WO2025242909A1 - CD30-targeting antibody-radioligand conjugates and their therapeutic use - Google Patents

Abstract

The present invention relates to an antibody-radionuclide conjugate comprising a) an antibody or antigen-binding fragment thereof specifically binding to CD30; and b) terbium-161, as well as to a pharmaceutical composition comprising the conjugate of the invention as well as at least one pharmaceutically acceptable carrier. The present invention further relates to a kit of parts comprising a) an antibody or antigen-binding fragment thereof specifically binding to CD30; and b) terbium-161, wherein said antibody or antigen-binding fragment thereof is capable of binding terbium-161. The present invention further encompasses therapeutic uses of the provided conjugates. Accordingly, the antibody-radionuclide conjugates of the present invention are particularly useful in the treatment of cancer, in particular lymphoma.

Description

CD30-targeting antibody-radioligand conjugates and their therapeutic use

Field of the invention

The present invention relates to an antibody-radionuclide conjugate comprising a) an antibody or antigenbinding fragment thereof specifically binding to CD30; and b) terbium-161 , as well as to a pharmaceutical composition comprising the conjugate of the invention as well as at least one pharmaceutically acceptable carrier. The present invention further relates to a kit of parts comprising a) an antibody or antigen-binding fragment thereof specifically binding to CD30; and b) terbium-161, wherein said antibody or antigenbinding fragment thereof is capable of binding terbium-161. The present invention further encompasses therapeutic uses of the provided conjugates. Accordingly, the antibody-radionuclide conjugates of the present invention are particularly useful in the treatment of cancer, in particular lymphoma.

Background of the invention

Lymphoma impacts over half a million new patients worldwide each year, constituting a significant health concern. In the specific context of Switzerland, approximately 1 ,800 new cases are identified annually (www.nicer.org). Additionally, this disease results in over 200,000 deaths worldwide every year [1], The World Health Organization (WHO) projects a clear trend, estimating a 1 .5-fold increase in incidence by the year 2040. Lymphomas are extremely heterogeneous diseases, broadly categorized into Hodgkin’s (HL) and non-Hodgkin’s lymphomas (NHL), creating a significant challenge in the identification of viable targets for the development of effective therapies.

Current lymphoma treatment is still inefficient, especially for some aggressive types of the disease that become resistant to standard therapies, leading to relapse rates as high as 60%. These entities harbour significant unmet medical needs. Lymphoma patients are usually treated with conventional chemo and radiotherapy, which do not target specifically the cancer cells thus inducing important side effects in healthy tissues. In addition, the use of external beam radiotherapy (EBRT) is greatly limited to the initial stages of the disease, when the tumor is localized. In terms of targeted therapy, the antibody-drug conjugate (ADC) brentuximab vedotin (Adcetris) represents the only approved targeted drug for lymphoma (HL, anaplastic large-cell lymphoma (ALCL), and cutaneous T-cell lymphoma (CTCL)) [2]— [4]. Adcetris targets CD30, a receptor highly and uniformly expressed on lymphoma cells but with limited expression in healthy tissues [5]. Adcetris combines an anti-CD30 antibody (cAC10), serving as the targeting agent, with the chemotherapeutic drug MMAE, which disrupts microtubules, thus acting as the therapeutic payload. While Adcetris has demonstrated remarkable efficacy in inducing tumor regression, it phases two critical challenges: the development of resistance and severe toxicities. A substantial subset of patients, up to 50% in HL, exhibits primary or acquired resistance to the treatment, resulting in relapse and refractory disease [6], [7], Additionally, the toxic side effects of Adcetris, most notably peripheral neuropathy, represent severe limitations on its therapeutic use. This side effect is also common to other ADCs and has been related to the unspecific drug payload release in peripheral nerves, thus hindering ADCs’ clinical use [8]— [10]. In addition, ADCs still rely on the use of chemotherapeutic drugs as their cytotoxic payloads. Altogether, it is evident that there is an urgent need for alternative and improved targeted therapies for lymphoma patients.

I n this context, radioimmunotherapy (RIT), in which antibodies are coupled with a therapeutic radionuclide, emerges as a promising alternative treatment. When the RIT agent (i.e., a radioimmunoconjugate, or in other words - antibody-radionuclide conjugate) is injected into the patient, the antibody guides the radionuclide directly to the cancer cells, where the radionuclide delivers the tumoricidal dose to kill them [11], Lymphoma cells are extremely radiosensitive: EBRT represents one of the most effective treatments for lymphoma [12], However, many lymphoma patients progress to an advanced stage of the disease, in which the malignant cells disseminate throughout the body, greatly limiting the use of EBRT. RIT overcomes this problem by delivering high doses of radiation directly to the tumor cells while preventing radiotoxicity in healthy tissues. In addition, since its mechanism of action differs from the ones of chemotherapy and ADCs, it holds the potential to treat relapsed and recurrent patients. An example of the use of RIT in cancer treatment is Zevalin (ibritumomab tiuxetan), an anti-CD20 RIT used to treat follicular lymphoma [13], [14], Zevalin, labeled with the therapeutic radionuclide yttrium-90, has proven to be effective, particularly in patients who have relapsed after previous treatments, leading in some cases to long-term remission. However, the long range of beta-particles emitted by yttrium-90 in tissue (approx.10 mm) leads to a limited targeting precision which increases the risk of radiation damage to nontargeted cells, compromising its safety. Nevertheless, this limitation is due to the physical properties of yttrium-90 and could be easily overcome using better therapeutic radionuclides with shorter-range electron emission, like the innovative terbium-161 . Zhang and coworkers disclose the use of an anti-CF30 antibody radiolabeled with ⁹⁰Y (Zhang et al., “Effective therapy of murine models of human leukemia and lymphoma with radiolabeled anti-CD30 antibody, HeFi-1”, Proceedings ofthe National Academy of Sciences, vol. 104, no. 20, pages 8444-8448).

Lepareur and others review clinical advances and perspectives in targeted radionuclide therapy (Lepareur et al., “Clinical Advances and Perspectives in Targeted Radionuclide Therapy”, Pharmaceutics, vol. 15, no. 6, page 1733).

Summary of the invention

Accordingly, it was an objective technical problem of the present invention to provide an improved antibody-radionuclide conjugate against CD30 and improved uses thereof in therapy of cancer, in particular lymphoma. The problem is solved by the embodiments disclosed herein, and as characterized by the claims.

In particular, the present invention combines CD30 targeting, which has already been validated and probed to be an effective target in the treatment of a cancer patient, in particular a lymphoma patient, with the use of a therapeutic radionuclide, terbium-161 , that due to its unique physical properties (0 - and conversion and Auger electrons emission) can eliminate both tumor masses and single cells without affecting the neighboring healthy tissues.

It has been shown by the present inventors that the antibody-radionuclide conjugates of the invention are not only active against the CD30+ lymphoma cells (see Figure 1) and act by specifically causing DNA damage, thus synergizing with the effects ofthe CD30+ antibody alone (see Figure 2), but also specifically localize to tumor site, as demonstrated in Figure 3. Importantly, the exceptional therapeutic efficacy was not only demonstrated in the cellular model, but also in the animal models (see e.g. Figure 4), where it was demonstrated to cause no further side effects, as for example demonstrated by monitoring the mouse body weight upon administration of the conjugates in the animal studies.

Thus, the antibody-radionuclide conjugates of the invention are likely to address the issue of severe side effects of existing therapies, in particular therapies based on antibody-drug conjugates. Such side effect is most notably peripheral neuropathy. As a targeted therapy, provided antibody-radioligand conjugates aim at selectively eliminating the lymphoma cells while reducing the toxicity to other organs compared to non-targeted treatments such as chemotherapy. In the case of ADCs, peripheral neuropathy due to the unspecific release of the drug payload represents a huge safety concern. It affects up to 67% of patients treated with Adcetris according to different clinical studies and it can be severe enough to require dose reductions and even treatment discontinuation [8], [9]. The implementation of targeted therapies other than ADCs will avoid this concerning side effect. Importantly, the therapeutic radionuclide terbium-161 forms stable complexes with the chelator-antibody system, ensuring no unspecific release of the therapeutic payload. In addition, preliminary SPECT/CT imaging experiments have shown a remarkably high tumor uptake as compared to healthy tissues (see Figure 3). To date few studies have been published regarding potential toxicity of terbium-161 as a therapeutic radionuclide. Compared to lutetium- 177, the most clinically used therapeutic radionuclide, both radionuclides share similar chemical and radio-decay properties. However, terbium-161 additionally to the 0- emission, also co-emits conversion and Auger electrons. Due to the short range of conversion and Auger electrons in tissue, terbium-161 radiolabeled compounds would need to bind to the cells to induce toxicity. As the beta minus emissions of both terbium-161 and lutetium-177 are very similar, it is reasonable not to expect higher toxicities with terbium-161 if the compound is passing by or accumulating non-specifically in an organ or tissue.

Recently, a preclinical study with somatostatin analogues has shown no significant differences in hematological toxicity of terbium-161 compared to lutetium-177 [19]. In this specific case, the somatostatin receptor is also expressed in some bone marrow cells, leading to high doses to this organ, thus explaining the hematological toxicities observed in the study. In the present case, since CD30 is not constitutively expressed in bone marrow cells, hematological toxicities are expected to be even lower.

Of note, the antibody-radionuclide conjugates of the present invention may likely address the issue of primary or acquired resistance to the treatment, as they act by inducing cell death by direct irradiation of the cancer cells. This mechanism of action differs from one of standard therapies such as chemotherapy or Adcetris and therefore holds the potential to eliminate lymphoma cells that have become resistant to previous therapies. In addition, lymphoma cells are about twice as sensitive to radiation compared to solid tumors.

Furthermore, the present antibody-radionuclide conjugates may likely address the issue of high relapse rates in lymphoma patients. Minimal residual disease (MRD) describes a small population of cancer cells that survive after treatment and eventually lead to recurrence. In lymphoma, MRD can be found in bone marrow and blood and strongly correlates with recurrence. The eradication of this small subset of cells is extremely challenging. Thanks to the unique properties of terbium-161 , the present conjugates could eliminate the remaining single lymphoma cells and small cell clusters after standard treatment, thus reducing the chances of relapse.

The examples supporting the present invention provide unequivocal support for superiority of the use of terbium-161 over lutetium-177, in particular through superior and CD30-specific cytotoxicity, favorable biodistribution, high tumor uptake and absorption, with a more pronounced on target effect with regard to alteration in pathways regulating DNA damage response and cell cycle. Lutetium-177 is considered nowadays to be the standard of care when radiotherapy is to be applied. In certain studies, Lutetium-177 and Yttrium-90 have been compared side by side (see for example, Forrer et al., Eur. J. Nucl. Med. Mol. Imaging, 2009, 36:1443-1452). It is concluded that while ⁹⁰Y remains valuable in certain contexts, for example when it comes to targeting of large bulky tumors, ¹⁷⁷Lu offers superior precision, safety, theranostic capability, and logistical advantages, making it the preferred radionuclide for most modern targeted radionuclide therapies. Thus, the skilled person would immediately recognize that demonstrated superiority of Terbium-161 over Lutetium-177 is also strongly indicative of superiority of this radionuclide over Yttrium-90.

In particular, as presented herein, it has been demonstrated that a representative terbium-161 -charged antibody-radionuclide conjugate of the present invention, namely, [¹⁶¹Tb]Tb-cAC10 exhibits more potent cytotoxicity and induces more DNA damage than analogous lutetium-177-charged radioimmunoconjugate, i.e., [¹⁷⁷Lu]Lu-cAC10. Of note, both [¹⁶¹Tb]Tb-cAC10 and [¹⁷⁷Lu]Lu-cAC10 show high tumor uptake and low accumulation in non-targeted organs and tissues. Furthermore, [¹⁶¹Tb]Tb- cAC10 treatment prolongs survival in the absence of severe side effects. Finally, phosphoproteomic analysis reveals increased therapeutic response to [¹⁶¹Tb]Tb-cAC10 in comparison to [¹⁷⁷Lu]Lu-cAC10.

Experimental examples presented herein showcase the efficacy of the terbium-161 radioimmunotherapy according to the present invention for the treatment of CD30+ T-cell lymphomas. It has been demonstrated that terbium-161 outperforms the current benchmark lutetium-177, likely due to the coemission of conversion and Auger electrons, and consequently also outperforms therapies based on yttrium-90. The presented in vivo phosphoproteomics study comparing terbium-161 with the benchmark lutetium-177 reveals significant alterations in signaling pathways and biological responses that confirm the superior efficacy of terbium-161 .

The invention is summarized in the following embodiments.

In a first embodiment, the present invention relates to an antibody-radionuclide conjugate, comprising a) an antibody or antigen-binding fragment thereof specifically binding to CD30; and b) terbium-161.

In a second embodiment, the present invention relates to a kit of parts comprising a) an antibody or antigen-binding fragment thereof specifically binding to CD30; and b) terbium-161 wherein said antibody or antigen-binding fragment thereof is capable of binding terbi um- 161 .

In a third embodiment, the present invention relates to a pharmaceutical composition comprising the antibody-radionuclide conjugate of the present invention and at least one pharmaceutically acceptable carrier.

In a fourth embodiment, the present invention relates to the antibody-radionuclide conjugate of the present invention, the kit of parts of the present invention, or the pharmaceutical composition of the present invention, for use in therapy.

In a fifth embodiment, the present invention relates to the antibody-radionuclide conjugate of the present invention, the kit of parts of the present invention, or the pharmaceutical composition of the present invention, for use in treatment or prevention of cancer.

In a sixth embodiment, the present invention relates to the antibody-radionuclide conjugate of the present invention any, the kit of parts of the present invention or the pharmaceutical composition of the present invention in the manufacture of a medicament for treating or preventing cancer.

In a seventh embodiment, the present invention relates to a method of treating cancer, comprising the step of administering the antibody-radionuclide conjugate of the present invention, parts of the kit of parts of the present invention, or the pharmaceutical composition of the present invention to a subject in need thereof. It is to be understood that a therapeutically effective amount of said antibody-radionuclide conjugate, said parts of the kit of parts or said pharmaceutical composition is to be administered.

Brief description of figures

The invention is further illustrated by the appended figures. These are not meant to be construed as limiting the scope of the invention in any way, which is defined by the hereto appended claims. Fig. 1 shows: A-C) Cell viability of the CD30+ lymphoma cell lines Karpas 299 (A), Mac2A (B), and Myla (C) after treatment with different activities of [¹⁷⁷Lu]Lu- or [¹⁶¹Tb]Tb-cAC10 (0-20 MBq/mL). D) Comparison of the ICso values calculated for the three cell lines after treatment. E-F) Cell survival (clonogenic assay) of Karpas 299 (E) and Mac2A (F) cell lines after treatment with increasing activities (0-5 MBq/mL) of lutetium-177 and terbium-161 radioimmunoconjugates. * p < 0.05, ** p < 0.01 , ns = non-significant. Statistical analysis performed by Student t-test (cell viability) and oneway ANOVA, Tukey’s multiple comparison test (cell survival). Data represented as mean ± SD.

Fig. 2 shows DNA damage assessment (yH2AX) in Karpas 299 cells. A) Representative DAPI (blue) and yH2AX (green) stainings in control (cAC10) and radioimmunoconjugate-treated cells ([¹⁷⁷Lu]Lu- or [¹⁶¹Tb]Tb-cAC10). Scale bar = 10 pm. B) Quantification of yH2AX-positive stained area in the control and radioimmunoconjugate-treated cells. Results are expressed as percentage of the area normalized by DAPI area. * p < 0.05, ** p < 0.01. Statistical analysis performed by one-way ANOVA, Tukey’s multiple comparison test. Data represented as mean ± SD.

Fig. 3 shows biodistribution data in Karpas 299 tumor-bearing mice after i.v. injection of [¹⁷⁷Lu]Lu- cAC10 (1 MBq, 30 ug) (A) and [¹⁶¹Tb]Tb-cAC10 (1 MBq, 30 pg) (B) represented as percentage of injected activity per mass of tissue (% lA/g). Blocking condition was achieved by co-injection of 500 ug of cold cAC10. * p < 0.05, ** p < 0.01 , *** p < 0.001 , ns = non-significant; n = 5 per group. Statistical analysis performed by Student t-test. Data represented as mean ± SD. C) SPECT/CT images of Karpas 299 tumor-bearing mice shown as maximum intensity projections 72 h after injection of the radioimmunoconjugates (10MBq, 30 ug). Mice were injected i.v. with cAC10 or control IgG radiolabeled either with lutetium-177 or terbium-161. T = tumor, Li = liver, Sp = spleen.

Fig. 4 presents a therapeutic study comparing [¹⁷⁷Lu]Lu- cAC10 and [¹⁶¹Tb]Tb-cAC10 (2 MBq, 30 pg) in Karpas 299 tumor-bearing mice. A) Kaplan-Meier plot of control (PBS, cAC10, [¹⁷⁷Lu]Lu-lgG, and [¹⁶¹Tb]Tb-lgG) and experimental groups ([¹⁷⁷Lu]Lu- cAC10, [¹⁶¹Tb]Tb-cAC10). Median survival is indicated in days. B-G) Relative tumor volume of each mouse in PBS (B), cAC10 (C), [¹⁷⁷Lu]Lu- IgG (D), [¹⁶¹Tb]Tb-lgG (E), [¹⁷⁷Lu]Lu- cAC10 (F), and [¹⁶¹Tb]Tb-cAC10 (G) groups. The green line represents the average relative tumor volume. H) Relative tumor volume in the six groups at day 11 (time at which the first animal in the study reached an endpoint). I) T umor doubling time in days of each group until day 11. * p < 0.05, ** p < 0.01, *** p < 0.001 ; n = 10 per group. Statistical analysis performed by one-way ANOVA, Tukey’s multiple comparison test. Data shown as mean ± SD.

Fig. 5 presents a representative HPLC chromatograms of the lutetium-177 and terbi um- 161 radiolabeled antibodies after purification. A) [¹⁷⁷Lu]Lu- cAC10 B) [¹⁶¹Tb]Tb-cAC10 C) [¹⁷⁷Lu]Lu-lgG D) [¹⁶¹Tb]Tb- IgG. Free lutetium-177 and terbium-161 appear at a retention time of around 24 min.

Fig. 6 shows binding and cell uptake and internalization results in Karpas 299 cells. A and B) Affinity assay with increasing concentrations of [¹⁷⁷Lu]Lu- cAC10 (A) or [¹⁶¹Tb]Tb-cAC10 (B). C and D) Cell uptake and internalization of [¹⁷⁷Lu]Lu- cAC10 (C) or [¹⁶¹Tb]Tb-cAC10 (D) after different time points. Statistical analysis performed by Student-t test. Data shown as mean ± SD.

Fig. 7 shows A) Cell viability of the CD30- Jurkat cells after incubation with increasing concentrations of [¹⁷⁷Lu]Lu- cAC10 or [¹⁶¹Tb]Tb-cAC10 (0-20 MBq/mL). B) Cell viability of Karpas 299 cells after treatment with the lutetium-177 or terbium-161 radiolabeled-control antibody at different concentrations (0-20 MBq/mL).

Fig. 8 presents relative body weight of each individual mouse from the therapy study in the six groups. A) PBS B) cAC10 C) [¹⁷⁷Lu]Lu-lgG D) [¹⁶¹Tb]Tb-lgG E) [¹⁷⁷Lu]Lu- cAC10 F) [¹⁶¹Tb]Tb-cAC10. Mouse body weight is normalized to their initial weight on the day of the treatment.

Fig. 9 presents comparative phosphoproteomic analysis of [¹⁷⁷Lu]Lu-cAC10 and [¹⁶¹Tb]Tb-cAC1 fl- treated tumors: (A) Volcano plots representing the phosphopeptide abundance in response to [¹⁷⁷Lu]Lu-cAC10 or [¹⁶¹Tb]Tb-cAC10 relative to the cAC10 control. Red and blue dots indicate significant changes (FDR < 0.25, 1.5 fold change). (B) Venn diagram summarizing the overlap between significantly altered phosphoproteins in response to [¹⁷⁷Lu]Lu-cAC10 or [¹⁶¹Tb]Tb-cAC10 treatment; as well as (C) enrichment analysis for selected relevant and common biological processes and pathway analysis (Reactome) of the differentially altered phosphorylated proteins upon [177Lu]Lu-cAC10 and [161Tb]Tb-cAC10 treatment.

Fig. 10 shows complete blood count (CBC) values after radioimmunotherapy. CBC analysis was evaluated before treatment (Day 0) and on days 7, 14, 21, 28, and 35 after i.v. injection of [¹⁶¹Tb]Tb-cAC10 or [¹⁷⁷Lu]Lu-cAC10 (2 MBq, 30 pg) in non-tumor-bearing mice. Results are normalized (fold-change) to baseline values before treatment (Day 0) (mean ± SD, n=4). Fig. 11 presents in vitro plasma stability results for [¹⁷⁷Lu]Lu-cAC10 and [¹⁶¹Tb]Tb-cAC10 after incubation at 37 °C in human plasma for 7 days.

Fig. 12 shows CD30 membrane expression in the three different CD30-positive T-cell lymphoma cell lines by flow cytometry. (A) Histograms of the CD30 expression in Karpas 299, Mac2A, and Myla cells in comparison with the unstained controls. (B) Comparison of the CD30 expression (MFI) within the three cell lines.

Fig. 13 shows subcellular localization of the radioimmunoconjugates in Myla cells. Results are expressed as percentage of the total radioimmunoconjugate uptake. Statistical analysis performed by Student-t test. Data shown as mean ± SD.

Fig. 14 presents complete blood count (CBC) values after treatment with [¹⁶¹Tb]Tb-cAC10 or [¹⁷⁷Lu]Lu- cAC10 (2 MBq, 30 pg) in non-tumor-bearing mice. Results are represented as mean ± SD. Dashed line indicates the average values of a healthy athymic nude mouse.

Fig. 15 shows study evaluating the adequate [¹⁷⁷Lu]Lu-cAC10 injected activity in Karpas 299 tumorbearing mice. Kaplan-Meier plot of control (PBS) and experimental group ([¹⁷⁷Lu]Lu-cAC10) (n=4).

Fig. 16 shows A-C) Volcano plots representing the protein (A), phosphopeptide (B), and integrated phosphopeptide (C) abundance comparing [¹⁶¹Tb]Tb-cAC10 to [¹⁷⁷Lu]Lu-cAC10 treated tumors. D) Significantly altered phosphopeptides represented as fold change relative to the cAC10 control for the [¹⁶¹Tb]Tb-cAC10 versus [¹⁷⁷Lu]Lu-cAC10 comparison. Red and blue dots indicate significantly altered abundance of proteins or phosphopeptides.

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[19] S. D. Busslinger, A. K. Mapanao, K. Kegler, P. Bernhardt, F. Fliihmann, J. Fricke, J. R. Zeevaart, U. Koster, N. P. Meulen, R. Schibli, and C. Muller, “Comparison of the tolerability of terbium-161 and lutetium-177 in combination with somatostatin analogues in the preclinical setting,” bioRxiv, p. 2024.03.29.587329, Jan. 2024, doi: 10.1101/2024.03.29.587329.

Detailed description of the invention

As previously mentioned, in one embodiment, the present invention relates to an antibody-radionuclide conjugate, comprising a) an antibody or antigen-binding fragment thereof specifically binding to CD30; and b) a therapeutic radionuclide.

In a particularly preferred embodiment, the present invention relates to an antibody-radionuclide conjugate, comprising a) an antibody or antigen-binding fragment thereof specifically binding to CD30; and b) terbium-161

As apparent to the skilled person, the term “antibody radionuclide conjugate” (ARC) is preferably defined as a variant of an antibody-drug conjugate where the drug molecule or active molecule represents a radionuclide/radioisotope. Said radionuclide/radioisotope may either be covalently bound to the antibody e.g. in case of radioactive iodine or may be bound by a metal chelator complex appended to the antibody e.g. as in the case of radioactive lutetium, actinium or terbium. The so formed antibody-radionuclide conjugate is capable to deliver a high amount of radiation to the tumor tissue thereby killing the tumor cells due to the damaging of DNA, essential enzymes etc.

As preferably referred to herein, an antibody is defined in the following.

In general, the term "antibody" is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), fully-human antibodies and antibody constructs so long as they exhibit the desired antigen-binding activity. Unless explicitly indicated to the contrary, when discussing the antibody or the properties thereof, whenever a reference is made to an antibody, any form of the antibody as apparent to the skilled person (such as a form selected from monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and fully-h uman antibodies) is meant.

As apparent to the skilled person, the term “antibody” may also refer to an antigen-binding fragment thereof. As preferably referred to herein, an "antigen-binding fragment" of an antibody refers to a molecule other than an intact antibody that comprises a portion of an intact antibody and that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab' -SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.

In one embodiment, the antibody comprised in the antibody-radionuclide conjugate of the invention may be a full-length antibody and not an antigen-binding fragment of an antibody.

Preferably, the antibody is a monoclonal antibody, a chimeric antibody, a recombinant antibody, a single chain antibody, a humanized antibody, a bispecific antibody, and/or a multi-specific antibody.

The term “monoclonal antibody” as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Monoclonal antibodies are advantageous in that they may be synthesized by a hybridoma culture, essentially uncontaminated by other immunoglobulins. The modified "monoclonal" indicates the character of the antibody as being amongst a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. As mentioned above, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method described by Kohler, Nature 256 (1975), 495.

The term “chimeric antibodies”, refers to an antibody which comprises a variable region of the present invention fused or chimerized with an antibody region (e.g., constant region) from another, human or nonhuman species (e.g., mouse, horse, rabbit, dog, cow, chicken). The term “recombinant antibody” includes all antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal (e.g. a mouse) that is transgenic for human immunoglobulin genes, antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial human antibody library, or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Accordingly, the term antibody also relates to recombinant human antibodies, heterologous antibodies and heterohybrid antibodies. Such recombinant human antibodies have variable and constant regions (if present) derived from human germline immunoglobulin sequences. Such antibodies can, however, be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

A "heterologous antibody" is defined in relation to the transgenic non-human organism producing such an antibody. This term refers to an antibody having an amino acid sequence or an encoding nucleic acid sequence corresponding to that found in an organism not consisting of the transgenic non-human animal, and generally from a species other than that of the transgenic non-human animal.

The term "heterohybrid antibody" refers to an antibody having light and heavy chains of different organismal origins. For example, an antibody having a human heavy chain associated with a murine light chain is a heterohybrid antibody. Examples of heterohybrid antibodies include chimeric and humanized antibodies.

The term antibody also relates to humanized antibodies. "Humanized" forms of non-human (e.g. murine or rabbit) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Often, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibody may comprise residues, which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see: JonesNature 321 (1986), 522-525; Reichmann Nature 332 (1998), 323-327 and Presta Curr Op Struct Biol 2 (1992), 593-596.

A single chain antibody, i.e. , “single-chain Fv” or “scFv” antibody fragments have, in the context of the invention, the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. Techniques described for the production of single chain antibodies are described, e.g., in Pliickthun in The Pharmacology of Monoclonal Antibodies, Rosenburg and Moore eds. Springer-Verlag, N.Y. (1994), 269- 315.

A “Fab fragment” as used herein is comprised of one light chain and the CH1 and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

An "Fc" region contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.

A "Fab¹ fragment" contains one light chain and a portion of one heavy chain that contains the VH domain and the C H1 domain and also the region between the CH1 and C H2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form a F(ab')2 molecule.

A "F(ab')2 fragment" contains two light chains and two heavy chains containing a portion of the constant region between the CH1 and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains. A F (ab')2 fragment thus is composed of two Fab' fragments that are held together by a disulfide bond between the two heavy chains. The "Fv region" comprises the variable regions from both the heavy and light chains, but lacks the constant regions.

A bispecific antibody, as referred to herein, is an antibody that can simultaneously bind to two different types of antigen, or to two different epitopes of the same antigen. Upon development, bispecific antibodies can be manufactured in several structural formats, which are known to the skilled person.

A multi-specific antibody, as referred to herein, is an antibody that can simultaneously bind to more than two different types of antigen, or to more than two different epitopes of the same antigen.

Antibodies, antibody constructs, antibody fragments, antibody derivatives (all being Ig-derived) to be employed in accordance with the invention or their corresponding immunoglobulin chain(s) can be further modified using conventional techniques known in the art, for example, by using amino acid deletion(s), insertion(s), substitution(s), addition(s), and/or recombination(s) and/or any other modification(s) known in the art either alone or in combination. Methods for introducing such modifications in the DNA sequence underlying the amino acid sequence of an immunoglobulin chain are well known to the person skilled in the art; see, e.g., Sambrook (1989), loc. cit. The term “Ig-derived domain” particularly relates to (poly) peptide constructs comprising at least one CDR. Fragments or derivatives of the recited Ig-derived domains define (poly) peptides which are parts of the above antibody molecules and/or which are modified by chemical/biochemical or molecular biological methods. Corresponding methods are known in the art and described inter alia in laboratory manuals (see Sambrook et al., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press, 2nd edition (1989) and 3rd edition (2001); Gerhardt et al., Methods for General and Molecular Bacteriology ASM Press (1994); Lefkovits, Immunology Methods Manual: The Comprehensive Sourcebook of Techniques; Academic Press (1997); Golemis, Protein- Protein Interactions: A Molecular Cloning Manual Cold Spring Harbor Laboratory Press (2002)).

More preferably, the antibody as referred to herein is a monoclonal antibody.

The antibody as referred to herein may be an lgG1 , lgG2a or lgG2b, lgG3, lgG4, IgM, lgA1 , lgA2, IgAsec, IgD, IgE. As used herein, "isotype" refers to the antibody class (e.g., IgM or lgG1) that is encoded by heavy chain constant region genes. The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., lgG1 , lgG2, lgG3, lgG4, lgA1, and lgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, 5, £, y, and p, respectively.

Preferably, the monoclonal antibody as described herein is an lgG1 antibody.

The term “binding to” as used in the context of the present invention defines a binding (interaction) of at least two “antigen-interaction-sites” with each other. The term “antigen-interaction-site” defines, in accordance with the present invention, a motif of a polypeptide, i.e. , a part of the antibody of the present invention, which shows the capacity of specific interaction with a specific antigen or a specific group of antigens displayed on the surface of the cancer cell.

Herein, said specific antigen displayed on the surface of the cancer cell is CD30.

CD30 is known to the skilled person as a protein available in UniProt database under entry P28908. It is a cell membrane protein of the tumor necrosis factor receptor family and a tumor marker. This receptor is expressed by activated, but not by resting, T and B cells. TRAF2 and TRAF5 can interact with this receptor, and mediate the signal transduction that leads to the activation of NF-kappaB. It is a positive regulator of apoptosis, and also has been shown to limit the proliferative potential of autoreactive CD8 effector T cells and protect the body against autoimmunity. Two alternatively spliced transcript variants of this gene encoding distinct isoforms have been reported. CD30 is associated with anaplastic large cell lymphoma. It is expressed in embryonal carcinoma but not in seminoma and is thus a useful marker in distinguishing between these germ cell tumors. CD30 and CD 15 are also expressed on Reed-Sternberg cells typical for Hodgkin's lymphoma. CD30 is the target of the FDA approved therapeutic brentuximab vedotin (Adcetris). This active agent is also referred to as SGN-35, and has been previously known as cAC10-vcMMAE

As preferably referred to herein, specific binding to CD30 refers to the situation, wherein, the antibody or the fragment thereof is capable of binding CD30 with sufficient affinity that said antibody is useful as a diagnostic and/or therapeutic agent in targeting CD30. Accordingly, and preferably, the extent of binding of such antibody to an unrelated, non-CD30 protein is less than about 10% of the binding of the antibody to CD30, as measured e.g., in an immunoassay (for example a radioimmunoassay) or in an SPR assay (surface-plasmon resonance). More preferably, the extent of binding of such antibody to an unrelated, non-CD30 protein is less than about 5% of the binding of the antibody to CD30, as measured e.g. in an immunoassay (for example a radioimmunoassay), in an assay based on radioactivity measurement, or in an SPR assay (surface-plasmon resonance, e.g. performed used Biacore).

The antibody or the antigen-binding fragment thereof, as used herein, binds to an epitope on CD30 enabling treatment and/or prevention effects of the radioligand. Preferably, the antibody or antigen-binding fragment thereof of a) binds to the same epitope of CD30 as cAC10 antibody.

According to the present invention, it is preferred that the antibody or antigen-binding fragment thereof of a) is an antibody comprising the CDR sequences of the cAC10 antibody or an antibody comprising VH and VL sequences of the cAC10 antibody or corresponds to the cAC10 antibody.

As understood herein the cAC10 antibody is the antibody comprised in brentuximab vedotin. The cAC10 antibody comprises HC and LC sequences of SEQ ID NOs 2 and 5, respectively.

Accordingly, the antibody comprised in the antibody-radionuclide conjugate of the invention may be an antibody comprising the CDR sequences of the cAC10 antibody, in particular the CDR sequences comprised in the HC and LC sequences of SEQ ID NOs 2 and 5, respectively. The skilled person is aware of methods to determine CDR sequences in a given HC or LC sequence, for example using the Kabat numbering scheme.

The antibody comprised in the antibody-radionuclide conjugate of the invention may be an antibody comprising the variable region of the heavy chain (VH) of the antibody cAC10 as shown SEQ ID NO.: 1 :

CAC10 VH (SEQ ID NO.: 1):

QIQLQQSGPEWKPGASVKISCKASGYTFTDYYITWVKQKPGQGLEWIGWIYPGSGNTKYNE KFKGKATLTVDTSSSTAFMQLSSLTSEDTAVYFCANYGNYWFAYWGQGTQVTVSA

The antibody comprised in the antibody-radionuclide conjugate of the invention may be an antibody comprising the heavy chain of the antibody cAC10 as shown SEQ ID NO.: 2: cAC HC (SEQ ID NO.: 2):

QIQLQQSGPEWKPGASVKISCKASGYTFTDYYITWVKQKPGQGLEWIGWIYPGSGNTKYNE

KFKGKATLTVDTSSSTAFMQLSSLTSEDTAVYFCANYGNYWFAYWGQGTQVTVSAASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSW TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK

The antibody comprised in the antibody-radionuclide conjugate of the invention may be an antibody comprising the heavy chain of the antibody cAC10 together with the signal sequence as shown SEQ ID NO.: 3 cACI O FL HC (SEQ ID NO.: 3)

MKHLWFFLLLVAAPRWVLSQIQLQQSGPEWKPGASVKISCKASGYTFTDYYITWVKQKPGQ GLEWIGWI YPGSGNTKYNEKFKGKATLTVDTSSSTAFMQLSSLTSEDTAVYFCANYGNYWFA YWGQGTQVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKP

REEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The antibody comprised in the antibody-radionuclide conjugate of the invention may be an antibody comprising the variable region of the light chain (VL) of the antibody cAC10 as shown SEQ ID NO.: 4:

CAC10 VL (SEQ ID NO.: 4):

DIVLTQSPASLAVSLGQRATISCKASQSVDFDGDSYMNWYQQKPGQPPKVLIYAASNLESGI PARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPWTFGGGTKLEIK

The antibody comprised in the antibody-radionuclide conjugate of the invention may be an antibody comprising the light chain of the antibody cACIO as shown SEQ ID NO.: 5: cACI O LC (SEQ DI NO.: 5)

DIVLTQSPASLAVSLGQRATISCKASQSVDFDGDSYMNWYQQKPGQPPKVLIYAASNLESGI

PARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPWTFGGGTKLEIKRTVAAPSVFI FPP SDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS

KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

The antibody comprised in the antibody-radionuclide conjugate of the invention may be an antibody comprising the heavy chain of the antibody cAC10 together with the signal sequence as shown SEQ ID NO.: 6. cACI O FL HC (SEQ ID NO.: 6):

MVLQTQVFISLLLWISGAYGDIVLTQSPASLAVSLGQRATISCKASQSVDFDGDSYMNWYQQ KPGQPPKVLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPWTFGG GTKLEIKRTVAAPSVFI FPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQES VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Preferably, the antibody comprised in the antibody-radionuclide conjugate of the invention is the cAC10 antibody, as defined herein, and as comprised in brentuximab-vedotin.

Alternatively, an antibody comprising the CDR sequences or the VH and/or VL sequences or the heavy and/or light chain of an antibody comprising a heavy chain having an amino acid sequence according to SEQ ID NO.: 7, and comprising a light chain having an amino acid sequence according to SEQ DI NO.: 8.

Alternative FL HC (SEQ ID NO.: 7)

MEFGLSWVFLVALFRGVQCQIQLQQSGPEWKPGASVKISCKASGYTFTDYYITWVKQKPGQ GLEWIGWI YPGSGNTKYNEKFKGKATLTVDTSSSTAFMQLSSLTSEDTAVYFCANYGNYWFA YWGQGTQVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Alternative FL LC (SEQ ID NO.: 8)

MKYLLPTAAAGLLLLAAQPAMADIVLTQSPASLAVSLGQRATISCKASQSVDFDGDSYMNWY QQKPGQPPKVLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPWTF GGGTKLEIKRTVAAPSVFI FPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

The amino acid sequences disclosed herein are also provided in the appended sequence listing (sequence protocol), compliant with WIPO ST. 26. In case of a conflict between the sequences disclosed in the description and those recited in the appended sequence listing, the present invention relates to both versions of a sequence individually, preferably to a version recited in appended sequence listing.

"Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

Amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigenbinding.

In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the CDRs and FRs. Conservative substitutions are shown in Table D1 under the heading of "preferred substitutions." More substantial changes are provided in Table D1 under the heading of "exemplary substitutions," and as further described below in reference to amino acid side chain classes. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC. Table D1.

Amino acids may be grouped according to common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, lie;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one of these classes for another class. One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g. a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and/or will have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more CDR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity ( e.g. binding affinity).

Alterations (e.g., substitutions) may be made in CDRs, e.g., to improve antibody affinity. Such alterations may be made in CDR "hotspots," i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and/or SDRs (a-CDRs), with the resulting variant VH or VL being tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001).) In some embodiments of affinity maturation, diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method to introduce diversity involves CDR-directed approaches, in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR H3 and CDR-L3 in particular are often targeted.

In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in CDRs. Such alterations may be outside of CDR "hotspots" or SDRs. In certain embodiments of the variant VH and VL sequences provided above, each CDR either is unaltered, or contains no more than one, two or three amino acid substitutions.

A useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with antigen is affected. Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions. Alternatively, or additionally, a crystal structure of an antigen-antibody complex is used to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N- terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g. for ADEPT) or a polypeptide which increases the serum half-life of the antibody.

In certain embodiments, an antibody provided herein is altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.

Where the antibody comprises an Fc region, the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GIcNAc), galactose, and sialic acid, as well as a fucose attached to a GIcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in an antibody of the invention may be made in order to create antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibody may be from 1 % to 80%, from 1 % to 65%, from 5% to 65% or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e. g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fe region residues); however, Asn297 may also be located about ± 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to "defucosylated" or "fucose deficient" antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621 ; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; W02005/053742; W02002/031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Led 3 OHO cells deficient in protein fucosylation (Ripka etal. Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US 2003/0157108 Al, Presta, L; and WO 2004/056312 Al, Adams et al., especially at Example 11), and knockout cell lines, such as alpha-1 , 6-fucosyltransferase gene, FUT8, knockout OHO cells (see, e.g., Yamane-Ohnuki et al. Bioteeh. Bioeng. 87: 614 (2004); Kanda, Y. et al., Bioteehnol. Bioeng., 94(4):680-688 (2006); and W02003/085 I07).

Antibodies variants are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GIcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet et al.); US Patent No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.). Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).

In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human lgG1 , lgG2, lgG3 or lgG4 Fc region) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions.

In certain embodiments, the invention contemplates an antibody variant that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half-life of the antibody in vivo is important yet certain effector functions (such as complement and antibody-dependent cellular cytotoxicity) are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC (complement-dependent cytotoxicity) and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcyR binding (hence likely lacking ADCC activity), but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express Fc(RI II only, whereas monocytes express Fc(RI, Fc(RII and Fc(RIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 (see, e.g. Hellstrom, I. et al. Proc. Nat’l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat’l Acad. Sci. USA 82:1499- 1502 (1985); 5,821 ,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)).

The antibody may in one embodiment include further a domain or an amino acid sequence used for cotargeting to tumor microenvironment (e.g., targeting Fibroblast activation protein-a (FAP)); to overcome blood brain barrier (BBB) (e.g., targeting transferrin receptors); or to overcome endothelial cells (EC) barrier, (e.g., caveolae targeting of aminopeptidase P2 (APP2). Said additional targeting domain may thereby be directly included into the sequence of the antibody or the antigen-binding fragment to the C or N-terminus using a spacer peptide (fusion construct) or later attached e.g., by a site-specific functionalization.

The antibody (or the antigen-binding fragment thereof, as and if applicable) may in one embodiment include a further domain or a further amino acid sequence. For example, the antibody may include a localization sequence.

Antibodies may be produced using recombinant methods and compositions, e.g., as described in U.S. Patent No. 4,816,567. These methods are well known to the skilled person. The expression of antibodies (or particular constructs, are applicable) is followed by a chemical modification, as described herein. Alternatively or additionally, the antibody-radionuclide conjugates can be obtained according to or as per analogy to the protocols described in the Examples section.

As mentioned before, the antibody-radionuclide conjugate of the present invention comprises as b) a radionuclide, in particular a therapeutic radionuclide. More preferably, said therapeutic radionuclide is also a radionuclide that is useful in diagnosis. In other words, said radionuclide is preferably a theragnostic radionuclide. Preferably, as defined in the present invention, b) is terbium-161 .

The terms “radioisotope/radionuclide” in the scope of this invention are preferably used synonymically and preferably represent an atom that has excess nuclear energy, making it unstable. This excess energy can be used in one of three ways: emitted from the nucleus as gamma radiation; transferred to one of its electrons to release it as a conversion electron; or used to create and emit a new particle (alpha particle or beta particle) from the nucleus. Radioisotope/radionuclide is herein preferably defined as an isotope which has a half-life of less than 10¹⁹ years.

Accordingly, the therapeutic radionuclide is preferably selected from ¹⁶¹Tb, ⁶⁷Cu, ⁸⁹Sr, ⁹⁰Y, ¹³¹l, ¹⁵³Sm, ¹⁷⁷Lu, ²²³Ra, ²²⁵Ac, ⁴⁷Sc, ¹⁴⁹Tb, and ²¹²Pb. The radionuclide is preferably ¹⁶¹Tb.

As recognizable to the skilled person, certain antibody-radionuclide conjugate may also be used for diagnostic purposes. Certain radionuclides as disclosed herein, can be monitored, for example terbium 161 due to its y-emission can be visualized with gamma camera and hence used for detection of cancer tissue or cell-type, as targeted to by the antibody. Terbium-149 which can be used for targeted alpha therapy, has visibility in PET scans and thus can be monitored. According to the present disclosure, fluorine-18, scandium-43, scandium-44, copper-61 , copper-64, gallium-68, zirconium-89, indium-11 1 , iodine-123, terbium-152, terbium-155, as well as terbium-161 are particularly useful in diagnostic applications as described herein and may be referred to as radionuclides useful in diagnosis. As known to the skilled person, the radionuclides useful in diagnosis can be monitored by using a suitable method, for example Scintigraphy, Single Photon Emission Computed Tomography (SPE-CT); or Positron emission tomography Computed Tomography (PET-CT).

The skilled person will appreciate that an immunoconjugate wherein the active agent comprises a radionuclide useful for therapeutic application, for example selected from terbium-161 , copper-67, strontium-89, yttrium-90, iodine-131 , samarium-153, lutetium-177, radium-223 and actinium 225, (these radionuclides may be referred to as radionuclides useful in therapy) will have substantially the same biodistribution as the immunoconjugate wherein the active agent is a radionuclide useful for diagnostic application. Therefore, the immunoconjugates of the present invention can preferably be used for monitoring of biodistribution of therapeutic immunoconjugates during therapy. For example, an immunoconjugate comprising an active agent being a radionuclide useful in therapy can be supplemented for this purpose preferably with less than 10 weight% of an immunoconjugate wherein the active agent comprises a radionuclide useful in diagnosis, as defined herein. Further preferably, the immunoconjugate of the present invention for use in the combined therapeutic and diagnostic application may comprise two radionuclides, one radionuclide useful in therapy and one radionuclide useful in diagnosis, for example attached to a single polymeric carrier, to different repeating units thereof. Preferable are combinations wherein a radionuclide useful in therapy and a radionuclide useful in diagnosis are isotopes of the same element. Therefore, preferred combinations include scandium-43 and scandium-47, copper-61 and copper-67, copper-64 and copper-67, iodine-123 and iodine-131 , terbium-152 and terbium-161 , zirconium-89 and terbium-161 and terbium-155 and terbium-161. Further preferred combinations include isotopes of two different elements, for example indium-1 11 and lutetium-177, and indium-1 11 and terbium- 161.

Accordingly, in one embodiment, b) may comprise more than one radionuclide, as described herein.

In a particularly preferred embodiment of the invention, the radionuclide in b) is a radionuclide usable in a theragnostic application, i.e. suitable both for therapeutic application as well as suitable for being monitored. Particularly preferred such radionuclide is terbium-161.

It has been demonstrated by the present inventors that, despite apparent similarities between lutetium- 177 and terbium-161, the latter has shown surprisingly improved in vivo efficacy, as shown e.g. in Figure 4. Accordingly, terbium-161 conjugates of the invention have shown unexpectedly improved anti-tumour effect, compared e.g. to lutetium-177. This surprising synergy between CD30-targeting antibody, such as cAC10, and terbium-161 , as demonstrated in Figure 4, could not be predicted in any way.

The present invention is not limited with respect to the form of attachment of the radionuclide to antibody. However, as previously mentioned, it is particularly preferred that the antibody in the antibody-radionuclide conjugate of the invention comprises a metal chelator. It is to be understood that said metal chelator is charged ((i.e., being charged) with a radionuclide as described herein, preferably with terbium-161 , or is capable of being charged with said radionuclide, in particular with terbium-161. As understood herein, preferably the metal chelator is charged with a radionuclide when said radionuclide binds to metal chelator, i.e., colocalizes with it. Thus, preferably the stoichiometry of the interaction between the metal chelator and the radionuclide is 1 :1. As understood herein, preferably the metal chelator is capable of being charged with a radionuclide if, upon contacting the metal chelator with said radionuclide, obtained is a metal chelator charged with said radionuclide, as defined herein. It is to be understood, as also is apparent to the skilled person, that the radionuclide is preferably present as a metal ion. Different metal chelators suitable for use in the conjugates of the present invention are known to the skilled person. Accordingly, the metal chelator in the antibody-radioligand conjugate of the present invention is preferably selected from 1 ,4,7,10-tetraazacyclododecane-1 ,4,7,10-tetraacetic acid (DOTA), diethylenetriaminepentaacetic (DTPA), desferrioxamine (DFO) and triethylenetetramine (TETA), 1 ,4,8,11- tetraazabicyclo[6.6.2]hexadecane-4, 11 -diacetic acid (CB-TE2A); ethylenediaminetetraacetic acid (EDTA); ethylene glycolbis(2-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA); 1 ,4,8,11- tetraazacyclotetradecane-1, 4, 8, 11 -tetraacetic acid (TETA); ethylenebis-(2-4 hydroxy-phenylglycine) (EHPG); 5-CI-EHPG; 5BrEHPG; 5-Me-EHPG; 5t-Bu-EHPG; 5-sec-Bu-EHPG; benzodiethylenetriamine pentaacetic acid (benzo-DTPA); dibenzo-DTPA; phenyl-DTPA, diphenyl-DTPA; benzyl-DTPA; dibenzyl- DTPA; bis-2(hydroxybenzyl)-ethylene-diaminediacetic acid (HBED) and derivatives thereof; Ac-DOTA; benzo-DOTA; dibenzo-DOTA; 1 ,4,7-triazacyclononane N,N',N"-triacetic acid(NOTA); benzo-NOTA; 1 ,4,7-triazacyclononane N,N'-diacetic acid N”-glutaminic acid(NODAGA), benzo-TETA, benzo-DOTMA, where DOTMA is 1 ,4,7, 10-tetraazacyclotetradecane-1 ,4,7,1 O-tetra(methyl tetraacetic acid), benzo- TETMA, where TETMA is 1 ,4, 8,11-tetraazacyclotetradecane-1 ,4,8,11-(methyl tetraacetic acid); derivatives of 1 ,3-propylenediaminetetraacetic acid (PDTA); triethylenetetraaminehexaacetic acid (TTHA); derivatives of 1 ,5,10-N,N',N"-tris(2,3- dihydroxybenzoyl)-tricatecholate (LICAM); and 1 ,3,5- N,N',N"-tris(2,3- dihydroxybenzoyl)aminomethylbenzene (MECAM),

More preferably, the metal chelator (or, in other words, the metal chelator moiety) is 1 ,4,7,10- tetraazacyclododecane-1 ,4,7,10-tetraacetic acid (DOTA).

As previously mentioned, the way of attachment of the metal chelator to the antibody is not particularly limited and any feasibly way of attachment can be used in the antibody-radioligand conjugate of the present invention.

In one preferred embodiment, the metal chelator is derivatized with para-SCN-benzyl group. Upon incubation with the protein, an adduct is formed upon reaction of the -NCS group with amino group of lysine side chains (and/or N-terminal free amino group), the adduct comprising the -NH-C(=S)-NH- moiety. Accordingly, in one embodiment of the present invention, and as exemplified herein, the metal chelator moiety is attached to the antibody or antigen-binding fragment thereof through a linker comprising - NHC(=S)-NH- moiety.

In a particularly preferred embodiment, a p-SCN-benzyl-DOTA is used. Accordingly, the antibodyradionuclide conjugate comprises in this case a following moiety: wherein said moiety is attached to the antibody or antigen-binding fragment thereof via amino group of lysine residue. It is further to be understood that the metal chelator may be charged with radionuclide, but may also be present in a radionuclide-free form.

Alternatively, a strategy of attachment based on labeling -of SH groups of cysteine residues in the antibody in a reaction with maleimide moiety may be employed. To this end, the metal chelator is to be derivatized with the maleimide moiety, and contacted with the antibody, so that the following reaction occurs: wherein Cys is a cysteine residue within the antibody, presenting an -SH moiety, as shown in the scheme. As it is apparent to the skilled person a further reaction of the opening of the maleimide ring, for example upon treatment with buffer at pH higher than 7, may occur.

Further labeling strategies may be based on introducing particular sequences in the protein that can undergo further modification. The examples of such suitable sequences include aldehyde tag and sortase recognition motif.

The aldehyde tag is an artificial peptide tag recognized by the formylglycine-generating enzyme (FGE). A suitable example of an aldehyde tag is a tag according to sequence LCTPSR (SEQ ID NO.: 9), wherein upon FGE acting on said sequence, the cysteine residue is converted to formylglycine, which can be used in further attachment strategies.

The sortase recognition motif is according to sequence LPXTG (SEQ ID NO.: 10), wherein X can be any natural amino acid residue. The sortase enzyme, for example Staphylococcus aureus sortase, is a transpeptidase that attaches surface proteins to the cell wall; it cleaves between the Gly and Thr of the LPXTG motif and catalyses the formation of an amide bond between the carboxyl-group of threonine and the amino-group of the cell-wall peptidoglycan. As known to the skilled person, sortase recognition motif allows for attachment of further peptidic moieties, and can also be used for conjugating a metal chelator to the antibody, to yield an antibody-radionuclide conjugate.

It is further apparent to the skilled person that labeling strategy based on transglutaminase can be used for attaching the metal chelator to the antibody. Transglutaminases catalyze an acyl-transfer reaction to the side chain of glutamine residues of their protein substrate. Depending on the acyl donor, this can result in an amide bond between the glutamine and a primary amine, crosslinking between two proteins via a side chain lysine of the donor protein or the deamidation of glutamine. For protein labelling purposes, the acyl-transfer reaction is preferred. While transglutaminases are specific for glutamine on the target protein, the flexibility in terms of the amine containing acyl-donor offers diverse possibilities for modification. In contrast to other protein-ligation strategies, the probe containing reactant is not required to be a peptide and can simply be an alkylamine or an oligoamine as long as it contains a primary amine. For example, the metal chelator such as DOTA, can be attached to a peptide sequence suitable for attachment to the antibody via transglutaminase-catalyzed reaction. One example of such sequence is RAKAR sequence (SEQ ID NO. 11). Further such sequences are disclosed in WO 2019/057772.

The antibody may also optionally include one or more non-canonical amino acids to be used for coupling of said antibody with another chemical entity. Said amino acids may include residues that would be reactive in addition reactions known to the skilled person as click chemistry. Suitable examples of such residues include residues comprising azide moiety, or cyclooctyne moiety or a moiety being capable to perform an inverse-demand Diels-Alder cycloaddition reaction e.g., a trans-cyclooctene / tetrazine reaction pair. However, the invention is not meant to be limited to any of these examples, and other such residues known to the skilled person may also be used. The methods of producing antibodies (or fragments thereof, as applicable) comprising non-canonical amino acid residue(s) using recombinant methods are known to the skilled person.

Additional strategies for modifying an antibody with a metal chelator are described in the literature, for example by Falck and Mueller (10.3390/antib7010004).

The present invention is further not limited in any way with respect to the radionuclide-antibody ratio, and any obtainable ratio can be used in the antibody-radionuclide conjugate of the present invention. For example, the radionuclide/antibody ratio may be between 1 and 8, preferably between 2 and 4, such as 2.0, 2.1 , 2.2, .2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1 , 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9 or 4.0. Preferably, said ratio is between 2.5 and 3.5. Particularly preferred is ratio of about 3.0, such as 3.0.

As preferably understood herein, the term “about” when, referring to a numerical value, refers to said value ± 10% thereof, more preferably said value ± 5% thereof, even more preferably said value ± 1 % thereof, again more preferably to said value.

The present invention further relates to a kit of parts comprising a) an antibody or antigen-binding fragment thereof specifically binding to CD30; and b) a therapeutic radionuclide wherein said antibody or antigen-binding fragment thereof of a) is capable of binding the therapeutic radionuclide.

The present invention further relates to a kit of parts comprising a) an antibody or antigen-binding fragment thereof specifically binding to CD30; and b) terbium-161 wherein said antibody or antigen-binding fragment thereof of a) is capable of binding terbi um-161 .

The antibody or antigen-binding thereof is as described hereinabove. In particular, it is preferred that said antibody or antigen-binding thereof is functionalized with a metal chelator, as described herein. Said metal chelator is capable of binding a radionuclide, for example terbium-161. It is to be understood that said radionuclide can be formulated according to any way deemed suitable by the skilled person, for example as a salt, in particular a pharmaceutically acceptable salt. Accordingly, the presence of the metal chelator comprised in a) realizes the feature of said antibody or antigen-binding fragment thereof being capable of binding terbium-161 , as said metal chelator is capable of being charged with metal chelator. Thus, said antibody in a), which is capable of binding terbium-161, may also be referred to as a conjugate of the antibody, as described herein, with an uncharged metal chelator, as described herein. As apparent to the skilled person, upon contacting of a) an antibody or antigen-binding fragment thereof specifically binding to CD30; and b) terbium-161 , wherein said antibody or antigen-binding fragment thereof of a) is capable of binding terbium-161 (i.e. being charged with terbium-161 ), said a) will become charged with terbium- 161.

It is further to be understood that the present invention relates to the antibody or antigen-binding fragment thereof, specifically binding to CD30 wherein said antibody or antigen-binding fragment thereof is capable of binding terbium-161. Accordingly, the present invention also explicitly covers the cold version of the antibody-radionuclide conjugate of the present invention, which has not yet been charged with said radionuclide.

As immediately apparent to the skilled person, the antibody or antigen-binding fragment thereof is capable of binding terbium-161 when, upon contacting said antibody or its fragment with terbium-161 a complex between the antibody or antigen-binding fragment thereof and Terbium-161 is formed. In other words, the antibody or antigen-binding fragment thereof is capable of binding terbium-161 when said antibody or antigen-binding fragment thereof binds (or can bind) terbium-161.

The present invention further relates to a pharmaceutical composition comprising the antibodyradionuclide conjugate of the present invention and at least one pharmaceutically acceptable carrier.

Pharmaceutical formulations of an antibody-radionuclide conjugate of the present invention as described herein are prepared by mixing such antibody-radionuclide conjugate having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA or GLDA; sugars such as sucrose, mannitol, trehalose or sorbitol; osmo-protectants like ectoin; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include insterstitial drug dispersion agents such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH- 20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in US Patent Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.

The term "pharmaceutical formulation" or “pharmaceutical composition” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.

A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

Exemplary lyophilized immunoconjugate formulations are described in US Patent No. 6,267,958. Aqueous immunoconjugate formulations include those described in US Patent No. 6,171,586 and W02006/044908, the latter formulations including a histidine-acetate buffer.

The formulation herein may also contain more than one active ingredient as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.

Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody or immunoconjugate, such as antibody-radionuclide conjugate, which matrices are in the form of shaped articles, e.g. films, or microcapsules.

The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.

In one embodiment, the present invention relates to the antibody-radionuclide conjugate of the present invention or pharmaceutical composition of the present invention or the kit of parts of the present invention for use as a medicament. In other words, the present invention relates to the antibody-radionuclide conjugate of the present invention or pharmaceutical composition of the present invention or the kit of parts of the present invention for use in therapy. It is to be understood that the antibody-radionuclide conjugate or the pharmaceutical compositions or the kits of parts of the present invention can be used in the treatment of a disease or a disorder.

As used herein, "treatment" (and grammatical variations thereof such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, antibody-radionuclide conjugate of the invention are used to delay development of a disease or to slow the progression of a disease.

An antibody-radionuclide conjugate of the invention (and any additional therapeutic agent) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional, intrauterine or intravesical administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g. by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.

Antibody-radionuclide conjugate of the invention would be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The antibodyradionuclide conjugate need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antibody-radionuclide conjugate present in the formulation, the type of disorder or treatment, and otherfactors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of an antibody-radionuclide conjugate of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody-radionuclide conjugate, the severity and course of the disease, whether the antibody-radionuclide conjugate is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibodyradionuclide conjugate, and the discretion of the attending physician. The antibody-radionuclide conjugate is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 pg/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of antibody or antibodyradionuclide conjugate can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. One typical daily dosage might range from about 1 pg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs. One exemplary dosage of the antibody-radionuclide conjugate would be in the range from about 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, e.g. every week or every three weeks (e.g. such that the patient receives from about two to about twenty, or e.g. about six doses of the antibody). An initial higher loading dose, followed by one or more lower doses may be administered. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

For the antibody-radionuclide conjugates, in particular those comprising a radionuclide useful in therapy, the dose of radionuclide may also be described in the units of radioactivity, preferably in MBq/kg body weight. The total daily dose of the compound comprising a radionuclide, wherein said radionuclide is a 0- emitter administered to a human subject or patient in single dose or in divided doses preferably is between 3 and 300 MBq/kg body weight. In case of radionuclide being a-emitter, typical doses are lower, for example less than 30 MBq in total (e.g., in a typical administration of a radiopharmaceutical including ²²⁵Ac, it can be 18.5 MBq/patient). As it is known to the skilled person, the further preferred dosing regimens depend on the used radionuclide. For the compounds of the present invention comprising yttrium-90 the total daily dose administered to a human subject or patient in single dose or in divided doses preferably is between 5 and 35 MBq/kg body weight, even more preferably between 7 and 25 MBq/kg body weight, most preferably between 10 and 15 MBq/kg body weight. For the compounds of the present invention comprising ¹⁷⁷Lu the total daily dose administered to a human subject or patient in single dose or in divided doses preferably is between 5 and 150 MBq/kg body weight, even more preferably between 10 and 80 MBq/kg body weight, most preferably between 10 and 60 MBq/kg body weight. Accordingly, a typical dose of ¹⁷⁷Lu administrated to a patient can be, e.g., 7.4 GBq per session. As it is to be understood herein, a daily dose is preferably understood as a dose administered in a single session, to be concluded in a single day, which according to the therapy schedule may be administered with intervals of several days, for example every two weeks, every four weeks, or every eight weeks. As understood herein, single dose compositions may contain such amounts or submultiples thereof to make up the daily dose. As understood herein, the skilled person will be able to determine the preferred dosage, depending on the radionuclide and on the desired application (for example treatment of a solid tumor). In particularly preferred case, where the radionuclide is terbium-161 , total daily dose to be administered to a human is between 1 and 100 MBq/kg body weight, preferably between 5 and 30 MBq/kg body weight, more preferably between 10 and 20 MBq/kg body weight.

In one embodiment, the present invention relates to the antibody-radionuclide conjugate of the present invention or the pharmaceutical composition of the present invention for use in the treatment of cancer. It is preferred that the cancer is CD30-positive.

As apparent to the skilled person, CD30 positive cancers include:

• Anaplastic large cell lymphoma, ALK positive and ALK negative

• ALK positive large B-cell lymphoma

• Classical Hodgkin Lymphoma

• EBV positive large B-cell lymphoma, NOS

• Lymphomatoid Papulosis

• Primary cutaneous CD30 positive T-cell Lymphoproliferative disorder

• Breast-implants associated ALCL As further apparent to the skilled person, commonly CD30-positive or partly CD30-positive cancers include:

• Angioimmunoblastic T-cell lymphoma

• Primary effusion lymphoma

• Primary mediastinal grey zone lymphoma

• Large cell transformation of mycosis fungoides

• Peripheral T-cell lymphoma, NOS

It is further apparent to the skilled person that CD30-positive non-lymphoma entities include embryonal carcinoma (testis) and advanced systemic mastocytosis.

Accordingly, it is preferred in the present invention that cancer is lymphoma, preferably T-cell lymphoma. It is further preferred that the T-cell lymphoma is selected from peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma, anaplastic large cell lymphoma, adult T-cell lymphoma, extranodal NK/T-cell lymphoma, and cutaneous T-cell lymphoma.

Antibody-radionuclide conjugates of the invention can be used either alone or in combination with other agents in a therapy. For instance, an antibody-radionuclide conjugate of the invention may be coadministered with at least one additional therapeutic agent.

Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case, administration of the antibody-radionuclide conjugate of the invention can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent and/or adjuvant. Antibody-radionuclide conjugates of the invention can also be used in combination with radiation therapy.

The antibody-radionuclide conjugate may be administered to a subject with an additional therapeutic agent, selected from alkylating agents, platinum agents, taxanes, vinca agents, anti-estrogen drugs, aromatase inhibitors, ovarian suppression agents, VEGFA/EGFR inhibitors, EGF/EGFR inhibitors, PARP inhibitors, cytostatic alkaloids, cytotoxic antibiotics, antimetabolites, endocrine/hormonal agents, immune checkpoint inhibitors and bisphosphonate therapy agent.

The antibody-radionuclide conjugate of the invention may beadministered to a subject with an additional therapeutic agent, wherein said additional therapeutic agent may be an agent sensitizing the cells to radiotherapy, for example selected from protein kinase inhibitor and DNA intercalating agent.

Preferably, protein kinase inhibitor is selected from Alisertib, MK1775, MK2206, Saracatinib, Temsirolimus, Crizotinib, Ceritinib, Alectinib, Brigatinib, Bosutinib, Dasatinib, Imatinib, Nilotinib, Ponatinib, Vemurafenib, Dabrafenib, Ibrutinib, Ibrutinib, Palbociclib, Sorafenib, Ribociclib, Crizotinib, Cabozantinib, Gefitinib, Erlotinib, Lapatinib, Vandetanib, Afatinib, Osimertinib, Ruxolitinib, Tofacitinib, Trametinib, Axitinib, Gefitinib, Imatinib, Lenvatinib, Nintedanib, Pazopanib, Regorafenib, Sorafenib, Sunitinib, Vandetanib, Bosutinib, Dasatinib, Ponatinib, Vandetanib, Axitinib, Lenvatinib, Nintedanib, Regorafenib, Pazopanib, Sorafenib, and Sunitinib, more preferably selected from Alisertib, MK1775, MK2206, Saracatinib, and Temsirolimus.

Preferably, the DNA intercalating agent is selected from Doxorubicin and Nemorubicin.

An agent sensitizing the cells to radiotherapy may also be AZD7648:

As it will be clear to the skilled person in view of the disclosure hereinabove, the present invention further relates to the antibody-radionuclide conjugate of the present invention or the pharmaceutical composition of the present invention for use in the manufacture of a medicament for treating cancer.

As it will further be clear to the skilled person in view of the disclosure hereinabove, the present invention further relates to a method of treatment of cancer, the method comprising administering to an individual in need thereof of the antibody-radionuclide conjugate of the present invention or the pharmaceutical composition of the present invention. It is to be understood that the antibody-radionuclide conjugate of the invention or the pharmaceutical composition of the invention, are to be administered in a therapeutically effective amount.

An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals ( e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non human primates such as macaques), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

The patient to be treated (or the subject to be treated) may be a subject that has developed resistance to at least one other treatment (such as treatment with Adcetris), or a subject with a relapsed cancer, in particular relapsed lymphoma, such as a subject with a minimal residual disease.

An "effective amount" of an agent, e.g., a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

In a further embodiment, the present invention further relates to an antibody-radionuclide conjugate of the present invention, or the kit of parts of the present invention, for use in diagnosis.

As referred to herein, diagnosis relates to any efforts for determining whether or not the subject suffers from or is affected by a particular disease. In particular, diagnosis encompasses the diagnosis for curative purposes, which includes the steps using particular reagents, referred to herein, to make necessary determination, and represents the deductive medical (or veterinary, as applicable) decision phase as a purely intellectual exercise. The diagnosis may also include the preceding steps which are constitutive for making that diagnosis, as mentioned here any steps involving the use of provided reagents. The diagnosis, unless provided to the contrary, may also include specific interactions with the human or animal body, such as administration of the reagent to said human or said animal.

Accordingly, in a further embodiment, the present invention relates to use of the antibody-radionuclide conjugate of the present invention, or the kit of parts of the present invention, in the manufacture of a reagent for diagnosis.

To this end, particularly useful are conjugates comprising a radionuclide selected from terbium-161 , fluorine-18, scandium-43, scandium-44, copper-61 , copper-64, gallium-68, zirconium-89, indium-11 1 , iodine-123, terbium-152, and terbium-155, which radionuclides are particularly useful in diagnostic application, as described herein.

The antibody-drug conjugates of the present invention are particularly useful in the diagnosis of cancer, in particular cancer associated with CD30. In other words, the cancer, as referred to herein, is preferably a CD30-positive cancer. Thus, according to the present invention, preferably the diagnosis is diagnosis of cancer, preferably diagnosis of CD30-positive cancer.

As mentioned previously, the cancer is preferably lymphoma. More preferably, the lymphoma is a T-cell lymphoma. T-cell lymphoma is preferably selected from peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma, anaplastic large cell lymphoma, adult T-cell lymphoma, extranodal NK/T-cell lymphoma, and cutaneous T-cell lymphoma.

Alternatively, as encompassed by the present invention, insofar diagnostic methods and reagents are concerned, the cancer is Anaplastic large cell lymphoma, ALK positive large B-cell lymphoma, Classical Hodgkin Lymphoma, EBV positive large B-cell lymphoma, Lymphomatoid Papulosis, Primary cutaneous CD30 positive T-cell Lymphoproliferative disorder, Breast-implants associated ALCL, Angioimmunoblastic T-cell lymphoma, Primary effusion lymphoma, Primary mediastinal grey zone lymphoma, Large cell transformation of mycosis fungoides, Peripheral T-cell lymphoma, embryonal carcinoma (testis) or advanced systemic mastocytosis.

Further examples and/or embodiments of the present invention are disclosed as the following numbered items.

1 . An antibody-radionuclide conjugate, comprising a) an antibody or antigen-binding fragment thereof specifically binding to CD30; and b) terbium-161.

2. A kit of parts comprising a) an antibody or antigen-binding fragment thereof specifically binding to CD30; and b) terbium-161 wherein said antibody or antigen-binding fragment thereof is capable of binding terbi um-161 .

3. The antibody-radionuclide conjugate of item 1 or the kit of parts of item 2, wherein the antibody or the antigen binding fragment thereof of a) is a monoclonal antibody, a chimeric antibody, a recombinant antibody, an antigen-binding fragment of a recombinant antibody, a single chain antibody, a humanized antibody, a bispecific antibody, a multi-specific antibody.

4. The antibody-radionuclide conjugate of item 3 or the kit of parts of item 3, wherein the antibody or the antigen binding fragment thereof of a) is a monoclonal antibody. 5. The antibody-radionuclide conjugate of item 3 or 4, or the kit of parts of item 3 or 4, wherein the antibody or the antigen binding fragment thereof of a) is an lgG1 antibody.

6. The antibody-radionuclide conjugate of any one of items 1 or 3 to 5, or the kit of parts of any one of items 2 to 5, wherein the antibody or antigen-binding fragment thereof of a) binds to the same epitope of CD30 as cAC10 antibody.

7. The antibody-radionuclide conjugate of any one of items 1 or 3 to 6, or the kit of parts of any one of items 2 to 6, wherein the antibody or antigen-binding fragment thereof of a) is cAC10 antibody

8. The antibody-radionuclide conjugate of any one of items 1 or 3 to 7, or the kit of parts of any one of items 2 to 7, wherein the antibody-radionuclide conjugate comprises a metal chelator, said metal chelator being charged with terbi um-161 or capable of being charged with terbi um-161 .

9. The antibody-radionuclide conjugate of item 8, or the kit of parts of item 8, wherein the metal chelator is selected from 1 ,4,7,10-tetraazacyclododecane-1 ,4,7,10-tetraacetic acid (DOTA), diethylenetriaminepentaacetic (DTPA), desferrioxamine (DFO) and triethylenetetramine (TETA), 1 ,4,8, 11 -tetraazabicyclo[6.6.2]hexadecane-4, 11 -diacetic acid (CB-TE2A); ethylenediaminetetraacetic acid (EDTA); ethylene glycolbis(2-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA); 1 ,4,8, 11 -tetraazacyclotetradecane-1 ,4,8,11 -tetraacetic acid (TETA); ethylenebis- (2-4 hydroxy-phenylglycine) (EHPG); 5-CI-EHPG; 5BrEHPG; 5-Me-EHPG; 5t-Bu-EHPG; 5-sec- Bu-EHPG; benzodiethylenetriamine pentaacetic acid (benzo-DTPA); dibenzo-DTPA; phenyl- DTPA, diphenyl-DTPA; benzyl-DTPA; dibenzyl-DTPA; bis-2(hydroxybenzyl)-ethylene- diaminediacetic acid (HBED) and derivatives thereof; Ac-DOTA; benzo-DOTA; dibenzo-DOTA; 1 ,4,7-triazacyclononane N,N',N"-triacetic acid(NOTA); benzo-NOTA; 1 ,4,7-triazacyclononane N, N'-diacetic acid N”-glutaminic acid(NODAGA), benzo-TETA, benzo-DOTMA, where DOTMA is 1 ,4,7, 10-tetraazacyclotetradecane-1 ,4,7, 1 O-tetra(methyl tetraacetic acid), benzo-TETMA, where TETMA is 1 ,4, 8, 11 -tetraazacyclotetradecane-1 ,4, 8, 11 -(methyl tetraacetic acid); derivatives of 1 ,3-propylenediaminetetraacetic acid (PDTA); triethylenetetraaminehexaacetic acid (TTHA); derivatives of 1 ,5,10-N,N',N"-tris(2,3- dihydroxybenzoyl)-tricatecholate (LICAM); and 1 ,3,5- N,N',N"-tris(2,3- dihydroxybenzoyl)aminomethylbenzene (MECAM), 10. The antibody-radionuclide conjugate of item 8 or 9, or the kit of parts of item 8 or 9, wherein the metal chelator is 1 ,4,7,10-tetraazacyclododecane-1 ,4,7,10-tetraacetic acid (DOTA).

11 . The antibody-radionuclide conjugate of any one of items 8 to 10, or the kit of parts of any one of items 8 to 10, wherein the metal chelator is attached to the antibody or antigen-binding fragment thereof through a linker comprising -NHC(=S)-NH- moiety.

12. The antibody-radionuclide conjugate of any one of items 8 to 11 , or the kit of parts of any one of items 8 to 11 , comprising the following moiety: wherein said moiety is attached to the antibody or antigen-binding fragment thereof via amino group of lysine residue.

13. The antibody-radionuclide conjugate of any one of claims 8 to 12, or the kit of parts of any one of items 8 to 12, wherein the chelator/antibody stoichiometric ratio is between 2.5 and 3.5.

14. The antibody-radionuclide conjugate of item 13, or the kit of parts of item 13, wherein the chelator/antibody stoichiometric ratio is about 3.0.

15. A pharmaceutical composition comprising the antibody-radionuclide conjugate of any one of items 1 or 3 to 14, and at least one pharmaceutically acceptable carrier.

16. The antibody-radionuclide conjugate of any one of items 1 or 3 to 14, the kit of parts of any one of items 2 to 14, or the pharmaceutical composition of item 15, for use in therapy. 17. The antibody-radionuclide conjugate of any one of items 1 or 3 to 14, the kit of parts of any one of items 2 to 14, or the pharmaceutical composition of item 15, for use in treatment or prevention of cancer.

18. Use of the antibody-radionuclide conjugate of any one of claims 1 or 3 to 14 or the kit of parts of any one of claims 2 to 14 or the pharmaceutical composition of claim 15, in the manufacture of a medicament for treating or preventing cancer.

19. A method of treating cancer, comprising the step of administering a therapeutically effective amount of the antibody-radionuclide conjugate of any one of items 1 or 3 to 14, parts of the kit of parts of any one of items 2 to 14, or the pharmaceutical composition of item 15 to a subject in need thereof.

20. The antibody-radionuclide conjugate for use of item 17, the kit of parts for use of item 17, the pharmaceutical composition for use of item 17, the use of item 18 or the method of item 19, wherein the cancer is lymphoma.

21 . The antibody-radionuclide conjugate for use of item 17 or 20, the kit of parts for use of item 17 or 20, the pharmaceutical composition for use of item 17 or 20, the use of item 18 or 20, or the method of item 19 or 20, wherein lymphoma is a T-cell lymphoma.

22. The antibody-radionuclide conjugate for use of item 21 , the kit of parts for use of item 21 , the pharmaceutical composition for use of item 21 , the use of item 21 , or the method of item 21 , wherein T-cell lymphoma is selected from peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma, anaplastic large cell lymphoma, adult T-cell lymphoma, extranodal NK/T-cell lymphoma, and cutaneous T-cell lymphoma.

23. The antibody-radionuclide conjugate for use of item 17, the kit of parts for use of item 17, the pharmaceutical composition for use of item 17, the use of item 18 or the method of item 19, wherein the cancer is Anaplastic large cell lymphoma, ALK positive large B-cell lymphoma, Classical Hodgkin Lymphoma, EBV positive large B-cell lymphoma, Lymphomatoid Papulosis, Primary cutaneous CD30 positive T-cell Lymphoproliferative disorder, Breast-implants associated ALCL, Angioimmunoblastic T-cell lymphoma, Primary effusion lymphoma, Primary mediastinal grey zone lymphoma, Large cell transformation of mycosis fungoides, Peripheral T- cell lymphoma, embryonal carcinoma (testis) or advanced systemic mastocytosis.

24. The antibody-radionuclide conjugate for use of any one items 17 or 20 to 23, the kit of parts for use of any one items 17 or 20 to 23, the pharmaceutical composition for use of any one items 17 or 20 to 23, the use of any one of items 18 or 20 to 23, or the method of any one of items 19 to

23, wherein said antibody-radioligand conjugate or the parts of the kit of parts or the pharmaceutical composition is to be administered to a subject with an additional therapeutic agent, selected from alkylating agents, platinum agents, taxanes, vinca agents, anti-estrogen drugs, aromatase inhibitors, ovarian suppression agents, VEGF/VEGFR inhibitors, EGF/EGFR inhibitors, PARP inhibitors, cytostatic alkaloids, cytotoxic antibiotics, antimetabolites, endocrine/hormonal agents, immune checkpoint inhibitors and bisphosphonate therapy agent.

25. The antibody-radionuclide conjugate for use of any one items 17 or 20 to 24, the kit of parts for use of any one items 17 or 20 to 24, the pharmaceutical composition for use of any one items 17 or 20 to 24, the use of any one of items 18 or 20 to 24, or the method of any one of items 19 to

24, wherein total daily dose to be administered to a human is between 1 and 100 MBq/kg body weight, preferably between 5 and 30 MBq/kg body weight, more preferably between 10 and 20 MBq/kg body weight.

26. The antibody-radionuclide conjugate of any one of items 1 or 3 to 14, or the kit of parts of any one of items 2 to 14, for use in diagnosis.

27. Use of the antibody-radionuclide conjugate of any one of items 1 or 3 to 14, or the kit of parts of any one of items 3 to 14, in the manufacture of a reagent for diagnosis.

28. The antibody drug conjugate for use, or the kit of parts for use of item 26 or the use of item 27, wherein the diagnosis is diagnosis of cancer.

29. The antibody drug conjugate for use or the kit of parts for use of item 28 or the use of item 28, wherein the cancer is lymphoma.

30. The antibody-radionuclide conjugate for use or the kit of parts for use of item 28 or 29, or the use of claim 28 or 29, wherein the lymphoma is a T-cell lymphoma. 31 . The antibody-radionuclide conjugate for use or the kit of parts for use of item 30, or the use of item 30, wherein the T-cell lymphoma is selected from peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma, anaplastic large cell lymphoma, adult T-cell lymphoma, extranodal NK/T-cell lymphoma, and cutaneous T-cell lymphoma.

32. The antibody drug conjugate for use or the kit of parts for use of item 28 or the use of item 28, wherein the cancer is Anaplastic large cell lymphoma, ALK positive large B-cell lymphoma, Classical Hodgkin Lymphoma, EBV positive large B-cell lymphoma, Lymphomatoid Papulosis, Primary cutaneous CD30 positive T-cell Lymphoproliferative disorder, Breast-implants associated ALCL, Angioimmunoblastic T-cell lymphoma, Primary effusion lymphoma, Primary mediastinal grey zone lymphoma, Large cell transformation of mycosis fungoides, Peripheral T- cell lymphoma, embryonal carcinoma (testis) or advanced systemic mastocytosis.

The invention is illustrated in the following examples, which are not meant to be construed as limiting.

Examples

Materials and methods

Antibody conjugation, radiolabeling, and quality control cAC10 antibody (lgG1 kappa) was produced by Proteogenix (Schiltigheim, France). Conjugation of the antibody to DOTA was performed by incubation with p-SCN-Bn-DOTA (Macrocyclics, USA) in 0.1 M boric acid, pH 9.0, at an antibody-to-chelator molar ratio of 1 :5 for 15 h at RT. Ligand excess was removed by ultrafiltration (Vivaspin 6 ultrafiltration tube, Sartorius, Germany) and the purified immunoconjugates were buffer-exchanged into PBS, pH 7.4. The number of chelators coupled per antibody was determined by mass spectrometry.

The radiometal chelator DOTA was coupled to the cAC10 antibody at a ratio of 3.1 chelators per antibody. DOTA-functionalized [¹⁷⁷Lu]Lu-cAC10 and [¹⁶¹Tb]Tb-cAC10 were obtained at high specific activities (> 0.3 MBq/ g) and radiochemical purities (> 90%) (Fig. 5). Both radioimmunoconjugates showed high plasma stability, with > 90% of the radioactivity coupled to the immunoconjugates after 7 days. The binding affinity was in the lower nanomolar range (Fig. 6), consistent with previous reports. No-carrier-added (n.c.a.) lutetium-177 was purchased from ITM Medical Isotopes (Munich, Germany). N.c.a terbium-161 was produced at Paul Scherrer Institute, Switzerland as previously reported (doi.org/10.1186/s41181-019-0063-6.). For radiolabeling, 40 MBq or lutetium-177 or terbium-161 solutions per microgram of immunoconjugates were reacted in 0.25 M ammonium acetate buffer, pH 5.5. After 15 h at 37°C, EDTA was added to a final concentration of 100 pM and further incubated for 5 min. Purification of the radiolabeled antibody was performed by size exclusion chromatography (PD10 columns, Cytiva, Marlborough, USA) in PBS, pH 7.4. Specific activity and radiochemical purity were analyzed by HPLC size exclusion chromatography on a Superose® 6 10/300 GL column (Cytiva) (Supplementary figure 1). [¹⁷⁷Lu]Lu-cAC10 and [¹⁶¹Tb]Tb-cAC10 radioimmunoconjugates immunoreactive fraction was calculated using the Lindmo method with slight modifications (10.1016/0022- 1759(84)90435-6).

For some experiments, an unspecific IgG (trastuzumab, Cantonal Pharmacy Zurich) was used as a control. DOTA-conjugation and radiolabeling were performed following the same steps described for cAC10.

Cell lines and cell culture

Karpas 299 (Anaplastic Large T-Cell Lymphoma, ALTCL), Mac2A, and Myla (Cutaneous T-Cell lymphoma, CTCL) cell lines were kindly provided by Dr Christoph Schlapbach. All cell lines were CD30+ as analyzed by flow cytometry. Jurkat cells were used as a negative control. Cell lines were cultured in Roswell Park Memorial Institute medium (RPM1 1640) (BioConcept, Allschwil, Switzerland) supplemented with 10% Fetal Bovine Serum (FBS), 100 U/mL penicillin/ streptomycin, and 2 mM glutamine (BioConcept) and incubated at 37 °C and 5% CO2 in a humidified atmosphere.

Binding, and cell uptake and internalization assays

To assay affinity, 1 million Karpas 299 cells were incubated with increasing concentrations of [¹⁷⁷Lu]Lu- cAC10 or [¹⁶¹Tb]Tb-cAC10 (0.5-50 nM) without or with 10 pg of cAC10 (blocking condition). After incubation for 4 h at4°C, cells were centrifuged and washed with PBS. Supernatants and cell pellets were y-counted (Packard Model 5003 Cobra II Auto Gamma Counter, Packard, USA).

To study the cell uptake and internalization of the radioimmunoconjugates, 1 million Karpas 299, Mac2A, or Myla cells were incubated with 8 ng of either [¹⁷⁷L u] Lu-cAC 10 or [¹⁶¹ Tb]Tb-cAC 10 without or with 10 pg of cAC10 (blocking condition). Cells were incubated for different times (1 , 2, 5, 15, and 24 h) at 37°C in a humidified incubator. Cells were pulled and washed with PBS and an acidic buffer (Glycine buffer, pH 2.2). Supernatants, washes, and cell pellets were y-counted (Packard Model 5003 Cobra II Auto Gamma Counter) to determine the total uptake and internalized fractions. All experiments were performed at least in triplicate. Statistical analysis was performed by Student t-test.

Cell viability and survival assays

Cell viability after exposure to [¹⁷⁷Lu]Lu-cAC10 or [¹⁶¹Tb]Tb-cAC10 was evaluated using the MTT assay. Cells were seeded in 96-well plates (10,000 cells/well) and treated with different concentrations of the radioimmunoconjugates (0.005-20 MBq/mL) for 15 h. After incubation, cells were washed and resuspended in fresh medium. After 4 days, MTT reagent was added to the cells and further incubated at 37 °C for 4 h. After solubilization, the absorbance at 560 nm, which correlates to the number of viable cells, was measured using a multi-well spectrophotometer (Victor™ X3, Perkin Elmer, Waltham, MA, USA). Cell viability was expressed as percentage compared to the viability of control cells (treated with cAC10).

The survival of the cells after treatment was assessed via clonogenic assay in MethoCult methylcellulose- based medium (MethoCult™ H4434, Stemcell Technologies, Vancouver, BC, Canada). After 15 h treatment with [¹⁷⁷Lu]Lu-cAC10 or [¹⁶¹Tb]Tb-cAC10 (0.01-5 MBq/mL), cells were seeded in 24-well plates in MethoCult medium and incubated for 1 week, until colonies were visible. Viable colonies were stained with MTT solution and counted under the microscope. Cell survival was normalized to the number of control cells (treated with cAC10) and expressed as a percentage. All experiments were performed at least in triplicate. Statistical analysis was performed by Student t-test and one-way ANOVA, Tukey’s multiple comparison test.

DNA damage

The DNA damage induced by exposure to the radioimmunoconjugates was studied by immunocytochemistry (ICC) of phosphorylated yH2AX, triggered by the formation of DNA double-strand breaks. Karpas 299 cells were exposed to 0.5 MBq/mL of either [¹⁷⁷Lu]Lu-cAC10 or [¹⁶¹Tb]Tb-cAC10 for 15 h. After incubation, cells were washed with PBS and fixed with 4% PFA for 15 minutes at RT. 30,000 cells per condition were placed in microscope slides and dried on a hot plate (40°C). Cells were permeabilized with 0.5% Triton X in PBS for 10 minutes, blocked with 10% goat serum for 1 h at RT, and incubated with the anti-yH2AX primary antibody (1 :400, Cell Signaling Techonology, Danvers, MA, USA) ON at4°C. After incubation, the secondary antibody was added (1 : 1000, Alexa 488 anti-rabbit, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) for 1 h. After, DAPI (2 pg/mL, Invitrogen) was added for 10 min and slides were mounted (Prolong mounting medium, Invitrogen). Immunostainings were visualized by confocal microscopy (Leica Stellaris, Leica, Wetzlar, Germany). 15 images from different fields were taken from each condition and quantified using Fiji, Imaged software. The percentage of the yH2AX- positive area and staining intensity were normalized by the DAPI staining (cell nuclei). Statistical analysis was performed by one-way ANOVA, Tukey’s multiple comparison test.

In vivo experiments

Four-week-old female athymic nude mice (Crl:NU(NCr)-Foxn1 ^nu) were purchased from Charles River (Sulzfeld, Germany). All animal experiments were carried out following the Swiss Regulations for Animal Welfare guidelines and approved by the Cantonal Committee of Animal Experimentation (licenses N° 75721 and 75666). The subcutaneous tumor model was generated by subcutaneous injection of 5 million Karpas 299 cells in 1 :1 PBS/Matrigel in the right flank of the animals. This mouse model was used for biodistribution, SPECT/CT imaging, and therapy studies.

Biodistribution and SPECT/CT imaging

To assess the radioimmunoconjugates’ biodistribution, animals bearing tumors around 250-350 mm³ were randomized into groups (n= 4 per group). Mice were intravenously administered [¹⁷⁷Lu]Lu-cAC10 or [¹⁶¹Tb]Tb-cAC10 (1 MBq, 30 ug). The blocking condition was achieved by co-injection of 500 ug of cAC10. Mice were euthanized at different time points (24, 48, 96, and 144 h), and relevant tissues and organs were collected, weighed, and y-counted. The decay-corrected data was expressed as percentage of the injected activity per gram of tissue (% lA/g). Statistical analysis was performed by Student t-test.

SPECT/CT imaging was performed after intravenous injection of lutetium-177 and terbium-161- radiolabeled cAC10 or control IgG (10MBq, 30 ug). Mice were imaged 72 h after injection under anesthesia in a SPECT/CT scanner (NanoSPECT/CT, Mediso Medical Imaging Systems, Budapest, Hungary). SPECT data were acquired by Nucline software (version 1 .02, Bioscan, Washington DC, USA). SPECT data were reconstructed iteratively with HiSPECT software (version 1.4.3049, Scivis, Gottingen, Germany). The fused SPECT and CT datasets were analyzed with the InVivoScope postprocessing software (version 1.44, Bioscan, Washington DC, USA).

Therapy study

For the radioimmunotherapy study, mice bearing tumors around 50 mm³ were randomized into 6 groups (n=10). Treatment mice were intravenously administered a single injection of [¹⁷⁷Lu]Lu-cAC10 or [¹⁶¹Tb]Tb-cAC10 (2 MBq, 30 ug). For controls, PBS, cAC10, lutetium or terbium-radiolabeled control IgG were injected (2 MBq, 30 ug). Tumor growth and animal body weight were evaluated every 2-3 days throughout the study. Mice were euthanized when reached any humane endpoint criteria (more than 20% weight loss, tumors larger than 1 ,500 mm³, or tumor ulceration). T umor volume was calculated using the equation V= (L x W²)/2, where W is the tumor width and L is the tumor length. The mice survival was represented as Kaplan-Meier curves and analyzed using a log-rank test (Mantel-Cox). The doubling time of each growing tumor until day 11 was calculated based on a fitted exponential tumor growth curve. Statistical analysis was performed by one-way ANOVA, Tukey’s multiple comparison test.

Plasma stability

The stability of ¹⁷⁷Lu and ¹⁶¹Tb-labeled cAC10 radioimmunoconjugates was assessed in vitro by diluting in human plasma (Kantonspital Baden, Baden, Switzerland) at an activity concentration of around 50 MBq/mL and incubating it at 37°C. Samples were analyzed at 0, 24, 48, 72, and 168 h by HPLC size exclusion chromatography using a Superose® 6 10/300 GL column (Cytiva), with PBS + 0.01 % sodium azide and 200 mM EDTA as eluent.

Flow cytometry

CD30 membrane expression in Karpas 299, Mac2A, and Myla cell lines was determined by flow cytometry (CytoFlex, Beckman Coulter) using APC anti-human CD30 monoclonal antibody (clone BY88, BioLegend). Data was analyzed and plotted using FloJo software v10.9.0 (BD Life Sciences).

Subcellular localization of the radioimmunoconjugates

Karpas 299, Mac2A, and Myla cells (10 x 10⁶) were incubated with 8 ng of either [¹⁷⁷Lu]Lu-cAC10 or [¹⁶¹Tb]Tb-cAC10 without or with 10 pg of cAC10 (blocking condition). After 15 h of incubation, cells were pulled and washed with PBS and an acidic buffer (Glycine buffer, pH 2.2). Subsequently, the cell nuclei and cytoplasm/membrane fractions were separated using the Nuclei EZ Prep Nuclei Isolation Kit (Sigma Aldrich, USA) following the manufacturer’s instructions. Membrane, cytoplasm, and nuclei fractions were collected and the activity was measured in a y-counter (Packard Model 5003 Cobra II Auto Gamma Counter). Membrane, cytoplasmics, and nuclear localization were expressed as percentage of total cellular uptake, defined as the total measured activity in all fractions. The collected fractions were stained with 0.4% trypan blue solution and analyzed under a microscope to confirm proper nuclei separation from other cell fragments.

Dosimetry

The dosimetry for organs and tumor tissue was calculated by fitting an exponential curve to non-decay- corrected mean activity concentration versus time data using the Curve Fitting Toolbox in MATLAB (MATLAB R2024a, MathWorks, Natick, MA). Each fitted curve was integrated to infinity to obtain the time- integrated activity concentration (TIAC). The absorbed doses were then determined by multiplying the TIAC with the corresponding absorbed energy for all tissues.

Histopathological analysis

Tumors and relevant organs were collected after euthanasia from the therapy study mice to perform histopathological analysis. Following fixation in 4% neutral buffered formaldehyde solution and embedding in paraffin, 2-pm sections were stained with hematoxylin and eosin using an autostainer (Leica BOND RX, Leica Biosystems, Nussloch, Germany). Slides were observed under a microscope and scanned with an automatic brightfield scanner (Panoramic 250 FLASH III, 3DHistech, Budapest, Hungary).

Hematotoxicity study

Non-tumor-bearing athymic nude mice were intravenously administered with a single injection of [¹⁷⁷Lu]Lu-cAC10 or [¹⁶¹Tb]Tb-cAC10 (2 MBq, 30 pg), matching the treatment for the radioimmunotherapy study (n=4). Control mice were left untreated or injected with PBS. Blood was drawn from the tail once per week and analyzed using a hematology analyzer (VetScan HM5, Abaxis, United States).

Proteomics and phosphoproteomics analysis

For the in vivo phosphoproteomics study, mice bearing tumors around 100 mm³ were randomized into 4 groups (n=4). Treatment mice were intravenously administered a single injection of [¹⁷⁷Lu]Lu-cAC10 or [¹⁶¹Tb]Tb-cAC10 (2 MBq, 30 ug). Control mice were injected with PBS or cAC10 (30 ug). 72 h after treatment, when the highest compound tumor accumulation was detected, mice were euthanized and tumors collected and deep-frozen in liquid nitrogen for later analysis.

Sample digestion and clean-up

Tumor samples were lysed in 100 pL 4% SDS/Tris-HCI, pH 8.5, and protein extraction was performed using a tissue homogenizer (TissueLyser II, QIAGEN) with 2x2min cycles at 30 Hz. Samples were boiled for 10 min at 95 °C, followed by two 1 -minute rounds of High Intensity Focused Ultrasound (HIFU) at 100% amplitude. They were then treated with 5 units of benzonase for 15 min at 30 °C and centrifuged at 20000 x g for 10 min. Protein concentration was estimated using the Lunatic UV/Vis spectrophotometer (Unchained Labs), and 60 pg of total protein per sample was used for digestion. Proteins were reduced with 2 mM TCEP and alkylated with 15 mM chloroacetamide at 30 °C for 30 min in the dark. Single-pot solid-phase enhanced sample preparation (SP3) was used for protein processing, and purification, digestion, and peptide clean-up were done using the KingFisher Flex System and Carboxylate-Modified Magnetic Particles. Beads were conditioned, and samples diluted with ethanol to 60% were processed on the robot. Steps included protein binding, washing, overnight digestion at 37 °C with trypsin, and peptide elution with MilliQ water. The digest solution and water elution were combined and dried completely.

TMT labeling and peptide fractionation

250 pg of TMT 18-plex reagent (Thermo Fisher Scientific) was dissolved in 15 pL of anhydrous acetonitrile (Sigma-Aldrich) and added to peptides in 45 pL of 50 mM TEAB, pH 8.5. The solution was gently mixed and incubated for 60 min at room temperature. The reaction was quenched with 3.5 pL of 5% hydroxylamine (Thermo Fisher Scientific). A pooled TMT sample was created by mixing equal amounts from each TMT channel. Labeled peptides were pre-fractionated using high pH reverse phase chromatography on an XBridge Peptide BEH C18 column (Waters) with a 72-min gradient from 5-40% acetonitrile/9 mM NH4HCO2. Fractions were collected every minute and concatenated into 36 final fractions, then dried.

Phosphopeptide enrichment

Phosphopeptide enrichment was performed using a KingFisher Flex System (Thermo Fisher Scientific) and Ti-IMAC HP MagBeads (ReSyn Biosciences). Beads were conditioned with 3 washes of 200 pl binding buffer (80% acetonitrile, 5% TFA, 0.1 M glycolic acid). Each fraction was dissolved in 200 pl binding buffer, with 2 pg per sample reserved for whole proteome analysis. Phosphopeptide enrichment was carried out on the KingFisher, following these steps: washing beads in binding buffer (5 min), binding phosphopeptides to beads (30 min), washing beads in wash buffers 1 and 2 (80% acetonitrile, 1 % TFA, and 10% acetonitrile, 0.2% TFA, 3 min each), and eluting peptides (80 pl 1 % NH4OH in water, 10 min). Eluates were mixed with 10 pL of 10% formic acid. Phospho-enriched samples and whole proteome analysis fractions were loaded onto Evotips according to the manufacturer’s instructions.

LC-MS/MS analysis

Mass spectrometry analysis was conducted on an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific) with a Flex source, coupled to an Evosep One (Evosep). Solvent composition was 0.1 % formic acid for channel A and 0.1 % formic acid, 99.9% acetonitrile for channel B. Peptides were separated on a PepSep C18 Column (Bruker) using the 30SPD method at 50°C. The mass spectrometer operated in data-dependent mode (DDA) with a 3 s cycle time. Full-scan MS spectra were acquired at 120,000 resolution for proteome samples (350-1 ,500 m/z) and enriched samples (350-1800 m/z), with a target value of 3,000,000 or maximum injection time of 45 ms. Precursors above 2,000 intensity were selected for MS/MS, isolated with a 0.7 m/z window, and fragmented by HCD at 32% collision energy. HCD spectra were acquired at 45,000 resolution, with an Auto injection time for proteome samples and 200 ms for enriched samples. AGC was set to 100%, with charge state screening enabled. Precursors were excluded for 8 s, with a 10 ppm window, and internal lock mass calibration was used. Data were managed using the local LIMS system.

Data analysis

The acquired shotgun MS data were processed for identification and quantification using Fragpipe 21.1 (Philosopher 5.1.0). Spectra were searched against a concatenated Uniprot human reference proteome using MSFragger 4.0 and Percolator. TMT modifications on peptide N-termini and lysine side chains, as well as carbamidomethylation of cysteine, were set as fixed modifications, while methionine oxidation was set as variable. Enzyme specificity was set to trypsin/P with a minimum peptide length of 7 amino acids and a maximum of two missed cleavages. Reporter ion intensities were extracted with 20 ppm integration tolerance. Peptide and protein quantification used a 50% co-isolation filter.

For statistical evaluation of the total proteome dataset, the FragPipe output "psm.tsv" was used as input to the prolfqua R-package. A minimum abundance of 1 was required for each TMT-channel. TMT- abundances for all PSMs of the same protein were aggregated using the median-polish method, Iog2- transformed, and normalized with a robust z-score transformation. A linear model was fitted to compute significant differences between conditions, evaluated with a moderated Wald-test and adjusted for multiple testing using the BH method.

For the phospho-enriched dataset, the TMT-report "abundance_multi-site_None.tsv" was used. TMT- integrator reports were prefiltered with a localization probability of at least 0.75. Phosphosite-centric TMT abundances were Iog2-transformed, normalized, fitted, and contrasted as described for the total proteome dataset. Results of the total proteome analysis were joined with the phosphosite-centric results, filtering out contaminant proteins. The estimated log2FC for phosphosites was adjusted for protein-level changes using the procedure suggested by Kohler et al (Kohler D, Tsai T-H, Verschueren E, et al. MSstatsPTM: Statistical Relative Quantification of Posttranslational Modifications in Bottom-Up Mass Spectrometry- Based Proteomics. Mol Cell Proteomics. 2023;22: 100477). Adjusted log2FC and new p-values were again adjusted for multiple testing using the BH method. Proteins and phosphopeptides with an FDR <0.25 and 1.5-fold change were defined as significantly altered proteins and phosphoproteins. Bioinformatics and statistical analysis

Phosphoproteins identified through integrated proteomics and phosphoproteomics with significant changes in abundance were submitted to enrichment analysis of Gene Ontology (GO) for biological processes and pathway analysis (Reactome) using DAVID (EASE threshold 0.05) (htps://david.ncifcrf.gov/). Venn diagrams were drawn by online software BioVenn (htp://www.biovenn.nl/).

Statistical analysis was performed using GraphPad Prism 8 (version 8.3.1). Data are represented as mean ± SD. A p-value < 0.05 was considered statistically significant. Comparisons between groups were analyzed by Student’s t-test, one-way ANOVA with T ukey’s multiple comparison test, or log-rank (Mantel- Cox) test. Correlation between multiple variables was analyzed by multiple Pearson’s correlation.

Results

[¹⁶¹Tb]Tb-cAC10 exhibits more potent cytotoxicity than [¹⁷⁷Lu]Lu-cAC10

The cytotoxic effect of [¹⁶¹Tb]Tb-cAC10 compared to [¹⁷⁷Lu]Lu-cAC10, was evaluated in three CD30+T- cell lymphoma cell lines (Karpas 299, Mac2A, and Myla). In all tested cell lines, [¹⁶¹Tb]Tb-cAC10 was more potent in reducing cell viability compared to the lutetium-177-radiolabeled counterpart, as reflected in the calculated ICso values (1.29 ± 0.63 and 0.14 ± 0.09 MBq/mL for Karpas 299, 1.29 ± 0.69 and 0.03 ± 0.02 MBq/mL for Mac2A, and 2.36 ± 0.88 and 1.23 ± 0.77 MBq/mL for Myla, respectively) (Fig. 1A-C). The largest difference was observed in the Mac2A cell line, where [¹⁶¹Tb]Tb-cAC10 was 43-fold more potent than [¹⁷⁷Lu]Lu-cAC10. For Karpas 299 and Myla the ICso for terbium-161 were 9.2- and 1.9-fold lower compared to lutetium-177, respectively. Importantly, these differences in cytotoxicity were not due to differences in cell uptake or subcellular localization between both radiolanthanides (Supplemental Fig. 4 and 5). The cellular uptake and cytotoxicity of both radioimmunoconjugates correlated with the different CD30 membrane expression in the three cell lines (Fig. 12). In addition, the potent cytotoxicity was CD30- dependent, as demonstrated by two independent control experiments (Supplemental Fig. 8). [¹⁶¹Tb]Tb- cAC10 and [¹⁷⁷Lu]Lu-cAC10 were tested in the CD30-negative Jurkat cell line (Fig. 7A), whereas a radiolabeled control IgG was tested in Karpas 299 cells (Supplemental Fig. 7B). The calculated ICso values were 249- and 760-fold higher for terbium-161 and 19- and 32-fold higher for lutetium-177, respectively when compared to the most sensitive cell line, Mac2A.

Colony-forming assays demonstrated that the terbium-161-labeled antibody was also more effective in reducing cell survival compared to the lutetium-177 radioimmunoconjugate in the Karpas 299 and Mac2A cell lines (Fig. 1 E-F). The most pronounced difference between both radioimmunoconjugates was, again, observed in the Mac2A cell line, where <10% of the cells treated with 0.5 MBq/mL of [¹⁶¹Tb]Tb-cAC10 survived, whereas a ten-fold higher activity concentration of [¹⁷⁷Lu]Lu-cAC10 was needed to obtain a similar effect.

[¹⁶¹Tb]Tb-cAC10 induces more DNA damage compared to [¹⁷⁷Lu]Lu-cAC10 yH2AX ICC showed a 1.9-fold increase in the percentage of DNA DSB in Karpas 299 cells treated with the terbium-labeled compound compared to its lutetium-labeled counterpart (Fig 2A and B). These findings confirm terbium-161 ’s higher DSB induction.

[¹⁶¹Tb]Tb-cAC10 and [¹⁷⁷Lu]Lu-cAC10 show high tumor uptake and low accumulation in nontargeted organs and tissues

[¹⁷⁷Lu]Lu-cAC10 and [¹⁶¹Tb]Tb-cAC10 biodistribution in Karpas 299-derived xenografts was assessed in a time-dependent study. Both compounds showed a favorable and similar biodistribution, with high tumor uptake (up to 30% lA/g after 48 h), this tissue exhibiting the highest compound accumulation at all time points (Fig. 3 and Supplemental Table 1).

Supplemental Table 1. Biodistribution data in Karpas 299 tumor-bearing mice after i.v. injection of 1 MBq, 30 ug of [¹⁷⁷Lu]Lu-cAC10 or [¹⁶¹Tb]Tb-cAC10 represented as percentage of injected activity per mass of tissue (% lA/g). Blocking condition was achieved by co-injection of 500 ug of cold cAC10. n = 5 per group. Data represented as mean ± SD. Liver and spleen uptake was around 20 and 12% lA/g after 48 h, decreasing after 144 h. Tumor accumulation was CD30-dependent, as demonstrated by the significant reduction in tumor uptake in the blocking condition (Fig. 3). Dosimetry calculations showed an averaged 1.6-fold higher absorbed dose to all organs, including the tumor, for [¹⁶¹Tb]Tb-cAC10 compared to the lutetium-radiolabeled counterpart (Supplemental Table 2).

Supplemental Table 2. Absorbed organ doses calculated based on non-decay-corrected biodistribution data obtained for [¹⁷⁷Lu]Lu-cAC10 and [¹⁶¹Tb]Tb-cAC10.

[¹⁶¹Tb]Tb-cAC10 treatment prolongs survival in the absence of severe side effects

The therapeutic efficacy of [¹⁷⁷Lu]Lu-cAC10 and [¹⁶¹Tb]Tb-cAC10 radioimmunoconjugates was evaluated in vivo following the administration of 2 MBq of either radiolabeled compound. The injected activity was selected based on prior study results (Fig. 15) to enable the direct comparison of both radionuclides and evaluate the potential contribution of terbium-161 's additional co-emission of conversion and Auger electrons. Control mice (PBS, cAC10, [¹⁷⁷Lu]Lu-lgG, and [¹⁶¹Tb]Tb-lgG) presented a rapid tumor growth leading to euthanasia between Days 11-24 in all cases (Fig. 4). Eleven days after the start of the treatment, mice in the control groups presented significantly higher relative tumor volumes and lower tumor doubling times as compared to both [¹⁷⁷Lu]Lu-cAC10 and [¹⁶¹Tb]Tb-cAC10-treated groups (Fig. 4H and I). No significant differences were observed in the median survivals between control groups (Fig. 4A). Treatment with a single dose of 2 MBq [¹⁷⁷Lu]Lu-cAC10 led to a significant tumor growth arrest (Fig. 4H and I) and the consequent prolongation of survival (Fig. 4A). However, eventually, all tumors managed to escape the treatment and no mice survived by the end of the experiment. In contrast, treatment with the same activity of [¹⁶¹Tb]Tb-cAC10 significantly prolonged survival in the mouse model as compared to the ¹⁷⁷Lu-treated group (41 days vs. 21 days median survival) (Fig. 4A). Importantly, [¹⁶¹Tb]Tb-cAC10 treatment led to complete tumor elimination in 4 mice that remained tumor-free until the end of the experiment.

In terms of toxicity, conventional histomorphological analysis of H&E-stained tissue sections of selected organs revealed no obvious signs of early side effects in any of the treated groups. Animal body weight increased similarly across all groups, with no differences between control and treated mice (Fig. 8). To further evaluate the potential acute toxicity effects derived from the radioimmunotherapy, a hematotoxicity experiment was conducted in non-tumor-bearing mice treated with conditions matching the therapeutic study. No differences in any cell blood populations or analyzed parameters were observed after treatment with either of the two radioimmunoconjugates (Fig. 10, Fig. 14). Moreover, these values were within the normal range for a healthy athymic nude mouse (Fig. 14).

Phosphoproteomic analysis reveals increased therapeutic response to [¹⁶¹Tb]Tb-cAC10 in comparison to [¹⁷⁷Lu]Lu-cAC10

To compare terbium-161 and lutetium-177-induced signaling networks, tumors from mice treated with 2 MBq [¹⁷⁷Lu]Lu-cAC10 or [¹⁶¹Tb]Tb-cAC10 were submitted to quantitative phosphoproteomics analysis. Phosphoproteomics and proteomics quantified the abundance of 12548 phosphopeptides and 7763 proteins. Upon integration of protein expression and phosphorylation profiles, the abundance of 21 and 63 unique phosphopeptides, representing 14 and 38 proteins, was significantly altered in response to [¹⁷⁷Lu]Lu-cAC10 and [¹⁶¹Tb]Tb-cAC10 treatment, respectively, as compared to the cAC10 control (Fig. 9A). No significant changes were identified at the proteome level. Among the significantly altered phosphopeptides, 9 phosphoproteins were common for both radiolanthanides (Fig. 9B).

Enrichment analysis revealed that both [¹⁷⁷Lu] Lu-cAC 10 and [¹⁶¹Tb]Tb-cAC10 induced alterations in DDR and replication stress response (RSR), cell cycle arrest, and protein metabolism regulation (Fig. 9C). Terbium-161 treatment specifically altered pathways involved in SUMOylation and chromatin organization in our model. To further investigate the potential differences between both radionuclides, we directly compared the phosphopeptide abundance in [¹⁷⁷Lu]Lu-cAC10 and [¹⁶¹Tb]Tb-cAC10-treated tumors, revealing significant alterations in only 18 phosphopeptides (corresponding to 12 proteins), which confirmed that the vast majority of phosphorylations are similarly affected by both radiolanthanides (Fig. 16). Upon these 18 phosphopeptides, 17 showed a significantly increased abundance in the terbium-161 dataset compared to lutetium-177 (Fig. 16).

Claims

1 . An antibody-radionuclide conjugate, comprising a) an antibody or antigen-binding fragment thereof specifically binding to CD30; and b) terbium-161.

2. A kit of parts comprising a) an antibody or antigen-binding fragment thereof specifically binding to CD30; and b) terbium-161 wherein said antibody or antigen-binding fragment thereof is capable of binding terbi um-161 .

3. The kit of parts of claim 2, wherein said antibody or antigen-binding fragment thereof comprises a metal chelator capable of being charged with terbi um-161 .

4. The antibody-radionuclide conjugate of claim 1 or the kit of parts of claim 2 or 3, wherein the antibody or the antigen binding fragment thereof of a) is a monoclonal antibody.

5. The antibody-radionuclide conjugate or the kit of parts of claim 4, wherein the antibody or the antigen binding fragment thereof of a) is an lgG1 antibody.

6. The antibody-radionuclide conjugate of any one of claims 1 , 4 or 5, or the kit of parts of any one of claims 2 to 5, wherein the antibody or antigen-binding fragmentthereof of a) binds to the same epitope of CD30 as cAC10 antibody.

7. The antibody-radionuclide conjugate of any one of claims 1 or 4 to 6, or the kit of parts of any one of claims 2 to 6, wherein the antibody or antigen-binding fragment thereof of a) is an antibody comprising the CDR sequences of the cAC10 antibody or an antibody comprising VH and VL sequences of the cAC10 antibody or corresponds to the cAC10 antibody.

8. The antibody-radionuclide conjugate of any one of claims 1 or 4 to 7, or the kit of parts of any one of claims 2 to 7, wherein the antibody-radionuclide conjugate comprises a metal chelator, said metal chelator being charged with terbium-161 or being capable of being charged with terbium- 161.

9. The antibody-radionuclide conjugate of claim 8 or the kit of parts of claim 8, wherein the metal chelator is 1 ,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA).

10. The antibody-radionuclide conjugate of claim 8 or 9, or the kit of parts of claim 8 or 9, wherein the metal chelator is attached to the antibody or antigen-binding fragment thereof through a linker comprising -NHC(=S)-NH- moiety.

11. The antibody-radionuclide conjugate of claim 10 or the kit of parts of claim 10, wherein the antibody-radionuclide conjugate or the kit of parts comprises the following moiety: wherein said moiety is attached to the antibody or antigen-binding fragment thereof via amino group of lysine residue.

12. The antibody-radionuclide conjugate of any one of claims 8 to 11 , or the kit of parts of any one of claims 8 to 11 , wherein the metal chelator/antibody stoichiometric ratio is between 2.5 and 3.5.

13. The antibody-radionuclide conjugate or the kit of parts of claim 12, wherein the metal chelator/antibody stoichiometric ratio is about 3.0.

14. A pharmaceutical composition comprising the antibody-radionuclide conjugate of any one of claims 1 or 4 to 13, and at least one pharmaceutically acceptable carrier.

15. The antibody-radionuclide conjugate of any one of claims 1 or 4 to 13, the kit of parts of any one of claims 2 to 13, or the pharmaceutical composition of claim 14, for use in therapy.

16. The antibody-radionuclide conjugate of any one of claims 1 or 4 to 13, the kit of parts of any one of claims 2 to 13, or the pharmaceutical composition of claim 14, for use in treatmentor prevention of cancer.

17. The antibody-radionuclide conjugate for use of claim 16, the kit of parts for use of claim 16, or the pharmaceutical composition for use of claim 16, wherein the cancer is lymphoma.

18. The antibody-radionuclide conjugate for use of claim 17, the kit of parts for use of claim 17, or the pharmaceutical composition for use of claim 17, wherein lymphoma is a T-cell lymphoma.

19. The antibody-radionuclide conjugate for use of claim 18, the kit of parts for use of claim 18, or the pharmaceutical composition for use of claim 18, wherein T-cell lymphoma is selected from peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma, anaplastic large cell lymphoma, adult T-cell lymphoma, extranodal NK/T-cell lymphoma, and cutaneous T-cell lymphoma.

20. The antibody-radionuclide conjugate for use of any one of claims 17 to 19, the kit of parts for use of any one of claims 17 to 19, or the pharmaceutical composition for use of claim 17 to 19, wherein said antibody-radioligand conjugate or the parts of the kit of parts or the pharmaceutical composition is to be administered to a subject with an additional therapeutic agent, selected from alkylating agents, platinum agents, taxanes, vinca agents, anti-estrogen drugs, aromatase inhibitors, ovarian suppression agents, VEGF/VEGFR inhibitors, EGF/EGFR inhibitors, PARP inhibitors, cytostatic alkaloids, cytotoxic antibiotics, antimetabolites, endocrine/hormonal agents, immune checkpoint inhibitors and bisphosphonate therapy agent.

21 . The antibody-radionuclide conjugate for use of any one claims 16 to 20, the kit of parts for use of any one claims 16 to 20, or the pharmaceutical composition for use of any one claims 16 to 20, wherein total daily dose to be administered to a human is between 1 and 100 MBq/kg body weight, preferably between 5 and 30 MBq/kg body weight, more preferably between 10 and 20 MBq/kg body weight.

22. The antibody-radionuclide conjugate of any one of claims 1 or 4 to 13, or the kit of parts of any one of claims 2 to 14, for use in diagnosis.

23. Use of the antibody-radionuclide conjugate of any one of claims 1 or 4 to 13, the kit of parts of any one of claims 2 to 13, or the pharmaceutical composition of claim 14, for manufacture of a medicament for the treatment or prevention of cancer.

24. The use of claim 23, wherein the cancer is lymphoma.

25. The use of claim 24, wherein lymphoma is a T-cell lymphoma.

26. The use of claim 25, wherein T-cell lymphoma is selected from peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma, anaplastic large cell lymphoma, adult T-cell lymphoma, extranodal NK/T-cell lymphoma, and cutaneous T-cell lymphoma.

27. The use of any one of claims 24 to 26, wherein said antibody-radioligand conjugate or the parts of the kit of parts or the pharmaceutical composition is to be administered to a subject with an additional therapeutic agent, selected from alkylating agents, platinum agents, taxanes, vinca agents, anti-estrogen drugs, aromatase inhibitors, ovarian suppression agents, VEGF/VEGFR inhibitors, EGF/EGFR inhibitors, PARP inhibitors, cytostatic alkaloids, cytotoxic antibiotics, antimetabolites, endocrine/hormonal agents, immune checkpoint inhibitors and bisphosphonate therapy agent.

28. The use of any one of claims 24 to 27, wherein total daily dose to be administered to a human is between 1 and 100 MBq/kg body weight, preferably between 5 and 30 MBq/kg body weight, more preferably between 10 and 20 MBq/kg body weight.

29. A method of treating cancer, comprising the step of administering the therapeutically effective amount of antibody-radionuclide conjugate of claim 1 to a subject in need thereof.