Serum albumin binding nanobody compositions and methods of use thereof

Abstract

To provide compositions and methods for increasing the stability of recombinant nanobodies. Also provided are recombinant Nanobodies comprising an IL-2 polypeptide that binds serum albumin, and uses thereof for treating cancer.SOLUTION: There is provided a recombinant Nanobody comprising a human serum albumin (HSA) binding polypeptide and an IL-2 polypeptide, wherein the HSA binding polypeptide specifically binds to an HSA epitope selected from the group consisting of epitope 1, epitope 2, epitope 3 and epitope 4.SELECTED DRAWING: Figure 16

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is the benefit of U.S. Provisional Application No. 62 / 986,180, filed March 6, 2020. No. 6,239,693, which is expressly incorporated herein by reference in its entirety.

[0002] The present disclosure relates to human serum albumin-binding nanobodies. [Background technology]

[0003] Nanobodies (Nb) are heavy chain-only antibodies (V) from camelids. H H) derived from Nb is a small, intact, natural antigen-binding domain. Nb has good solubility, thermal stability, It is characterized by its tissue penetration and low toxicity to humans due to its high sequence similarity to human IgG2 / 3. However, its therapeutic potential is marked by a short T1 / 2 (usually less than a few hours). Its small size (approximately 1.5%), the absence of heavy-light chain pairing and glycosylation, 15 kDa), Nb is a recombinant protein for rapid bulk production from E. coli. The ability to bioengineer Nb can be achieved by synthesizing it as a DNA fragment. The ease of use of Nbs facilitates the development of multifunctional agents. Similar to IgG, the antigen binding specificity of Nbs is Mainly, the complementarity determining regions (CDRs, 3 and 6 in IgG) - CDR3 is the most variable, This is achieved by a hypervariable loop, which is a "fingerprint." The CDR loop consists of four It is presented by a robust tertiary core composed of conserved framework regions. With smaller paratopes, Nbs may have impaired affinity for antigen engagement. It has been hypothesized that Nb may be able to identify "secret" epitope-like kinases on antigens. The cavities that IgG cannot access due to steric hindrance are accessible. Several pilot structural studies indicate that binding to these epitopes may be possible. The small size and "convex" shape make it easy to insert the CDR loop(s) into the groove. This is achieved by both the "shape" and "shape."

[0004] Although many Nbs have been developed for specific targets, the field is still in the process of developing high performance Nb-based drugs for drug delivery. The availability of quality and versatile Nb agents remains limited. Qualitative, cross-species binding, and bioengineering robustness are likely to be important. However, whether other biophysical, physicochemical, and structural features are important, and if so, It remains unclear how important these factors are. Formulations often have a changing therapeutic window, allowing for more effective drug delivery while reducing side effects. To maximize efficacy, a sustainable and fine-tuned individual half-life is most desirable. Systematic and comprehensive evaluation of these factors will be useful in designing next-generation drugs for precision medicine. stand.

[0005] Human serum albumin (HSA) is the most stable serum protein, with a half-life of approximately 3 weeks. Unlike small molecules that are primarily removed by glomerular filtration, HSA (67 kD a) cannot be directly removed by the kidney. If internalization continues, HSA becomes HSA forms a stable complex with cellular FcRn, a co-receptor for Fc They can be efficiently recycled by Rn-mediated endocytosis, which This has been shown to prevent the rapid lysosomal degradation common to large serum proteins. The location of HSA-FcRn interaction within the endosome, where the complex is recycled, Although the specific concentration(s) remains unclear, the tolerance of the acidic environment of the endosome is unclear. A can be a prerequisite for successful recycling and stabilization of A.

[0006] However, the interaction of HSA with Nb has not yet been characterized, and cytokine therapy There is a continuing need for effective nanobody therapeutics in many areas, including Interleukin-2 (IL-2) is an important component of immune regulation. is a central immune cytokine important in the regulation of homeostasis and T cell activity. By controlling the proliferation of cytotoxic T cells, IL-2 upregulates the immune response and contributes to tumor progression. Proleukin (aldesleukin, Novartis) is a human interleukin inhibitor. It is a recombinant form of IL-2 and was the first approved cancer immunotherapy agent. IL-2 is used in the treatment of advanced melanoma and metastatic renal cell carcinoma. Treatment can, in some cases, result in up to 25 years without recurrence in patients with metastatic tumors. It has been reported that IL-2 therapy results in a complete remission in approximately 10% of patients. 70% have complete tumor regression. However, the main drawback of IL-2 is its insufficient These include poor pharmacokinetics, poor drug efficacy, and high toxicity. 5 kDa), IL-2 is rapidly removed from the circulation by glomerular filtration and is administered to humans. After administration, it has a median half-life of less than 30 minutes. Consequently, high doses and repeated administration (usually 7 hours per dose) is often required, which can lead to serious toxicity and vascular leakage. may result in side effects including steroid syndrome.

[0007] For drug delivery, the availability of high quality and multifunctional agents is limited. Qualitative, cross-species binding, and bioengineering robustness are likely to be important. However, whether other biophysical, physicochemical, and structural features are important, and if so, It remains unclear how important these factors are. Formulations often vary significantly in their therapeutic window, reducing side effects. However, to maximize the potential of drugs, fine-tuned, sustainable, personalized A reduced life is most desirable.

[0008] Therefore, compositions to maximize the potency and half-life of therapeutic compositions such as IL-2 are The compositions and methods disclosed herein meet these and other needs. Respond to the need. Summary of the Invention

[0009] In some embodiments, a human serum albumin (HSA) binding polypeptide and an IL-2 polypeptide are Disclosed herein are recombinant Nanobodies comprising the polypeptide. The HSA-binding polypeptide comprises epitope 1, epitope 2, epitope 3, and epitope specifically binds to an HSA epitope selected from the group consisting of epitope 4, Peptide 1 is composed of amino acid residues 298 to 307, 311, 332 to 341, and 37 of SEQ ID NO: 1. 1 to 386 of SEQ ID NO: 1, and epitope 2 comprises amino acid residues 5 to 13, 62 to 67, and 93-99 and 228-266 of SEQ ID NO: 1, and epitope 3 comprises amino acid residues 2 Epitope 4 comprises amino acid residues 26 to 230 and 298 to 337 of SEQ ID NO: 1. 33-38, and 111-145. In some embodiments, the HSA-binding polypeptide The peptide has an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. Nanobodies comprising human serum albumin (HSA) binding polypeptides are also included in the present invention. As disclosed herein, the HSA-binding polypeptide comprises a complementarity-determining region (CDR) 3, R3 is an amino acid sequence having at least 50% similarity to any of SEQ ID NOs: 10 to 100. In some embodiments, the recombinant Nanobodies disclosed herein comprise the sequence In some embodiments, the nanobody is an IL-2 polypeptide. Such nanobodies surprisingly increase the stability of the IL-2 polypeptide. Although nanobodies can improve the IL-2 polypeptide and IL-2 receptor The affinity with

[0010] In the subject being treated, administration of the recombinant Nanobodies disclosed herein It is shown herein that the use of agonists in the treatment of rheumatoid arthritis can reduce tumor volume and improve subject survival. Thus, in some embodiments, the present invention provides a method for the treatment of cancer in a subject in need thereof. A method of treatment comprising administering a human serum albumin (HSA) binding polypeptide and an IL-2 polypeptide. the method comprising administering a therapeutically effective amount of a recombinant nanobody comprising a peptide. Disclosed herein. [Brief explanation of the drawings]

[0011] [Figure 1a] Identification and characterization of HSA-Nb Figure 1a shows the schematic structure and amino acid composition of HSA-Nb. [Figure 1b] Figure 1b shows the identification and characterization of HSA-Nbs. Figure 1b shows circos and logo plots showing CDR3 diversity. [Figure 1c] Figure 1c shows the identification and characterization of HSA-Nbs. Figure 1c shows the ELISA heat map of albumin cross-species binding of 89 different Nbs. [Figure 1d] Figure 1d shows the identification and characterization of HSA-Nbs. Figure 1d shows the KD (including K and Kd) of three representative Nbs by surface plasmon resonance. [Figure 1e] Figure 1e shows the correlation of ELISA OD and KD affinity by SPR. [Figure 1f] Figure 1f shows the identification and characterization of HSA-Nb. Figure 1f shows the bead binding assay of HSA-Nb13 complexes at various Nb concentrations. [Figure 1g] Figure 1g shows the identification and characterization of HSA-Nbs. The variation in Nb cross-reactivity by pull-down assay is shown in Figure 1g. Three representative Nbs (Nb3, Nb6, and Nb13) were immunoprecipitated with affinity resins coupled to albumin from different species, including human, monkey, mouse, bovine, and llama. [Figure 1h] Figure 1h shows the identification and characterization of HSA-Nbs. Figure 1h shows the heat map of Nb thermal stability by differential scanning fluorimetry. [Figure 2a] Figure 2a shows the structural docking and cross-linking of HSA-Nb complexes. Figure 2a shows the major HSA epitopes identified by structural docking. [Figure 2b] Figure 2b shows a pictorial representation of HSA and its four major epitopes. [Figure 2c] Figure 2c shows the structural docking and cross-linking of the HSA-Nb complex. Figure 2c shows the electrostatic surface co-localization of HSA and the epitope. Figure 2d shows the relative abundance of the epitope based on the cross-linking model of the HSA-Nb complex. [Figure 2d]Figure 2d shows the structural docking and cross-linking of the HSA-Nb complex. Figure 2d shows the relative abundance of epitopes based on the cross-linking model of the HSA-Nb complex. [Figure 3a] Figure 3 shows the overall structural characterization of the tetrameric HSA-Nb complex. Figure 3a shows size-exclusion chromatography (SEC) analysis of the reconstituted tetrameric complex composed of Nb13, Nb29, Nb80, and HSA. [Figure 3b] Figure 3b shows the overall structural characterization of the tetrameric HSA-Nb complex. Figure 3b shows a negative stain image of the complex. [Figure 3c] Figure 3c shows the integrated structural characterization of the tetrameric HSA-Nb complex. Figure 3c shows the hybrid structural model (best score) of the complex overlaid with negative stain EM. [Figure 3d] Figure 3d shows the X-ray structure of the HSA-FcRn complex. [Figure 3e] Figures 3e-3g show a close-up of the interface and cross-linking constraints in the model. [Figure 3f] Figures 3e-3g show a close-up of the interface and cross-linking constraints in the model. [Figure 3g] Figures 3e-3g show a close-up of the interface and cross-linking constraints in the model. [Figure 3h] Figure 3h shows the integrated structural characterization of the tetrameric HSA-Nb complex. Figure 3h lists the cross-linking satisfaction scores for the model. [Figure 3i] Figure 3i-3k shows the complete structural characterization of the tetrameric HSA-Nb complex. Figure 3i-3k shows the site-directed mutagenesis analysis of two charged residues (K383 and D400) that cross and form stable salt bridges with corresponding residues on Nb80. [Figure 3j]Figure 3i-3k shows the complete structural characterization of the tetrameric HSA-Nb complex. Figure 3i-3k shows the site-directed mutagenesis analysis of two charged residues (K383 and D400) that cross and form stable salt bridges with corresponding residues on Nb80. [Figure 3k] Figure 3i-3k shows the complete structural characterization of the tetrameric HSA-Nb complex. Figure 3i-3k shows the site-directed mutagenesis analysis of two charged residues (K383 and D400) that cross and form stable salt bridges with corresponding residues on Nb80. [Figure 4a] Figure 4a shows a schematic of the MS-based assay for multiplexed PK measurements. [Figure 4b] High-throughput Nb pharmacokinetics in a humanized mouse model is shown. Figure 4b shows the PK analysis of 22 Nbs in a humanized mouse model. A single bolus of an equimolar mixture of 22 Nbs, including 20 HSA-Nbs and two non-binder controls, was administered intravenously to three animals. Serum samples were collected at different time points and proteolyzed. The resulting peptides were separated by LC, and these molecules and their fragment ion products were quantified using an Orbitrap QE HFX mass spectrometer. Each data point represents the median Nb amount from three different animals. The data were then fitted to a biphasic model to calculate the half-lives of the Nbs. [Figure 4c] Figure 4c shows high-throughput Nb pharmacokinetics in a humanized mouse model. Figure 4c shows a heatmap summary of PK distribution and elimination. [Figure 4d] Figure 4d shows the correlation analysis of Nb PK and disposition in a humanized mouse model. [Figure 5a] Figure 5 shows the development of a new class of Nb-fusion cytokines, the duraleukins. Figure 5a shows the schematic design of duraleukins. [Figure 5b] Figure 5b shows the development of a new class of Nb-fusion cytokines, the duraleukins. Figure 5b shows the protocol for producing duraleukins. [Figure 5c]Figure 5c shows the development of a new class of Nb-fusion cytokine, duraleukin. Figure 5c shows the thermal stability of duraleukin by differential scanning fluorimetry. [Figure 5d] Figure 5d shows the development of a new class of Nb fusion cytokines, duraleukins. Figure 5d shows an in vitro CTL-2 cell proliferation assay of duraleukins and IL-2. [Figure 5e] Figure 5 shows the development of a new class of Nb fusion cytokine, duraleukin. Figure 5e shows the in vitro stability of duraleukin in the presence of human serum. [Figure 5f] Figure 5f shows the development of a new class of Nb fusion cytokines, duraleukins. Figure 5f shows the KD affinity measurements of DL80 for HSA binding. Ka(1 / Ms)=1.66e5; Kd(1 / s)=2.75e-5; KD=1.66pM. [Figure 5g] We demonstrate the development of a new class of Nb-fusion cytokine, duraleukin. Figure 5g shows the bead-binding assay (pH dependence) of DL80 for HSA binding. DL80 was pulled down using HSA-conjugated agarose resin at different pH buffers (pH 2 to pH 12). The relative intensity of affinity-isolated DL80 protein on SDS-PAGE was quantified using Image J. [Figure 6] Figure 6 shows the in vivo efficacy of Duraleukin in a melanoma mouse model. Figure 6a shows the tumor growth curve. C57BL / 6J mice bearing subcutaneous B16F10 tumors were treated with a combination of TA99 and Duraleukin or hIL-2 (n=8) at different doses / intervals. PBS treatment was used as a control. Figure 6b shows the animal survival curve after treatment. Figure 6c shows flow cytometry analysis of tumor-infiltrating immune cells. [Figure 7a] Surface plasmon resonance (SPR) measurements of exemplary HSA Nbs (100 and 113 counted Nbs) are shown. Ka (association rate), Kd (dissociation rate), and affinity KD were documented. Figure 7a: Nb77: Ka(1 / Ms) = 5.68e6; Kd(1 / s) = 7.68e-5; KD = 1.35 pM. [Figure 7b] Surface plasmon resonance (SPR) measurements of exemplary HSA Nbs (100 and 113 counted Nbs) are shown. Ka (association rate), Kd (dissociation rate), and affinity KD were documented. Figure 7b: Nb29: Ka(1 / Ms) = 9.73e6; Kd(1 / s) = 1.9e-3; KD = 1.22 nM. [Figure 7c] Surface plasmon resonance (SPR) measurements of exemplary HSA Nbs (counted Nbs 100 and 113) are shown. Ka (association rate), Kd (dissociation rate), and affinity KD were documented. Figure 7c: Nb13: Ka(1 / Ms) = 2.85e5; Kd(1 / s) = 5.73e-5; KD = 201 pM. [Figure 7d] Surface plasmon resonance (SPR) measurements of exemplary HSA Nbs (counted Nbs 100 and 113) are shown. Ka (association rate), Kd (dissociation rate), and affinity KD were documented. Figure 7d: Nb158: Ka(1 / Ms) = 1.04e5; Kd(1 / s) = 3.51e-5; KD = 339 pM. [Figure 7e] Surface plasmon resonance (SPR) measurements of exemplary HSA Nbs (counted Nbs 100 and 113) are shown. Ka (association rate), Kd (dissociation rate), and affinity KD were documented. Figure 7e: Nb80: Ka(1 / Ms) = 1.66e5; Kd(1 / s) = 2.75e-5; KD = 166 pM. [Figure 7f] Surface plasmon resonance (SPR) measurements of exemplary HSA Nbs (100 and 113 counted Nbs) are shown. Ka (association rate), Kd (dissociation rate), and affinity KD were documented. Figure 7f: Nb26: Ka(1 / Ms) = 3.38e5; Kd(1 / s) = 6.97e-5; KD = 206 pM. [Figure 7g] Surface plasmon resonance (SPR) measurements of exemplary HSA Nbs (100 and 113 counted Nbs) are shown. Ka (association rate), Kd (dissociation rate), and affinity KD were documented. Figure 7g: Nb69: Ka(1 / Ms) = 2.7e5; Kd(1 / s) = 1.97e-4; KD = 730 pM. [Figure 7h]Surface plasmon resonance (SPR) measurements of exemplary HSA Nbs (100 and 113 counted Nbs) are shown. Ka (association rate), Kd (dissociation rate), and affinity KD were documented. Figure 7h: Nb78: Ka(1 / Ms) = 2.34e5; Kd(1 / s) = 3.99e-5; KD = 170 pM. [Figure 7i] Surface plasmon resonance (SPR) measurements of exemplary HSA Nbs (100 and 113 counted Nbs) are shown. Ka (association rate), Kd (dissociation rate), and affinity KD were documented. Figure 7i: Nb85: Ka(1 / Ms) = 1.11e6; Kd(1 / s) = 5.04e-4; KD = 454 pM. [Figure 7j] Surface plasmon resonance (SPR) measurements of exemplary HSA Nbs (counted Nbs 100 and 113) are shown. Ka (association rate), Kd (dissociation rate), and affinity KD were documented. Figure 7j: Nb129: Ka(1 / Ms) = 1.03e6; Kd(1 / s) = 1.14e-4; KD = 108 pM. [Figure 7k] Surface plasmon resonance (SPR) measurements of exemplary HSA Nbs (counted Nbs 100 and 113) are shown. Ka (association rate), Kd (dissociation rate), and affinity KD were documented. Figure 7k: Nb132: Ka(1 / Ms) = 3.03e5; Kd(1 / s) = 5.6e-5; KD = 185 pM. [Figure 7l] Surface plasmon resonance (SPR) measurements of exemplary HSA Nbs (counted Nbs 100 and 113) are shown. Ka (association rate), Kd (dissociation rate), and affinity KD were documented. Figure 7l: Nb93: Ka(1 / Ms) = 1.37e6; Kd(1 / s) = 1.36e-4; KD = 99 pM. [Figure 7m] Surface plasmon resonance (SPR) measurements of exemplary HSA Nbs (100 and 113 counted) are shown. Ka (association rate), Kd (dissociation rate), and affinity KD were documented. Figure 7m: Nb81: Ka(1 / Ms) = 8.82e4; Kd(1 / s) = 1.42e-5; KD = 161 pM. [Figure 7n]Surface plasmon resonance (SPR) measurements of exemplary HSA Nbs (100 and 113 counted Nbs) are shown. Ka (association rate), Kd (dissociation rate), and affinity KD were documented. Figure 7n: Nb64: Ka(1 / Ms) = 3.83e5; Kd(1 / s) = 2.06e-4; KD = 538 pM. [Figure 7o] Surface plasmon resonance (SPR) measurements of exemplary HSA Nbs (100 and 113 counted Nbs) are shown. Ka (association rate), Kd (dissociation rate), and affinity KD were documented. Figure 7o: Nb75: Ka(1 / Ms) = 1.03e6; Kd(1 / s) = 1.02e-4; KD = 100 pM. [Figure 7p] Surface plasmon resonance (SPR) measurements of exemplary HSA Nbs (counted Nbs 100 and 113) are shown. Ka (association rate), Kd (dissociation rate), and affinity KD were documented. Figure 7p: Nb 126: Ka (1 / Ms) = 6.92e6; Kd (1 / s) = 1.74e-3; KD = 251 pM. [Figure 7q] Surface plasmon resonance (SPR) measurements of exemplary HSA Nbs (100 and 113 counted Nbs) are shown. Ka (association rate), Kd (dissociation rate), and affinity KD were documented. Figure 7q: Nb68: Ka(1 / Ms) = 2.06e6; Kd(1 / s) = 2.9e-2; KD = 14 nM. [Figure 7r] Surface plasmon resonance (SPR) measurements of exemplary HSA Nbs (100 and 113 counted Nbs) are shown. Ka (association rate), Kd (dissociation rate), and affinity KD were documented. Figure 7r: Nb98: Ka(1 / Ms) = 1.16e6; Kd(1 / s) = 2.7e-2; KD = 23 nM. [Figure 8a] The thermal stability melting temperatures (Tm) of representative HSA Nb were measured by differential scanning fluorimetry (DSF). Figure 8a: Nb100, Tm = 63.37 °C. [Figure 8b] The thermal stability melting temperatures (Tm) of representative HSA Nbs are shown. Measurements were performed by differential scanning fluorimetry (DSF). Figure 8b: Nb113, Tm = 64.12 °C. [Figure 8c]The thermal stability melting temperatures (Tm) of representative HSA Nbs are shown. Measurements were performed by differential scanning fluorimetry (DSF). Figure 8c: Nb68, Tm = 44 °C. [Figure 8d] The thermal stability melting temperatures (Tm) of representative HSA Nbs are shown. Measurements were performed by differential scanning fluorimetry (DSF). Figure 8d: Nb69, Tm = 38.02 °C. [Figure 8e] The thermal stability melting temperatures (Tm) of representative HSA Nbs are shown. Measurements were performed by differential scanning fluorimetry (DSF). Figure 8e: Nb125, Tm = 47.07 °C. [Figure 8f] The thermal stability melting temperatures (Tm) of representative HSA Nbs are shown. Measurements were performed by differential scanning fluorimetry (DSF). Figure 8f: Nb126, Tm = 45.89 °C. [Figure 8g] The thermal stability melting temperatures (Tm) of representative HSA Nb were measured by differential scanning fluorimetry (DSF). Figure 8g: Nb75, Tm = 49.34 °C. [Figure 8h] The thermal stability melting temperatures (Tm) of representative HSA Nbs are shown. Measurements were performed by differential scanning fluorimetry (DSF). Figure 8h: Nb77, Tm = 54.29 °C. [Figure 8i] The thermal stability melting temperatures (Tm) of representative HSA Nbs are shown. Measurements were performed by differential scanning fluorometry (DSF). Figure 8i: Nb129, Tm = 44.12 °C; Figure 8j: Nb13, Tm = 53.42 °C. [Figure 8j] The thermal stability melting temperatures (Tm) of representative HSA Nbs are shown. Measurements were performed by differential scanning fluorimetry (DSF). Figure 8j: Nb13, Tm = 53.42 °C. [Figure 8k] The thermal stability melting temperatures (Tm) of representative HSA Nbs are shown. Measurements were performed by differential scanning fluorimetry (DSF). Figure 8k: Nb78, Tm = 70.52 °C. [Figure 8l] The thermal stability melting temperatures (Tm) of representative HSA Nbs are shown. Measurements were performed by differential scanning fluorometry (DSF). Figure 8l: Nb80, Tm = 50.82 °C; Figure 8m: Nb132, Tm = 54 °C. [Figure 8m]The thermal stability melting temperatures (Tm) of representative HSA Nbs are shown. Measurements were performed by differential scanning fluorimetry (DSF). Figure 8m: Nb132: Tm = 54°C. [Figure 8n] The thermal stability melting temperatures (Tm) of representative HSA Nbs are shown. Measurements were performed by differential scanning fluorimetry (DSF). Figure 8n: Nb158, Tm = 59.97 °C; Figure 8o: Nb81, Tm = 42.68 °C. [Figure 8o] The thermal stability melting temperatures (Tm) of representative HSA Nbs are shown. Measurements were performed by differential scanning fluorimetry (DSF). Figure 8o: Nb81, Tm = 42.68 °C. [Figure 8p] The thermal stability melting temperatures (Tm) of representative HSA Nbs are shown. Measurements were performed by differential scanning fluorimetry (DSF). Figure 8p: Nb85, Tm = 53.17 °C. [Figure 8q] The thermal stability melting temperatures (Tm) of representative HSA Nbs are shown. Measurements were performed by differential scanning fluorimetry (DSF). Figure 8p: Nb29, Tm = 42.16 °C. [Figure 8r] The thermal stability melting temperatures (Tm) of representative HSA Nbs are shown. Measurements were performed by differential scanning fluorimetry (DSF). Figure 8r: Nb64, Tm = 45.17 °C. [Figure 8s] The thermal stability melting temperatures (Tm) of representative HSA Nbs are shown. Measurements were performed by differential scanning fluorimetry (DSF). Figure 8s: Nb93, Tm = 61.18 °C. [Figure 8t] The thermal stability melting temperatures (Tm) of representative HSA Nbs are shown. Measurements were performed by differential scanning fluorimetry (DSF). Figure 8t: Nb98, Tm = 64.91 °C. [Figure 9] A representative cross-linking model of HSA-Nb complexes is shown. [Figure 10]Figure 10 shows the purification and negative staining electron microscopy (EM) particle selection of tetrameric Nb-HSA complexes. Figure 10a shows size-exclusion chromatography and SDS-PAGE analysis of the reconstituted tetrameric HSA-Nb complexes. Figures 10b-10d show EM images of HSA-Nb complexes with or without MBP (maltose-binding protein) tag and HSA. Figure 10b shows the Nb80-Nb13-Nb29-HSA complex, Figure 10c shows the MBP-Nb80-Nb13-Nb29-MBP-HSA complex, and Figure 10d shows HSA. [Figure 11] Figure 11 shows a schematic diagram of a novel fragment-ion-based method for multiplexed quantification of Nb PK. Figure 11a is a schematic diagram of the digestion and LC / MS analysis steps. Figure 11b is a chart showing spike-in nanobodies. The black square indicates Nb1-MS1, the black circle indicates Nb2-MS1, the white square indicates Nb3-MS1, and the white circle indicates Nb4-MS1. Figure 11c is a chart showing the steps. Figure 11d is a schematic diagram of the isolation, quadrupole, fragmentation, HCD cell, and quantification steps. Figure 11e is a chart showing spike-in nanobodies. The black circle indicates Nb1-PRM, the black square indicates Nb1-MS1, the black inverted triangle indicates Nb2-PRM, the black circle indicates Nb2-MS1, the white circle indicates Nb3-PRM, the white square indicates Nb3-MS1, the white equilateral triangle indicates Nb4-PRM, and the white inverted triangle indicates Nb4-MS1. [Figure 12] The precision (median coefficient of variation or CV) of Nb PK measurements is shown for three different mice (12a (mouse 1)), 12b (mouse 2), and 12c (mouse 3)). [Figure 13]Figure 13 shows validation of the B6.Cg-Tg(FCGRT)32Dcr Albem12Mvw Fcgrttm1Dcr / MvwJ mouse model. Figure 13a shows validation of Album12Mvw homozygous mice by DNA electrophoresis. Figure 13b shows Fcgrttm1Dcr homozygous mice by Sanger sequencing and SDS-PAGE analysis. Figure 13c shows the pharmacokinetics (PK) of HSA in the mouse model. Half-life (slow) = 8.08 (days); half-life (fast) = 0.53 (days); R2 = 0.9547. Half-life (slow) = 8.08 (days); half-life (fast) = 0.53 (days); R2 = 0.9547. [Figure 14] Serum protein quantification in a humanized mouse model after administration of HSA and Nb. A significant increase in mouse IgG was confirmed on day 7. [Figure 15] Pharmacokinetic analysis of IL-2 (open circles) and DL80 (closed squares) in a wild-type C57BL / 6J mouse model. The half-life (slow) for IL-2 is 0.0086, and the half-life (fast) for IL-2 is approximately 2.7e-0.06. The half-life (slow) for duraleukin is 0.40, and the half-life (fast) for duraleukin is 0.039. [Figure 16] Four clusters on HSA where Nb-binding epitopes are located are shown: Cluster 1 (Nb80): 298-307, 311, 332-341, and 371-386 (aa); Cluster 2 (Nb13): 5-13, 62-67, 93-99, and 228-266 (aa); Cluster 3: 226-230 and 298-337; Cluster 4 (Nb29): 33-38 and 111-145. DETAILED DESCRIPTION OF THE INVENTION

[0012] Recently, the use of immunization of camelids and an integrated proteomics pipeline has led to A large repertoire of nanobodies (Nb) for HSA binding has been identified. By using a multidisciplinary approach, a cohort of high-quality HSA-Nbs was developed, In some embodiments, some HSA-Nbs were used in cytokine In combination with human IL-2, it can be used to treat melanoma in mouse models. They have developed highly stable compositions collectively known as "lareukins."

[0013] Duraleukin has been described by four notable features:

[0014] First, it is easy to produce and manufacture. The Nb fusion product of IL-2 (duraleukin) is As used herein, highly purified and interferon-like proteins are produced in bacteria, e.g., E. coli cells. It has been shown that it can be readily produced in bulk as a clean and functional protein. High doses of IL-2 (Proleukin (Aldesleukin, Novartis)) Note that this is a benefit of the drug's efficacy. Genome Sciences) production is primarily limited in more expensive mammalian cells. This limits its practical utility.

[0015] Second, it is easy to bioengineer. The ring is simple and convenient. In one example, another polypeptide drug is fused at the C-terminus. This allows the creation of "trifunctional drugs," which is a promising area for bioengineering. This can be very difficult, says Albroukin (Human Genome Sciences ) shows a stark contrast.

[0016] Third, significant flexibility for optimized / tailored development and clinical use. Using a humanized albumin mouse model, different It is shown herein that different albumin Nbs have different in vivo pharmacokinetics (PK). This means that different duraleukins with different HSAs have unique PK. This has been shown to be effective in treating the disease, and by utilizing this, it is possible to The main problem facing the company (Kim, Novartis) is optimizing drug efficacy and Side effects can be minimized

[0017] Fourth, it has excellent potency. The recombinant polypeptide has excellent resistance to serum protease activity. It inherits the remarkable physicochemical properties of Nb, such as thermal stability and resistance (almost immunity) Albroukin (Human Genome Sciences) (30kDa Because duraleukin is three times smaller than leukocytes (90 kDa vs 90 kDa), it is structurally normal and It retains the full functionality of the carrier protein IL-2. It has limited drug uptake capacity. Due to its small size, duraleukin is a maximally efficient drug delivery system. The molar concentration of Nb(15kJ / 2000kcal) in intact small domains can be easily reached. Fusion with Da) inhibits the interaction of IL-2 with its receptor, the FcRn complex. Although it does not significantly affect the Large recombinant proteins such as IL-2 receptor complexes are particularly bulky and large. When the compound is mixed with the hydroxyl group, it may be affected by steric hindrance in the structure.

[0018] Therefore, the stability and / or efficacy of IL-2 in the treatment of diseases (e.g., cancer) may be improved. A composition for improving efficacy, the composition comprising a human serum albumin (HSA)-binding polynucleotide. The present invention provides the above composition, which is a recombinant nanobody comprising a peptide and an IL-2 polypeptide. In some embodiments, the recombinant Nanobody is a natural or recombinant Nanobody. Increases the half-life of IL-2 compared to IL-2 polypeptide. Human serum albumin Administration of a recombinant nanobody comprising a (HSA)-binding polypeptide and an IL-2 polypeptide. This surprisingly reduces tumor volume and improves survival in treated subjects.

[0019] Terms used throughout this application should be interpreted in the ordinary and typical sense of those skilled in the art. However, applicants wish to give the following terms the specific definitions set forth below. do.

[0020] term As used in this specification and claims, the singular forms "a," "an," and "the" " includes plural referents unless the context clearly dictates otherwise. For example, the term " A "cell" includes a plurality of cells, including mixtures thereof.

[0021] As used herein, "about" refers to a measurable value such as an amount, percentage, etc. The term encompasses variations of ±20%, ±10%, ±5%, or ±1% from the measurable value. This means that...

[0022] "Administration" or "administering" to a subject refers to introducing or delivering an agent to a subject. Administration can be by any suitable route, including oral, intravenous, intraperitoneal, nasal, inhalation, etc. Administration can be by any route. Administration can be by self-administration or administration by another person. do.

[0023] The term "antibody / antibodies" is used herein in a broad sense. Antibodies that can be used include polyclonal, monoclonal, and bispecific antibodies. In addition to the immunoglobulin molecules, the term "antibody" also includes those immunoglobulins fragments or polymers of immunoglobulin molecules, as well as immunoglobulin molecules or fragments thereof Antibodies are human or humanized fragments of the antibody. Antibodies usually consist of two identical light (L) chains and A heterotetrameric glycoprotein of approximately 150,000 daltons consisting of two identical heavy (H) chains. Each heavy chain contains a variable domain (V H ), followed by several Each light chain has a variable domain (V L ) and a stationary drive It has a main.

[0024] Antibodies may be identified using the in vitro assays described herein or by similar methods. , can be tested for their desired activity, and then their in vivo therapeutic and and / or prophylactic activity is tested according to known clinical trial methods. There are three major classes: IgA, IgD, IgE, IgG, and IgM. Some are further subdivided into subclasses (isotypes), e.g., IgG-1, IgG-2, I The antibodies can be divided into IgA-1 and IgA-2; IgG-3 and IgG-4; and IgA-1 and IgA-2. The heavy chain constants corresponding to the different classes of immunoglobulins will be recognized. The normal domains are called alpha, delta, epsilon, gamma, and mu, respectively. do.

[0025] The terms "antigenic determinant" and "epitope" may also be used interchangeably herein. and the position on the antigen or target recognized by the antigen-binding molecule (such as the Nanobody of the invention). An epitope is a continuous amino acid sequence juxtaposed by tertiary folding of a protein. Epitopes can be formed both from amino acids ("linear epitopes") or non-contiguous amino acids. The latter epitopes are defined herein as being made up of at least some non-contiguous amino acids. In literature, these are referred to as "conformational epitopes." Epitopes are usually at least three, more An epitope usually contains at least 5 or 8-10 amino acids in a unique spatial structure. Methods for measuring the spatial structure of, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance For example, Epitope Mapping Protocols in Methods in Molecular Biology,Vol.66,Glen See E. Morris, Ed. (1996).

[0026] The terms "antigen-binding site," "binding site," and "binding domain" refer to an antigenic determinant or A specific element, portion, or antigen of a polypeptide, such as a nanobody, that binds to an epitope. It means an amino acid residue.

[0027] The term "cancer" refers to a relatively autonomous growth that results in a significant loss of control over cell proliferation (i.e. refers to cells that exhibit an abnormal growth phenotype characterized by abnormal cell division (i.e., uncontrolled cell division) Cancer cells can be malignant or benign. Examples of various cancers include melanoma, Breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, These include, but are not limited to, lymphoma, leukemia, lung cancer, and the like. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is renal cancer.

[0028] The terms "CDR" and "complementarity determining region" are used interchangeably and refer to regions involved in binding to an antigen. CDRs refer to the portions of the variable chains of an antibody that bind to the CDRs. Thus, CDRs are part of the "antigen-binding site." or "antigen-binding site". In some embodiments, nanobodies are It contains three CDRs that together form the antigen-binding site.

[0029] As used herein, the term "comprising" and variations thereof ", "including" and variations thereof, and "comprising" and "including" are non-limiting terms. Although the term "(ng)" has been used herein to describe various embodiments, The terms "consisting essentially of" and "consisting of" provide more specific embodiments. Instead of "comprising" and "including" may be used and disclosed.

[0030] "Composition" means any agent that has a beneficial biological effect. The results may include both therapeutic effects, such as treatment of a disorder or other undesirable physiological condition, and non-therapeutic effects, such as This includes both preventative effects, such as prevention of a disorder or other undesirable physiological condition. These terms also include, but are not limited to, bacteria, vectors, polynucleotides, cells, Salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs pharmaceutically acceptable, pharmacologically active derivatives of the beneficial agents specifically mentioned herein, such as When the term "composition" is used, then or a particular composition When a compound is specifically identified, the term includes the compound itself as well as any pharmaceutically acceptable salt thereof. pharmacologically active vectors, polynucleotides, salts, esters, amides, enhancers, co- It is understood that the term "antibody" includes conjugates, active metabolites, isomers, fragments, analogs, and the like. In some embodiments, the compositions disclosed herein contain human serum albumin. Recombinant polypeptides, including heparin (HSA) binding polypeptides and IL-2 polypeptides Includes:

[0031] "Effective amount" includes, but is not limited to, a dose effective to treat a medical condition or disease (e.g., cancer) includes an amount capable of reducing, reversing, mitigating, preventing, or diagnosing the symptoms or signs of Unless otherwise indicated, expressly or by context, an "effective amount" is an amount that alleviates a condition. The severity of the disease or disorder and whether the treatment is effective or ineffective will determine the severity of the disease or disorder. The ability to prevent, treat, or alleviate a disease or disorder may be determined by biomarkers or clinical The floor parameters can be measured without implying any limitations. In some embodiments, the term "effective amount of a recombinant Nanobody" refers to an amount of a recombinant Nanobody that is effective to prevent, treat, or or an amount of recombinant Nanobody sufficient to alleviate the symptoms.

[0032] A "fragment" or "functional fragment" may or may not be linked to other sequences. Regardless of the insertion, deletion, substitution, or other selection of specific regions or specific amino acid residues. provided that the activity of the fragment is comparable to that of the unmodified peptide or protein. These modifications are not significantly altered or worsened compared to the quality of the Additional properties, e.g., removal or addition of disulfide bond-capable amino acids, increased biological lifespan, In either case, the functional fragment can be a fragment of H It must have bioactive properties, such as binding to SA and / or ameliorating cancer. do not have.

[0033] The "half-life" of an amino acid sequence, compound, or polypeptide of the invention is generally determined, e.g., Decomposition of sequences or compounds and / or recombination of sequences or compounds by natural mechanisms The clearance or separation of the amino acid sequence, compound, or polypeptide It can be defined as the time it takes for the serum concentration to decrease by 50% in vivo. The in vivo half-life of the nanobody, amino acid sequence, compound, or polypeptide may be determined by the drug. These can be measured in any known manner, such as by kinetic analysis. Kenneth, A et al., Chemical Stability of Pharmaceuticals:A Handbook for Pharmaci sts;Peters et al., Pharmacokinete analysis s:A Practical Approach(1996);Marcel Dekk Published by er, "Pharmacokinetics", M Gibaldi & D Perron (revised 2nd edition) (1982).

[0034] The terms "identity" or "homology" refer to sequences that are aligned, introducing gaps and, if necessary, If so, after achieving maximum percent identity for the entire sequence, and As part of the comparison, the bases or residues of the corresponding sequences are compared, without taking into account any conservative substitutions. The percentage of nucleotide bases or amino acid residues in a candidate sequence that are identical to the base The percentage of a specific sequence (e.g., 80%, 85%, 90%, or 95%) of a "sequence identity" between a polynucleotide or The polynucleotide region (or polypeptide or polypeptide region) is aligned. When the two sequences are compared, the ratio of bases (or amino acids) is the same. This alignment and the percentage homology or sequence identity Cents may be measured using software programs known in the art. Such alignments can be performed using, for example, the Align program (DNAsta Need for leman et al. (1970) J.Mol.Biol.48:443-453 The method can be used to

[0035] As used herein, the term "increased" or "increase" generally refers to a statistical For the avoidance of doubt, "increased" means an increase in the amount of at least a 10% increase, for example, at least about 20%, or less, compared to the level at least about 30%, or at least about 40%, or at least about 50%, or less At least about 60%, or at least about 70%, or at least about 80%, or at least an increase of approximately 90% or up to 100% (inclusive) or compared to the reference level Any increase between 10 and 100% or at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or more means.

[0036] As used herein, the terms "nanobody," "V H H," "V H H antibody fragment "Single domain antibody" and "single domain antibody" are used interchangeably and are incorporated by reference in their entirety. and those derived from the family Camelidae, which are described in PCT Publication No. WO94 / 04678. The single heavy chain variable region of antibodies typically found in camelids, without any light chains, such as It means domain.

[0037] As used herein, "operably linked" refers to polynucleotides within a single polypeptide chain. refers to an arrangement of polypeptide segments, where the individual polypeptide segments are non- Specifically, proteins, fragments thereof, binding peptides, and / or signal peptides. The term "operably linked" refers to a single polypeptide or fragment thereof. Within a protein, there are different individual polypeptides with no intervening amino acids between the different segments. In addition, individual polypeptides may be fused together via a "linker" that includes one or more intervening amino acids. "-" can mean that the two are connected to each other.

[0038] As used herein, the terms "decreased," "decreasing," "decreasing," or "reducing" "Reduce" usually means a statistically significant reduction. However, for the avoidance of doubt, For clarity, "decreased" means a decrease of at least 10% compared to the reference level, e.g. For example, at least about 20%, or at least about 30%, or at least about 40%, or or at least about 50%, or at least about 60%, or at least about 70%, or is at least about 80%, or at least about 90%, or up to 100% (inclusive) a decrease in the level (i.e., absence of levels compared to the reference sample) or This refers to any reduction between 10 and 100% compared to the baseline.

[0039] As used herein, the term "nucleic acid" refers to a nucleotide, e.g., a deoxyribonucleic acid. refers to a polymer composed of nucleotides (DNA) or ribonucleotides (RNA) As used herein, the terms "ribonucleic acid" and "RNA" refer to ribonucleic acid. As used herein, "deoxyribonucleic acid" and "deoxyribonucleic acid" refer to a polymer composed of deoxyribonucleic acids. The term "DNA" refers to a polymer composed of deoxyribonucleotides .

[0040] The terms "polynucleotide" and "oligonucleotide" are used interchangeably. It is either a silibonucleotide or a ribonucleotide, or an analog thereof. A polynucleotide refers to a polymeric form of nucleotides of any length. It may have any basic structure and may perform any function, known or unknown. Non-limiting examples of domains include: genes or gene fragments, exons, introns, messages mRNA, transcriptional RNA, ribosomal RNA, ribozyme, cDNA, Recombinant polynucleotides, branched polynucleotides, plasmids, vectors, any sequence isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be made prior to assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double- and single-stranded molecules. Unless otherwise specified, any embodiment of the present invention that is a polynucleotide may be in double-stranded form and its double-stranded form. Two complementary single-stranded oligonucleotides known or predicted to form a single-stranded oligonucleotide It encompasses both of the forms.

[0041] The term "polypeptide" is used in the broadest sense to refer to a polypeptide consisting of two or more subunit amino acids, It refers to compounds that are amino acid analogs or peptidomimetics. The subunits are peptide bonds. In other embodiments, the subunits may be linked by other bonds, e.g. For example, the bond can be by an ester, an ether, or the like. The term "amino acid" refers to glycine and both the D or L optical isomers, as well as the amino acids Any natural and / or unnatural or synthetic amino acids, including analogs and peptidomimetics When the peptide chain is short, a peptide of three or more amino acids is generally called an "o If the peptide chain is long, the peptide is generally called a "polypeptide." " or "protein."

[0042] A "pharmaceutically acceptable carrier" (sometimes called a "carrier") is generally a substance that is safe and toxic. means a carrier or excipient useful in preparing a non-steroidal pharmaceutical or therapeutic composition. , including carriers acceptable for veterinary and / or human pharmaceutical or therapeutic use. The term "pharmaceutically acceptable carrier" includes, but is not limited to, phosphate buffered saline. saline, water, emulsions (such as oil / water or water / oil emulsions) and / or can contain various types of wetting agents.

[0043] As used herein, the term "carrier" refers to any excipient, diluent, filler, salts, buffers, stabilizers, solubilizers, lipids, stabilizers, or other compounds of the present invention for use in pharmaceutical formulations. and other materials well known in the art. Selection of Carriers for Use in the Compositions The dosage will depend on the intended route of administration of the composition. The preparation of therapeutically acceptable carriers and formulations can be found, for example, in Remington's Pharmaceuticals. ceutical Sciences,21st Edition,ed.Univer city ​​of the Sciences in Philadelphia,Lip pincott,Williams & Wilkins,Philadelphia, PA, 2005. Examples of physiologically acceptable carriers include saline, Buffers with glycerol, DMSO, phosphate buffers, citrate buffers, and other organic acids buffer solutions such as HCl; antioxidants containing ascorbic acid; low molecular weight (less than about 10 residues) polypeptides Proteins such as serum albumin, gelatin, or immunoglobulins; polyvinyl Hydrophilic polymers such as thiazolinone; glycine, glutamine, asparagine, arginine , or amino acids such as lysine; monosaccharides, disaccharides, and glucose, mannose, if or other carbohydrates, including dextrin; chelating agents such as EDTA; mannitol or is a sugar alcohol such as sorbitol; a salt-forming counterion such as sodium; and / or T WEEN® (ICI, Inc.; Bridgewater, New Jersey) ey), polyethylene glycol (PEG), and PLURONICS® (B ASF (Florham Park, NJ) and other nonionic surfactants. To provide for administration of such doses for the desired therapeutic treatment, the compositions disclosed herein are The composition has a total weight of about 0.1 to 99g, based on the weight of the entire composition including the carrier and diluent. % of one or more of the subject compounds.

[0044] "Recombinant" as used herein in reference to a polypeptide means a polypeptide that is not naturally occurring. By "polypeptides" is meant a combination of two or more polypeptides, each of which is a suitable combination.

[0045] The term "specificity" refers to the ability of a particular antigen-binding molecule (such as a Nanobody of the invention) to bind to Nanobodies with low specificity are those with only a single antigen or antigenic determinant. While it binds to multiple different epitopes via a single antigen-binding site or binding domain, Highly specific nanobodies bind to a single antigen-binding site or domain. Binds to one to several epitopes. In some embodiments, several epitopes are similar. Similar or very similar, e.g., cross-species epitopes. In this case, the term "specifically binds to" as used herein with respect to Nanobodies. In this case, the nanobody binds preferentially to one epitope compared to other epitopes. Specific binding refers to the binding affinity and stringency under which binding occurs. In one example, high affinity binding under stringent conditions may occur. When present, the nanobody specifically binds to the epitope. The HSA-binding polypeptides or Nanobodies described herein bind to human serum albumin. It specifically binds to

[0046] The term "subject" refers to any animal, including primates (e.g., humans), cattle, sheep, goats, horses, dogs, This includes animals such as mammals, including but not limited to cats, rabbits, rats, mice, etc. In some embodiments, the subject is a human.

[0047] A "therapeutically effective amount" is an amount that is administered to a researcher, veterinarian, physician, or other biological or medical studies of tissues, systems, animals, or humans that have been pursued by clinicians Human serum albumin (HSA)-binding polypeptide and IL-2 polypeptide, which induce a cellular response In some embodiments, the amount of a composition, such as a recombinant Nanobody, comprising a peptide. In some embodiments, the desired response may be a decrease in tumor size, a decrease in the size of a tumor associated with a cancer (e.g., melanoma) and / or a related In other embodiments, the desired response is, for example, the induction of anti-tumor cytotoxic T lymphocytes. Enhancement of antitumor immune responses, including activation of lymphocytes, natural killer cells, and / or B cells and / or an increase in the level of tumor antigen-specific antibodies in the subject. In some cases, the desired biological or medical response may occur over a period of days, weeks, or years. A therapeutically effective amount is achieved after administering multiple dosages of the composition to the subject. The disorder or condition and its severity, route of administration, time of administration, rate of excretion, drug combinations, treatment The use of any of these drugs will depend on the judgment of the treating physician, the dosage form, and the age, weight, general health, sex, and other factors of the subject being treated. and / or diet-dependent changes. Human serum albumin (HSA) binding polypeptide and and a recombinant nanobody comprising an IL-2 polypeptide as described herein, which is effective for the treatment of The amount can be determined by one skilled in the art.

[0048] A significant therapeutic reduction in symptoms is a significant reduction in the measured parameter compared to controls or untreated subjects. For example, at least about 10%, at least about 20%, at least about 30% %, at least about 40%, at least about 50%, at least about 60%, at least about 70 %, at least about 80%, at least about 90%, at least about 100%, at least about 1 25%, at least about 150%, or more. Parameters include clinically detectable markers of disease, e.g., biological markers Increase or decrease in levels, e.g., reduction in tumor size; e.g., anti-tumor cytotoxic T lymphocytes, Increased anti-tumor immune response, including activation of neural killer cells and / or B cells; Increased levels of tumor antigen-specific antibodies in the circulation and / or tumors, and / or The total daily dose of the compositions and formulations disclosed herein is It will be understood that the exact amount of time required will be determined by the attending physician within the bounds of good medical practice. The exact amount will vary depending on factors such as the type of disease being treated.

[0049] As used herein, the terms "treat," "treating," "treatment," and the like These grammatical variants express the intensity of one or more of the accompanying symptoms of cancer or a condition in part or in part. completely delay, mitigate, alleviate, or reduce the risk of cancer and / or one or more of the causes of cancer Treatment according to the present invention includes preventing, alleviating, or inhibiting the causes of the disease. ventively, prophylactically, palliatively, or It may be applied therapeutically. In some embodiments, the treatment is a reduction in tumor size. In some embodiments, the treatment is the reduction of cancer metastasis or cancer lesions. In embodiments, the treatment is a reduction in tumor burden.

[0050] Prophylactic compositions can be used to treat cancer before onset (e.g., before overt signs of cancer), during early onset (e.g., During early signs and symptoms of cancer, after established progression of cancer, or in the later stages of cancer , is administered to the subject. Prophylactic administration can occur minutes to months before the appearance of cancer.

[0051] composition A human serum albumin (HSA)-binding polypeptide and an IL-2 polypeptide. A recombinant nanobody, wherein the HSA-binding polypeptide is specific for an HSA epitope. As mentioned above, the combination of this group of nanobodies is Recombinant nanobodies are surprisingly effective in extending the half-life of IL-2 polypeptides In some embodiments, the IL-2 polypeptide of the Nanobody The affinity of the nanobody for the IL-2 receptor is not part of the nanobody or Not significantly reduced or not reduced compared to unbound IL-2 .

[0052] With respect to the human serum albumin portion of a Nanobody, "serum albumin" refers to the amount of serum albumin found in the blood of vertebrates. Serum albumin is a type of globular protein in blood. It is produced in the liver. "Serum albumin" or "HSA" as used herein refers to a protein containing cyclic adenosine 5'-diphosphate It refers to a polypeptide that hydrolyzes tri-ribose and is coded for by the ALB gene. In some embodiments, the HSA polypeptide comprises one or more of the following: Publicly available databases: HGNC: 399, Entrez Gene: 213, Ensembl:ENSG00000163631, OMIM:103600, UniP In some embodiments, H The SA polypeptide may have a sequence similar to SEQ ID NO: 1 or a sequence similar to SEQ ID NO: 1 but which is about 80%, about 85%, about 90%, about 95%, or about 98%, or about 80%, about 85%, about 90%, about a polypeptide sequence having greater than 95%, or about 98%, homology to the sequence of SEQ ID NO: The HSA polypeptide of SEQ ID NO: 1 includes a polypeptide comprising a portion of immature HSA. or may be pre-processed from mature HSA, and therefore are referred to herein as SEQ ID NO: The present invention includes mature or processed portions of the HSA polypeptide of No. 1.

[0053] As mentioned above, an "epitope" is a molecule that is bound to an antigen by an antigen-binding molecule (such as a nanobody of the invention). The location on an antigen or target that is recognized. The term "epitope" refers to a linear epitope and Figure 16 shows some epitopes on HSA, including both conformational and conformational epitopes. These epitopes are called "clusters."

[0054] Thus, in some embodiments, a human serum albumin (HSA) binding polypeptide and and an IL-2 polypeptide, and The polypeptide comprises epitope 1, epitope 2, epitope 3, and epitope 4. specifically binds to an HSA epitope selected from the group consisting of: Amino acid residues 298 to 307, 311, 332 to 341, and 371 to 386 of No. 1 epitope 2 comprises amino acid residues 5-13, 62-67, 93-99 of SEQ ID NO: 1; and 228 to 266, and epitope 3 comprises amino acid residues 226 to 230 of SEQ ID NO: 1. and 298 to 337 of SEQ ID NO: 1, and epitope 4 comprises amino acid residues 33 to 38 of SEQ ID NO: 1, and 111-145. In some embodiments, the HSA binding polypeptide comprises the sequence Amino acid residues 298 to 307, 311, 332 to 341, and 371 to 386 of No. 1 In some embodiments, the HSA-binding polypeptide specifically binds to an HSA epitope comprising The peptides include 5 to 13, 62 to 67, 93 to 99, and 228 to 266 of SEQ ID NO: 1. In some embodiments, the HSA-binding polypeptide specifically binds to an HSA epitope. The peptide is an HSA epitope containing amino acid residues 33 to 38 and 111 to 145 of SEQ ID NO: 1. In some embodiments, the HSA binding polypeptide specifically binds to SEQ ID NO: 1, including amino acid residues 298-307, 311, 332-341, and 371-386 Binds specifically to HSA epitopes.

[0055] The specificity of an antigen-binding molecule (e.g., HSA-binding polypeptide, Nanobody of the invention) can be determined by the parent It should be understood that the degree of affinity can be measured based on affinity and / or avidity. The dissociation equilibrium constant (K D The affinity, expressed by the formula (I), is the affinity between the antigenic determinant and the antigen. It is an index of binding strength with the antigen-binding site on the original binding molecule, and K D The smaller the value, the better the antigen determination. The binding strength between the base and the antigen-binding molecule becomes stronger (or the affinity is 1 / K D That is, Affinity constant (K A Methods for measuring affinity are well known in the art. The binding activity of the antigen-binding molecules (HSA-binding polypeptides and the nanoparticles of the present invention) is well known to those skilled in the art. Avidity is a measure of the strength of binding between a specific antigen (e.g., a chemoattractant, a chemoattractant, or a chemoattractant) and the appropriate antigen. The affinity of the antigen-binding site on the antigen-binding molecule and the appropriate binding site on the antigen-binding molecule The number of target sites is related to the number of target sites. and the Nanobodies of the invention) are directed to their antigens at 10 -5 ~10 -12 mol / L or less, and preferably 10 -7 ~10 -12 mol / L or less, and more preferably 10 -8 ~ 10 -12 Dissociation constant (K in mol / L D ) (i.e., 10 5 ~10 12 L / mol or more, and Preferably, 10 7 ~10 12 L / mol or more, and more preferably 10 8 ~10 12 L / mol binding rate constant (K A In some embodiments, the Ka (binding rate) , 1Ms) is about 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 1010 , or 10 11 In some embodiments, the Ka is about 10 7 In some embodiments, K D (dissociation rate, s) is approximately 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 - 10 , or 10 -11 In some embodiments, K D is about 10 -7 It is. In some embodiments, the antigen binding proteins disclosed herein are -9 mol / L Less than K D Any K greater than 10 μM binds to its antigen. D The values ​​are generally non-singular. Dissociation constants are considered to represent actual or apparent binding, as will be apparent to those skilled in the art. The dissociation constant of the crosslink may be the dissociation constant of the crosslink.

[0056] In some embodiments, the HSA binding polypeptide of the Nanobody is specific for human HSA. In some embodiments, the HSA-binding polypeptide of the Nanobody binds to human HSA. Specifically binds to SA and mouse serum albumin. In some embodiments, the nanobody The specificity of antibody binding is due, or largely due, to the CDR3 region of the nanobody. can be done.

[0057] Thus, in some embodiments, the HSA-binding polypeptide of the Nanobody is a complementary The CDR3 comprises a sex-determining region (CDR) 3, and the CDR3 is represented by SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 1 2, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, Sequence number 24, sequence number 25, sequence number 26, sequence number 27, sequence number 28, sequence number 29 , SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: No. 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 5 2, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, Sequence number 64, sequence number 65, sequence number 66, sequence number 67, sequence number 68, sequence number 69 , SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: No. 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 9 2, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: SEQ ID NO: 98, SEQ ID NO: 99, or SEQ ID NO: 100. Contains the amino acid sequence.

[0058] "50% similarity" is calculated as follows: Residue Groups: Amino acid residues are grouped according to their biophysical properties as follows: To be checked: Group 1: A, V, I, L, F, M, W, P → V (hydrophobic residues) Group 2: S, G, C, N, Q, Y, T → T (polar residues) Group 3: K, R, H → R (positively charged residues) Group 4: D, E → E (negatively charged residues) Seq_1 is a sequence selected from the group consisting of SEQ ID NOs: 10 to 100. Seq_2 is the incoming sequence. 1. Representing amino acids as groups of residues 2. Generate tetramer sets, Set_1 and Set_2, for both sequences. 3. Score =

number

[0059] In the present disclosure, "score threshold ≧0.5: that is" refers to the sequences described in SEQ ID NOs: 10 to 100. , 50% similarity (e.g., at least 60% similarity) to 91 CDR3 sequences , at least 65% similarity, at least 70% similarity, at least 75% similarity, At least 80% similarity, at least 85% similarity, at least 90% similarity, At least 95% similarity, at least 98% similarity, or at least 99% similarity ) and all amino acid sequences (including the CDR3 finger of the Nanobody) that are equal to or exceed the above. -print arrangement).

[0060] In some embodiments, the HSA-binding polypeptide of the Nanobody has the sequence SEQ ID NO: 10, Sequence number 11, sequence number 12, sequence number 13, sequence number 14, sequence number 15, sequence number 16 , SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: No. 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 3 9, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, Sequence number 51, sequence number 52, sequence number 53, sequence number 54, sequence number 55, sequence number 56 , SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: No. 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 7 9, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, Sequence number 91, sequence number 92, sequence number 93, sequence number 94, sequence number 95, sequence number 96 , SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, and SEQ ID NO: 100. The complementarity determining region (CDR) 3 contains an amino acid sequence corresponding to the target sequence.

[0061] Thus, in some embodiments, the HSA binding polypeptide of the Nanobody has the sequence No. 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 2 1, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, Sequence number 33, sequence number 34, sequence number 35, sequence number 36, sequence number 37, sequence number 38 , SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: No. 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 6 1, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, Sequence number 73, sequence number 74, sequence number 75, sequence number 76, sequence number 77, sequence number 78 , SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: No. 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, and SEQ ID NO: 100 and a CDR3 comprising an amino acid sequence selected from the group consisting of epitope 1, epitope 2, epitope 3, epitope 4, epitope 5, epitope 6, epitope 7, epitope 8, epitope 9, epitope 10, epitope 11, epitope 12, epitope 13, epitope 14, epitope 15, epitope 16, epitope 17, epi an HSA epitope selected from the group consisting of epitope 2, epitope 3, and epitope 4; wherein epitope 1 specifically binds to amino acid residues 298-30 of SEQ ID NO: 1. epitope 2 comprises SEQ ID NO: 1 and amino acid residues 5 to 13, 62 to 67, 93 to 99, and 228 to 266 of the epitope. Group 3 contains amino acid residues 226 to 230 and 298 to 337 of SEQ ID NO: 1, and Tope 4 comprises amino acid residues 33 to 38 and 111 to 145 of SEQ ID NO:1.

[0062] Regarding the IL-2 portion of the nanobody, IL-2 binds to the IL-2 receptor on the cell surface. Binding induces activation, proliferation, and proliferation of B lymphocytes, T lymphocytes, and natural killer cells. "IL-2" is used herein to refer to cyclic adenovirus-associated interleukin (IL-2). Synthetic 5'-diphosphate-ribose hydrolyzing polypeptide, human In some embodiments, it is encoded by the IL2 gene. The polypeptides may be identified in one or more of the following publicly available databases: HGNC:6001, Entrez Gene:3558, Ensembl: ENSG00000109471, OMIM:147680, UniProtKB:P6 In some embodiments, the IL-2 polypeptide has the sequence of SEQ ID NO: 5, or is about 80%, about 85%, about 90%, about 95%, or about 98% identical to SEQ ID NO:5, or more than about 80%, about 85%, about 90%, about 95%, or about 98% homology. or a polypeptide comprising a portion of SEQ ID NO: 5. The IL-2 polypeptide of the fifth embodiment may represent immature IL-2 or may be derived from mature IL-2. The mature portion of the HSA polypeptide of SEQ ID NO: 5 can be processed and is therefore referred to herein as the mature portion of the HSA polypeptide of SEQ ID NO: 5. Or a processing part is included.

[0063] Thus, in some embodiments, the IL-2 polypeptide has the amino acid sequence of SEQ ID NO:5. The "functional flag" of IL-2 is used herein to refer to a sequence of the IL-2 amino acid sequence or a functional fragment thereof. "ment" refers to, for example, binding to the IL-2 receptor and / or activation of NK cells, B cells, and / or or fragments of IL-2 that have biologically active properties, including activation of T lymphocytes. It should be understood that in some embodiments, the HSA-binding polypeptide In some embodiments, the IL-2 polypeptide is linked to the IL-2 polypeptide via a linker. The linker comprises the amino acid sequence of SEQ ID NO:6.

[0064] In some embodiments, the IL-2 polypeptide includes, but is not limited to, aldesleukin. Aldesleukin is a recombinant human IL-2 polypeptide, but is not limited to the amino acid sequence At position 125, it may have a serine instead of a cysteine. In some embodiments, the aldesleukin comprises the amino acid sequence of SEQ ID NO: 103. In some cases, aldesleukin is Proleukin (Novartis).

[0065] Thus, in some embodiments, an HSA-binding polypeptide and an IL-2 polypeptide are and a recombinant Nanobody comprising the sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4; The IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 5 or a functional fragment thereof. The HSA-binding polypeptide is linked to the IL-2 polypeptide via a linker. The car comprises the amino acid sequence of SEQ ID NO:6.

[0066] In some embodiments, the nanobody is a natural IL-2 or albroukin (H Increased thermostability compared to IL-2 such as Human Genome Sciences Increased resistance to serum protease activity and / or increased sensitivity to steroids. In embodiments, the nanobody is a natural IL-2 or albroukin (Human Ge It reaches maximum activity in vivo more rapidly than other IL-2s, such as IL-2 (Immune Sciences) The effective molar concentration is reached.

[0067] In some embodiments, the recombinant Nanobody of any of the foregoing aspects has a molecular mass of less than about 50 kDa. For example, less than about 45 kDa, less than about 40 kDa, less than about 35 kDa, or less than about In some embodiments, the combination of any of the foregoing aspects can be less than 30 kDa. The recombinant nanobody is selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9. Contains the amino acid sequence.

[0068] The recombinant nanobody is designed to bind to natural IL-2 or albroukin (Human Gen Compared with IL-2 such as those from Home Sciences, the IL-2 polypeptide of the nanobody Nearly identical or increased affinity between the peptide moiety and the IL-2 receptor. The IL-2 receptor is often referred to as the α subunit (also called IL-2Rα or CD25), β subunit (or IL-2Rβ or CD122), and γ subunit Three subunits, called IL-2γ, common γ chain, or CD132, It is a heterotrimeric receptor with β and γ subunits. Intermediate affinity binding (approximately 10 -9 While forming a complex with α, β The γ and γ subunits form a high-affinity IL-2 receptor binding to IL-2 (and Other 10 -11 In some embodiments, the recombinant nanobody is and the IL-2 polypeptide portion of the antibody, e.g., the α subunit, the β and γ subunits, or or a nearly identical parental gene between the IL-2 receptor and the IL-2 receptor, which contains α, β, and γ subunits. In some embodiments, the recombinant Nanobody has affinity or increased affinity for the , the IL-2 polypeptide portion of the nanobody and the IL-1 polypeptide portion containing the α, β, and γ subunits. It has nearly identical or increased affinity between the two receptors.

[0069] Herein, affinity at acidic pH correlates well with excretion rate (Spearman ρ = 0. 78), which is subsequently understood to be well correlated with affinity (especially dissociation rate) at neutral pH. Therefore, both the slow bond dissociation rate and the acidic pH-dependent interaction are Important for extending the stability of the nanobody-HSA-FcRn tertiary complex within the endosome In some embodiments, the HSA binding polypeptide of the recombinant Nanobody has a molecular weight of about It specifically binds to HSA in a conformationally stable form at pH values ​​ranging from 3.5 to approximately 7.5. .

[0070] Native IL-2 has a half-life of approximately 30-60 minutes in humans. By using nanobodies to target endogenous human serum albumin, I Therefore, in some embodiments, the half-life of L-2 can be extended. The recombinant IL-2 Nanobody described in 1. above is a natural or non-nanobody recombinant IL-2 at least 50-fold (e.g., at least 1 00x, at least 200x, at least 300x, at least 400x, at least 5 00 times, at least 600 times, at least 700 times, at least 800 times, at least 9 In some embodiments, the antibody has a half-life that is (at least 1000 times, or at least 1000 times) longer. The recombinant IL-2 nanobodies described herein may be used in combination with recombinant IL-2 nanobodies such as aldesleukin. at least 50-fold (e.g., at least 100-fold, at least 200-fold, fold, at least 300 times, at least 400 times, at least 500 times, at least 600 times times, at least 700 times, at least 800 times, at least 900 times, or at least In some embodiments, the recombinant proteins described herein have a half-life that is (1000-fold) longer. The IL-2 nanobody is a novel antibody against IL-2. s) (HSA / IL-2 fusion protein) than recombinant human IL-2 Also 2x, 5x, 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 9x 0 or 100 times longer half-lives.

[0071] In some embodiments, nanobodies comprising human serum albumin (HSA) binding polypeptides are The present invention provides a method for the preparation of an HSA-binding polypeptide comprising: CDR3 is SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: No. 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 3 1, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, Sequence number 43, sequence number 44, sequence number 45, sequence number 46, sequence number 47, sequence number 48 , SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: No. 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 7 1, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, Sequence number 83, sequence number 84, sequence number 85, sequence number 86, sequence number 87, sequence number 88 , SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, or A sequence having at least 50% similarity (e.g., at least 60% similarity, at least At least 65% similarity, at least 70% similarity, at least 75% similarity, at least At least 80% similarity, at least 85% similarity, at least 90% similarity, at least (at least 95% similarity, at least 98% similarity, or at least 99% similarity) Similarity is calculated as described above.

[0072] In some embodiments, polypeptides comprising human serum albumin (HSA) binding polypeptides The HSA-binding polypeptide has a complementarity-determining region (CDR) 3 of and CDR3 is SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, Sequence number 20, sequence number 21, sequence number 22, sequence number 23, sequence number 24, sequence number 25 , SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: No. 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 4 8, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, Sequence number 60, sequence number 61, sequence number 62, sequence number 63, sequence number 64, sequence number 65 , SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: No. 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 8 8, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: No. 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, and and SEQ ID NO: 100.

[0073] In some embodiments, the recombinant Nanobody or polypeptide of any of the preceding aspects is formulated in a pharmaceutically acceptable carrier.

[0074] Treatment method 1. A method of treating cancer in a subject in need thereof, comprising administering to a subject a therapeutically effective amount of a compound described herein. The method is provided herein, comprising administering a therapeutically effective amount of a recombinant Nanobody to a subject. The present disclosure provides a human serum albumin (HSA) binding polypeptide and an IL-2 polypeptide. and administering a therapeutically effective amount of a recombinant human ovarian protein containing a peptide to treat cancer. This is a surprising discovery that treatment can be achieved.

[0075] Accordingly, provided herein are methods for treating cancer in a subject in need thereof. and administering a therapeutically effective amount of a recombinant nanobody, wherein the nanobody is a human Contains a serum albumin (HSA) binding polypeptide and an IL-2 polypeptide, The conjugated polypeptide specifically binds to the HSA epitope, and the HSA epitope is an epitope epitope 1, epitope 2, epitope 3, and epitope 4; Peptide 1 is amino acid residues 298 to 307, 311, 332 to 341, and 37 of SEQ ID NO: 1. 1 to 386 of SEQ ID NO: 1, and epitope 2 comprises amino acid residues 5 to 13, 62 to 67, and 93-99 and 228-266 of SEQ ID NO: 1, and epitope 3 is 26 to 230 and 298 to 337, and epitope 4 comprises amino acid residues of SEQ ID NO: 1 The above methods include those described above, including 33 to 38 and 111 to 145.

[0076] In some embodiments of the method, the HSA-binding polypeptide portion of the Nanobody has SEQ ID NO: No. 1, containing amino acid residues 298-307, 311, 332-341, and 371-386 In some embodiments, the HSA-binding polypeptide specifically binds to an HSA epitope containing the HSA epitope. The peptide is a H including 5 to 13, 62 to 67, 93 to 99, and 228 to 266 of SEQ ID NO: 1. In some embodiments, the HSA-binding polypeptide specifically binds to an HSA epitope. is an HSA epitope comprising amino acid residues 33 to 38 and 111 to 145 of SEQ ID NO: 1 In some embodiments, the HSA binding polypeptide specifically binds to SEQ ID NO: 1. H containing amino acid residues 298-307, 311, 332-341, and 371-386 Binds specifically to the SA epitope.

[0077] Examples of cancers that can be treated using the methods described herein include melanoma, breast cancer, and prostate cancer. Cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain tumor, lymphoma, white In some embodiments, cancer includes, but is not limited to, hematologic malignancies, lung cancer, and the like. is melanoma. In some embodiments, the cancer is renal carcinoma.

[0078] In some embodiments, administering a recombinant Nanobody described herein results in: Metastasis is reduced, tumor growth slows and / or terminates, tumor size decreases, and T cells and NK cell activation, proliferation, and cytotoxic function are promoted, and the levels of antitumor antibodies are increased. The term "antibody" as used herein refers to a compound that enhances the survival and / or survival rate of a subject. "Increase," "promote," "extend," and "decrease," "reduce" The incidence of such symptoms in subjects compared to before treatment, or in the overall or study population It is understood to mean an increase or decrease compared to the original, and is not intended herein. It must be.

[0079] In some embodiments of the method, the HSA binding polypeptide of any of the preceding aspects is a complementary The CDR3 comprises a sex-determining region (CDR) 3, and the CDR3 is represented by SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 1 2, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, Sequence number 24, sequence number 25, sequence number 26, sequence number 27, sequence number 28, sequence number 29 , SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: No. 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 5 2, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, Sequence number 64, sequence number 65, sequence number 66, sequence number 67, sequence number 68, sequence number 69 , SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: No. 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 9 2, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: SEQ ID NO: 98, SEQ ID NO: 99, or SEQ ID NO: 100. At least 60% similarity, at least 65% similarity, at least 70% similarity, at least At least 75% similarity, at least 80% similarity, at least 85% similarity, at least at least 90% similarity, at least 95% similarity, at least 98% similarity, or at least It contains amino acid sequences that share at least 99% similarity with the corresponding amino acid sequence.

[0080] "50% similarity" is calculated as follows: Residue Groups: Amino acid residues are grouped according to their biophysical properties as follows: To be checked: Group 1: A, V, I, L, F, M, W, P → V (hydrophobic residues) Group 2: S, G, C, N, Q, Y, T → T (polar residues) Group 3: K, R, H → R (positively charged residues) Group 4: D, E → E (negatively charged residues) Seq_1 is a sequence selected from the group consisting of SEQ ID NOs: 10 to 100. Seq_2 is the incoming sequence. 1. Representing amino acids as groups of residues 2. Generate tetramer sets, Set_1 and Set_2, for both sequences. 3. Score =

number

[0081] In the present disclosure, "score threshold ≧0.5: that is" refers to the sequences described in SEQ ID NOs: 10 to 100. , 91 CDR3 master sequences, 50% similarity (e.g., at least 60% similarity) Sex, at least 65% similarity, at least 70% similarity, at least 75% similarity , at least 80% similarity, at least 85% similarity, at least 90% similarity, At least 95% similarity, at least 98% similarity, or at least 99% similarity All amino acid sequences (CDR3 fragments of nanobodies) that are equal to or exceed the This means the garprint sequence.

[0082] In some embodiments of the method, the HSA binding polypeptide of any of the preceding aspects has the sequence A complementarity determining region (CDR) comprising an amino acid sequence selected from the group consisting of numbers 10 to 100 )3 included.

[0083] In some embodiments, the method of treating cancer comprises administering to a subject a human serum albumin (HSA)-binding polypeptide. administering a therapeutically effective amount of a recombinant nanobody comprising a peptide and an IL-2 polypeptide. and the HSA-binding polypeptide is selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4. In some embodiments, the Nanobody comprises an amino acid sequence selected from the group consisting of: The IL-2 polypeptide portion has the amino acid sequence of SEQ ID NO: 5 or a functional fragment thereof. In some embodiments, the HSA-binding polypeptide is linked to an IL- In some embodiments, the linker is an amino acid sequence of SEQ ID NO: 6. Contains the acid sequence.

[0084] In some embodiments of the method, the recombinant Nanobody is less than about 50 kDa, e.g. For example, less than about 45 kDa, less than about 40 kDa, less than about 35 kDa, or less than about 30 kDa. In one example, the recombinant polypeptide can be SEQ ID NO: 7, SEQ ID NO: 8, and and SEQ ID NO: 9.

[0085] In some embodiments of the invention, the recombinant Nanobody inhibits natural IL- 2 polypeptide, aldesleukin (e.g., Proleukin, Novartis), and is about 10 times more potent than Albroukin (Human Genome Sciences), Approximately 20 times, approximately 30 times, approximately 40 times, 50 times, approximately 100 times, approximately 200 times, approximately 300 times, approximately 40 0x, approximately 500x, approximately 600x, approximately 700x, approximately 800x, approximately 900x, or approximately 100x 0 times longer half-life.

[0086] Thus, in some embodiments, the frequency of administration of the recombinant Nanobody is greater than that of the natural IL- 2 polypeptide, aldesleukin, or albroukin (Human Genome Less than about 2 times, less than about 3 times, less than about 4 times, or less than about the frequency of administration of Less than 5x, Less than approximately 6x, Less than approximately 7x, Less than approximately 8x, Less than approximately 9x, Less than approximately 10x, Approximately 15x less than, less than about 20 times, less than about 30 times, less than about 40 times, less than about 50 times, less than about 80 times, less than about 1 Less than 00 times, less than about 150 times, less than about 200 times, less than about 300 times, less than about 500 times, less than about 8 00-fold or less than about 1000-fold.

[0087] The administration frequency of the recombinant Nanobody of any of the aforementioned embodiments may be at least once a month, Once a week, once every two weeks, once a week, twice a week, three times a week, four times a week, five times a week, six times a week , once a day, or, 2 times a day, 3 times a day, 4 times a day, 5 times a day, 6 times a day, 7 times a day, Examples include, but are not limited to, eight times per day or nine times per day. In such cases, the interval between each administration is less than about 2 months, less than about 1 month, less than about 3 weeks, less than about 2 weeks, or is less than about 1 week, e.g., less than about 6, 5, 4, 3, 2, or 1 day(s) In some embodiments, the frequency of administration of the recombinant Nanobody is at least These include, but are not limited to, once a day, twice a day, or three times a day. In some embodiments, the interval between each administration is about 48 hours, about 36 hours, about 24 hours, or about 22 hours. , about 20 hours, about 18 hours, about 16 hours, about 14 hours, about 12 hours, about 10 hours, about 9 hours In some embodiments, the interval between each administration is less than about 8 hours, about 7 hours, or about 8 hours. Approximately 24 hours, approximately 22 hours, approximately 20 hours, approximately 18 hours, approximately 16 hours, approximately 14 hours, approximately 12 hours about 10 hours, about 9 hours, about 8 hours, about 7 hours, or less than about 6 hours. In this embodiment, the interval between each administration is constant. For example, administration may be daily, every other day, every third day, or every 3 days. Administration can be once every four days, once every five days, or weekly. Administration can also be continuous. and adjusted to maintain levels of recombinant Nanobodies within any desired and predetermined range. It is possible.

[0088] The recombinant nanobodies described herein are capable of extending the half-life of IL-2. In some embodiments, the recombinant Nanobody binds to a naturally occurring IL-2 protein. Compared to lipeptide or albroukin (Human Genome Sciences) In comparison, this increases the duration of the therapeutic effect of IL-2 and / or reduces the amount of IL- Therefore, the amount of 2 can be reduced as part of the recombinant nanobody. The amount of IL-2 in a therapeutically effective dose is determined by the amount of IL-2 (e.g., a naturally occurring IL-2 polypeptide or is recommended for a single dose of Albroukin (Human Genome Sciences) For example, the conventional recommended dose of IL-2 can be administered in an amount of X. In some cases, the nanoparticles or recombinant polypeptide composition may be at least about 0.9X, about 0.8X, Approximately 0.7X, approximately 0.6X, approximately 0.5X, approximately 0.4X, approximately 0.3X, approximately 0.2X, or approximately In some embodiments, these more A therapeutically effective dose of IL-2 may be present for a period of time after administration. In this case, the side effects of IL-2 can be reduced and / or the subject's tolerance to IL-2 can be improved. This can reduce the likelihood of resistance developing.

[0089] The disclosed methods can be performed at any time before the onset of cancer. In such cases, the disclosed methods may be used to treat cancer at the onset of 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 4 0, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27 , 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 year; 12, 11 , 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 month; 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 1 3, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 days; 60, 48, 36, 3 0, 24, 18, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, or 2 hours before , or cancer started 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 2 5, 30, 35, 40, 45, 50, 55, 60, 75, 90, 105, 120 minutes; 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, 24, 30, 36, 48, 6 0 hours; 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 1 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 , 45, 60, 90 days or more; 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more; 60, 59, 58, 57, 56, 55, 54, 53, 52, 5 1, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38 , 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 1 It can be used after 1, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 year. The method is divided into stages 0, 1, 2, 3, and 4. It can be done at any stage of cancer, including V.

[0090] The recombinant Nanobodies described herein can be present in any suitable formulation. The dosage form can be adapted for administration by any suitable route. are administered orally (including buccal or sublingually), rectally, epidurally, intracranially, intraocularly, by inhalation, intranasally, or topically (including buccal). , sublingual, or transdermal), vaginal, intraurethral, ​​parenteral, intracranial, subcutaneous, intramuscular, intravenous, Intraperitoneal, intracutaneous, intraosseous, intracardiac, intraarticular, intravenous, intrathecal, intravitreal, intracerebral, gingival, subgingival Such formulations include, but are not limited to, intravenous, intracerebroventricular, and intradermal. It can be prepared by any method known in the art.

[0091] The compounds and compositions disclosed herein can be used to treat cancer and / or metastasis in a subject. Treatment, inhibition, alleviation, reduction, amelioration, and / or prevention of the above-mentioned subjects, administering an amount of the compound of any of the preceding embodiments, or the pharmaceutical composition of any of the preceding embodiments, and / or prevent the above, including: In some embodiments, the cancer is lymphoma, B-cell lymphoma, T-cell lymphoma, mycosis fungoides, or , Hodgkin's disease, myeloid leukemia, bladder cancer, brain tumors, nervous system cancers, head and neck cancer, head and neck squamous cell carcinoma , lung cancer such as small cell lung cancer and non-small cell lung cancer, neuroblastoma / glioblastoma, ovarian cancer, skin cancer , liver cancer, melanoma, squamous cell carcinoma of the mouth, throat, larynx, and lung, cervical cancer, breast cancer, epithelial cancer, kidney cancer Cancer, genitourinary cancer, esophageal carcinoma, head and neck carcinoma, colorectal cancer, hematopoietic organ cancer, testicular cancer, colorectal cancer, rectal cancer, The cancer is selected from the group consisting of prostate cancer and pancreatic cancer.

[0092] In some embodiments, the recombinant Nanobody of any of the foregoing aspects is pharmaceutically acceptable. The compound is formulated in a carrier that can be used. [Example]

[0093] The following examples are provided below to illustrate compositions, methods, and results according to the disclosed subject matter. These examples are intended to encompass all aspects of the subject matter disclosed herein. It is not intended to be exhaustive, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention that are apparent to one skilled in the art. It is not intended to be a diagram.

[0094] Example 1: Synthetic characterization of HSA-Nbs. We expressed 98 recombinant soluble nanobodies (Nb) with unique CDR3 sequences. , purified from E. coli using His-cobalt resin. The amino acid composition on the DR loop is shown in Figure 1a. These Nbs have highly diverse CDR It has a triplet sequence and forms an intramolecular disulfide bond that is important for isoelectric point, hydrophobicity, and thermal stability. The physicochemical properties of the CDR3, including the CDR3 cysteine ​​residue, are significantly altered (Fig. 1b). The amino acids were aligned and the occurrence at each position was calculated to generate a sequence weblogo (Figure 1b). Enzyme-linked immunosorbent assay (ELISA) was performed to confirm that Nb was present in humans, monkeys, and The relative affinities of 89 Nbs binding to albumin from different species, including mouse, were evaluated. While binding to HSA, an OD affinity of over three orders of magnitude was observed. However, less than 50% of HSA-Nbs bind to the highly conserved cynomolgus albumin. Only approximately 9% (8 Nbs) appreciably cross-reacted with mouse albumin. Although HSA-specific Nbs showed remarkable specificity, these cross-species binding The data are also useful for experiments in corresponding animal models.

[0095] Twenty HSA-Nbs spanning different ELISA ranges were randomly selected and surface plasma By SPR, the K D Affinity (K on and K. off Carefully measure both 75% (15 / 20) of the selected Nbs were affinity matured and showed enhanced activity for HSA binding. Although they can achieve sub-nM affinities, they have inherent and often diverse binding and dissociation rates. For example, Nb126 has a density of 1.7 × 10 3 (1 / s)K off have This is two orders of magnitude faster than Nb26, but due to different binding rates, both have similar KD (251 pM and 206 pM, respectively) was obtained. Other Nb molecules, such as Nb13 (201 pM) and Nb80 (166 pM), exhibit rapid binding kinetics and A bead binding assay was performed to confirm the SPR measurements. In addition, there was a good correlation between the calculated ELISA affinity and the SPR KD. Seki (R 2 = 0.78, p < 0.001) was found (Fig. 1e). The results were confirmed by bead-binding assay (Fig. 1g). was evaluated using a differential scanning calorimeter (DSC). A wide range of melting temperatures was observed, from 30 to 70°C. In conclusion, this rich repertoire is of exceptionally high quality. and diversified Nb.

[0096] Example 2. HSA immunogenicity landscape revealed by cross-linking and modeling. The fundamental property of humoral immunity is to generate large cohorts of antibodies to protect against any foreign body to which one is exposed. The ability to recognize structure, but the structural substrate that dictates such universality is not fully understood. Here, we present a perspective and a better understanding of the nature of immunogenicity. Next, a rapid docking approach was used to identify the epitope(s) (Fig. 2a). This was then verified by chemical cross-linking and mass spectrometry (CXMS) (Figure 2b , Figure 9). Several features were learned: 1) Highly divergent CDR3 sequences and HS Although other physicochemical properties of A-Nb exist, only four dominant epitopes are identified. 2a-2d and Fig. 16), indicating the presence of immunodominance. Epitopes are highly selective by the mammalian immune system and share sequences between humans and camelids. 3) Surface electrostatic charge and epitope shape are important.

[0097] Example 3. Hybrid structural characterization of tetrameric HSA-Nb complexes. To validate dominant epitopes and better understand the structural substrates of immunogenicity, The antibody titration was performed with HSA and three Nbs (Nb8) corresponding to different major epitopes. The hybrid structure of the tetrameric complex consisting of 0, 13, and 29 was measured. Size exclusion chromatography (SEC) was used to identify the three Nb-free nuclei. We confirmed that these do not compete with HSA interaction (Fig. 3a). A clear size shift was observed upon each Nb addition. The results were analyzed by staining electron microscopy (EM) (Figure 3b, Figures 10b-10d). 3D density maps were reconstructed by averaging over 0.000 EM particles (Figures 3b-3c). Computationally process the structural models and use cross-reactivity to screen for the lowest energy models; The placement and localization of Nb were suppressed. EM, cross-linking suppression, and mutagenesis were simultaneously achieved. The final model was then verified (Fig. 3e-3h).

[0098] The overall structure of the tetrameric complex confirmed the major epitope, which is the " The "pine tree" is decorated with three conformational Nb "gifts" in different locations. This is a Christmas tree memory. Beyond the overall structure, there are a few observations: 1) Nb is a process important for HSA stability by not simultaneously occupying the HSA FcRn binding site. 2) It does not interfere with the FcRn-mediated endocytosis process. A good complementarity was confirmed between the cavity and the concave paratope of Nb. Notably, shape complementarity correlates with affinity, with a maximum Nb80 (160 pM) being impressive. The lowest Nb29 (approximately 1 nM) gave relatively poor agreement. Importantly, none of these Nb was found to be as effective as the reported HSA-Nb (#5VNW, ca. K of 450 nM D ) did not colocalize.

[0099] At the interface, sufficient hydrophobic and charged residues are observed, which may explain the high affinity. To further investigate these interactions, two charged residues on HSA were (K383 and E400). Based on the model described herein, these The residues form two stable salt bridges across the Nb CDR with corresponding opposite charges. The resulting structure mimics the Camelidae residues well (Fig. 3i and 3j). In particular, a mutation E400 R double mutant K383 D Nb80-HSA interaction The E400 R completely lost such strong interactions (Fig. 3k). This further confirms the accuracy of the model described herein. The sequence shown in Figure 3j is YETT LEKCCAAADPHECYAKVFDEF (SEQ ID NO: 101) and YEATLEDC CAKDDPHACYATVFDKL (sequence number 102).

[0100] Example 4. Development of an accurate MS assay for multiplexed Nb pharmacokinetic analysis. Next, new MS assays were developed to reduce the bias and challenges of comparisons across large numbers of animals. This enables high-throughput, accurate, and specific PK analysis of Nb while maintaining the same level of sensitivity. The fragment-ion-based MS method demonstrated good linearity for detecting Nb in serum. Most of these HSA-Nbs cross-react with mouse albumin (Figs. 11a-11e). Therefore, the double knockout of mAlb and its receptor mFcRn was performed. , a humanized albumin mouse model (Tg32 -Alb - / - mFcRn - / - hFcRn Tg / Tg ) was selected. HSA introduction It has been reported that when this was done, the half-life of native HSA in humans was reproduced.

[0101] After HSA injection, 22 mice were treated with 20 HSA binders and 2 non-binder controls. of Nb were mixed at equimolar concentrations in PBS and administered by a single bolus intravenous (i.v.) injection. , was administered to this model (n=3). A molar ratio of Nb to HSA of 1:5 was used to measure binding. The presence of excessive amounts of HSA due to the injection was confirmed. Blood was sampled at different time points after injection. The serum proteins containing Nb were proteolyzed. The peptides were separated by advanced liquid chromatography (LC) and analyzed for Nb signatures. Peptides, along with their fragment ions, were identified by high-resolution Orbitrap MS. Based on hundreds of LC runs, the average quantification CV was , 15.3% (Figs. 12a-12c).

[0102] As shown in Fig. 4b, compared with the control Nb, whose serum half-life was approximately 26–60 min, H SA-Nb showed significantly improved half-life – up to 771-fold reduced blood clearance Surprisingly, a unique and significantly altered T1 / 2 (1 A 21-day incubation period (0.6–7.6 days) was observed (Fig. 4c), which is in line with the previously reported 21-day incubation period for this model. Instead, it correlated well with the half-life of 8.1 days for HSA (w / o Nb coadministration). Label-free quantitative serum proteomics revealed that 5–6 days after HSA injection, serum I We identified a several hundred-fold increase in IgG levels, which indicates a significant increase in the anti-HSA immune response in the model. The results show that the α-glucan complex is amplified at relatively high doses (but still at full physiological levels) (Figure 14). When administered at a dose below 100mg / kg, such well-folded foreign proteins This is not uncommon for protein.

[0103] Example 5. Correlation of Nb pharmacokinetics with its physicochemical properties. To better understand the differences in HSA-Nb PK, we investigated the PK and affinity (7.4 and The melting temperature, isoelectric point, and hydrophobicity (at both CDR and total protein levels) were also investigated. Correlation analysis was performed between the physicochemical properties, including the solubility in both HCl and HCl solutions (Fig. 4d). Interestingly, the affinity at acidic pH correlated well with the excretion rate (Spearman ρ = 0. 78), followed by the affinity at neutral pH (particularly the dissociation rate) and CDR3 hydrophobicity and sufficient Correlated. Reasonable correlations were found between the three parameters: slow bond dissociation rate, Both the Nb-HSA-FcR and acidic pH-dependent interactions are involved in the formation of Nb-HSA-FcR in endosomes. It is thought to be important for extending the stability of the ntertiary complex, and by weakening the binding, This resulted in early termination of the "Chhike" journal and faster removal of piggybacked Nb cargo. Increased hydrophobicity on the paratope may be important for high affinity binding. The data presented herein demonstrates.

[0104] Example 6. Development of Duraleukins: A new class of Nb fusion antibodies for cancer immunotherapy Itokine. For proof-of-concept, the present Nb toolkit was applied to the model therapeutic molecule interleukin-2 ( IL-2 was administered to stimulate both innate and adaptive (e.g., CD8+ T and naive) immune responses. It has the function of suppressing the progression of cancer by enhancing both immune responses (neural killer cells) and immune responses. Human IL-2 (approximately 15kJ / mL) is a key cytokine in the development of cancer immunotherapy. Da) has been applied to effectively treat multiple cancers, including kidney cancer and advanced melanoma. However, its clinical efficacy is limited due to its short half-life (less than 30-60 minutes in humans). Furthermore, frequent administration of high doses of hIL-2 has been shown to significantly limit its efficacy. Although necessary for anticancer activity, it has significant side effects, including liver toxicity and vascular leak syndrome. This can result.

[0105] Following hIL-2, various N-terminal HSA-Nb fusion constructs were designed to These compositions are commonly referred to as "duraleukins" (Figure 5a Three types of duraleukin (DL77, DL80, and DL158) were 80, and Nb158, and bulk purified from E. coli inclusion bodies. All constructs showed good thermostability at approximately 60°C (Fig. 5c). These were compared to hIL-2 as shown in an in vitro CD8+ T cell proliferation assay. The antibody maintained comparable biological activity (Fig. 5d) and was incubated with mouse serum for several days in vitro. Furthermore, these duraleukins (DL 80) maintain high affinity for HSA binding (273 pM) (Figure 5f). 80 shows a decrease in affinity at acidic pH (2-8 fold, Figure 5g), but nevertheless It has one of the highest affinities for mouse albumin and is therefore suitable for in vivo evaluation. I chose it for that reason.

[0106] Example 7. Evaluation of the therapeutic efficacy of DL80 for melanoma treatment. Using a wild-type B6 mouse model, DL80 significantly inhibited the growth of hIL-2 in blood-depleted mice compared to hIL-2. It was confirmed that the stability was increased by approximately 46-fold compared to the control (FIG. 15). Next, the therapeutic effect of DL80 was evaluated in a B16F10 melanoma mouse model. Mice were treated with equimolar concentrations of DL80 or hIL-2 in each injection for a total of 24 days of treatment. The tumor markers were administered subcutaneously at different frequencies (DL80 every 6 days, hIL-2 daily). TA99, a mAb that recognizes carcinoma TRP1 and is well known for its synergistic activity with IL-2, was used. Both groups were treated simultaneously with a dose of 150 μg daily. After treatment, the animals were allowed to recover. Tumor size (Fig. 6a) and survival (Fig. 6b) were analyzed simultaneously for one month. Only 1 / 6 of the therapeutic dose was used, and the fusion construct DL80 showed moderate PK. Despite the fact that only improvements were observed in the WT B6 mouse model, DL80 and h The IL-2 treatment group showed a significant reduction in tumor burden and overall survival compared to the PBS control. compared to 25% (2 mice) in the hIL-2 group and 0% in the PBS group. Fifty percent (4 mice) of the DL80-treated melanoma animals survived. Near complete tumor regression was observed. A clear recurrence of tumor formation was observed in hIL-2-treated mice, whereas it was observed in the DL80-responsive model. This indicates the superiority of duraleukin in cancer treatment. To further explore the potential mechanism(s) of antitumor efficacy, we performed different post-treatment In this step, tumors were isolated and corresponding immune cells were analyzed by flow cytometry. Although no significant changes were detected 1 day after treatment, they were significantly altered 3 days after injection. In DL80-treated tissue, CD8+ T cells and natural killer (NK) A significant increase in both the number of immune cells was observed, indicating that duraleukin stimulates the activation of these immune cells. These results indicate that the IL-16 receptor agonist (IL-16) efficiently penetrated tumors and exerted its antitumor activity.

[0107] Example 8. HSA-binding Nb improves drug stability and therapeutic efficacy. Many small biomolecules and therapeutic agents (including Nb) are difficult to treat due to their poor stability in vivo. Here, the remarkable physicochemical and biophysical properties of camelid Nb To solve this fundamental problem in drug delivery, we have developed a robust and A general approach was developed. A large cohort of high-quality HSA-Nbs was Cutting-edge interdisciplinary approaches spanning proteomics, structural biology, and computational modeling The accurate and fast throughput of Nb PK has been systematically characterized using We developed a novel proteomic method to measure the stable Nb fusion site. The tocainide duraleukin has been developed and its antitumor efficacy has been preferentially characterized.

[0108] Four dominant epitopes on HSA support the immunodominance hypothesis (decorated clitoris). A preference for concave recognition by Nb was observed, which is due to the fact that convex This can be explained by the Nb structure. However, most of our Nbs still show significant affinity for antigen engagement comparable to IgG. Consistency and specificity can be achieved.

[0109] Considering the high serum concentration of albumin, the high affinity of Nb is not necessary for the increased PK. The above results show the opposite: affinity and pH dependence Both FcRn-mediated endocytosis and efficient cargo transport were positively correlated. The importance of the microenvironment to the local concentration of receptor complexes for delivery is emphasized.

[0110] Example 9. Methods. Nb DNA synthesis and cloning. The Nb gene was synthesized from Escherichia coli The vector was codon-optimized for expression in vitro and nucleotides were synthesized in vitro (Synbi). After verification by Sanger sequencing, the Nb gene was cloned using the BamHI and X pET-21b(+) at the hoI or EcoRI and NotI restriction enzyme cleavage sites It was cloned into a vector.

[0111] Purification of recombinant Nb. Nb DNA constructs were transformed into BL21(DE3) cells. The cells were transformed and plated on agar with 50 μg / mL ampicillin overnight at 37°C. One bacterial colony was picked for LB broth culture and IPTG Nb protein induction. Briefly, when the OD reached approximately 0.4-0.6, 0.5 mM IPTG was added to the E. coli cell culture and Nb was induced overnight at 16 °C. Then, the cells Collect the cells, sonicate briefly, and lyse them in lysis buffer (1x PBS, 150 mM N, The cells were lysed using TX-100 containing aCl and 0.2% protease inhibitors. The soluble protein extract was collected at 15,000 x g for 10 min, and the recombinant Nb was purified by Hi The product was purified on s6-cobalt resin (Thermo) and eluted with imidazole. The eluted Nb was dialyzed against dialysis buffer (e.g., 1X DPBS, pH 7.4) and used. Previously stored at -80.

[0112] ELISA (Enzyme-Linked Immunosorbent Assay). Indirect ELISA is performed to measure immune responses and Nanobody affinity was evaluated. Antigens were plated onto 96-well ELISA plates (R&D syst Coating buffer (15 mM sodium carbonate, 35 mM sodium bicarbonate) The plates were coated with 1-10 ng / well of PBS (pH 9.6) overnight at 4°C and incubated for 2 h at room temperature. Blocking was performed with blocking buffer (DPBS, 0.05% Tween 20, 5% milk) for 1 h. Immune sera or nanobodies were serially diluted in blocking buffer and incubated at 2°C with the antigen. The cells were incubated for 1 h. Llama Fc (Thermo) or his tag (Genscript HRP-conjugated secondary antibodies against pt) were diluted 1:5,0 in blocking buffer. The solution was diluted to 10,000 and incubated at room temperature for 1 hour in 1x PBST (DP Nonspecific absorbance was removed by washing three times with BS, 0.05% Tween 20). After washing, the samples were analyzed by ion exchange using freshly prepared 3,3',5,5'-tetramethylbenzidine (TM B) Signal was generated by incubation with substrate for 10 minutes at room temperature in the dark. After the stop solution (R%D system), the plate was read in a plate reader (Multis At multiple wavelengths (density at 450 nm, kan GO, Thermo Fisher) The raw data was then read at Pr Processed by ism7 (GraphPad) and fitted to 4PL curves to obtain logIC50, Alternatively, if the results could not be fitted to a curve, the mean value was calculated.

[0113] In vitro bead pull-down. Surface plasmon resonance (SPR) surface plasmon resonance ( Nb by SPR, Biacore 3000 system (GE Healthcare) Affinity assays were used to measure the affinity of Nbs. Briefly, GST, OMP25 PDZ domains or protein antigens such as human serum albumin are bound to activated CM5 cells. The protein sample was immobilized in the flow channel of the sensor chip. 5) diluted to 10-30 μg / mL and measured at 5 μL / min for 420 seconds using the SPR system. The surface was then blotting with 1 M ethanolamine-HCl (pH 8.5). For each Nb sample, a roughly 1000-fold range of 2 mM DTT-containing A series of dilutions were run using HBS-EP+ running buffer (GE-Healthcare). The dilution was injected in duplicate at a flow rate of 20-30 μL / min for 120-180 seconds, and then the dilution was Dissociation times of 5 to 20 minutes were allowed based on dissociation rate. , a low pH buffer containing 10 mM glycine-HCl (pH 1.5-2.5), 20-40 mM NaOH (pH 12-13) at a flow rate of 40-50 μL / min for 2 seconds. The binding sites for each Nb were regenerated with a high pH buffer solution (pH 7.0) at 40-50 μL / min for 30 seconds. The scangram is processed to generate a 1:1 Langmuir model or a 1:1 run with mass transfer. Analysis was performed using BIA evaluation by fitting with the Gummi model.

Claims

1. A human serum albumin (HSA)-binding polypeptide and an IL-2 polypeptide. A recombinant Nanobody, wherein the HSA-binding polypeptide comprises epitope 1, epitope 2, epitope 3, epitope 4, epitope 5, epitope 6, epitope 7, epitope 8, epitope 9, epitope 10, epitope 11, epitope 12, epitope 13, epitope 14, epitope 15, epitope 16, epitope 17, epitope The present invention relates to an HSA epitope selected from the group consisting of epitope 2, epitope 3, and epitope 4. heterogeneously bound, Epitope 1 is amino acid residues 298-307, 311, and 332-341 of SEQ ID NO:1 , and 371 to 386, Epitope 2 is amino acid residues 5-13, 62-67, 93-99, and 228 to 266, inclusive; Epitope 3 comprises amino acid residues 226-230 and 298-337 of SEQ ID NO:

1. See and Epitope 4 comprises amino acid residues 33-38 and 111-145 of SEQ ID NO: 1; The recombinant nanobody.

2. The HSA-binding polypeptide specifically binds to human HSA and mouse serum albumin.

2. The recombinant nanobody of claim 1 .

3. The HSA-binding polypeptide is selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO:

4.

3. The recombinant Nanobody of claim 1 or 2, comprising an amino acid sequence selected from:

4. The IL-2 polypeptide has the amino acid sequence of SEQ ID NO:5, SEQ ID NO:103, or 4. The recombinant Nanobody of claim 1, comprising a functional fragment of 。

5. The HSA-binding polypeptide is linked to the IL-2 polypeptide via a linker.

5. The recombinant Nanobody of claim 1 ,

6. The recombinant nanobody of claim 5, wherein the linker comprises the amino acid sequence of SEQ ID NO:

6. Day.

7. 7. The method of claim 1, wherein the recombinant Nanobody is less than about 50 kDa. Recombinant nanobodies described.

8. The HSA-binding polypeptide binds to the IL-2 polypeptide and its IL-2 receptor. The recombinant Nanobody of any one of claims 1 to 7, wherein the recombinant Nanobody does not reduce the affinity between 。

9. the recombinant Nanobody is selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 The recombinant nanobody of any one of claims 1 to 8, comprising a selected amino acid sequence. Yes.

10. 1. A method for treating cancer in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of human serum albumin (e.g., a therapeutically effective amount of a recombinant human IL-2 polypeptide comprising an HSA-binding polypeptide and an IL-2 polypeptide. and administering a nanobody to a subject, the nanobody comprising the HSA-binding polypeptide binding to epitope 1, epitope 2, epitope 3, epitope 4, epitope 5, epitope 6, epitope 7, epitope 8, epitope 9, epitope 10, epitope 11, epitope 12, epitope 13, epitope 14, epitope 15, epitope 16, epit an HSA epitope selected from the group consisting of epitope 2, epitope 3, and epitope 4; specifically binds to the Epitope 1 is amino acid residues 298-307, 311, and 332-341 of SEQ ID NO:1 , and 371 to 386, Epitope 2 is amino acid residues 5-13, 62-67, 93-99, and 228 to 266, inclusive; Epitope 3 comprises amino acid residues 226-230 and 298-337 of SEQ ID NO:

1. See and Epitope 4 comprises amino acid residues 33-38 and 111-145 of SEQ ID NO: 1; The method.

11. The HSA-binding polypeptide specifically binds to human HSA and mouse serum albumin. The method of claim 10 .

12. The HSA-binding polypeptide is selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO:

4. The method of claim 11 or 12, comprising an amino acid sequence selected from:

13. The IL-2 polypeptide has the amino acid sequence of SEQ ID NO:5, SEQ ID NO:103, or The method according to any one of claims 10 to 12, comprising a functional fragment of

14. The HSA-binding polypeptide is linked to the IL-2 polypeptide via a linker. The method according to any one of claims 10 to 13,

15. 15. The method of claim 14, wherein the linker comprises the amino acid sequence of SEQ ID NO:

6.

16. 16. Any one of claims 10 to 15, wherein the recombinant Nanobody is less than about 50 kDa. The method described below.

17. The HSA-binding polypeptide binds to the IL-2 polypeptide and its IL-2 receptor. The method according to any one of claims 10 to 16, wherein the method does not reduce the affinity between

18. the recombinant Nanobody is selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 The method of any one of claims 10 to 17, comprising a selected amino acid sequence.

19. The recombinant Nanobody exhibits approximately 100-fold higher in vivo activity than the native IL-2 polypeptide. The method of any one of claims 10 to 18, wherein the compound has a half-life.

20. Any of claims 10 to 19, wherein the cancer is selected from the group consisting of melanoma and renal cancer.

2. The method according to claim 1.

21. 1. A Nanobody comprising a human serum albumin (HSA) binding polypeptide, The HSA-binding polypeptide comprises a complementarity-determining region (CDR) 3, the CDR3 comprising: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 1 5, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, Sequence number 27, sequence number 28, sequence number 29, sequence number 30, sequence number 31, sequence number 32 , SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO: No. 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:5 5, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, Sequence number 67, sequence number 68, sequence number 69, sequence number 70, sequence number 71, sequence number 72 , SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO: No. 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 9 5, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, or SEQ ID NO:100 The Nanobody comprises an amino acid sequence having at least 50% similarity to

22. The CDR3 is SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, Sequence number 20, sequence number 21, sequence number 22, sequence number 23, sequence number 24, sequence number 25 , SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: No. 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 4 8, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, Sequence number 60, sequence number 61, sequence number 62, sequence number 63, sequence number 64, sequence number 65 , SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: No. 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 8 8, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: No. 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, and and SEQ ID NO:

100. Nobody.

23. The HSA-binding polypeptide comprises epitope 1, epitope 2, epitope 3, and epitope 4. specifically binds to an HSA epitope selected from the group consisting of epitope 4; Epitope 1 is amino acid residues 298-307, 311, and 332-341 of SEQ ID NO:1 , and 371 to 386, Epitope 2 is amino acid residues 5-13, 62-67, 93-99, and 228 to 266, inclusive; Epitope 3 comprises amino acid residues 226-230 and 298-337 of SEQ ID NO:

1. See and Epitope 4 comprises amino acid residues 33-38 and 111-145 of SEQ ID NO: 1; 23. A nanobody according to claim 21 or 22.

24. The nanoparticles according to any one of claims 21 to 23, further comprising an IL-2 polypeptide. Day.

25. The IL-2 polypeptide has the amino acid sequence of SEQ ID NO:5, SEQ ID NO:103, or 25. The nanobody of claim 24, comprising a functional fragment of

26. The HSA-binding polypeptide is linked to the IL-2 polypeptide via a linker.

26. The nanobody of claim 24 or 25.

27. 27. The nanobody of claim 26, wherein the linker comprises the amino acid sequence of SEQ ID NO:

6.

28. The nanobody of any one of claims 21 to 27, wherein the nanobody is less than 50 kDa. Nobody.

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