CD163 binding complex

Abstract

The present invention relates to polypeptides, more particularly polypeptides comprising an immunoglobulin domain that binds to human and mouse CD163 proteins, and applications of such polypeptides, such as for use as an immunotracer. The present invention also relates to complexes, more particularly complexes of polypeptides comprising an immunoglobulin domain that binds to human and mouse CD163 proteins and further parts such as a therapeutic moiety, and applications of such complexes.

Description

[Technical Field]

[0001] The present invention relates to polypeptides, more particularly polypeptides comprising an immunoglobulin domain that binds to human and mouse CD163 proteins, and applications of such polypeptides, such as for use as an immunotracer. The present invention also relates to complexes, more particularly complexes of polypeptides comprising an immunoglobulin domain that binds to human and mouse CD163 proteins and further parts such as a therapeutic moiety, and applications of such complexes. [Background technology]

[0002] Immunomodulatory drugs, and in particular immune checkpoint inhibitors (ICIs), have revolutionized cancer treatment. While highly effective in subsets of cancer and cancer patients, ICIs remain ineffective in a relatively large fraction of cancer and cancer patients. Much effort is currently being devoted to understanding the underlying under-response or non-response to ICI therapy and discovering ways to overcome the under-response to ICI therapy. Predicting or following up on cancer patients' responses to ICI therapy, or to therapies generally involving immunomodulatory drugs, both pre- and during treatment, would be ideal for both patients (in terms of helping to discover the best possible treatment) and socioeconomics (in terms of helping to optimize the allocation of available healthcare resources). However, treatment prediction or follow-up using invasive methods such as biopsy or blood biomarkers has so far proven difficult because they do not provide a complete overview of the tumor microenvironment and / or lack spatial information. Therefore, a valuable alternative would be to have non-invasive means for detecting a response or non-response to treatment, i.e., means for detecting treatment outcomes as early as possible after treatment disclosure.

[0003] Non-invasive immunomonitoring, applicable to in vivo monitoring of immune responses, provides a means to detect therapeutic outcomes and help understand the reasons for a subject's response or non-response to treatment. Many different immunomonitoring imaging techniques have been developed, one of which is antibody-based tracers as part of a group of molecular tracers (e.g., outlined in McCarthy et al. 2020, Front Immunol 11:1067).

[0004] The differentiated protein 163 (CD163) cluster is a protein belonging to the cysteine-rich (SRCR) superfamily of scavenger receptors and is known to be expressed in monocytes and macrophages. CD163 is an endocytosis receptor for the hemoglobin-haptoglobin complex. Another CD protein, CD206 (or macrophage mannose receptor, MMR), is also expressed in macrophages. The presence of tumor-associated macrophages (TAMs) in tumors is usually correlated with poor patient outcomes. TAMs may account for less than 50% of the tumor mass.

[0005] Efforts using CD206 antibody-based tracers have been reported, for example, as early markers for tumor recurrence and metastasis (Zhang et al. 2017, Theranostics 7:4276-4288), or for example, tracking repair macrophages in the myocardium after myocardial infarction (Varasteh et al. 2022, Front Cardiovasc Med 9:889963). Clinical trials using CD206 single-domain antibody-based tracers have been reported in relation to positron emission tomography (PET / CT scanning) of breast cancer, head and neck cancer, or melanoma (NCT04168528).

[0006] Many studies on PET tracers developed for in vivo imaging of macrophages associated with different diseases are summarized in Fernandes et al. 2022 (EJNMMI Radiopharmacy and Chemistry 7:11), one of which involves a CD163 antibody-based tracer as a means for tracking macrophages in collagen-induced arthritis as an example of an inflammatory disease (Eichendorff et al. 2015, Mol Imaging Biol 17:87-93). Tarin et al. 2015 (Scientific Reports 5:17135) disclosed guiding gold-coated iron oxide nanoparticles to macrophages with an anti-CD163 antibody for the purpose of detecting atherosclerosis by MRI means.

[0007] Clearly, there is a need to expand the range of equipment available for non-invasive immunomonitoring, particularly in the field of cancer. [Overview of the project]

[0008] The present invention relates to a polypeptide that binds to human and mouse CD163, wherein the amino acid sequence of the polypeptide comprises a CDR1 region, a CDR2 region, and a CDR3 region, the CDR1, CDR2, and CDR3 regions being located within an immunoglobulin variable domain (IVD) as defined by Sequence ID No. 1, and selected from these CDR1, CDR2, and CDR3 regions as determined by the Kabat, Chothia, Martin, or IMGT methods. In one embodiment, the CDR1 region is defined by Sequence ID No. 2, the CDR2 region is defined by Sequence ID No. 3, and the CDR3 region is defined by Sequence ID No. 4. In another embodiment, the polypeptide that binds to human and mouse CD163 further comprises, more specifically, FR1, FR2, FR3, or FR4 regions present in the IVD, as defined by SEQ ID NO: 1; in particular, such FR1 region is defined by SEQ ID NO: 5, such FR2 region is defined by SEQ ID NO: 6, such FR3 region is defined by SEQ ID NO: 7, and such FR4 region is defined by SEQ ID NO: 8.

[0009] In a further embodiment, the CDR region and / or the FR region are humanized, and / or the IVD is humanized.

[0010] In further embodiments, any of the polypeptides defined above may further include a functional moiety; in particular, such a functional moiety may be a peptide motif recognized by a His tag or a peptide ligase, or a detectable moiety. In contrast, in certain embodiments, such a detectable moiety may be bound to a specific site contained in the polypeptide.

[0011] The present invention relates to isolated nucleic acids encoding the polypeptides defined above; vectors containing such nucleic acids; and further to host cells that express the polypeptides defined above, or contain nucleic acids encoding the polypeptides defined above, or contain vectors containing such nucleic acids.

[0012] The present invention further relates to a pharmaceutical composition comprising the polypeptide defined above.

[0013] The present invention further relates to the defined polypeptides, or pharmaceutical compositions comprising such polypeptides, for use in diagnostic, surgical, therapeutic monitoring, or contrast agents.

[0014] The present invention relates to a method for producing the polypeptide defined above, wherein the method is: -Expressing the polypeptide in the host cells defined above, or synthesizing and manufacturing the polypeptide; - Purifying the expressed or manufactured polypeptide; - If necessary, a detectable moiety is attached to the purified polypeptide, This includes further information about the methods.

[0015] The present invention further relates to polypeptides that conjugate to human and mouse CD163, wherein the polypeptide is conjugated to a prophylactic or therapeutic agent, a cytotoxic moiety or cytotoxic drug, an immunostimulant or immunosuppressant, a Toll-like receptor agonist, a photon absorber, and liposomes or nanoparticles. In one embodiment, such an ISVD complex is intended for use as a pharmaceutical product (selecting an appropriate payload) in the treatment or inhibition of cancer, for use in combination with additional anticancer agents as needed. Pharmaceutical compositions containing such a complex are also included. [Brief explanation of the drawing]

[0016] [Figure 1] Affinity of CD163-targeted single-domain antibody (23766) to hCD163 and mCD163. A-B) Surface plasmon resonance plots of binding of single-domain antibody 23766 at different concentrations. [Figure 2]Binding of a CD163-targeted single-domain antibody (23766) to HEK293T cells overexpressing hCD163 or mCD163 protein. A) Binding of a CD163-specific single-domain antibody 23766 or an unrelated single-domain antibody ("Irr Nb"; R3b23) to HEK293T cells overexpressing (A) hCD163 or (B) mCD163 protein. Binding was detected as mean fluorescence intensity (MFI) using flow cytometry with a C-terminal His tag and a fluorescently labeled anti-His antibody. [Figure 3] SPECT / CT imaging of 99mTc-labeled single-domain antibody 23766. Representative SPECT / CT images of C57BL / 6J WT or CD163- / - mice intravenously injected with 99mTc-labeled single-domain antibody 23766 ("Nb 23766") or an unrelated single-domain antibody ("unrelated Nb"). High accumulation of single-domain antibody 23766 is observed in the cervical lymph nodes (upper arrow), liver (center arrow), and bone marrow (lower arrow) of WT mice. [Figure 4] Ex vivo in vivo distribution analysis of cross-reactive CD163-targeted single-domain antibody 23766 in naive animals. Ex vivo γ-counting of organs isolated from naive C57BL / 6 WT mice and C57BL / 6J CD163 KO mice 90 minutes after injection of 99mTc-labeled lead CD163-targeted single-domain antibody or an unrelated single-domain antibody ("Irr Nb", R3b23). The in vivo distribution of the CD163-specific single-domain antibody in the three mice is shown and expressed as the mean ± standard deviation of the percentage of injected activity per gram of organ or tissue (%IA / g). Statistical analysis was performed using Student's t-test. ns, p>0.05;**, p<0.01;***, p<0.001;***, p<0.0001. [Figure 5]SPECT / CT imaging of 99mTc-labeled single-domain antibody 23766 in macrophage-depleted and untreated naive WT mice. Representative SPECT / CT images of treated (600 mg / kg PLX3397 in food) WT or untreated WT mice intravenously injected with 99mTc-labeled single-domain antibody 23766 ("Nb 23766"), anti-MMR single-domain antibody ("anti-MMR Nb"), and unrelated single-domain antibody ("unrelated Nb", R3b23). No accumulation was observed in organs other than the kidneys and bladder in both untreated and macrophage-depleted mice injected with the unrelated single-domain antibody. Both single-domain antibody 23766 and anti-MMR single-domain antibody showed high accumulation in the cervical lymph nodes (upper arrow), liver (center arrow), and bone marrow (lower arrow) of non-depleted WT mice. In macrophage-depleted mice injected with the 99mTc-labeled single-domain antibody 23766, signal uptake was absent or minimal in the lymph nodes and liver, indicating that this signal domain antibody is strictly macrophage-dependent. High hepatic uptake was observed in macrophage-depleted WT mice injected with the 99mTc-labeled anti-MMR single-domain antibody, demonstrating that the MMR receptor is expressed not only on macrophages but also on other cells within the liver, such as hepatic endothelial cells. [Figure 6]SPECT / CT imaging of 99mTc-labeled single-domain antibody 23766 in mice carrying MC38 tumors, B16-F10 tumors, or LLC-OVA tumors. Representative whole-body SPECT / CT images of A) MC38 tumor-carrying mice, B) B16-F10 tumor-carrying mice, and C) LLC-OVA tumor-carrying mice that were intravenously injected with either 99mTc-labeled single-domain antibody 23766 ("Nb 23766") or an unrelated single-domain antibody ("unrelated Nb"). Uptake of both single-domain antibodies 23766 is observed in the lymph nodes (upper arrow), liver (center arrow), and bone marrow (lower arrow) in MC38, B16-F10, and LLC-OVA tumor-carrying mice. In MC38 tumors, accumulation of CD163-specific single-domain antibodies was not observed at all or only slightly; in B16-F10 tumors, only peripheral uptake of single-domain antibodies was observed; and in LLC-OVA tumor models, central tumor uptake of single-domain antibodies was observed. D) Cross-section of LLC-OVA tumors intravenously injected with 99mTc-labeled single-domain antibody 23766 or an unrelated single-domain antibody. Tumors are outlined by dotted lines. [Figure 7]Ex vivo in vivo distribution analysis of CD163-specific single-domain antibody 23766 in mice carrying MC38 tumors, B16-F10 tumors, or LLC-OVA tumors. Ex vivo γ-counting of tumors from A) MC38 tumor-carrying, C) B16-F10 tumor-carrying, or E) LLC-OVA tumor-carrying mice 90 minutes after injection of 99mTc-labeled single-domain antibody. Tumor-to-blood ratio of different 99mTc-labeled single-domain antibodies in B) MC38 tumor-carrying mice, D) B16-F10 tumor-carrying mice, and F) LLC-OVA tumor-carrying mice, calculated by dividing the percentage of activity per gram in tumors by the percentage of injected activity per gram in blood. The in vivo distribution of single-domain antibodies in 5 mice is shown and expressed as the mean ± standard deviation of the percentage of injected activity per gram (%IA / g) in organs or tissues. Statistical analysis was performed using two-way ANOVA. ns, p>0.05;**, p<0.01;***, p<0.001;***, p<0.0001. [99mTc]-Tc-23766Nb: 99mTc-labeled CD163-specific single-domain antibody 23766. [99mTc]Tc-Irr Nb: 99mTc-labeled unrelated single-domain antibody. [Figure 8] Ex vivo flow cytometry data of CD163-specific single-domain antibody 23766 in MC38 tumor, B16-F10 tumor, or LLC-OVA tumor-bearing mice. CD163 expression was detected as mean fluorescence intensity (ΔMFI). The LLC-OVA tumor model exhibits the highest CD163 expression on all macrophage types, verifying the highest uptake of anti-CD163 immune tracers in this tumor model. Data are presented as mean ± standard deviation. [Figure 9]Determination of single-domain antibody binding affinity after NOTA binding by ELISA and cell binding assays. (A) Binding of 68-gallium labeled anti-CD163 single-domain antibody ("[68Ga]Ga-NOTA-anti-CD163 Nb") at different concentrations to human CD163-Avi-hexahistine protein compared to binding of a control unrelated 68-gallium labeled single-domain antibody ("[68Ga]Ga-NOTA-Irr Nb"). (B) Single-domain antibody binding of 67-gallium labeled anti-CD163 single-domain antibody at different concentrations to mouse CD163-overexpressing HEK293T cells. [Figure 10] The 68-gallium labeled anti-CD163 monoimmunotracer demonstrates specificity in in vivo binding to CD163+ cells. A-B) Representative μPET / CT images of CD163 knockout (CD163- / -) and wild-type (WT) mice intravenously injected with (A) a 68-gallium labeled unrelated single-domain antibody ("[68Ga]Ga-NOTA-Irr Nb") and (B) a 68-gallium labeled anti-CD163 immunotracer ("[68Ga]Ga-NOTA-anti-CD163 Nb"). Cervical lymph nodes (upper arrow), liver (center arrow), and bone marrow (lower arrow) are highlighted in wild-type (WT) mice. (C~F) Ex vivo uptake values ​​of anti-CD163 immunotracer ("aCD163") or unrelated immunotracer ("Irr") are shown in the liver, spleen, cervical lymph nodes, and bone marrow of CD163 knockout (CD163- / -) and wild-type (WT) mice. All data are plotted as the mean ± standard deviation of the percentage of injected activity per gram of organ or tissue (%IA / g). Statistical analysis was performed using unpaired two-tailed t-tests. **, p<0.01; ***, p<0.001; ****, p<0.0001. [Figure 11]The 68-gallium-labeled anti-CD163 immunotracer can visualize macrophage distribution during anti-macrophage therapy. A) Individual growth curves of PLX-treated LLC-OVA tumor-bearing mice showing responders with suppressed tumor growth (R), partially suppressed tumor growth (PR), and non-responders (NR) with no effect on tumor growth. B-C) ​​Representative whole-body μPET / CT images of (B) untreated mice and (C) macrophage-depleted mice (non-responders (NR) and responders (R)) intravenously injected with the 68-gallium-labeled anti-CD163 immunotracer on the final day of the treatment experiment. No or minimal signal uptake was observed in the lymph nodes (upper arrow), bone marrow (center arrow), and liver (lower arrow) of depleted mice, and single-domain antibody uptake in the tumors (lower arrow) was also lower compared to untreated mice. Tumors are outlined by dotted lines. D-E)(D) Correlation plot showing a significant correlation between %IA of tumor and tumor volume, and (E) the percentage of CD163+ MHC-II low F4 / 80 representing macrophages and tumor volume. F-G)(F) Graph showing flow cytometry results of the percentage ratio of MHC-II high / MHC-II low cells as a portion of hepatocytes, and (G) the percentage of CD163+ cells as a portion of macrophages. PLX: Macrophage depletion compound PLX3397. [Modes for carrying out the invention]

[0017] For diagnostic or in vivo molecular imaging purposes, contrast agents must be able to reach their targets with high efficiency. This requires a combination of a sufficiently small size to obtain adequate tissue permeability, selective binding to the target to obtain a high signal-to-noise ratio at the target site, and low overall retention or accumulation in the body (as a result of elimination from the body; usually in the liver or kidneys) to avoid areas with high background signal that would adversely affect the signal at the target site.

[0018] In the process leading to the present invention, we first identified immunoglobulin single variable domain (ISVD) molecules, also referred to herein as single-domain antibodies (sdAbs), that bind with high specificity to human and mouse CD163 (hCD163 and mCD163, respectively).

[0019] After screening the rama immunoassay library, anti-CD163 sdAbs were identified and their binding (in CD163-expressing cells) and affinity were evaluated using enzyme-linked immunosorbent assay (ELISA), flow cytometry, and surface plasmon resonance (SPR). Technetium-99m( 99m Tc) label, 68 Ga label or 67 Single-photon emission computed tomography imaging in mice after intravenous injection of a Ga-labeled anti-CD163 sdAb revealed several properties that make this sdAb an interesting diagnostic tool: (i) cross-reactivity to human and mouse CD163; (ii) superior in vivo visualization of macrophage-enriched organs (lymph nodes, liver, intestine, bone marrow) in healthy mice (compared to unrelated ISVDs); and (ii) the ability to distinguish between the presence or absence of CD163+ macrophage uptake in tumors, which has a significantly higher tumor-to-blood ratio compared to unrelated ISVDs. Surprisingly, compared to CD206 sdAb-based immunotracers, CD163 sdAb-based immunotracers exhibit higher macrophage specificity and lower background in liver tissue. These combined properties provide a well-suited identified CD163-conjugated ISVD as a diagnostic agent for molecular imaging, for example.

[0020] Based on this, the present invention is defined in the following aspects and embodiments, which will be described in more detail thereafter. Since the present invention relates to polypeptides containing complementarity-determining regions (CDRs), first, how such CDRs are determined will be explained.

[0021] The determination of the CDR region in antibody / immunoglobulin sequences generally depends on the algorithm / method applied (Kabat-, Chothia-, Martin (extended Chothia), IMGT (ImMunoGeneTics Information System) numbering schemes; see, e.g., http: / / www.bioinf.org.Uk / abs / index.html#kabatnum and http: / / www.imgt.org / IMGTScientificChart / Numbering / IMGTnumbering.html). The application of different methods to the same antibody / immunoglobulin sequence may result in different CDR amino acid sequences, and this difference may be present in the depiction of the CDR sequence length and / or contour within the antibody / immunoglobulin / I(S)VD sequence. Accordingly, the CDR of the CD163-conjugated polypeptide of the present invention can be described as the CDR sequence present in the single variable-domain anti-CD163 antibody characterized herein, or as determined or described by well-known methods, such as following the Kabat-, Chothia-, Martin(extended Chothia), or IMGT numbering schemes or methods. For example, the CDR sequences defined in SEQ ID NOs. 2-4 were described by the Kabat method from the CD163 single-domain antibody defined by SEQ ID NO. 1. The application of other methods may result in CDR sequences that are (slightly) different from those defined in SEQ ID NOs. 2-4 (thus the FR sequences are consequently different, see further).

[0022] In a first aspect, the present invention relates to a polypeptide that specifically binds to human and mouse CD163 (hereinafter also referred to herein as a polypeptide that specifically binds to CD163 or a CD163-binding polypeptide), wherein the amino acid sequence of the polypeptide comprises a CDR1 region, a CDR2 region, and a CDR3 region, and the CDR1, CDR2, and CDR3 regions are selected from these CDR1, CDR2, and CDR3 regions, respectively, and are present in a CD163-binding single-domain antibody or an immunoglobulin variable domain (IVD) or immunoglobulin single variable domain (ISVD) as defined by Sequence ID No. 1.

[0023] More specifically, the polypeptide that specifically binds to human and mouse CD163 comprises an immunoglobulin (single) variable domain that carries the specificity of the polypeptide for binding to human and mouse CD163, and I(S)VD comprises a CDR1 region, a CDR2 region, and a CDR3 region, the CDR1, CDR2, and CDR3 regions being the CDR1, CDR2, and CDR3 regions present in the CD163-binding single-domain antibody as defined by Sequence ID No. 1.

[0024] In an embodiment to this effect, the CDR region is determined by applying the Kabat, Chothia, Martin, or IMGT method to Sequence ID 1. In a more specific embodiment, the CDR region is determined by the Kabat method, and the CDR1 region is defined by Sequence ID 2; the CDR2 region is defined by Sequence ID 3; and the CDR3 region is defined by Sequence ID 4.

[0025] In further embodiments, polypeptides that specifically bind to human and mouse CD163, such as polypeptides that specifically bind to human and mouse CD163 containing a CD163-specific immunoglobulin (single) variable domain (I(S)VD), are characterized by further comprising at least a framework region (FR), such as a framework region from the immunoglobulin (single) variable domain, wherein the (I(S)VD) polypeptide may contain four or fewer FR regions (FR1 preceding CDR1; FR2 scattered between CDR1 and CDR2; FR3 scattered between CDR2 and CDR3; FR4 after CDR3; the relative positions mentioned are in the direction from the amino terminus to the carboxy terminus of the (I(S)VD)). In particular, the FR1 region can be defined by SEQ ID NO: 5. The FR2 region can be defined by SEQ ID NO: 6. The FR3 region can be defined by SEQ ID NO: 7. And the FR4 region can be defined by SEQ ID NO: 8. In this specification, sequence-defined FR regions are outlined based on the depiction of the contours of each CDR region determined by the Kabat method; therefore, these FR regions may differ slightly when the CDR region is determined by a method other than the Kabat method.

[0026] In any of the above-mentioned regions associated with the FR region, lysine can be replaced with alanine, which is useful for binding to NOTA chelators (see further reference) or other imaging moieties; such substitution is obviously only permitted under conditions where the binding affinity between the CD163-binding polypeptide and CD163 is not significantly affected.

[0027] In further embodiments, any of the polypeptides that specifically bind to human and mouse CD163, such as any of the polypeptides that specifically bind to human and mouse CD163, including any of the polypeptides that specifically bind to human and mouse CD163, further comprises a portion that extends the half-life of the polypeptide once administered to a subject. Such a half-life extending portion may be, for example, serum albumin-bound I(S)VD, or albumin itself. Other half-life extending modalities include PEGylation (or any modification such as glycol-PEGylation or biotinylated PEG), binding peptides (regardless of the form of the fusion protein) such as XTEN, PAS ("Pro Ala Ser"), ELP (elastin-like polypeptide), GLK (gelatin-like protein), HAP (addition of (Gly4Ser)n peptide), and addition of polysaccharide moieties (for example, outlined in Zaman et al. 2019, J Controlled Release 301:176-189).

[0028] In any of the above, the CDR region and / or the FR region and / or I(S)VD may be humanized. The humanized CDR and / or FR and / or I(S)VD can be obtained by any known suitable method and is therefore not strictly limited to polypeptides obtained using polypeptides containing the natural VHH domain as starting materials. Humanized immunoglobulin monovariate domains may have several advantages compared to the corresponding natural VHH domain, such as reduced immunogenicity. Such humanization generally involves substituting one or more amino acid residues in the sequence of the natural CDR and / or framework region (FR) with amino acid residues present at the same position in a human VH domain, such as a human VH3 domain. The humanization substitution should be selected such that the resulting humanized immunoglobulin domain still retains (or is further improved, for example, by affinity maturation) the desirable properties of the original immunoglobulin. Those skilled in the art will be able to select a suitable combination of humanization substitutions that strikes a good balance between the desirable properties obtained by the humanization substitution on the one hand and optimizes or acquires the desirable properties of the natural VHH domain on the other hand. Generally, the specificity of binding to the target is not significantly affected by humanized antibodies / immunoglobulins / I(S)VD (or polypeptides containing such antibodies / immunoglobulins / I(S)VD), and generally, the affinity and / or affinity of binding to the target is not significantly affected by humanized antibodies / immunoglobulins / I(S)VD (or polypeptides containing such antibodies / immunoglobulins / I(S)VD).

[0029] The CD163-conjugated polypeptide of the present invention (in the form of a fusion, a conjugate therewith, or a complex therewith) one or more non(poly)peptide components such as a detectable moiety (see further reference) or PEGylated (e.g., International Publication No. 2017 / 059397), one or more further polypeptides or polypeptide domains such as a His tag, or one or more further peptides or polypeptide domains such as a sortag motif (soltase peptide ligase amino acid substrate motif LPXTG (SEQ ID NO: 9), e.g., LPETG (SEQ ID NO: 10); Mao et al. 2004, J Am Chem Soc 126:2670-2671), or a peptide asparaginyl ligase motif (e.g., recognized by buterase 1 or VyPAL2; the motif sequence is NXL, where X is, for example, Gly, Ser, Ala, or Gln; Zhang et al. 2022, Int J Mol Sci 23:458; Hu et al. 2022, Plant Cell 34:4936-4949) may include such portions / tags / motifs, which are referred to herein as “functional portions.” In one example, the CD163-binding polypeptide or the CD163-specific I(S)VD itself may be replicated or multiple-replicated (the monomers are linked by mobile linkers, such as linkers based on Gly-Pro repeats, Pro-Ala repeats, Gly-Ser repeats, or combinations thereof) to produce a polyvalent (but single-specific) binding molecule. In another case, a further polypeptide or polypeptide domain (linked to the CD163-binding polypeptide by mobile linkers, such as linkers based on Gly-Pro repeats, Pro-Ala repeats, Gly-Ser repeats, or combinations thereof; or may be contained in the CD163-binding polypeptide as a fusion protein) may extend the serum half-life (e.g., serum albumin-binding proteins or peptides; see above).

[0030] Therefore, in any of the above, the CD163-conjugated polypeptide may further include a functional moiety. In one embodiment, the functional moiety is a detectable moiety. A CD163-conjugated polypeptide, as defined herein, carrying a detectable moiety together with it, can be an immunotracer; if the detectable moiety is radiolabeled, the CD163-conjugated polypeptide can be a radioimmunotracer.

[0031] Both the naked CD163-conjugated polypeptide (without detectable moiety) and the CD163-conjugated polypeptide containing the detectable moiety described herein are useful when considering in vivo imaging applications. In fact, the naked CD163-conjugated polypeptide may be administered to the subject co-administered with the CD163-conjugated polypeptide containing the detectable moiety, or administered to the subject before administering the CD163-conjugated polypeptide containing the detectable moiety to mask the sink of the CD163-conjugated polypeptide, more specifically, renal sink; as a result, the sink background signal can be reduced. Furthermore, it has been reported that preloading with an unlabeled antibody may extend the imaging frame of the labeled antibody (Nishio et al. 2020, Mol Imaging Biol 22:156-164).

[0032] Generally, a “detectable portion” refers to a portion that emits or can emit a signal upon sufficient stimulation and is detectable by any means, preferably non-invasive means, once it enters the human body. Furthermore, a detectable portion may enable computer typesetting of an image, and as a result, the detectable portion may be called a contrast agent. Examples of detectable portions include fluorescent emitters, positron emitters, radioactive emitters, and others.

[0033] The measurement of the amount of detectable moiety / contrast agent (carried, bound, chelated, contained on a CD163 binding polypeptide) is typically performed using an instrument that counts radioactivity or determines radiation (which may be in the nature of photons) density or radiation concentration. The counted or determined radioactivity can be converted into an image. Depending on the nature of the luminescence by the detectable moiety, it may be detectable by techniques such as PET (positron emission tomography), SPECT (single photon emission computed tomography), fluorescence imaging, fluorescence tomography, near-infrared imaging, near-infrared tomography, optical tomography, and others.

[0034] Examples of radioluminescent substances / radioactive labels include 68 Ga, 110m In, 18 F, 45 Ti, 44 Sc, 47 Sc, 61 Cu, 60 Cu, 62 Cu, 66 Ga, 64 Cu, 55 Ca, 72 As, 86 Y, 90 Y, 89 Zr, 125 I, 74 Br, 75 Br, 76 Br, 77 Br, 78 Br, 111 In, 114m In, 114 In, 99m Tc, 11 C, 32 Cl, 33 Cl, 34 Cl, 123 I, 124 I, 131 I, 186 Re, 188 Re, 177 Lu, 99 Tc, 212 Bi, 213 Bi, 212 Pb, 225 Ac, 153 Sm, and 67Examples of fluorescent luminescent materials include cyanine dyes (e.g., Cy5, Cy5.5, Cy7, Cy7.5), indorenine dyes, benzoindorenine dyes, phenoxazines, BODIPY dyes, rhodamines, Si-rhodamines, Alexa dyes, and derivatives of any of these.

[0035] Many radionuclides possess metallic properties and typically cannot form stable covalent bonds with proteins or peptides. One solution is to label proteins or peptides with radioactive metals using chelating agents, i.e., polydentate ligands, that form non-covalent compounds, so-called chelates, with metal ions. Therefore, CD163-conjugated polypeptides may be conjugated in any way with such chelating agents that enable the incorporation of radionuclides; this allows the radionuclides to coordinate, chelate, or complex with the CD163-conjugated polypeptide. Examples of chelating agents include polyaminopolycarboxylate chelating agents, which may be macrocyclic or non-dry. Polyaminopolycarboxylate chelating agents can be conjugated to CD163-conjugated polypeptides, for example, by the thiol group of a cysteine ​​residue or the ε-amino group of a lysine residue. Examples of macrocyclic chelating agents for radioactive isotopes such as iridium, gallium, yttrium, and bismuth, as well as radioactive actinides and radioactive lanthanides, include the polypeptides and their derivatives such as DOTA(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) and maleimidomonoamide-DOTA(1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid-10-maleimidoethylacetamide), and DOTAGA(2,2',2''-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid). Other chelating agents include NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) and its derivatives such as NODAGA (2,2'-(7-(1-carboxy-4-((2,5-dioxopyrrolidine-1-yl)oxy)-4-oxobutyl)-1,4,7-triazonane-1,4-diyl)diacetic acid). Non-dry polyaminopolycarboxylate chelating agents include different derivatives of DTPA (diethylenetriaminepentaacetic acid).Further chelating agents include DFO, CB-DO2A, 3p-C-DEPA, TCMC, Oxo-DO3A, TE2A, CB-TE2A, CB-TE1A1P, CB-TE2P, MM-TE2A, DM-TE2A, diamsar, NODASA, NETA, TACN-TM, 1B4M-DTPA, CHX-A''-DTPA, TRAP, NOPO, AAZTA, DATA, H2dedpa, H4octapa, H2azapa, H5decapa, H6phospa, HBED, SHBED, BPCA, CP256, PCTA, HEHA, PEPA, EDTA, TETA, and TRITA.

[0036] The detectable portion in the CD163-conjugated polypeptide is itself included in the prosthetic group, and the prosthetic group may be bound to the polypeptide by a binding moiety such as cyclooctin containing a chelating agent or a reactive group that forms a covalent bond with an amine, carboxyl, carbonyl, or thiol functional group on the CD163-conjugated polypeptide. Examples of cyclooctin include dibenzocyclooctin (DIBO), biarylazacyclooctinone (BARAC), dimethoxyazacyclooctin (DIMAC), and dibenzocyclooctin (DBCO), DBCO-PEG4-NHS-ester, DBCO-sulfo-NHS-ester, DBCO-PEG4-acid, DBCO-PEG4-amine, or DBCO-PEG4-maleimide. 18 Examples of F-labeled prosthetic groups include 18 F-3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-fluoropyridine( 18 It is F-FFPEGA. 18 As an F-labeled prosthetic group, N-succinimidyl-4-[ 18 F] Fluorobenzoate ([ 18F]SFB) (e.g., Li et al. 2014, Applied Radiation and Isotopes 94:113-117); Examples of I-labeled prosthetic groups include 4-guanidinomethyl-3-[(*)I]iodobenzoate N-succinimidyl ([(*)I]SGMIB) and 3-guanidinomethyl-5-[(*)I]iodobenzoate N-succinimidyl (iso-[(*)I]SGMIB), where (*)I is, for example, 131I (see, for example, Choi et al. 2014, Nucl Med Biol 41:802-812).

[0037] The above binding methods may result in heterogeneous tracer populations. Site-specific binding strategies attempt to overcome this drawback and include chemoenzymatic methods for binding polypeptides such as antibodies / immunoglobulins / I(S)VD to chelating agents or detectable moieties, such as by saltase-mediated peptide transfer (Antos et al. 2009, Curr Protoc Protein Sci, Chapter 15:unti-15.3) (e.g., Massa et al. 2016, Exp Opin Drug Deliv 13:1149-1163) or peptide ligase-mediated binding (see above). Accordingly, the CD163-bound polypeptides described herein may have detectable moieties bound to specific sites within the polypeptide, such as forming homogeneous or nearly homogeneous populations of tracer molecules.

[0038] Other embodiments relate to isolated nucleic acids encoding the CD163-binding polypeptide described herein; vectors comprising such nucleic acid; and host cells comprising such nucleic acid or vector and / or expressing the CD163-binding polypeptide described herein.

[0039] Further embodiments relate to pharmaceutical compositions comprising the CD163-conjugated polypeptide described herein (without / not comprising a functional moiety, with / comprising a functional moiety, or with / comprising a detectable moiety). Such pharmaceutical compositions comprise the CD163-conjugated polypeptide described herein formulated in a suitable excipient. The suitable excipient is suitable for administration to a subject and, for example, is non-toxic. On the other hand, the excipient may have a function of stabilizing or solubilizing the CD163-conjugated polypeptide, such as with / comprising a functional moiety.

[0040] Further embodiments relate to the CD163-conjugated polypeptides described herein, or pharmaceutical compositions containing them for use as contrast agents, such as for diagnostic, surgical or guided surgery, therapeutic monitoring, and particularly as described herein. Alternatively, the present invention relates to a diagnostic method or therapeutic monitoring method, wherein the method comprises administering the CD163-conjugated polypeptides described herein, or pharmaceutical compositions containing them, to a subject. As a result of such administration, the presence of CD163+ cells can be diagnosed or fluctuations of such cells can be tracked before, after, or during treatment, such as immunomodulatory therapy. Alternatively, the present invention relates to a surgical resection method of a tumor, wherein the method comprises administering the CD163-conjugated polypeptides described herein, or pharmaceutical compositions containing them, to a subject, wherein the CD163-conjugated polypeptides can assist in contouring the tumor during resection, particularly if they include a detectable portion. In certain embodiments, the CD163-conjugated polypeptides described herein are applied in the fields of cancer or tumor imaging, cancer or tumor therapy monitoring, cancer or tumor diagnosis, or cancer or tumor surgery or guidance for cancer or tumor surgery.

[0041] The specificity or selectivity of a cell target, particularly a macrophage cell target, describes a state in which, at a given concentration, the composition interacts (e.g., binds, etc.) with the target cells of interest with a higher efficacy than the composition interacts with other cells (not intended as target cells) (e.g., at least 2, 5, or 10 times higher efficacy, or at least 20, 50, or 100 times higher efficacy). The exclusivity of a cell target describes a state in which the composition interacts only with the target cells of interest.

[0042] diagnosis In general, as used herein, “diagnosis” refers to the detection of human or mouse CD163 or cells exhibiting human or mouse CD163. This may be ex vivo or in vitro, such as in a sample from a (human) subject (and, for example, by ELISA, immunocytochemistry (ICH), Western blotting, or surface plasmon resonance). This may also be in vivo diagnosis, in particular non-invasive in vivo diagnosis, such as by the medical imaging or molecular imaging described herein. Whether by a sample from a (human) subject or by an in vivo (imaging) method, the diagnosis enables monitoring of responses to treatment, such as responses to immunomodulatory therapies, such as immunotherapy or treatment of subjects with tumors or cancer. Diagnosis, and imaging in particular, can be helpful in assisting surgical or guided surgery, for example, by outlining the contours of tumors requiring surgical resection.

[0043] Treatment monitoring As examples of immunomodulation of therapeutic compounds, the FDA approved the anti-PD-1 mAbs pembrolizumab, nivolumab, and semiprimab; the anti-PD-L1 mAbs durvalumab, atezolizumab, and avelumab; the anti-CTLA4 mAb ipilimumab; and the combination of the anti-LAG3 mAbs reratrimab and nivolumab, which have since become available as standard treatment for several types of cancer. The negative aspects of this success story are the high cost of such treatments, easily exceeding $100,000 per patient (e.g., Aguiar et al. 2017, Ann Oncol 28:2256-2263), and the observation that these immune checkpoint blockers are only beneficial for a subset of patients (e.g., Alsaab et al. 2017, Front Pharmacol 8:561). The high failure rate, combined with the high societal cost, is driving the search for predictive biomarkers that may help in selecting the right treatment for the right patients. The most commonly used predictive biomarker currently is PD-L1 expression, assessed by IHC for tumor biopsies, although its limitations are evident. These limitations include heterologous expression, the role of expression outside the tumor, and its dynamic expression during the disease process. Such limitations would be overcome by non-invasive molecular imaging using radiolabeled tracers, which would allow for deep tumor invasion and reliable, repeated quantification of the marker—this would enable mapping of the immune distribution of both primary tumors and metastatic lesions, or even within such tumors or lesions, both pre- and post-treatment.

[0044] In vivo tumor imaging based on immune tracers can support disease diagnosis, patient stratification (determining which patients are more likely to respond to immunotherapy), disease monitoring (changes in tumor images obtained during treatment reflect responses or non-responses to both immune and immunotherapy), and the design and development of novel immunotherapies (throughout preclinical or clinical development). More specifically, imaging of immune cells based on the labeled anti-CD163 moieties of the present invention, particularly CD163+ immune cells (such as immunoPET imaging), can similarly support monitoring the efficacy of immunotherapies, immunogenic or immunomodulatory therapies, as well as further support patient stratification and provide valuable information when designing and / or developing novel immunotherapies, immunogenic or immunomodulatory therapies.

[0045] Immunotherapy and immunogenic therapy Immunotherapy is generally defined, in relation to the present invention, as a treatment that uses the body's own immune system to help fight disease, more particularly cancer. As used herein, immunotherapy treatment represents the reactivation and / or stimulation and / or restoration of the mammalian immune response toward pathological conditions such as tumors, cancers, or neoplasms that evade and / or suppress normal immune surveillance. The reactivation and / or stimulation and / or restoration of the mammalian immune response then results in increased removal of tumor, cancer, or neoplasm cells by the mammalian immune system (anti-cancer, anti-tumor, or anti-neoplasmic immune response; an immune response adaptable to tumors, cancers, or neoplasms). Particularly interesting immunotherapeutic agents include immune checkpoint inhibitors (such as anti-PD-1, anti-PD-L1, or anti-CTLA-4 antibodies), bispecific antibodies that bridge cancer cells and immune cells, dendritic cell vaccines, oncolytic viruses, and cell-based therapies (e.g., CAR-T). Immunotherapy is a promising new area of ​​cancer treatment, and several immunotherapies have been evaluated in preclinical and clinical trials, demonstrating promising activity (Callahan et al. 2013, J Leukoc Biol 94:41-53; Page et al. 2014, Annu Rev Med 65:185-202). However, not all patients are necessarily sensitive to immune checkpoint blockade, and PD-1 or PD-L1 blocking antibodies can accelerate tumor progression. An overview of clinical development in the field of immune checkpoint therapy can be found in Fan et al. 2019 (Oncology Reports 41:3-14). Monoclonal antibodies that target and inhibit PD-1 include pembrolizumab, nivolumab, and cemiprimab. Monoclonal antibodies that target and inhibit PD-L1 include atezolizumab, avelumab, and durvalumab. Ipilimumab is an example of a monoclonal antibody that targets and inhibits CTLA-4. Combination cancer therapies, including chemotherapy, can achieve a higher rate of disease suppression by acting on different elements of the tumor ecosystem to obtain a synergistic antitumor effect.Certain chemotherapy regimens can enhance tumor immunity by inducing immunogenic cell death and promoting cancer immunoediting evasion; therefore, such treatments are now accepted to be called immunogenic therapies because they induce an immunogenic response. Drug parts known to induce immunogenic cell death include bleomycin, bortezomib, cyclophosphamide, doxorubicin, epirubicin, idarubicin, maphosphamide, mitoxantrone, oxaliplatin, and patupirone (Bezu et al. 2015, Front Immunol 6:187). Another form of immunotherapy is chimeric antigen receptor (CAR) T-cell therapy, in which allogeneic T cells are designed to recognize tumor neoantigens and oncolytic viruses that selectively affect and kill cancer cells. For example, therapy using RNA encoding MLKL is a further means of inducing an immunogenic response (Van Hoecke et al. 2018, Nat Commun 9:3417), as is vaccination with neoepitope (Brennick et al. 2017, Immunotherapy 9:361-371).

[0046] Applications of CD163 single-domain antibodies Several applications relating to targeting CD163 have been described in the art. For example, U.S. Patent No. 9,724,426(B2) claims a drug combining a CD163-binding moiety and a cytotoxic moiety or drug, which enables the drug to be internalized into cells when it binds to cells that have CD163 exposed on their surface. International Publication No. 2011 / 039510 refers to a CD163-binding molecule conjugated to an immunostimulant, which may be, for example, a Toll-like receptor (TLR) ligand. U.S. Patent No. 9,476,890(B2) claims a CD163-binding antibody conjugated to a prophylactic or therapeutic drug. International Publication No. 2017 / 158436 refers to a fusion protein of an immunostimulant and a target unit that guides the immunostimulant to tumor-associated macrophages. The target unit may, for example, bind to CD163 and may be, for example, a monovariate immunoglobulin domain. U.S. Patent Application Publication 2018 / 236076 refers to an anti-CD25 antibody (e.g., lacking an Fc receptor region) conjugated with a photon absorber (IR-700), the antibody targeting, for example, CD25+ cells (e.g., Tregs), which can then be selectively eliminated by photoimmunotherapy. This concept has been theoretically extended, in particular, to CD163+ cells. International Publication 2018 / 156725 refers to tumor therapy using an antibody (or antigen-binding fragment) conjugated with a cytotoxic compound, the antibody conjugating to CD163, CD204, or CD206. U.S. Patent Application Publication 2022 / 0073638 refers to a method for increasing CD8+ T cell infiltration in tumors by administration of an anti-CD163 antibody; and a method for treating cancer by combining an anti-CD163 antibody with an immune checkpoint inhibitor, the anti-CD163 antibody may be an antibody-drug conjugate as described in the specification. U.S. Patent No. 10,751,284 refers to tumor-view macrophage-targeting liposomes loaded with cytotoxic agents, having liposomes that target macrophages with a CD163-conjugated antibody.A CD206 monodomain antibody conjugated to the Toll-like receptor 7 / 8 agonist imidazoquinoline IMDQ has been reported to repolarize tumor-associated macrophages into a tumor-killing state and reduce tumor growth (Bolli et al. 2021, Adv Sci 2021:2004574).

[0047] The advantages of the CD163-conjugated polypeptide according to the present invention extend equally well to such applications. In fact, for diagnostic or in vivo molecular imaging purposes, as well as for therapeutic purposes, contrast agents or therapeutic agents must be able to reach their targets with high efficiency. This requires a combination of sizes small enough to obtain sufficient tissue permeability. Selective binding to the target to obtain a high signal-to-noise ratio at the target site (contrast agent) also contributes to the specificity of the therapeutic agent. Furthermore, the cell selectivity profile of the CD163-conjugated polypeptide according to the present invention is ideal in avoiding undesirable side effects as much as possible.

[0048] Accordingly, in further embodiments, the present invention relates to preventive or therapeutic cytotoxic moieties or cytotoxic drugs, immunostimulants, immunosuppressants, Toll-like receptor agonists, photon absorbers, CD163-conjugated polypeptides according to the present invention conjugated to liposomes or nanoparticles; and compositions such as pharmaceutical compositions comprising such conjugated molecules. In one embodiment, the CD163-conjugated polypeptide is a CD163-conjugated mono-variable domain of a CD163-conjugated immunoglobulin as defined herein, such as a CD163-conjugated mono-domain antibody or one defined by a CDR region contained therein, or one defined by a CDR and FR region contained therein. Such conjugated molecules, or compositions containing them, are particularly intended for use as pharmaceuticals or in the manufacture of pharmaceuticals; or, depending on their payloads, for use in / in the treatment of cancer or tumors or for inhibiting (the progression of) cancer or tumors, or for use in such treatment or inhibition methods; for use in / in the treatment of inflammatory or autoimmune diseases or for inhibiting (the progression of) inflammatory or autoimmune diseases, or for use in such treatment or inhibition methods; for use in / in the treatment of infectious diseases or for inhibiting (the progression of) infectious diseases, or for use in such treatment or inhibition methods. In the case of use in the treatment of cancer or tumors or for inhibiting (the progression of) cancer, it may be used in combination with or as part of combination therapy with further anticancer or antitumor agents. Such further anticancer or antitumor agents may be, for example, immune checkpoint inhibitors (see above under immunotherapy) or cytotoxic agents (see below).

[0049] The CD163-conjugated polypeptide according to the present invention can generally be conjugated to any prophylactic or therapeutic agent; in particular, such prophylactic or therapeutic agents can be selected based on their efficacy when targeted against CD163-positive cells, especially macrophages. In some embodiments, the prophylactic or therapeutic agent is conjugated to the CD163-conjugated polypeptide by a spacer arm, the length of which is designed to avoid or reduce potential steric hindrance. Alternatively, the prophylactic or therapeutic agent is loaded onto nanoparticles, liposomes, lipid nanoparticles, or the like, and the loaded nanoparticles or liposomes conjugate to the CD163-conjugated polypeptide according to the present invention.

[0050] Some examples of preventive or therapeutic drugs include cytotoxic agents (for use in the treatment of cancer, etc.), immunostimulants (for use in the treatment of cancer, etc.), immunosuppressants (for use in the treatment of inflammatory or autoimmune diseases, etc.), and antibacterial agents (for use in the treatment of infectious diseases, etc.).

[0051] Examples of cytotoxic agents or parts thereof include alkylating agents (e.g., cisplatin, carboplatin), antimetabolites (e.g., methotrexate, azathioprine), antimitotics (e.g., vincristine), topoisomerase inhibitors (e.g., doxorubicin, etoposide), and toxins (e.g., calicheamycin).

[0052] Immunosuppressants include anti-inflammatory drugs. Such drugs include: glucocorticoids (e.g., cortisone and its derivatives; prednisone and its derivatives; dexamethasone and its derivatives; triamcinolone and its derivatives; paramethasone; betamethasone; fludrocortisone; fluocinolone); methotrexate; cyclophosphamide; 6-mercaptopurine; cyclosporine; tacrolimus; mycophenolate mofetil; sirolimus; everolimus; nonsteroidal anti-inflammatory drugs (NSAIDs, aspirin, ibuprofen, etc.); steroids (vitamin D, etc.); and disease-modifying antirheumatic drugs (DMARDs, penicillamine, sulfasalazine, cyclosporine, etc.).

[0053] Immunosuppressants can be used to treat inflammatory and / or autoimmune conditions or disorders. Inflammatory and autoimmune conditions or disorders include arthritis (rheumatoid arthritis, spondylitis, osteoarthritis, etc.); chronic inflammatory bowel disease (IBD, Crohn's disease, ulcerative colitis); periodontitis; psoriasis; asthma; systemic lupus erythematosus; multiple sclerosis; autoimmune chronic inflammatory diseases; connective tissue diseases; autoimmune liver diseases (biliary cirrhosis, etc.); sepsis; hemophagocytic syndrome; liver disease; liver failure; hepatitis; atherosclerosis; diabetes mellitus; obesity; non-alcoholic fatty liver disease; non-alcoholic steatohepatitis (NASH); alcoholic steatohepatitis (ASH); acute alcoholic hepatitis; arthritis; inflammation-induced cartilage destruction; cirrhosis; organ transplantation; idiopathic thrombocytopenic purpura (ITP); sarcoidosis; uveitis; HLA-B27 positive uveitis; acute uveitis; macrophage activation syndrome; and giant cell arteritis.

[0054] Immunostimulants may be drugs that can stimulate one or more antitumor activities in macrophages. Examples of immunostimulants include cytokines and interleukins (e.g., interleukin-2), Toll-like receptor (TLR) agonists such as TLR7 / 8 ligands or agonists (e.g., IMDQ, imidazoquinoline variant 1-(4-(aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-c]quinoline-4-amine), bacterial polysaccharides, and costimulatory ligands (e.g., 41bb, CD80, CD86).

[0055] Antimicrobial agents include: antibiotics, antituberculosis antibiotics (such as isoniazid and ethambutol), antiretroviral drugs (e.g., reverse transcription inhibitors (such as zidovudine) or protease inhibitors (such as indinavir)), and drugs effective against leishmaniasis (such as meglumine antimonium). Antimicrobial agents can be used in the treatment of microorganism-induced conditions or disorders such as tuberculosis, AIDS, HIV infection, and leishmaniasis.

[0056] The CD163-conjugated polypeptide according to the present invention can generally be conjugated with a photon absorber, for example, so that the obtained CD163-conjugated polypeptide complex can be used in near-infrared photoimmunotherapy (NIR-PIT) by activating the photon absorber with near-infrared light. An example of a photon absorber is a photoactivated silica phthalocyanine dye (IRDye700DX).

[0057] In its final form, the present invention relates to a method for producing a CD163-conjugated polypeptide according to the present invention, wherein such method is: - A step of expressing the CD163-conjugated polypeptide in a suitable host cell (including nucleic acids or vectors as described herein), or a step of synthesizing and producing the CD163-conjugated polypeptide; - A step of purifying the expressed or synthesized / produced CD163-conjugated polypeptide, This includes methods.

[0058] Such a method may further include the steps of coupling, incorporating, binding, ligating, bonding, complexing, chelating, conjugating (e.g., site-specific binding), or covalently or noncovalently binding the detectable portion with a purified CD163-conjugated polypeptide, or a prophylactic or therapeutic cytotoxic portion or cytotoxic drug, immunostimulant, immunosuppressant, Toll-like receptor agonist, photon absorber, liposome or nanoparticle.

[0059] Other definitions The present invention will be described with reference to specific embodiments and specific drawings, but the present invention is not limited thereto and is limited only by the claims. None of the reference symbols in the claims should be construed as limiting. The drawings described are schematic only and not limiting. In the drawings, some sizes of elements may be exaggerated for illustrative purposes and may not be drawn to scale. The term “comprising,” when used herein and in the claims, does not exclude any other elements or processes. Where the singular form, e.g., “a” or “an,” or the indefinite or definite article is used when saying “the,” this includes plural nouns unless otherwise specifically designated. Furthermore, the terms first, second, third, and similar terms herein and in the claims are used to distinguish similar elements and not necessarily to describe an order or chronological order. It should be understood that the terms used in this manner are interchangeable under appropriate circumstances and that embodiments of the present invention described herein may be operated in an order other than that described or illustrated herein. Unless otherwise defined herein, all terms used herein have the same meaning as those understood by those skilled in the art. The practitioner directs, with respect to definitions and terms in the art, particularly to Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 100), John Wiley & Sons, New York (2012). The definitions provided herein should not be construed as having any scope unintelligible to those skilled in the art.

[0060] As used herein, the term “defined by SEQ ID NO: X” refers to a biological sequence consisting of the amino acid or nucleotide sequence given by SEQ ID NO: X. For example, a CDR defined by SEQ ID NO: X consists of the amino acid sequence given by SEQ ID NO: X. Further examples include an amino acid sequence containing SEQ ID NO: X, which is longer than the amino acid sequence given by SEQ ID NO: X but completely contains the amino acid sequence given by SEQ ID NO: X (the amino acid sequence given by SEQ ID NO: X may be located at the N-terminus or C-terminus of the longer amino acid sequence, or may be embedded within the longer amino acid sequence), or an amino acid sequence consisting of the amino acid sequence given by SEQ ID NO: X.

[0061] As used herein, the term “antibody” refers to an immunoglobulin (Ig) molecule that specifically binds to an antigen. Antibodies can be whole immunoglobulins of natural or recombinant origin, or they can be the immunoreactive portion of whole immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. As used herein, the term “immunoglobulin domain” refers to a globular region of an antibody chain (e.g., the chains of a conventional four-chain antibody or a heavy-chain antibody), or a polypeptide essentially derived from such a globular region. An immunoglobulin domain is characterized by holding an immunoglobulin fold comprising an antibody molecule consisting of a bilayer sandwich of about seven antiparallel β-chains arranged in a 2β-sheet stabilized by conserved disulfide bonds as needed.

[0062] The specificity of an antibody / immunoglobulin / I(S)VD to an antigen is defined by the composition of the antigen-binding domain in the antibody / immunoglobulin / I(S)VD (usually one or more of the CDRs, specific amino acids of the antibody / immunoglobulin / I(S)VD that interact with the paratope-forming antigen) and the composition of the antigen (the portion of the antigen that interacts with the antibody / immunoglobulin / I(S)VD that forms the epitope). Binding specificity is understood to represent the binding of the antibody / immunoglobulin / I(S)VD to a single target molecule or a small number of target molecules that (coincidentally) share an epitope recognized by the antibody / immunoglobulin / I(S)VD.

[0063] The affinity of an antibody / immunoglobulin / I(S)VD for a target is a measure of the strength of the interaction between the epitope on the target (antigen) and the epitope / antigen binding site in the antibody / immunoglobulin / I(S)VD. This can be defined as follows: K A = [Ab-Ag] / [Ab][Ag] When KA has a certain affinity, [Ab] is the molar concentration of the unoccupied binding site on the antibody / immunoglobulin / I(S)VD, [Ag] is the molar concentration of the unoccupied binding site on the antigen, and [Ab-Ag] is the molar concentration of the antibody-antigen complex.

[0064] Affinity provides information about the overall strength of the antibody / immunoglobulin / I(S)VD-antigen complex and generally depends on the affinity, the titer of the antibody / immunoglobulin / I(S)VD and the antigen, and the structural interaction of the binding partner.

[0065] As used herein, the term “immunoglobulin variable domain” (abbreviated as “I(S)VD”) means an immunoglobulin domain essentially consisting of four “framework regions” referred to as “framework region 1” or “FR1”; “framework region 2” or “FR2”; “framework region 3” or “FR3”; and “framework region 4” or “FR4”; respectively, these framework regions being interspersed with three “complementarity-determining regions” or “CDR” referred to as “complementarity-determining region 1” or “CDR1”; “complementarity-determining region 2” or “CDR2”; and “complementarity-determining region 3” or “CDR3”; respectively. Thus, the general structure or sequence of the immunoglobulin variable domain can be shown as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. This is an immunoglobulin variable domain (IVD) that confers specificity to the antibody to the antigen by carrying the antigen-binding site. The method for contouring / restricting the CDR in the antibody / immunoglobulin / IVD is described herein above.

[0066] The term "immunoglobulin single variable domain" (abbreviated as "ISVD"), equivalent to the term "single variable domain," defines a molecule in which an antigen-binding site resides on and is formed by a single immunoglobulin domain. This sets an immunoglobulin single variable domain apart from "conventional" immunoglobulins or fragments, where two immunoglobulin domains, particularly two variable domains, interact to form an antigen-binding site. Typically, in conventional immunoglobulins, the heavy chain variable domain (VH) and light chain variable domain (VL) interact to form an antigen-binding site. In this case, the complementarity-determining regions (CDRs) of both the VH and VL contribute to the antigen-binding site; i.e., all six CDRs are involved in antigen-binding site formation. From the perspective of the above definition, the antigen-binding sites of conventional four-chain antibodies (IgG, IgM, IgA, IgD, or IgE molecules; known in the art, etc.) or Fab fragments, F(ab')2 fragments, disulfide-bonded Fv fragments, or scFv fragments, or diabodies derived from such conventional four-chain antibodies (all known in the art) are not typically considered immunoglobulin monovariate domains. In these cases, binding to each epitope of the antigen does not typically occur by a single (mono) immunoglobulin domain, but rather by a pair of (related) immunoglobulin domains, such as light-chain and heavy-chain variable domains, i.e., a VH-VL pair of immunoglobulin domains, which co-bind with the epitopes of each antigen. In contrast, immunoglobulin monovariate domains can specifically bind to antigen epitopes without pairing with additional immunoglobulin variable domains. The binding site of an immunoglobulin monovariate domain is formed by a single VH / VHH or VL domain. Therefore, the antigen-binding site of the immunoglobulin monovariate domain is formed by 3 or fewer CDRs.As a result, the single variable domain may be a light chain variable domain sequence (e.g., a VL sequence) or a suitable fragment thereof, insofar as it can form a single antigen-binding unit (i.e., a functional antigen-binding unit essentially consisting of a single variable domain; as a result, the single antigen-binding domain does not need to interact with another variable domain to form a functional antigen-binding unit); or a heavy chain variable domain sequence (e.g., a VH sequence or VHH sequence) or a suitable fragment thereof. In one embodiment of the present invention, the immunoglobulin single variable domain is a heavy chain variable domain sequence (e.g., a VH sequence): more specifically, the immunoglobulin single variable domain may be a heavy chain variable domain sequence derived from a conventional four-chain antibody or a heavy chain variable domain sequence derived from a heavy chain antibody. For example, the immunoglobulin single variable domain may be a (single) domain antibody (or an amino acid sequence suitable for use as a (single) domain antibody), "dAb" or dAb (or an amino acid sequence suitable for use as dAb) or Nanobody® (as defined herein, but not limited to VHH); another single variable domain, or a suitable fragment of any one of them. In particular, the immunoglobulin single variable domain may be a nanobody (as defined herein) or a suitable fragment thereof. Note: Nanobody (registered trademark), Nanobodies (registered trademark), and Nanoclone (registered trademark) are registered trademarks of Ablynx NV. For a general description of Nanobodies (registered trademark), see the further description below, as well as the prior art cited herein, such as in International Publication No. 2008 / 020079.

[0067] The "VHH domain," also known as VHH, VHH domains, VHH antibody fragments, and VHH antibodies were originally described as the antigen-binding immunoglobulin (variable) domain of "heavy-chain antibodies" (i.e., "antibodies without light chains"; Hamers-Casterman et al (1993) Nature 363:446-448). The term "VHH domain" was chosen to distinguish these variable domains from the heavy-chain variable domains present in conventional four-chain antibodies (also referred to herein as "VH domains") and the light-chain variable domains present in conventional four-chain antibodies (also referred to herein as "VL domains"). For further explanation of VHH and Nanobody (registered trademark), see Muyldermans 2001 (Molecular Biotechnology). (Review of 74:277-302), and the following patent documents mentioned as general background art: International Publication Nos. 94 / 04678, 95 / 04079 and 96 / 34103; International Publication Nos. 94 / 25591, 99 / 37681, 00 / 40968, 00 / 43507, 00 / 65057, 01 / 40310, 01 / 44301, European Patent No. 1134231 and 02 / 48193; International Publication Nos. 97 / 49805, 01 / 21817, 03 / 035694 and 03 / 054016 See also International Publications 03 / 055527; 03 / 050531; 01 / 90190; 03 / 025020; and International Publications 04 / 041867, 04 / 041862, 04 / 041865, 04 / 041863, 04 / 062551, 05 / 044858, 06 / 40153, 06 / 079372, 06 / 122786, 06 / 122787 and 06 / 122825.As described in these references, Nanobody® (more specifically, VHH sequences and partially humanized Nanobody®) can be characterized in particular by the presence of one or more “Hallmark residues” in one or more framework sequences. Further descriptions of Nanobody®, including humanization and / or camelization of Nanobody®, as well as other modifications, parts or fragments, derivatives or “Nanobody® fusions,” multivalent constructs (including some non-limiting examples of linker sequences), and different modifications and preparations thereof that extend the half-life of Nanobody®, can be found, for example, in International Publication No. 08 / 101985 and International Publication No. 08 / 142164.

[0068] Domain antibodies, also known as "dAb" (the terms "domain antibody" and "dAb" are trademarks used by GlaxoSmithKline Group companies), are described, for example, in European Patent No. 0368684, Ward et al. 1989 (Nature 341:544-546), Holt et al. 2003 (Trends in Biotechnology 21:484-490), and International Publication No. 03 / 002609, as well as, for example, International Publication No. 04 / 068820, International Publication No. 06 / 030220, and International Publication No. 06 / 003388. Domain antibodies essentially correspond to the VH or VL domains of non-camelid mammals, in particular to human quadrilateral antibodies. As single antigen-binding domains, that is, to bind to epitopes without forming pairs with VL or VH domains, specific selection for such antigen-binding properties is required, for example, by using a library of human single VH or VL domain sequences. Domain antibodies, like VHH, have a molecular weight of approximately 13–16 kDa and, if derived from a fully human sequence, do not require humanization for therapeutic use in humans, for example. It should be noted that single variable domains can be derived from specific shark species (e.g., the so-called "IgNAR domain," see, for example, International Publication No. 05 / 18629).

[0069] Immunoglobulin monovariate domains such as domain antibodies and Nanobody (registered trademark) (including VHH domains and humanized VHH domains) can be subjected to affinity maturation by introducing one or more modifications into the amino acid sequence of one or more CDRs, and these modifications result in improved affinity of the obtained immunoglobulin monovariate domain to its respective antigen compared to its respective parent molecule. The affinity-matured immunoglobulin single variable domain molecule of the present invention may be prepared by methods known in the art, for example, by methods described by Marks et al. 1992 (Biotechnology 10:779-783), Barbas et al. 1994 (Proc Natl Acad Sci USA 91:3809-3813), Shier et al. 1995 (Gene 169:147-155), Yelton et al. 1995 (Immunol 155:1994-2004), Jackson et al. 1995 (J Immunol 154:3310-3319), Hawkins et al. 1992 (J MoI Biol 226:889-896), and Johnson and Hawkins (Affinity maturation of antibodies using phage display, Oxford University Press, 1996). Methods for designing / selecting and / or preparing polypeptides starting from immunoglobulin monovariable domains such as domain antibodies and Nanobody® are also referred to herein as “formatting” the immunoglobulin monovariable domain, and an immunoglobulin monovariable domain comprising as part of a polypeptide is “formatted” or “in the format of” the polypeptide. Examples of methods by which immunoglobulin monovariable domains can be formatted, and examples of formatting to avoid glycosylation, for example, will be obvious to those skilled in the art based on this disclosure.

[0070] Immunoglobulin monovariate domains, such as domain antibodies and Nanobody® (including the VHH domain), can be humanized, i.e., their degree of sequence identity with the nearest human germline sequence can be increased. Specifically, a humanized immunoglobulin monovariate domain, such as Nanobody® (including the VHH domain), may be an immunoglobulin monovariate domain as generally defined in the preceding paragraph, but may contain at least one amino acid residue (specifically, at least one framework residue) that is a humanization substitution (as defined herein) and / or equivalent. Potentially useful humanization substitutions can be identified by comparing the sequence of the framework region of a native VHH sequence with the corresponding framework sequence of one or more closely related human VH sequences, and then one or more of the thus determined potential useful humanization substitutions (or combinations thereof) can be introduced into the VHH sequence (furthermore, as described herein, in any way that is itself high), and the resulting humanized VHH sequence can be tested for affinity to a target, stability, ease and level of expression, and / or other desired properties. In this method, other suitable humanized substitutions (or suitable combinations thereof) can be determined by those skilled in the art through a limited degree of trial and error. Furthermore, based on the foregoing, immunoglobulin monovariate domains (or their framework regions), such as Nanobody® (including the VHH domain), may be partially or fully humanized.

[0071] As used herein, “serum albumin conjugate” or “serum albumin-binding polypeptide” is a protein-based agent capable of specifically binding to serum albumin. In various embodiments, the serum albumin conjugate may be conjugated to the full-length and / or mature and / or isoform and / or splice variant and / or fragment and / or other natural or synthetic analogs, variants or variants of serum albumin. In various embodiments, the serum albumin conjugate of the present invention may be conjugated to any form of serum albumin, including monomers, dimers, trimers, tetramers, heterodimers, multimers and related forms. In embodiments, the serum albumin conjugate conjugates to a monomer of serum albumin. In embodiments, the present serum albumin-binding polypeptide comprises an immunoglobulin variable domain having an antigen-binding site comprising three complementarity-determining regions (CDR1, CDR2, and CDR3). In embodiments, the antigen-binding site recognizes one or more epitopes present on serum albumin. In various embodiments, the serum albumin conjugate comprises a full-length antibody or a fragment thereof. In embodiments, the serum albumin conjugate comprises a single-domain antibody or an immunoglobulin single variable domain (ISVD). In specific embodiments, the serum albumin conjugate binds to rat serum albumin (Uniprot P02770). In specific embodiments, the serum albumin conjugate binds to mouse serum albumin (Uniprot P07724). In specific embodiments, the serum albumin conjugate binds to human serum albumin (Uniprot P02768).

[0072] The above embodiments and examples may generally involve administering a CD163-conjugated polypeptide or a pharmaceutical composition containing the same to a mammal in need, i.e., a mammal with a tumor, cancer or neoplasm requiring (non-invasive) medical imaging, diagnosis, surgery (or guided surgery) or therapeutic monitoring. Generally, an effective amount of the CD163-conjugated polypeptide or a pharmaceutical composition containing the same is administered to the mammal in need to satisfy the desired effect. The effective amount depends on many factors, such as the route of administration, and must be determined by a physician on a case-by-case basis. “Administration” means any form of contact that results in an interaction between the drug (the CD163-conjugated polypeptide described herein) or a pharmaceutical composition containing the drug (such as a pharmaceutical or pharmaceutical composition) and the object (e.g., cells, tissues, organs, body cavities) that the drug or composition comes into contact with. The interaction between the drug or composition and the object may occur immediately or almost immediately after administration of the drug or composition, may occur over a long period of time (starting immediately or almost immediately after administration of the drug or composition), or may be delayed relative to the time of administration of the drug or composition. More specifically, "contact" results in the delivery of an effective amount of the agent or a composition containing the agent to the subject.

[0073] The term “effective dose” refers to a dosage regimen of the drug (the CD163-conjugated polypeptide described herein) or a composition containing the drug (e.g., a pharmaceutical composition). The effective dose will generally depend on and / or require adjustment to the mode of contact or administration. To obtain or maintain the effective dose, the drug or a composition containing the drug may be administered as a single dose or as repeated doses. The effective dose may further vary depending on the diagnosis, imaging, or severity of the condition to be manipulated; this may depend on the overall health and physical condition of the mammal or patient and will usually require assessment by a physician or doctor to determine the effective dose. The effective dose may further be obtained by combinations of different types of contact or administration.

[0074] While specific embodiments, configurations, and materials and / or molecules are discussed with respect to the cells and methods according to the present invention, it should be understood that various changes or modifications in form and detail may be made without departing from the scope and nature of the invention. The following examples are provided to better illustrate specific embodiments and should not be considered limiting to applications. Applications are limited only by the claims.

[0075] The content of the references cited herein is incorporated by reference. [Examples]

[0076] Example 1. Anti-CD163 single-domain antibody that binds with high affinity to both human and mouse CD163 receptors. Two llamas were immunized with recombinant human CD163 (hCD163) protein and mouse CD163 (mCD163) protein to obtain cross-reactive CD163-specific single-domain antibodies (sdAbs). After phage display planning and initiation screening, only 9 clones were considered cross-reactive. After performing a ThermoFluor assay to determine thermal stability, surface plasmon resonance (SPR) to analyze affinity to both recombinant hCD163 and mCD163 proteins, and flow cytometry experiments to test affinity to HEK293T cells overexpressing either hCD163 or mCD163, one read cross-reactive CD163-targeted sdAb (sdAb 23766) was selected. This sdAb showed high binding affinity to both hCD163 and mCD163 by SPR (Figure 1 and Table 1). HEK293T hCD163 + Cells and HEK293T mCD163 + Strong binding was also observed in cells (Figure 2 and Table 1), and sdAb 23766 has a melting point of 78.0°C, which means that this sdAb can be incubated at high temperatures (<50°C) for radiolabeling (Tables 1A and 1B).

[0077] [Table 1A]

[0078] [Table 1B]

[0079] The amino acid sequences of sdAb23766 and its CDR and FR regions were determined. These sequences are shown below. sdAb23766 DVQLVESGGG LVQPGGSLRL SCAASGITFS SYAVAWFRQA SGKEREFVAF IGWDGDTTYY VDSVKGRFTI SRDNAKNMVY LQMNSLKPDD TAIYYCARHK TLWRSSWDNR PVQYDYWGQG TQVTVSS(Sequence ID 1) sdAb23766 CDR1:GITFSSYA(Sequence ID 2) sdAb23766 CDR2:IGWDGDTT(Sequence ID 3) sdAb23766 CDR3:ARHKTLWRSSWDNRPVQYDY(Sequence ID 4) sdAb23766 FR1:DVQLVESGGGLVQPGGSLRLSCAAS(Sequence ID 5) sdAb23766 FR2:VAWFRQASGKEREFVAF(Sequence ID 6) sdAb23766 FR3:YYVDSVKGRFTISRDNAKNMVYLQMNSLKPDDTAIYYC(Sequence ID 7) sdAb23766 FR4:WGQGTQVTVSS(Sequence ID 8)

[0080] The human CD163 gene is located at chr12:7,470,811-7,503,893 (GRCh38 / hg38; minus strand), or chr12:7,623,407-7,656,373 (GRCh37 / hg19; minus strand according to Entrez Gene), or chr12:7,623,409-7,656,489 (GRCh37 / hg19; minus strand according to Ensembl). Reference mRNA sequences: GenBank accession numbers NM_001370145.1;NM_001370146.1;NM_004244.6; and NM_203416.4. The coding sequence for human CD163 can be found further, for example, at GenBank accession number DQ058615.1.

[0081] The code sequence for mouse CD163 can be found further, for example, under GenBank accession number BC145793.1.

[0082] Example 2. Anti-CD163 single-domain antibody specifically targets macrophages in naive and tumor-bearing mice. Next, the inventors tested sdAb 23766 for selectivity, in vivo distribution, background signaling, and in vivo tracer accumulation. First, sdAb 23766 and the unrelated sdAb R3b23 were tested using their C-terminal His tags to technetium-99m( 99m Site-specific labeling was performed using Tc) in naive C57BL / 6J wild-type (WT) mice (n=3) and C57BL / 6J CD163 knockout mice. - / - The drug was administered intravenously to mice (n=3). One hour after injection, the mice were imaged using a SPECT / CT camera, and these organs were harvested for ex vivo in vivo analysis by gamma (γ) counting. 99m Tc-labeled sdAb 23766 showed high uptake in macrophage-enriched organs such as cervical lymph nodes, liver, intestines, and bone marrow in naive WT mice, but CD163 - / -Uptake was not observed in mice (Figure 3). This suggests that signal uptake is specific to cells expressing the mCD163 receptor. CD163 was also observed in WT mice. - / - No signal for unrelated sdAbs was observed in mice. Specific uptake of sdAb 23766 was confirmed by ex vivo γ counting, based on uptake observed in the spleen (Figure 4). Although sdAbs are removed by the renal system, they are reabsorbed by proximal tubular cells and retained in the renal cortex. Therefore, splenic uptake was masked in SPECT / CT images by the high signal in the kidney (Chigoho et al. 2021, Curr Opin Chem Biol 63:219-228).

[0083] Next, the inventors determined the macrophage specificity of sdAb 23766 in untreated or macrophage-depleted mice. For comparison, the inventors also included sdAb 3.49, which targets the macrophage mannose receptor (MMR or CD206) and is currently in clinical trials (NCT04168528). One week prior to the in vivo distribution experiment, three mice received a diet containing the colony-stimulating factor 1 receptor (CSF1R) inhibitor PLX3397 to induce macrophage depletion, while the other three mice received a control diet. Untreated mice showed high uptake of both anti-MMR and anti-CD163 sdAbs in the cervical lymph nodes, liver, and bone marrow. Interestingly, sdAb 23766 signaling in the liver was significantly reduced in mice treated with the PLX3397 compound, while anti-MMR sdAb signaling in the liver retained the same signal under these conditions. Therefore, anti-MMR sdAbs target not only macrophages but also other cells expressing the MMR receptor in the liver (e.g., LSECs), while anti-CD163 sdAbs are macrophage-specific (Figure 5).

[0084] Next, the inventors aimed to visualize CD163-expressing tumor-associated macrophages (TAMs) using sdAb 23766, and conducted in vivo distribution experiments in three tumor models (MC38, B16-F10, and LLC-OVA). Tumor cells were subcutaneously inoculated into the right flank of animals, and after 2-2.5 weeks, at 500-1000 mm. 3 Imaging experiments were conducted to induce tumor growth. SPECT / CT images and ex vivo in vivo distribution data showed that 99m Limited uptake of Tc-labeled sdAb 23766 was observed within MC38 tumors (Figures 6A and 7A). The B16-F10 tumor model showed uptake of radiolabeled sdAb around the tumor (Figure 6B) and ex vivo uptake similar to that of MC38 tumors (Figure 7C). In the case of LLC-OVA tumors, 99m Tc-labeled sdAb 23766 uptake was observed in the center of tumors with higher mean ex vivo uptake compared to the other two tumor models (2% IA / g) (Figure 7E). Furthermore, 99m Tc label sdAb 23766 is, 99m Compared to Tc-unlabeled sdAb, all three tumor models showed significantly higher tumor-to-blood ratios (Figures 7B, 7D, 7F). Results obtained from SPECT / CT imaging and ex vivo γ counting could be validated by flow cytometry to show CD163 expression in different tumors, with LLC-OVA tumors demonstrating the highest CD163 expression levels on TAMs recognized by the marker F4 / 80. The marker MHC-II was included to determine MHC-II-low and MHC-II-high TAM populations in the immune cell compartment.

[0085] Example 3. Converting lead anti-CD163 tracers toward PET tracers. When the inventors wish to use an anti-CD163 immunotracer for nuclear imaging of patients, ideally the tracer needs to be converted into a PET tracer. This involves a single-domain antibody and a radioactive metal 68The binding of Ga to the chelating agent is required for radiolabeling. The final binding method yielded a chelating agent:single-domain antibody ratio of 2.32 ± 0.12, when determined by mass spectrometry. Immediately after labeling, the stability of the injection buffer and human serum was assessed at different time points at room temperature and 37°C. After 60 minutes, [ 68 The Ga]Ga-NOTA-anti-CD163 single-domain antibody remained stable in injection buffer (RCP; 95.5±1.2%) and human serum (RCP; 92.2±2.9%) (Table 3). The NOTA-single-domain antibody was stable against recombinant hCD163 protein (K D (1.55±0.33nM) and HEK293T mCD163 + Cell(K D It showed similar binding affinity to (12.0±0.8nM), and did not show the effect of NOTA binding on binding properties. After radiation labeling, gallium-68 ( 68 Ga)(t 1 / 2 =68 minutes) or longer-lived surrogate isotope gallium-67 ( 67 Ga)(t 1 / 2 Using either of the following (=78.3 hours): 68 Ga]Ga-NOTA-anti-CD163 single-domain antibody and [ 67 Both Ga]Ga-NOTA-anti-CD163 single-domain antibody and hCD163 recombinant protein (K D :9.11±3.32nM) (Figure 9A) and HEK293T mCD163 + Cell(K D It still showed binding affinity in the low nanomolar range (7.82 ± 1.13 nM) (Figure 9B).

[0086] CD163 was identified by μPET / CT imaging and ex vivo analysis. + The PET tracer was re-tested for cell specificity. 68 Ga]Ga-NOTA-anti-CD163 single-domain antibody and [ 68 Ga]Ga-NOTA-Irr single-domain antibody (±10 MBq) was used to test on naive C57BL / 6J WT and CD163 - / -The antibody was administered intravenously to mice (n=3). The anti-CD163 tracer showed specific radioactive uptake for PET / CT images (Figure 10A-B) and ex vivo (γ) counting of lymph nodes in the neck and groin, liver, intestines, and bone marrow (Figure 10C-F). Therefore, we were able to conclude that the CD163 single-domain antibody was successfully converted into a PET tracer.

[0087] Example 4. The lead anti-CD163 single-domain antibody can visualize TAM dynamics in the tumor microenvironment using longitudinal imaging during CSF1R therapy. The ultimate goal is to track TAMs within the tumor microenvironment (TME) longitudinally using radiolabeled sdAb 23766 during immunotherapy. Therefore, LLC-OVA tumor-bearing mice receive a diet of the macrophage-depleting compound PLX3397 (600 mg / kg AIN-76A diet) for 21 days. Control mice receive a standard AIN-76A diet. During this period, mice are scanned at three different time points to determine the presence of CD163-expressing TAMs. Significantly lower uptake is observed in SPECT / CT images and ex vivo γ counting data in mice treated with PLX3397 in lymph nodes, liver, and tumors. Flow cytometry data confirms a significant reduction in macrophages in tumors and liver in the PLX3397-treated group. Radiolabeled anti-CD163 sdAb 23766 can visualize the distribution of TAMs during anti-macrophage immunotherapy.

[0088] Example 5. Nuclear imaging using a lead anti-CD163 single-domain antibody tracer can non-invasively monitor macrophage depletion in the tumor microenvironment during anti-macrophage therapy. The inventors then attempted to visualize treatment outcomes by imaging the tumor microenvironment (TME) during immunotherapy using a radiolabeled CD163 immunotracer. For this purpose, LLC-OVA tumor-bearing mice were treated for 16 days with either the macrophage depletion compound PLX3397 or a control diet. Interestingly, the treated mice could be classified into different groups based on tumor growth: non-responders (showing no effect on tumor growth), partial responders (showing partial suppression of tumor growth), or responders (showing a reduction in tumor growth) (Figure 11A). As expected, the anti-CD163 tracer showed high uptake in lymph nodes, bone marrow, liver, and tumors in untreated mice (Figure 11B). Compared to untreated mice, PET / CT images showed lower uptake of the anti-CD163 tracer in lymph nodes, bone marrow, and liver in PLX-treated mice (Figures 11B-C). Responder (R) mice showed less overall uptake compared to non-responder mice (Figure 11C). The relationship between tumor volume and radioactive uptake (%IA), as well as between tumor volume and CD163, was observed. + MHC-II low A significant correlation was observed between macrophage percentage and PET imaging data (Figure 11D-E). Furthermore, flow cytometry analysis showed that tumors in responders correlated more strongly with the antitumor phenotype of macrophages compared to partial responders and non-responders, indicating a higher MCH-II level. high / MHC-II low The ratio of TAMs was shown to be included (Wang et al. 2011, BMC immunology 12:43) (Figure 11F). Responders also showed lower CD163 expression compared to partial responders and non-responders (Figure 11G).

[0089] Example 6. Materials and Methods 6.1. DNA constructs The lentiviral pHR vector, the packaging vector pCMV which packages plasmid pCMVΔR8.9, and VSV.G which encodes plasmid pMD.G were gifts from D. Trono (University of Geneva, Switzerland). PHR vectors encoding hCD163 or mCD163 proteins were prepared by infusion cloning (Takara Bio, Kusatsu, Japan).

[0090] 6.2.Cell culture HEK293T cells and B16-F10 cells were purchased from ATCC (Bösel, Germany). LLC-OVA cells were kindly provided by Dmitry Gabrilovich (The Wistar Institute, Philadelphia, USA). MC38 cells were kindly provided by Massimiliano Mazzone (VIB-KU, Leuven, Belgium). All cells were grown in 5% CO2 and at 37°C. LLC-OVA cells were grown in Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher Scientific, Waltham, Massachusetts, USA) supplemented with 1% penicillin / streptomycin (Gibco, Thermo Fisher Scientific) and 10% fetal bovine serum (FBS, Serana, Pessin, Germany). HEK293T, MC38, and B16-F10 cells were grown in Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher Scientific) supplemented with 1% penicillin / streptomycin and 10% FBS.

[0091] 6.3. Animal Models I purchased a female wild-type C57BL6 / J mouse from Charles River (Ecurie, France). CD163 - / -The mice were previously described (Fischer-Riepe et al. 2020, J Allergy Clin Immunol 146:1137-1151) and kindly provided by Johannes Roth (WWU Münster, Germany). For imaging of tumor-bearing mice, MC38, B16-F10, or LLC-OVA tumor cells were subcutaneously injected into the right flank of the mice. The mice were examined daily, and tumor growth was measured using digital calipers. Tumor volume was calculated using the formula (length × width). 2 The calculation was performed using ) / 2. All experiments using mice were approved by the Ethical Committee for laboratory animals at Vrije Universiteit Brussel and conducted in accordance with the European Guidelines for Animal Experiments (ethical document numbers 21-272-14, 21-272-23, and 22-272-28).

[0092] 6.4. sdAb preparation, selection, and manufacturing Two llamas were subcutaneously injected six times with 100 μg recombinant human (h) CD163-Avi-His6 (U-Protein Express BV), 100 μg recombinant human CD163-His6 (Acro Biosystems, Newark, Delaware, USA), 100 μg recombinant mouse (m) CD163-Avi-His6 (U-Protein Express BV), and 100 μg recombinant mCD163-His6 (provided by Johannes Roth, WWU Münster), and mixed with Gerbu adjuvant P (Gerbu Biotechnik) on a weekly basis. After immunization, peripheral blood was collected from both llamas, and peripheral blood mononuclear cells were isolated using lymphhoprep tubes (Greiner Bio-one, Kremsmünster, Austria). RNA was isolated from peripheral blood lymphocytes using an RNA extraction kit (Qiagen, Hilden, Germany) and reverse transcribed into cDNA. Genes encoding the variable domain of heavy-chain-only antibodies were amplified and ligated into pMECS phage vectors (Muyldermans 2021, FEBS J 288:2084-2102) to obtain two separate phage display libraries. These libraries were then biopanned by infection with the M13K07 helper phage to produce phages. For each library, three pannings in solution were performed using in-house site-directed biotinylated hCD163-Avi-His6 or mCD163-Avi-His6. 100 nM antigen was used for the first and second pannings, while 10 nM antigen was used during the final panning. In total, 190 unique clones (95 from the second panning and 95 from the third panning) were randomly selected and screened by ELISA for their ability to specifically bind to hCD163 and mCD163. Site-directed biotinylated hCD163-Avi-His6 and mCD163-Avi-His6 were used to determine specific binding by ELISA, and the cells were immobilized on streptavidin-coated 96-well plates. Positive hits were sent for sequencing (Eurofins genomics) and grouped into different B cell lineages based on their CDR3 sequences.sdAb was prepared and purified as described above (Pardon et al. 2014, Nat Protoc 9:674-693).

[0093] 6.5. Surface Plasmon Resonance (SPR) The affinity of purified CD163-targeted sdAb and NOTA-conjugated anti-CD163 sdAb to recombinant hCD163 and mCD163 proteins (U-Protein Express BV) was determined using BIACORE-T200 (GE Healthcare, Freiburg, Germany). Surface plasmon resonance (SPR) measurements were performed at 25°C using HEPES-buffered saline (HBS, 20 mM HEPES pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.05% Tween-20) Lanning buffer. SdAb was injected sequentially in 2-fold serial dilutions from 250 to 1 nM. The association step was 100 seconds, the dissociation step was 200 seconds, and a two-step regeneration step was performed using 100 mM glycine at pH 3.0 at a rate of 30 μL / min for 35 seconds. Using BIAcORE evaluation software (GE Healthcare), local curve fitting was performed by fitting the obtained sensorgrams to the theoretical curve, and the 1-1 bond geometry was inferred. To determine the parallel dissociation constant, the ratio of the association and dissociation rate constants was determined.

[0094] 6.6. Affinity determination by flow cytometry Serial dilutions of CD163-targeted sdAb and NOTA-bound anti-CD163 sdAb were incubated with 500,000 HEK293T cells overexpressing hCD163 or mCD163 in FACS buffer (HBSS (Gibco) supplemented with 1% FBS and 2 mM EDTA (Duchefa Biochemie, Haarlem, Netherlands)) for 1 hour at 4°C. NOTA-sdAb binding was detected by incubation of the cells with PE-tagged anti-VHH Ab (1:500 in FACS buffer, Genscript, Piscataway, New Jersey, USA) for 30 minutes at 4°C. The cells were washed once with FACS buffer. Next, sdAb binding was detected by incubation of the cells with Alexa Fluor®-488 tagged anti-HA antibody (1:1000 in FACS buffer, clone 16B12, Biolegend, San Diego, California, USA) at 4°C for 30 minutes. The cells were washed once again with FACS buffer. sdAb was determined using a FACS CANTO II analyzer (BD Biosciences, Franklin Lakes, New Jersey, USA). The mean fluorescence intensity of sdAb was determined using FlowJo version 10.

[0095] 6.7. Thermal Shift Assay sdAb (concentrations ranging from 0.2 mg / ml to 0.5 mg / ml) was mixed with 1×SYPRO® Orange Protein Gel Stain (Thermo Fisher Scientific) in PBS and added to a white 96-well PCR plate (BiOrad, Pleasanton, California, USA). The fluorescence signal was measured using CFX connect® Real-Time PCR (Biorad) with a 0.5°C stepwise increase, during a temperature increase from 20 to 95°C. The melting point of sdAb was calculated using the Boltzmann equation.

[0096] 6.8. Anti-CSF1R treatment in naive and tumor-bearing mice To deplete macrophages in naive and tumor-bearing mice, the CSF1R inhibitor pexidartinib (PLX3397) is used. PLX3397 (Advanced Chemblock, Inc.) is added to the AIN-76A diet by Research Diets, Inc. at a concentration of 600 mg / kg diet. To achieve depletion, naive mice are fed PLX3397 diet ad libitum for 7 days and LLC-OVA tumor-bearing mice are fed PLX3397 diet ad libitum for 21 days. Control mice receive the AIN-76A standard diet during the same period (n = 3 / group).

[0097] 6.9.sdAb's 99m Tc radiolabeling The sdAb was labeled with 99m Tc as described above (Xavier et al. 2012, Methods Mol Biol 911:485 - 490). Briefly, 99m Tc-tricarbonyl was prepared by adding 150 mCi 99m TcO4 - to the Isolink® labeling kit (Scherrer Institute, Filigen, Switzerland). Next, 50 μg of His-tagged sdAb was added and incubated at 50 °C for 90 min. 99m The 99m Tc-labeled sdAb was purified by gel filtration from unbound

[0098] 6.10. Pinhole SPECT-microCT imaging and image analysis Approximately 5 μg of radiolabeled sdAb was injected into the mice. One hour after the injection, the mice were intraperitoneally injected with 75 mg / kg of ketamine and 1 mg / kg of medetomidine (Ketamidor, Richter Pharma AG, Weise, Austria), and SPECT-microCT imaging was performed using a + vector scanner (MiLAbs, Utrecht, Netherlands). The imaging setup consisted of a 1.5 mm 75-pinhole general-purpose collimator in spiral mode with 6 gantry positions. The total SPECT scan time was 150 seconds per position for 15 minutes, and the CT scan (60 kV and 615 mA) was 2 minutes. After imaging, the mice were sacrificed and the organs were harvested. The radioactivity of each organ was determined using a 2 Wizard γ counter (Perkin-Elmer, Waltham, MA, USA). The uptake in each organ was corrected for radioactive decay and calculated as a percentage of the injected activity per gram of organ. SPECT / CT image analysis was performed using AMIDE (UCLA, Los Angeles, CA, USA) and OsiriX (Pixmea, Geneva, Switzerland).

[0099] 6.11. Organ processing and flow cytometry analysis Single-cell preparations of tumors and livers were prepared as described above (Van Damme et al. 2021, J Immunother Cancer 9:e001749). The antibodies used for staining of the single-cell preparations can be seen in Table 2. The Δ median fluorescence intensity (ΔMFI) was determined by subtracting the MFI of the staining and the MFI of the isotype control. Data were obtained using a FACS CANTO II analyzer and analyzed using FlowJo.

[0100] 6.12. Random binding and ion exchange of NOTA chelator with lysine of sdAb Random binding of sdAb to p-SCN-Bn-NOTA (NOTA-NCS, Macrocyclics, Inc., Plano, Texas, USA) was adjusted to obtain the optimal chelator-to-sdAb ratio, compared to a standard protocol (Xavier et al. 2013, J Nucl Med 54:776-784). 5.5 mg / mL of anti-CD163 sdAb (7 mg, 0.49 μmol) was first buffer-exchanged with 0.25 M sodium carbonate (anhydrous sodium carbonate; sodium bicarbonate; sodium chloride, VWR Chemicals, Leuven, Belgium) adjusted to pH 9.75 using a PD-10 size exclusion column (GE Healthcare, Buckinghamshire, UK). A 30-fold molar excess of NOTA-NCS was then added to the sdAb solution (7.98 mg, 1.43 μmol). NOTA-sdAb was added to a μmol (1 / 2) solution and incubated at room temperature for 3.5 hours. After incubation, NOTA-sdAb was purified by size exclusion chromatography (SEC) using 0.1 M NaOAc as the mobile phase (0.8 mL / min) on a Hiload® 16 / 600 Superdex® 30 pg column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The concentration of the collected NOTA-sdAb fraction was spectroscopically measured using a Nanodrop 2000 by UV absorption at 280 nm (ε=72287 M-1 cm-1, MW=15309 Da). SEC using a Superdex Peptide 10 / 300 GL column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) was also used for quality control of NOTA-sdAb. The number of chelating agents per sdAb was determined by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS).

[0101] 6.13.NOTA-CD163 sdAb 68 / 67 Ga labeling and radioactive metal chelation stability evaluation Randomly bound NOTA-CD163 sdAb (6.5 nmol) in 0.1 M HCl (Galli Eo (trademark), IRE ELiT, Fleurus, Belgium)68 Ga / 68 1 mL of 1 M NaOAc buffer pH 5 and 1 mL of eluted from the Ga generator 68 The solution was added to Ga eluate (374-618 MBq) and incubated at room temperature for 10 minutes. For stability and in vivo testing, the solution was purified using a PD-10 desalting column pre-equilibrated with 1×PBS in 0.9% NaCl pH 5.8-6.1 (injection buffer) containing 5 mg / mL vitamin C, or with test labeling. After purification, the radioactive sdAb solution was filtered through a 0.22 μm filter (Millipore, Belgium). Radiochemical purity was measured before and after purification using radio-iTLC ([ 68 Ga]Ga-NOTA-sdAb Rf=0, [ 68 The cell binding test was evaluated using Ga]Ga-citrate (Rf=1). Since the cell binding test requires a long incubation and washing step, NOTA-sdAb was used. 68 A longer-lived isotope as an alternative to Ga 67 Labeled using Ga, [ 67 Ga]GaCl3 is diluted with metal-free water (TraceSELECT®, Honeywell Riedel-de Haen®, Fisher Scientific) and the final solution is added to a Waters Sep-Pak® Reservoir adapter. 67 Obtained from Ga]Ga-citrate. sdAb labeling was performed using 6.5 nmol NOTA-sdAb, 5 M NH4OAc pH 5~5.2, and ±111 MBq [ 67 [Ga]GaCl3 was required. The radiolabeled sdAb solution was purified using a NAP-5 column (GE Healthcare, Belgium) and then filtered.

[0102] [ ELISA 68 The affinity of Ga]Ga-NOTA-anti-CD163 sdAb was determined as described in the previous paragraph. The radioactive sdAb binding to the hCD163-Avi-His6 protein was measured using a γ counter.

[0103] [ 68 The radioactive metal chelate stability of Ga]Ga-NOTA-anti-CD163 sdAb (5-69 MBq, after filtration) was evaluated at 30, 60, 120, and 180 minutes after labeling under different conditions (injection buffer RT, 37°C; human serum 37°C; mouse serum 37°C). The stability of the radiolabeled compound was analyzed at these time points by radio-iTLC and radio-SEC.

[0104] 6.14.[ 67 Cell binding assay using Ga]Ga-NOTA-anti-CD163 sdAb 5 × 10 in 1 mL of DMEM medium per well 4 HEK293T mCD163 + Cells were seeded in a 24-well plate, and a radioactive cell binding test was performed 2 days later. The plate was cooled to 4°C, and the experiment was performed after 1 hour. After 30 minutes, the plate was removed and the medium was replaced with 400 μL of unrefilled medium or blocking unrefilled medium (unmodified sdAb in excess of 100 molars). Radiolabeled sdAb at different concentrations ranging from 300 nM to 0.1 nM was added to the cells. After incubation at 4°C for 1 hour, the wells were washed twice with 0.75 mL of ice-cold 1×PBS, and lysis was performed at room temperature for 5 minutes by adding 0.75 mL of 1 M NaOH. All fractions were collected and Wizard 2 The count was performed using a gamma counter (PerkinElmer, Mechelen, Belgium).

[0105] 6.15. μPET / CT Imaging and Image Analysis The timing of injections and the use of anesthetics are the same as in the chapter "Pinhole μSPECT / CT Imaging and Image Analysis". C57BL / 6J WT and C57BL / 6J CD163 - / - In mice, in a volume of 130-170 μL [ 68Mice were injected with Ga]Ga-NOTA-sdAb (92.7±17.5 MBq / mL, 0.33 nmol). Mice were imaged in a prone position and fixed to a mouse hotel or single-mouse examination table with iFIX Fleece 5 tape (Interventional systems, Kitzbühel, Austria). Imaging was performed for 12-20 minutes at a resolution of 850 μm using a Molecubes PET β-cube / CT-X-cube system (Molecubes, Ghent, Belgium). The Molecubes PET system features 45 PET detectors arranged in 5 rings to obtain a scanner diameter of 7.6 cm and an axial length of 13 cm (Krishnamoorthy et al. 2018, Phys Med Biol 63:155013). The energy peak was set to 511 keV and the energy resolution to 30%. Reconstruction was performed using Molecubes software with the OSEM algorithm, and attenuation was based on the CT images. A mouse tumor in the right flank was positioned 7–7.5 cm within the field of view (FOV). Phantom base correction of the scanner was performed using a 500 μL syringe containing a minimum value of 20 μCi at the axis center of the FOV. Post-processing was performed using VivoQuant® 2022 (Invicro, Needham, USA). The obtained radioactivity concentration was measured per tissue volume (Becquerels / cubic centimeter) with decay correction and expressed as a percentage of injected dose per cubic centimeter (%ID / cc).

[0106] 6.16.Statistical analysis Quantitative data were expressed as mean ± standard deviation and analyzed using GraphPad Prism (version 9.5.1) software. Statistical analysis was performed using an unpaired two-tailed t-test, one-way ANOVA including Dunnett's multiple comparison test, or two-way ANOVA including Dunnett's multiple comparison test, and is indicated in the figure captions. P<0.05 was considered statistically significant, ns, p>0.05;*, p<0.05;**, p<0.01;***, p<0.001;****, p<0.0001.

[0107] Table 2

[0108] Table 3

Claims

1. A polypeptide that binds to human and mouse CD163, wherein the amino acid sequence of the polypeptide comprises a CDR1 region, a CDR2 region, and a CDR3 region, the CDR1, CDR2, and CDR3 regions being located within an immunoglobulin variable domain (IVD) as defined by Sequence ID No. 1, and selected from these CDR1, CDR2, and CDR3 regions determined by the Kabat, Chothia, Martin, or IMGT methods.

2. The polypeptide according to claim 1, wherein the CDR1 region is defined by sequence number 2, the CDR2 region is defined by sequence number 3, and the CDR3 region is defined by sequence number 4.

3. The polypeptide according to claim 1 or 2, wherein the polypeptide further comprises at least the FR1, FR2, FR3, or FR4 regions present in the IVD as defined by Sequence ID No.

1.

4. The polypeptide according to claim 3, wherein the FR1 region is defined by sequence number 5, the FR2 region is defined by sequence number 6, the FR3 region is defined by sequence number 7, and the FR4 region is defined by sequence number 8.

5. The polypeptide according to any one of claims 1 to 4, wherein the CDR region and / or FR region are humanized and / or the IVD is humanized.

6. The polypeptide according to any one of claims 1 to 5, further comprising a functional portion such as a His tag, a peptide motif recognized by a peptide ligase, or a detectable portion.

7. The polypeptide according to claim 6, wherein the detectable portion is bound to a specific site contained in the polypeptide.

8. An isolated nucleic acid encoding a polypeptide according to any one of claims 1 to 7.

9. A vector comprising the nucleic acid described in claim 8.

10. A host cell that expresses a polypeptide according to any one of claims 1 to 7, or a host cell comprising the nucleic acid according to claim 8, or a host cell comprising the vector according to claim 9.

11. The polypeptide is the polypeptide according to any one of claims 1 to 7, which is used for diagnostic purposes, surgical procedures, therapeutic monitoring, or as a contrast agent.

12. A method for producing a polypeptide according to any one of claims 1 to 7, wherein the method is: - Expressing the polypeptide in a host cell as described in claim 10, or synthesizing and producing the polypeptide; - Purifying the expressed or manufactured polypeptide; - If necessary, a detectable moiety is attached to the purified polypeptide, Methods that include...

13. The polypeptide according to any one of claims 1 to 5, wherein the polypeptide is conjugated with a preventive or therapeutic agent, a cytotoxic moiety or cytotoxic drug, an immunostimulant or immunosuppressant, a Toll-like receptor agonist, a photon absorber, and liposomes or nanoparticles.

14. The polypeptide according to claim 13, wherein the polypeptide is a polypeptide intended for use as a pharmaceutical, such as for use in combination with further anticancer agents as necessary in the treatment or inhibition of cancer.

15. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 7, or a polypeptide according to claim 13 or 14.

Images