Novel polypeptide

Abstract

The present disclosure relates to certain classes of artificial polypeptides having binding affinity for B7-H3, with the sequence EKX3X4ALX7EIX 10 X 11 LPNLX 16 X 17 X 18 QX 20 X 21 AFIX 25 X 26 LNX 29 X 30 or a B7-H3 binding polypeptide comprising a sequence having at least 93% identity thereto. The present disclosure relates to the use of such B7-H3 binding polypeptides as therapeutic, prognostic, diagnostic, and / or antitumor agents.

Description

[Technical Field]

[0001] Field of Invention This disclosure relates to a group of artificial polypeptides having binding affinity to B7-H3. This disclosure also relates to the use of such B7-H3 binding polypeptides as therapeutic agents, prognostic agents, diagnostic agents, and / or oncological agents. [Background technology]

[0002] background Biology of B7-H3 B7-H3 (CD276), a member of the B7 family, is a type I transmembrane protein. Other members of this family include CD80 (B7-1), CD86 (B7-2), and PD-L1 (B7-H1). The overall role of B7 family proteins and their receptors is to act as coagulators in the immune response. B7-H3 has been reported to possess both costimulatory and coin inhibitory functions in the immune system and to play a role in both innate and adaptive immune responses. However, most reports consider B7-H3 to be primarily a co-inhibitor playing a role in the innate immune response. B7-H3 has been shown to inhibit the TH1 response, CD4 / CD8 T cell activation and proliferation, IFNγ production, and reduce transcription factor activity, thereby turning off the T cell response (Kontos et al., 2021, Clin Cancer Res 27, 1227-1235).

[0003] Structurally, B7-H3 (45-66 kDa) consists of an extracellular Ig-like domain, a transmembrane region, and a short intracellular region. B7-H3 has two isoforms, called 4Ig-B7-H3 and 2Ig-B7-H3, based on the number of extracellular Ig-like domains. In humans, the extracellular domain consists of either a pair of immunoglobulin variable domains (IgV) and immunoglobulin constant domains (IgC) (2IgB7-H3), or two pairs of identical immunoglobulins due to exon duplication (4IgB7-H3). Mice only possess the 2IgB7-H3 variant. Soluble B7-H3 (sB7-H3; approximately 37 kDa) is produced by alternative splicing or, more commonly, by cleavage from the cell surface via metalloproteinases. The short intracellular domain does not carry signaling pathways. Further research is needed to define the interactions of the B7-H3 receptor and elucidate its physiological role, which may be useful in designing B7-H3 interactome inhibitors. (Kanchan et al., 2022, Biochim Biophys Acta Rev Cancer 1877(5),188783).

[0004] While B7-H3 exhibits a broad mRNA expression profile, its steady-state protein expression is limited, suggesting the existence of important post-transcriptional regulatory mechanisms. Protein expression is observed in both immune and non-immune cells. Expression in immune cells is quite low, but can be induced, particularly in antigen-presenting cells (APCs). Expression in normal non-immune cells has been detected in resting fibroblasts, osteoblasts, endothelial cells, and tissues of the adrenal gland, pancreas, liver, colon, stomach, placenta, testis, and prostate (Du et al., 2019 Cancer Cell 35, 221-237). Although expression is low in normal tissues, elevated B7-H3 levels are often seen in tumor tissues. This difference between tumor and healthy tissues is important for utilizing B7-H3-binding molecules as targeted therapies and diagnostic tools (Kontos et al., supra and its references).

[0005] B7-H3 in cancer B7-H3 has been reported to contribute to tumorigenesis through several different mechanisms, including promoting invasion, promoting angiogenesis, promoting proliferation, anti-apoptosis, and metabolic reprogramming (see review in Castellanos et al., 2017, Immunol 6, 66-75).

[0006] Overexpression of B7-H3 is found in 60-90% of tumor tissues in patients with conditions such as neuroblastoma, medulloblastoma, glioma, melanoma, and leukemia, as well as breast cancer, prostate cancer, ovarian cancer, cervical cancer, gastric cancer, pancreatic cancer, kidney cancer, and colorectal cancer. High expression correlates with tumor progression and poor clinical outcomes. Expression is observed not only on tumor cells but also in the stroma, including the tumor-associated vascular system. Histological staining has shown that B7-H3 is expressed in the membrane, cytoplasm, and nucleus of tumor cells and tumor stroma. In colorectal cancer, nuclear staining of B7-H3 is strongly correlated with poor clinical outcomes (Ingebrigtsen et al., 2012, Int J Cancer 131, 2528-2536), suggesting that the expression pattern of B7-H3 in tumor tissue may be complex.

[0007] In an analysis of 1342 tumor tissues and 245 normal tissues, Seaman et al. found B7-H3 expression in various tumor tissues, and importantly, even higher expression in the tumor stroma. In healthy tissues, B7-H3 expression was absent or moderate, with the highest expression observed in the liver (Seaman et al., 2017, Cancer Cell 31, 501-515). In this study, 65% of the normal tissues examined were B7-H3 negative, 33% weakly positive, and only 1% moderately positive. In tumor tissues, only 27% of tumor cells and 6% of stromal cells were B7-H3 negative. On the other hand, 30% of tumor cells and 51% of stromal cells showed the highest expression scores, with the remaining proportions distributed between weak and moderate expression. This highlights that B7-H3 is generally highly expressed in various types of tumors, as well as its marked expression in tumor stromal cells. Furthermore, high expression of B7-H3 is strongly correlated with metastasis, and sB7-H3 has been observed in the serum of patients with advanced-stage cancer (Kanchan el. al., supra). Thus, based on the available information regarding B7-H3 overexpression in many different tumor types, B7-H3, as a potential marker of disease invasiveness and metastasis, appears to be an attractive protein for targeted radiotherapy and in vivo diagnostic applications, as well as for use as a non-invasive target for diagnosis and prognosis.

[0008] Clinical and preclinical development of B7-H3 target molecules Several anti-B7-H3 monoclonal antibodies or antibody-drug conjugates are currently in clinical development for the treatment of various tumors (see review in Kontos et al., supra and Kasten et al., 2020, Curr Med Chem 27, 4016-4038). These include enoblituzumab (an Fc-optimized B7-H3 targeted monoclonal antibody that utilizes antibody-dependent cytotoxicity to kill tumor cells); MGC018, DS-7300a, BAT8009 (an antibody-drug conjugate with a cytotoxic payload against B7-H3 expressing tumors); MVC-280 / TAK280 (a bispecific antibody targeting B7-H3 and CD3); and ombrutamab (8H9, also known as Omblastys®; a radiolabeled monoclonal B7-H3 antibody that received priority review from the FDA for the treatment of pediatric patients with central nervous system / septal metastases in neuroblastoma). In clinical trials, ombrutamab has been administered intraventricularly, by positive pressure infusion, intraperitoneally for the treatment of CNS tumors, intradermally for the treatment of dysplastic round cell tumors, and intradermally for the treatment of patients with refractory, relapsed, or progressive CNS or leptomeningeal diseases.

[0009] Orlotamab, a bispecific antibody targeting B7-H3 and CD3, was in clinical development but was discontinued due to the observation of elevated liver enzymes, which were suggested to be a result of cytokine release induced by the interaction between orlotamab and CD3 on T cells.

[0010] The potential for using B7-H3 targeted antibodies for diagnostic purposes is supported by the results of Burvenich et al., who demonstrated the specific tumor localization of the zirconium-89-labeled B7-H3 targeted humanized monoclonal antibody DS-5573a (Burvenich et al., 2018, Theranostics 8, 4199-4209).

[0011] To date, no radiotherapy targeting B7-H3 other than the monoclonal antibody Omblastys® has been identified as being clinically investigated. As mentioned above, it has not been developed for systemic administration.

[0012] Because molecular tissue penetration is negatively correlated with its size, relatively large antibody molecules inherently have poor tissue distribution and penetration capabilities. Therefore, they may not be optimal target sites for delivering radioisotopes to solid tumors in a therapeutic setting. Furthermore, while antibodies are widely used in a variety of routine situations due to their high affinity and specificity for numerous antigens—including for analysis, purification, diagnosis, and therapeutic purposes—they still have drawbacks. These include a tendency to aggregate and a long systemic half-life, increasing the risk of damaging healthy tissue in targeted radiotherapy. When used for diagnostics (such as imaging), monoclonal antibodies have a long half-life, resulting in poor contrast if there isn't enough time for the level of unbound antibody to decrease. Thus, monoclonal antibodies are not always optimal for therapeutic or diagnostic applications.

[0013] B7-H3 binding polypeptides based on the Z scaffold have also been reported. Using a cell-based selection system, Stern et al. identified a moderately B7-H3 affinity B7-H3 binding polypeptide (denoted AC2; Kd 310nM) that can be further optimized to produce B7-H3 conjugates with higher affinity (Kd 0.9–20nM) (Stern et al., 2019, ACS Comb Sci 21, 207-222; see also WO2020041626). By binding AC2 to either indocyanine green (Bam et al., 2019, Bioconjug Chem 30, 1677-1689) or microbubbles (Bam et al., 2020, Clin Cancer Res 26, 2140-2150), the feasibility of using B7-H3 targeted Z scaffold variants for tumor visualization may be demonstrated. Furthermore, it is modified to include a peptide-based cysteine-containing chelator at the C-terminus, 99 AC12 labeled with mTc has recently been used for preclinical imaging of B7-H3 expressing tumors (Oroujeni et al., 2022, Pharmaceutics, 14(9):1780).

[0014] Preclinical in vivo evaluations of AC2 and AC12 suggest that their affinity for B7-H3 is insufficient to translate into improved sensitivity and specificity for clinical use, particularly in clinical imaging (Bam et al., 2019 supra, Bam et al., 2020 supra, and Oroujeni et al., 2022 supra). These polypeptides also have unsatisfactory immunogenicity profiles.

[0015] From the above, it is clear that there is a continued need for drugs with high affinity and specificity for B7-H3 to enable high-contrast imaging and to avoid off-target effects, as well as for drugs with low immunogenicity to ensure safe and well-tolerated therapeutic agents. [Overview of the project]

[0016] The object of this disclosure is to provide novel B7-H3 binders that can be used, for example, in therapeutic, prognostic, and diagnostic applications.

[0017] The purpose of this disclosure is to provide molecules that enable efficient treatment, including theranotic applications, for example, of various forms of cancer, while mitigating the aforementioned and other shortcomings of current therapies.

[0018] A further objective of this disclosure is to provide molecules suitable for prognostic and diagnostic applications, such as prognostic and diagnostic applications for various forms of cancer.

[0019] These and other objectives, which are obvious to those skilled in the art from this disclosure, are claimed in the appended claims and are satisfied by different embodiments as generally disclosed herein.

[0020] Accordingly, in a first aspect of this disclosure, a B7-H3 linked polypeptide comprising the B7-H3 linked motif BM is provided, which is as follows: i) EKX3X4ALX7EIX 10 X 11 LPNLX16 X 17 X 18 QX 20 X 21 AFIX 25 X 26 LNX 29 X 30 (Array number 567) Here, independently of each other X3 is selected from I and V; X4 is selected from A, D, E, G, H, K, L, M, N, Q, S, T, and Y; X7 is selected from A, G, H, and S; X 10 is selected from I and V; X 11 is selected from N and W; X 16 is selected from N and T; X 17 is selected from H and Y; X 18 is selected from A, D, E, G, H, N, Q, S, and T; X 20 is selected from I and V; X 21 is selected from K and R; X 25 is selected from A, E, F, H, L, and W; X 26 is selected from K and S; X 29 is selected from A, D, and E; X 30 is selected from A, D, and H; and ii) an amino acid sequence having at least 93% identity to the array defined in i) selected from the amino acid sequences consisting of.

[0021] In one embodiment, a B7-H3 binding polypeptide comprising the B7-H3 binding motif BM is provided, and this motif is as follows: i) EKX3X4ALX7EIX 10 X 11 LPNLX 16 X17 X 18 QX 20 X 21 AFIX 25 X 26 LNX 29 X 30 (Array number 567) Here, independently of each other X3 is selected from I and V; X4 is selected from A, D, E, G, H, K, L, M, N, Q, S, T, and Y; X7 is selected from A, G, H, and S; X 10 is selected from I and V; X 11 is selected from N and W; X 16 is selected from N and T; X 17 is selected from H and Y; X 18 is selected from A, D, E, G, H, N, Q, S, and T; X 20 is selected from I and V; X 21 is selected from K and R; X 25 is selected from A, E, F, H, L, and W; X 26 is selected from K and S; X 29 is selected from A, D, and E; X 30 is selected from A, D, and H; and ii) When X4 is Y, or X 18 is G, an amino acid sequence having at least 93% identity to the array defined in i), provided that X 26 is K consisting of an amino acid sequence selected from.<00009​​​​​​X4 is selected from D, E, G, H, K, L, M, N, Q, S, T, and Y; X7 is selected from A, G, H, and S; X 10 It is selected from I and V; X 11 It is selected from N and W; X 16 It is selected from N and T; X 17 is selected from H and Y; X 18 It is selected from A, D, E, G, H, N, Q, S, and T; X 20 It is selected from I and V; X 21 is selected from K and R; X 25 It is selected from A, E, F, H, L, and W; X 26 is selected from K and S; X 29 is selected from A, D and E; and X 30 This is a B7-H3 linked polypeptide as described in item 1, selected from A, D, and H.

[0023] In one embodiment, a B7-H3 binding polypeptide is provided, in sequence i) X3 is selected from I and V; X4 is selected from D, E, G, H, K, L, M, N, Q, S, T, and Y; X7 is selected from A, G, H, and S; X 10 It is selected from I and V; X 11 It is selected from N and W; X 16 It is selected from N and T; X 17 is selected from H and Y; X 18 It is selected from A, D, E, G, H, N, Q, S, and T; X 20 It is selected from I and V; X 21 is selected from K and R; X 25 It is selected from A, E, F, H, L, and W; X 26 is selected from K and S; X 29 is selected from A, D and E; and X 30 This is selected from A, D, and H.

[0024] In one embodiment, a B7-H3 binding polypeptide is provided, in sequence i) X3 is selected from I and V; X4 is selected from D, G, K, L, M, N, S, T, and Y; X7 is selected from A, G, H, and S; X 10 is I; X 11 It is selected from N and W; X 16 is T; X 17 is Y; X 18 It is selected from A, D, E, H, N, Q, S, and T; X 20 It is selected from I and V; X 21 is selected from K and R; X 25 is selected from A, E, H, and W; X 26 is K; X 29 is selected from A, D and E; and X 30 This is selected from A, D, and H.

[0025] In yet another embodiment, a B7-H3 binding polypeptide is provided, in sequence i) X3 is I; X4 is selected from K and S; The X7 is available in G and S versions; X 10 is I; X 11 is W; X 16 is T; X 17 is Y; X 18 is selected from E, N and Q; X 20 is I; X 21 is K; X 25 is selected from A and H; X 26 is K; X 29 is selected from A and D; and X 30 is selected from A and D.

[0026] The symbols "X n " and "X m " are used herein to denote the amino acids at positions n and m in various amino acid sequences defined herein, where n and m are often integers indicating the positions of the amino acids within the sequence, counted from the N-terminus of the said sequence. In other amino acid sequences, n and m are in lower case and "X n " and "X m " residues only indicate the internal order. As an example of the former, X4 and X7 denote the amino acids at positions 4 and 7 respectively from the N-terminus of sequence i).

[0027] In an embodiment according to the first aspect, there is provided a polypeptide in which X (n ) is independently selected from the group of possible residues according to Table 1. Those skilled in the art will understand that X n can be selected from any one of the listed groups of possible residues, and this selection is independent of the selection of the amino acid in X m , where n≠m. Thus, X (n)のAny of the listed possible residues at a given position can be combined independently with any of the listed possible residues at any other variable position in Table 1.

[0028] Those skilled in the art will understand that Table 1 is read as follows: In one embodiment according to the first aspect, the amino acid residue "X" in sequence i) n A polypeptide is provided in which X7 in sequence i) is selected from "possible residues". Accordingly, Table 1 discloses some specific and individual embodiments of the first aspect of this disclosure. For example, in one embodiment according to the first aspect, a polypeptide is provided in which X7 in sequence i) is selected from A, G, H and S, and in another embodiment according to the first aspect, a polypeptide is provided in which X7 in sequence i) is selected from G and S. For example, one such combined embodiment is provided in which X7 is selected from G and S, and X 18 is selected from E, N, and Q, and X 25 This includes polypeptides selected from A and H.

[0029] [Table 1-1]

[0030] [Table 1-2]

[0031] In a more specific embodiment, a subclass of B7-H3 linked polypeptides is defined. Array i) satisfies at least 5 of the 9 conditions I to IX: I.X3 is I; II.X 10 is I; III.X 11 It is selected from N and W; IV.X 16 is T; VX 17 is Y; VI.X 20 is I; VII.X 21 is K; VIII.X 25 is selected from A, E and H; and IX.X 26 is K.

[0032] In some examples of B7-H3 linked polypeptides according to the first embodiment, sequence i) satisfies at least six of the nine conditions I to IX. More specifically, sequence i) may satisfy at least seven of the nine conditions I to IX, for example, at least eight of the nine conditions I to IX, for example, all of the nine conditions I to IX.

[0033] In some embodiments of the B7-H3 linked polypeptide according to the first aspect, X 10 is I, X 17 Y is X 21 In some embodiments, X 10 is I, X 17 Y is X 20 is I. In some embodiments, X 11 is W, and X 17 Y is X 20 is I. In some embodiments, X3 is I and X 10 is I, X 17 It is Y.

[0034] As will be described in detail in the following experimental section, the selection of B7-H3 binding polypeptide variants led to the identification of numerous individual B7-H3 binding motif (BM) sequences belonging to a class defined in the first aspect of this disclosure. These sequences constitute individual embodiments of sequence i) or ii) according to this aspect. The sequences of individual B7-H3 binding motifs correspond to amino acid positions 8-37 of SEQ ID NOs: 1-535. In one embodiment of the B7-H3 binding polypeptide according to this first aspect, sequence i) corresponds to the sequence from positions 8 to 37 of a sequence selected from the group consisting of SEQ ID NOs: 1-535. In another embodiment of the B7-H3 binding polypeptide according to this first aspect, sequence i) corresponds to the sequence from positions 8 to 37 in a sequence selected from the group consisting of SEQ ID NOs: 1-21 and 480-535. In one embodiment, sequence i) corresponds to the sequence from positions 8 to 37 in a sequence selected from the group consisting of SEQ ID NOs: 1-21. In one embodiment, sequence i) corresponds to the sequence from position 8 to 37 in a sequence selected from the group consisting of sequence numbers 1 to 3. In one embodiment, sequence i) corresponds to the sequence from position 8 to 37 of sequence number 1. In one embodiment, sequence i) corresponds to the sequence from position 8 to 37 of sequence number 2. In one embodiment, sequence i) corresponds to the sequence from position 8 to 37 of sequence number 3.

[0035] As those skilled in the art will understand, the functionality of any polypeptide, such as the B7-H3 binding ability of the polypeptides of this disclosure, depends on the tertiary structure of the polypeptide. Therefore, it is possible to make slight changes to the amino acid sequence in the polypeptide without affecting the functionality of the polypeptide. Accordingly, this disclosure encompasses modified variants of B7-H3 binding polypeptides that retain their B7-H3 binding properties.

[0036] Thus, the present disclosure includes B7-H3 linked polypeptides having an amino acid sequence with 93% or more identity, e.g., 96% or more identity, to the polypeptide defined in i). For example, an amino acid residue belonging to a specific functional group of an amino acid residue (e.g., hydrophobic, hydrophilic, polar, etc.) can be exchanged for another amino acid residue from the same functional group.

[0037] In some embodiments, such modifications may be made at any position in the sequence of the B7-H3 binding polypeptide as disclosed herein. In other embodiments, such modifications may be made only at non-variable positions, also referred to as scaffold amino acid residues. In such cases, modifications at variable positions are not permitted. In other embodiments, such modifications may be made only at variable positions.

[0038] According to one possible definition of such "variable position," these are the positions indicated by "X" in array i) as defined above.

[0039] According to another intended definition, “variable positions” are positions that are randomized in the selection library of Z variants before selection, and therefore, for example, positions 2, 3, 4, 6, 7, 10, 11, 17, 18, 20, 21, 25, 28, 29, and 30 of sequence i). Positions 16 and 26 are not included in this definition of “variable positions,” although they are scaffolding positions in this context, and it is acknowledged that each position can be one of two options. See Nord et al. (1995), Prot Eng 8:601-608, and Lofblom et al. (2010), FEBS Letters, 584:2670-2680. Similar to the B7-H3 binding Z variants in this disclosure, the polypeptides disclosed by Nord et al. and Lofblom et al. are also based on a scaffold of Z derivatives of domain B of protein A derived from Staphylococcus aureus, although directed to other targets. As shown by Nord et al. (see, for example, Figure 4), the amino acids at position 23 (corresponding to position 16 of the B7-H3 binding motif) and position 33 (corresponding to position 26 of the B7-H3 binding motif) are N and S, respectively. As also shown by Lofblom et al., polypeptides having amino acid residues N and S at positions 23 and 33 (corresponding to positions 16 and 26 of the B7-H3 binding motif; see Figure 2 by Lofblom et al.), and polypeptides having amino acid residues T and K at positions 23 and 33, respectively, all maintain their basic structure and function. Therefore, in the context of this definition of "variable position," the amino acid residues at positions 16 and 26 are intended to form part of a common scaffold, with either N or T at scaffold position 16 and either S or K at scaffold position 26.

[0040] The term "identity %" as used throughout this specification may be calculated, for example, as follows: The query sequence is calculated using the CLUSTAL W algorithm (Thompson et al., 1994, Nucleic Acids Research, 22: 4673-4680). The comparison is performed on a window corresponding to the shortest sequence in the aligned sequence. The shortest sequence in the aligned sequence may also be the target sequence. In other examples, the shortest sequence in the aligned sequence is the query sequence. Amino acid residues at each position are compared and reported as identity % of the corresponding query sequence positions in the target sequence.

[0041] In another embodiment, a B7-H3 binding polypeptide is provided that includes a binding motif sequence corresponding to the sequence from position 8 to 37 in a sequence selected from the group consisting of SEQ ID NOs: 1 to 535; or a sequence having 93% or more identity thereto, for example, 96% identity thereto.

[0042] In some embodiments, the BM as defined above “forms part of” a trihelical protein domain. This is understood to mean that the sequence of the BM is “inserted” or “grafted” into the sequence of the original trihelical domain so as to replace a similar structural motif in the original domain. For example, without wishing to be bound by theory, it can be thought that the BM constitutes two of the three helices of a trihelical bundle, and therefore such a two-helical motif can be replaced within any trihelical bundle. As those skilled in the art will understand, replacing two helices of a trihelical domain with two BM helices must be done in such a way that it does not affect the basic structure of the polypeptide. That is, the overall folding of the Cα backbone of the polypeptide according to this embodiment of the invention is substantially the same as the folding of the trihelical protein domain from which it forms part, for example having the same elements of the secondary structure in the same order. Therefore, the BM according to this disclosure “forms part of” the three-helical bundle domain when the polypeptide according to this embodiment has the same fold as the original domain, meaning that the basic structural properties are shared, and these properties result in, for example, similar CD spectra. Those skilled in the art will know the other relevant parameters.

[0043] In certain embodiments, the B7-H3 binding motif (BM) thus forms part of a trihelical protein domain. For example, BM may essentially constitute two alpha helices having interconnected loops within the trihelical protein domain. In certain embodiments, the trihelical protein domain is selected from a bacterial receptor domain. In some embodiments, the trihelical protein domain is selected from a domain or derivative thereof of Staphylococcus aureus protein A. Non-limiting examples of such domains include five different trihelical domains of Staphylococcus aureus protein A, such as domain B, and their derivatives. In some embodiments, the trihelical protein domain is a variant of protein Z derived from domain B of Staphylococcus protein A (Wahlberg E et al., 2003, PNAS 100(6):3185-3190).

[0044] In some embodiments, such as those disclosed herein, where a B7-H3 binding polypeptide forms part of a three-helical protein domain, the B7-H3 binding polypeptide comprises a binding module (BMod), the amino acid sequence of which is selected from the following: iii) K-[BM]-PSQSX a X b LLX c EAKKLX d X e X f Selected from Q (sequence number 568); Here, [BM] is a B7-H3 bond motif according to any definition herein; X a It is selected from A and S; X b is selected from E and N; X c is selected from A, S, and C; X d is selected from E, N, and S; X e is selected from D, E, and S; X fis selected from A and S; and An amino acid sequence having at least 93% identity with the sequence defined in iv)iii).

[0045] In some embodiments, the polypeptide may exhibit beneficial high structural stability, such as resistance to chemical modification, resistance to changes in physical conditions, and resistance to proteolysis, during manufacturing and storage, as well as in vivo.

[0046] As described above, polypeptides containing minor changes compared to the above-described amino acid sequences, which do not significantly affect the tertiary structure and function of the polypeptide, are also within the scope of this disclosure. Accordingly, in some embodiments, sequence iv) has at least 93%, for example, at least 95%, for example, at least 97% identity with the sequence defined by iii).

[0047] In one embodiment, X in sequence iii) a It is A. In one embodiment, X in sequence iii) a S is. In one embodiment, X in sequence iii) b It is N. In one embodiment, X in sequence iii) b It is E. In one embodiment, X in sequence iii) c It is A.

[0048] In one embodiment, X in sequence iii) c S is. In one embodiment, X in sequence iii) c C is C. In one embodiment, X in sequence iii) d It is E. In one embodiment, X in sequence iii) d It is N. In one embodiment, X in sequence iii) d S is.

[0049] In one embodiment, X in sequence iii) e It is D. In one embodiment, X in sequence iii) e It is E. In one embodiment, X in sequence iii) e S is. In one embodiment, X in sequence iii) d X e The following are selected from EE, ES, SD, SE, and SS. In one embodiment, X in sequence iii) d X e It is ES.

[0050] In one embodiment, X in sequence iii) d X e He is a systems engineer. In one embodiment, X in sequence iii) d X e It is SD. In one embodiment, X in sequence iii) f It is A. In one embodiment, X in sequence iii) f S is. In one embodiment, X in sequence iii) a A is X b is N and X c A is X f It is A.

[0051] In one embodiment, in sequence iii), X a is S; X b is E; X c A is X f It is A. In one embodiment, in sequence iii), X a A is X b is N and X c C is X f It is A. In one embodiment, in sequence iii), X a is S; X bis E; X c is S, X f S is. In one embodiment, in sequence iii), X a is S; X b is E; X c C is X f S is. In one embodiment, in sequence iii), X a A is X b is N and X c A is X d X e ND and X f It is A.

[0052] In one embodiment, in sequence iii), X a is S; X b is E; X c A is X d X e ND and X f It is A. In one embodiment, in sequence iii), X a A is X b is N and X c is C and X d X e ND and X f It is A. In one embodiment, in sequence iii), X a is S; X b is E; X c is S; X d X e ND and X f S is. In one embodiment, in sequence iii), X a is S; X b is E; X c is C and X d X e ND and X f S is. In one embodiment, in sequence iii), X a A is X b is N and Xc A is X d X e is SE, X f It is A.

[0053] In one embodiment, in sequence iii), X a is S; X b is E; X c A is X d X e is SE, X f It is A. In one embodiment, in sequence iii), X a A is X b is N and X c is C and X d X e is SE, X f It is A. In one embodiment, in sequence iii), X a is S; X b is E; X c is S; X d X e is SE, X f S is. In one embodiment, in sequence iii), X a is S; X b is E; X c is C and X d X eは SE and X f S is. In one embodiment, in sequence iii), X a A is X b is N, and X c A is X d X e It is SD, X f It is A.

[0054] In one embodiment, in sequence iii), X a is S; X b is E; X c A is X d X e It is SD, X fIt is A. In one embodiment, in sequence iii), X a A is X b is N and X c is C and X d X e It is SD, X f It is A. In one embodiment, in sequence iii), X a is S; X b is E; X c is S; X d X e It is SD, X f S is. In one embodiment, in sequence iii), X a is S; X b is E; X c is C and X d X e It is SD, X f S is.

[0055] In a further embodiment, sequence iii) corresponds to the amino acid sequence from position 7 to 55 of a sequence selected from the group consisting of SEQ ID NOs: 1 to 535. In one embodiment, sequence iii) corresponds to the amino acid sequence from position 7 to 55 in a sequence selected from the group consisting of SEQ ID NOs: 1 to 21 and 480 to 535. In another embodiment, sequence iii) corresponds to the amino acid sequence from position 7 to 55 in a sequence selected from the group consisting of SEQ ID NOs: 1 to 21. In one embodiment, sequence iii) corresponds to the amino acid sequence from position 7 to 55 in a sequence selected from the group consisting of SEQ ID NOs: 1 to 3. In yet another embodiment, sequence iii) corresponds to the amino acid sequence from position 7 to 55 of SEQ ID NO: 1. In yet another embodiment, sequence iii) corresponds to the amino acid sequence from position 7 to 55 of SEQ ID NO: 2. In yet another embodiment, sequence iii) corresponds to the amino acid sequence from position 7 to 55 of SEQ ID NO: 3.

[0056] In further embodiments, B7-H3 linked polypeptides described herein are provided, comprising an amino acid sequence selected from the following: v)YA-[BMod]-AP(Sequence ID 569) Here, [BMod] is a B7-H3 bonded molecule as defined above; and An amino acid sequence having at least 86% identity with the sequence defined by vi)v).

[0057] As described above, polypeptides that include minor changes compared to the above amino acid sequences without significantly affecting the tertiary structure and its function also fall within the scope of this disclosure. Therefore, in some embodiments, the B7-H3 binding polypeptide defined above may have sequence vi) which is, for example, at least 86%, e.g., at least 88%, e.g., at least 90%, e.g., at least 92%, e.g., at least 94%, e.g., at least 96%, e.g., at least 98% identical to the sequence defined by v).

[0058] In some embodiments, the B7-H3 binding motif may form part of a polypeptide comprising an amino acid sequence selected from the following: VDAKYAK-[BM]-PSQSSELLSEAKKLNDSQAPK(Sequence ID 570); AEAKFAK-[BM]-PSQSSELLSEAKKLSESQAPK(Sequence ID 571); AEAKYAK-[BM]-PSQSSELLSEAKKLNDSQAPK(Sequence ID 572; AEAKFAK-[BM]-PSQSSELLSEAKKLNDSQAPK(Sequence ID 573); AEAKFAK-[BM]-PSQSSELLSEAKKLNESQAPK(Sequence ID 574); AEAKYAK-[BM]-PSQSSELLSEAKKLSESQAPK(Sequence ID 575); ADNNFNK-[BM]-PSQSANLLSEAKKLNESQAPK(Sequence ID 576); ADNKFNK-[BM]-PSQSANLLAEAKKLNDAQAPK(Sequence ID 577); ADNKFNK-[BM]-PSVSKEILAEAKKLNDAQAPK(Sequence ID 578); ADAQQNNFNK-[BM]-PSQSTNVLGEAKKLNESQAPK(Sequence ID 579); AQHDE-[BM]-PSQSANVLGEAQKLNDSQAPK(Sequence ID 580); VDNKFNK-[BM]-PSQSANLLAEAKKLNDAQAPK(Sequence ID 581); AEAKYAK-[BM]-PSESSELLSEAKKLNKSQAPK (sequence number 582); VDAKYAK-[BM]-PSQSSELLAEAKKLNDAQAPK(Sequence ID 583); VDAKYAK-[BM]-PSQSSELLAEAKKLNDSQAPK(Sequence ID 584); VDAKYAK-[BM]-PSQSSELLSEAKKLSESQAPK(Sequence ID 585); VDAKYAK-[BM]-PSQSSELLSEAKKLESSQAPK(Sequence ID 586); VDAKYAK-[BM]-PSQSSELLAEAKKLNKAQAPK(sequence code 587); and AEAKYAK-[BM]-PSQSSELLAEAKKLNKAQAPK(Sequence ID 588) Here, [BM] is the B7-H3 bond motif as defined above.

[0059] In one embodiment, a B7-H3 linked polypeptide described herein is provided, which comprises an amino acid sequence selected from the following: vii)VDAKYAK-[BM]-PSQSSELLSEAKKLNDSQAPK(Sequence ID 570) Here, [BM] is a B7-H3 bond motif as defined herein; An amino acid sequence having at least 86% identity with the sequence defined by viii).

[0060] In further embodiments, B7-H3 linked polypeptides described herein are provided, comprising an amino acid sequence selected from the following: ix)AEAKFAK-[BM]-PSQSSELLSEAKKLSESQAPK(Sequence ID 571) Here, [BM] is a B7-H3 bond motif as defined herein; and An amino acid sequence having at least 86% identity with the sequence defined by x).

[0061] In a further embodiment, sequence ix) is selected from the group consisting of sequence numbers 15-16, 420-424, 427-428, 430-436, 438-444 and 480-535.

[0062] In another further embodiment, a B7-H3 linked polypeptide described herein is provided, comprising an amino acid sequence selected from the following: xi)AEAKYAK-[BM]-PSQSSELLSEAKKLNDSQAPK(Sequence ID 572) Here, [BM] is a B7-H3 bond motif as defined herein; and An amino acid sequence having at least 86% identity with the sequence defined by xii)xi).

[0063] In another further embodiment, sequence xi) is selected from the group consisting of sequence numbers 1-13, 17-418, and 446-479. In another embodiment, sequence xi) is selected from the group consisting of sequence numbers 1-13 and 17-21. In another embodiment, sequence xi) is selected from the group consisting of sequence numbers 1-3. In another embodiment, sequence xi) is sequence number 1. In another embodiment, sequence number xi) is sequence number 2. In another embodiment, sequence number xi) is sequence number 3.

[0064] In another further embodiment, the B7-H3 binding polypeptide comprises an amino acid sequence selected from the following: xiii)AEAKFAK-[BM]-PSQSSELSEAKKLNESQAPK(Sequence ID 573) Here, [BM] is a B7-H3 bond motif as defined herein; and An amino acid sequence having at least 86% identity with the sequence defined by xiv)xiii).

[0065] In another further embodiment, sequence xiii) is selected from sequence numbers 14, 425-426, 429, 437 and 445.

[0066] In one embodiment, the interaction with B7-H3 is K D The value is at most 1 × 10 -6 M, for example, at most 5 x 10 -7 M, for example, at most 1 × 10 -7 M, for example, at most 5 x 10 -8 M, for example, at most 1 × 10 -8 A B7-H3-binding polypeptide of the first embodiment described herein is provided, which can be bound to B7-H3 as M.

[0067] As used herein, the terms “B7-H3 binding” and “binding affinity to B7-H3” refer to polypeptide properties that can be tested by various sensor-based techniques, such as ELISA, Kinetic Exclusion Assay (KinExA®) and / or surface plasmon resonance (SPR) techniques, biolayer interferometry (BLI; e.g., Octet®) and quartz crystal microbalancing (QCM) techniques.

[0068] For example, as described in the experimental section below, the B7-H3 binding affinity can be tested in an experiment in which a polypeptide sample is captured on an antibody-coated ELISA plate, biotinylated B7-H3 is added, and then streptavidin-labeled HRP is added. A TMB substrate is added, and the absorbance at 450 nm is measured using a multi-well plate reader. Those skilled in the art can interpret the results obtained by such an experiment to establish at least a qualitative measure of the polypeptide's binding affinity to B7-H3. If quantitative measurement is required, ELISA can also be used, for example, to determine the EC50 value (half-polar effective concentration) of the interaction. The response of polypeptides to a dilution series of biotinylated B7-H3 is measured using ELISA as described above. Those skilled in the art can then interpret the results obtained by such an experiment, and the EC50 value can be calculated from the results, for example, using GraphPad Prism 5 and nonlinear regression.

[0069] The affinity of the binding interaction, for example, the equilibrium dissociation constant (K D What is defined as the coupling rate (k) can also be determined by different sensor-based methods, as mentioned above. Importantly, these techniques measure coupling in real time, so the coupling rate (k) can be determined. a ) and dissociation rate (k d ) allows for the evaluation of the dynamic characteristics of the interaction. One such technique is based on surface plasmon resonance (SPR). Here, B7-H3 or a fragment thereof is immobilized on a sensor chip, and a sample of the polypeptide whose affinity is to be determined is prepared by serial dilution and injected onto the chip. Alternatively, the polypeptide to be tested is immobilized on the sensor chip of the instrument, and a sample containing B7-H3 or a fragment thereof is passed over the chip. The binding value can be defined, for example, with a Biacore (Cytiva), Sierra (Bruker), Carterra (Carterra), or ProteOn XPR 36 (Bio-Rad) instrument. DThe values ​​and / or dynamical constants can then be calculated from the results, for example, using the 1:1 Langmuir binding model in Biacore Insight Evaluation software, or other appropriate models and software provided by the instrument manufacturer. Similarly, the binding rate and affinity of B7-H3 can also be evaluated by BLI, an optical analysis technique that analyzes the interference pattern of white light reflected from two surfaces on the biosensor tip: the immobilized protein layer and the internal reference layer. Measurements using BLI can be performed, for example, using the Octet® HTX system (manufactured by Sartorius), and the data are monitored and analyzed using appropriate models and software provided by the instrument manufacturer.

[0070] B7-H3 binding affinity can also be obtained using a continuous flow system based on QCM technology. To monitor the binding interaction, one or a fragment of the interacting molecule is immobilized on the sensor surface, and a sample containing the other molecule is injected onto the sensor surface. The signal output is given in frequency (Hz) and is directly related to the change in mass on the sensor surface. Kinematic measurements using QCM technology can be performed, for example, with an Attana A200® (Attana) instrument. Data are collected by Attester software, and the KD value is calculated from the results using, for example, a 1:1 Langmuir binding model, and then processed with evaluation software or other appropriate software provided by the instrument manufacturer.

[0071] Another method for determining the binding affinity to B7-H3 is the Kinetic Exclusion Assay (KinExA®; Sapidyne Instruments Inc; Darling and Brault, 2004, Assay and Drug Dev Tech 2(6):647-657) for measuring the equilibrium binding affinity and dynamics between unmodified molecules in solution. In affinity analysis, the equilibrium dissociation constant K D and bonding rate k a The dissociation rate kd was determined experimentally, and the dissociation rate kd is k d =KD * k a It is calculated based on the formula.

[0072] K of KinExA (registered trademark) D In the analysis, one interaction partner (e.g., titration-binding partner) needs to be immobilized on a solid phase and used as a probe to capture the other interaction partner (e.g., constant-binding partner) that is released in the solution once equilibrium is reached. In each experiment, a series of solutions in which one binding partner is at a constant concentration and the other binding partner is titrated are equilibrated. The solutions are then briefly exposed to the solid phase to capture some of the released constant-binding partners and label them with fluorescent secondary molecules. The short contact time with the solid phase is shorter than the time required for the dissociation of the complex pre-formed in the solution, meaning that competition between the solution and the titrated binding partner in the solid phase is "kinetically eliminated." Since the solid phase is used only as a probe for the free-constant binding partner of each sample, the solution equilibrium does not change during the measurement. D The value is calculated from the signal generated from the captured free constant binding partner and is directly proportional to the concentration of the free constant binding partner in the equilibrated sample. The data was analyzed using KinExA® Pro software and the least squares method. D The optimal solution for the active binding site concentration (ABC) can be fitted to a curve representative of a stoichiometric relationship model, such as a 1:1 reversible bimolecule interaction.

[0073] Determining the binding rate using KinExA® can be performed in a similar manner to equilibrium analysis, except that the measurements are collected "pre-equilibrium" and the binding signal is a function of time and the total concentration of the titration binding partners. aThere are two methods that can be used to determine the amount of free constant binding partner. The "direct method" fixes the concentrations of the titrated binding partner and the constant binding partner and probes the solution over time. The amount of free constant binding partner in the solution decreases as the sample approaches equilibrium. The "infusion method" fixes the incubation time and the concentration of one partner and titrates the concentration of the other partner. As the concentration of the titrated binding partner increases, more complexes are formed, so the amount of free constant binding partner decreases.

[0074] As used in this disclosure, the terms “albumin binding” and “binding affinity to albumin” refer to the properties of a polypeptide that can be tested for B7-H3 in a manner similar to the examples described above, for example, by ELISA, SPR, BLI, QCM and / or KinExA®.

[0075] Those skilled in the art will understand that, without departing from the scope of this disclosure, various modifications and / or additions can be made to B7-H3 conjugated polypeptides in any form disclosed herein to adapt them to specific applications. For example, in one embodiment, a B7-H3 conjugated polypeptide as described herein is provided, which is extended and / or contains additional amino acids at the C-terminus and / or N-terminus. Such a polypeptide should be understood as a polypeptide having one or more amino acid residues at the first and / or last positions of the polypeptide chain. Thus, a B7-H3 conjugated polypeptide may contain any appropriate number of additional amino acid residues, for example, at least one additional amino acid residue. Each additional amino acid residue can be added individually or collectively, for example, to improve and / or simplify the production, purification, in vivo or in vitro stabilization, coupling, or detection of the polypeptide. Such additional amino acid residues may include one or more amino acid residues added for the purpose of chemical coupling. One example is the addition of a cysteine ​​residue. Additional amino acid residues can also provide "tags" for polypeptide purification or detection, such as His6 tags, (HisGlu)3 tags ("HEHEHE" tags), or "myc" (c-myc) or "FLAG" tags for interaction with tag-specific antibodies, or, in the case of His6 tags, immobilized metal affinity chromatography (IMAC).

[0076] In one embodiment, a B7-H3 binding polypeptide as described herein is provided, comprising additional amino acids at the C-terminus and / or N-terminus. For example, in one embodiment of a B7-H3 binding polypeptide as disclosed herein, it comprises any one of the sequences disclosed herein, having 0 to 30 additional C-terminal and / or N-terminal residues, e.g., 0 to 25, e.g., 0 to 20, e.g., 0 to 15, e.g., 0 to 10, e.g., 0 to 7 additional C-terminal and / or N-terminal residues. In one embodiment, the B7-H3 binding polypeptide comprises any one of the sequences disclosed herein, having 0 to 25, e.g., 0 to 15, e.g., 0 to 4, e.g., 3 additional C-terminal residues.

[0077] In one further embodiment, a B7-H3 linked polypeptide is provided herein, comprising an additional amino acid at the C-terminus and / or N-terminus, wherein the additional amino acid improves polypeptide production, purification, in vitro or in vivo stabilization, coupling, or detection.

[0078] Further amino acids as described above can be conjugated to the B7-H3 conjugated polypeptide by chemical conjugation (using known organic chemical methods), by expressing the B7-H3 conjugated polypeptide as a fusion protein, or by any other means, such as directly or via a linker, for example, an amino acid linker.

[0079] Further polypeptide domains may provide yet another B7-H3 binding moiety. Thus, in further embodiments, a B7-H3 binding polypeptide in the form of a polymer is provided. The polymer is understood to contain at least two B7-H3 binding polypeptides, as disclosed herein as monomer units, whose amino acid sequences may be identical or different. The polypeptide polymer may contain a suitable number of domains, each having a B7-H3 binding motif, and each forming a monomer within the polymer. These domains may have the same amino acid sequence or they may have different amino acid sequences. In other words, the B7-H3 binding polypeptide of the present invention may form homo- or hetero-polymers, for example, homo- or hetero-dimers. In one embodiment, a B7-H3 binding polypeptide is provided in which the monomer units are covalently bonded. In another embodiment, the B7-H3 binding polypeptide monomer units are expressed as a fusion protein. In one embodiment, a B7-H3 binding polypeptide in the form of a dimer is provided. In one particular embodiment, the dimer form is a homo-dimer form. In another embodiment, the dimer form is a heterodimer form. To clarify this point, throughout this disclosure, the term “B7-H3 conjugated polypeptide” is used to encompass all forms of B7-H3 conjugated polypeptide, i.e., both monomeric and polymeric forms.

[0080] The additional amino acids described above may, for example, comprise one or more additional polypeptide domains. These additional polypeptide domains may provide the B7-H3 binding polypeptide with other functions, such as another binding function, or an enzymatic function, or a toxic function, or a combination thereof.

[0081] Furthermore, the B7-H3 binding polypeptide as defined herein may be beneficial as part of a fusion protein or a conjugate containing a second or further portion. The second and further portions / sites of the fusion polypeptide or conjugate in such a protein may preferably possess the desired biological activity.

[0082] Accordingly, in a second aspect of this disclosure, a fusion protein or conjugate comprising the following is provided: -A first portion comprising a B7-H3 linked polypeptide according to the first embodiment; and - A second part comprising a polypeptide having the desired biological activity.

[0083] In one embodiment, the fusion protein or conjugate may further include a further portion having a desired biological activity that may be the same as or different from the biological activity of the second portion.

[0084] Non-limiting examples of desired biological activity include therapeutic activity, binding activity, and enzymatic activity. In one embodiment, a fusion protein according to a second embodiment is provided in which the biological activity is therapeutic activity. In one embodiment, a fusion protein according to a second embodiment is provided in which the biological activity is binding activity. In one embodiment, a fusion protein according to a second embodiment is provided in which the biological activity is enzymatic activity.

[0085] In one embodiment, the second portion having the desired biological activity is a therapeutically active polypeptide. Non-limiting examples of therapeutically active polypeptides are biomolecules, such as molecules selected from the group consisting of human endogenous enzymes, hormones, growth factors, chemokines, cytokines, and lymphakines.

[0086] In one embodiment of the first or second aspect of this disclosure, a B7-H3 linked polypeptide, fusion protein, or conjugate comprising an anticancer agent is provided. In one embodiment of the second aspect, the second portion in the fusion protein or conjugate is an anticancer agent. Non-limiting examples of anticancer agents for use in this context include agents selected from the group consisting of auristatin, anthracyclines, calicheamycin, combretastatin, doxorubicin, duocalmycin, CC-1065 antitumor antibiotic, ecteinsascidin, geldanamycin, meitansinoids, methotrexate, mycotoxins, taxol, lysine, bougain, geronin, Pseudomonas exotoxin 38 (PE38), diphtheria toxin (DT), and their analogues, as well as their derivatives and combinations thereof. Those skilled in the art will understand that an unrestricted example of an anticancer agent is intended to include all possible variants of the said agent, for example, the agent auristatin is intended to include, for example, auristatin E, auristatin F, auristatin PE, and their derivatives.

[0087] In one embodiment of a second aspect of this disclosure, a B7-H3 binding polypeptide, fusion protein, or conjugate is provided, where the biological activity in the second part is binding activity. In one embodiment, the binding activity is albumin-binding activity that increases the in vivo half-life of the fusion protein or conjugate and / or alters the biodistribution characteristics of the fusion protein or conjugate. In one embodiment, a fusion protein described herein is provided, where the binding activity increases the in vivo half-life of the fusion protein or conjugate. In another embodiment, a fusion protein described herein is provided, where the binding activity alters the biodistribution characteristics of the fusion protein or conjugate. In one embodiment, the binding activity acts to block biological activity. Non-limiting examples of binding activity include binding activity that increases the in vivo half-life of the fusion protein or conjugate, binding activity that alters the biodistribution characteristics of the fusion protein or conjugate, and / or binding activity that acts to block, inhibit, activate, increase, antagonize, or stimulate biological activity, for example. One example of such binding activity is binding activity that increases the in vivo half-life of a fusion protein or conjugate. In one embodiment of the fusion protein or conjugate, the in vivo half-life of the fusion protein or conjugate is longer than the in vivo half-life of the B7-H3 binding polypeptide itself. In one embodiment, the in vivo half-life is increased by at least 10 times, e.g., at least 25 times, e.g., at least 50 times, e.g., at least 75 times, e.g., at least 100 times, compared to the in vivo half-life of the B7-H3 binding polypeptide itself. Another example of such binding activity is binding activity that alters the in vivo distribution characteristics of a fusion protein or conjugate. In one embodiment of the fusion protein or conjugate, the in vivo distribution characteristics of the fusion protein or conjugate are modified so that a reduced amount of the fusion protein or conjugate is retained in the kidney compared to the B7-H3 binding polypeptide itself.In one embodiment, the retention is reduced by at least 2 times, for example, at least 4 times, for example, at least 6 times, for example, at least 8 times, for example, at least 10 times, for example, at least 15 times compared to the retention of the B7-H3 bound polypeptide itself.

[0088] As discussed, the fusion protein or conjugate may include at least one further portion having binding activity to a target. In one particular embodiment, the target is albumin, and binding to albumin increases the in vivo half-life of the fusion protein or conjugate. In one such embodiment, the albumin-binding activity is provided by the albumin-binding domain (ABD) of streptococcal protein G or a derivative thereof. Thus, the fusion protein may comprise, for example, a B7-H3 binding polypeptide in monomeric or multimeric form (such as homodimer or heterodimer form) as defined herein, and the ABD of streptococcal protein G or a derivative thereof. Derivatives of the ABD of streptococcal protein G are known to those skilled in the art, for example, from WO2009 / 016043, WO2012 / 004384 and WO2014 / 048977, all of which are incorporated herein by reference. The ABD may include an amino acid sequence selected from the group consisting of, for example, SEQ ID NOs: 547, 548, and 631. In one embodiment, the ABD includes SEQ ID NO: 547. In another embodiment, the ABD includes SEQ ID NO: 548. In a further embodiment, the ABD includes SEQ ID NO: 631. It will be understood that the albumin-binding domain (ABD) may be located at the C-terminus and / or N-terminus of the B7-H3 binding polypeptide.

[0089] In specific embodiments of this aspect of the Disclosure, a particular polypeptide of the Disclosure is used as the B7-H3 binding site, albumin binding site, and linker sequence (see further below), and the fusion protein or conjugate comprises (or comprises) an amino acid sequence selected from the group consisting of SEQ ID NOs: 550-566 and SEQ ID NOs: 632-634. In one embodiment, the amino acid sequence is selected from the group consisting of SEQ ID NOs: 550-557. In another embodiment, the amino acid sequence is selected from the group consisting of SEQ ID NOs: 558-560. In another embodiment, the amino acid sequence is selected from the group consisting of SEQ ID NOs: 561-563. In another embodiment, the amino acid sequence is selected from the group consisting of SEQ ID NOs: 564-566. In another embodiment, the amino acid sequence is selected from the group consisting of SEQ ID NOs: 632-634. In one embodiment, the amino acid sequence is selected from the group consisting of SEQ ID NOs: 557, 560, 563, and 566. In a specific embodiment, the amino acid sequence is SEQ ID NO: 557. In another specific embodiment, the amino acid sequence is SEQ ID NO: 560. In a specific embodiment, the amino acid sequence is SEQ ID NO: 563. In a specific embodiment, the amino acid sequence is SEQ ID NO: 566. In one embodiment, the amino acid sequence is selected from the group consisting of SEQ ID NOs: 555, 558, 561, and 564. In a specific embodiment, the amino acid sequence is SEQ ID NO: 555. In another specific embodiment, the amino acid sequence is SEQ ID NO: 558. In a specific embodiment, the amino acid sequence is SEQ ID NO: 561. In a specific embodiment, the amino acid sequence is SEQ ID NO: 564. In one embodiment, the amino acid sequence is selected from the group consisting of SEQ ID NOs: 556, 559, 562, and 565. In a specific embodiment, the amino acid sequence is SEQ ID NO: 556. In another specific embodiment, the amino acid sequence is SEQ ID NO: 559. In a specific embodiment, the amino acid sequence is SEQ ID NO: 562. In a particular embodiment, the amino acid sequence is SEQ ID NO: 565.In one embodiment, the amino acid sequence is selected from the group consisting of SEQ ID NOs: 632, 633, and 634. In a specific embodiment, the amino acid sequence is SEQ ID NO: 632. In another specific embodiment, the amino acid sequence is SEQ ID NO: 633. In a particular embodiment, the amino acid sequence is SEQ ID NO: 634.

[0090] In another embodiment, a fusion protein or conjugate is provided in which the second portion having the desired binding activity is a protein based on protein Z derived from the B domain of protein A from Staphylococcus aureus, having a binding affinity to targets other than B7-H3.

[0091] In another embodiment, the fusion protein or conjugate described herein is provided, wherein the second portion is selected from the group consisting of human endogenous enzymes, hormones, growth factors, chemokines, cytokines, and lymphakines.

[0092] In another embodiment, a fusion protein or conjugate is provided in which the second portion having the desired binding activity is an antibody or its antigen-binding fragment. As is well known, an antibody is an immunoglobulin molecule that can specifically bind to a target (antigen) such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., via at least one antigen-recognition site on the immunoglobulin molecule. As used herein, the term “antibody or its antigen-binding fragment” includes not only full-length or intact polyclonal or monoclonal antibodies, but also their antigen-binding fragments, e.g., Fab, Fab', F(ab')2, Fab3, Fv and its variants, fusion proteins containing one or more antibody portions, humanized antibodies, chimeric antibodies, minibodies, diabodies, triabodies, tetrabodies, linear antibodies, single-chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified configuration of an immunoglobulin molecule containing an antigen-recognition site of the desired specificity, such as glycosylated variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. Further examples of modified antibodies and their antigen-binding fragments include nanobodies, AlbudAbs, DART (biaffinity retargeting), BiTE (bispecific T-cell engager), TandAb (tandem diabody), DAF (dual-acting Fab), two-in-one o-antibodies, SMIP (small molecule immunotherapy), FynomAbs (antibody-fused finomer), DVD-Ig (bivariable domain immunoglobulin), CovX-bodies (peptide-modified antibodies), duobodies, and triomAbs. This list of antibody and antigen-binding fragment variants is not intended to be limiting, and those skilled in the art will be aware of other suitable variants.

[0093] In one embodiment, the at least one antibody or its antigen-binding fragment is selected from the group consisting of full-length antibodies, Fab fragments, Fab' fragments, F(ab')2 fragments, single-chain Fab(scFab) fragments, Fc fragments, Fv fragments, single-chain Fv(scFv) fragments, (scFv)2, scFv-Fc constructs, and domain antibodies. In one embodiment, the at least one antibody or its antigen-binding fragment is selected from full-length antibodies, Fab fragments, and scFv fragments. In one particular embodiment, the at least one antibody or its antigen-binding fragment is a full-length antibody.

[0094] In one embodiment, the antibody or its antigen-binding fragment is selected from the group consisting of monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, and their antigen-binding fragments.

[0095] In one embodiment, the antibody or its antigen-binding fragment has affinity for an antigen, such as an antigen associated with cancer.

[0096] Conjugates such as those disclosed herein can be produced by conjugating at least one B7-H3 linked polypeptide or fusion protein, as described herein, to at least one additional moiety. Those skilled in the art are aware of conjugation methods, such as conventional chemical conjugation methods using charged succinimidyl esters or carbodiimides.

[0097] Those skilled in the art recognize that the construction of fusion proteins often involves the use of linkers between fused functional sites, and that there are different types of linkers with different properties, such as flexible amino acid linkers, rigid amino acid linkers, and cleavable amino acid linkers. Linkers are useful, for example, to increase the stability or improve folding of fusion proteins, increase expression, improve biological activity, enable targeting, and alter the pharmacokinetics of fusion proteins. Accordingly, in one embodiment, a B7-H3-binding polypeptide, fusion protein, or conjugate in any form disclosed herein is provided, further comprising at least one linker, at least one selected from flexible amino acid linkers, rigid amino acid linkers, and cleavable amino acid linkers, and so on. In one embodiment, the linker is positioned between the B7-H3-binding polypeptide and further polypeptide domains. In one embodiment, the linker is positioned between two B7-H3 binding polypeptides or domains as disclosed herein; for example, between a B7-H3 binding domain and an albumin binding domain as disclosed herein; or between a B7-H3 binding domain and a Z variant binding to a different target other than B7-H3 as disclosed herein; or, for example, between a B7-H3 binding domain and an antibody or its antigen-binding fragment as disclosed herein. Flexible linkers are often used in the art when the bound domains require some degree of movement or interaction, and may be particularly useful in some embodiments. Such linkers generally consist of small nonpolar (e.g., G) or polar (e.g., S or T) amino acids. Some flexible linkers are, for example, (GGGGS) pAs shown above, it mainly consists of stretches of G and S residues. By adjusting the copy number "p", the linker can be optimized to achieve proper separation between functional sites or to maintain necessary intersite interactions. In addition to G and S linkers, other flexible linkers are known in the art, such as G and S linkers with added amino acid residues such as T and A to maintain flexibility, and G and S linkers with added polar amino acid residues to improve solubility. Non-limiting examples of linker additions include AGS, ASGS (SEQ ID NO: 589), GAPGGGGS (SEQ ID NO: 590), GAPGGGGSGGGGSGGGGSTSA (SEQ ID NO: 591), GGGGSLVPRGSGGGGS (SEQ ID NO: 592), (GS)3 (SEQ ID NO: 593), (GS)4 (SEQ ID NO: 594), (GS)8 (SEQ ID NO: 595), GGSGGHMGSGG (SEQ ID NO: 596), GGSGGSGGSGG (SEQ ID NO: 597), GGSGG (SEQ ID NO: 599) 8) Examples include GGSGGGGG (SEQ ID NO: 599), GGGSEGGGSEGGGSEGGG (SEQ ID NO: 600), AAGAATAA (SEQ ID NO: 601), GGGGG (SEQ ID NO: 602), GGSSG (SEQ ID NO: 603), GSGGGTGGGSSG (SEQ ID NO: 604), GSGSGSGSGGSG (SEQ ID NO: 605), GSGGSGGSGGSGGS (SEQ ID NO: 606), GSGGSGSGGSGGSG (SEQ ID NO: 607), GGGGSAS (SEQ ID NO: 608), and GT. Those skilled in the art are aware of other suitable linkers.

[0098] In one embodiment, the linker is a flexible linker comprising glycine (G), serine (S), and / or threonine (T) residues. In one embodiment, the linker is (G n S m ) p and (S n G m ) pThere is a general formula selected from, where independently, n=1 to 7, m=0 to 7, n+m≦8 and p=1 to 7. In one embodiment, n=1 to 5. In one embodiment, m=0 to 5. In one embodiment, p=1 to 5. In a more specific embodiment, n=4, m=1 and p=1 to 5. In one embodiment, the linker is selected from the group consisting of S4G (sequence number 609), (S4G)3 (sequence number 610) and (S4G)4 (sequence number 611). In one embodiment, the linker is selected from the group consisting of G4S (sequence number 612), (G4S)2 (sequence number 613), (G4S)3 (sequence number 614), (G4S)4 (sequence number 615) and (G4S)5 (sequence number 616). In one particular embodiment, the linker is G4S, and in another embodiment, the linker is (G4S)2. In another embodiment, the linker is (G4S)3. In another embodiment, the linker is (G4S)5.

[0099] In another embodiment, the linker is a rigid linker comprising alanine (A), glutamic acid (E), lysine (K), proline (P), and / or threonine (T) residues. Non-limiting examples of linkers include A(EAAAK)4A, (SEQ ID NO: 635), A(EAAAK)5A (SEQ ID NO: 617), (KEAAA)4KAK (SEQ ID NO: 636), (KEAAA)5KAK (SEQ ID NO: 618), and (TP)8 (SEQ ID NO: 619).

[0100] With regard to the above-mentioned fusion proteins or conjugates incorporating B7-H3-binding polypeptides according to this disclosure, it should be noted that the designations of the first, second, and further parts are made for clarity to distinguish, on the one hand, the B7-H3-binding polypeptide or polypeptide according to the present invention from, on the other hand, the identical or other functional parts. These designations are not intended to refer to the actual order of different domains in the polypeptide chain of the fusion protein or conjugate. Similarly, the designations of the first monomer unit and the second monomer unit are made for clarity to distinguish the units. Therefore, for example, the first part (or monomer unit) may appear at the N-terminus, middle, or C-terminus of the fusion protein or conjugate of this disclosure without limitation.

[0101] The above embodiments further include polypeptides further comprising a label, such as a B7-H3-conjugated polypeptide according to the first embodiment or a B7-H3-conjugated polypeptide contained in a fusion protein or conjugate according to the second embodiment. In further embodiments, the label is selected from the group consisting of fluorescent dyes and metals, chromogenic dyes, chemiluminescent compounds, bioluminescent proteins, enzymes, radionuclides, radioactive particles and pretargeting recognition tags. Such labels can be used, for example, for the detection of polypeptides, as will be well known to those skilled in the art.

[0102] In some embodiments, the labeled B7-H3 binding polypeptide exists as a portion in a fusion protein or conjugate that also includes a second or further portion having the desired biological activity. In some embodiments, the label is bound only to the B7-H3 binding polypeptide, and in other embodiments, it is bound to both the B7-H3 binding polypeptide and the second portion of the fusion protein or conjugate. Furthermore, it is also possible that the label is bound to the second portion rather than the B7-H3 binding portion. Thus, in yet another embodiment, a B7-H3 binding polypeptide including a second site is provided, where the label is bound only to the second site. Accordingly, when referring to a labeled polypeptide, this should be understood as a reference to all aspects of polypeptides as described herein, including B7-H3 binding polypeptides, fusion proteins, and conjugates containing B7-H3 binding polypeptides.

[0103] Indirect labeling of Z variant polypeptides has recently been demonstrated using pretargeting recognition tags (Westerlund et al, 2015, Bioconjugate Chem 26:1724-1736). Similarly, this disclosure provides a B7-H3 conjugated polypeptide according to this specification labeled with a pretargeting moiety, which can then be used for indirect labeling with a moiety complementary to the pretargeting moiety. In one embodiment, a B7-H3 conjugated polypeptide, fusion protein, or conjugate according to this specification is provided, which is selected from oligonucleotides / complementary oligonucleotides, such as DNA / complementary DNA, RNA / complementary RNA, phosphorothioate nucleic acid / complementary phosphorothioate nucleic acid, and peptide nucleic acid / complementary peptide nucleic acid, and morpholino / complementary morpholino, and includes a pretargeting recognition tag that can associate with a complement to form a complementary pair of a pretargeting moiety selected from, for example, strepto(avidin) / biotin. In a further embodiment, the pretargeting recognition tag is a peptide nucleic acid tag. In a further embodiment, the pretargeting recognition tag is a 10-20 mer peptide nucleic acid, such as a 15 mer peptide nucleic acid sequence. If a pretargeting moiety is included, the B7-H3 binder of this disclosure can associate with a complementary pretargeting moiety, such complementary pretargeting moiety may then contain or bind to a suitable radionuclide. Those skilled in the art will recognize a radionuclide suitable for therapeutic, diagnostic, and / or prognostic purposes. Such a radionuclide may be chelated to the complementary pretargeting moiety via a chelation environment, as is generally described for the B7-H3 binders below.

[0104] Most radionuclides have metallic properties, and metals typically cannot form stable covalent bonds with elements presented in proteins and peptides. Therefore, labeling proteins and peptides with radioactive metals is performed using chelating agents, i.e., polydentate ligands, which form non-covalent compounds called chelates with metal ions. In one embodiment of a B7-H3 linked polypeptide, fusion protein, or conjugate, the incorporation of a radionuclide is made possible by providing a chelate environment in which the radionuclide can coordinate, chelate, or complexe with the polypeptide.

[0105] One example of a chelating agent is a polyaminopolycarboxylic acid type chelating agent. These polyaminopolycarboxylate chelating agents are divided into two classes: macrocyclic chelating agents and acyclic chelating agents.

[0106] In one embodiment, a B7-H3 linked polypeptide, fusion protein, or conjugate is provided, which includes a chelate environment provided by a polyaminopolycarboxylic acid chelate linked to a B7-H3 linked polypeptide via a thiol group of a cysteine ​​residue or an amine group of a lysine residue.

[0107] The most commonly used macrocyclic chelating agents for indium, gallium, yttrium, bismuth, radioactive actinides, and radioactive lanthanides are different derivatives of DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). In one embodiment, the chelating environment for a B7-H3 linked polypeptide, a B7-H3 linked polypeptide in heterodimeric form, a fusion protein, or a conjugate is provided by DOTA or a derivative thereof. More specifically, in one embodiment, the chelated polypeptides included in this disclosure are obtained by reacting the DOTA derivative 1,4,7,10-tetraazacyclododecane-1,4,7-trisacetic acid-10-maleimidoethylacetamide (maleimidonomonoamide-DOTA) with the polypeptide. In one embodiment, the chelated polypeptides included in this disclosure are obtained by reacting the DOTA derivative DOTAGA(2,2',2''-(10-(2,6-dioxotetrahydro-2H2O-pyran-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid) with the polypeptide. Furthermore, 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and its derivatives can be used as chelating agents. Thus, in one embodiment, the chelate environment for a B7-H3-binding polypeptide, a B7-H3-binding polypeptide in heterodimeric form, a fusion protein, or a conjugate is provided by NOTA or its derivatives. In one embodiment, the chelated polypeptides included in the present disclosure are obtained by reacting the NOTA derivative NODAGA(2,2'-(7-(1-carboxy-4-((2,5-dioxopyrrolidine-1-yl)oxy)-4-oxobutyl)-1,4,7-triazonan-1,4-diyl)diacetic acid) with the polypeptide.

[0108] The most commonly used acyclic polyaminopolycarboxylic acid chelating agents are different derivatives of DTPA (diethylenetriaminepentaacetic acid). Therefore, polypeptides, fusion proteins, or conjugates having a chelating environment provided by diethylenetriaminepentaacetic acid or its derivatives are also included in this disclosure.

[0109] Another example of a chelating agent is the N3S chelating agent, which is a type of peptide-based tetradentate chelating agent. As the term N3S suggests, the four attachment or coordinating groups of such a chelator are formed from three nitrogen atoms and one sulfur atom, preferably provided by consecutive amino acid residues of a polypeptide chain. In an N3S chelating agent, the N and S atoms are spatially arranged to provide a “pocket” suitable for complex formation or attachment of radioactive metals.

[0110] In one embodiment, a B7-H3 linked polypeptide, fusion protein, or conjugate of a first embodiment is provided, comprising a chelating environment provided by a peptide-based chelating agent, wherein the peptide sequence encoding the peptide-based chelating agent is located at the C-terminus of the B7-H3 linked polypeptide, fusion protein, or conjugate.

[0111] Those skilled in the art will recognize peptide-based chelators suitable for inclusion in the polypeptide chain of the B7-H3 binding molecule according to this disclosure. For example, in one specific embodiment, the peptide sequence encoding the peptide-based chelator is selected from the group consisting of -GGGC (SEQ ID NO: 620), -GSEC (SEQ ID NO: 621), -GGSC (SEQ ID NO: 622), -GGEC (SEQ ID NO: 623), -GGKC (SEQ ID NO: 624), and -KVDC (SEQ ID NO: 625). In a specific embodiment, the peptide sequence encoding the peptide-based chelator is -GGGC.

[0112] In embodiments in which a polypeptide, fusion protein, or conjugate is directly or indirectly labeled with an imaging agent (e.g., a radiopharmaceutical) (e.g., via pretargeting as described above), the amount of labeled polypeptide present in tissue such as tumor tissue can be measured using an imaging device, such as by acquiring radioactivity counts or radiation density images, or derivatives thereof such as radiation concentration. In one embodiment, a radiolabeled B7-H3-conjugated polypeptide, fusion protein, or conjugate is provided, comprising a radiochelate of the B7-H3-conjugated polypeptide, fusion protein, or conjugate described herein and a radionuclide. In a more specific embodiment, the radionuclide is suitable for medical imaging. In another specific embodiment, the radionuclide is suitable for therapeutic use. Non-limiting examples of radionuclides suitable for either direct labeling of the B7-H3 binder or indirect labeling by labeling of a complementary pretargeting portion according to any embodiment disclosed herein include: 225 Ac, 72 As, 212 Bi, 213 Bi, 76 Br, 55 Co, 61 Cu, 64 Cu, 67 Cu, 18 F, [ 18 F]AIF, 19 F, 66 Ga, 67 Ga, 68 Ga, 166 Ho, 110m In, 111 In, 123 I, 124 I, 131 I, 177 Lu, 51 Mn, 52m Mn, 52 Mn, 212 Pb, 149 PM, 186 Re, 188 Re, 44 Sc, 153 Sm, 149 Tb, 152 Tb, 155 Tb, 161 Tb, 99mTc, 45 Ti, 227 Th, 86 Y, 90 Y and 89 Zr is one example.

[0113] In one embodiment, if the radionuclide is suitable for medical imaging, the radionuclide is, 72 As, 76 Br, 55 Co, 61 Cu, 64 Cu, 18 F, [ 18 F]AIF, 19 F, 66 Ga, 67 Ga, 68 Ga, 110m In, 111 In, 123 I, 124 I, 131 I, 177 Lu, 51 Mn, 52m Mn, 52 Mn, 186 Re, 188 Re, 44 Sc, 149 Tb, 152 Tb, 155 Tb, 161 Tb, 99m Tc, 45 Ti, 86 Y and 89 Selected from the group consisting of Zr.

[0114] In another embodiment, if the radionuclide is suitable for treatment, the radionuclide is, 225 Ac, 212 Bi, 213 Bi, 67 Cu, 166 Ho, 177 Lu, 212 Pb, 149 PM, 186 Re, 188 Re, 153 Sm, 149 Tb, 161 Tb, 227 Th and 90Selected from the group consisting of Y.

[0115] In one embodiment, the imaging device used for such measurement is a positron emission tomography (PET) scanner, in which case the radionuclide is selected to be suitable for PET. Those skilled in the art know radionuclides suitable for use in PET. For example, radionuclides for PET are: 72 As, 76 Br, 55 Co, 61 Cu, 64 Cu, 18 F, [ 18 F]AIF, 66 Ga, 68 Ga, 110m In, 44 Sc, 152 Tb, 45 Ti, 86 Y and 89 Selected from the group consisting of Zr. In one embodiment, a radiolabeled B7-H3-conjugated polypeptide, fusion protein, or conjugate described herein is provided, wherein the nuclide is suitable for use in PET. 72 As, 76 Br, 55 Co, 61 Cu, 64 Cu, 18 F, [ 18 F]AIF, 66 Ga, 68 Ga, 110m In, 44 Sc, (52 Tb, 45 Ti, 86 Y and 89 Selected from the group consisting of Zr.

[0116] In another embodiment, the imaging apparatus used is a single-photon emission computed tomography (SPECT) apparatus, in which case the radionuclides are selected to be suitable for SPECT. Those skilled in the art know which radionuclides are suitable for use in SPECT. For example, SPECT radionuclides are: 67 Ga, 111 In, 123 I, 131 I, 177 Lu,155 Tb, 99m Selected from the group consisting of Tc. In another embodiment, a radiolabeled B7-H3-binding polypeptide, fusion protein, or conjugate described herein is provided, wherein the nuclide is suitable for use in SPECT. 67 Ga, 111 In, 123 I, 131 I, 177 Lu, 155 Tb, 99m Selected from the group consisting of Tc.

[0117] In another embodiment, the nuclide is suitable for medical imaging, and the chelate environment is provided by a peptide-based chelating agent as described herein. In a more specific embodiment, the nuclide is 99m Tc, 51 Mn, 52m Mn, 52 Mn, 186 Re and 188 Selected from the group consisting of Re. In a particular embodiment, radionuclides suitable for medical imaging are, 99m It is Tc.

[0118] In an alternative embodiment, the radionuclide is suitable for therapeutic use, and the chelate environment is provided by a peptide-based chelating agent as described herein. In a more specific embodiment, the radionuclide is 186 Re and 188 Selected from the group consisting of Re. In a particular embodiment, the radionuclide suitable for treatment is, 188 It is Re.

[0119] In these embodiments of the radiolabeled B7-H3 binding polypeptide, fusion protein, or conjugate according to the second embodiment, the radionuclide is complexed with the B7-H3 binding polypeptide, fusion protein, or conjugate via a chelate environment provided by the C-terminal GGGC sequence.

[0120] Therefore, in one embodiment, direct or indirect radionuclide labeling, for example, 72 As, 76 Br, 55 Co, 61 Cu, 64 Cu, 18 F, [ 18 F]AIF, 19 F, 66 Ga, 67 Ga, 68 Ga, 110m In, 111 In, 123 I, 124 I, 131 I, 177 Lu, 44 Sc, 99m Tc, 45 Ti, 86 Y and 89 A radioactive nuclide selected from the group consisting of Zr, for example, 68 Ga, 110m In, 18 F, 152 Tb, 155 Tb, 45 Ti, 44 Sc, 61 Cu, 66 Ga, 64 Cu, 55 Co, 72 As, 86 Y, 89 Zr, 124 I and 76 A radionuclide selected from the group consisting of Br, for example, 18 The following B7-H3 binding polypeptides, fusion proteins, or conjugates, comprising F, are provided herein.

[0121] In some embodiments, the labeled B7-H3 binding polypeptide exists as a portion in a fusion protein or conjugate that also includes a second portion having the desired biological activity. In some examples, the label is coupled only to the B7-H3 binding polypeptide, and in some examples, it is coupled to both the B7-H3 binding polypeptide and the second portion of the fusion protein or conjugate. Furthermore, it is also possible for the label to be conjugated only to the second portion and not to the B7-H3 binding portion. Thus, in yet another embodiment, a B7-H3 binding polypeptide is provided which includes a second portion, wherein the label is coupled only to the second portion.

[0122] Where a labeled polypeptide is mentioned, it should be understood as a reference to all embodiments of the polypeptides described herein, including B7-H3 conjugate polypeptides, fusion proteins, and conjugates containing B7-H3 conjugate polypeptides. Therefore, a labeled polypeptide may comprise only a B7-H3 conjugate polypeptide and, for example, a radionuclide chelated or covalently bonded to the B7-H3 conjugate polypeptide, or it may comprise a B7-H3 conjugate polypeptide, a radionuclide, and a second portion such as a small molecule with desired biological activity, e.g., a small molecule with therapeutic effect. A labeled polypeptide may comprise a B7-H3 conjugate polypeptide in heterodimer form and, for example, a radionuclide chelated or covalently bonded to the B7-H3 conjugate polypeptide, or a B7-H3 conjugate polypeptide in heterodimer form, a therapeutic radionuclide, and a second portion such as a small molecule with desired biological activity, e.g., a small molecule with therapeutic effect. Furthermore, the radionuclides described herein can serve multiple purposes; that is, they can satisfy both the capabilities of a radionuclide suitable for therapy and the capabilities of a radionuclide suitable for medical imaging. Thus, molecules labeled with radionuclides are said to be suitable as theranostics, which are molecules suitable for both therapy and medical imaging. The term “theranostic” as used herein can be used interchangeably with the term “ceragnostic.” Furthermore, as those skilled in the art will understand, a theranostic molecule or theranognostic molecule can also be understood as: 1) the same molecule, such as a polypeptide or fusion protein, labeled with the same radionuclide for both imaging and therapy, as described above; 2) the use of the same molecule, such as a polypeptide or fusion protein, but labeled with different radionuclides for imaging and therapy; and / or 3) the use of different molecules, such as different polypeptides or different fusion proteins, labeled with different radionuclides for imaging and therapy, respectively. In this way, the theranostic characteristic can also be satisfied, such as one radionuclide being suitable for therapy and the other radionuclide being suitable for medical imaging. Those skilled in the art will be able to make many other possible modifications based on the teachings herein.Labeled polypeptides or fusion proteins are suitable for both therapeutic applications and medical imaging for detection and monitoring of therapeutic responses. Furthermore, it is possible to attach payloads other than radionuclides via cysteine ​​residues. Examples of cytotoxic payloads that can be bound to cysteine ​​residues include: maytansinoids, e.g., DM1 and DM4; auristatins, e.g., monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF); DNA damaging agents, e.g., duocalmycin, pyrrolobenzodiazepine (PBD), and caliciamycin; camptothecin such as SN-38; taxanes; tubulidine; STING agonists; TLR agonists; topoisomerase II inhibitors; small interfering RNAs; antisense oligonucleotides; and proteolytic payloads.

[0123] Further embodiments of this disclosure provide a polynucleotide encoding a B7-H3-binding polypeptide or fusion protein as described herein; an expression vector containing the polynucleotide; and a host cell containing the expression vector. Also included in this disclosure is a method for producing such a B7-H3-binding polypeptide or fusion protein, comprising culturing the host cell under conditions that allow expression of the polypeptide from its expression vector, and isolating the polypeptide.

[0124] The B7-H3 binding polypeptides or fusion proteins of this disclosure may, alternatively, be produced by non-biological peptide synthesis using amino acids and / or amino acid derivatives having protected reactive side chains, said non-biological peptide synthesis includes: - Stepwise coupling of amino acids and / or amino acid derivatives to form polypeptides or fusion proteins according to this specification having protected reactive side chains, - Removing protecting groups from the reactive side chains of polypeptides or fusion proteins, and - Folding polypeptides or fusion proteins in an aqueous solution.

[0125] In another embodiment, a composition is provided comprising a B7-H3 binding polypeptide, fusion protein, or conjugate described herein and at least one pharmaceutically acceptable excipient or carrier. In one embodiment, the composition further comprises at least one additional activator, e.g., at least two additional activators, e.g., at least three additional activators. Non-limiting examples of additional activators that may prove useful in such combinations are anticancer agents, such as those described herein in relation to the first and second embodiments of this disclosure.

[0126] The small size and robustness of the B7-H3 conjugated polypeptides of this disclosure offer several advantages compared to conventional monoclonal antibody-based therapies. Such advantages include advantages in formulation, modes of administration such as alternative routes of administration, administration at higher molar doses than antibodies, and the absence of Fc-mediated side effects.

[0127] It should be understood that the B7-H3 conjugated polypeptides described herein are typically useful as therapeutic agents, diagnostic agents, and / or prognostic agents on their own. Therapeutic effects may be achieved, for example, by antagonizing the action of B7-H3.

[0128] Accordingly, in one aspect of this disclosure, a B7-H3 linked polypeptide, fusion protein, conjugate or composition described herein is provided for use, for example, in vivo, as a pharmaceutical, diagnostic and / or prognostic agent.

[0129] In one embodiment, the B7-H3 conjugated polypeptide, fusion protein, conjugate, or composition is provided for use as a pharmaceutical. In a more specific embodiment, the B7-H3 conjugated polypeptide, fusion protein, conjugate, or composition described herein is provided for use as a pharmaceutical, wherein the polypeptide, fusion protein, conjugate, or composition modulates B7-H3 function in vivo. As used herein, the term “modulate” means altering the activity, such as partially or completely inhibiting B7-H3 function.

[0130] In one embodiment, the B7-H3 conjugated polypeptide, fusion protein, conjugate, or composition is provided for use as a diagnostic agent and / or prognostic agent. In another embodiment, the B7-H3 conjugated polypeptide, fusion protein, conjugate, or composition is provided for use as an in vivo diagnostic agent and / or prognostic agent. In a more specific embodiment, the B7-H3 conjugated polypeptide, fusion protein, conjugate, or composition described herein is provided for use as an in vivo diagnostic agent. In another more specific embodiment, the B7-H3 conjugated polypeptide, fusion protein, conjugate, or composition described herein is provided for use as an in vivo prognostic diagnostic agent. In another specific embodiment, the B7-H3 conjugated polypeptide, fusion protein, conjugate, or composition described herein is provided for use as a diagnostic agent, wherein the use in diagnosis is carried out in vitro. In another specific embodiment, the B7-H3 conjugated polypeptide, fusion protein, conjugate, or composition described herein is provided for use as a prognostic diagnostic agent, wherein the use in prognostic diagnosis is carried out in vitro.

[0131] In one embodiment, a B7-H3-binding polypeptide, fusion protein, conjugate, or composition described herein is provided for use in the treatment, prognosis, or diagnosis of B7-H3-related disorders or diseases.

[0132] As used herein, the term “B7-H3 related disorder or disease” means any disorder, disease or condition in which the action of B7-H3 plays a role, and / or any disorder, disease or condition in which targeting or modulation (e.g., inhibition) of B7-H3 may be beneficial. Such disorders, diseases or conditions may be selected from the group consisting of cancers. Non-limiting examples include breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, liver cancer including hepatocellular carcinoma, lung cancer including non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, sarcomas including osteosarcoma, urothelial cell carcinoma, gliomas including neuroblastoma, medulloblastoma, glioblastoma and diffuse pontine glioma, melanoma, leukemia and mesothelioma.

[0133] In one embodiment, a B7-H3-binding polypeptide, fusion protein, conjugate, or composition is provided for use in the treatment, diagnosis, or prognosis of a B7-H3-related disorder or disease, wherein the B7-H3-related disorder or disease is cancer. In one embodiment, the cancer is selected from the group consisting of breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, liver cancer including hepatocellular carcinoma, lung cancer including non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, prostate cancer, pancreatic cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, sarcoma including osteosarcoma, urothelial cell carcinoma, neuroblastoma, medulloblastoma, glioblastoma, and diffuse pontine glioma, melanoma, leukemia, and mesothelioma. In a particular embodiment, the cancer is selected from the group consisting of breast cancer, pancreatic cancer, and sarcoma.

[0134] It is understood that the B7-H3 linked polypeptide, fusion protein, conjugate, or composition may be used as a standalone therapeutic agent, diagnostic agent, or prognostic agent, or as a companion therapeutic agent, companion diagnostic agent, and / or companion prognostic agent.

[0135] Thus, in one embodiment of therapeutic use, it is beneficial to administer a therapeutically effective amount of the B7-H3 linked polypeptide, fusion protein, conjugate, or composition described herein together with at least one second drug substance, such as an anticancer agent.

[0136] The agents of this disclosure are intended to be administered orally, topically, intravenously, intraperitoneally, subcutaneously, intrapulmonaryly, transdermally, intramuscularly, intranasally, buccally, sublingually, or via suppository. In particular, intravenous or subcutaneous administration is preferred for imaging and radiotherapy applications. Accordingly, in another aspect of this disclosure, B7-H3 linked polypeptides, fusion proteins, conjugates or compositions for use as described herein are provided, where the administration is selected from the group consisting of oral administration, topical administration, intravenous administration, intraperitoneally, subcutaneously, intrapulmonaryly, transdermally, intramuscularly, intranasally, buccally, sublingually, or via suppository, for example, intravenously.

[0137] In another aspect of this disclosure, a method for treating a B7-H3 related disorder is provided, comprising administering an effective amount of a B7-H3 binding polypeptide, fusion protein, conjugate, or composition described herein to a subject in need thereof.

[0138] In one embodiment, a method is provided for treating a B7-H3-related disorder, comprising administering an effective amount of a B7-H3-binding polypeptide, fusion protein, conjugate, or composition described herein to a subject in need thereof, wherein the B7-H3-binding polypeptide, fusion protein, or conjugate modulates B7-H3 function in vivo.

[0139] In a further aspect of this disclosure, a method is provided for detecting the presence of B7-H3 in a sample, comprising: providing a sample suspected to contain B7-H3; contacting the sample with a B7-H3-binding polypeptide, fusion protein, conjugate, or composition as defined herein; and detecting the binding of the B7-H3-binding polypeptide, fusion protein, conjugate, or composition to indicate the presence of B7-H3 in the sample.

[0140] In another aspect of this disclosure, a method for determining the presence of B7-H3 in a subject is provided, comprising the following steps: a) A step of contacting a subject, or a sample isolated from a subject, with a B7-H3 binding polypeptide, fusion protein, conjugate, or composition described herein; and b) A step of obtaining a value corresponding to the amount of B7-H3 bound polypeptide, fusion protein, conjugate, or composition bound to the subject or sample.

[0141] In another aspect of this disclosure, a method is provided for determining the presence of B7-H3 in a subject, as described herein, the method being a medical imaging method: -Step a) comprises systemically administering the B7-H3 linked polypeptide, fusion protein, conjugate, or composition; - The B7-H3 linked polypeptide, fusion protein, conjugate, or composition comprises a radionuclide label suitable for medical imaging; and -Step b) includes obtaining one or more images of at least a portion of the object using a medical imaging device, wherein the images indicate the presence of radionuclides in the body.

[0142] In one embodiment of the method for medical imaging, the B7-H3 binding polypeptide, fusion protein, or conjugate comprises a pretargeting recognition tag as described herein, or the composition comprises such a B7-H3 binding polypeptide, fusion protein, or conjugate, and step a) further comprises bringing a target into contact with a complementary pretargeting moiety labeled with a detectable label such as a radionuclide label.

[0143] In a related embodiment, an in vivo diagnostic method is provided, comprising the following steps: - The step of contacting the B7-H3 linked polypeptide, fusion protein, conjugate, or composition described herein; - A step of detecting the binding of a B7-H3-binding polypeptide, fusion protein, conjugate, or composition to indicate the presence of B7-H3 in the subject; and - The process of confirming the diagnosis using the information obtained.

[0144] In another related embodiment, an in vivo prognostic diagnostic method is provided, comprising the following steps: - The step of contacting the B7-H3 linked polypeptide, fusion protein, conjugate, or composition described herein; - A step of detecting the binding of a B7-H3-binding polypeptide, fusion protein, conjugate, or composition to indicate the presence of B7-H3 in the subject; and - The process of determining the prognosis using the information obtained.

[0145] In a related embodiment, an in vitro diagnostic method comprising the following steps is provided: - A step of providing a sample suspected to contain B7-H3; - The step of contacting the sample with a B7-H3 linked polypeptide, fusion protein, conjugate, or composition described herein; - A step of detecting the binding of a B7-H3-binding polypeptide, fusion protein, conjugate, or composition to indicate the presence of B7-H3 in the sample; and - The process of confirming the diagnosis using the information obtained.

[0146] In another related embodiment, an in vitro prognostic diagnostic method is provided, comprising the following steps: - A step of providing a sample suspected to contain B7-H3; - The step of contacting the sample with a B7-H3 linked polypeptide, fusion protein, conjugate, or composition described herein; - A step of detecting the binding of a B7-H3-binding polypeptide, fusion protein, conjugate, or composition to indicate the presence of B7-H3 in the sample; and - The process of determining the prognosis using the information obtained.

[0147] The embodiments described above may include further steps such that the described method is part of a monitoring process for a subject before, during, or after treatment. Accordingly, relevant embodiments provide an in vivo or in vitro diagnostic or prognostic method, as described herein, further comprising the following steps: - A step of repeating a detection step, wherein the detection is performed at several time points in time, at the same provided sample or at different provided samples, as part of monitoring of the subject before, during, or after treatment.

[0148] In another embodiment, the method for in vitro diagnosis or prognosis described herein is provided, further comprising the step of comparing a value corresponding to the amount of a bound B7-H3 bound polypeptide with a reference value.

[0149] In another embodiment, the diagnosis or prognosis relates to a B7-H3 related disorder or disease.

[0150] In one embodiment of each method for in vivo diagnosis and in vivo prognosis, the B7-H3 binding polypeptide, fusion protein, or conjugate comprises a pretargeting recognition tag as described herein, or the composition comprises such a B7-H3 binding polypeptide, fusion protein, or conjugate, and the contact step further comprises contacting the subject with a complementary pretargeting moiety labeled with a detectable label such as a radionuclide label.

[0151] Those skilled in the art will understand that any statements relating to the use of the B7-H3 linked polypeptides, fusion proteins, conjugates, or compositions described herein for the treatment, diagnosis, or prognosis of a disease or disorder are equally relevant to the relevant therapeutic, diagnostic, imaging, prognostic, or theranostic methods of this disclosure. For brevity, such statements will not be repeated here.

[0152] While the present invention has been described with reference to various exemplary aspects and embodiments, it will be understood by those skilled in the art that various modifications can be made and equivalents can be substituted for its elements without departing from the scope of the invention. In addition, many modifications can be made to adapt specific situations or molecules to the teachings of the invention without departing from the essential scope of the invention. Accordingly, the present invention is not limited to any particular embodiment intended, but is intended to include all embodiments that fall within the scope of the appended claims. [Brief explanation of the drawing]

[0153] [Figure 1] Figure 1 shows an example of sensorgrams of four His6-Z variants bound to B7-H3(2Ig)-Fc, analyzed by surface plasmon resonance on a single-cycle kinetic screen, as described in Example 4: ZBH480 (top solid line), ZBH482 (middle solid line), and ZBH481 (bottom solid line), with reference sequence ZAC12 (dashed line). [Figure 2]Figure 2 shows the binding of the indicated Z variant to B7-H3 expressing SKOV-3 cells, as evaluated by fluorescence intensity as described in Example 4. Cells were incubated with (A) the recombinantly generated His6-Z variant and (B) the synthesized Z variant, with decreasing concentrations (from 500 nM to 32 pM). [Figure 3] Figure 3 shows an example of circular dichroism (CD) spectra collected as described in Example 4. (A) Collected CD spectra of ZBH480 before (dashed line) and after (solid line) thermal induction modification, (B) Melting curve of ZBH480. [Figure 4-1] Figure 4 shows examples of sensorgrams of the Z variant ZBH538 that binds to Fc-fusion B7-H3 from (A) human (B7-H3(2Ig)-Fc), (B) cynomolgus monkey (cB7-H3(4Ig)-Fc), (C) mouse (mB7-H3(2Ig)-Fc), and (D) rat (rB7-H3(2Ig)-Fc), analyzed by surface plasmon resonance as described in Example 4. The Z variant was injected at four concentrations (0.56 nM (lower dashed line), 1.67 nM, 5 nM, and 15 nM (upper solid line)) onto Fc-fusion B7-H3 proteins trapped on the surface of a protein A chip. [Figure 4-2] Figure 4 shows examples of sensorgrams of the Z variant ZBH538 that binds to Fc-fusion B7-H3 from (A) human (B7-H3(2Ig)-Fc), (B) cynomolgus monkey (cB7-H3(4Ig)-Fc), (C) mouse (mB7-H3(2Ig)-Fc), and (D) rat (rB7-H3(2Ig)-Fc), analyzed by surface plasmon resonance as described in Example 4. The Z variant was injected at four concentrations (0.56 nM (lower dashed line), 1.67 nM, 5 nM, and 15 nM (upper solid line)) onto Fc-fusion B7-H3 proteins trapped on the surface of a protein A chip. [Figure 5]Figure 5 shows an example of sensorgrams of six His6-Z variants from the second maturation binding to B7-H3(2Ig)-Fc (solid gray lines; ZBH001, ZBH002, ZBH003, ZBH007, ZBH009, and ZBH011) analyzed by surface plasmon resonance on a single-cycle kinetic screen, as described in Example 7. A reference variant ZAC12 (dashed black line) and ZBH481 (solid black line) from the first maturation were included for comparison, analyzed in parallel. [Figure 6] Figure 6 shows the binding of six His6-Z variants to B7-H3 expressing SKOV-3 cells, as evaluated by fluorescence intensity as described in Example 7. The cells were incubated with decreasing concentrations of each indicated His6-Z variant (from 500 nM to 21 pM). For comparison, the reference variant ZAC12 (open-round) and ZBH481 (open-square) from initial maturation were analyzed in parallel. [Figure 7] Figure 7 shows an example of circular dichroism (CD) spectra recovered as described in Example 7. (A) CD spectra recovered before (dashed line) and after (solid line) thermal induction modification of ZBH001, and (B) melting curve of ZBH001. [Figure 8] Figure 8 shows the SP analysis of the binding of the dimeric polypeptide ZBHD01 (dashed gray line) to B7-H3 and the binding of the monomer Z variant ZBH538 (solid black line), as described in Example 8. The variant was injected onto B7-H3 (B7-H3(4Ig)-Fc) captured on a protein A chip. [Figure 9] Figure 9 shows the binding of the dimeric polypeptide ZBHD01 to the monomer Z variant ZBH538 in SKOV-3 cells, both incubated at reduced concentrations (from 555 nM to 28 pM) as described in Example 8. [Figure 10]Figure 10 shows the in vitro binding specificity of (A) 99mTc-ZBH536, (B) 99mTc-ZBH538, (C) 99mTc-ZBH539, and (D) reference 99mTc-ZAC12c to BT474 and SKOV-3 cells, as described in Example 10. Due to pre-saturation of B7-H3, a 200-fold molar excess of unlabeled Z variant was added before the addition of the labeled conjugate. Data are normalized to the mean cell-associated radioactivity of non-blocked cells of each cell line. Data are shown as mean ± SD of the three samples. [Figure 11] Figure 11 shows interaction maps of the binding of (A) 99mTc-ZBH536, (B) 99mTc-ZBH538, (C) 99mTc-ZBH539, and (D) reference 99mTc-ZAC12c to SKOV-3 cells. Input data were obtained from ligand tracer measurements of cell binding activity during binding of labeled conjugates to SKOV-3 cells and dissociation from cells. Binding was measured at two concentrations (1 nM and 3 nM) for 99mTc-ZBH536, 99mTc-ZBH538, and 99mTc-ZBH539, and at three concentrations (2 nM, 6 nM, and 18 nM) for 99mTc-ZAC12c. Measurements were performed in duplicate. [Figure 12] Figure 12 shows the comparative in vivo distribution of labeled 99mTc-labeled Z variants in various organs and tissues of female NMRI mice 4 hours after injection. 3 μg (60 kBq) of the labeled conjugate was injected into the tail vein. Data are expressed as the percentage of administered activity (injection probe) per gram of tissue (%ID / g), representing the mean ± SD of four mice. [Figure 13] Figure 13 shows (A) in vivo distribution and (B) tumor-to-organ ratio of the labeled 99mTc-labeled Z variant 4 hours after injection in BALB / C nu / nu mice carrying SKOV-3 xenografts. 3 μg of the labeled conjugate (60 kBq) was injected into the tail vein. Data are expressed as %ID / g and represent the mean ± SD of four mice. [Figure 14]Figure 14 shows the uptake of 99mTc-ZBH538 4 hours after injection in SKOV-3 (B7-H3 positive) and Ramos (B7-H3 negative) xenografts. Data are expressed in %ID / g and represent the mean ± SD of four mice. P-values ​​were obtained using an unpaired t-test. [Figure 15] Figure 15 shows imaging of (A) 99mTc-ZBH536, (B) 99mTc-ZBH538, (C) 99mTc-ZBH539, and (D) reference 99mTc-ZAC12c in BALB / C nu / nu mice with B7-H3-positive SKOV-3 xenografts 4 hours after injection. 3 μg of labeled Z variant (6 MBq) was injected into the tail vein. Arrows indicate tumor (T) and liver (L). [Figure 16] Figure 16 shows imaging of (A) 99mTc-ZBH536, (B) 99mTc-ZBH538, and (C) 99mTc-ZBH539 in BALB / C nu / nu mice with B7-H3 negative Ramos xenografts 4 hours after injection. 3 μg of labeled Z variant (6 MBq) was injected into the tail vein. Arrows point to tumors (T). [Figure 17] Figure 17 shows the interaction map of binding to SKOV-3 cells by (A) 111In-ZBH538 and (B) reference 111In-ZAC12c. Input data were obtained from ligand tracer measurements of cell binding activity during binding of labeled conjugates to SKOV-3 cells and dissociation from SKOV-3 cells. Binding was measured at three different concentrations: 111In-ZBH538 at 1, 3, and 9 nM, and 111In-ZAC12c at 2, 6, and 18 nM. Measurements were performed in duplicate. [Figure 18] Figure 18 shows the in vivo distribution of 111In-ZBH538 and reference 111In-ZAC12c 4 hours (A) and 24 hours (B) after injection in BALB / C nu / nu mice carrying SKOV-3 xenografts. [Figure 19] Figure 19 shows the tumor-to-organ ratios of 111In-ZBH538 and reference 111In-ZAC12c 4 hours (A) and 24 hours (B) after injection in BALB / C nu / nu mice carrying SKOV-3 xenografts. [Figure 20] Figure 20 shows (A) biodistribution and (B) tumor-to-organ ratio of a direct comparison of 111In-labeled and 99mTc-labeled ZBH538 and reference ZAC12c 4 hours after injection in BALB / C nu / nu mice with SKOV-3 xenografts. [Figure 21] Figure 21 shows imaging of 111In-ZBH538 and reference 111In-ZAC12c 4 hours after injection in BALB / C nu / nu mice with (A) B7-H3 positive SKOV-3 xenografts and (B) B7-H3 negative Ramos xenografts. Arrows point to tumor (T) and liver (L). [Figure 22] Figure 22 shows the SPR analysis of binding of the ABD fusion polypeptide ZBHD02-ZBHD09 to B7-H3. The sensorgram shows that a 45 nM variant was injected onto immobilized B7-H3(4Ig)-His. [Figure 23] Figure 23 shows the SPR analysis results for binding to HSA and MSA, respectively, by ABD fusion polypeptides ZBHD02, ZBHD07, ZBHD08, and ZBHD09. [Figure 24] Figure 24 shows the fluorescence intensity of the binding of the ABD fusion polypeptide ZBHD02-ZBHD09 to B7-H3 expressing SKOV-3 cells. The cells were incubated with decreasing concentrations of the specified polypeptides (from 500 nM to 0.16 nM). [Figure 25] Figure 25 shows the in vivo distribution of ABD-fused Z variants in BALB / C nu / nu mice carrying SKOV3 xenografts. (A) 177Lu-ZBHD02, 177Lu-ZBHD07, 177Lu-ZBHD08, and 177Lu-ZBHD09 48 hours after injection. (B) 177Lu-ZBHD02, 24, 48, and 168 hours after injection. [Figure 26] Figure 26 shows images of (A) 177Lu-ZBHD02, (B) 177Lu-ZBHD07, (C) 177Lu-ZBHD08, and (D) 177Lu-ZBHD09 in BALB / C nu / nu mice carrying a B7-H3 positive SKOV-3 xenograft, 48 hours after injection. [Figure 27] Figure 27 shows imaging of 177Lu-ZBHD02 48 hours after injection in BALB / C nu / nu mice with (A) B7-H3 positive SKOV-3 xenografts and (B) B7-H3 negative Ramos xenografts. Arrows point to tumors (T). [Figure 28] Figure 28 shows the in vivo B7-H3 specificity of the Z variants. Uptake of (A) 68Ga-ZBH001, (B) 68Ga-ZBH002, (C) 68Ga-ZBH003, and (D) reference 68Ga-ZAC12 at 2 hours post-injection in SKOV-3 (B7-H3 positive) and Ramos (B7-H3 negative) xenografts. Data are expressed as %ID / g, mean ± SD of four mice. [Figure 29] Figure 29 shows (A) in vivo distribution and (B) tumor-to-organ ratio 2 hours after injection of labeled 68Ga-labeled Z variant into BALB / C nu / nu mice carrying SKOV-3 xenografts. 2 μg of labeled conjugate (400 kBq) was injected into the tail vein. Data are expressed as %ID / g and represent the mean ± SD of four mice. [Figure 30] Figure 30 shows imaging 2 hours after injection of (A) 68Ga-ZBH003, (B) 68Ga-ZBH001, (C) 68Ga-ZBH002, and (D) reference 68Ga-ZAC12 into BALB / C nu / nu mice carrying B7-H3 positive SKOV-3 xenografts. [Figure 31] Figure 31 shows imaging of 68Ga-ZBH003 after injection in BALB / C nu / nu mice with (A) B7-H3 positive SKOV-3 xenografts and (B) B7-H3 negative Ramos xenografts. Arrows indicate tumors (T). [Modes for carrying out the invention]

[0154] [Examples]

[0155] overview The following examples disclose the development of novel Z variant molecules targeting B7-H3 based on phage display technology. Polypeptides selected as described herein are sequenced, and their amino acid sequences are listed in the sequence listing along with the indicated sequence identifiers. The examples further describe the characterization of these selected B7-H3 binding polypeptides, their variants and derivatives, and fusion proteins containing them, and demonstrate their in vitro and in vivo functionality.

[0156] Table 2 shows the various B7-H3 proteins used in the examples.

[0157] [Table 2]

[0158] Example 1 Design and construction of a matured library of B7-H3-bound Z variants overview In this example, a phage selection library was partially designed based on four B7-H3 binding variants previously identified by yeast display technology, as described by Stern et al. (op.) and WO2020041626. The mature library was approximately 6 × 10⁶ 9 It contained several clones.

[0159] Materials and methods Design and construction of affinity-mature B7-H3 libraries The library was partially designed based on the four B7-H3 binding sequences described by Stern et al., as previously cited. Following a strategy based primarily on the binding motif of the Z variant defined in SEQ ID NOs. 541-544, the 13 surface-exposed positions of the Z molecular scaffold were biased towards specific amino acid residues. The library design is shown in Table 3, indicating the percentage of amino acids used at each of the 13 randomized positions.

[0160] [Table 3]

[0161] Using TRIM technology, we synthesized two oligonucleotides with complementary 3' ends, one forward and the other reverse complementary. These oligonucleotides were ordered from Ella Biotech GmbH (Martinsried, Germany).

[0162] The library was constructed basically as described above (for example, PCT publication WO2017 / 072280), using the vector indicated as pAY04242 in this case, with the following exceptions: 1) Transformation was performed on electrocompetent Escherichia coli (E. coli) XL-1 blue cells (Agilent Technologies, cat.), after electroporation, the cells were pooled and incubated in recovery medium (Lucigen) at 37°C for 60 minutes, and then cultured for approximately 8 hours in 2L TSB-YE medium [30g / L trypsin soybean broth, 5.0g / L yeast extract] supplemented with 10μg / mL tetracycline and 100μg / mL carbenicillin. The quality and amino acid distribution of the library were verified by sequencing, basically as described in WO2009 / 077175. In this library, the albumin-binding domain (ABD, Streptococcus strain The GA3 domain (SEQ ID NO: 548) of protein G derived from G148 is used as a fusion partner for the Z variant.

[0163] Preparation of library phage stocks: Cells from a glycerol stock containing a phagemid library were inoculated into 3L TSB [30g / L trypsin-containing soybean broth] supplemented with 100μg / mL carbenicillin, 10μg / mL tetracycline, and 1% glucose, and analyzed at an optical density of 600nm (OD). 600The cells were cultured at 70 rpm and 37°C until the ratio reached 0.79. These cultures were infected with a 50 × molar excess of M13K07 helper phage (New England Biolabs), and the cells were cultured at 37°C for 1.5 hours. The cells were pelletized and resuspended in 3 L TSB+YE supplemented with 100 μg / mL carbenicillin, 25 μg / mL neomycin, and 0.1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG). Culture was carried out at 30°C and 70 rpm, and harvested after 20 hours. Cells in culture were removed by centrifugation. The phage particles were precipitated twice from the supernatant using polyethylene glycol / sodium chloride (PEG / NaCl), essentially filtered as described in Gronwall et al., 2007 J Biotechnol 128:162-183, and finally dissolved in phosphate-buffered saline (PBS: Dulbecco's Gibco DPBS; 9.6 mM phosphate, 138 mM NaCl, 2.67 mM KCl, pH 7.4) and glycerol. The phage stock was stored at -80°C until use for selection.

[0164] result Building a Library The library was designed based on the four B7-H3 binding mutants described by Stern et al., cited above. The theoretical size of the designed library is 7.3 × 10⁻⁶. 15 It was the Z variant. The actual library size was determined by titration after transformation into E. coli. The actual library size, determined by titration after transformation into XL-1 Blue cells, was 6 × 10⁶. 9 The organisms were transformants. Library quality was tested by determining the nucleotide sequences of 96 transformants and comparing their actual sequences to the theoretical design. Analysis of the nucleotide sequences of individual libraries confirmed that the codon distribution was in line with the theoretical design. The library was named Zlib008B7-H3.I.

[0165] Example 2 Selection and screening of B7-H3 bound Z variants overview In this example, two different recombinantly produced extracellular domains of human B7-H3 were used as targets in phage display selection using a mature phage library of Z variants. The selected clones were bulk cloned into an expression vector driven by a T7 promoter, sequenced, produced in E. coli, and assayed against B7-H3 by ELISA (enzyme-linked immunosorbent assay).

[0166] Materials and methods Phage display selection for B7-H3-bound Z variants Phage stocks of the library Zlib008B7-H3.I, designed and constructed as described in Example 1, were used for selection against two different biotinylated B7-H3 target proteins, b-B7-H3(4Ig)-His_1 and b-B7-H3(4Ig)-His_2 (see Table 2). In the first cycle, selection was performed using streptavidin solid-phase beads (SA beads, Speedbead magnetic SA, Cytiva) on tracks 1-2 and solution on tracks 3-10. In the third cycle, neutravidin beads (NA beads, Speedbead magnetic NA, Cytiva) were used. As selection progressed, tracks 1-4 were further divided by target concentration, number of washes, and / or time.

[0167] To reduce the amount of background binder, pre-selection was performed using SA beads in the first and second cycles, and NA beads in the third cycle. For pre-selection, phage stocks were incubated with coated beads at RT for 30-40 minutes. The beads used for pre-selection or selection were pre-blocked with PBS supplemented with 3% bovine serum albumin (BSA, Sigma-A) and 0.1% Tween® 20 (PBSTB), and all tubes used were non-stick type (pre-block 1.5 ml tubes (Protein LoBind), Eppendorf).

[0168] Selection was performed in PBSTB at 37°C. Selection times ranged from 20 to 120 minutes, with target-phage particle complexes on SA and NA beads being captured in the third cycle. For solid-phase selection, biotinylated B7-H3 was immobilized on SA beads prior to selection. Finally, different beads immobilized with target phage particle complexes were washed with PBS (PBST 0.1%) containing 0.1% Tween-20 for approximately 30 seconds per wash. The selection, bead blocking, pre-selection, and washing steps were performed manually in Eppendorf tubes, or using a Kingfisher Duo apparatus (Thermo Fisher) with a Kingfisher Deepwell 96 plate (Thermo Fisher), or a combination of both. In addition to the above, some tracks of phage stock were heated to 70°C before use for selection.

[0169] Table 4 outlines the selection strategy of increasing stringency in a continuous cycle while lowering the target concentration and increasing the number of washes. Elution was performed as described in WO2009 / 077175.

[0170] [Table 4-1]

[0171] [Table 4-2]

[0172] Phage particle amplification and preparationThe amplification of phage particles between different selection cycles was performed as follows: Escherichia coli XL-1 Blue strain was used for phage amplification, with M13K07 helper phage in an excess of 55-65 ×. The XL-1 Blue strain was cultured at 37°C until the early logarithmic phase in TSB medium (Tryptic Soy Broth, 30 g / L) supplemented with 1% glucose and 1 μg / mL tetracycline, and then infected with phage particles. Bacteria were used in excess of 22-2500 × relative to the amount of phage particles. Subsequently, TSB medium supplemented with 1% glucose, 1 μg / mL tetracycline, and 200 μg / mL carbenicillin was added, doubling the volume of medium. After culturing at 37°C for approximately 1 hour, helper phage was added, and the culture was incubated at 37°C for 1.5 hours. Hyperinfecting bacteria were pelletized by centrifugation and resuspended in 50 mL of TSB+YE medium supplemented with 25 μg / mL neomycin and 0.1 mM IPTG (isopropyl-β-D-1-thiogalactopyranoside), and incubated overnight at 30°C. The cultured cells were pelletized, and the phage particles in the supernatant were precipitated twice with PEG / NaCl. Finally, the phage particles were resuspended in selection buffer before proceeding to the next selection cycle.

[0173] Bulk cloning In the final selection cycle, logarithmic-phase bacteria were infected with eluate and cultured overnight at 37°C in TSB supplemented with 0.1 μg / mL carbenicillin. Each culture medium was pelleted, and plasmid DNA was prepared using QIAprep Spin Miniprep (Qiagen). Each plasmid preparation was used separately, and phage-selective mutants with an N-terminal His6-tag were subcloned together into a T7 promoter-driven expression vector using standard molecular cloning methods. Transformed E. coli T7E2 cells (GeneBridges) were plated on TBAB agar plates (30 g / L tryptothenic blood agar base, Oxoid) supplemented with 0.2 g / L neomycin and 1% glucose. The Z gene fragment was subcloned into the T7 promoter-driven expression vector to obtain the coding sequence MGSSHHHHHHLQ-[ZBH##] (SEQ ID NO: 626). ZBH#### refers to the sequence of individual, 58-amino acid residues of the B7-H3-binding Z variant.

[0174] Preparation of Z variants for ELISA Subcloned and transformed single colonies of the selected variant were inoculated into 1.2 mL of TSB-YE medium supplemented with 100 μg / mL ampicillin and 0.175 mM IPTG, and Z variants were prepared in a deep-well plate (Rainin, Mettler Toledo Liquidator pyramidal well bottom, 2.2 mL). The plates were incubated at 37°C for 17-19 hours with rotation. The cells were pelleted by centrifugation and resuspended in 250 μL of 2×PBST 0.05% (2×PBS supplemented with 0.05% Tween20), and incubated at 90°C for 7 minutes. The heat-treated suspension was filtered using a 96-well deep-filter plate (AcroPrep filter plate, Pall Laboratories). The filtered supernatant (Heat Treated (HT) lysate), along with the soluble portion of the extract, contained the Z variant as a fusion to His6 represented as MGSSHHHHHLQ-[ZBH####] (SEQ ID NO: 626). All individually isolated clones were subjected to DNA sequencing.

[0175] ELISA screening of Z variants for B7-H320 μL of 3 μg / mL anti-His6 mouse antibody (Abcam cat.no.18184), diluted in PBS, was coated overnight at 4°C in a 384-well ELISA plate (Greiner). The wells were washed four times with 0.05% PBST, and then blocked at RT for 1.5 hours with 50 μL of Blocker® casein in PBS (Thermo Scientific). After washing the wells four times with 0.05% PBST, 20 μL of HT lysate diluted 1:20 in PBST was added to each well, and incubated at RT for 1 hour and 20 minutes. As a positive control, HT lysate prepared using ZAC12 (SEQ ID NO: 543), cloned using the same method as the tested mutant, was added in double doses to each plate. As a negative control, HT lysate prepared using a Z variant that bound to an unrelated target and cloned as Z-His6 was added. The supernatant was flushed, and the wells were washed four times with 0.05% PBST. Subsequently, 20 μL of biotinylated Fc-fusion B7-H3 (b-B7-H3(4Ig)-Fc) at concentrations of 5 nM, 1.7 nM, 0.56 nM, and 0 nM, diluted with Blocker® casein in PBS, was added to each well. The plate was incubated at RT for 1 hour and 20 minutes, and then washed as described above. HRP (Thermo Scientific) conjugated with streptavidin diluted 1:30000 in Blocker® casein in PBS was added to each well, and the plate was incubated for 45 minutes. After washing as described above, 20 μL of TMB substrate (1-Step Ultra TMB-ELISA Pierce ThermoFisher Scientific) was added to the wells, and the plate was processed according to the manufacturer's recommendations. Absorbance at 450 nm was measured using a multi-well plate reader (EnSpire, Perkin Elmer).

[0176] result Phage display selection for B7-H3-bound Z variants Using the mature library Zlib008B7-H3.I, phage display selection for biotinylated B7-H3 was performed for one or four cycles, after which individual clones were obtained.

[0177] Sequence determination Sequencing was performed on clones obtained after one or four cycles of selection. Each variant was assigned a unique identification number ####, and each variant is referred to herein as ZBH####. The amino acid sequences of the selected 58-amino acid long Z variants are listed in the sequence listing as SEQ ID NOs. 480–535. The inferred B7-H3 binding motif extends from residue 8 to residue 37 in each sequence. The amino acid sequence of the 49-amino acid long polypeptide predicted to constitute a complete three-helical bundle within each of these Z variants extends from residue 7 to residue 55.

[0178] ELISA screening of Z variants for B7-H3 Clones obtained after 1-cycle and 4-cycle selection were produced as soluble crude samples by heat treatment in 96-well plates and screened for B7-H3 binding activity by ELISA. The Z variants listed as (SEQ ID NOs. 480-535) were shown to give responses between 0.55 and 3.2 AU at a target concentration of 5 nM B7-H3, corresponding to at least 3 × blank controls. The corresponding responses of the binders to biotinylated B7-H3 concentrations of 1.7 nM and 0.57 nM were shown to give responses between 0.28 and 3.1 AU, and between 0.15 and 2.0 AU, respectively. The mean responses for ZAC12 were 5 nM: 2.25 AU, 1.7 nM: 1.53 AU, and 0.57 nM: 0.76 AU, respectively.

[0179] Example 3 Production of B7-H3 bonded Z variant overview This example describes the general procedure for subcloning and production of His-tagged Z variants, Z variants fused with the His tag, Z variants fused with the TEV (tobacco etch virus) protease cleavage site, and Z variants fused with ABD. These will be used in subsequent characterization experiments. Production of Z variants using peptide synthesis is also described.

[0180] Materials and methods His 6 Subcloning of Z variants with tags The DNA encoding monomer reference Z variants (sequence numbers 541-545) was obtained from ATUM (Newark, California) as codon-optimized and synthesized genes. The synthesized Z gene fragments were subcloned into a T7 promoter-driven expression vector, resulting in the encoded gene MGSSHHHHHHLQ-[ZBH####] (sequence number 626).

[0181] Cuttable His 6 - Subcloning of Z variants fused with tags The monomeric version of the B7-H3 binding Z variant was subcloned into a T7 promoter-driven vector using standard molecular biological strategies, resulting in the encoded sequence MGSSHHHHHHSSGVDLGTENLYFQG-[ZBH###]-C (SEQ ID NO: 627). After TEV protease treatment, the resulting sequence was in the format G-[ZBH###]-C (SEQ ID NO: 628).

[0182] The gene encoding a dimerized version of the Z variant, designated ZBHD01 (SEQ ID NO: 549), was ordered from GeneArt (Thermo Fisher Scientific, MA, USA) as a synthetic gene with optimized codons and cloned into a custom plasmid. The Z variant was cloned as a fusion protein with a His6 tag and a TEV protease cleavage site. The synthesized dimerized Z gene fragment was subcloned into a T7 promoter-driven expression vector to obtain the coding sequence MGSSHHHHHHSSGVDLGTENLYFQG-[ZAC12m]-GAPGGGGSGGGGSGGGGSTS-[ZAC12mc] (SEQ ID NO: 629). After TEV protease treatment, the resulting sequence was in the format G-[ZAC12m]-GAPGGGGSGGGGSGGGGSTS-[ZAC12mc] (SEQ ID NO: 630).

[0183] Subcloning of the Z variant in fusion with ABDA set of variants were ordered from GeneArt (TermoFisher Scientific GENEART GmbH, Regensburg, Germany) as cloned genes in custom vectors. These variants involved incorporating the ABD portion (PP013; SEQ ID NO: 547) at different positions within the polypeptide (N-terminus, C-terminus, or between two Z-parts) and inserting different linkers (of different lengths and types) between the parts. All constructs contained C-terminal cysteine ​​for subsequent conjugation. The encoded proteins are in the following formats: ZBH481-G4SAS-PP013-C(ZBHD02; SEQ ID NO: 550), GS-PP013-(G4S)2-ZBH481-C(ZBHD03; SEQ ID NO: 551), ZBH481-(G4S)5-ZBH481-G4SAS-PP013-C(ZBHD04; SEQ ID NO: 552), ZBH481-(G4S)3-ZBH481-G4SAS-PP013-C(ZBHD05; SEQ ID NO: 553), and GS-PP013-(G4S). These were 2-ZBH481-(G4S)5-ZBH481-C(ZBHD06; SEQ ID NO: 554), ZBH481-(G4S)3AS-PP013-(G4S)3-ZBH481-C(ZBHD07; SEQ ID NO: 555), ZBH481-A(EAAAK)4AGS-PP013-(KEAAA)4KAKGS-ZBH481-C(ZBHD08; SEQ ID NO: 556), and ZBH481-(TP)8-PP013-(TP)8-ZBH481-C(ZBHD09; SEQ ID NO: 557).

[0184] Z variant expression Generally, Z variants were expressed in autoinduction medium (Overnight Express TB, Novagen) inoculated with pre-cultures of E. coli T7E2 clones containing plasmids with confirmed gene fragment sequences for each B7-H3 binding Z variant. The culture medium (3-200 ml) was incubated at 37°C at 150 rpm or 300 rpm for 16 hours, after which cells were collected by centrifugation.

[0185] The Z variant fused with ABD was produced in 0.6L scale fed-batch cultures. E. coli T7E2 clones with plasmids in which the sequences of each polypeptide gene fragment were confirmed were then processed into final OD. 600 After pre-culturing to a value of 0.1, the cells were cultured at 37°C. 17.5 hours after the start of culture, the temperature was lowered to 31°C, and after 18 hours, induction with IPTG was performed, followed by 9 hours of expression. After a total of 27 hours, the culture was cooled to below 18°C, and the cells were harvested by centrifugation.

[0186] Cell disruption Cells were disrupted using known methods, including heat treatment, sonication, and FastPrep-24® (MP Biomedicals). Cell debris and, if applicable, residual silica spheres from FastPrep-24® were removed by centrifugation. The supernatant was either used immediately for purification or stored at -20°C until purification.

[0187] His 6 - Z variants fused with tags (+ / -TEV cleavage sites) were purified in a 96-well plate format. The lysate, clarified by centrifugation, was prepacked with 75 μL of Ni Sepharose HP (Cytiva) in a His MultiTrap HP plate (Cytiva) or a 1 mL filter plate (1.2 μm, Acroprep), and equilibrated with binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole, pH 7.4). After washing with washing buffer (20 mM sodium phosphate, 0.5 M NaCl, 60 mM imidazole, pH 7.4), the His6-tagged Z variant was eluted with elution buffer (20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH 7.4). All washing and elution steps were performed using centrifugal force. Buffer exchange to PBS was performed using a PD MultiTrap G-25 desalting plate (Cytiva).

[0188] After TEV protease cleavage, reverse IMAC purification was performed in a 96-well plate format.A B7-H3-bound Z variant, a fusion of a His6-tag and a TEV protease cleavage site, was incubated overnight at 4°C with His6-tagged TEV protease in a molar ratio of Z variant:TEV protease = 25:1, and DTT was added to a final concentration of 2 mM. The incubation sample, with 20 mM imidazole added, was applied to a 1 mL filter plate (1.2 μm, Acroprep) prepacked with 150 μL of Ni Sepharose HP (Cytiva) equilibrated with binding buffer. The Z variant cleaved by the TEV protease was recovered via flow-through using centrifugal force, and the His-tagged material bound to IMAC resin. Buffer exchange to the relevant buffer (PBS or 0.1 M HAc) was performed by centrifugal force using a PD Multi-Trap G-25 desalting plate (Cytiva).

[0189] His 6 Refinement of Z variants fused with tags Lysate clarified by centrifugation was applied to a 1 mL His GraviTrap IMAC column (Cytiva). Contaminants were removed by washing with washing buffer, and the Z variant was eluted with elution buffer. Constructs with a purity of 95% or less (SDS-PAGE based), or other constructs deemed to be in this condition, underwent a second purification step using reverse-phase chromatography (RPC). Each Z variant was loaded onto a 1 mL or 3 mL Resource 15 RPC column (Cytiva) pre-equilibrated with RPC solvent A (0.1% trifluoroacetic acid (TFA), 10% acetonitrile (ACN), 90% water). After washing the column with RPC solvent A, the bound protein was eluted 18 CVs using a linear gradient of 0-60% RPC solvent B (0.1% TFA, 80% ACN, 20% water). Subsequently, the buffer was replaced with PBS using a PD-10 desalting column (Cytiva).

[0190] His 6 - Purification of a fusion Z variant having a tag and a TEV protease cleavage site.The B7-H3-bound Z variant of the fusion having a :His6-tag and a TEV protease cleavage site was purified as described in the previous section, but 1 mM DTT was added to all buffers used for the Z variant containing the C-terminal cysteine. Furthermore, the :Z variant, which had undergone a tag cleavage step before purification by RPC, was rebuffered in PBS and incubated with His-tagged TEV protease overnight at 4°C to a Z variant:TEV protease molar ratio of 25:1 or 30:1, with DTT added to a final concentration of 2 mM. The incubation samples, with 20 mM imidazole added, were applied to a 1 mL His GraviTrap IMAC column (Cytiva) equilibrated with binding buffer. The Z variant cleaved by TEV protease was recovered by flow-through, and the His-tagged material was bound to the IMAC resin. The untagged Z variant was further purified by reverse-phase chromatography (RPC) and then buffered in PBS as described in the previous section, or in 0.2 M NaAc pH 6.0 if subsequently conjugated.

[0191] Purification of the Z variant fused with ABD Each cell pellet was resuspended in a purification buffer (50 mM sodium phosphate, 200 mM NaCl, 1 mM EDTA, pH 7.0), and DENARASE® (c-Lecta; 1 μL / g pellet) and DTT (final concentration 20 mM) were added. The cells were lysed by sonication, clarified by centrifugation and filtration, and the supernatant was run on a HiScale 10 column. The HiScale 10 column was packed with resin immobilized with an anti-ABD ligand (proprietary development) and connected to an AKTA Avant 25 system (Cytiva). After washing with purification buffer, the ABD-fused Z variant was eluted with 0.1 M HAc, 1 mM EDTA, pH 3.1. After adding 10% ACN to each eluate, purification by RPC was performed using a 20 ml SOURCE 30 RPC column (Cytiva) in essentially the same manner as above.

[0192] DOTA conjugation of the Z variant, with or without ABDEach purified polypeptide possessing a unique C-terminal cysteine ​​was buffer-exchanged using a PD-10 desalting column to conjugation buffer (0.2M NaAc, 2mM EDTA, pH 6, treated with Chelex® 100 resin). Maleimide-mono-amide-DOTA (Macrocyclics, cat. No. B-272) was added in a 4-fold molar excess, and each sample was incubated at 22°C and 600 rpm for 60 minutes. After that, the buffer was buffer-exchanged using a PD-10 desalting column to 0.2M NaAc, pH 6 (treated with Chelex® 100 resin). Samples used for radiolabeling were conjugated in the same manner, but the EDTA in the conjugation buffer was omitted, and two additional buffer exchanges were performed.

[0193] NOTA conjugation of Z variant: A Z variant with a unique C-terminal cysteine ​​was conjugated with NOTA (CheMatech, cat.no.C101) according to the methodology described above for DOTA conjugation, with incubation performed at 24°C and 450 rpm for 90 minutes.

[0194] Z variant peptide synthesis A subset of the Z variant (ZBH536-ZBH539, SEQ ID NOs. 536-539, and reference variants ZAC12c and SEQ ID NO. 540) was produced by contract manufacturer Bachem (St Helens, UK) using solid-phase peptide synthesis with the Fmoc / tBu strategy as synthetic peptides. The C-terminal amino acids at positions 56-58 of the synthesized Z variants (originally A, P, and K, respectively) were mutated to amino acid residue G, and an amino acid C was added to the C-terminus.

[0195] General protein characterization: Protein concentration was determined by absorbance measurement at 280 nm. Purity was analyzed by SDS-PAGE stained with Coomassie blue, and the identity of each purified Z variant was confirmed by HPLC-MS analysis.

[0196] result Generation of B7-H3 bonded Z variantsHis6-tagged Z variants, or B7-H3-bound Z variants possessing both a His6-tagged Z variant and a TEV protease cleavage site, and Z variants fused with ABD were expressed as soluble gene products in E. coli and purified using affinity chromatography, sometimes followed by RPC. Where applicable, the His6-tagged Z variant was removed in the TEV protease cleavage step. Monomeric Z variants containing a C-terminal GGGC sequence were successfully produced by chemical synthesis. SDS-PAGE analysis of each final protein preparation indicated that they primarily contained B7-H3-bound Z variants. The correct identity and molecular weight of each Z variant, ABD-fused Z variant, and bound variant were confirmed by HPLC-MS analysis.

[0197] Example 4 Characterization of purified B7-H3 bonded Z variants overview In this example, N-terminal His6-tagged Z variants (SEQ ID NOs. 480-535) prepared as described in Example 3 were evaluated for their binding properties, and a subset was also subjected to stability testing. Interactions between the Z variants and human B7-H3 were evaluated using surface plasmon resonance (SPR) and biolayer interferometry (BLI). In vitro cell binding was evaluated using MCF-7 and SKOV-3 cells expressing B7-H3. Melting temperature and secondary structure content were analyzed by circular dichroism (CD) spectroscopy. Chemically synthesized Z variants (SEQ ID NOs. 536-539 and REF.S. ID NOs. 540) were also used to measure interactions with B7-H3 from other species.

[0198] Materials and methods SPR k for B7-H3 off screening : Dissociation rate constant (k) of the His6-Z variant for Fc fusion B7-H3 (B7-H3(2Ig)-Fc) offThe protein was measured using a Biacore 8K instrument (Cytiva). B7-H3(2Ig)-Fc was diluted to 10 nM with HBS-EP+ and captured in flow cell 2 of the Protein A tip (Cytiva), resulting in a capture level of approximately 430 RU. His6-Z variants at concentrations of 1 nM and 5 nM were used as analytes and injected into both flow cells 1 and 2. The association time was 120 seconds (30 μL / min) and the dissociation time was 300 seconds (30 μL / min). HBS-EP+ was used as the running buffer, and 10 mM glycine-HCl pH 1.5 (two pulses of 30 seconds / 30 μL / min) was used as the regeneration buffer. The assay temperature was 30°C. The reference cell (flow cell 1) and blank cycle injection (HBS-EP+) were subtracted from the sensorgram before evaluation using Biacore Insight evaluation software.

[0199] SPR single-cycle kinetic screen for B7-H3 Affinity of the His6-Z variant for Fc-fused B7-H3 (B7-H3(2Ig)-Fc) (K D The concentration of B7-H3(2Ig)-Fc was determined by a single-cycle kinetic run using a Biacore 8K instrument (Cytiva). B7-H3(2Ig)-Fc was diluted to 10 nM with HBS-EP+ and captured in flow cell 2 of a Protein A tip (Cytiva), resulting in a capture level of approximately 430 RU. His6-Z variants at concentrations of 0.56, 1.67, 5, and 15 nM were used as analytes and injected into both flow cells 1 and 2. The association time was 120 seconds (50 μL / min) and the dissociation time was 300 seconds (50 μL / min). HBS-EP+ was used as the running buffer, and 10 mM glycine-HCl pH 1.5 (2 pulses of 30 seconds each / 30 μL / min) was used as the regeneration buffer. The assay temperature was 25°C. The reference cell (flow cell 1) and blank cycle injection (HBS-EP+) were subtracted from the sensorgram before evaluation using Biacore Insight evaluation software.

[0200] BLI Kinetic Screen : Affinity of the B7-H3 bound His6-Z variant (K D ) and kinetic value (kon and k off The ) was determined using BLI on an Octet HTX instrument (Sartorius). In the experiment, the His6-Z variant was diluted to a concentration of 0.5 μg / mL with 1×KB buffer (10× Octet Kinetics buffer diluted with PBS) and loaded onto an Anti-His biosensor (Octet® Anti-Penta-HIS (HIS1K) Biosensors) for 300 seconds. Binding was performed for 400 seconds using 8 nM hB7-H3(2Ig)-Fc or 1×KB buffer (for reference sample). Dissociation was performed for 400 seconds using 1×KB buffer. Between each cycle, the anti-His biosensor was regenerated with 10 mM glycine-hydrochloride pH 1.5 (6 × 2 seconds). Throughout the experiment, a shaking speed of 1000 rpm and an assay temperature of 30°C were used. The reference sample (1 × KB buffer) was subtracted from the sensorgram before evaluation using Octet Analysis Studio 12.2 software.

[0201] Cell binding assay In the initial screening experiment, the His6-Z variant was tested for binding to B7-H3 expressing MCF-7 cells. The cells were placed in a V-bottom 96-well plate (0.2 × 10⁶). 6 Cells were placed in a well and incubated with a single concentration of 50 nM His6-Z variant at 4°C for 1 hour. Each variant was tested in duplicate. After 1× washing with PBS containing 1% fetal bovine serum (FBS), His6-Z binding was identified by adding anti-Z polyclonal antibody at a concentration of 4 μg / mL and incubating at 4°C for 1 hour, followed by Alexa488-conjugated goat-anti-rabbit IgG diluted 1:2000 and incubating at 4°C for 1 hour. After two washes with FBS, fluorescence intensity was measured using a multimode plate reader (Enspire).

[0202] In the second experiment, a subset of mature His6-Z variants (n=7) were tested for binding to SKOV-3 cells expressing B7-H3. The experiment was performed on MCF-7 cells as described above, but with the His6-Z variant incubated at concentrations reduced from 500 nM to 32 pM. Each variant was tested in triplicates. Binding curves were plotted, and EC50 values ​​were determined using GraphPad Prism software.

[0203] In the third experiment, chemically synthesized Z variants (SEQ ID NOs. 536-539 and reference variant SEQ ID NO. 540) were tested for binding to B7-H3 expressing SKOV-3 cells. The same method as described above was used for the SKOV-3 cells. Each Z variant was tested in quadruple repeats.

[0204] Circular dichroism (CD) spectroscopy A portion of the His6-Z variant was diluted to 0.5 mg / mL with PBS. A CD spectrum from 250–195 nm was obtained at 20°C. Furthermore, variable temperature measurements (VTM) were performed to determine the melting temperature (Tm). In VTM, the absorbance at 221 nm was measured while increasing the temperature from 20°C to 90°C with a temperature gradient of 5°C / min. To investigate the refolding ability of the Z variant, a new CD spectrum was obtained at 20°C after the heating procedure. CD measurements were performed using a Jasco J-810 spectropolarimeter (Jasco Scandinavia AB) with a cell having a path length of 1 mm.

[0205] SPR analysis of B7-H3 from different speciesThe binding of chemically synthesized Z variants (SEQ ID NOs. 536-539 and reference variant SEQ ID NO. 540) to Fc-fused B7-H3 derived from human (B7-H3(2Ig)-Fc), cynomolgus monkey (cB7-H3(4Ig)-Fc), mouse (mB7-H3(2Ig)-Fc), and rat (rB7-H3(2Ig)-Fc) was analyzed using a Biacore 8K instrument (Cytiva). The Fc-fused B7-H3 variants were diluted to 10 nM with HBS-EP+ and captured in flow cell 2 of a Protein A tip (Cytiva), resulting in capture levels of approximately 270, 250, 260, and 320 RU in human, cynomolgus monkey, mouse, and rat, respectively. His6-Z variants were used as analytes at concentrations of 0.56 nM, 1.67 nM, 5 nM, and 15 nM and injected into both flow cells 1 and 2. The association time was 120 seconds (50 μL / min) and the dissociation time was 300 seconds (50 μL / min). HBS-EP+ was used as the running buffer, and 10 mM glycine-HCl pH 1.5 (two pulses of 30 seconds / 30 μL / min) was used as the regeneration buffer. The assay temperature was 30°C. The reference cell (flow cell 1) and blank cycle injection (HBS-EP+) were subtracted from the sensorgram before evaluation using Biacore Insight evaluation software.

[0206] result SPR k for B7-H3 off screening The interaction between the His6-Z variant and B7-H3 was analyzed using a Biacore 8K instrument by injecting two concentrations of purified Z variant onto a surface immobilized with B7-H3(2Ig)-Fc. The dissociation rate constant (k) was determined based on data from each selected B7-H3 binder at a 5 nM concentration injected onto the B7-H3 surface. off ) are shown in Table 5.

[0207] SPR single-cycle kinematic screen for B7-H3A single-cycle ketic screen for B7-H3 of a subset of purified Z variants was analyzed using SPR on a Biacore 8K. B7-H3(2Ig)-Fc was captured on a Protein A tip and injected onto the Z variant surface at four concentrations. Affinity (K D The values ​​were estimated and are shown in Table 5. Examples of sensograms obtained for four B7-H3 binding Z variants (ZBH480-ZBH482, SEQ ID NOs. 480-482, and reference variants ZAC12 and SEQ ID NOs. 543) for B7-H3(2Ig)-Fc are shown in Figure 1.

[0208] BLI Kytic Screen: The interaction between the His6-Z variant and B7-H3 was analyzed using an Octet HTX instrument by loading the His6-Z variant onto an anti-His biosensor and then associating it with hB7-H3(2Ig)-Fc. The dissociation rate constant (k off ) are shown in Table 5.

[0209] Cell binding assayThe binding of His6-Z variants to B7-H3-expressing MCF-7 cells was evaluated by fluorescence intensity in the initial screening experiment. Cells were incubated with one concentration of purified Z variant and detected with anti-Z pAb and Alexa488-conjugated anti-rabbit IgG. Fluorescence intensity varied between 488 and 6677 (arbitrary units). A summary of the mean fluorescence values ​​of selected conjugates (improved from ZAC12 in either the cell assay, Biacore, or Octet screening assay) is shown in Table 5. In the second experiment, the binding of seven mature His6-Z variants to B7-H3-expressing SKOV-3 cells was evaluated by fluorescence intensity. Cells were incubated with decreasing concentrations of purified Z variant (ranging from 500 nM to 32 pM) and detected with anti-Z pAb and Alexa488-conjugated anti-rabbit IgG. The calculated mean EC50 values ​​are summarized in Table 6, and representative experimental curves are shown in Figure 2A. In the third experiment, the binding of four synthetic Z variants to B7-H3 expressing cells was evaluated by fluorescence intensity. Cells were incubated with anti-Z pAb and Alexa488-labeled anti-rabbit IgG while decreasing the concentration of the synthetic Z variants (ranging from 500 nM to 32 pM). Mean EC50 values ​​are summarized in Table 7, and a curve from one representative experiment is shown in Figure 2B.

[0210] [Table 5-1]

[0211] [Table 5-2]

[0212] [Table 5-3]

[0213] [Table 6]

[0214] [Table 7]

[0215] CD analysis The CD spectra determined for nine B7-H3 bonded Z variants with the :His6 tag indicated that all variants possessed an α-helical structure at 20°C, judging from the typical minimum wavelengths of 208 nm and 222 nm. When the spectra before and after heating to 90°C were superimposed, reversible folding was observed in all Z variants. The melting temperatures (Tm) are summarized in Table 8. An example of the CD spectrum of His6-ZBH480 before and after heating to 90°C is shown in Figure 3A, and its melting curve is shown in Figure 3B.

[0216] [Table 8]

[0217] SPR analysis of B7-H3 from different species The binding of chemically synthesized Z variants (ZBH536-ZBH539, SEQ ID NOs. 536-539 and reference variant ZAC12c, SEQ ID NO. 540) to human, cynomolgus monkey, mouse, and rat-derived B7-H3 was analyzed using a Biacore 8K instrument by injecting four concentrations of purified Z variants onto B7-H3(2Ig)-Fc, cB7-H3(4Ig)-Fc, mB7-H3(2Ig)-Fc, and rB7-H3(2Ig)-Fc captured on the surface of a protein A chip. The Z variants showed nearly identical binding to B7-H3 from different species. Figure 4 shows sensorgrams obtained for one B7-H3-binding polypeptide (ZBH538, SEQ ID NO. 538) against human, cynomolgus monkey, mouse, and rat B7-H3.

[0218] Example 5 Design and construction of two mature libraries of the B7-H3-bound Z variant. overview In this example, two new libraries were designed based on the B7-H3 binding variant identified in the first selection described above. The new mature libraries each have a size of approximately 5.2 × 10⁶. 8 and 5.9 × 10 8It contained individual clones of [the creature].

[0219] Materials and methods Design of two B7-H3 affinity matured libraries Two novel libraries were designed based on the sequences of the B7-H3 binding Z variants selected, produced, and characterized as described in Examples 1-4. Following a strategy based on the binding motif of the Z variants identified in the initial maturation, 13 surface-exposed positions of the Z molecular scaffold were biased to specific amino acid residues, including those defined in SEQ ID NOs. 480-535. The variations within the 13 altered positions were the same between the two libraries. However, they differed in three scaffold positions. One library was designed at Y5, N52, D53 (numbers relative to the full-length Z variant sequence), and the other library was designed at F5, S52, and E53. Furthermore, both libraries also included two scaffold positions within BM:X 29 and X 30 The numbering (for BM as defined herein) included variability. The new library design is shown in Table 9, showing the percentage of amino acids used for each of the 15 randomized positions.

[0220] [Table 9]

[0221] Two oligonucleotides with complementary 3' ends, one forward-complementary and the other reverse-complementary, were synthesized using TRIM technology. These oligonucleotides were ordered from Ella Biotech GmbH (Martinsried, Germany).

[0222] The libraries were constructed using vectors pAY02592 and pAY04242 as described in Example 1, with the following exceptions: 1) Library transformation was performed on electrocompetent XL-1 Blue cells; 2) Transformed cells were then pooled and cultured for 6.5 hours at 37°C in 1 L of TSB-YE medium supplemented with 2% glucose, 10 g / mL tetracycline, and 100 g / mL ampicillin. The quality of the libraries and the amino acid distribution were confirmed by sequencing as described in Example 1.

[0223] Preparation of phage stocks: Phage stocks were cultured and prepared in 1.5 L of medium for each library, basically as described in Example 1. The phage particles were precipitated twice from the supernatant using PEG / NaCl, filtered as described in Example 1, and finally dissolved in PBS and glycerol. The phage stocks were stored at -80°C until use in selection.

[0224] result Building a Library Based on the set of B7-H3 binding variants described in Examples 1-4, two new libraries were designed. The theoretical size of the designed libraries is 4.5 × 10⁶. 7 It was the Z variant. The actual size of the two libraries, determined by titration after transformation into E. coli, was approximately 5.2 × 10⁶ each. 8 and 5.9 × 10 8 The cells were transformed into XL-1 Blue cells. The quality of the libraries was verified by determining the nucleotide sequences of 192 transformants per library and comparing the actual sequences with the theoretical design. Sequence analysis of individual library members confirmed the codon distribution according to the theoretical design. The libraries were named Zlib009B7-H3.I and Zlib008B7-H3.II.

[0225] Example 6 Second selection and screening of affinity-mature B7-H3 binding Z variants overview In this example, B7-H3 was used as the target in phage display selection using two different B7-H3 mature phage libraries of the Z variant. The selected clones were cloned in large quantities into expression vectors driven by the T7 promoter, sequenced, produced in E. coli, and assayed against different target proteins by ELISA and SPR.

[0226] Materials and methods Phage display selection for B7-H3 bound Z variant: Phage display selection was performed using phage stocks from newly prepared mature libraries. Selection for biotinylated b-B7-H3(4Ig)-Fc and b-B7-H3(4Ig)-His_1 was performed in essentially solution for all tracks, as described in Example 2, using the specific conditions described in Table 10.

[0227] For washing, streptavidin-coated SpeedBeads were used as the solid phase in cycles 1, 2, and 4, while neutravidin beads were used in cycle 3. Washing was performed manually in cycle 1, and manually and / or using a KingFisher Duo instrument in cycles 2-4. Selection was performed at RT, 37°C, or 50°C, using pre-blocked tubes (Protein LowBind, Eppendorf). To reduce the degree of nonspecific binding, pre-selection was performed for 1-2 cycles using streptavidin-coated SpeedBeads as the solid phase. In some tracks, biotinylated Fc (Jackson ImmunoResearch Laboratories) was coated onto the beads before use in pre-selection (tracks 15, 21, and 22). Furthermore, in some tracks, the phage stock was preheated at 70°C for 15 minutes, then centrifuged at 13,000 rpm before being used for selection (tracks 9, 16, 25, 32, 41, and 50).

[0228] As the selection process progressed, the tracks were further divided according to target concentration, number of washes, and / or duration.

[0229] [Table 10-1]

[0230] [Table 10-2]

[0231] Phage particle amplification and preparation Phage stock preparation and phage amplification between selection cycles were carried out as described in Examples 1 and 2. Logarithmic-stage bacteria were infected with elutes from the selected tracks (cycles 2-4), and the culture pool was used for bulk cloning of the selected mutants, essentially as described in Example 2.

[0232] Generating Z variants for join analysis: Z variants were prepared, and HT lysates of each variant were prepared as described in Example 2. The supernatant obtained by finally filtering the soluble portion of the extract contained the Z variant as a fusion with His6, represented as MGSSHHHHHHLQ-[ZBH###] (SEQ ID NO: 626). ZBH### refers to the sequence of each individual, 58-amino acid residue, B7-H3-binding Z variant.

[0233] ELISA screening of Z variants:The binding of Z variants to B7-H3 was analyzed using a series of ELISA assays. In the first assay, each Z variant was analyzed against biotinylated B7-H3 (b-B7-H3(4Ig)-Fc) at a target concentration of 0.6 nM. The ELISA was performed as described in Example 2. In the second assay, a selected subset of conjugates was subjected to ELISA against biotinylated b-B7-H3(4Ig)-Fc at concentrations of 1.8, 0.6, 0.2, and 0 nM. In the third assay, another selected subset of conjugates was subjected to ELISA using a 96-well plate at a target concentration of 0.6 nM. In this assay, the subset of conjugates was also analyzed against an unrelated target protein biotinylated at a concentration of 12 nM. As a positive control, ZAC12 (SEQ ID NO: 543) was used at four concentrations in the ELISA experiments against biotinylated b-B7-H3(4Ig)-Fc. The absorbance at 450 nm was measured using a multi-well plate reader, EnSpire (Perkin Elmer).

[0234] SPR k for captured B7-H3 using HT dissolving solution off screening : For the purpose of ranking, the dissociation rate constant (k) of a subset (n=1674) of the His6-Z variant produced as HT solubles. off A screening experiment was set up to estimate ). The experiment was essentially performed on Fc-fused B7-H3 (B7-H3(2Ig)-Fc) captured on a protein A chip, using a Biacore 8K instrument.

[0235] SPR kinetic screen of captured Z variant against B7-H3The binding of a subset of Z variants (n=93) to B7-H3 was analyzed using SPR on a Biacore 8K. Each Z variant in the HT lysis solution was captured on anti-His Ab (Abcam cat.No.ab18184) immobilized on the surface of a CM5 chip and analyzed against B7-H3(4Ig)-Fc diluted to 30 nM with HBS-EP+. HBS-EP+ was used as the running buffer, and 10 mM glycine-HCl pH 2.0 (2 pulses of 20 seconds / 30 μL / min) was used as the regeneration buffer. The assay temperature was 30°C.

[0236] Sequence determination In parallel with ELISA and SPR screening, the DNA sequences of all clones were determined.

[0237] result Phage display selection of mature B7-H3-binding Z variants Individual clones were obtained after 2, 3, and 4 cycles of phage display selection against biotinylated B7-H3.

[0238] ELISA screening of Z variants Clones obtained after 2-4 cycles of selection were generated in 96-well plates and screened for binding activity to B7-H3. In the first assay, a conjugate was considered positive if it showed a response three times the background level at 0.6 nM B7-H3 (≥0.2 AU). In the second and third assays, a conjugate was considered positive if it showed a mean response three times the background level (≥0.2 AU) at 0.6 nM, along with a healthy target-dependent response at other concentrations.

[0239] SPR k for captured B7-H3 using HT dissolving solution off screening Using a Biacore 8K instrument, captured Fc-fusion B7-H3 proteins were injected with HT lysate, and the interaction between Z variants and B7-H3 was analyzed using off-rate screening experiments. Z variants with off-rate curves at least twice as good as the control Z variant ZAC12 were identified as candidates.

[0240] SPR kinetic screen of captured Z variant against B7-H3Affinity screening of a subset of Z variants in HT lysates for B7-H3 was analyzed using SPR on Biacore 8K. Z variants in HT lysates were captured on immobilized anti-His6 antibody, B7-H3 was injected as the analyte, and the kinetic response was evaluated. This assay method was used to rank conjugates in terms of affinity. Z variants found to have off-rate curves ranked at or above the control Z (ZBH481; SEQ ID NO: 481) from initial maturation were considered candidates.

[0241] Sequence determination Sequencing was performed on clones obtained after 3-4 cycles of selection. Each variant was given a unique identification number ###, and each variant is referred to as ZBH###. The amino acid sequences of the 58-amino acid long Z variants that were shortlisted after the ranking SPR screen described above are listed in the sequence list as SEQ ID NOs. 1-479. The inferred B7-H3 binding motif extends from residue 8 to residue 37 in each sequence. The amino acid sequence of the 49-amino acid long polypeptide predicted to constitute the complete 3-helix bundle within each of these Z variants is from residue 7 to residue 55. The primers used in bulk cloning appear to have produced some unintended, but functional, Z scaffold variants. Variants having amino acids Y5, N52, and D53 (SEQ ID NOs: 1-13, 17-418, 446-479) and amino acids F5, S52, and E53 (SEQ ID NOs: 15-16, 420-424, 427-428, 430-436, 438-444, 480-535), respectively, a variant having F5, N52, and E53 (SEQ ID NOs: 14, 425-426, 429, 437, 445), and one variant having F5, N52, and D53 (SEQ ID NO: 419) were identified.

[0242] Example 7 Characterization of the second generation of mature B7-H3-linked Z variants overview In this example, a subset (n=21) from the mature Z variants selected as described in Example 6 was further characterized. N-terminal His6-tagged Z variants (SEQ ID NOs: 1-21), produced similarly to Example 3, were evaluated for binding to B7-H3 using SPR, and further subsets were tested by cell binding assays and / or subjected to stability and secondary structure evaluation using CD spectroscopy.

[0243] Materials and methods SPR binding kinetics of the Z variant to B7-H3 A single-cycle kinetic screen of a subset (n=21) of Z variants against B7-H3 was performed using SPR on a Biacore 8K instrument (Cytiva). ZBH481 (SEQ ID NO: 481) from the first maturation and the reference variant ZAC12 (SEQ ID NO: 543) were included in the assay for comparison. B7-H3(4Ig)-His was immobilized by amine coupling to a carboxylated dextran layer on the surface of a CM5 chip (Cytiva) according to the manufacturer's recommendation. Surface 1 was activated and then deactivated and used as a reference. Each Z variant was injected onto the chip surface at concentrations of 0.33, 1, 3, 9, and 27 nM for 165 seconds, followed by dissociation for 720 seconds (flow rate 50 μl / min). The chip surface was regenerated with 50 mM NaOH (two pulses of 20 seconds / 30 μL / min). HBS-EP+ was used as the running buffer and dilution buffer. The assay temperature was 25°C. The reference surface and blank cycle injection (HBS-EP+) were subtracted, and the curve was fitted to the kinetic 1:1 binding model in Biacore Insight Evaluation software (Cytiva) to determine the binding characteristics (k on , k off We estimated the KD (Knowledge, Disability, and Cost).

[0244] Cell assayThe experiment was performed on SKOV-3 cells as described in Example 4, but the His6-Z variant was incubated at concentrations reduced from 500 nM to 21 pM. Each variant was tested in triplicates. ZBH481 (SEQ ID NO: 481) from the first maturation and the reference variant ZAC12 (SEQ ID NO: 543) were included in the assay for comparison. Binding curves were plotted and EC50 values ​​were determined using GraphPad Prism software.

[0245] CD spectral analysis For a subset of His6-Z variants (n=9), VTM was performed for each Z variant to determine Tm, and CD spectra in the 250-195 nm range were collected at 20°C before heating to 90°C and after heating to 90°C, as described in Example 4.

[0246] result Biacore binding kinetics of the Z variant to B7-H3 A single-cycle kinetic screen of a subset of Z variants for B7-H3 was analyzed using SPR on Biacore 8K. The Z variants were implanted onto immobilized B7-H3 at five concentrations, and their binding properties (k on , k off The following were estimated (and KD). The results are summarized in Table 11. Examples of sensorgrams obtained for six B7-H3 binding polypeptides to B7-H3(4Ig)-His are shown in Figure 5. All Z variants analyzed showed improved binding compared to the reference variant ZAC12 and the previously matured variant ZBH481.

[0247] Cell assayThe binding of six His6-Z variants to B7-H3-expressing SKOV-3 cells was evaluated by fluorescence intensity. Cells were incubated with decreasing concentrations of purified Z variants (ranging from 500 nM to 21 pM) and then detected with anti-Z pAb and Alexa488-labeled anti-rabbit IgG. The calculated mean EC50 values ​​are summarized in Table 11, and the binding curves are shown in Figure 6. All new, matured Z variants showed improved cell binding compared to the reference variant ZAC12 and the previously matured variant ZBH481.

[0248] [Table 11]

[0249] CD analysis CD spectra determined for nine B7-H3 bonded Z variants with the :His6 tag indicated that all variants possessed an α-helical structure at 20°C, judging from typical minimums at 208 nm and 222 nm. Upon heating to 90°C, five Z variants (ZBH001-ZBH004 and ZBH008) showed fully reversible folding, two Z variants (ZBH007 and ZBH009) showed nearly reversible folding, while two Z variants (ZBH011 and ZBH012) reacted sensitively to the harsh heating to 90°C. The melting temperatures (Tm) are summarized in Table 12. An example of the CD spectrum of ZBH001 before and after heating to 90°C is shown in Figure 7A. The corresponding melting curve for ZBH001 is shown in Figure 7B.

[0250] [Table 12]

[0251] Example 8 In vitro evaluation of dimeric polypeptides overview To investigate whether improved efficacy could be achieved by incorporating additional B7-H3 binding sites, the dimeric polypeptide ZBHD01 (SEQ ID NO: 549) was constructed and prepared as described in Example 3 and studied in SPR analysis and binding assays by binding to SKOV-3 cells.

[0252] Materials and methods SPR analysis The binding of the dimer polypeptide ZBHD01 and the monomer Z variant ZBH538 (SEQ ID NO: 538; included for comparison) to B7-H3(4Ig)-Fc was investigated using a Biacore 8K instrument (Cytiva). B7-H3(4Ig)-Fc was diluted to 2 nM with HBS-EP+ and captured in flow cell 2 of a Protein A tip (Cytiva), resulting in a capture level of approximately 45 RU. The dimer polypeptide and monomer Z variant were each used as analytes at a concentration of 40 nM and injected into both flow cells 1 and 2. The association time was 120 seconds (50 μL / min) and the dissociation time was 600 seconds (50 μL / min). HBS-EP+ was used as the running buffer, and 10 mM glycine-HCl pH 1.5 (2 pulses of 30 seconds each / 30 μL / min) was used as the regeneration buffer. The assay temperature was 25°C. The reference cell (flow cell 1) and blank cycle injection (HBS-EP+) were subtracted from the sensorgram before evaluation using Biacore Insight evaluation software.

[0253] Cell assay The experiment was performed on SKOV-3 cells as described in Example 4, but the polypeptide ZBHD01 or ZBH538 was incubated at concentrations reduced from 555 nM to 28 pM. Each variant was tested repeatedly. Binding curves were plotted, and EC50 values ​​were determined using GraphPad Prism software.

[0254] result SPR analysisThe binding of the dimer polypeptide ZBHD01 and the monomer Z variant ZBH538 to B7-H3 was qualitatively analyzed using SPR on a Biacore 8K instrument. Both variants were injected onto B7-H3 captured on a protein A chip, and their binding curves were compared. Figure 8 shows the sensorgrams obtained when ZBHD01 and ZBH538 bound to B7-H3(4Ig)-Fc, respectively.

[0255] Cell assay The binding of ZBHD01 and ZBH538 to B7-H3 expressing SKOV-3 cells was evaluated by fluorescence intensity. Cells were incubated with dimerized or monomeric variants at reduced concentrations (ranging from 555 nM to 28 pM), followed by the addition of anti-Z pAb and Alexa488-labeled anti-rabbit IgG for detection. The calculated mean EC50 values ​​are summarized in Table 13, and the binding curves are shown in Figure 9, demonstrating approximately a 3.8-fold improvement in EC50 values ​​compared to the dimerized format.

[0256] [Table 13]

[0257] Example 9 Radiolabeling of B7-H3 bonded Z variant overview This example describes the radiolabeling of B7-H3 bound Z variants used following the experiments described in Examples 10-12 and 14-15.

[0258] Materials and methods 99m Tc marking : Z variants ZBH536 (SEQ ID NO: 536), ZBH538 (SEQ ID NO: 538), ZBH539 (SEQ ID NO: 539) and reference Z variant ZAC12c (SEQ ID NO: 540) have a unique C-terminal cysteine. 99mSite-specific labeling with Tc was performed. A lyophilized labeling kit containing 75 μg of tin(II) chloride dihydrate (Fluka Chemika), 5 mg of sodium gluconate (Celsus Laboratories), and 100 μg of tetrasodium ethylenediaminetetraacetate (EDTANa4) (Sigma-Aldrich) was prepared as previously described (Ahlgren et al., 2010, Nucl Med Biol 37:539-546). 99m Tc was obtained as pertechnetium salt by eluting Ultra TechneKow generator (Mallinckrodt) with sterile 0.9% sodium chloride (Mallinckrodt). Radiolabeling of each Z variant was performed by adding the contents of the lyophilized kit dissolved in 120 μL of degassed PBS to 100 μg of the Z variant. 80 μL (200-300 MBq) 99m Tc-pertechnetate was added to the reaction mixture, and the vial was degassed to protect the mixture from oxidation. The reaction vial was thoroughly vortexed and incubated at 90°C for 1 hour.

[0259] 111 Signage by In The Z variant ZBH538 (SEQ ID NO: 538) and the reference Z variant ZAC12c (SEQ ID NO: 540) are joined using DOTA as described in Example 3. 111 The molecules were site-specifically labeled with In. 30 μL of 0.2 M NH4OAc (pH 5.5) was added to ZBH538-DOTA and ZAC12c-DOTA (40 μg / 22 μL, dissolved in 0.2 M NH4OAc, pH 5.5). Loosely bound 111 To remove In, EDTANa4 (5 mg / mL in Milli-Q water) was added to the reaction mixture in a 500-fold molar excess and incubated at 90°C for 10 minutes. Purification was performed using a NAP-5 column that had been pre-equilibriumized and eluted with 1% BSA in PBS.

[0260] 68 Signage by Ga :Gallium-68 is 68 Ge / 68The Ga generator (Eckert and Ziegler AG) was fractionated with 0.1 M HCl. The eluate with the highest radioactivity concentration was used for labeling. In the format G-[ZBH###]-C (SEQ ID NO: 628), 40 μg each of the NOTA-bound Z variants ZBH001 (SEQ ID NO: 1), ZBH002 (SEQ ID NO: 2), ZBH003 (SEQ ID NO: 3), and the reference Z variant ZAC12 (SEQ ID NO: 543) were mixed with 100 μL of 1.25 M NaAc, pH 3.6 in 0.2 M NaAc pH 6. The generator eluate (100 μl, 80 MBq) was added, and the mixture was incubated at 60°C for 10 minutes. Purification was performed using a pre-equilibriumized NAP-5 column eluted with 1% BSA in PBS.

[0261] 177 Signage by Lu 50 μg each of the DOTA-bound ABD fusion Z variants ZBHD02 (SEQ ID NO: 550), ZBHD07 (SEQ ID NO: 555), ZBHD08 (SEQ ID NO: 556), and ZBHD09 (SEQ ID NO: 557) in 0.2 M NaAc pH 6 was mixed with 70 μL of 0.2 M sodium acetate, pH 6.25 (metal-free). 50 MBq 177 LuCl3 (PerkinElmer) was added, and the mixture was incubated at 75°C for 1 hour. To remove loosely bound radionuclides, 1000-fold excess EDTANa4 was added, and the mixture was incubated at 75°C for 10 minutes. Purification was performed using a pre-equilibriumized NAP-5 column eluted with 1% BSA in PBS.

[0262] Evaluation of radioactive labels: The radiochemical yield of each Z variant was analyzed using instant thin-layer chromatography (ITLC-SG) (Agilent Technologies).

[0263] To perform cross-validation of Radio-ITLC data, RP-HPLC was performed using a LaChrom Elite® system (VWR Hitachi) consisting of an L-2130 pump, a UV detector (L-2400), and a radiation flow detector (Bioscan) connected in series. Purity analysis of the labeled compound was performed using an analytical column (Vydac RP C18 column, 300 Å; 3 × 150 mm; 5 μm). The RP-HPLC conditions were as follows: solvent A = 10 mM TFA in H2O, solvent B = 10 mM TFA in acetonitrile, UV detection at 220 nm, gradient elution: 5% to 70% B at 0-15 mins, 70% to 95% B at 15-18 mins, 5% B at 19-20 mins; and flow rate of 1.0 mL / min.

[0264] 99m To evaluate the in vitro stability of the Tc-labeled conjugate, each fraction (10 μL, 4 μg) of the freshly labeled Z variant was incubated with an excess volume of PBS (40 μL) at 37°C for 1 hour. 68 Ga and 177 To evaluate the in vitro stability of the Lu-labeled compounds, fractions of each labeled compound were incubated with an excess amount of EDTA. 68 The Ga-labeled material was incubated with 1000 molar excess EDTA at 37°C for 2 hours. 177 Lu-labeled samples were incubated with a 500 molar excess of EDTA in RT for 1 hour. Incubation was also performed in PBS as a control. The experiment was conducted in three consecutive sets. The emission of radionuclides was monitored by ITLC-SG as described above.

[0265] result 99m Tc marking : 99m Tc labeling: The B7-H3 bonded Z variant was obtained in radiochemical yields exceeding 95%. 99m Labeling with Tc was successful, and the RHT content was less than 5%.

[0266] 99m The specific activity of the Tc-labeled conjugate was 3 MBq / μg (molar activity 18.9 GBq / μmol).99m The Tc-labeled conjugate was stable during incubation at 37°C for 1 hour in the presence of excess PBS. 99m Tc release was less than 5%.

[0267] The results of radiolabeling and in vitro stability testing are summarized in Table 14A. In RP-HPLC analysis, the retention time of the labeled Z variant (measured with a radioactivity detector) was 11–12 minutes, the same as that of the unlabeled polypeptide (measured with a UV detector). This confirms the authenticity of the labeled conjugate.

[0268] [Table 14]

[0269] 111 Signage by In DOTA-bound ZBH538 and the reference Z variant ZAC12c were produced in radiochemical yields exceeding 91%. 111 Labeling with In was successful. After purification using a NAP-5 column, the radiochemical purity was over 98%.

[0270] 68 Signage by Ga The NOTA-bonded B7-H3-bonded Z variant yields a radiochemical yield of over 88%. 68 We successfully labeled the samples with Ga. After purification using a NAP-5 column, the radiochemical purity was over 99%. 68 The specific activity of the Ga-labeled compound was 1.2 MBq / μg. The Z variant showed high stability even after incubation in EDTA with a 1000-fold molar excess for up to 2 hours. The results of radiolabeling and in vitro stability tests are summarized in Table 14B. In RP-HPLC analysis, only one major peak was observed for each Z variant at retention times of 10-12 minutes.

[0271] [Table 15]

[0272] 177 Signage by Lu The DOTA-bound ABD fusion Z variant achieved a radiochemical yield of over 86% after EDTA treatment at 75°C for 10 minutes. 177 Labeling with Lu was successful. After purification using a NAP-5 column, the radiochemical purity was over 98%. 177 The specific activity of the Lu-labeled compound was 1 MBq / μg. The Z variant showed high stability even after incubation in EDTA with a 500-fold molar excess for up to 1 hour. The results of radiolabeling and in vitro stability tests are summarized in Table 14C. In RP-HPLC analysis, only one major peak was observed for each ABD-fused Z variant with a retention time of approximately 12 minutes.

[0273] [Table 16]

[0274] Example 10 99m In vitro evaluation of Tc-labeled B7-H3-bound Z variant. overview In this embodiment, before using the variant in the animal test described in Example 11, 99m This document describes an in vitro cell-based assay to evaluate the binding affinity and specificity of Tc-labeled conjugates.

[0275] Materials and methods cell cultureAs B7-H3 expression-positive cell lines, we used the ovarian cancer SKOV-3 cell line and the breast cancer BT-474 cell line obtained from the American Type Culture Collection (ATCC). As a non-B7-H3 expression-negative control cell line, we used the Ramos lymphoma cell line (ATCC). Using a B7-H3-specific antibody (mouse IgG1; R&D Systems, cat. No. MAB1027), we ranked the receptor expression levels of different cell lines by titration and expression quantification using BD Quantibrite beads (BD Biosciences). The estimated B7-H3 expression levels for SKOV-3, BT-474, and Ramos were 68,000 (high), 45,000 (intermediate), and 250 (low) receptors per cell, respectively (Oroujeni el al., 2022, Pharmaceutics 14:1780). The cells were cultured in RPMI medium (Flow Laboratories) supplemented with 10% fetal calf serum (BT-474 was 20% fetal calf serum), 2 mM L-glutamine, 100 IU / mL penicillin, and 100 mg / mL streptomycin. The cells were placed in cell culture dishes (35 mm in diameter) in batches of 10. 6 Cells were seeded at a cell / dish density. A set of three dishes was used to evaluate in vitro binding specificity.

[0276] In vitro binding specificity:In three control dishes, SKOV-3 and BT-474 cells were pre-saturated with a 200-fold excess of unlabeled Z variant for 15 minutes before adding the labeled conjugate. Cells from both the pre-saturated and unsaturated dishes were incubated with the labeled conjugate (10 nM) in a humidified incubator (5% CO2, 37°C) for 1 hour. After discarding the medium and washing the cells with cold serum-free medium, trypsin-EDTA solution (0.5 mL per dish) was added, and the cells were incubated for a further 10 minutes. The detached cells were diluted in 0.5 mL of complete medium, resuspended, and transferred to fractionation tubes. Cellular radioactivity was measured using an automated gamma spectrometer equipped with a 3-inch NaI(TI) well detector (2480 Wizard, Wallac), and cell-bound radioactivity was calculated. Data were analyzed using unpaired two-sided t-tests.

[0277] In vitro binding affinity As previously described (Bjorke et al., 2006 Appl. Radiat. Isot., 64:901-905), using the LigandTracer Yellow instrument (Ridgeview Instruments), the B7-H3 receptor on cells 99m The binding kinetics of the Tc-labeled Z variant were measured. SKOV-3 cells were seeded in a localized area of ​​a cell culture dish (89 mm diameter, Nunclon®, NUNC A / S). Measurements were performed in RT mode to prevent internalization. The uptake curve is as follows: 99m Tc-ZBH536, 99m Tc-ZBH538 and 99m For Tc-ZBH539, the values ​​are 1 and 3 nM. 99m For Tc-ZAC12c, recordings were made at 2, 6, and 18 nM. The radioactive culture medium was then removed and replaced with fresh, non-radioactive medium, and the dissociation curve was recorded. The data were analyzed using Interaction Map software (Ridgeview Diagnostics) to calculate the association rate, dissociation rate, and equilibrium dissociation constant (KD). The analysis was performed in duplicate.

[0278] result In vitro binding specificity : 99m The B7-H3 binding specificity of the Tc-labeled conjugate was tested using saturation experiments. When cells were pre-saturated with an excess amount of unlabeled anti-B7-H3 Z variant, binding was significantly improved (p<5×10⁻¹⁰). -5 The amount decreased (Figure 10), indicating that the binding was via B7-H3.

[0279] In vitro binding affinity : Real-time measurement of SKOV-3 cells using the LigandTracer Yellow instrument. 99m The binding dynamics of the Tc-labeled conjugate are shown in Figure 11 and Table 15. The binding of the radiolabeled conjugate to the SKOV-3 cell line was best matched using a 1:2 model, suggesting two types of interactions with B7-H3. K was present in all variants. D2 The values ​​were in the low nanomolar range. 99m Tc-ZBH538 is in the low picomolar region. D1 Value (K D1 It showed a value of 28±1.3pM (weight %=12), meaning it was the variant with the apparent highest affinity.

[0280] [Table 17]

[0281] Example 11 99m In vivo distribution and imaging studies of Tc-labeled B7-H3-bound Z variants. overview The use of radionuclide molecular imaging to monitor B7-H3 expression may replace biopsy sampling, providing a non-invasive and repeatable alternative method that also enables simultaneous detection of B7-H3 in metastatic lesions. This example describes in vivo in vivo distribution studies performed in both non-tumor and tumor-bearing mice using the B7-H3-binding Z variants ZBH536 (SEQ ID NO: 536), ZBH538 (SEQ ID NO: 538), ZBH539 (SEQ ID NO: 539), and the reference Z variant ZAC12c (SEQ ID NO: 540).

[0282] Materials and methods Handling of animals: The animal experiments were conducted in accordance with the national laws regarding laboratory animals. Approval was obtained from the Uppsala Animal Research Ethics Committee.

[0283] In vivo distribution in non-tumor mice : 3 μg of female NMRI mice (average body weight 36.5 ± 6.4 g) 99m A Tc-labeled Z variant (60 kBq, 100 μL in PBS) was injected via tail vein. Four hours later, the mice were euthanized by overdose of anesthetic solution (20 μL per g of body weight: ketamine, 10 mg / mL; xylazine, 1 mg / mL). Subsequently, the heart was punctured and blood samples were collected. Organ and tissue samples were collected and weighed. Organ radioactivity was measured using a gamma spectrometer with a NaI(TI) detector (2480 Wizard, Wallac), along with three standards and an empty syringe for each animal. Organ uptake was calculated as the injection dose percentage per g of tissue (%ID / g). Data were analyzed using unpaired two-sided t-tests and ANOVA with GraphPad Prism (Windows version 6; GraphPad Saltware) to determine statistical significance (p<0.05).

[0284] In vivo distribution in tumor-bearing mice In BALB / C nu / nu mice carrying B7-H3-positive SKOV-3 xenografts, the in vivo distribution and targeting characteristics were evaluated. To establish the xenografts, SKOV-3 cells (10 7 Cells (mouse) were subcutaneously injected into the right hind limb of female BALB / c nu / nu mice. As a specificity control, B7-H3 negative Ramos cells (5 × 10) were used. 6 Cells ( / mouse) were subcutaneously transplanted into the left hind limb of female BALB / c nu / nu mice. The experiment was performed 3 weeks after cell transplantation. The average animal body weight was 19.1 ± 1.9 g, and the average tumor weight was 0.10 ± 0.08 g and 0.09 ± 0.07 g for SKOV-3 and Ramos xenografts, respectively. In a group of 4 mice with tumors, 99mTc-labeled Z variant (3 μg, 60 kBq, 100 μL in PBS) was injected into the tail vein. To investigate B7-H3 specific accumulation, a group of animals with B7-H3-negative Ramos xenografts were injected with the same peptide and active dose for each conjugate. Biological distribution was measured 4 hours after injection and performed and analyzed as described above for NMRI mice.

[0285] In vivo imaging To confirm the results of in vivo distribution, SPECT / CT imaging was performed on small animals. One SKOV-3 xenograft mouse and one Ramos xenograft mouse were given 6 MBq / 3 μg. 99m A Tc-labeled Z variant was intravenously injected into the mice. Mice were imaged 4 hours after injection using a nanoScan SPECT / CT scanner (Mediso Medical Imaging Systems). The mice were euthanized by CO2 asphyxiation immediately before being placed in the camera. CT scans were performed with the following parameters: energy peak 50kV, 670μA, 480 projection, and scanning time 2.29 minutes. CT images were reconstructed in real time using Nucline 2.03 software (Mediso Medical Imaging Systems). SPECT raw data was reconstructed using TeraTomo® 3DSPECT reconstruction technology.

[0286] result In vivo distribution in non-tumor mice :In NMRI mice 4 hours after injection 99m The in vivo distribution results of the Tc-labeled Z variant are shown in Figure 12 and Table 16. The in vivo distribution data showed no significant difference in blood concentration. 99m Tc-ZBH538 showed low uptake in almost all organs and tissues. 99m Tc-ZBH536, 99m Tc-ZBH538 and 99m Tc-ZBH539 is a reference variant. 99m Compared to Tc-ZAC12c, hepatic absorption and splenic uptake were significantly lower; renal uptake was at the same level for all radioactive isotopes.

[0287] [Table 18]

[0288] In vivo distribution in tumor-bearing mice : In BALB / C nu / nu mice with SKOV-3 xenografts 4 hours after injection 99m The in vivo distribution of the Tc-labeled Z variant is shown in Figure 13A and Table 17. The tumor-to-organ ratio is shown in Figure 13B and Table 18. 99m Tc-ZBH536 (2.15 ± 1.01% ID / g), 99m Tc-ZBH538 (1.54 ± 0.19% ID / g) and 99m Tumor uptake of Tc-ZBH539 (3.19±0.21% ID / g) is compared to the reference variant. 99m It was higher than Tc-ZAC12c (1.04±0.08%ID / g). 99m Tc-ZBH538 had the lowest blood concentration (0.06±0.01%ID / g), and its uptake in the liver (0.26±0.02%ID / g) and bone (0.006±0.001%ID / g) was significantly lower (p<0.05) compared to other radioactive conjugates. 99m Tc-ZBH539 is, 99m Tc-ZBH538 (10.37 ± 1.34% ID / g) and 99m Compared to Tc-ZAC12c (10.30±1.13%ID / g), renal uptake was significantly lower (p<0.05) (5.85±0.28%ID / g). In the in vivo distribution profile, 99m The tumor-blood ratio (25.7±2.5) for Tc-ZBH538 was: 99m Tc-ZBH536 (11.3 ± 4.2) and 99m The levels were significantly higher compared to Tc-ZAC12c (11.0±0.5) (p<0.05). 99m The tumor-to-liver ratio for Tc-ZBH538 (5.9±0.8) was significantly higher (p<0.05) than for other radioisotopes.

[0289] Furthermore, in B7-H3 positive SKOV-3 xenografts 99mUptake of Tc-ZBH538 was significantly higher 4 hours after injection than in B7-H3-negative Ramos xenografts (p<5×10⁻¹⁰). -5 The tumor uptake was high (Figure 14), which supports the idea that tumor uptake is B7-H3 specific in vivo.

[0290] [Table 19]

[0291] [Table 20]

[0292] In vivo imaging :NanoSPECT / CT imaging results (Figure 15) show that in SKOV-3 tumors expressing B7-H3, 99m B7-H3 expression was visualized with high contrast 4 hours after injection of the Tc-labeled Z variant. 99m The activity accumulation of Tc-ZBH538 in the liver was visualized to be low. The uptake of activity in B7-H3-negative Ramos xenografts was considerably lower than in SKOV-3 xenografts (Figure 16). This confirms the B7-H3-mediated binding of these tracers in vivo.

[0293] Example 12 111 In vitro and in vivo evaluation of In-labeled B7-H3-bound Z variant. overview This example is, 111 This paper describes in vitro characterization and in vivo in vivo distribution studies in tumor-bearing mice using the B7-H3-binding Z variant ZBH538 (SEQ ID NO: 538) and the reference Z variant ZAC12c (SEQ ID NO: 540), both labeled with In. 99m The Tc-labeled conjugate was essentially carried out as described in Examples 10 and 11. 99mSide-by-side evaluations with the same variant labeled with Tc were performed in vivo for a more reliable comparison of differences associated with such radiolabeling, independent of inter-batch variability in animal physiology of mice that could otherwise affect biodistribution.

[0294] Materials and methods In vitro evaluation : Binding specificity and 111 An in vitro cell-based assay to evaluate the binding affinity of the In-labeled conjugate was performed essentially as described in Example 10. For determining the binding affinity by LigandTracer measurement, the uptake curve was obtained. 111 For In-ZBH538, the values ​​are 1, 3, and 9 nM. 111 In-ZAC12c was recorded at 2, 6, and 18 nM.

[0295] In vivo distribution in tumor-bearing mice BALB / C nu / nu mice, each carrying SKOV-3 and Ramos xenografts respectively, were prepared as described in Example 11. The average animal body weight was 17.8 ± 1.3 g, and the average tumor weights were 0.10 ± 0.06 g and 0.7 ± 0.4 g for SKOV-3 and Ramos xenografts, respectively. In a group of four mice with tumors, 111 In-labeled Z variant (3 μg, 20 kBq, 100 μL in PBS) was injected into the tail vein. To investigate B7-H3 specific accumulation, one group of animals with B7-H3-negative Ramos xenografts were injected with the same peptide and active dose for each conjugate. In vivo distribution was measured at 4 hours and 24 hours post-injection in mice with SKOV-3 xenografts, and at 4 hours post-injection in mice with Ramos xenografts. For comparison, the two groups of mice were... 99m Tc-ZBH538 and 99m Tc-ZAC12c (3 μg, 60 kBq, 100 μL, in PBS) was injected into the tail, and its biodistribution was measured 4 hours after injection.

[0296] In vivo imaging Two xenograft mice carrying SKOV-3 were each given 1-2 MBq / 3 μg 111In-labeled ZBH538 and ZAC12c were administered intravenously. To confirm in vivo specificity, two mice carrying Ramos xenografts were intravenously injected with the same active dose of each radiolabeled Z variant. As described in Example 11, the mice were imaged 4 hours after injection using a nanoScan SPECT / CT scanner.

[0297] result In vitro binding specificity : 111 The B7-H3 binding specificity of the In-labeled conjugate was tested using a saturation experiment. When cells were pre-saturated with an excess amount of unlabeled anti-B7-H3 Z variant, binding was significantly improved (p<5×10⁻¹⁰). -5 ) decreased, and it was shown that the bond was mediated through B7-H3.

[0298] In vitro binding affinity: Real-time measurement of SKOV-3 cells using the LigandTracer Yellow instrument. 111 The binding dynamics of the In-labeled conjugate are shown in Figure 17 and Table 19. 111 The binding of In-labeled conjugates is best fitted using a 1:1 model, and K in the sub-nanomole region. D This was obtained.

[0299] [Table 21]

[0300] In vivo distribution in tumor-bearing mice : In BALB / C nu / nu mice with SKOV-3 xenografts 4 hours and 24 hours after injection. 111 The in vivo distribution of the In-labeled Z variant is shown in Figures 18A-B and Table 20. The tumor-to-organ ratio is shown in Figures 19A-B and Table 21. 111 Tumor uptake of In-ZBH538 was 3.63±0.31% ID / g at 4 hours and 0.78±0.18% ID / g at 24 hours after injection, respectively. 111The tumor uptake was significantly higher (p<0.05) than that of In-ZAC12c (1.80±0.49 and 0.37±0.13%ID / g at 4 and 24 hours after injection, respectively). 111 In-ZBH538 is 111 Compared to In-ZAC12c (6.43±1.05 and 4.07±0.21% ID / g, respectively), the hepatic absorption rates were significantly lower (p<0.05) at both time points of the study (1.07±0.08 and 0.80±0.02% ID / g, respectively). Both radiolabeled compounds were observed to rapidly lose activity from the blood and almost all organs and tissues over time.

[0301] [Table 22]

[0302] The measured tumor-to-organ ratio was, 111 Compared to In-ZAC12c (20.60±7.10 and 20.41±6.75 respectively), both 4 hours and 24 hours after injection. 111 In-ZBH538 (31.09±2.9 and 43.18±13.82, respectively) showed a significantly higher tumor-to-blood ratio (p<0.05). Because uptake decreased over time in almost all organs and tissues, the tumor-to-organ ratio was higher at 4 hours compared to 24 hours for both radioactive conjugates. 111 In-ZBH538 is, 111 Compared to In-ZAC12c, it generally showed a higher tumor-to-organ ratio 4 hours after injection. For example, 111 The tumor-to-liver ratio (3.40±0.49) and tumor-to-bone ratio (168.57±29.09) for In-ZBH538 were: 111 These values ​​were significantly higher than those of In-ZAC12c (0.28±0.05 and 100.00±29.43, respectively). 111 In-ZBH538 was advantageous for imaging bone and liver metastases at an early stage of imaging.

[0303] [Table 23]

[0304] In tumor-bearing mice 4 hours after injection 111 In sign ZBH538 and 99m Figure 20 and Tables 22-23 show the results of comparing the in vivo distribution of Tc-labeled ZBH538. From this data, it can be seen that uptake in tumors is as follows: 99m Tc-labeled conjugate ( 99m Tc-ZBH538: 1.59±0.19% ID / g and 99m Compared to Tc-ZAC12c (0.84±0.18%ID / g), 111 In-ZBH538 (3.63±0.31%ID / g) showed significantly higher uptake (p<0.05). Liver uptake was 111 In-ZBH538 (1.07±0.08% ID / g) 111 In-ZAC12c (6.43 ± 1.05% ID / g) and 99m It was significantly lower compared to Tc-ZAC12c (2.70±0.30%ID / g) (p<0.05). 99m Tc-ZBH538 showed the lowest uptake in most organs and tissues, including tumors. 111 In-labels and 99m The difference in renal uptake of Tc-labels is due to the difference in the persistence and non-persistence of radioisotopes.

[0305] [Table 24]

[0306] As a result of these in vivo distribution profiles, 111 The tumor-to-blood ratio of In-ZBH538 is: 99m Tc-labeled conjugate ( 99m Tc-ZBH538: 15.74±4.13 99m Compared to Tc-ZAC12c: 4.11±0.87, it was significantly higher (p<0.05) (31.09±2.91). The tumor liver ratio was 111 In-ZBH538 (3.40 ± 0.49) and 99m There was no significant difference in Tc-ZBH538 (4.17±0.60).

[0307] [Table 25]

[0308] In vivo imaging : In BALB / C nu / nu mice with B7-H3 positive SKOV-3 xenografts 4 hours after injection 111 In-ZBH538 and 111 The results of nanoSPECT / CT imaging of In-ZAC12c (Figure 21A) confirmed the in vitro and intravitreal distribution data. 111 Compared to In-ZAC12c, 111 In-ZBH538 showed low activity accumulation in the liver and high accumulation in tumors. Furthermore, the uptake of activity in B7-H3 negative Ramos xenografts was considerably lower than in SKOV-3 xenografts (Figure 21B), which suggests that these 111 We confirmed the in vivo binding of the In-labeled conjugate via B7-H3.

[0309] Example 13 In vitro characterization of ABD-fused B7-H3 bonded Z variant overview For in vivo use, it is desirable to extend the half-life of the polypeptide, and one means of achieving this is fusion with an ABD moiety. This example describes the in vitro characterization of eight DOTA-binding ABD-fusion Z variants (ZBHD02-ZBHD09; SEQ ID NOs. 550-557) constructed in different formats with respect to 1) the arrangement of ABD moieties, 2) the number of B7-H3 binding Z moieties, and 3) the design of the linkers between these moieties. The polypeptides were cloned and prepared as described in Example 3 and studied by binding assays by SPR analysis and binding to SKOV-3 cells.

[0310] Materials and methods SPR analysis - binding to B7-H3Multicycle kinetic (MCK) screening of polypeptides binding to B7-H3 was performed using SPR on a Biacore 8K. B7-H3(4Ig)-His was immobilized by amine coupling to a carboxylated dextran layer on the surface of a CM5 chip (Cytiva) according to the manufacturer's recommendations. Surface 1 was activated and then deactivated and used as a reference. Each polypeptide was injected into the chip surface at concentrations of 15, 45, and 135 nM for 150 seconds, followed by dissociation for 720 seconds (flow rate 50 μl / min). The chip surface was regenerated with 50 mM NaOH (two pulses of 20 seconds / 30 μL / min). HBS-EP+ was used as the running buffer and dilution buffer. The assay temperature was 25°C. The reference cell (flow cell 1) and blank cycle injection (HBS-EP+) were subtracted from the sensorgram before evaluation using Biacore Insight evaluation software.

[0311] SPR analysis - binding to albumin MCK analysis was performed as described in the previous section. HSA (Albumedix, cat. No. 205-005) and MSA (Sigma Aldrich, cat. No. A3559) were immobilized on CM5 chips. Polypeptides ZBHD02 (SEQ ID NO: 550), ZBHD07 (SEQ ID NO: 555), ZBHD08 (SEQ ID NO: 556), and ZBHD09 (SEQ ID NO: 557) were injected onto the chip surface at concentrations of 1.6, 8, 40, 200, and 1000 nM for 400 seconds, and then dissociated for 4 hours (flow rate 30 μl / min).

[0312] Cell assay The binding of the polypeptide to B7-H3 expressing SKOV-3 cells and its fluorescence intensity were evaluated. The experiment was performed on SKOV-3 cells as described in Example 4, but the polypeptide concentration was reduced from 500 nM to 0.16 nM and incubated in the presence of 1.5 μM HSA.

[0313] result SPR analysis - binding to B7-H3Multicycle kinetic screens of polypeptides binding to B7-H3 were analyzed using SPR analysis with Biacore 8K. Polypeptides were injected at three concentrations onto immobilized B7-H3. An example of sensorgrams obtained for the binding of eight B7-H3 binding polypeptides, ZBHD02-ZBHD09 (SEQ ID NOs. 550-557), to B7-H3(4Ig)-His is shown in Figure 22.

[0314] SPR analysis - binding to albumin All four polypeptides tested showed similar binding kinetic values ​​retained for HSA and similar binding kinetic values ​​for MSA (Figure 23). As expected, the off-rate was faster for binding to MSA compared to binding to HSA. D The result was low.

[0315] Cell assay Table 24 summarizes the calculated EC50 values ​​for the binding of ABD fusion polypeptides to B7-H3 expressing SKOV-3 cells, and Figure 24 shows the binding curve.

[0316] [Table 26]

[0317] Consistent with the observations described in Example 8, both SPR and cell assay analyses show that the ABD fusion polypeptides (ZBHD04-ZBHD09), which are dimers with respect to the Z moiety, exhibit stronger binding to B7-H3 compared to the ABD fusion polypeptides (ZBHD02 and ZBHD03) which contain only one Z moiety.

[0318] Example 14 177 In vitro and in vivo evaluation of Lu-labeled ABD-fused B7-H3-linked Z variant. summary This example is a subset of the ABD-fused B7-H3 bonded Z variant evaluated in Example 13: as described in Example 9. 177This report describes in vitro characterization and in vivo in vivo distribution studies in tumor-bearing mice using ZBHD02 (SEQ ID NO: 550), ZBHD07 (SEQ ID NO: 555), ZBHD08 (SEQ ID NO: 556), and ZBHD09 (SEQ ID NO: 557), each labeled with Lu.

[0319] Materials and methods In vitro evaluation : 177 In vitro cell-based assays to evaluate the binding specificity and binding affinity of Lu-labeled ABD-fusion Z variants were performed essentially as described in Example 10. For determining binding affinity by LigandTracer measurement, uptake curves were recorded at 1 nM and 3 nM in both the presence and absence of 100 nM HSA.

[0320] In vivo distribution in tumor-bearing mice BALB / C nu / nu mice were prepared, each containing SKOV-3 and Ramos xenografts, respectively, and in vivo distribution measurements were performed two weeks after cell transplantation, basically as described in Example 11. The mean body weight of the animals was 18.5 ± 0.7 g. The mean tumor weight was 0.4 ± 0.2 g. In a group of SKOV-3 xenograft mice... 177 Lu-labeled ABD-fused Z variant (468 pmol, 260 kBq, 100 μL in PBS) was injected via tail vein. In vivo distribution was measured 48 hours after injection.

[0321] 177 An additional in vivo distribution study was also conducted on Lu-ZBHD02 (SEQ ID NO: 550), and evaluations were performed at 24 hours and 168 hours. Two groups of SKOV3 xenograft mice, each consisting of four mice, were used. 177 Lu-ZBHD02 (468 pmol, 260 kBq, 100 μL of PBS solution) was injected.

[0322] In vivo imaging : Four SKOV-3 xenograft mice were each given 3 MBq 177 Lu-ZBHD02, 177 Lu-ZBHD07, 177 Lu-ZBHD08 and 177Lu-ZBHD09 was administered intravenously. To confirm in vivo specificity, 3 MBq was administered to one Ramos xenograft mouse and one SKOV-3 xenograft mouse. 177 Lu-ZBHD02 was administered intravenously. In both experiments, nanoSPECT / CT images were acquired 48 hours after injection using nanoScan SC. CT acquisition was performed using an X-ray energy of 50 keV, and 20-minute SPECT helical scans were acquired using energy windows of 50-62, 103-124, and 188-230 keV. The data were reconstructed using Tera-Tomo™ 3D SPECT software.

[0323] result In vitro binding specificity : 177 The B7-H3 binding specificity of the Lu-labeled ABD-fusion Z variant was tested using saturation experiments. When cells were pre-saturated with a 200-fold excess of unlabeled anti-B7-H3 Z variant, binding was significantly reduced (p<0.05), indicating that binding is mediated by B7-H3.

[0324] In vitro binding affinity: Measurements were taken in real time using the LigandTracer Yellow instrument. 177 Table 25 shows the binding dynamics of Lu-labeled ABD-fused Z variants to SKOV-3 cells. The best binding match was achieved using a 1:2 model. Higher affinity binding (lower K) was observed compared to variants containing two Z sites (ZBHD07, ZBHD08, and ZBHD09) and variants sharing a single Z site (ZBHD02). D ) was observed. This is consistent with the results shown in Example 13 for the corresponding unlabeled variant.

[0325] [Table 27]

[0326] In vivo distribution in tumor-bearing mice : Four types of SKOV-3 tumor-bearing mice 48 hours after injection 177The results of a frontal comparison of the biological distribution of Lu-labeled ABD-fused Z variants are shown in Figure 25A and Table 26. The data are as follows: 177 Tumor uptake of Lu-ZBHD02 (19.64±0.77% ID / g) 177 Lu-ZBHD08 (13.13 ± 1.15% ID / g) and 177 This indicates that it was significantly higher (p<0.05) than Lu-ZBHD09 (13.95±0.52%ID / g). 177 Renal uptake of Lu-ZBHD02 (16.44±1.46% ID / g) is as follows: 177 Lu-ZBHD08 (19.77±2.07% ID / g) and 177 It was significantly lower (p<0.05) than Lu-ZBHD09 (23.99±2.30%ID / g). No significant difference was observed in liver uptake. 177 Lu-ZBHD08 177 Lu-ZBHD02 and 177 Lu-ZBHD07 showed significantly higher blood concentrations (p<0.05).

[0327] [Table 28]

[0328] At the additional time points of 24 hours and 168 hours after injection 177 The in vivo distribution of Lu-ZBHD02 is shown in Figure 25B and Table 27. Clearance from blood and normal organs and tissues such as lungs, stomach, muscle, and bone was rapid over time. Tumor uptake was 20.29±3.14%ID / g 24 hours after injection and was maintained until 48 hours (19.64±0.77%ID / g), before decreasing to 12.61±2.46%ID / g 168 hours after injection. Renal uptake did not show a significant difference between 24 hours (14.84±0.61%ID / g) and 48 hours (16.44±1.46%ID / g) after injection, but renal activity was significantly reduced at 168 hours (5.97±1.03%ID / g) (p<0.05). Based on the area under the curves for blood, kidney, tumor, and bone, the tumor-to-blood, tumor-to-kidney, and tumor-to-bone ratios were 3.5, 1.4, and 10.6, respectively.

[0329] [Table 29]

[0330] In vivo imaging NanoSPECT / CT imaging (Figure 26) was performed on BALB / C nu / nu mice carrying B7-H3 positive SKOV-3 xenografts 48 hours after injection, confirming the in vivo distribution data of the ex vivo drug. 177 Lu-ZBHD02 showed higher accumulation in tumors and lower accumulation in the kidneys compared to other variants. Furthermore, active uptake in B7-H3-positive SKOV-3 xenografts (Figure 27A) was considerably higher than in B7-H3-negative Ramos xenografts (Figure 27B), confirming in vivo B7-H3-mediated binding.

[0331] In summary, it has a single Z portion 177 Lu-ZBHD02 contains two Z moieties despite binding to B7-H3 with lower affinity. 177 Lu-ZBHD07, 177 Lu-ZBHD08 and 177 Compared to Lu-ZBHD09, it showed a better in vivo distribution profile.

[0332] Example 15 Second-generation in vitro and in vivo evaluation of mature B7-H3 binding Z variants. overview In this embodiment, NOTA conjugate is performed as described in Example 3, and as described in Example 9. 68 This paper describes in vitro cell binding specificity and in vivo in vivo distribution studies in tumor-bearing mice using Ga-labeled B7-H3 binding Z variants ZBH001 (SEQ ID NO: 1), ZBH002 (SEQ ID NO: 2), ZBH003 (SEQ ID NO: 3), and the reference Z variant ZAC12 (SEQ ID NO: 543), each having the format G-[ZBH###]-C (SEQ ID NO: 628).

[0333] Materials and methods In vitro binding specificity: 68 An in vitro cell-based assay to evaluate the binding specificity of the Ga-labeled Z variant was essentially performed as described in Example 10.

[0334] Biological distribution in tumor-bearing mice BALB / C nu / nu mice were prepared with SKOV-3 and Ramos xenografts, respectively, and in vivo distribution measurements were performed 3 weeks after cell transplantation, as essentially described in Example 11. The mean animal body weight was 18.2 ± 1.2 g, and the mean tumor weights were 0.11 ± 0.04 g and 0.41 ± 0.17 g for SKOV-3 and Ramos xenografts, respectively. In a group of four SKOV-3 xenograft mice... 68 Ga-labeled Z variant (2 μg, 400 kBq, 100 μL in PBS) was injected into the tail vein. To investigate B7-H3 specific accumulation, the same peptide and dose were injected into a group of mice carrying B7-H3-negative Ramos xenografts. In vivo distribution was measured 2 hours after injection.

[0335] In vivo imaging To confirm the results of in vivo distribution, small animal PET / CT imaging was performed. Each Z variant was intravenously injected at a dose of 2.5 MBq into one SKOV-3 xenograft mouse. Additionally, 2.5 MBq was injected into one Ramos xenograft mouse. 68Ga ZBH003 was injected into the mice. The mice were imaged two hours after injection using a PET / CT scanner (Mediso Medical Imaging Systems). The mice were euthanized by CO2 asphyxiation immediately before being placed in the camera. CT scans were performed using a nanoScan SPECT / CT (Mediso Medical Imaging Systems Ltd) in the same bed position immediately after the PET scan. The PET scan lasted 30 minutes, followed by a CT scan with the following parameters: CT energy peak 50 keV, 670 A, 480 projections, scanning time 2.29 minutes. The PET data was reconstructed into still images using the Tera-Tomo™ 3D reconstruction engine. The raw CT files were reconstructed in real time using Filter Back Projection in Nucline 2.03 software (Mediso Medical Imaging Systems). The PET and CT files were fused and displayed as Maximum Intensity Projection (MIP).

[0336] result In vitro binding specificity: In vitro 68 The B7-H3 binding specificity of the Ga-labeled Z variant was verified by saturation experiments. When cells were pre-saturated with a 200-fold excess of unlabeled anti-B7-H3 Z variant, binding was significantly reduced (p<0.05), indicating that the binding was B7-H3 specific.

[0337] In vivo distribution in tumor-bearing mice: The in vivo distribution in nude mice carrying human cancer xenografts is similar to that of B7-H3-positive SKOV-3 xenografts. 68 The tumor uptake of the Ga-labeled Z variant was significantly higher (p<0.05) than that of B7-H3-negative Ramos xenografts 2 hours after injection (Figure 28), demonstrating that tumor uptake is B7-H3 specific in vivo.

[0338] The results of the in vivo distribution in mice carrying SKOV-3 xenografts two hours after injection are shown in Figure 29A and Table 28. All three new variants were... 68Ga-ZBH003 (8.96 ± 1.16% ID / g), 68 Ga-ZBH001 (6.61 ± 0.41% ID / g) and 68 Tumor uptake of Ga-ZBH002 (7.25±1.56% ID / g) is as follows: 68 It was significantly higher (p<0.05) than Ga-ZAC12 (3.24±0.77%ID / g). (Reference variant) 68 Ga-ZAC12 showed significantly lower hepatic uptake (p<0.05) compared to the new variant. Tumor-to-organ ratios are shown in Figure 29B and Table 29.

[0339] [Table 30]

[0340] [Table 31]

[0341] In vivo imaging :BALB / C nu / nu mice fitted with B7-H3 positive SKOV-3 xenografts 68 When the Ga-labeled Z variant was injected and nanoPET / CT imaging was performed 2 hours after injection (Figure 30), in vitro in vivo distribution data was confirmed. Compared to other variants, 68 Significantly high uptake of Ga ZBH003 activity was observed in tumors. Furthermore, in B7-H3-positive SKOV-3 xenografts (Figure 31A) 68 The activity uptake of Ga ZBH003 was considerably higher than that of B7-H3-negative Ramos xenografts (Figure 31B), confirming B7-H3-mediated binding in vivo.

[0342] List of embodiments by category 1. A B7-H3 linked polypeptide comprising a B7-H3 linked motif BM, wherein the motif is as follows: i) EKX3X4ALX7EIX 10 X 11 LPNLX 16 X17 X 18 QX 20 X 21 AFIX 25 X 26 LNX 29 X 30 (Sequence ID 567) Here, independently of each other X3 is selected from I and V; X4 is selected from A, D, E, G, H, K, L, M, N, Q, S, T, and Y; X7 is selected from A, G, H, and S; X 10 It is selected from I and V; X 11 It is selected from N and W; X 16 It is selected from N and T; X 17 is selected from H and Y; X 18 It is selected from A, D, E, G, H, N, Q, S, and T; X 20 It is selected from I and V; X 21 is selected from K and R; X 25 It is selected from A, E, F, H, L, and W; X 26 is selected from K and S; X 29 It is selected from A, D, and E; X 30 is selected from A, D and H; and ii) An amino acid sequence having at least 93% identity with the sequence defined in i) The above B7-H3 linked polypeptide, comprising an amino acid sequence selected from the above.

[0343] 2. In array i), X3 is selected from I and V; X4 is selected from D, E, G, H, K, L, M, N, Q, S, T, and Y; X7 is selected from A, G, H, and S; X10 It is selected from I and V; X 11 It is selected from N and W; X 16 It is selected from N and T; X 17 is selected from H and Y; X 18 It is selected from A, D, E, G, H, N, Q, S, and T; X 20 It is selected from I and V; X 21 is selected from K and R; X 25 It is selected from A, E, F, H, L, and W; X 26 is selected from K and S; X 29 is selected from A, D and E; and X 30 This is a B7-H3 linked polypeptide as described in item 1, selected from A, D, and H.

[0344] 3. In array i), X3 is selected from I and V; X4 is selected from D, G, K, L, M, N, S, T, and Y; X7 is selected from A, G, H, and S; X 10 is I; X 11 It is selected from N and W; X 16 is T; X 17 is Y; X 18 It is selected from A, D, E, H, N, Q, S, and T; X 20 It is selected from I and V; X 21 is selected from K and R; X 25 is selected from A, E, H, and W; X 26 is K; X29 is selected from A, D and E; and X 30 This is a B7-H3 conjugated polypeptide selected from A, D, and H, as described in any preceding item.

[0345] 4. In array i), X3 is I; X4 is selected from K and S; The X7 is available in G and S versions; X 10 is I; X 11 is W; X 16 is T; X 17 is Y; X 18 is selected from E, N, and Q; X 20 is I; X 21 is K; X 25 is selected from A and H; X 26 is K; X 29 is selected from A and D; and X 30 This is a B7-H3 conjugated polypeptide selected from A and D, as described in any preceding item.

[0346] 5. A B7-H3 binding polypeptide as described in item 1, wherein sequence i) corresponds to the sequence from position 8 to 37 of a sequence selected from the group consisting of sequence numbers 1 to 535.

[0347] 6. A B7-H3 binding polypeptide as described in item 5, wherein sequence i) corresponds to the sequence from position 8 to 37 in a sequence selected from the group consisting of sequence numbers 1-21 and 480-535.

[0348] 7. A B7-H3 binding polypeptide as described in item 6, wherein sequence i) corresponds to the sequence from position 8 to 37 of a sequence selected from the group consisting of sequence numbers 1 to 21.

[0349] 8. A B7-H3 binding polypeptide as described in item 7, wherein sequence i) corresponds to the sequence from position 8 to 37 of a sequence selected from the group consisting of sequence numbers 1 to 3.

[0350] 9. The B7-H3 linked polypeptide described in item 8, where sequence i) corresponds to the sequence from position 8 to 37 of sequence number 1.

[0351] 10. The B7-H3 binding polypeptide described in item 8, where sequence i) corresponds to the sequence from position 8 to 37 of sequence number 2.

[0352] 11. The B7-H3 binding polypeptide described in item 8, wherein sequence i) corresponds to the sequence from position 8 to 37 of sequence number 3.

[0353] 12. A B7-H3 binding polypeptide as described in any preceding item, wherein the binding motif forms part of a 3-helix bundle protein domain.

[0354] 13. The B7-H3 binding polypeptide according to item 12, wherein the binding motif essentially constitutes two alpha helices having interconnected loops within the 3-helix bundle protein domain.

[0355] 14. The B7-H3 binding polypeptide according to item 12 or 13, wherein the 3-helix bundle protein domain is selected from a bacterial receptor domain.

[0356] 15. The B7-H3 binding polypeptide according to any one of items 12 to 14, wherein the 3-helix bundle protein domain is selected from the domain of protein A derived from Staphylococcus aureus or a derivative thereof.

[0357] 16. Containing a binding module (BMod), its amino acid sequence is as follows: iii) K-[BM]-PSQSX a X b LLXc EAKKLX d X e X f Q (Sequence ID 568) Here, [BM] is a B7-H3 bond motif defined in any one of items 1 through 11; X a It is selected from A and S; X b is selected from E and N; X c is selected from A, S, and C; X d is selected from E, N, and S; X e is selected from D, E and S; and X f is selected from A and S; and an amino acid sequence having at least 93% identity with the sequence defined in iv)iii) A B7-H3 linked polypeptide, selected from any of the preceding items.

[0358] 17. A B7-H3 binding polypeptide as described in item 16, wherein sequence iii) corresponds to the amino acid sequence from position 7 to 55 of a sequence selected from the group consisting of sequence numbers 1 to 535.

[0359] 18. A B7-H3 linked polypeptide as described in item 17, wherein sequence iii) corresponds to the amino acid sequence from position 7 to 55 of a sequence selected from the group consisting of SEQ ID NOs: 1-21 and 480-535.

[0360] 19. A B7-H3 linked polypeptide as described in item 18, wherein sequence iii) corresponds to the amino acid sequence from position 7 to 55 of a sequence selected from the group consisting of sequence numbers 1 to 21.

[0361] 20. A B7-H3 binding polypeptide as described in item 19, wherein sequence iii) corresponds to the amino acid sequence from position 7 to 55 of a sequence selected from the group consisting of sequence numbers 1 to 3.

[0362] 21. Sequence iii) corresponds to the amino acid sequence from position 7 to 55 of sequence number 1, and is the B7-H3 linked polypeptide described in item 20.

[0363] 22. Sequence iii) corresponds to the amino acid sequence from position 7 to 55 of sequence number 2, and is the B7-H3 linked polypeptide described in item 20.

[0364] 23. Sequence iii) corresponds to the amino acid sequence from position 7 to 55 of sequence number 3, and is the B7-H3 linked polypeptide described in item 20.

[0365] 24. Below: v)YA-[BMod]-AP(Sequence ID 569) Here, [BMod] is defined as follows: and Amino acid sequences having at least 86% identity with the sequences defined by vi)v). A B7-H3 linked polypeptide, comprising an amino acid sequence selected from any of the preceding items, as described in any preceding item.

[0366] 25. Below: vii)VDAKYAK-[BM]-PSQSSELLSEAKKLNDSQAPK(Sequence ID 570) Here, [BM] is as defined in any one of items 1-11; and an amino acid sequence having at least 86% identity with the sequence defined by viii)vii). A B7-H3 linked polypeptide, comprising an amino acid sequence selected from any of the preceding items, as described in any preceding item.

[0367] 26. Below: ix)AEAKFAK-[BM]-PSQSSELLSEAKKLSESQAPK(Sequence ID 571); where [BM] is as defined in any one of items 1-11; and Amino acid sequences having at least 86% identity with the sequence defined by x)ix). A B7-H3 linked polypeptide described in any one of items 1-23, containing an amino acid sequence selected from the above.

[0368] 27. A B7-H3 linked polypeptide as described in item 26, wherein sequence ix) is selected from the group consisting of sequence numbers 15-16, 420-424, 427-428, 430-436, 438-444 and 480-535.

[0369] 28. Below: xi)AEAKYAK-[BM]-PSQSSELLSEAKKLNDSQAPK(sequence ID 572); where [BM] is as defined in any one of items 1-11; and Amino acid sequences having at least 86% identity with the sequence defined by xii)xi). A B7-H3 linked polypeptide described in any one of items 1-23, containing an amino acid sequence selected from the above.

[0370] 29. A B7-H3 linked polypeptide as described in item 28, wherein sequence xi) is selected from the group consisting of sequence numbers 1-13, 17-418, and 446-479.

[0371] 30. A B7-H3 binding polypeptide as described in item 29, wherein sequence xi) is selected from the group consisting of sequence numbers 1-13 and 17-21.

[0372] 31. A B7-H3 binding polypeptide as described in item 30, wherein sequence xi) is selected from the group consisting of sequence numbers 1 to 3.

[0373] 32. The B7-H3 linked polypeptide described in item 31, wherein sequence xi) is sequence number 1.

[0374] 33. The B7-H3 linked polypeptide described in item 31, wherein sequence xi) is sequence number 2.

[0375] 34. The B7-H3 linked polypeptide described in item 31, wherein sequence xi) is sequence number 3.

[0376] 35. Below: xiii)AEAKFAK-[BM]-PSQSSELLSEAKKLNESQAPK(Sequence ID 574); where [BM] is as defined in any one of items 1-11; and Amino acid sequences having at least 86% identity with the sequences defined by xiv)xiii). A B7-H3 linked polypeptide described in any one of items 1-23, containing an amino acid sequence selected from the above.

[0377] 36. The B7-H3 linked polypeptide described in item 35, wherein sequence number xiii) is sequence numbers 14, 425-426, 429, 437 and 445.

[0378] 37. K interaction with B7-H3 D The value is at most 1 × 10 -6 M, for example, at most 5 x 10 -7 M, for example, at most 1 × 10 -7 M, for example, at most 5 x 10 -8 M, for example, at most 1 × 10 -8 A B7-H3-binding polypeptide described in any preceding item, which is capable of binding to B7-H3 as M.

[0379] 38. A B7-H3 linked polypeptide as described in any preceding item, comprising additional amino acids at the C-terminus and / or N-terminus.

[0380] 39. The B7-H3 linked polypeptide as described in item 38, wherein the additional amino acids (or more) improve polypeptide production, purification, in vitro or in vivo stabilization, coupling, or detection.

[0381] 40. A B7-H3 linked polypeptide described in any preceding item in a polymer form, comprising at least two B7-H3 linked polypeptide monomer units, the amino acids of which may be identical or different.

[0382] 41. The B7-H3 linked polypeptide according to item 40, wherein the B7-H3 linked polypeptide monomer units are covalently coupled to one another.

[0383] 42. The B7-H3 linked polypeptide described in item 40, wherein the B7-H3 linked polypeptide monomer unit is expressed as a fusion protein.

[0384] 43. A B7-H3 linked polypeptide described in any one of items 40-42, in the form of a dimer.

[0385] 44. Below: - The first part consisting of a B7-H3 linked polypeptide as described in any of the preceding items; and - A second part consisting of a polypeptide having the desired biological activity. A fusion protein or conjugate containing such a protein.

[0386] 45. A fusion protein or conjugate according to item 44, wherein the desired biological activity is therapeutic activity.

[0387] 46. ​​A fusion protein or conjugate according to item 44, wherein the desired biological activity is binding activity.

[0388] 47. A fusion protein or conjugate as described in item 44, wherein the desired biological activity is enzymatic activity.

[0389] 48. The fusion protein or conjugate according to item 46, wherein the binding activity is albumin-binding activity that increases the in vivo half-life of the fusion protein or conjugate and / or alters the in vivo distribution characteristics of the fusion protein or conjugate.

[0390] 49. The fusion protein or conjugate described in item 48, wherein the second portion comprises the albumin-binding domain of streptococcal protein G or a derivative thereof.

[0391] 50. The fusion protein or conjugate described in item 49, wherein the albumi domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 547, 548, and 631.

[0392] 51. A fusion protein or conjugate as described in item 50, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 550-566 and SEQ ID NOs. 632-634.

[0393] 52. A fusion protein or conjugate as described in item 51, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 550 to 557.

[0394] 53. A fusion protein or conjugate as described in item 51, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 558-560.

[0395] 54. A fusion protein or conjugate as described in item 51, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 561-563.

[0396] 55. A fusion protein or conjugate as described in item 51, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 564-566.

[0397] 56. A fusion protein or conjugate as described in item 51, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 632-634.

[0398] 57. A fusion protein or conjugate as described in item 51, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 557, 560, 563, and 566.

[0399] 58. A fusion protein or conjugate as described in item 51, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 555, 558, 561, and 564.

[0400] 59. A fusion protein or conjugate as described in item 51, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 556, 559, 562, and 565.

[0401] 60. A fusion protein or conjugate as described in item 51, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 632, 633, and 634.

[0402] 61. The fusion protein or conjugate described in item 46, wherein the binding activity acts to block biological activity.

[0403] 62. The fusion protein or conjugate described in item 45, wherein the second portion is a therapeutically active polypeptide.

[0404] 63. A fusion protein or conjugate as described in item 62, wherein the second part is an anticancer drug.

[0405] 64. A fusion protein or conjugate described in any one of items 44-47 and 61-63, wherein the second part is selected from the group consisting of human endogenous enzymes, hormones, growth factors, chemokines, cytokines, and lymphokines.

[0406] 65. A fusion protein or conjugate as described in any one of items 44-47, wherein the second part is selected from the group consisting of an antibody and its antigen-binding fragment.

[0407] 66. The fusion protein or conjugate described in item 65, wherein the antibody or its antigen-binding fragment is selected from the group consisting of full-length antibody, Fab fragment, Fab' fragment, F(ab')2 fragment, single-stranded Fab(scFab) fragment, Fc fragment, Fv fragment, single-stranded Fv(scFv) fragment, (scFv)2, scFv-Fc construct, and domain antibody.

[0408] 67. The fusion protein or conjugate according to item 66, wherein the at least one antibody or its antigen-binding fragment is selected from the group consisting of a full-length antibody, a Fab fragment, and an scFv fragment.

[0409] 68. The fusion protein or conjugate described in item 67, wherein at least one antibody or its antigen-binding fragment is a full-length antibody.

[0410] 69. A fusion protein or conjugate according to any one of items 65 to 68, wherein the antibody or its antigen-binding fragment has affinity for an antigen, for example, an antigen associated with cancer.

[0411] 70. A B7-H3 linked polypeptide, fusion protein, or conjugate as described in any preceding item, further comprising at least one linker, e.g., selected from a flexible amino acid linker, a rigid amino acid linker, and a cleavable amino acid linker.

[0412] 71. A B7-H3 linked polypeptide, fusion protein, or conjugate as described in any preceding item, further comprising a label.

[0413] 72. The B7-H3 binding polypeptide, fusion protein, or conjugate described in item 71, wherein the label is selected from the group consisting of fluorescent dyes and metals, chromogenic dyes, chemiluminescent compounds and bioluminescent proteins, enzymes, radionuclides, radioactive particles and pretargeting recognition tags.

[0414] 73. A B7-H3 linked polypeptide, fusion protein, or conjugate according to any preceding item, comprising a chelate environment provided by a polyaminopolycarboxylic acid chelating agent conjugated to a B7-H3 linked polypeptide via a thiol group of a cysteine ​​residue or an amine group of a lysine residue.

[0415] 74. A B7-H3 linked polypeptide, fusion protein, or conjugate as described in item 71, wherein the polyaminopolycarboxylic acid chelating agent is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, 1,4,7-triazacyclononane-1,4,7-triacetic acid, diethylenetriaminepentaacetic acid, or a derivative thereof.

[0416] 75. A B7-H3-linked polypeptide, fusion protein, or conjugate according to any one of items 1 to 72, comprising a chelating environment provided by a peptide-based chelating agent, wherein the peptide sequence representing the peptide-based chelating agent is located at the C-terminus of the B7-H3-linked polypeptide, fusion protein, or conjugate.

[0417] 76. The B7-H3 binding polypeptide, fusion protein, or conjugate described in item 75, wherein the C-terminal peptide sequence representing the peptide-based chelator is selected from the group consisting of -GGGC (SEQ ID NO: 620), -GSEC (SEQ ID NO: 621), -GGSC (SEQ ID NO: 622), -GGEC (SEQ ID NO: 623), -GGKC (SEQ ID NO: 624), and -KVDC (SEQ ID NO: 625).

[0418] 77. The B7-H3 binding polypeptide, fusion protein, or conjugate described in item 76, wherein the C-terminal peptide sequence representing the peptide-based chelator is -GGGC (SEQ ID NO: 620).

[0419] 78. A B7-H3 linked polypeptide, fusion protein, or conjugate as described in item 72, comprising a pretargeting recognition tag that can bind to a complement to form a complementary pair of the pretargeting moiety, the pair being selected from, for example, strepto(avidin) / biotin, oligonucleotide / complementary oligonucleotide, such as DNA / complementary DNA, RNA / complementary RNA, phosphorothioate nucleic acid / complementary phosphorothioate nucleic acid, and peptide nucleic acid / complementary peptide nucleic acid, and morpholino / complementary morpholino.

[0420] 79. The pretargeting recognition tag is a peptide nucleic acid tag, as described in item 78, a B7-H3 linked polypeptide, fusion protein, or conjugate.

[0421] 80. The pretargeting recognition tag is a 10-20 mer peptide nucleic acid sequence, such as a 15 mer peptide nucleic acid sequence, as described in item 79, a B7-H3 linked polypeptide, fusion protein, or conjugate.

[0422] 81. A radiolabeled B7-H3-binding polypeptide, fusion protein, or conjugate comprising a radiochelate of a B7-H3-binding polypeptide, fusion protein, or conjugate described in any one of items 73-77 and a radionuclide.

[0423] 82. Radiolabeled B7-H3 linked polypeptides, fusion proteins, or conjugates as described in item 81, which are suitable for medical imaging.

[0424] 83. The radioactive nuclide is 72 As, 76 Br, 55 Co, 61 Cu, 64 Cu, 18 F, [ 18 F]AIF, 19 F, 66 Ga, 67 Ga, 68 Ga, 110m In, 111 In, 123 I, 124 I, 131 I, 177 Lu, 51 Mn, 52m Mn, 52 Mn, 186 Re, 188 Re, 44 Sc, 149 Tb, 152 Tb, 155 Tb, 161 Tb, 99m Tc, 45 Ti, 86 Y, and89 A radiolabeled B7-H3 binding polypeptide, fusion protein, or conjugate as described in item 82, selected from the group consisting of Zr.

[0425] 84. A radiolabeled B7-H3 linked polypeptide, fusion protein, or conjugate as described in item 81, wherein the radionuclide is suitable for therapeutic use.

[0426] 85. The radioactive nuclide is 225 Ac, 212 Bi, 213 Bi, 67 Cu, 166 Ho, 177 Lu, 212 Pb, 149 PM, 186 Re, 188 Re, 153 Sm, 149 Tb, 161 Tb, 227 Th and 90 A radiolabeled B7-H3-binding polypeptide, fusion protein, or conjugate selected from the group consisting of Y, as described in item 84.

[0427] 86. A polynucleotide encoding a B7-H3 linked polypeptide or fusion protein as described in any preceding item.

[0428] 87. An expression vector containing the polynucleotides described in item 86.

[0429] 88. Host cells containing the expression vector described in item 87.

[0430] 89. Below: - A step of culturing the host cells described in item 88 under conditions that allow the expression of the polypeptide from the expression vector, and - Step of isolating the polypeptide A method for producing a B7-H3 linked polypeptide or fusion protein as described in any one of items 1 to 80, including the above.

[0431] 90. A method for producing a B7-H3 linked polypeptide, fusion protein or conjugate described in any one of items 1 to 85 by nonbiological peptide synthesis using amino acids and / or amino acid derivatives having protected reactive side chains, wherein the nonbiological peptide synthesis is - A step of coupling amino acids and / or amino acid derivatives in a stepwise manner to form a polypeptide or fusion protein as described herein having a protected reactive side chain, - A step of removing a protecting group from the reactive side chain of a polypeptide or fusion protein, and - A step of folding polypeptides, fusion proteins, or conjugates in an aqueous solution. The above method, including.

[0432] 91. A composition comprising a B7-H3 linked polypeptide, fusion protein, or conjugate described in any one of items 1 to 85, and at least one pharmaceutically acceptable excipient or carrier.

[0433] 92. The composition according to item 91, further comprising at least one additional activator, for example, at least two additional active ingredients, for example, at least three additional active ingredients.

[0434] 93. The composition according to item 92, wherein the at least one additional activator is an anticancer agent.

[0435] 94. A B7-H3 linked polypeptide, fusion protein or conjugate as described in any one of items 1 to 85, or a composition as described in any one of items 91 to 93, for use as a pharmaceutical, diagnostic, oncological diagnostic and / or prognostic agent.

[0436] 95. As a pharmaceutical product, B7-H3 linked polypeptides, fusion proteins, conjugates, or compositions for use as described in item 94.

[0437] 96. The B7-H3 binding polypeptide, fusion protein, conjugate, or composition for use as described in item 95, wherein the polypeptide, fusion protein, conjugate, or composition modulates B7-H3 function in vivo.

[0438] 97. B7-H3 linked polypeptides, fusion proteins, conjugates, or compositions for use as in vivo diagnostic agents and / or in vivo prognostic diagnostic agents, as described in item 94.

[0439] 98. B7-H3 linked polypeptides, fusion proteins, conjugates, or compositions for use as in vivo diagnostic agents, as described in item 97.

[0440] 99. B7-H3 conjugated polypeptides, fusion proteins, conjugates, or compositions for use as in vivo prognostic diagnostic agents, as described in item 97.

[0441] 100. B7-H3-conjugated polypeptides, fusion proteins, conjugates, or compositions for use in the treatment, prognosis, or diagnosis of B7-H3-related disorders or diseases, as described in any one of items 94-99.

[0442] 101. The B7-H3-conjugated polypeptide, fusion protein, conjugate, or composition for use as described in item 100, wherein the B7-H3-related disorder or disease is cancer.

[0443] 102. The aforementioned cancer is as follows: Breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, stomach cancer, liver cancer including hepatocellular carcinoma, lung cancer including non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, sarcomas including osteosarcoma, urothelial cell carcinoma, neuroblastoma, medulloblastoma, glioblastoma and glioma including diffuse pontine glioma, melanoma, leukemia and mesothelioma A B7-H3 linked polypeptide, fusion protein, conjugate, or composition for use as described in item 101, selected from the group consisting of the following:

[0444] 103. The B7-H3 linked polypeptide, fusion protein, conjugate or composition for use as described in item 102, wherein the cancer is selected from the group consisting of breast cancer, pancreatic cancer and sarcoma.

[0445] 104. Administration is as follows: Oral administration, topical administration, intravenous administration, intraperitoneal administration, subcutaneous administration, pulmonary administration, transdermal administration, intramuscular administration, intranasal administration, buccal administration, sublingual administration, or suppository administration, for example, subcutaneous administration such as intravenous administration. A B7-H3 linked polypeptide, fusion protein, conjugate, or composition for use as described in any one of items 94 to 103, selected from the group consisting of the following:

[0446] 105. A method of diagnosis performed in vitro, - A step of providing a sample suspected to contain B7-H3; - A step of contacting the sample with a B7-H3 linked polypeptide, fusion protein, or conjugate described in any one of items 1 to 83, or a composition described in any one of items 91 to 93; -A step of detecting the binding of a B7-H3-binding polypeptide, fusion protein, conjugate, or composition to indicate the presence of B7-H3 in the sample; and - The process of confirming the diagnosis using the obtained information. The above method, including.

[0447] 106. A method for prognostic diagnosis in vitro, - A step of providing a sample suspected to contain B7-H3; - A step of contacting the sample with a B7-H3 linked polypeptide, fusion protein, or conjugate described in any one of items 1 to 83, or a composition described in any one of items 91 to 93; -A step of detecting the binding of a B7-H3-binding polypeptide, fusion protein, conjugate, or composition to indicate the presence of B7-H3 in the sample; and - The process of determining the prognosis using the information obtained. The above method, including.

[0448] 107. Furthermore, - A process of repeating the detection process. The detection is performed at multiple time points at intervals in the same or different provided samples as part of monitoring of the subject before, during, or after treatment, according to any one of the in vitro diagnostic or prognostic methods described in any one of items 105 to 106.

[0449] 108. An in vitro diagnostic or prognostic method according to any one of items 106 to 107, further comprising the step of obtaining a value corresponding to the amount of B7-H3 bound polypeptide, fusion protein, conjugate, or composition in or bound to the sample.

[0450] 109. An in vitro diagnostic or prognostic method as described in item 108, further comprising the step of comparing the aforementioned value to a reference.

[0451] 110. An in vitro diagnostic or in vitro prognostic method described in any one of items 105 to 109, wherein the diagnosis or prognosis relates to a B7-H3 related disorder or disease.

[0452] 111. The method of in vitro diagnosis or prognosis described in item 110, wherein the B7-H3 related disorder or disease is cancer.

[0453] 112. The aforementioned cancer is as follows: Breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, stomach cancer, liver cancer including hepatocellular carcinoma, lung cancer including non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, sarcomas including osteosarcoma, urothelial cell carcinoma, neuroblastoma, medulloblastoma, glioblastoma and glioma including diffuse pontine glioma, melanoma, leukemia and mesothelioma A method of in vitro diagnosis or prognosis described in item 111, selected from the group consisting of the following.

[0454] 113. An in vitro diagnostic or prognostic method as described in item 112, wherein the cancer is selected from the group consisting of breast cancer, pancreatic cancer, and sarcoma.

[0455] 114. A method for detecting the presence of B7-H3 in a sample, comprising the steps of: providing a sample suspected to contain B7-H3; contacting the sample with a B7-H3-binding polypeptide, fusion protein, or conjugate described in any one of items 1 to 85, or a composition described in any one of items 91 to 93; and detecting the binding of the B7-H3-binding polypeptide, fusion protein, conjugate, or composition to indicate the presence of B7-H3 in the sample.

[0456] 115. A method for determining the presence of B7-H3 in a subject, the following: a) A step of contacting the subject, or a sample isolated from the subject, with a B7-H3 linked polypeptide, fusion protein, or conjugate described in any one of items 1 to 85, or a composition described in any one of items 91 to 93; and b) A step to obtain a value corresponding to the amount of B7-H3 bound polypeptide, fusion protein, conjugate, or composition bound to the subject or sample. The above method, including.

[0457] 116. The method of item 115, wherein the B7-H3 binding polypeptide, fusion protein, or conjugate conforms to any one of items 78-80, or the composition comprises such a B7-H3 binding polypeptide, fusion protein, or conjugate, and step a) further comprises bringing a subject into contact with a complementary pretargeting moiety labeled with a detectable label such as a radionuclide label.

[0458] 117. A method according to any one of items 114 to 116, further comprising the step of comparing the aforementioned value to a reference.

[0459] 118. A method for treating a B7-H3 related disorder, comprising the step of administering to a subject in need of such treatment an effective amount of a B7-H3 binding polypeptide, fusion protein or conjugate described in any one of items 1 to 85, or a composition described in any one of items 91 to 93.

[0460] 119. The method according to item 118, wherein the B7-H3 binding polypeptide, fusion protein, or conjugate modulates B7-H3 function in vivo.

[0461] 120. A method relating to any one of items 115-119, which is related to B7-H3 related disorders or diseases.

[0462] 121. The method according to item 120, wherein the B7-H3 related disorder or disease is cancer.

[0463] 122. The aforementioned cancer, Breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, stomach cancer, liver cancer including hepatocellular carcinoma, lung cancer including non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, sarcomas including osteosarcoma, urothelial cell carcinoma, neuroblastoma, medulloblastoma, glioblastoma and glioma including diffuse pontine glioma, melanoma, leukemia and mesothelioma The method described in item 121, selected from the group consisting of the following.

[0464] 123. The method according to item 122, wherein the cancer is selected from the group consisting of breast cancer, pancreatic cancer, and sarcoma.

[0465] 124. Below: - A step of contacting the subject with a B7-H3 conjugated polypeptide, fusion protein, or conjugate described in any one of items 78 to 80, including a pre-targeting recognition tag, or a composition containing such a B7-H3 conjugated polypeptide, fusion protein, or conjugate, and - A process of bringing a target into contact with a complementary pre-targeting portion containing radionuclides. The method described in item 123, further including the method described in item 123.

[0466] 125. The method according to any one of items 115 to 124, wherein the subject is a mammal such as a human subject.

[0467] 126. An in vivo procedure using one of the methods described in items 115-125.

[0468] 127. Below: -Step a) comprises systemically administering the B7-H3 linked polypeptide, fusion protein, conjugate, or composition; - The B7-H3 linked polypeptide, fusion protein, conjugate, or composition contains radionuclide labeling suitable for medical imaging; and -The method according to item 115, wherein step b) includes obtaining one or more images of at least a portion of a subject using a medical imaging device, the images being a method for medical imaging that indicates the presence of radionuclides in the body.

[0469] 128. Below: -Step a) includes administering the B7-H3 linked polypeptide, fusion protein, conjugate, or composition systemically; - The B7-H3 linked polypeptide, fusion protein, conjugate, composition, or pretargeting portion comprises a radionuclide label suitable for medical imaging; and -The method according to item 115, wherein step b) includes obtaining one or more images of at least a portion of a subject using a medical imaging device, the images being a method for medical imaging that shows the presence of a radionuclide in the body.

Claims

1. A B7-H3 linked polypeptide comprising a B7-H3 linked motif BM, wherein the motif is as follows: i) EXX ３ X ４ ALX ７ EIX １０ X １１ LPNLX １６ X １７ X １８ QX ２０ X ２１ AFIX ２５ X ２６ LNX ２９ X ３０ (Allocation number 567) Here, independently of each other X ３ is selected from I and V; X ４ is selected from A, D, E, G, H, K, L, M, N, Q, S, T, and Y; X ７ is selected from A, G, H, and S; X １０ is selected from I and V; X １１ It is selected from N and W; X １６ is selected from N and T; X １７ is selected from H and Y; X １８ is selected from A, D, E, G, H, N, Q, S, and T; X ２０ is selected from I and V; X ２１ is selected from K and R; X ２５ is selected from A, E, F, H, L, and W; X ２６ is selected from K and S; X ２９ It is selected from A, D, and E; X ３０ is selected from A, D, and H; and ii) an amino acid sequence having at least 93% identity with the sequence defined in i) The above B7-H3 linked polypeptide comprising an amino acid sequence selected from the above.

2. The B7-H3 linked polypeptide according to claim 1, wherein sequence i) corresponds to the sequence from position 8 to position 37 of a sequence selected from the group consisting of sequence numbers 1 to 535.

3. The B7-H3 binding polypeptide according to any prior claim, wherein the binding motif forms part of a 3-helix bundle protein domain, selected, for example, from a bacterial receptor domain, or selected, for example, from a domain or derivative thereof of protein A derived from Staphylococcus aureus.

4. below: xi) AEAKYAK-[BM]-PSQSSELLSEAKKLNDSQAPK (Sequence ID 572) Here, [BM] is as defined in any one of claims 1 to 2; and An amino acid sequence having at least 86% identity with the sequence defined by xi)xi). A B7-H3 linked polypeptide according to any one of claims 1 to 3, comprising an amino acid sequence selected from the above.

5. The B7-H3-binding polypeptide according to claim 4, wherein sequence xi) is selected from the group consisting of sequence numbers 1 to 13, 17 to 418 and 446 to 479.

6. K of the interaction with B7-H3 Ｄ The value is at most 1 x 10 －６ M, for example, at most 5 x 10 －７ M, for example, at most 1 x 10 －７ M, for example, at most 5 x 10 －８ M, for example, at most 1 x 10 －８ A B7-H3-binding polypeptide according to any of the prior claims, which is capable of binding to B7-H3 in such a manner as M.

7. A fusion protein or conjugate, - A first portion comprising a B7-H3 linked polypeptide as described in any of the preceding claims; and - A second part comprising a polypeptide having the desired biological activity. The above-mentioned fusion protein or conjugate, including the above-mentioned fusion protein or conjugate.

8. The fusion protein or conjugate according to claim 7, wherein the desired biological activity is a binding activity, for example, an albumin-binding activity that increases the in vivo half-life of the fusion protein or conjugate and / or alters the in vivo distribution characteristics of the fusion protein or conjugate.

9. The fusion protein or conjugate according to claim 8, wherein the second portion comprises, for example, an albumin-binding domain of streptococcal protein G or a derivative thereof, which comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 547, 548, and 631.

10. A fusion protein or conjugate according to claim 9, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 550-566 and 632-634.

11. For example, a B7-H3 linked polypeptide, fusion protein, or conjugate according to any prior claim, further comprising a label selected from the group consisting of fluorescent dyes and metals, chromogenic dyes, chemiluminescent compounds and bioluminescent proteins, enzymes, radionuclides, radioactive particles, and pretargeting recognition tags.

12. below: - A polyaminopolycarboxylic acid chelating agent conjugated to a B7-H3 linked polypeptide via a thiol group of a cysteine ​​residue or an amine group of a lysine residue, or - A peptide-based chelating agent wherein the peptide sequence representing the peptide-based chelating agent is located at the C-terminus of the B7-H3 linked polypeptide, fusion protein, or conjugate. A B7-H3 linked polypeptide, fusion protein, or conjugate according to any prior claim, comprising a chelate environment provided by.

13. A radiolabeled B7-H3-linked polypeptide, fusion protein, or conjugate comprising a radiochelate of the B7-H3-linked polypeptide, fusion protein, or conjugate described in claim 12, and a radionuclide.

14. A polynucleotide encoding a B7-H3 linked polypeptide or fusion protein as described in any prior claim.

15. A composition comprising a B7-H3 linked polypeptide, fusion protein, or conjugate according to any one of claims 1 to 13, and at least one pharmaceutically acceptable excipient or carrier.

16. A B7-H3 linked polypeptide, fusion protein, or conjugate according to any one of claims 1 to 13, or a composition according to claim 15, for use as a pharmaceutical product, an in vivo diagnostic agent, an in vivo theranostic diagnostic agent, and / or an in vivo prognostic diagnostic agent.

Images