Serum albumin-binding nanobody compositions and methods of use thereof

Abstract

Disclosed herein are compositions and methods for increasing the stability of recombinant Nanobodies. Also disclosed herein are recombinant Nanobodies comprising an IL-2 polypeptide that binds to serum albumin, and their use for treating cancer.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 986,180, filed March 6, 2020, 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 Nbs are the smallest intact, natural antigen-binding domains derived from human IgG (H). Nbs are characterized by good solubility, thermal stability, tissue penetration, and low toxicity to humans due to their high sequence similarity to human IgG2 / 3. However, their therapeutic potential is significantly limited by their short T1 / 2 (usually less than a few hours). Due to their small size (approximately 15 kDa) and the absence of heavy-light chain pairing and glycosylation, Nbs can be synthesized as recombinant DNA fragments for rapid bulk production from E. coli. The ease of bioengineering Nbs facilitates the development of multifunctional agents. Similar to IgG, the antigen-binding specificity of Nbs is primarily mediated by the hypervariable loops of the complementarity-determining regions (CDRs, 3 and 6 in IgG)—CDR3 being the most variable "fingerprint." The CDR loops are presented by a robust tertiary core composed of four highly conserved framework regions. It has been hypothesized that with smaller paratopes, Nbs may have impaired affinity for antigen engagement. Several pilot structural studies indicate that Nbs can access "secret" epitope-like cavities on antigens that may be inaccessible to IgG due to steric hindrance. Binding to these epitopes is achieved by both their small size and their "convex" shape, which together facilitate insertion of the CDR loop(s) into the groove.

[0004] Although many Nb have been developed for specific targets, the field remains limited by the availability of high-quality and multifunctional Nb agents for drug delivery. While solubility, thermal stability, cross-species binding, and robustness to bioengineering are likely important, it remains unclear whether and to what extent other biophysical, physicochemical, and structural characteristics are important. Furthermore, because biologics often have varying therapeutic windows, a sustainable, fine-tuned, and individualized half-life is highly desirable to maximize drug efficacy while reducing side effects. A systematic and comprehensive evaluation of these factors will aid in the design of next-generation drugs for precision medicine.

[0005] Human serum albumin (HSA) is the most stable serum protein, with a half-life of approximately 3 weeks. Unlike small molecules, which are primarily removed by glomerular filtration, HSA (67 kDa) cannot be directly removed by the kidney. Following internalization, HSA forms a stable complex with cellular FcRn, a coreceptor for IgG. It has been shown that HSA can be efficiently recycled by FcRn-mediated endocytosis, thereby avoiding the rapid lysosomal degradation common to large serum proteins. Although the local concentration(s) of HSA-FcRn interactions within endosomes where the complex is recycled remain unclear, the resistance of the acidic environment of the endosome may be a prerequisite for successful HSA recycling and its stabilization.

[0006] However, the interaction of HSA with Nb has not yet been characterized, and there is a continuing need for effective nanobody therapy in many areas, including cytokine therapy. Cytokine therapy is a key component of immune regulation. Interleukin-2 (IL-2) is a central immune cytokine important for homeostasis and the control of T cell activity. By controlling the proliferation of cytotoxic T cells, IL-2 upregulates immune responses and suppresses tumor progression. Proleukin (aldesleukin, Novartis) is a recombinant form of human interleukin-2 (IL-2) and was the first approved cancer immunotherapy agent. IL-2 is used to treat advanced melanoma and metastatic renal cell carcinoma. Such treatment has been reported to result in complete remission in up to 10% of patients with metastatic tumors, sometimes without recurrence for up to 25 years. Approximately 70% of patients treated with IL-2 experience complete tumor regression. However, major drawbacks of IL-2 include its poor pharmacokinetics, low drug efficacy, and high toxicity. Due to its small size (approximately 15 kDa), IL-2 is rapidly cleared from the circulation by glomerular filtration and has a median half-life of less than 30 minutes after administration to humans. Consequently, high doses and repeated administration (often 7 hours per administration) are often required, which can result in serious toxicity and side effects, including vascular leak syndrome.

[0007] For drug delivery, the availability of high-quality and multifunctional agents is limited. While solubility, thermal stability, cross-species binding, and bioengineering robustness are likely important, it remains unclear whether and to what extent other biophysical, physicochemical, and structural characteristics are important. Furthermore, because biologics often vary significantly in their therapeutic window, a fine-tuned, sustainable, and personalized half-life is highly desirable to maximize drug potential while reducing side effects.

[0008] Thus, there is a need for compositions and methods for maximizing the potency and half-life of therapeutic compositions such as IL-2. The compositions and methods disclosed herein address these and other needs. Summary of the Invention

[0009] In some aspects, disclosed herein are recombinant Nanobodies comprising a human serum albumin (HSA)-binding polypeptide and an IL-2 polypeptide. In some embodiments, 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, where epitope 1 comprises amino acid residues 298-307, 311, 332-341, and 371-386 of SEQ ID NO: 1; epitope 2 comprises amino acid residues 5-13, 62-67, 93-99, and 228-266 of SEQ ID NO: 1; epitope 3 comprises amino acid residues 226-230 and 298-337 of SEQ ID NO: 1; and epitope 4 comprises amino acid residues 33-38 and 111-145 of SEQ ID NO: 1. In some embodiments, the HSA-binding polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4. Also disclosed herein are Nanobodies comprising a human serum albumin (HSA)-binding polypeptide, wherein the HSA-binding polypeptide comprises a complementarity-determining region (CDR) 3, wherein the CDR3 comprises an amino acid sequence having at least 50% similarity to any of SEQ ID NOs: 10-100. In some embodiments, the recombinant Nanobodies disclosed herein are less than 50 kDa in size. In some embodiments, the Nanobodies further comprise an IL-2 polypeptide. Such Nanobodies can surprisingly improve the stability of the IL-2 polypeptide without significantly reducing the affinity of the IL-2 polypeptide of the Nanobody for the IL-2 receptor.

[0010] It is shown herein that administration of the recombinant Nanobodies disclosed herein can reduce tumor volume and improve survival in treated subjects. Accordingly, in some aspects, disclosed herein are methods of treating cancer in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant Nanobody comprising a human serum albumin (HSA) binding polypeptide and an IL-2 polypeptide. [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 in 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 Nbs are shown. Measurements were performed 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, a large repertoire of nanobodies (Nbs) for HSA binding has been identified by immunization of camelids and using an integrated proteomics pipeline. Here, a cohort of high-quality HSA-Nbs was developed and systematically characterized using a multidisciplinary approach. In some embodiments, several HSA-Nbs were combined with the cytokine human IL-2 to develop a highly stable composition, collectively referred to as "duraleukins," that can be used to treat melanoma in mouse models.

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

[0014] First, there is the ease of production and manufacturing. IL-2 Nb fusions (Duraleukin) are shown herein to be readily producible in bulk in bacteria, e.g., E. coli cells, as highly purified, intact, and functional proteins. Note that the high doses of IL-2 (Proleukin (Aldesleukin, Novartis)) are a perk of this drug's efficacy. In contrast, production of Albroukin (Human Genome Sciences) is primarily limited in more expensive mammalian cells, limiting its practical utility.

[0015] Second, there is the ease of bioengineering. Bioengineering of duraleukin is simple and straightforward. In one example, it can be fused at the C-terminus to another polypeptide drug, creating a "trifunctional drug." This is in stark contrast to albroukin (Human Genome Sciences), which can be very difficult to bioengineer.

[0016] Third, there is significant flexibility and different in vivo half-lives for optimized / tailored development and clinical use. Using a humanized albumin mouse model, it is shown herein that different albumin Nbs have different in vivo pharmacokinetics (PK). This indicates that different duraleukins with different HSAs can have unique PK, which can be exploited to optimize drug efficacy and minimize drug side effects, which is a major problem with the commercial drug Proleukin (aldesleukin, Novartis).

[0017] Fourth, it has excellent efficacy. The recombinant polypeptide inherits the remarkable physicochemical properties of Nb, such as thermostability and resistance (almost immune) to serum protease activity. Being three times smaller than albroukin (Human Genome Sciences) (30 kDa vs. 90 kDa), duraleukin is structurally intact and retains the full functionality of the carrier protein IL-2. Due to its limited drug-loading capacity, its small size allows duraleukin to easily reach the maximally efficient molar concentration for drug administration. While fusion of the intact small domain of Nb (15 kDa) does not significantly affect the interaction of IL-2 with its receptor, the FcRn complex, large recombinant proteins such as albroukin (Human Genome Sciences) may be affected by structural steric hindrance, especially when combined with the large and bulky IL-2 receptor complex.

[0018] Thus, disclosed herein are compositions that improve the stability and / or efficacy of IL-2 in the treatment of disease (e.g., cancer), the composition being a recombinant Nanobody comprising a human serum albumin (HSA)-binding polypeptide and an IL-2 polypeptide. In some embodiments, the recombinant Nanobody increases the half-life of IL-2 compared to native or recombinant IL-2 polypeptide. Administration of the recombinant Nanobody comprising a human serum albumin (HSA)-binding polypeptide and an IL-2 polypeptide 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 specific definitions as set forth below.

[0020] term As used in this specification and claims, the singular forms "a," "an," and "the" include 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, the term "about" when referring to a measurable value, such as an amount, percentage, etc., is meant to encompass a variation of ±20%, ±10%, ±5%, or ±1% from the measurable value.

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

[0023] The term "antibody" is used broadly herein and includes polyclonal, monoclonal, and bispecific antibodies. In addition to intact immunoglobulin molecules, the term "antibody" also includes fragments or polymers of those immunoglobulin molecules, as well as human or humanized forms of immunoglobulin molecules or fragments thereof. Antibodies are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain contains at one end a variable domain (V H ), followed by several constant domains. Each light chain has a variable domain (V) at one end. L ) at one end and a constant domain at the other end.

[0024] Antibodies can be tested for their desired activity using the in vitro assays described herein or by similar methods, and then their in vivo therapeutic and / or prophylactic activity is tested according to known clinical testing methods. There are five major classes of human immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. Those skilled in the art will recognize the equivalent classes in mice. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.

[0025] The terms "antigenic determinant" and "epitope" are also used interchangeably herein and refer to a location on an antigen or target that is recognized by an antigen-binding molecule (such as a Nanobody of the invention). Epitopes can be formed from both contiguous amino acids ("linear epitopes") or non-contiguous amino acids juxtaposed by tertiary folding of a protein. The latter epitopes, when formed by at least some non-contiguous amino acids, are referred to herein as "conformational epitopes." Epitopes typically comprise at least three, more usually at least five or eight to ten amino acids, in a unique spatial structure. Methods for determining the spatial structure of an epitope include, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, "Epitope Mapping Protocols in Methods in Molecular Biology," Vol. 66, Glenn E. Morris, Ed. (1996).

[0026] The terms "antigen-binding site," "binding site," and "binding domain" refer to specific elements, portions, or amino acid residues of a polypeptide, such as a nanobody, that bind to an antigenic determinant or epitope.

[0027] The term "cancer" refers to cells that exhibit a relatively autonomous growth and thus exhibit an abnormal growth phenotype characterized by a significant loss of control of cell proliferation (i.e., uncontrolled cell division). Cancer cells can be malignant or benign. Examples of various cancers include, but are not limited to, melanoma, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, 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 the portion of the variable chain of an antibody that is involved in binding to the antigen. Thus, a CDR is part of or is the "antigen binding site." In some embodiments, a nanobody comprises three CDRs that together form the antigen binding site.

[0029] As used herein, the term "comprising" and variations thereof are used synonymously with the term "including" and variations thereof and are open, non-limiting terms. Although the terms "comprising" and "including" have been used herein to describe various embodiments, the terms "consisting essentially of" and "consisting of" may be used in place of "comprising" and "including" to provide more specific embodiments, and are disclosed.

[0030] "Composition" refers to any agent that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, such as, for example, treating a disorder or other undesirable physiological condition, and prophylactic effects, such as, for example, preventing a disorder or other undesirable physiological condition. These terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of the beneficial agents specifically mentioned herein, including, but not limited to, bacteria, vectors, polynucleotides, cells, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the term "composition" is used, or when a particular composition is specifically identified, it should be understood that the term includes the composition itself as well as pharmaceutically acceptable, pharmacologically active vectors, polynucleotides, salts, esters, amides, enhancers, conjugates, active metabolites, isomers, fragments, analogs, and the like. In some aspects, the compositions disclosed herein comprise recombinant polypeptides, including human serum albumin (HSA)-binding polypeptides and IL-2 polypeptides.

[0031] An "effective amount" includes, but is not limited to, an amount that is capable of alleviating, reversing, mitigating, preventing, or diagnosing a symptom or sign of a medical condition or disease (e.g., cancer). Unless otherwise indicated explicitly or by context, an "effective amount" is not limited to the minimum amount sufficient to alleviate a condition. The severity of a disease or disorder, as well as the ability of a treatment to prevent, treat, or alleviate a disease or disorder, can be measured by biomarkers or by clinical parameters, without implying any limitations. In some embodiments, the term "effective amount of a recombinant Nanobody" refers to an amount of a recombinant Nanobody sufficient to prevent, treat, or alleviate cancer.

[0032] A "fragment," or "functional fragment," may include insertions, deletions, substitutions, or other selected modifications of specific regions or specific amino acid residues, whether or not linked to other sequences, provided that the activity of the fragment is not significantly altered or impaired compared to the unmodified peptide or protein. These modifications may confer some additional property, such as removal or addition of disulfide-bond-capable amino acids, increased biological lifespan, or altered secretion characteristics. In all cases, the functional fragment must retain the property of biological activity, such as binding to HSA and / or ameliorating cancer.

[0033] The "half-life" of an amino acid sequence, compound, or polypeptide of the invention can generally be defined as the time it takes for the serum concentration of the amino acid sequence, compound, or polypeptide to decline by 50% in vivo, for example due to degradation of the sequence or compound and / or clearance or segregation of the sequence or compound by natural mechanisms. The in vivo half-life of a Nanobody, amino acid sequence, compound, or polypeptide of the invention can be measured in any known manner, such as by pharmacokinetic analysis. These include, for example, Kenneth, A. et al., Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists; Peters et al., Pharmacokinete analysis: A Practical Approach (1996); "Pharmacokinetics", M. Gibaldi & D. Perron (revised 2nd ed.) published by Marcel Dekker (1982).

[0034] The term "identity" or "homology" should be interpreted to mean the percentage of nucleotide bases or amino acid residues in a candidate sequence that are identical to the bases or residues of the corresponding sequence being compared, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity for the entire sequence, and without considering any conservative substitutions as part of the sequence identity. A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) having a particular percentage (e.g., 80%, 85%, 90%, or 95%) of "sequence identity" to another sequence means that, when aligned, that percentage of bases (or amino acids) are identical in the comparison of the two sequences. This alignment and percent homology or percent sequence identity can be measured using software programs known in the art. Such alignments can be produced, for example, using the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, conveniently implemented by computer programs such as the Align program (DNAstar, Inc.).

[0035] As used herein, the term "increased" or "increase" generally refers to a statistically significant increase. For the avoidance of doubt, "increased" refers to an increase of at least 10% compared to the reference level, such as at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to 100% (inclusive), or any increase between 10 and 100% compared to the reference level, or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold, or any increase between 2-fold and 10-fold or more compared to the reference level.

[0036] As used herein, the terms "nanobody," "V H H," "V H "H antibody fragment" and "single domain antibody" are used interchangeably and refer to a single heavy chain variable domain of an antibody typically found in Camelidae, without any light chains, such as those from Camelidae, as described in PCT Publication No. WO 94 / 04678, which is incorporated by reference in its entirety.

[0037] As used herein, "operably linked" refers to the arrangement of polypeptide segments within a single polypeptide chain, where the individual polypeptide segments are, but are not limited to, proteins, fragments thereof, connecting peptides, and / or signal peptides. The term "operably linked" can refer to the direct fusion of different individual polypeptides within a single polypeptide or fragment thereof, with no intervening amino acids between the different segments, as well as when the individual polypeptides are joined to each other by a "linker" that comprises one or more intervening amino acids.

[0038] As used herein, the terms "decreased," "decreasing," "reduction," or "reducing" generally refer to a statistically significant decrease. However, for the avoidance of doubt, "decreased" refers to a decrease of at least 10% compared to a reference level, e.g., at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including a 100% decrease (i.e., no level compared to a reference sample), or any decrease between 10 and 100% compared to a reference level.

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

[0040] The terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: genes or gene fragments, exons, introns, messenger RNA (mRNA), transcribed RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, 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 imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides may be further modified after polymerization, such as by conjugation with a labeling component. The term refers to both double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention that is a polynucleotide encompasses both the double-stranded form and each of the two complementary single-stranded forms that are known to constitute or are predicted to constitute that double-stranded form.

[0041] The term "polypeptide" is used in the broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics. The subunits can be linked by peptide bonds. In alternative embodiments, the subunits can be linked by other bonds, such as esters, ethers, etc. As used herein, the term "amino acid" refers to any natural and / or unnatural or synthetic amino acid, including glycine and both the D or L optical isomers, as well as amino acid analogs and peptidomimetics. Peptides of three or more amino acids are commonly called "oligopeptides" when the peptide chain is short. When the peptide chain is long, the peptide is commonly called a "polypeptide" or a "protein."

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

[0043] As used herein, the term "carrier" includes any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material known in the art for use in pharmaceutical formulations. The choice of carrier for use in a composition will depend on the intended route of administration of the composition. The preparation of pharmaceutically acceptable carriers and formulations containing these materials is described, for example, in Remington's Pharmaceutical Sciences, 21st Edition, ed. University of the Sciences in Philadelphia, Lippincott, Williams & Wilkins, Philadelphia, PA, 2005. Examples of physiologically acceptable carriers include buffers such as saline, glycerol, DMSO, phosphate buffer, citrate buffer, and buffers with other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or non-ionic surfactants such as TWEEN® (ICI, Inc.; Bridgewater, New Jersey), polyethylene glycol (PEG), and PLURONICS® (BASF; Florham Park, NJ). To provide for administration of such dosages for the desired therapeutic treatment, the compositions disclosed herein may advantageously contain from about 0.1 to 99% by total weight of one or more of the subject compounds, based on the weight of the total composition including carriers and diluents.

[0044] "Recombinant" as used herein in reference to polypeptides means a combination of two or more polypeptides that is not a combination found in nature.

[0045] The term "specificity" refers to the number of different antigens or antigenic determinants to which a particular antigen-binding molecule (such as a Nanobody of the invention) can bind. Nanobodies with low specificity bind to multiple different epitopes via a single antigen-binding site or binding domain, while Nanobodies with high specificity bind to one to several epitopes via a single antigen-binding site or binding domain. In some embodiments, several epitopes are similar or very similar, e.g., cross-species epitopes. As used herein, the term "specifically binds to," as used herein with respect to Nanobodies, means that a Nanobody binds preferentially to one epitope over other epitopes. Specific binding can vary depending on the binding affinity and stringency of the binding conditions. In one example, a Nanobody specifically binds to an epitope when high-affinity binding exists under stringent conditions. In some embodiments, an HSA-binding polypeptide or Nanobody described herein specifically binds to human serum albumin.

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

[0047] A "therapeutically effective amount" refers to an amount of a composition, such as a recombinant Nanobody comprising a human serum albumin (HSA)-binding polypeptide and an IL-2 polypeptide, that elicits a biological or medical response in a tissue, system, animal, or human that is being sought by a researcher, veterinarian, physician, or other clinician over a generalized period of time. In some embodiments, the desired response is a decrease in tumor size, alleviation of cancer (e.g., melanoma) and / or associated symptoms. In other embodiments, the desired response is an increase in an anti-tumor immune response, including, for example, activation of anti-tumor cytotoxic T 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 is achieved after administering multiple dosages of the composition to the subject over a period of days, weeks, or years. A therapeutically effective amount will vary depending on the composition, the disorder or condition and its severity, the route of administration, the time of administration, the rate of excretion, the drug combination, the judgment of the treating physician, the dosage form, and the age, weight, general health, sex, and / or diet of the subject being treated. A therapeutically effective amount of a recombinant Nanobody, comprising a human serum albumin (HSA) binding polypeptide and an IL-2 polypeptide described herein can be determined by one of skill in the art.

[0048] A therapeutically significant reduction in symptoms is, 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 125%, at least about 150%, or more in a measured parameter compared to a control or untreated subject. Measured or measurable parameters include an increase or decrease in the level of a clinically detectable disease marker, e.g., a biological marker, such as a reduction in tumor size; an increase in an anti-tumor immune response, including, for example, activation of anti-tumor cytotoxic T lymphocytes, natural killer cells, and / or B cells; an increase in the level of tumor antigen-specific antibodies in the subject's circulation and / or tumor; and / or prolongation of the subject's survival. It will be understood that the total daily usage of the compositions and formulations disclosed herein will be determined by the attending physician within the bounds of good medical practice. The exact amount required will vary depending on factors such as the type of disease being treated.

[0049] As used herein, the terms "treat," "treating," "treatment," and grammatical variations thereof include partially or completely delaying, alleviating, ameliorating, or reducing the intensity of one or more associated symptoms of cancer or a condition, and / or alleviating, alleviating, or inhibiting one or more causes of cancer. Treatment according to the present invention may be applied preventively, prophylactically, palliatively, or therapeutically. In some embodiments, the treatment is a reduction in tumor size. In some embodiments, the treatment is a reduction in cancer metastasis or cancer lesions. In some embodiments, the treatment is a reduction in tumor count.

[0050] The prophylactic composition can be administered to a subject before onset (e.g., before overt signs of cancer), during early onset (e.g., at the earliest signs and symptoms of cancer), after established progression of cancer, or at a later stage of cancer. Prophylactic administration can occur minutes to months before the appearance of cancer.

[0051] composition Disclosed herein are recombinant Nanobodies comprising a human serum albumin (HSA)-binding polypeptide and an IL-2 polypeptide, wherein the HSA-binding polypeptide specifically binds to an HSA epitope. As described above, this group of recombinant Nanobodies is shown herein to be surprisingly effective in extending the half-life of the IL-2 polypeptide. In some embodiments, the affinity of the IL-2 polypeptide of the Nanobody for the IL-2 receptor is not, or is not, significantly reduced compared to IL-2 that is not part of or bound to the Nanobody.

[0052] With respect to the human serum albumin portion of a Nanobody, "serum albumin" refers to a type of globular protein found in the blood of vertebrates. Serum albumin is produced in the liver. "Human serum albumin" or "HSA," as used herein, refers to a polypeptide that hydrolyzes cyclic adenosine 5'-diphosphate-ribose and is encoded by the ALB gene. In some embodiments, the HSA polypeptide is identified in one or more publicly available databases, as follows: HGNC:399, Entrez Gene:213, Ensembl:ENSG00000163631, OMIM:103600, UniProtKB:P02768. In some embodiments, the HSA polypeptide comprises the sequence of SEQ ID NO: 1, or a polypeptide sequence having about 80%, about 85%, about 90%, about 95%, or about 98% homology to SEQ ID NO: 1, or greater than about 80%, about 85%, about 90%, about 95%, or about 98% homology thereto, or a polypeptide comprising a portion of SEQ ID NO: 1. The HSA polypeptide of SEQ ID NO: 1 may represent immature HSA or may be pre-processed from mature HSA, and thus, as used herein, includes mature or processed portions of the HSA polypeptide of SEQ ID NO: 1.

[0053] As mentioned above, an "epitope" is a location on an antigen or target that is recognized by an antigen-binding molecule (such as a Nanobody of the invention). The term "epitope" includes both linear and conformational epitopes. Figure 16 shows several epitopes on HSA; these epitopes are referred to as "clusters."

[0054] Thus, in some embodiments, disclosed herein are recombinant Nanobodies 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, wherein epitope 1 comprises amino acid residues 298-307, 311, 332-341, and 371-386 of SEQ ID NO:1; epitope 2 comprises amino acid residues 5-13, 62-67, 93-99, and 228-266 of SEQ ID NO:1; epitope 3 comprises amino acid residues 226-230 and 298-337 of SEQ ID NO:1; and epitope 4 comprises amino acid residues 33-38 and 111-145 of SEQ ID NO:1. In some embodiments, the HSA binding polypeptide specifically binds to an HSA epitope comprising amino acid residues 298-307, 311, 332-341, and 371-386 of SEQ ID NO: 1. In some embodiments, the HSA binding polypeptide specifically binds to an HSA epitope comprising amino acid residues 5-13, 62-67, 93-99, and 228-266 of SEQ ID NO: 1. In some embodiments, the HSA binding polypeptide specifically binds to an HSA epitope comprising amino acid residues 33-38 and 111-145 of SEQ ID NO: 1. In some embodiments, the HSA binding polypeptide specifically binds to an HSA epitope comprising amino acid residues 298-307, 311, 332-341, and 371-386 of SEQ ID NO: 1.

[0055] It should be understood that the specificity of an antigen-binding molecule (e.g., an HSA-binding polypeptide, a Nanobody of the invention) can be measured based on affinity and / or avidity. The dissociation equilibrium constant (K DThe affinity, expressed by K ), is an index of the binding strength between an antigenic determinant and an antigen-binding site on an antigen-binding molecule. D The smaller the value of K, the stronger the binding strength between the antigenic determinant and the antigen-binding molecule (or the affinity is expressed as 1 / K D is the affinity constant (K A )). Methods for measuring affinity are well known to those skilled in the art. Avidity is an indicator of the strength of binding between an antigen-binding molecule (such as an HSA-binding polypeptide and a Nanobody of the invention) and an appropriate antigen. Avidity is related to both the affinity between an antigenic determinant and its antigen-binding site on the antigen-binding molecule and the number of appropriate sites present on the antigen-binding molecule. Typically, an antigen-binding protein (such as an HSA-binding polypeptide and a Nanobody of the invention) binds to its antigen 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 (association rate, 1 Ms) is about 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , or 10 11 In some embodiments, the Ka is about 10 7 In some embodiments, the KD (dissociation rate, s) is about 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 , or 10 -11In some embodiments, K D is about 10 -7 In some embodiments, the antigen binding proteins disclosed herein have a nucleotide sequence of about 10 -9 K in moles / L D Any K greater than 10 μM binds to its antigen. D The value is generally considered to represent non-specific binding. The dissociation constant may be the actual or apparent dissociation constant, as will be apparent to one of skill in the art.

[0056] In some embodiments, the HSA-binding polypeptide of a Nanobody specifically binds to human HSA. In some embodiments, the HSA-binding polypeptide of a Nanobody specifically binds to human HSA and mouse serum albumin. In some embodiments, the specificity of Nanobody binding can be attributed, or largely attributed, to the CDR3 region of the Nanobody.

[0057] Thus, in some embodiments, the HSA-binding polypeptide of the Nanobody comprises a complementarity-determining region (CDR) 3, wherein CDR3 is selected from the group consisting of 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: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: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: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: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: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 and / or 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: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: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: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: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 SEQ ID NO:100.

[0058] "50% similarity" is calculated as follows: Residue groups: Amino acid residues are grouped according to their biophysical properties as follows: 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: i.e." means all amino acid sequences (CDR3 fingerprint sequences of Nanobodies) that are equal to or exceed 50% similarity (e.g., at least 60% similarity, 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) to the 91 CDR3 sequences set forth in SEQ ID NOs: 10-100.

[0060] In some embodiments, the HSA-binding polypeptide of a Nanobody is selected from the group consisting of 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: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: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: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: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: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: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: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: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.

[0061] Thus, in some embodiments, the HSA-binding polypeptide of a Nanobody is selected from the group consisting of 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: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: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: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: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: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:71, SEQ ID NO:72, and a CDR3 comprising an amino acid sequence selected from the group consisting of 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: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:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, and SEQ ID NO:100, wherein the CDR3 comprises an amino acid sequence selected from the group consisting of epitope 1. , epitope 2, epitope 3, and epitope 4, wherein epitope 1 comprises amino acid residues 298-307, 311, 332-341, and 371-386 of SEQ ID NO:1; epitope 2 comprises amino acid residues 5-13, 62-67, 93-99, and 228-266 of SEQ ID NO:1; epitope 3 comprises amino acid residues 226-230 and 298-337 of SEQ ID NO:1; and epitope 4 comprises amino acid residues 33-38 and 111-145 of SEQ ID NO:1.

[0062] With respect to the IL-2 portion of the Nanobody, IL-2 is an interleukin that is involved in the activation, proliferation, and survival of B lymphocytes, T lymphocytes, and natural killer cells by binding to the IL-2 receptor on the cell surface. By "IL-2" herein is meant a polypeptide that hydrolyzes cyclic adenosine 5'-diphosphate-ribose, and in humans is encoded by the IL2 gene. In some embodiments, the IL-2 polypeptide is one identified in one or more of the following publicly available databases: HGNC:6001, Entrez Gene:3558, Ensembl:ENSG00000109471, OMIM:147680, UniProtKB:P60568. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 5, or a polypeptide sequence having about 80%, about 85%, about 90%, about 95%, or about 98% homology to SEQ ID NO: 5, or greater than about 80%, about 85%, about 90%, about 95%, or about 98% homology thereto, or a polypeptide comprising a portion of SEQ ID NO: 5. The IL-2 polypeptide of SEQ ID NO: 5 may represent immature IL-2 or may be pre-processed from mature IL-2, and thus, as used herein, includes mature or processed portions of the HSA polypeptide of SEQ ID NO: 5.

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

[0064] In some embodiments, the IL-2 polypeptide is a recombinant human IL-2 polypeptide, including but not limited to aldesleukin. The aldesleukin can have a serine instead of a cysteine ​​at amino acid position 125. In some embodiments, the aldesleukin comprises the amino acid sequence of SEQ ID NO: 103. In some embodiments, the aldesleukin is Proleukin (Novartis).

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

[0066] In some embodiments, the Nanobody has increased thermostability and / or increased resistance to serum protease activity compared to native IL-2 or other IL-2, such as Albroukin (Human Genome Sciences). In some embodiments, the Nanobody reaches its maximum effective molar concentration in vivo more quickly than native IL-2 or other IL-2, such as Albroukin (Human Genome Sciences).

[0067] In some embodiments, the recombinant Nanobody of any of the foregoing aspects is less than about 50 kDa, e.g., less than about 45 kDa, less than about 40 kDa, less than about 35 kDa, or less than about 30 kDa. In some embodiments, the recombinant Nanobody of any of the foregoing aspects comprises an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9.

[0068] Recombinant Nanobodies have approximately the same or increased affinity between the IL-2 polypeptide portion of the Nanobody and the IL-2 receptor compared to native IL-2 or an IL-2 such as Albroukin (Human Genome Sciences). The IL-2 receptor is a heterotrimeric receptor with three subunits, often referred to as the α subunit (or IL-2Rα or CD25), the β subunit (or IL-2Rβ or CD122), and the γ subunit (or IL-2γ, common γ chain, or CD132). The β and γ subunits exhibit intermediate affinity binding for IL-2 (approximately 10 -9 While the α, β, and γ subunits form a complex with a KD of 10 M, the α, β, and γ subunits form a high-affinity IL-2 receptor binding to IL-2 (approximately 10 -11 In some embodiments, the recombinant Nanobody has about the same affinity or increased affinity between the IL-2 polypeptide portion of the Nanobody and an IL-2 receptor comprising, for example, an α subunit, a β and γ subunit, or an α, β and γ subunit. In some embodiments, the recombinant Nanobody has about the same affinity or increased affinity between the IL-2 polypeptide portion of the Nanobody and an IL-2 receptor comprising, for example, an α, β and γ subunit.

[0069] It is understood herein that affinity at acidic pH correlates well with excretion rate (Spearman ρ=0.78), which in turn correlates well with affinity (particularly dissociation rate) at neutral pH. Thus, both slow binding and dissociation rates and acidic pH-dependent interactions are important for prolonging the stability of Nanobody-HSA-FcRn tertiary complexes within endosomes. In some embodiments, recombinant Nanobody HSA-binding polypeptides specifically bind to HSA in a conformationally stable form at a pH ranging from about 3.5 to about 7.5.

[0070] Native IL-2 has a half-life of less than about 30-60 minutes in humans. The use of recombinant Nanobodies can extend the half-life of IL-2 by targeting endogenous human serum albumin. Thus, in some embodiments, the recombinant IL-2 Nanobodies described herein have a half-life that is at least 50-fold (e.g., at least 100-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, or at least 1000-fold) longer than that of native or non-Nanobody recombinant IL-2 polypeptides or control Nanobodies. In some embodiments, the recombinant IL-2 Nanobodies described herein have a half-life that is at least 50-fold (e.g., at least 100-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, or at least 1000-fold) longer than recombinant human IL-2, such as aldesleukin. In some embodiments, the recombinant IL-2 Nanobodies described herein have a half-life that is at least 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold longer than recombinant human IL-2, such as aldesleukin (Human Genome Sciences) (HSA / IL-2 fusion protein).

[0071] In some aspects, disclosed herein are Nanobodies comprising a human serum albumin (HSA) binding polypeptide, wherein the HSA binding polypeptide comprises a complementarity determining region (CDR) 3, wherein CDR3 is selected from the group consisting of 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: 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: 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: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: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: 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: 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: 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: 92, The present invention includes amino acid sequences having at least 50% similarity (e.g., at least 60% similarity, 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) to 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 SEQ ID NO:100. Similarity is calculated as described above.

[0072] In some aspects, disclosed herein is a polypeptide comprising a human serum albumin (HSA) binding polypeptide, wherein the HSA binding polypeptide comprises a complementarity determining region (CDR) 3, wherein CDR3 is selected from the group consisting of 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: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: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: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: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: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: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: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: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.

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

[0074] Treatment method Provided herein are methods of treating cancer in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant Nanobody as described herein. The present disclosure demonstrates the surprising discovery that administering a therapeutically effective amount of a recombinant Nanobody comprising a human serum albumin (HSA) binding polypeptide and an IL-2 polypeptide results in the treatment of cancer.

[0075] Accordingly, provided herein is a method for treating cancer in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant Nanobody, wherein the Nanobody comprises a human serum albumin (HSA)-binding polypeptide and an IL-2 polypeptide, and wherein the HSA-binding polypeptide specifically binds to an HSA epitope, the HSA epitope being selected from the group consisting of epitope 1, epitope 2, epitope 3, and epitope 4, wherein epitope 1 comprises amino acid residues 298-307, 311, 332-341, and 371-386 of SEQ ID NO:1, epitope 2 comprises amino acid residues 5-13, 62-67, 93-99, and 228-266 of SEQ ID NO:1, epitope 3 comprises amino acid residues 226-230, and 298-337 of SEQ ID NO:1, and epitope 4 comprises amino acid residues 33-38, and 111-145 of SEQ ID NO:1. Includes:

[0076] In some embodiments of the methods, the HSA-binding polypeptide portion of the Nanobody specifically binds to an HSA epitope comprising amino acid residues 298-307, 311, 332-341, and 371-386 of SEQ ID NO: 1. In some embodiments, the HSA-binding polypeptide specifically binds to an HSA epitope comprising amino acid residues 5-13, 62-67, 93-99, and 228-266 of SEQ ID NO: 1. In some embodiments, the HSA-binding polypeptide specifically binds to an HSA epitope comprising amino acid residues 33-38 and 111-145 of SEQ ID NO: 1. In some embodiments, the HSA-binding polypeptide specifically binds to an HSA epitope comprising amino acid residues 298-307, 311, 332-341, and 371-386 of SEQ ID NO: 1.

[0077] Examples of cancers treatable using the methods described herein include, but are not limited to, melanoma, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is renal cancer.

[0078] In some embodiments, administration of a recombinant Nanobody described herein reduces metastasis, slows and / or terminates tumor growth, reduces tumor size, promotes T cell and NK cell activation, proliferation, and cytotoxic function, increases the level of anti-tumor antibodies, and prolongs and / or increases the survival rate of a subject. As used herein, the terms "increase," "promote," "prolong," and "reduce," "reduce" are understood and should be contemplated herein to mean an increase or decrease compared to before treatment in a subject, or compared to the occurrence of such condition in the overall or study population.

[0079] In some embodiments of the method, the HSA binding polypeptide of any of the aforementioned aspects comprises a complementarity determining region (CDR) 3, wherein CDR3 is selected from the group consisting of 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: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: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: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: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: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. 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: 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: 55, SEQ ID NO: 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: Column number 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: 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: 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: 92, SEQ ID NO: 93, Includes amino acid sequences having at least 50% similarity (e.g., at least 60% similarity, 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) to 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 SEQ ID NO:100.

[0080] "50% similarity" is calculated as follows: Residue groups: Amino acid residues are grouped according to their biophysical properties as follows: 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: i.e." means all amino acid sequences (CDR3 fingerprint sequences of Nanobodies) that are equal to or exceed 50% similarity (e.g., at least 60% similarity, 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) of the 91 CDR3 master sequences set forth in SEQ ID NOs: 10-100.

[0082] In some embodiments of the method, the HSA-binding polypeptide of any of the aforementioned aspects comprises a complementarity determining region (CDR) 3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10-100.

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

[0084] In some embodiments of the method, the recombinant Nanobody is less than about 50 kDa, e.g., 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 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9.

[0085] In some embodiments of the invention, the recombinant Nanobody has a half-life in a subject that is about 10-fold, about 20-fold, about 30-fold, about 40-fold, 50-fold, about 100-fold, about 200-fold, about 300-fold, about 400-fold, about 500-fold, about 600-fold, about 700-fold, about 800-fold, about 900-fold, or about 1000-fold longer than a naturally occurring IL-2 polypeptide, aldesleukin (e.g., Proleukin, Novartis), or albroukin (Human Genome Sciences).

[0086] Thus, in some embodiments, the frequency of administration of the recombinant Nanobody is less than about 2-fold, less than about 3-fold, less than about 4-fold, less than about 5-fold, less than about 6-fold, less than about 7-fold, less than about 8-fold, less than about 9-fold, less than about 10-fold, less than about 15-fold, less than about 20-fold, less than about 30-fold, less than about 40-fold, less than about 50-fold, less than about 80-fold, less than about 100-fold, less than about 150-fold, less than about 200-fold, less than about 300-fold, less than about 500-fold, less than about 800-fold, or less than about 1000-fold the frequency of administration of a native IL-2 polypeptide, aldesleukin, or albroukin (Human Genome Sciences).

[0087] The administration frequency of the recombinant Nanobody of any of the aforementioned aspects includes, but is not limited to, at least once a month, once every three weeks, 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 twice a day, three times a day, four times a day, five times a day, six times a day, seven times a day, eight times a day, or nine times a day. In some embodiments, the interval between each administration is less than about two months, less than about one month, less than about three weeks, less than about two weeks, or less than about one week, e.g., less than about 6, 5, 4, 3, 2, or 1 day(s). In some embodiments, the administration frequency of the recombinant Nanobody includes, but is not limited to, at least daily, once, twice a day, or three times a day. In some embodiments, the interval between each administration is less than about 48 hours, about 36 hours, about 24 hours, about 22 hours, about 20 hours, about 18 hours, about 16 hours, about 14 hours, about 12 hours, about 10 hours, about 9 hours, about 8 hours, or about 7 hours. In some embodiments, the interval between each administration is less than about 24 hours, about 22 hours, about 20 hours, about 18 hours, about 16 hours, about 14 hours, about 12 hours, about 10 hours, about 9 hours, about 8 hours, about 7 hours, or about 6 hours. In some embodiments, the interval between each administration is constant. For example, administration can be daily, every other day, every third day, every fourth day, every fifth day, or every week. Administration can also be continuous and adjusted to maintain levels of recombinant Nanobody within any desired and predetermined range.

[0088] The present disclosure demonstrates that the recombinant Nanobodies described herein can extend the half-life of IL-2. In some embodiments, the recombinant Nanobodies can increase the duration of the therapeutic effect of IL-2 and / or reduce the amount of IL-2 that needs to be administered compared to native IL-2 polypeptides or Albroukin (Human Genome Sciences). Thus, the amount of IL-2 in a therapeutically effective dose as part of a recombinant Nanobody can be less than the amount recommended for a single administration of IL-2 (e.g., native IL-2 polypeptides or Albroukin (Human Genome Sciences)). For example, if a conventionally recommended dose of IL-2 is amount X, then the nanoparticles, or recombinant polypeptide composition, can contain IL-2 in an amount of about 0.9X, about 0.8X, about 0.7X, about 0.6X, about 0.5X, about 0.4X, about 0.3X, about 0.2X, or about 0.1X. In some embodiments, these lower therapeutically effective doses of IL-2 may reduce the side effects of IL-2, if any, over a period of time following administration and / or may reduce the likelihood that a subject will develop resistance to IL-2.

[0089] The disclosed methods can be performed at any time before the onset of cancer. In some embodiments, the disclosed methods are performed within 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 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, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 year prior to the onset of cancer. 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, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 days; 60, 48, 36, 30, 24, 18, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, or 2 hours before, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 month before, or 1 month after ... 5, 20, 25, 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, 60 hours; 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 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, 51, 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, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 year. The disclosed methods can be used at any stage of cancer, including stage 0, stage I, stage II, stage III, and stage IV cancer.

[0090] The recombinant Nanobodies described herein can be present in any suitable dosage form. The dosage form can be adapted for administration by any suitable route. Suitable routes include, but are not limited to, oral (including buccal or sublingual), rectal, epidural, intracranial, intraocular, inhalation, nasal, topical (including buccal, sublingual, or transdermal), intravaginal, intraurethral, ​​parenteral, intracranial, subcutaneous, intramuscular, intravenous, intraperitoneal, intradermal, intraosseous, intracardiac, intraarticular, intravenous, intrathecal, intravitreal, intracerebral, gingival, subgingival, intraventricular, and intradermal. Such formulations can be prepared by any method known in the art.

[0091] The compounds and compositions disclosed herein are effective in treating, inhibiting, alleviating, reducing, ameliorating, and / or preventing cancer and / or metastasis in a subject, comprising administering to the subject a therapeutically effective amount of a compound of any of the aforementioned embodiments, or a pharmaceutical composition of any of the aforementioned embodiments. In some embodiments, the cancer is selected from the group consisting of lymphoma, B-cell lymphoma, T-cell lymphoma, mycosis fungoides, Hodgkin's disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, 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, renal cancer, genitourinary cancer, esophageal carcinoma, head and neck carcinoma, colon cancer, hematopoietic cancer, testicular cancer, colon cancer, rectal cancer, prostate cancer, and pancreatic cancer.

[0092] In some embodiments, the recombinant Nanobody of any of the foregoing aspects is formulated in a pharmaceutically acceptable carrier. [Example]

[0093] The following examples are set forth below to illustrate compositions, methods, and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention that would be apparent to one skilled in the art.

[0094] Example 1: Synthetic characterization of HSA-Nbs. Ninety-eight recombinant soluble nanobodies (Nbs) with unique CDR3 sequences were expressed and purified from E. coli using His-Cobalt resin. A schematic diagram of the Nbs and the amino acid composition of the CDR loops are shown in Figure 1a. These Nbs have highly diverse CDR3 sequences, exhibiting significant variations in physicochemical properties, including the CDR3 cysteines that form intramolecular disulfides, which are important for isoelectric point, hydrophobicity, and thermal stability (Figure 1b). The CDR3 amino acids were aligned, and the occurrence at each position was calculated to generate a sequence web logo (Figure 1b). Enzyme-linked immunosorbent assays (ELISAs) were performed to assess the relative binding affinities of the Nbs to albumin from different species, including human, monkey, and mouse. Eighty-nine Nbs bound to HSA, with affinities exceeding three orders of magnitude. Interestingly, less than 50% of HSA-Nbs bind to the highly conserved cynomolgus monkey albumin, and only approximately 9% (8 Nbs) can appreciably cross-react with mouse albumin. Although HSA-specific Nbs showed remarkable specificity, these cross-species binders are also useful for experiments in corresponding animal models.

[0095] Twenty HSA-Nbs spanning different ELISA ranges were randomly selected and their K values ​​were determined by surface plasmon resonance (SPR). D Affinity (K on and K. offBoth the binding affinity and dissociation affinity were carefully measured. 75% (15 / 20) of the selected Nbs were affinity matured, resulting in sub-nM affinities for HSA binding, but with unique and often diverse binding and dissociation kinetics (Fig. 1d). For example, Nb126 had a binding affinity of 1.7 × 10 3 (1 / s)K off Nb13 (13 pM), Nb13 (201 pM), and Nb80 (166 pM) exhibited both fast binding and slow dissociation rates. Bead binding assays were performed to confirm the SPR measurements (Fig. 1f). In addition, there was a good correlation (R ) between the calculated ELISA affinity and the SPR KD. 2 = 0.78, p < 0.001) was found (Fig. 1e). Albumin cross-species binding was confirmed by bead-binding assay (Fig. 1g). The thermal stability of 20 HSA-Nbs was assessed using differential scanning calorimetry (DSC). A wide range of melting temperatures was observed, ranging from 30 to 70 °C (Fig. 1h, Fig. 8). In conclusion, this rich repertoire is composed of exceptionally high-quality and diverse Nbs.

[0096] Example 2. HSA immunogenicity landscape revealed by cross-linking and modeling. A fundamental property of humoral immunity is the ability to generate large cohorts of antibodies to recognize any exposed external structure, but the structural substrates that direct such universality remain incompletely understood. Here, to better understand the landscape and nature of immunogenicity, we used a rapid docking approach to identify epitopes (Figure 2a), which were subsequently validated by chemical cross-linking and mass spectrometry (CXMS) (Figure 2b, Figure 9). Several features were learned: 1) Despite the highly diverse CDR3 sequences and other physicochemical properties of HSA-Nbs, only four dominant epitopes were identified (Figures 2a-2d and Figure 16), indicating the presence of immunodominance. 2) Epitopes are highly selective by the mammalian immune system, avoiding sequences shared 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 verify the dominant epitope and better understand the structural basis of immunogenicity, we evaluated and measured the hybrid structure of a tetrameric complex composed of HSA and three Nb molecules (Nb 80, 13, and 29) corresponding to different major epitopes. First, size-exclusion chromatography (SEC) was used to confirm that the three Nb molecules were not simultaneously occupied, i.e., they did not compete for HSA interaction (Figure 3a). As shown in Figure 3a, a distinct size shift was observed upon addition of each Nb. Homogeneous fractions of the complex were analyzed by negative-stain electron microscopy (EM) (Figure 3b, Figures 10b-10d). Approximately 22,000 EM particles were averaged to reconstruct 3D density maps (Figures 3b-3c). Next, the structural model was computationally processed, and the lowest-energy model was screened using cross-reactivity to constrain the placement and localization of the Nb molecules. The final model was validated by simultaneously satisfying EM, cross-linking suppression, and mutagenesis (Figures 3e–3h).

[0098] The overall structure of the tetrameric complex confirmed the major epitope, a Christmas tree reminiscent of the HSA "pine tree" decorated with three conformational Nb "gifts" at different locations. Beyond the overall structure, several observations exist: 1) Nbs do not simultaneously occupy the HSA FcRn-binding site and therefore do not interfere with FcRn-mediated endocytosis, a process critical for HSA stability. 2) Good complementarity was confirmed between the epitope cavity and the concave paratope of the Nbs. Interestingly, shape complementarity correlated with affinity, with the highest Nb80 (160 pM) showing excellent match and the lowest Nb29 (approximately 1 nM) showing relatively poor match. Importantly, none of these Nbs matched the reported HSA-Nb (#5VNW, K of approximately 450 nM). D ) did not colocalize.

[0099] At the interface, sufficient hydrophobic and charged residues were observed, which could explain the high affinity. To further investigate these interactions, two charged residues on HSA (K383 and E400) were mutated. Based on the model described herein, these residues form two crossed stable salt bridges with corresponding opposite charges on the Nb CDR, which can well mimic camelid residues (Figures 3i and 3j). Interestingly, one mutation, E400, R double mutant K383 D significantly weakened the Nb80-HSA interaction. R completely abolishes such strong interactions (Figure 3k), further confirming the accuracy of the model described herein. The sequences shown in Figure 3j are YETTLEKCCAAADPHECYAKVFDEF (SEQ ID NO: 101) and YEATLEDCCAKDDPHACYATVFDKL (SEQ ID NO: 102).

[0100] Example 4. Development of an accurate MS assay for multiplexed Nb pharmacokinetic analysis. Next, we developed a new MS assay to enable high-throughput, accurate, and specific PK analysis of Nbs while reducing the bias and challenges of comparisons across multiple animals. The label-free fragment-ion-based MS method has good linearity for detecting Nbs in serum (Figures 11a-11e). Because most of these HSA-Nbs do not cross-react with mouse albumin, we used a humanized albumin mouse model (Tg32-Alb) with a double knockout of mAb and its receptor, mFcRn, as well as an additional knockout of hFcRn. - / - mFcRn - / - hFcRn Tg / Tg ) was selected. It has been reported that the half-life of native HSA in humans was reproduced when HSA was introduced.

[0101] Following HSA injection, 22 Nb, including 20 HSA binders and two non-binder controls, were mixed at equimolar concentrations in PBS and administered to this model (n = 3) via a single bolus intravenous (iv) injection. A 1:5 molar ratio of Nb to HSA was used to ensure the presence of excess HSA for binding. Blood was sampled at different time points post-injection and stored as serum. Serum proteins containing Nb were proteolyzed. The resulting peptides were separated by advanced liquid chromatography (LC), and Nb signature peptides, along with their fragment ions, were quantified by high-resolution Orbitrap MS (Figure 4a, Figures 11a-11e). The average quantification CV, based on hundreds of LC runs, was 15.3% (Figures 12a-12c).

[0102] As shown in Figure 4b, compared with control Nbs, whose serum half-lives were approximately 26–60 min, HSA-Nbs exhibited significantly improved half-lives—up to a 771-fold reduction in blood clearance (Nb158) was observed. Surprisingly, a significantly altered T (1.6–7.6 days) was observed (Figure 4c), which correlated well with the half-life of 8.1 days for HSA, instead of the 21 days previously reported in this model (w / o Nb coadministration, Figure 13c). Label-free quantitative serum proteomics identified a several-hundred-fold increase in serum IgG levels 5–6 days after HSA injection. This indicated the development of an anti-HSA immune response in the model (Figure 14), which is not unusual for such well-folded foreign proteins when administered at relatively high doses (but still below full physiological levels).

[0103] Example 5. Correlation of Nb pharmacokinetics with its physicochemical properties. To better understand the differences in HSA-Nb PK, we performed a correlation analysis between Nb PK and physicochemical properties, including affinity (both at 7.4 and acidic pH), melting temperature, isoelectric point, and hydrophobicity (at both the CDR and total protein levels) (Figure 4d). Interestingly, affinity at acidic pH correlated well with excretion rate (Spearman ρ = 0.78), followed by affinity at neutral pH (particularly dissociation rate) and CDR3 hydrophobicity. A reasonable correlation was found between the three parameters. Both the slow binding / dissociation rate and the acidic pH-dependent interactions are likely important for prolonging the stability of the Nb-HSA-FcRn tertiary complex within endosomes, where weakened binding leads to premature termination of the "hitchhiking" journal and faster clearance of the piggybacked Nb cargo. The data described herein indicate that increased hydrophobicity on the paratope may be important for high-affinity binding.

[0104] Example 6. Development of Duraleukin: A new class of Nb-fusion cytokines for cancer immunotherapy. For proof-of-concept purposes, we applied this Nb toolkit to the model therapeutic molecule interleukin-2 (IL-2). IL-2 is a master cytokine that functions to suppress cancer progression by enhancing both innate and adaptive immune responses (e.g., CD8+ T and natural killer cells). Human IL-2 (approximately 15 kDa) was first applied as a cancer immunotherapy agent and has been used to effectively treat multiple cancers, including kidney cancer and advanced melanoma. However, its clinical efficacy remains significantly limited by its short half-life (less than 30–60 minutes in humans). Furthermore, frequent administration of high doses of hIL-2, although necessary for its anticancer activity, can result in significant side effects, including liver toxicity and vascular leak syndrome.

[0105] Following hIL-2, various N-terminal HSA-Nb fusion constructs were designed to generate novel compositions (these compositions are commonly referred to as "duraleukins") (Fig. 5a). Three duraleukins (DL77, DL80, and DL158) were rapidly produced using Nb77, Nb80, and Nb158 and bulk-purified from E. coli inclusion bodies (Fig. 5b). All constructs exhibited good thermal stability at approximately 60°C (Fig. 5c). They maintained bioactivity comparable to hIL-2, as demonstrated in an in vitro CD8+ T cell proliferation assay (Fig. 5d), and were highly stable when incubated in vitro with mouse serum for several days (Fig. 5e). Furthermore, these duraleukins (e.g., DL80) maintained high affinity for HSA binding (273 pM) (Fig. 5f). DL80 exhibits reduced affinity at acidic pH (2-8 fold, FIG. 5g), but was nevertheless chosen for in vivo evaluation as it has one of the greatest affinities for mouse albumin.

[0106] Example 7. Evaluation of the therapeutic efficacy of DL80 for melanoma treatment. Using a wild-type B6 mouse model, we confirmed that DL80 had increased stability, with an approximately 46-fold improvement in blood clearance compared to hIL-2 (Figure 15). Next, the therapeutic effect of DL80 was evaluated in a B16F10 melanoma mouse model. Tumor-bearing mice were subcutaneously administered equimolar concentrations of DL80 or hIL-2 at different frequencies (DL80 every 6 days, hIL-2 daily) for a total of 24 days of treatment. TA99, a mAb that recognizes the tumor marker TRP1 and has well-known synergistic activity with IL-2, was co-treated with both groups at a dose of 150 μg every 6 days. After treatment, animals were allowed to recover, and tumor size (Figure 6a) and survival (Figure 6b) were simultaneously analyzed for one month. Despite the fact that only one-sixth the therapeutic dose of hIL-2 was used and only modest PK improvements were observed relative to the fusion construct DL80 in the WT B6 mouse model, the DL80 and hIL-2 treatment groups demonstrated a significant reduction in tumor burden and overall survival compared to PBS controls. Fifty percent (four mice) of DL80-treated melanoma animals survived, compared with 25% (two mice) in the hIL-2 group and 0% in the PBS group. Near-complete tumor regression was observed in the DL80-responsive model, whereas a clear recurrence of tumor formation was observed in the hIL-2-treated mice (Figure 6a), demonstrating the superiority of duraleukin for cancer therapy. To further explore the potential mechanism(s) of the antitumor effect, tumors were isolated at different stages after treatment, and corresponding immune cells were analyzed by flow cytometry (Figure 6c). Although no significant changes were detected one day after treatment, a significant increase in both CD8+ T cells and natural killer (NK) cells was observed in DL80-treated tissues three days after injection, indicating that duraleukin efficiently penetrated tumors stimulating these immune cells to exert its antitumor activity.

[0107] Example 8. HSA-binding Nb improves drug stability and therapeutic efficacy. Many small biomolecules and therapeutic agents (including Nb) are limited by their poor stability in vivo. Here, we developed a robust and general approach to solve this fundamental problem in drug delivery, facilitated by the remarkable physicochemical and biophysical properties of camelid Nb. A large cohort of high-quality HSA-Nb has been systematically characterized using a cutting-edge interdisciplinary approach spanning biophysics, proteomics, structural biology, and computational modeling. A novel proteomic method was developed for accurate, high-throughput measurement of Nb PK. Next, we developed a stable Nb-fusion cytokine, duraleukin, and prioritized its antitumor efficacy.

[0108] Four dominant epitopes on HSA were discovered, supporting the immunodominance hypothesis (decorated Christmas tree). A preference for concave recognition by Nbs was observed, which can be explained by the convex Nb structure. Despite having relatively small paratopes, most of our Nbs are still able to achieve remarkable affinity and specificity for antigen engagement comparable to IgG.

[0109] Considering the high serum concentration of albumin, it can be imagined that high affinity of Nb is not necessary for increased PK. The above results show the opposite: both affinity and pH-dependent binding are positively correlated, emphasizing the importance of the microenvironment for FcRn-mediated endocytosis and the local concentration of receptor complexes for efficient cargo delivery.

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

[0111] Purification of recombinant Nb. Nb DNA constructs were transformed into BL21(DE3) cells and plated on agar plates overnight at 37°C with 50 μg / mL ampicillin. 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. Next, cells were harvested, briefly sonicated, and lysed on ice using lysis buffer (TX-100 containing 1X PBS, 150 mM NaCl, and 0.2% protease inhibitors). After lysis, soluble protein extracts were collected at 15,000 x g for 10 min. Recombinant Nb was purified on His6-cobalt resin (Thermo) and eluted with imidazole. The eluted Nb was then dialyzed against dialysis buffer (e.g., 1X DPBS, pH 7.4) and stored at −80°C before use.

[0112] ELISA (Enzyme-Linked Immunosorbent Assay). Indirect ELISA was performed to evaluate immune responses and nanobody affinity. Antigens were coated onto 96-well ELISA plates (R&D systems) at 1–10 ng / well in coating buffer (15 mM sodium carbonate, 35 mM sodium bicarbonate, pH 9.6) overnight at 4°C and blocked with blocking buffer (DPBS, 0.05% Tween 20, 5% milk) for 2 hours at room temperature. Immune serum or nanobodies were serially diluted in blocking buffer and incubated with antigen for 2 hours. HRP-conjugated secondary antibodies against llama Fc (Thermo) or his tag (Genscript) were diluted 1:5,000–10,000 in blocking buffer and incubated for 1 hour at room temperature. Nonspecific absorbance was removed by washing three times with 1x PBST (DPBS, 0.05% Tween 20). After washing, samples were incubated with freshly prepared 3,3',5,5'-tetramethylbenzidine (TMB) substrate for 10 minutes at room temperature in the dark to develop signal. After stop solution (R&D system), plates were read at multiple wavelengths (optical density at 550 nm subtracted from density at 450 nm) on a plate reader (Multiskan GO, Thermo Fisher). Raw data were then processed using Prism 7 (GraphPad) and fitted to a 4PL curve to calculate the logIC50 or the average value if the results did not fit the curve.

[0113] In vitro bead pull-down. Nb affinity was measured using surface plasmon resonance (SPR) on a Biacore 3000 system (GE Healthcare). Briefly, protein antigens such as GST, OMP25 PDZ domain, or human serum albumin were immobilized on the flow channel of an activated CM5 sensor chip. Protein analytes were diluted to 10–30 μg / mL in 10 mM sodium acetate (pH 4.5) and injected into the SPR system at 5 μL / min for 420 s. The surface was then blocked with 1 M ethanolamine-HCl (pH 8.5). For each Nb analyte, a dilution series spanning approximately 1000-fold was injected in duplicate at a flow rate of 20–30 μL / min for 120–180 s using HBS-EP+ running buffer (GE Healthcare) containing 2 mM DTT, followed by a dissociation time of 5–20 min based on dissociation rate. Between injections, the sensor chip surface was regenerated with either a low-pH buffer containing 10 mM glycine-HCl (pH 1.5–2.5) for 30 s at a flow rate of 40–50 μL / min or a high-pH buffer containing 20–40 mM NaOH (pH 12–13) for 30 s at a flow rate of 40–50 μL / min. Binding sensorgrams for each Nb were processed and analyzed using BIA analysis by fitting with a 1:1 Langmuir model or a 1:1 Langmuir model with mass transfer. The inventions described in the original claims are listed below. [Invention 1] A recombinant Nanobody comprising a human serum albumin (HSA) binding polypeptide and an IL-2 polypeptide, 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; Epitope 1 comprises amino acid residues 298 to 307, 311, 332 to 341, and 371 to 386 of SEQ ID NO: 1; epitope 2 comprises amino acid residues 5 to 13, 62 to 67, 93 to 99, and 228 to 266 of SEQ ID NO: 1; Epitope 3 comprises amino acid residues 226 to 230 and 298 to 337 of SEQ ID NO: 1; and Epitope 4 comprises amino acid residues 33 to 38 and 111 to 145 of SEQ ID NO: 1; The recombinant nanobody. [Invention 2] 2. The recombinant Nanobody according to claim 1, wherein the HSA-binding polypeptide specifically binds to human HSA and mouse serum albumin. [Invention 3] 3. The recombinant Nanobody according to claim 1 or 2, wherein the HSA-binding polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4. [Invention 4] 4. The recombinant Nanobody according to any one of claims 1 to 3, wherein the IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 103, or a functional fragment thereof. [Invention 5] 5. The recombinant Nanobody according to any one of claims 1 to 4, wherein the HSA-binding polypeptide is linked to the IL-2 polypeptide via a linker. [Invention 6] 6. The recombinant Nanobody according to claim 5, wherein the linker comprises the amino acid sequence of SEQ ID NO: 6. [Invention 7] 7. The recombinant Nanobody according to any one of claims 1 to 6, wherein said recombinant Nanobody is less than about 50 kDa. [Invention 8] 8. The recombinant Nanobody according to any one of claims 1 to 7, wherein the HSA-binding polypeptide does not reduce the affinity between the IL-2 polypeptide and its IL-2 receptor. [Invention 9] 9. The recombinant Nanobody according to any one of claims 1 to 8, wherein the recombinant Nanobody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9. [Invention 10] 1. A method for treating cancer in a subject in need thereof, comprising: administering a therapeutically effective amount of 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; Epitope 1 comprises amino acid residues 298 to 307, 311, 332 to 341, and 371 to 386 of SEQ ID NO: 1; epitope 2 comprises amino acid residues 5 to 13, 62 to 67, 93 to 99, and 228 to 266 of SEQ ID NO: 1; Epitope 3 comprises amino acid residues 226 to 230 and 298 to 337 of SEQ ID NO: 1; and Epitope 4 comprises amino acid residues 33 to 38 and 111 to 145 of SEQ ID NO: 1; The method. [Invention 11] 11. The method according to claim 10, wherein the HSA-binding polypeptide specifically binds to human HSA and mouse serum albumin. [Invention 12] 13. The method of claim 11 or 12, wherein the HSA-binding polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4. [Invention 13] 13. The method according to any one of inventions 10 to 12, wherein the IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 103, or a functional fragment thereof. [Invention 14] 14. The method according to any one of Inventions 10 to 13, wherein the HSA-binding polypeptide is linked to the IL-2 polypeptide via a linker. [Invention 15] 15. The method of claim 14, wherein the linker comprises the amino acid sequence of SEQ ID NO:6. [Invention 16] 16. The method according to any one of claims 10 to 15, wherein said recombinant Nanobody is less than about 50 kDa. [Invention 17] 17. The method according to any one of Inventions 10 to 16, wherein the HSA-binding polypeptide does not reduce the affinity between the IL-2 polypeptide and its IL-2 receptor. [Invention 18] 18. The method according to any one of inventions 10 to 17, wherein the recombinant Nanobody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9. [Invention 19] 19. The method according to any one of claims 10 to 18, wherein said recombinant Nanobody has an in vivo half-life that is about 100 times longer than that of the native IL-2 polypeptide. [Invention 20] 20. The method according to any one of Inventions 10 to 19, wherein the cancer is selected from the group consisting of melanoma and renal cancer. [Invention 21] 1. A Nanobody comprising a human serum albumin (HSA) binding polypeptide, The HSA-binding polypeptide comprises a complementarity determining region (CDR) 3, and the CDR3 is selected from the group consisting of 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: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: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: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:52, SEQ ID NO:53, SEQ ID NO:54 4, comprising an amino acid sequence having at least 50% similarity to 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: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: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: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: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 SEQ ID NO:100; The nanobody. [Invention 22] The 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: 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: 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: 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: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, 22. The Nanobody of claim 21, comprising an amino acid sequence selected from the group consisting of 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: 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: 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: 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. [Invention 23] 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; Epitope 1 comprises amino acid residues 298 to 307, 311, 332 to 341, and 371 to 386 of SEQ ID NO: 1; epitope 2 comprises amino acid residues 5 to 13, 62 to 67, 93 to 99, and 228 to 266 of SEQ ID NO: 1; Epitope 3 comprises amino acid residues 226 to 230 and 298 to 337 of SEQ ID NO: 1; and Epitope 4 comprises amino acid residues 33 to 38 and 111 to 145 of SEQ ID NO: 1; 23. A nanobody according to invention 21 or 22. [Invention 24] 24. The Nanobody of any one of inventions 21 to 23, further comprising an IL-2 polypeptide. [Invention 25] 25. The Nanobody according to invention 24, wherein said IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 103, or a functional fragment thereof. [Invention 26] 26. The Nanobody of invention 24 or 25, wherein the HSA-binding polypeptide is linked to the IL-2 polypeptide via a linker. [Invention 27] 27. The nanobody according to claim 26, wherein said linker comprises the amino acid sequence of SEQ ID NO: 6. [Invention 28] 28. The nanobody of any one of inventions 21 to 27, wherein the nanobody is less than 50 kDa.

Claims

1. A recombinant Nanobody comprising a human serum albumin (HSA) binding polypeptide and an IL-2 polypeptide, 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; Epitope 1 comprises amino acid residues 298-307, 311, 332-341, and 371-386 of SEQ ID NO: 1; Epitope 2 comprises amino acid residues 5-13, 62-67, 93-99, and 228-266 of SEQ ID NO:1; Epitope 3 comprises amino acid residues 226-230 and 298-337 of SEQ ID NO: 1; and Epitope 4 comprises amino acid residues 33-38 and 111-145 of SEQ ID NO: 1; The recombinant nanobody.

2. 2. The recombinant Nanobody of claim 1, wherein the HSA-binding polypeptide specifically binds to human HSA and mouse serum albumin.

3. 3. The recombinant Nanobody of claim 1, wherein the HSA-binding polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO:

4.

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

5. The recombinant Nanobody of any one of claims 1 to 4, wherein the HSA-binding polypeptide is linked to the IL-2 polypeptide via a linker.

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

6.

7. The recombinant Nanobody of any one of claims 1 to 6, wherein the recombinant Nanobody is less than about 50 kDa.

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

9. The recombinant Nanobody of any one of claims 1 to 8, wherein the recombinant Nanobody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO:

9.

10. 1. A pharmaceutical composition for use in a method for treating cancer in a subject in need thereof, comprising: the pharmaceutical composition comprises 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; Epitope 1 comprises amino acid residues 298-307, 311, 332-341, and 371-386 of SEQ ID NO: 1; Epitope 2 comprises amino acid residues 5-13, 62-67, 93-99, and 228-266 of SEQ ID NO:1; Epitope 3 comprises amino acid residues 226-230 and 298-337 of SEQ ID NO: 1; and Epitope 4 comprises amino acid residues 33-38 and 111-145 of SEQ ID NO: 1; The pharmaceutical composition.

11. The pharmaceutical composition of claim 10, wherein the HSA-binding polypeptide specifically binds to human HSA and mouse serum albumin.

12. 12. The pharmaceutical composition of claim 10 or 11, wherein the HSA-binding polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:

4.

13. The pharmaceutical composition of any one of claims 10 to 12, wherein the IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 103, or a functional fragment thereof.

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

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

6.

16. The pharmaceutical composition of any one of claims 10 to 15, wherein the recombinant Nanobody is less than about 50 kDa.

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

18. 18. The pharmaceutical composition of any one of claims 10 to 17, wherein the recombinant Nanobody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO:

9.

19. 19. The pharmaceutical composition of any one of claims 10 to 18, wherein the recombinant Nanobody has an in vivo half-life that is about 100 times longer than the native IL-2 polypeptide.

20. The pharmaceutical composition according to any one of claims 10 to 19, wherein the cancer is selected from the group consisting of melanoma and renal cancer.

Images