacterial cells, such as E. coli, Bacillus sp., such as B licheniformis and B. subtilis; fungal cells such as Saccharomyces cerevisiae, Pichia pastoris, Aspergillus niger, A. oryzae, Trichoderma reesei, Pencillium chrysogenum; insect cells, Mammalian cells such as HeLa cells, CHO cells, HEK cells.

[0069] 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 the two polypeptide chains, but they can also combine the two chains correctly and produce the complete molecule of the invention.

[0070] In order to produce compounds of the invention, it is advantageous to produce the compound using basically same technology as known in the art for producing recombinant 10335 / PC

[0071] 26 November 2025 antibodies. Using this approach, the two nucleic acids encoding the two strands of the compounds are provided with suitable expression signals and transformed into 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 consists of and are expressed as two separate polypeptide strands. It has turned out that this method is highly effective for producing compounds of the invention in high purity and yields.

[0072] After production the compound of the invention is recovered from the cell culture supernatant using methods known in the art, such as precipitation, affinity purification and other chromatographic methods. Since compounds of the invention comprises a Fab fragment, same recovery methods as used for complete antibodies may conveniently be used, such as anti-CHl, Protein A or Protein L affinity purification.

[0073] Compositions

[0074] The invention also relates to compositions comprising one or more compounds of the invention.

[0075] The compositions comprise in addition to the compound of the invention, one or more of diluents, salts, pH regulating agents, stabilizers, antioxidants, tonicity regulating agents etc.

[0076] In a preferred embodiment, the composition is a pharmaceutical composition comprising only pharmaceutically acceptable ingredients, such as ingredients disclosed in well recognized Pharmacopoeias, e.g., as described in European Pharmacopoeia 10thEdition; using methods and technologies known in the pharmaceutical or apothecary area.

[0077] Use of the compound

[0078] The bispecific antibodies of the invention are useful for immunotherapy, in particular for pretargeted radio immunotherapy (PRIT).

[0079] PRIT is used for treating cancers in methods where a bispecific antibody, comprising a first binding site capable of binding a tumor antigen and a second binding site capable of binding a radionuclide, a chelator binding a radionuclide or a molecule linked to a chelator, e.g. a peptide bound to a chelator group, where the first binding site is capable of binding the 10335 / PC

[0080] 26 November 2025 peptide part; is administered to a patient in need of treatment. After allowing the antibody to bind to the tumor and letting unbound antibody be cleared from the plasma, a radionuclide or a chelator binding a radionuclide, which radionuclide or chelator binding a radionuclide is recognized by the bispecific antibody, is administered to the patient and will be bound by the bispecific antibody localized at the tumor. Unbound radionuclide or chelator binding radionuclide will rapidly be cleared from the plasma via renal clearance.

[0081] It is preferred that the bispecific antibody is cleared from the plasma before the radionuclide is administered, 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 a radionuclide in order to improve clearance.

[0082] The bispecific antibodies of the invention comprise two identical tumor binding sites. Thus, the compounds will have a higher avidity to the tumor antigen due to the coordination between the two tumor antigen binding sites, compared with a similar compound containing the same binding sites, but only having one tumor binding site.

[0083] Thus, in one preferred aspect the invention relates to a method of treating or diagnosing cancer in a patient, comprising the steps of i. Administering a bispecific antibody according to the invention, which bispecific antibody is capable of binding a tumor antigen and further capable of binding a chelator with a bound radionuclide, to a subject in need of such treatment or diagnosis; wherein the bispecific antibody comprises: a. a first polypeptide comprising a first VHH capable of binding a tumor antigen and a light chain (VL) of a DOTA binding Fab; and b. a second polypeptide comprising a second VHH capable of binding the tumor antigen, and a heavy chain (VH) of the DOTA binding Fab. ii. After a holding period (interval), administering the chelator binding a radionuclide to the subject. 10335 / PC

[0084] 26 November 2025

[0085] The tumor antigen may be any such antigen associated with tumors. Examples of tumor antigens include: HER2, B7-H3, CA6, CD138, CD20, CD19, CD22, CD27L, CD30, CD33, CD37, CD38, CD47, CD56, CD66e, CD70, CD74, CD79b, EGFR, CEA, EGFRvlll, FRa, GCC, GPNMB, Mesothelin, MUC16, NaPi2b, Nectin 4, PSMA, STEAP1, Trop-2, 5T4, AGS-16, alpha v beta6, 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-l, Mucin 1, p-cadherin, peritosin, Fyn, SLTRK6, Tenascin c, VEGFR2, and PRLR.

[0086] Preferred tumor antigens are B7H3, CD38, HER2, and GD2.

[0087] The holding period may be selected in the range of 12 h to 7 days, e.g., 12 h, 18 h, 24 h, 36 h, 2 days, 3 days, 4, days, 5 days, 6 days, or 7 days.

[0088] One issue that always is necessary to consider when performing PRIT treatment is the unintended damage that occurs to healthy tissues that inevitably also are exposed to radioactivity when a chelator with a bound radionuclide is injected in the patient. Kidneys appear to be particularly exposed because non-bound radionuclides are mainly excreted from the body by renal clearance.

[0089] The inventors have realized that the bispecific antibodies of the invention have a very high tumor binding, in a mouse model, in comparison to prior art molecules used for PRIT, such as SADA-conjugates.

[0090] Further, the inventors have realized that the bispecific antibodies of the invention have a stable binding to tumor tissue.

[0091] This has led to the realization that if the holding period between the administration of the antibody and the administration of the chelator binding a radionuclide is selected to be more than 24 hours the binding of bispecific antibody to the tumor tissue represented by the amount of radioactivity located to the tumor tissue remains high, whereas the radioactivity located to other tissues declines fast when the holding period is above 24 hours

[0092] Thus, the inventors have realized that the bispecific antibodies of the invention provide for very high tumor to kidney ratios when the holding period is selected above 24 hours, in particular higher than the tumor to kidney ratios that can be achieved with bispecific antibodies that have been used for PRIT in the prior art, such as SADA-conjugates. 10335 / PC

[0093] 26 November 2025

[0094] Thus, in a particular preferred embodiment, the invention relates to a method of treating cancer in a patient, comprising the steps of i. Administering a bispecific antibody according to the invention, which bispecific antibody is capable of binding a tumor antigen and further capable of binding a chelator with a bound radionuclide, to a subject in need of such treatment or diagnosis; ii. After a holding period of at least 12 hours, administering the chelator binding a radionuclide to the subject.

[0095] In this embodiment, the holding period is preferably selected as at least 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72, hours, 96 hours or 120 hours.

[0096] Alternatively, due to the rapid clearing it will also be possible to administer the DOTA chelator binding a radionuclide after a short holding period and still not expose healthy tissues to undesirable levels of radiation.

[0097] Even though a satisfactory efficient clearing may be obtained using the bispecific antibody of the invention, it is possible to include a step of administering a clearing agent in order to improve clearance. However, in most situations it is superfluous and desirable to omit, in order to avoid a further administration step and the inconvenience for the patient connected to such an additional step.

[0098] Similar methods of treatment using bispecific antibodies have previously been disclosed in e.g., WO 2018 / 204873 with the significant difference that the methods of this prior art document use bispecific antibody constructs based on antigen binding sites in the scFv format and further comprising a SADA (Self Assembly DisAssembly) domain.

[0099] In one embodiment the cancer is selected among osteosarcoma, neuroblastoma, liposarcoma, fibrosarcoma, carcinoma, malignant fibrous histiocytoma, leiomyosarcoma, spindle cell sarcoma, brain tumor, small cell lung cancer, retinoblastoma, HTLV-l infected T cell leukemia, breast cancer, colon cancer, prostate cancer, T-cell and B-cell lymphomas, 10335 / PC

[0100] 26 November 2025 glioblastoma multiforme, malignant glioma, Head and Neck cancer, solid tumors and non- small-cell lung cancer.

[0101] The skilled person will appreciate that the chelator used in the method of treatment / diagnosis of the invention can be any chelator that is recognized and can be bound by the Fab fragment present in the bispecific antibody of the invention.

[0102] In one preferred embodiment, the Fab is capable of binding DOTA, or a derivative of DOTA. In this embodiment the chelator is DOTA or a derivative of DOTA such as the compounds comprising a DOTA ring system, to which the Fab can bind, e.g., compounds disclosed in WO 2010 / 099536, WO 2019 / 010299 and WO 2022 / 005998 (incorporated herein by reference).

[0103] The radionuclide may be any radionuclide that can be bound by a chelator, typically radionuclide cations. Examples of suitable radionuclides include:225Ac,227Ac,241Am,211At,215At,217At,218At,209Bi,211Bi,212Bi,213Bi,134Ce,249Cf,252Cf,244Cm,245Cm,248Cm,57Co,58Co,51Cr,61Cu,64Cu,67Cu,152Dy,165Dy,152Eu,59Fe,221Fr,67Ga,68Ga,66Ga,161Ho,110mln,mln,192lr,133La,177Lu,237Np,189mOs,231Pa,203Pb,212Pb,210Po,211Po,212Po,214Po,215Po,216Po,218Po,195mPt,238Pu,239Pu,240Pu,244Pu,223Ra,224Ra,226Ra,82Rb,186Re,188Re,103mRh,119Sb,75Se,89Sr,149Tb,151Tb,161Tb99mTc,94mTc,227Th,228Th,229Th,230Th,232Th,201TI,90Y,86Y,89Zr.

[0104] In one embodiment the method of the invention is a method for treating cancer. In this embodiment the skilled person will select a suitable radionuclide e.g., a radionuclide delivering a high energy, and preferably with a limited (low) penetration so only the targeted tissue is affected. The treating physician will be able to select a suitable radionuclide for treatment without exercising any inventive activity.

[0105] In another embodiment, the method of the invention is a method for diagnosing cancer. In this embodiment, the method typically comprises a subsequent step of detecting the radionuclides bound to the compound of the invention and localized at the surface of tumor cells using a suitable detection technology as known in the art.

[0106] In this embodiment, the skilled person may select a radionuclide having a long penetration range and which deposits a low energy in surrounding tissues. It is within the skills of the average practitioner to select a suitable radionuclide for the diagnosis. The detection may be performed using well known methods and equipment for detecting radionuclides, such as a PET or SPECT scanner. 10335 / PC

[0107] 26 November 2025

[0108] In some embodiments the method of the invention comprises a second and optional subsequent administration of chelator with bound radionuclide. Such a second administration of chelator with bound radionuclide is typically done 1-7 days after the first administration of chelator with bound radionuclide, and a subsequent administration of chelator with bound radionuclide is typically done 1-7 days after the previous administration of chelator with bound radionuclide. The radionuclide and / or chelator administered in the first, second and optional subsequent administration may be identical, or it may be a different radionuclide and / or chelator used in the second or subsequent administration of chelator binding a radionuclide. For example, an alpha-emitter may be administered in the first administration and a beta-emitter administered in the second and optional subsequent administration. Or in another example, a radionuclide suitable for PET or SPECT scanning is administered in the first administration and a scanning is performed in order to detect the tumor(s), and a radionuclide more suited to eradicate the tumor cells is administered in the second and optional subsequent administration.

[0109] In some embodiment the method of treating cancer according to the invention is performed as a multiple cycle treatment, where each cycle consist of administration of a bispecific antibody of the invention followed after the holding period by administration of the chelator binding a radionuclide. Such a multiple cycle treatment may consist of 2-5 cycles or more, according to the discretion of the doctor in charge of the treatment.

[0110] Benefits of the bispecific antibodies of the invention

[0111] The bispecific antibodies of the invention have been shown to have a fast clearance from plasma after administration. This has the effect of minimizing exposure of healthy tissue to radiation during diagnosing / treatment. Further the bispecific antibody can be used without need for clearing agent.

[0112] The fast clearing is further confirmed by comparison to SADA molecules, where the bispecific antibodies of the invention showed significantly faster clearance.

[0113] Fast clearance further allows shorter interval between dosing of the bispecific antibody and the radioactivity, leading to improved patient compliance and shorter treatment periods. 10335 / PC

[0114] 26 November 2025

[0115] The bispecific antibodies provide a very high tumor uptake and retention leading to that a patient having had administered the bispecific antibody will have a higher fraction of antibodies bound to the tumor tissue compared to prior art antibodies used in PRIT. This means that the tumor tissue will be exposed to an even higher radiation level compared with prior art antibodies used in PRIT, which will improve the efficacy in eradicating tumor cells. In one mouse experiment the antibodies of the invention showed a tumor binding of approximately 3-5 fold of the tumor binding of a corresponding bispecific antibody in the format of a SADA complex (WO 2018 / 204873).

[0116] A well-known problem when using PRIT is exposure of other organs, particularly the kidneys, to radiation.

[0117] The bispecific antibodies of the invention have the advantage of a high tumor to kidney ratio, thus providing a higher exposure of the tumor compared to the exposure of the kidney. This means that the risk of kidney damage is smaller compared to prior art PRIT methods.

[0118] Further, because of the fast plasma clearance of the bispecific antibody of the invention it may be possible to use same interval between dosing of bispecific antibody and dosing of chelator binding radionuclide, as typically used for bispecific antibodies of the prior art, such as the bispecific antibodies disclosed in WO 2018 / 204873, and thereby obtain an even higher clearing resulting in even less unintended exposure of healthy tissues such as the kidneys.

[0119] Figures

[0120] Fig. 1 A-C shows the alignment of the amino acid sequences of the selected VHH fragments and selected amino acid substitutions making the sequence more similar to the human sequence.

[0121] Figure 2 shows the plasma clearance of the 8 tested bispecific antibodies of the invention in comparison with the reference B7-H3-SADA molecule, 3BH-9. For more details see example 7.

[0122] Figure 3 A-C shows the Biodistribution of for the selected bispecific antibodies of the invention in comparison with the reference B7-H3-SADA molecule, 3BH-9. Figure 3A shows 10335 / PC

[0123] 26 November 2025 the tumor uptake after 24h, Figure 3B shows the kidney uptake after 24 h and figure 3C shows the calculated tumor to kidney ratios. For more details see example 8.

[0124] Short description of the sequences

[0125] SEQ ID NO: 1: the amino acid sequence of VHH fragment 22E01;

[0126] SEQ ID NO: 2: the amino acid sequence of VHH fragment 22D02;

[0127] SEQ ID NO: 3: the amino acid sequence of VHH fragment 22B03;

[0128] SEQ ID NO: 4: the amino acid sequence of VHH fragment 22C09;

[0129] SEQ ID NO: 5: the amino acid sequence of VHH fragment 23A03;

[0130] SEQ ID NO: 6: the amino acid sequence of VHH fragment 23B04;

[0131] SEQ ID NO: 7: the amino acid sequence of VHH fragment 23E04;

[0132] SEQ ID NO: 8: the amino acid sequence of VHH fragment 23A11;

[0133] SEQ ID NO: 9: The amino acid sequence of the first polypeptide of the bispecific antibody YM00155;

[0134] SEQ ID NO: 10: The amino acid sequence of the second polypeptide of the bispecific antibody YM00155;

[0135] SEQ ID NO: 11: The amino acid sequence of the first polypeptide of the bispecific antibody YM00156;

[0136] SEQ ID NO: 12: The amino acid sequence of the second polypeptide of the bispecific antibody YM00156;

[0137] SEQ ID NO: 13: The amino acid sequence of the first polypeptide of the bispecific antibody YM00157;

[0138] SEQ ID NO: 14: The amino acid sequence of the second polypeptide of the bispecific antibody YM00157;

[0139] SEQ ID NO: 15: The amino acid sequence of the first polypeptide of the bispecific antibody YM00160; 10335 / PC

[0140] 26 November 2025

[0141] SEQ ID NO: 16: The amino acid sequence of the second polypeptide of the bispecific antibody YM00160;

[0142] SEQ ID NO: 17: The amino acid sequence of the first polypeptide of the bispecific antibody YM00162;

[0143] SEQ ID NO: 18: The amino acid sequence of the second polypeptide of the bispecific antibody YM00162;

[0144] SEQ ID NO: 19: The amino acid sequence of the first polypeptide of the bispecific antibody YM00163;

[0145] SEQ ID NO: 20: The amino acid sequence of the second polypeptide of the bispecific antibody YM00163;

[0146] SEQ ID NO: 21: The amino acid sequence of the first polypeptide of the bispecific antibody YM00164;

[0147] SEQ ID NO: 22: The amino acid sequence of the second polypeptide of the bispecific antibody YM00164;

[0148] SEQ ID NO: 23: The amino acid sequence of the first polypeptide of the bispecific antibody YM00165;

[0149] SEQ ID NO: 24: The amino acid sequence of the second polypeptide of the bispecific antibody YM00165;

[0150] SEQ ID NO: 25: The amino acid sequence of the first polypeptide of the humanized bispecific antibody YM00172;

[0151] SEQ ID NO: 26: The amino acid sequence of the second polypeptide of the humanized bispecific antibody YM00172;

[0152] SEQ ID NO: 27: The amino acid sequence of the first polypeptide of the humanized bispecific antibody YM00177;

[0153] SEQ ID NO: 28: The amino acid sequence of the second polypeptide of the humanized bispecific antibody YM00177;

[0154] SEQ ID NO: 29: The amino acid sequence of the first polypeptide of the humanized bispecific antibody YM00189; 10335 / PC

[0155] 26 November 2025

[0156] SEQ ID NO: 30: The amino acid sequence of the second polypeptide of the humanized bispecific antibody YM00189;

[0157] SEQ ID NO: 31: The amino acid sequence of the first polypeptide of the humanized bispecific antibody YM00196;

[0158] SEQ ID NO: 32: The amino acid sequence of the second polypeptide of the humanized bispecific antibody YM00196;

[0159] SEQ ID NO: 33: The amino acid sequence of the first polypeptide of the humanized bispecific antibody YM00228;

[0160] SEQ ID NO: 34: The amino acid sequence of the second polypeptide of the humanized bispecific antibody YM00228;

[0161] SEQ ID NO: 35: The amino acid sequence of the first polypeptide of the humanized bispecific antibody YM00221;

[0162] SEQ ID NO: 36: The amino acid sequence of the second polypeptide of the humanized bispecific antibody YM00221;

[0163] SEQ ID NO: 37: The amino acid sequence of the first polypeptide of the humanized bispecific antibody YM00211;

[0164] SEQ ID NO: 38: The amino acid sequence of the second polypeptide of the humanized bispecific antibody YM00211;

[0165] SEQ ID NO: 39: The amino acid sequence of the first polypeptide of the humanized bispecific antibody YM00203;

[0166] SEQ ID NO: 40: The amino acid sequence of the second polypeptide of the humanized bispecific antibody YM00203;

[0167] SEQ ID NO: 41: the amino acid sequence of the first polypeptide of the CD38 binding bispecific antibody YM0005; and

[0168] SEQ ID NO: 42: The amino acid sequence of the second polypeptide of the CD38 binding bispecific antibody YM0005.

[0169] All cited references are incorporated by reference. 10335 / PC

[0170] 26 November 2025

[0171] The accompanying Figures and Examples are provided to explain rather than limit the present invention. It will be clear to the person skilled in the art that aspects, embodiments, claims and any items of the present invention may be combined.

[0172] Unless otherwise mentioned, all percentages are in weight / weight. Unless otherwise mentioned, all measurements are conducted under standard conditions (ambient temperature and pressure). Unless otherwise mentioned, test conditions are according to European Pharmacopoeia 10.0.

[0173] Examples

[0174] Materials and methods: Used B7H3 antigens: 10335 / PC

[0175] 26 November 2025

[0176] Cells:

[0177] FACS analysis: for EC50 determinations were performed using following parameters:

[0178] • Cells: HEK293T or CHO-K1 cell lines suspended in FACS buffer: • Cell preparation: cells were recovered and resuspended to a final concentration of lxlO6cells / ml in FACS buffer and aliquoted in a V-bottom 96 well plate (150pl / well corresponding to 1.5X105cells / well).

[0179] • Sample molecule: twelve steps 3-fold serial dilutions (300 nM to 0.002 nM) of the molecules were prepared in FACS buffer and added to the cells • Detection: Mouse anti-human IgG (CHI-specific) antibody (BD Pharmigen, cat. Nr.

[0180] 555784) diluted 1:1000 in FACS buffer followed by goat anti mouse IgG-APC conjugates (Invitrogen cat. Nr- A-865) diluted 1:500 in FACS buffer

[0181] • Controls: full detection control, secondary antibody control and unstained cells;

[0182] • Analysis was performed in iQUE3 screener (SARTURIUS, AnnArbor Ml, USA), bispecific antibody binding was detected on RL-1 fluorescence detector. 10335 / PC

[0183] 26 November 2025

[0184] • FACS buffer: 0.5% FBS / 0.5 mM EDTA / lxPBS.

[0185] SPR analysis using Biacore 8K+ (Cytiva Denmark) to determine the affinity of bispecific antibodies to huB7H3(4lg)-His was done using following parameters:

[0186] • Immobilization CFJB1301 sensor chip (Cytiva, cat. No 2914603) immobilized with anti-human CHI specific molecule by amine coupling at 3000 RU

[0187] • Antibody samples: Bispecific antibodies captured at 20 nM injected in flow cell 2 for 1 min at 30 pl / min for 1 min

[0188] • Positive control: A B7H3 binding antibody comprising the VH and VL domains of Omburtamab and a human Fc domain was used as control. The control antibody was captured at 10 nM injected in flow cell 2 for 1 min at 30 pl / min for 1 min

[0189] • Negative control: Human IgG isotype antibody (Sigma, cat. No. 14506-50) captured at 5 nM injected in flow cell 2 for 1 min at 30 pl / min for 1 min

[0190] • Antigen: 2-fold dilution of hyB7-H3(4lg)-His tag protein starting at 40 nM down to 2.5 nM, 500 nM down to 31.25 nM or 1000 nM down to 62.5 nM injected on both flow cells of CFJB1301 for 1 min at 30 pl / min

[0191] • Off-rate measurement: 300 seconds

[0192] • Regeneration: Baseline levels were restored to pre-antibody capture levels using two consecutive injections of 10 pl of regeneration solution

[0193] • SPR running buffer: lxHBS-EP+ pH 7.4 (Cytiva, cat no. BR100669)

[0194] • Analysis: Data was analysed using the single kinetics predefined evaluation method of Biacore Insight Evaluation. Kinetic parameters were calculated using the 1:1 binding or the steady state models.

[0195] Example 1. Screening for B7H3 binding VHH fragment

[0196] A commercial library of Llama VHH fragments was used for this example (FairJourney Biologies (FJB), Porto, Portugal; llama VHH naive library FL1951, FJB name L0020- L0029VHH_Naive). Further the B7H3 antigens bio-huB7H3 (4lg)-h is, bio-moB7H3-His and 10335 / PC

[0197] 26 November 2025 bio-cyB7H3 were purchased from Aero Biosystems (Newark, DE, USA) and used in this example.

[0198] The library was screened for VHH fragments binding to the B7H3 antigen (using phage display technology, master plates generation, primary screening by cells expression QC and by P.E. binding FACS on CHO-K1 WT and CHO-K1 cells transiently transfected with huB7H3, moB7H3 and cyB7H3) according to the manufacturer's instructions.

[0199] A total of 276 clones were selected in the initial screening. 186 clones showing binding to at least one B7H3 expressing cell line and no binding to CHO-K1 WT cells were sequenced in addition to 20 clones classified as possibly B7H3 specific (potential binding to at least one B7H3 expressing cell line when comparing to CHO-K1 WT). A total of 84 unique VHH sequences combined in 51 unique CDR combinations were identified.

[0200] After sequence analysis a secondary screening (P.E. off-rate determination by SPR on huB7H3 (41g and 21g), moB7H3 and cyB7H3, and P.E. binding FACS CHO-K1 cells and CHO-K1 cells transiently transfected with huB7H3, moB7H3 and cyB7H3) resulted in a lead panel of 14 VHH clones for production.

[0201] The 14 VHH antibodies were characterized by: binding and affinity determination by SPR on bio-huB7H3(4lg)-Fc, bio-moB7H3-His and bio-cyB7H3-His and EC50 determination via FACS on CHO-K1 WT and transiently transfected cells with hu-, mo- or cyB7H3 cells. 8 clones were binders to at least one of the proteins huB7-H3(4lgand2lg), moB7-H3 or cynoB7-H3.

[0202] The 8 selected VHHs are shown in table 1 below.

[0203] Table 1: The selected B7H3 VHH's 10335 / PC

[0204] 26 November 2025

[0205] Example 2. FACS analysis of the selected VHH fragments

[0206] The 8 selected VHH fragments from Example 1 were analysed by FACS analysis using CHO-K1 cells expressing huB7-H3, using wildtype CHO-K1 cells as reference. The analysis showed the following binding efficiencies expressed as EC50 values:

[0207] Table 2: EC50 determinations by FACS

[0208] Example 3. Preparation of bispecific antibodies

[0209] Bispecific antibodies comprising the 8 VHH fragments isolated in Example 1 were prepared, each molecule consisting of: 10335 / PC

[0210] 26 November 2025 a first polypeptide containing the VHH fragment, a G4S linker and the light chain of the DOTA binding Fab fragment derived from the 2D12.5 antibody (Corneille et al. J. Am. ChemSoc. 125:15039-15048), comprising the CDR substitutions of the C825 scFv disclosed in WO 2010 / 099536, and a second polypeptide containing the VHH fragment, a G4S linker and the heavy chain of the DOTA binding Fab fragment derived from the 2D12.5 antibody (Corneille et al. J. Am. ChemSoc. 125:15039-15048), comprising the CDR substitutions of the C825 scFv disclosed in WO 2010 / 099536. Thus, the generated bispecific molecules are bivalent with respect to the B7H3 binding site and monovalent with respect to the DOTA binding site.

[0211] The complete sequences of the generated bispecific molecules are shown in Table 3.

[0212] Table 3: Bispecific molecules comprising the B7H3 binding VHH fragments

[0213] The molecules were prepared essentially as described in example 1 of WO 2024 / 099526 by transforming HEK suspension cells with an expression cassette encoding the first polypeptide 10335 / PC

[0214] 26 November 2025 and an expression cassette encoding the second polypeptide, growing the transformants in expression medium and recovering the bispecific antibodies from the culture media.

[0215] Example 4: Analysis of bispecific antibodies The bispecific antibodies prepared in Example 3 were analysed by FACS analysis using HEK293T cells which are known to express the B7H3 antigen (Shi et al. (2016), Molecular medicine reports, Volume 14(1), pages 943-948) and by SPR analysis.

[0216] The results are shown in Table 4. Table 4: Analysis of bispecific antibodies

[0217] As a control FACS analysis was also performed using Jurkat E6-1 cells that are known not to overexpress B7H3. No binding to Jurkat cells were detected. The results show that the bispecific antibodies have excellent binding properties to HEK293T cells and the SPR analysis also confirms a high affinity to the B7H3 antigen. 10335 / PC

[0218] 26 November 2025

[0219] Example 5: Humanization of the bivalent antibodies

[0220] The bivalent antibodies of example 3 were humanized by aligning the VHH sequences with the human heavy chain sequence. Based on the alignment the expected CDR sequences could be identified.

[0221] A workflow based on two different approaches for humanization was used to prevent the long timelines associated with the phage display approach. The first was based on the method described by T. Sulea (Methods Mol Biol. 2022:2446:299-312. doi: 10.1007 / 978-1- 0716-2075-5_14) and the second was variants suggested by the Discovery Studio Software (dessault systems) using a VHH model generated by NanobodyBuilder(OPIG). This model was also used to identify residues within 5 A from any CDR sequence used as part of the Sulea workflow. The number of variants was limited to around 8 with 5-6 from the Sulea workflow (increasing number of backmutations from full human framework), and 2-3 (Best single mutations and best Machine learning variant) from the Discovery Studio approach.

[0222] The selected and performed substitutions are shown in figure 1.

[0223] The C825 VH and VL sequences were lined to human CHI and CL (lambda) respectively.

[0224] The affinity of the generated variants were analyzed by SPR and the best variant for each parental bispecific molecule were identified, as shown in table 5.

[0225] Included were all YPRITS [YM00155-YM00165] showing EC50<10 nM in FACS (HEK293) and KD in SPR in nM range.

[0226] Table 5. Selected Humanized bispecific antibodies 10335 / PC

[0227] 26 November 2025

[0228] Example 6: Affinity determination of B7H3 humanized variants The binding properties of the selected humanized variants were determined by SPR analysis against B7H3 and FACS analysis using HEK293T cells.

[0229] The results are shown in table 6. Table 6. Kinetic parameters and EC50 values for humanized variants.

[0230] The results showed that the humanized variants had excellent binding properties to B7H3 in line with, or even better than their non-humanized parents.

[0231] Example 7. Pharmacokinetic profile of bispecific antibodies 10335 / PC

[0232] 26 November 2025

[0233] A standard PK study was performed testing the clearing of the 8 bispecific antibodies prepared in example 3. As a control the B7H3-SADA molecule 3BH-9 was included.

[0234] The study was done using Balb / c AnNCrl female mice age 12-14 weeks.

[0235] Groups of 6 mice received a single IV bolus injection with a dose of 10 mg / kg of each of the 8 bispecific antibodies.

[0236] Blood samples were collected at the following time points after injections: 0.5, 2, 8, 24 and 72 hours.

[0237] Subsequently the mice were terminated by cardiac puncture after CO2 euthanasia.

[0238] The plasma concentration of the bispecific antibodies was determined by B7H3-ELISA.

[0239] The results are shown in figure 2 and in table 7 below.

[0240] Table 7: Plasma concentrations after 24 and 72 hours

[0241] The results showed that the bispecific antibodies of the invention had a very fast clearance resulting in low plasma concentration already after 24 hours and almost nothing left after 72 hours. In particular, the clearance was significantly faster than the reference molecule B7H3- SADA.

[0242] Example 8. Biodistribution of the bispecific antibodies of the invention 10335 / PC

[0243] 26 November 2025

[0244] A biodistribution experiment was conducted using a mouse xenograft model.

[0245] Balb / c Nude AnN / Rj female mice age 7 weeks were given 5xl07cells of a human colon adenocarcinoma cell line SW480 (ATCC-CCL-228) in 100 pl PBS:Matrigel (1:1).

[0246] The tumors were allowed to grow until they reached a size of 150-250 mm3(between 11 and 13 days) when treatment started.

[0247] Groups of 3 mice were given 10 mg / kg, single IV, 5 ml / kg, retro-orbital sinus, of the reference molecule 3BH-9 or one of the bispecific antibodies. After an interval of 24 h or 48 h 10 MBq,177LuDOTA was given IV single dose, 100 pl, retro-orbital sinus, according to table

[0248] 8.

[0249] Table 8: study groups for biodistribution study 10335 / PC

[0250] 26 November 2025

[0251] The mice were terminated 24 h after177LuDOTA administration, tissues were collected and bound activity determined using gamma counting.

[0252] The 3BH-9 tissue distribution was similar to previous data obtained in the same model validating the model and data for this study.

[0253] The distribution data for tumor and kidney update is shown in figure 3 and in table 9.

[0254] Table 9. Distribution data - Tumors and Kidneys 10335 / PC

[0255] 26 November 2025

[0256] The results showed that all bispecific antibodies of the invention showed a higher tumor uptake compared to the 3BH-9 reference compound. They also showed a higher kidney uptake when the177LuDOTA was administered after 24 h.

[0257] However, for the 3 compounds administered both with a 24h and 48h dosing interval the longer dosing interval reduced the kidney uptake by up to 86% with a reduced tumor uptake by a maximum of 30%, leading to a tumor to kidney ratio >1 for the best molecules in this experiment.

[0258] This experiment suggests a better tumor retention for the bispecific antibodies of the invention compared to the SADA compounds and might indicate that the radioactivity can be dosed later to spare the healthy tissue without impacting the tumor uptake significantly.

[0259] Example 9. Biodistribution of humanized B7H3 - binding bispecific antibodies of the invention.

[0260] A biodistribution study was performed using 5 humanized B7H3- binding bispecific antibodies as prepared in Example 5: YM00172, YM00189, YM00221, YM00211 and YM00203. The biodistribution study was done essentially as described in example 8.

[0261] The study confirmed that all tested humanized B7H3- binding bispecific antibodies had a fast clearing rate, in fact an even faster clearing rate than the corresponding not humanized bispecific antibodies. Due to the fast clearing the177Lu-DOTA was administered 24 hours after the administration of the bispecific antibody.

[0262] The study also confirmed that the tested molecules had a relative higher tumor binding compared with other tissues of the mice.

[0263] Example 10. Biodistribution of a CD38 binding bispecific antibodies of the invention.

[0264] A CD38 binding bispecific antibody consisting of a first polypeptide a first polypeptide comprising a VHH capable of binding the CD38 tumor antigen and a light chain of the DOTA binding Fab, with the amino acid sequence disclosed in SEQ ID NO: 41 , and a second polypeptide comprising the VHH capable of binding the CD38 tumor antigen and a heavy 10335 / PC

[0265] 26 November 2025 chain of the DOTA binding Fab, with the amino acid sequence disclosed in SEQ ID NO: 42; we prepared essentially as described in Example 3. The antibody was called YM0005.

[0266] A biodistribution experiment was conducted using a mouse xenograft model expressing CD38.

[0267] Balb / c Nude R2G2 Daudi female mice aged 8 weeks were given 106cells of a Daudi cell line (Daudi, human Burkitt's lymphoma, ATCC (reference ATCC-CCL-213)(in 100 pl PBS-Daudi cells suspension).

[0268] The tumors were allowed to grow until they reached a size of 200-400 mm3(between 21 and 24 days) when treatment started.

[0269] 2 groups of 3 mice were given the 10 mg / kg, single IV, 5 ml / kg, retro-orbital sinus, of the bispecific antibody YM0005. Following an interval of 24 h, or 48 h for the two groups respectively 10 MBq,177LuDOTA was given as an IV single dose, 100 pl, retro-orbital sinus.

[0270] The mice were terminated 24 h after177LuDOTA administration, tissues were collected and bound activity determined using gamma counting.

[0271] The distribution data for tumor and kidney uptake is shown in table 10.

[0272] Table 10. Uptake at different dosing intervals:

[0273] The results showed that the CD38 bispecific antibodies showed a high tumor uptake. They also showed a higher kidney uptake when the177LuDOTA was administered after 24 h.

[0274] Using the longer dosing interval of 48h before administration of the177LuDOTA the kidney uptake was reduced by a factor 20, and a lower kidney uptake was provided. 10335 / PC

[0275] 26 November 2025

[0276] This experiment demonstrates a beneficial tumor retention for the bispecific antibodies and might indicate that the radioactivity can be dosed later to spare the healthy tissue without impacting the tumor uptake significantly.

Claims

10335 / PC26 November 2025Claims1. A bispecific antibody comprising: i. a first polypeptide comprising a first VHH capable of binding B7H3, and a light chain (VL) of a DOTA binding Fab; and ii. a second polypeptide comprising a second VHH capable of binding B7H3, and a heavy chain (VH) of the DOTA binding Fab.

2. The bispecific antibody according to claim 1, wherein said bispecific antibody is a humanized antibody.

3. The bispecific antibody according to any of the preceding claims, further comprising at least one linker separating at least one VHH and the VL or VH chains of the Fab.

4. The bispecific antibody according to any of the preceding claims, wherein said bispecific antibody comprises a first polypeptide with a sequence of SEQ. ID: No. 9 and a second polypeptide with a sequence of SEQ. ID: NO. 10; a first polypeptide with a sequence of SEQ. ID: No. 11 and a second polypeptide with a sequence of SEQ. ID: NO. 12; a first polypeptide with a sequence of SEQ. ID: No. 13 and a second polypeptide with a sequence of SEQ. ID: NO. 14; a first polypeptide with a sequence of SEQ. ID: No. 15 and a second polypeptide with a sequence of SEQ. ID: NO. 16; a first polypeptide with a sequence of SEQ. ID: No. 17 and a second polypeptide with a sequence of SEQ. ID: NO. 18; a first polypeptide with a sequence of SEQ. ID: No. 19 and a second polypeptide with a sequence of SEQ. ID: NO. 20; a first polypeptide with a sequence of SEQ. ID: No. 21 and a second polypeptide with a sequence of SEQ. ID: NO. 22;10335 / PC26 November 2025 a first polypeptide with a sequence of SEQ. ID: No. 23 and a second polypeptide with a sequence of SEQ. ID: NO. 24; a first polypeptide with a sequence of SEQ. ID: No. 25 and a second polypeptide with a sequence of SEQ. ID: NO. 26; a first polypeptide with a sequence of SEQ. ID: No. 27 and a second polypeptide with a sequence of SEQ. ID: NO. 28; a first polypeptide with a sequence of SEQ. ID: No. 29 and a second polypeptide with a sequence of SEQ. ID: NO. 30; a first polypeptide with a sequence of SEQ. ID: No. 31 and a second polypeptide with a sequence of SEQ. ID: NO. 32; a first polypeptide with a sequence of SEQ. ID: No. 33 and a second polypeptide with a sequence of SEQ. ID: NO. 34; a first polypeptide with a sequence of SEQ. ID: No. 35 and a second polypeptide with a sequence of SEQ. ID: NO. 36; a first polypeptide with a sequence of SEQ. ID: No. 37 and a second polypeptide with a sequence of SEQ. ID: NO. 38; or a first polypeptide with a sequence of SEQ. ID: No. 39 and a second polypeptide with a sequence of SEQ. ID: NO. 40.

5. A VHH capable of binding B7H3 comprising a sequence which is at least 90% identical to one of the sequences selected among seq. ID. No. 1-86. The VHH according to claim 5, wherein said VHH is humanized.

7. A nucleic acid encoding a bispecific antibody according to any of claims 1-4 or a VHH according to any of claims 5-6.

8. An expression vector comprising the nucleic acid of claim 7.

9. A host cell comprising the expression vector of claim 8 or the nucleic acid of claim 7.10335 / PC26 November 202510. A composition comprising the bispecific antibody of any of claims 1-4.

11. The composition of claim 10, being a pharmaceutical composition.

12. The bispecific antibody of claim 1 or the composition of claim 10 or 11 for use in treatment or diagnosis of cancer.

13. A method for diagnosis or treatment of cancer comprising the steps of i. administering a bispecific antibody to a subject in need of such treatment of diagnosis, which bispecific antibody is capable of binding a tumor antigen and further capable of binding a chelator with a bound radionuclide, to a subject in need of such treatment or diagnosis; wherein the bispecific antibody comprises: a. a first polypeptide comprising a first VHH capable of binding a tumor antigen and a light chain (VL) of a DOTA binding Fab; and b. a second polypeptide comprising a second VHH capable of binding the tumor antigen, and a heavy chain (VH) of the DOTA binding Fab. ii. After an interval administering a chelator comprising a DOTA structure binding a radionuclide.

14. The method of claim 13 wherein the tumor antigen is selected among HER2, B7-H3, CA6, CD138, CD20, CD19, CD22, CD27L, CD30, CD33, CD37, CD38, CD47, CD56, CD66e, CD70, CD74, CD79b, EGFR, CEA, EGFRvlll, FRa, GCC, GPNMB, Mesothelin, MUC16, NaPi2b, Nectin 4, PSMA, STEAP1, Trop-2, 5T4, AGS-16, alpha v beta6, 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, peritosin, Fyn, SLTRK6, Tenascin c, VEGFR2, and PRLR.

15. The method of claim 13 or 14, wherein the tumor antigen is B7H3.10335 / PC26 November 202516. The method of claim 15, wherein the bispecific antibody is an antibody of claim 1.

17. The method of claim 13, being a method for diagnosing a cancer, further comprising the step of iii. Detecting the bound radionuclide.

18. The method according to any of claims 13 or 17, wherein said interval is selected between 8 hours - 7 days.

19. The method according to any of claims 13-18, wherein said cancer is selected among osteosarcoma, neuroblastoma, liposarcoma, fibrosarcoma, carcinoma, malignant fibrous histiocytoma, leiomyosarcoma, spindle cell sarcoma, brain tumor, small cell lung cancer, retinoblastoma, HTLV-l infected T cell leukemia, breast cancer, colon cancer, prostate cancer, T-cell and B-cell lymphomas, glioblastoma multiforme, malignant glioma, Head and Neck cancer, solid tumors and non-small-cell lung cancer.

20. The method according to any of claims 13-19, wherein the chelator is selected among DOTA and DOTA derivates such as BnDOTA, Proteus I, Proteus II.

21. The method according to any of claims 14-19, wherein the radionuclide is selected among225Ac, ™ Ac,241Am,211At,215At,217At,218At,209Bi,211Bi,212Bi,213Bi,249Cf,252Cf, 244Cm,245Cm,248Cm,57Co,58Co,51Cr,64Cu,67Cu,152Dy,165Dy,152Eu,59Fe,221Fr,67Ga, 68Ga,66Ga,161Ho,110mln,mln,192lr,133La,177Lu,237Np,189mOs,231Pa,203Pb,212Pb,210Po, 211Po,212Po,214Po,215Po,216Po,218Po,195mPt,238Pu,239Pu,240Pu,244Pu,223Ra,224Ra, 226Ra,82Rb,186Re,188Re,103mRh,119Sb,75Se,89Sr,149Tb,151Tb,161Tb99mTc,94mTc,227Th, 228Th,229Th,230Th,232Th,201TI,90Y,86Y, and89Zr.10335 / PC26 November 202522. A method of producing the bispecific antibody according to any of claims 1-4, comprising the steps of i. Providing a host cell according to claim 9, ii. Cultivating the host cell under conditions leading to expression of the nucleic acid encoding the compound, and iii. Recovering the compound from the cultivation broth.

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