Anti-ECM nanobody-cytokine fusions with enhanced affinity and their applications

Abstract

Provided herein are anti-FN-EIIIB nanobodies, conjugates thereof, and methods of using nanobodies and conjugates thereof. Provided are anti-ECM nanobodies coupled with cytokines, which can be used to target the tumor microenvironment and stimulate a local immune response against the tumor. It is recognized herein that several parameters affect efficacy, including increasing nanobody affinity to tumor ECM, increasing expression levels of ECM targets, and increasing delivery routes (systemic or intratumoral). The increased affinity of the nanobodies described herein provides significant advantages over prior art antibodies, including the "parent NJB2" antibody.

Description

[Technical field]

[0001] Related Applications This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 304,954, filed January 31, 2022, which is hereby incorporated by reference in its entirety.

[0002] Federally funded research This invention was made with Government support under W81XWH-14-1-0240 awarded by the U.S. Army Medical Research and Development Command. The U.S. Government has certain rights in the invention.

[0003] Reference to Electronic Sequence Listing The contents of the electronic format of the Sequence Listing (M065670517WO00-SEQ-GIC.xml; size: 106,337 bytes; and creation date: January 30, 2023) are incorporated herein by reference in their entirety. [Background technology]

[0004] background The extracellular matrix (ECM) and ECM proteins play important physiological roles in growth, wound healing, cell migration and differentiation, among other processes. Furthermore, in pathological conditions, the composition of the ECM is altered, and previous observations have indicated that these changes in ECM proteins may play a significant role in cancer progression and metastasis, in cardiovascular disease, and in fibrosis. There is a need for new molecules that target ECM proteins for use in imaging and targeting disease states. Summary of the Invention [Means for solving the problem]

[0005] Abstract Provided in the present disclosure are cytokine-coupled anti-ECM nanobodies that can be used to target cytokines to the tumor microenvironment and stimulate a local immune response against the tumor. It is recognized herein that several parameters affect efficacy, including increasing nanobody affinity to tumor ECM, increasing expression levels of ECM targets, and increasing the delivery route (systemic or intratumoral). The affinity-enhanced nanobodies described herein provide significant advantages over prior art antibodies, including the "parent NJB2" antibody. The parent nanobody NJB2 is specific for the EIIIB domain of fibronectin, which shows selective expression in disease sites such as tumors, metastases, and fibrosis, among others. The affinity-enhanced nanobodies described herein retain their specificity for EIIIB.

[0006] In one aspect, the disclosure provides a Nanobody that specifically binds to the EIIIB domain of fibronectin, comprising a) a complementarity determining region (CDR) 1 comprising G(S / R / I / N)TFSH(N / S / Y / K)AG(G / S); b) a CDR2 comprising (G / R)(I / V)(S / R / G / N)S(D / A / Y)GNI(N / S); and c) a CDR3 comprising NIRG(S / T)YG(N / S)TYY(S / R)R, and does not comprise SEQ ID NO:1.

[0007] In some embodiments, the Nanobody comprises a) a CDR1 selected from GSTFSHNAGG (SEQ ID NO: 54), GSTFSHSAGG (SEQ ID NO: 57), GSTFSHYAGG (SEQ ID NO: 107), GSTFSHKAGG (SEQ ID NO: 106), GRTFSHNAGG (SEQ ID NO: 105), GSTFSHNAGS (SEQ ID NO: 104), GITFSHNAGG (SEQ ID NO: 103), GRTFSHSAGG (SEQ ID NO: 63), and GNTFSHSAGG (SEQ ID NO: 102); and b) a CDR1 selected from GISSDGNIN (SEQ ID NO: 55), GIRSDGNIN (SEQ ID NO: 58), GISSDGNIN (SEQ ID NO: 59), GISSDGNIN (SEQ ID NO: 60), GISSDGNIN (SEQ ID NO: 61), GISSDGNIN (SEQ ID NO: 62), GISSDGNIN (SEQ ID NO: 63), GISSDGNIN (SEQ ID NO: 64), GISSDGNIN (SEQ ID NO: 65), GISSDGNIN (SEQ ID NO: 66), GISSDGNIN (SEQ ID NO: 67), GISSDGNIN (SEQ ID NO: 68), GISSDGNIN (SEQ ID NO: 69), GISSDGNIN (SEQ ID NO: 70), GISSDGNIN (SEQ ID NO: 71), GISSDGNIN (SEQ ID NO: 72), GISSDGNIN (SEQ ID NO: 73), GISSDGNIN (SEQ ID NO: 74), GISSDGNIN (SEQ ID NO: 75), GISSDGNIN (SEQ ID NO: 76), GISSDGNIN (SEQ ID NO: 77), GISSDGNIN (SEQ ID NO: 78), GISSDGNIN and c) a CDR2 selected from IGSDGNIN (SEQ ID NO:59), GISSAGNIN (SEQ ID NO:60), RISSDGNIN (SEQ ID NO:108), GINSYGNIN (SEQ ID NO:109), GIGSAGNIN (SEQ ID NO:62), GIRSDGNIS (SEQ ID NO:101), and GVRSDGNIN (SEQ ID NO:61); and b) a CDR3 selected from NIRGSYGNTYYSR (SEQ ID NO:56), NIRGTYGNTYYSR (SEQ ID NO:100), NIRGSYGNTYYRR (SEQ ID NO:99) and NIRGSYGSTYYSR (SEQ ID NO:98).

[0008] In some embodiments, the Nanobodies are: a) GSTFSHSAGG (SEQ ID NO:57), GIRSDGNIN (SEQ ID NO:58), and NIRGSYGNTYYSR (SEQ ID NO:56), respectively; b) GSTFSHSAGG (SEQ ID NO:57), GIGSDGNIN (SEQ ID NO:59), and NIRGSYGNTYYSR (SEQ ID NO:56), respectively; c) GSTFSHSAGG (SEQ ID NO:57), GISSAGNIN (SEQ ID NO:60), and NIRGSYGNTYYSR (SEQ ID NO:56), respectively; d) e) comprising CDR1, CDR2, and CDR3 which respectively comprise GSTFSHSAGG (SEQ ID NO:57), GIGSAGNIN (SEQ ID NO:62), and NIRGSYGNTYYSR (SEQ ID NO:56); or f) comprising CDR1, CDR2, and CDR3 which respectively comprise GRTFSHSAGG (SEQ ID NO:63), GIRSDGNIN (SEQ ID NO:58), and NIRGSYGNTYYSR (SEQ ID NO:56).

[0009] In some embodiments, the nanobody comprises CDR1, CDR2, and CDR3 comprising GSTFSHSAGG (SEQ ID NO: 57), GIRSDGNIN (SEQ ID NO: 58), and NIRGSYGNTYYSR (SEQ ID NO: 56), respectively.

[0010] In some embodiments, the nanobody comprises: a) QVQLVETGGGLVQAGGSLRLSCAAS (SEQ ID NO: 64); b) QVQLVGTGGGLVQAGGSLRLSCAAS (SEQ ID NO: 68); c) RVQLVETGGGLVQAGGSLRLSCAAS (SEQ ID NO: 85); d) QVRLVETGGGLVQAGGSLRLSCAVS (SEQ ID NO: 86); e) RVQLVETGGGLVQAGGSLRLSCAVS (SEQ ID NO: 66); f) QVQLVETGGGLVQAGGSLRLSCAVS (SEQ ID NO: 71); g) QVQLVETRGGLVQAGGSLRLSCAVS (SEQ ID NO: 72); h i) RVQLVETEGGLVQAGGSLRLSCAAS (SEQ ID NO: 87); j) QVRLVETGGGLVQAGGSLRLSCAAS (SEQ ID NO: 88); k) QVQLVEAGGGLVQAGGSLRLSCAAS (SEQ ID NO: 73); l) QVQLVETRGGLVQAGGSLRLSCAAS (SEQ ID NO: 89); m) RVQLVETRGGLVQAGGSLRLSCAAS (SEQ ID NO: 90); or n) QVQLVGTGGGLVQAGGSLRLSCAVS (SEQ ID NO: 91). The present invention further includes a framework 1 comprising:

[0011] In some embodiments, the nanobody comprises framework 1 comprising: a) RVQLVETGGGLVQAGGSLRLSCAVS (SEQ ID NO: 66); b) QVQLVETRGGLVQAGGSLRLSCAVS (SEQ ID NO: 72); c) QVQLVETGGGLVQAGGSLRLSCAVS (SEQ ID NO: 71); d) QVQLVETEGGLVQAGGSLRLSCAAS (SEQ ID NO: 70); e) QVQLVETGGGLVQAGGSLRLSCAAS (SEQ ID NO: 64); or f) QVQLVEAGGGLVQAGGSLRLSCAAS (SEQ ID NO: 73).

[0012] In some embodiments, the nanobody further comprises a framework 4 comprising: a) WGQGTQVTVSS (SEQ ID NO: 65); b) WGQGIQVTVSS (SEQ ID NO: 69); c) WSQGTQVTVSS (SEQ ID NO: 92); d) WGQRTQVTVSS (SEQ ID NO: 97); e) WGRGAQVTVSS (SEQ ID NO: 93); f) WGQGAQVTVSS (SEQ ID NO: 67); g) WGQGTQVTVFS (SEQ ID NO: 94); h) WGQGTRVTVSS (SEQ ID NO: 95); or i) GGQGTQVTVFS (SEQ ID NO: 96).

[0013] In some embodiments, the nanobody comprises framework 4 comprising a) WGQGAQVTVSS (SEQ ID NO: 67), b) WGQGTQVTVSS (SEQ ID NO: 65), or c) WGQGIQVTVSS (SEQ ID NO: 69).

[0014] In some embodiments, the Nanobody comprises the amino acid sequence of any one of SEQ ID NOs: 2-53. In some embodiments, the Nanobody comprises the amino acid sequence of SEQ ID NO:3.

[0015] In some embodiments, the nanobody is conjugated with a cargo molecule. In some embodiments, the cargo molecule is linked to the N-terminus or C-terminus of the nanobody. In some embodiments, the cargo molecule is a molecular imaging probe, a drug, a toxin, an antibody, a cytokine, an enzyme (e.g., an ECM-modifying enzyme), a CAR cell, e.g., a CAR-T cell, a radioisotope, a protein, or a nanoparticle. In some embodiments, the nanobody is attached to or associated with a carrier. In some embodiments, the carrier is a nanoparticle. In some embodiments, the carrier further comprises a cargo molecule. In some embodiments, the cargo molecule is selected from a drug, a toxin, an antibody, a cytokine, a nucleic acid, an siRNA, an shRNA, a modified mRNA, a DNA, an oligonucleotide, a CRISPR Cas9 reagent, and an imaging probe. In some embodiments, the cargo molecule is a cytokine selected from IL-2, IL-6, IL-8, IL-10, IL-12, IL-15, IL-18, TNF, IFNg, IFNb, IFNa, and a chemokine or a variant thereof.

[0016] In one aspect, the present disclosure provides a pharmaceutical composition comprising a Nanobody of the disclosure and a pharma- ceutically acceptable carrier.

[0017] In one aspect, the disclosure provides a nucleic acid comprising a nucleic acid sequence encoding a Nanobody of the disclosure. The disclosure also provides a vector comprising a nucleic acid of the disclosure, and a cell comprising a nucleic acid or vector of the disclosure. The disclosure further provides a method for producing a Nanobody, the method comprising (i) culturing a cell comprising a nucleic acid or vector of the disclosure under conditions suitable for expression of the Nanobody, and (ii) recovering the expressed Nanobody.

[0018] In one aspect, the present disclosure provides a method of treating a disease or condition associated with fibronectin, such as EIIIB-containing fibronectin, comprising administering to a subject in need thereof an effective amount of a Nanobody or pharmaceutical composition of the disclosure.

[0019] In one aspect, the present disclosure provides a method comprising administering to a subject having a fibronectin-associated disease or condition an effective amount of a nanobody of the disclosure conjugated to a cargo molecule, to deliver the cargo molecule to the extracellular matrix of the diseased tissue.

[0020] In some embodiments, the methods include determining the presence or absence of the EIIIB domain of fibronectin in the subject, and determining whether the subject has a disease or condition associated with fibronectin.

[0021] In some embodiments, the methods include tracking the progression of a fibronectin-associated disease or condition by measuring the presence or absence of the EIIIB domain of fibronectin over time.

[0022] In some embodiments, the method includes measuring the presence or absence of the EIIIB domain of fibronectin in tissue samples isolated from the subject at a first time point and a second time point, and determining progression to an advanced stage of the disease or condition based on a change in the presence or absence of the EIIIB domain of fibronectin at the first and second time points, where i) if the EIIIB domain of fibronectin is present at a higher level in the isolated tissue sample from the second time point, the disease or condition has progressed to an advanced stage, and ii) if the EIIIB domain of fibronectin is present at a lower level in the isolated tissue sample from the second time point, the disease or condition has regressed to a less advanced stage.

[0023] In some embodiments, the disease or condition is cancer, cardiovascular disease, inflammatory disorders, or fibrosis. In some embodiments, the cancer is metastatic cancer. In some embodiments, the cancer is melanoma, breast cancer, or pancreatic cancer.

[0024] In some embodiments, administration is intravenous, intratumor, subcutaneous, intramuscular, intradermal, intraperitoneal, intranasal, intratracheal, intrathecal, intracranial, intraperitoneal, rectal, nasal, or oral administration.

[0025] In some aspects, a nanobody cytokine fusion protein is provided that comprises a nanobody that specifically binds to the EIIIB domain of fibronectin fused to a cytokine, in some embodiments, the cytokine is selected from the group consisting of IL-2, IL-6, IL-8, IL-10, IL-12, IL-18, TNF, IFN-γ, IFN-β, chemokines, and IFN-α, and variants thereof.

[0026] In some embodiments, the Nanobody-cytokine fusion protein comprises at least two Nanobodies fused to a cytokine. In some embodiments, the Nanobody-cytokine fusion protein comprises between 2 and 25 Nanobodies fused to a cytokine. In some embodiments, the Nanobody comprises multiple copies of the same Nanobody. In some embodiments, the Nanobody comprises at least two different Nanobodies.

[0027] In some embodiments, the Nanobody-cytokine fusion protein comprises at least two cytokines fused to a Nanobody. In some embodiments, the at least two cytokines are attached to the distal end of the Nanobody. In some embodiments, the at least two cytokines are the same cytokine. In some embodiments, the at least two cytokines are two different cytokines.

[0028] In some embodiments, the nanobody cytokine fusion protein further comprises albumin. [Brief description of the drawings]

[0029] [Figure 1A-B]Figures 1A-1D show the effect of NJB2-IL-2 immunocytokine with nanomolar affinity for EIIIB in B16F10 tumors. (Figure 1A) Schematic of protein fusions with nanobody as rectangle and IL-2 as oval. IL2-mt: mutant IL2 inactivated by mutations described by Lombardi et al. Nat. Commun. 8, 15373 (2017) and Liu et al. J. Immunother. 32, 887-894 (2009). (Figure 1B) NJB2-IL2-mt, NJT6-IL2, and NJB2-IL2 on a non-reducing SDS-PAGE gel stained with Coomassie blue. (Figure 1C) Timeline of the B16F10 study. 1M: one million. (Figure 1D) Tumor growth (left) and survival (right). Tumor area (mean + SD) is shown until mice within that group were euthanized. Statistical significance of survival rates was generated by log-rank Mantel-Cox test. ns: non-significant. N=5 for all groups. [Figure 1C-D] Same as above. [Figure 2A-B] Figures 2A-2C show the evolution of nanobodies with picomolar affinity for EIIIB using yeast surface display. (Figure 2A) Schematic of yeast surface display. (Figure 2B) The structure of NJB2 was predicted using ABodyBuilder on the SAbPred server. The CDRs (grey) and framework (tan) are shown in ribbon format. The positions of mutations in LMJ1.2C and LMJ2.5I are colored and shown in sphere format. Diagram generated using UCSF Chimera. (Figure 2C) Affinities of 10 novel nanobodies generated by yeast surface display as determined using Biolayer Interferometry. KD values ​​are shown. [Figure 2C] Same as above. [Diagram 3]Figure 3 shows that NJB2-based nanobodies are specific for FN-EIIIB. Western blots show that sortase-biotin tagged nanobodies detect EIIIB-containing fragments, namely EIIIB-His and FN(7-15)-EIIIB. They do not bind to human or mouse plasma fibronectin lacking EIIIB or normal lung ECM. [Figure 4A-B] Figures 4A-4D show the effect on tumors after intravenous administration of IL-2 immunocytokine with nanomolar and picomolar affinity for FN-EIIIB. (Figure 4A) Schematic of protein fusion (top) and timeline of B16F10 study (bottom). 1M: million. (Figure 4B) Survival rates are shown until mice were euthanized within the group. Survival rate comparisons were generated by log-rank Mantel-Cox test. ns: non-significant. N=7-9 for two independent experiments. (Figure 4C) Timeline of 4T1 study. 1M, million. (Figure 4D) Tumor growth (left) and survival rate (right). Tumor area (mean + SD) is shown until mice were euthanized within the group. Survival rate comparisons were generated by log-rank Mantel-Cox test. ns: non-significant. N=6-7. [Figure 4C-D] Same as above. [Figure 5A-B] Figures 5A-5C show the effect on tumors after intratumoral administration of IL-2 immunocytokine with nanomolar and picomolar affinity for FN-EIIIB. (Figure 5A) Timeline of the B16F10 study. (Figure 5B) Tumor growth (left) and survival (right). Tumor area (mean + SD) is shown until mice within that group were euthanized. Survival comparisons were generated by log-rank Mantel-Cox test. ns: non-significant. N=8 for TA99+PBS, N=11 for all other groups for two independent experiments. (Figure 5C) Results of rechallenge experiments performed with N=1 for NJT6-IL2, N=6 for NJB2-IL2, and N=4 for LMJ2.5I-IL2. [Figure 5C] Same as above. [Figure 6]Figures 6A-6B show the effect after intravenous administration of IL-12 immunocytokine specific for FN-EIIIB. (Figure 6A) Timeline of the 4T1 study and details of dosing of IL-12-based immunocytokine in the 4T1 model. (Figure 6B) Survival (left) and tumor growth (right). Tumor area (mean + SD) is shown until mice in that group were euthanized. Survival comparisons were generated by log-rank Mantel-Cox test. ns: non-significant. N=6. IP: intraperitoneal. IV: intravenous. [Figure 7] Figures 7A-7B show NJT6-IFNα, NJB2-IFNα, and LMJ2.5I-IFNα on a non-reducing SDS-PAGE gel stained with Coomassie blue, and Figure 7B shows that all IFNα fusion proteins are active in vitro as tested by the RAW Lucia macrophage activation assay. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] Detailed Description The present disclosure provides nanobodies that bind to the EIIIB domain of fibronectin. These nanobodies are referred to herein as "anti-FN-EIIIB nanobodies". The present disclosure also provides nanobody conjugates, nucleic acids encoding anti-FN-EIIIB nanobodies or conjugates thereof, compositions comprising anti-FN-EIIIB nanobodies or conjugates thereof, methods of producing nanobodies (e.g., recombinant production methods), and methods of using nanobodies or conjugates thereof, e.g., methods of treating diseases or conditions associated with fibronectin.

[0031] We have developed nanobodies that are highly specific for epitopes in the EIIIB domain of fibronectin (FN), which are present in the ECM of diseased tissues but not in normal healthy adult tissues. The nanobodies thus generated can be used for in vivo and in vitro diagnostic imaging (such as, but not limited to, immunofluorescence (IF), immunohistochemistry (IHC), positron emission tomography / computed tomography (PET / CT)), as well as for targeted therapy across a wide range of diseases. Thus, the nanobodies of the present disclosure are useful for identifying diseased tissues (and pathological conditions of a subject) and for delivering therapeutic agents or other cargoes to the area.

[0032] As shown in the examples, a significant increase in the affinity of the new evolved nanobodies based on the parent anti-FN-EIIIB nanobody, NJB2, was observed. Studies on nanobody-cytokine fusions are also described herein. FN-EIIIB-specific NJB2-based immunocytokines (IL2 and IL12) mildly increased survival rates in melanoma and breast cancer models when administered systemically compared to non-targeted and size control NJT6. Intratumoral administration of NJB2-IL2 significantly increased cures in melanoma models compared to non-targeted NJT6. IL2 fusions of evolved nanobodies tested in vivo showed trends suggesting that enhanced affinity may be beneficial. Nanobody-cytokine fusions with cytokines may also bind to other non-disease sites where cytokine-receptors are expressed, sometimes referred to as "systemic cytokine sinks". Without wishing to be bound by theory, this is not the case when other drugs or toxins are attached to these higher affinity binding agents. Therefore, for applications such as nanobody-drug or nanobody-isotope conjugates, higher affinity versions are expected to be more beneficial than the parent NJB2.

[0033] The higher affinity anti-FN-EIIIB nanobodies of the present disclosure have several advantages.

[0034] 1. Advantages of higher affinity anti-FN-EIIIB nanobodies. One possible way to enhance tumor-specific binding of nanobody-drug conjugates is to increase the binding affinity of the nanobody to its target. These higher affinity binders may home more effectively to disease ECM and remain bound for longer periods, thus increasing therapeutic efficacy. They may also help to circumvent dosing-limiting problems, since it may be possible to provide effective local drug concentrations with smaller amounts of nanobody-drug conjugate, and furthermore to reduce systemic toxicity. For nanobody-cytokine fusions, this may be a major advantage. The biodistribution of an immunocytokine depends on (i) the affinity of the nanobody to its target antigen, (ii) the affinity of the cytokine to its receptor, and (iii) also on the site of expression of the target antigen and cytokine receptor in different cells / organs. This can result in the nanobody-cytokine fusion binding to the target antigen (in the tumor) and also to other non-disease sites where cytokine-receptors are expressed, sometimes referred to as "systemic cytokine sinks" (3). A higher affinity nanobody can help to outcompete the cytokine for binding to its receptor, thus reducing binding in the systemic sink and allowing a higher degree of localization at disease sites. In some embodiments, the target-sink balance is altered by decreasing the binding of the immunocytokine to the sink by lowering the affinity of the cytokine for its receptor.

[0035] The anti-FN-EIIIB nanobodies of the present disclosure are specific to proteins / domains that are specifically expressed in diseased tissues such as cancer and are generally absent in normal adult tissues. Cargo molecules can be site-specifically attached to these nanobodies for targeted delivery to diseased sites. This targeted approach offers advantages over systemic therapy by allowing selective accumulation in tumor and disease ECM. This results in increased specificity and sensitivity of detection / treatment, and reduced background and off-target toxicity.

[0036] 2. Advantages over other targeted approaches. The nanobodies of the present disclosure also have several advantages over other targeted approaches. Although other targeted therapies exist, they often suffer from systemic toxicity due to the broader expression of the targeted proteins (4). The selective expression of the EIIIB domain at disease sites and the specific binding of nanobodies to their target proteins allows for improvements over other targeting approaches. The rapid clearance of small (15 kDa) nanobodies also provides an advantage in terms of reduced systemic effects.

[0037] 3. Broad application in multiple cancer types and conditions (not limited to cancer type). Existing targeted therapy applications, such as HER2-specific nanobodies for breast and ovarian cancer, rituximab for lymphoid tumors, etc., are cancer type limited. Therapies and detection tools developed using the disclosed anti-FN-EIIIB nanobodies will have the advantage that they can be used as pan-cancer therapeutic / targeting agents, since they recognize antigens whose expression is not limited to cancer type. In addition to cancer, the target antigens are expressed in other conditions characterized by ECM remodeling. These include, but are not limited to, cardiovascular disorders, such as atheroma, aneurysm, myocardial infarction, fibrosis, autoimmune and inflammatory disorders. In some embodiments, LC-MS / MS data are presented that define the signatures of ECM proteins abundant in a variety of human tumors and metastases as well as ECM proteins expressed in more limited tumor types. These proteomics-defined ECM proteins can be explored as additional novel targets.

[0038] 4. Advantages of targeting tumor ECM over targeting tumor cells. Targeting extracellular matrix proteins such as the EIIIB domain of FN offers several advantages over existing methods. Therapies targeting tumor cells such as anti-CD30, anti-CD19 and anti-CD20 may have limited efficacy due to tumor cell heterogeneity and tumor cell genomic instability. In addition, resistance mechanisms may occur, such as reduced or heterogeneous target expression, target receptor internalization and drug efflux. ECM proteins are rarely mutated in cancer. Therefore, ECM is less heterogeneous and more stable. ECM is also highly abundant and accessible, with fibronectin being a major component of tumor ECM. Proteomic data also identified other tumor and / or metastasis specific ECM proteins and nanobodies as opposed to those that could be similarly isolated from the M13 library in a similar manner, thus broadening the scope of targeting reagents available using this approach.

[0039] 5. Advantages over local therapies. Local therapies, such as those used in the treatment of GBM, involve local and direct administration of drugs to the tumor. A key limitation of local therapies is that they are inefficient in treating widely dispersed metastases. Systemic administration of nanobodies allows access of the nanobodies to all diseased tissues, including both the primary tumor and metastatic sites throughout the body, where the nanobodies selectively bind to the tumor / disease ECM.

[0040] 6. NJB2 nanobodies recognize the EIIIB domain of both mouse and human FN. A major advantage of the NJB2-based nanobodies of the present disclosure is that they recognize both the mouse and human EIIIB domains, thereby enabling validation in immunocompetent rodent models and subsequent translation to preclinical studies and clinical trials.

[0041] I. Nanobodies In one aspect, the present disclosure provides nanobodies that specifically bind to the EIIIB domain of fibronectin. The terms "nanobody", "VHH", "VHH antibody fragment" and "single domain antibody" are used interchangeably and refer to the variable domain of a single heavy chain of an antibody that is of the type found in camelids, which naturally lack light chains. In the absence of light chains, each nanobody has three complementarity determining regions (CDRs), which are designated CDR1, CDR2 and CDR3, respectively. Nanobodies according to the present invention can be camel, dromedary, llama or alpaca nanobodies. Nanobodies have several advantages over antibody formats for applications such as, but not limited to, targeted delivery and in vivo diagnostic imaging. Some of the advantages include: (i) small size (2.5 nm diameter x 4 nm, and molecular weight of 15 kDa, allowing for much faster clearance compared to IgG (150 kDa) (renal cut-off for glomerular filtration is 60 kDa)) (5); (ii) good tissue penetration compared to full-sized antibodies; (iii) short circulating half-life compared to full-sized antibodies; (iv) camelid sdAbs (VHH) share a high degree of sequence identity with human VH and are not expected to elicit immunogenic responses in mice; (v) no adverse immunogenic responses have been reported in humans from nanobodies currently in clinical trials; (vi) nanobodies are a renewable source; (vii) nanobodies are affinity matured in vivo, which is a major advantage compared to scFvs or artificial scaffolds, and as shown in some embodiments, allows for the development of nanobodies that can be used to refine their affinity in vivo. (viii) nanobodies are better suited than scFvs for interacting with grooves on the surface of antigens, such as the catalytic site of an enzyme (6); (ix) nanobodies can be humanized if necessary (7); and (x) nanobodies are generally stable after addition of cargo, such as an imaging probe, and stable conjugated nanobodies can be rapidly and reproducibly generated and purified.

[0042] The present disclosure provides isolated Nanobodies. "Isolated Nanobodies", as used herein, refer to polypeptides that are substantially physically separated from other cellular material (e.g., separated from the cells that produce the Nanobody) or from other materials that would prevent their use in any of the diagnostic or therapeutic methods of the invention. In particular, the polypeptides are sufficiently pure and sufficiently free of other biological components of their host cells to be useful, for example, in the production or sequencing of pharmaceutical preparations. Isolated Nanobodies of the invention may be mixed with a pharma- ceutically acceptable carrier in a pharmaceutical preparation, so that they may constitute only a small percentage by weight of the preparation. Nevertheless, a Nanobody is substantially pure in that it has been substantially separated from materials with which it may be associated in an organism.

[0043] In some embodiments, the nanobodies or other peptides described herein specifically bind to the corresponding target epitope. A nanobody or peptide, such as an antibody, that "specifically binds" to an epitope is an expression well understood in the art. A molecule is said to exhibit "specific binding" if it reacts more frequently, rapidly, for a longer period and / or with greater affinity to a particular target antigen than it reacts with alternative targets. A nanobody "specifically binds" if it binds to a target antigen or epitope with greater affinity, avidity, ease, and / or duration than it binds to other substances. For example, a nanobody that specifically (or preferentially) binds to an antigen or an antigenic epitope therein is a nanobody that binds to this target antigen with greater affinity, avidity, ease, and / or duration than it binds to other antigens or other epitopes in the same antigen. It is also understood by this definition that, for example, a nanobody that specifically binds to a first target antigen may or may not specifically or preferentially bind to a second target antigen. Therefore, "specific binding" or "preferential binding" does not necessarily require (although it may include) exclusive binding. In some examples, a nanobody that "specifically binds" to a target antigen or epitope thereof may not bind to other antigens or other epitopes in the same antigen. In some embodiments, a nanobody that specifically binds to the EIIIB domain of fibronectin will bind with lower affinity (if at all) to other domains of fibronectin or other proteins. Lower affinity may include at least 10% lower, 20% lower, 30% lower, 40% lower, 50% lower, 60% lower, 70% lower, 80% lower, 90% lower, or 95% lower affinity.

[0044] In some embodiments, the nanobodies described herein have suitable binding affinity to a target antigen or antigenic epitope thereof (e.g., the EIIIB domain of fibronectin). As used herein, "binding affinity" refers to the apparent association constant or KA. KA is the reciprocal of the dissociation constant (KD). The nanobodies described herein have a binding affinity of at least 10 to a target antigen or antigenic epitope thereof (e.g., the EIIIB domain of fibronectin). -9 , 10 -10 M, 10 -11 , 10 -12 A nanobody may have a binding affinity (KD) of or less than 100 . An increase in binding affinity corresponds to a decrease in KD. Higher affinity binding of a nanobody to a first antigen compared to a second antigen may be indicated by a higher KA (or a lower numerical KD) for binding to the first antigen than the KA (or numerical KD) for binding to the second antigen. In such cases, the nanobody has specificity for a first antigen (e.g., a first protein or mimetic thereof in a first conformation) compared to a second antigen (e.g., the same first protein or mimetic thereof in a second conformation; or a second protein).

[0045] For example, in some embodiments, the anti-FN-EIIIB nanobodies described herein have a higher binding affinity (higher K or smaller K) to the EIIIB domain of fibronectin compared to the binding affinity to a second antigen or antigenic epitope. The difference in binding affinity (e.g., for specificity or other comparison) is at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 10,000 or 10 5 It could be double.

[0046] Binding affinity (or binding specificity) can be determined by a variety of methods, including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, biolayer interferometry or spectroscopy (e.g., using a fluorescence assay).

[0047] In some embodiments, the Nanobodies are "humanized", i.e. have an amino acid sequence by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring VHH sequence and / or an affinity matured VHH sequence (and in particular in the framework sequence) by one or more of the amino acid residues present at the corresponding positions in a VH domain from a conventional four-chain antibody from a human. Methods for humanization are well known. Humanized Nanobodies may have several advantages, such as reduced immunogenicity, compared to the corresponding naturally occurring VHH domain (or affinity matured VHH domain). "Humanization" can be performed by providing a nucleotide sequence encoding a naturally occurring VHH domain and then changing one or more codons in the nucleotide sequence such that the new nucleotide sequence encodes a "humanized" Nanobody. This nucleic acid can then be expressed to obtain a Nanobody. Alternatively, the amino acid sequence of a humanized Nanobody can be designed based on the amino acid sequence of a naturally occurring VHH domain and then synthesized de novo using techniques for peptide synthesis. The skilled artisan can also combine in an appropriate manner one or more parts of one or more naturally occurring VHH sequences (e.g. one or more FR or CDR sequences) and / or one or more synthetic or semi-synthetic sequences to obtain a Nanobody or a nucleotide sequence or a nucleic acid encoding same.

[0048] Nanobodies generally have an amino acid sequence with the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively.

[0049] The anti-FN-EIIIB nanobodies of the present disclosure are derived from the parent anti-FN-EIIIB nanobody NJB2. In some embodiments, the anti-FN-EIIIB nanobodies of the present disclosure have the following consensus CDR1 sequence: G(S / R / I / N)TFSH(N / S / Y / K)AG(G / S). In some embodiments, the anti-FN-EIIIB nanobodies of the present disclosure have the following consensus CDR2 sequence: (G / R)(I / V)(S / R / G / N)S(D / A / Y)GNI(N / S). In some embodiments, the anti-FN-EIIIB nanobodies of the present disclosure have the following consensus CDR3 sequence: NIRG(S / T)YG(N / S)TYY(S / R)R. Possible amino acid alternatives at each position are shown in brackets. In some embodiments, anti-FN-EIIIB nanobodies of the disclosure comprise a CDR1 comprising G(S / R / I / N)TFSH(N / S / Y / K)AG(G / S). In some embodiments, anti-FN-EIIIB nanobodies of the disclosure have a CDR2 comprising (G / R)(I / V)(S / R / G / N)S(D / A / Y)GNI(N / S). In some embodiments, anti-FN-EIIIB nanobodies of the disclosure have a CDR3 comprising NIRG(S / T)YG(N / S)TYY(S / R)R. In some embodiments, anti-FN-EIIIB Nanobodies of the disclosure have a) complementarity determining region (CDR)1 comprising G(S / R / I / N)TFSH(N / S / Y / K)AG(G / S), b) CDR2 comprising (G / R)(I / V)(S / R / G / N)S(D / A / Y)GNI(N / S), and c) CDR3 comprising NIRG(S / T)YG(N / S)TYY(S / R)R. In some embodiments, anti-FN-EIIIB Nanobodies of the disclosure do not comprise SEQ ID NO: 1, the sequence of the parent Nanobody NJB2.

[0050] In some embodiments, anti-FN-EIIIB nanobodies comprise a CDR1 selected from GSTFSHNAGG (SEQ ID NO: 54), GSTFSHSAGG (SEQ ID NO: 57), GSTFSHYAGG (SEQ ID NO: 107), GSTFSHKAGG (SEQ ID NO: 106), GRTFSHNAGG (SEQ ID NO: 105), GSTFSHNAGS (SEQ ID NO: 104), GITFSHNAGG (SEQ ID NO: 103), GRTFSHSAGG (SEQ ID NO: 63), and GNTFSHSAGG (SEQ ID NO: 102). In some embodiments, anti-FN-EIIIB nanobodies comprise a CDR2 selected from GISSDGNIN (SEQ ID NO:55), GIRSDGNIN (SEQ ID NO:58), GIGSDGNIN (SEQ ID NO:59), GISSAGNIN (SEQ ID NO:60), RISSDGNIN (SEQ ID NO:108), GINSYGNIN (SEQ ID NO:109), GIGSAGNIN (SEQ ID NO:62), GIRSDGNIS (SEQ ID NO:101), and GVRSDGNIN (SEQ ID NO:61). In some embodiments, anti-FN-EIIIB nanobodies comprise a CDR3 selected from NIRGSYGNTYYSR (SEQ ID NO:56), NIRGTYGNTYYSR (SEQ ID NO:100), NIRGSYGNTYYRR (SEQ ID NO:99), and NIRGSYGSTYYSR (SEQ ID NO:98).In some embodiments, the anti-FN-EIIIB Nanobodies comprise a) a CDR1 selected from GSTFSHNAGG (SEQ ID NO: 54), GSTFSHSAGG (SEQ ID NO: 57), GSTFSHYAGG (SEQ ID NO: 107), GSTFSHKAGG (SEQ ID NO: 106), GRTFSHNAGG (SEQ ID NO: 105), GSTFSHNAGS (SEQ ID NO: 104), GITFSHNAGG (SEQ ID NO: 103), GRTFSHSAGG (SEQ ID NO: 63), and GNTFSHSAGG (SEQ ID NO: 102); and b) a CDR1 selected from GISSDGNIN (SEQ ID NO: 55), GIRSDGNIN (SEQ ID NO: 5 8), GIGSDGNIN (SEQ ID NO: 59), GISSAGNIN (SEQ ID NO: 60), RISSDGNIN (SEQ ID NO: 108), GINSYGNIN (SEQ ID NO: 109), GIGSAGNIN (SEQ ID NO: 62), GIRSDGNIS (SEQ ID NO: 101), and GVRSDGNIN (SEQ ID NO: 61); and c) a CDR3 selected from NIRGSYGNTYYSR (SEQ ID NO: 56), NIRGTYGNTYYSR (SEQ ID NO: 100), NIRGSYGNTYYRR (SEQ ID NO: 99), and NIRGSYGSTYYSR (SEQ ID NO: 98). In some embodiments, the anti-FN-EIIIB nanobody does not comprise SEQ ID NO: 1.

[0051] In some embodiments, an anti-FN-EIIIB nanobody comprises a CDR1 comprising GSTFSHSAGG (SEQ ID NO: 57), a CDR2 comprising GIRSDGNIN (SEQ ID NO: 58), and a CDR3 comprising NIRGSYGNTYYSR (SEQ ID NO: 56). In some embodiments, an anti-FN-EIIIB nanobody does not comprise SEQ ID NO: 1.

[0052] In some embodiments, an anti-FN-EIIIB nanobody comprises a CDR1 comprising GSTFSHSAGG (SEQ ID NO: 57), a CDR2 comprising GIGSDGNIN (SEQ ID NO: 59), and a CDR3 comprising NIRGSYGNTYYSR (SEQ ID NO: 56). In some embodiments, an anti-FN-EIIIB nanobody does not comprise SEQ ID NO: 1.

[0053] In some embodiments, an anti-FN-EIIIB nanobody comprises a CDR1 comprising GSTFSHSAGG (SEQ ID NO: 57), a CDR2 comprising GISSAGNIN (SEQ ID NO: 60), and a CDR3 comprising NIRGSYGNTYYSR (SEQ ID NO: 56). In some embodiments, an anti-FN-EIIIB nanobody does not comprise SEQ ID NO: 1.

[0054] In some embodiments, an anti-FN-EIIIB nanobody comprises a CDR1 comprising GSTFSHSAGG (SEQ ID NO: 57), a CDR2 comprising GIGSAGNIN (SEQ ID NO: 62), and a CDR3 comprising NIRGSYGNTYYSR (SEQ ID NO: 56). In some embodiments, an anti-FN-EIIIB nanobody does not comprise SEQ ID NO: 1.

[0055] In some embodiments, an anti-FN-EIIIB nanobody comprises a CDR1 comprising GSTFSHSAGG (SEQ ID NO: 57), a CDR2 comprising GVRSDGNIN (SEQ ID NO: 61), and a CDR3 comprising NIRGSYGNTYYSR (SEQ ID NO: 56). In some embodiments, an anti-FN-EIIIB nanobody does not comprise SEQ ID NO: 1.

[0056] In some embodiments, an anti-FN-EIIIB nanobody comprises a CDR1 comprising GRTFSHSAGG (SEQ ID NO: 63), a CDR2 comprising GIRSDGNIN (SEQ ID NO: 58), and a CDR3 comprising NIRGSYGNTYYSR (SEQ ID NO: 56). In some embodiments, an anti-FN-EIIIB nanobody does not comprise SEQ ID NO: 1.

[0057] In some embodiments, an anti-FN-EIIIB Nanobody comprises CDR1, CDR2, and CDR3 of a Nanobody comprising the amino acid sequence of any one of SEQ ID NOs: 1-53. In some embodiments, an anti-FN-EIIIB Nanobody comprises CDR1, CDR2, and CDR3 of a Nanobody comprising the amino acid sequence of any one of SEQ ID NOs: 2-11. In some embodiments, an anti-FN-EIIIB Nanobody comprises CDR1, CDR2, and CDR3 of a Nanobody comprising the amino acid sequence of SEQ ID NO: 3.

[0058] In some embodiments, the anti-FN-EIIIB nanobody is a) QVQLVETGGGLVQAGGSLRLSCAAS (SEQ ID NO: 64); b) QVQLVGTGGGLVQAGGSLRLSCAAS (SEQ ID NO: 68); c) RVQLVETGGGLVQAGGSLRLSCAAS (SEQ ID NO: 85); d) QVRLVETGGGLVQAGGSLRLSCAVS (SEQ ID NO: 86); e) RVQLVETGGGLVQAGGSLRLSCAVS (SEQ ID NO: 66); f) QVQLVETGGGLVQAGGSLRLSCAVS (SEQ ID NO: 71); g) QVQLVETRGGLVQAGGSLRLSCAVS (SEQ ID NO: No. 72); h) QVQLVETEGGLVQAGGSLRLSCAAS (SEQ ID NO: 70); i) RVQLVETEGGLVQAGGSLRLSCAAS (SEQ ID NO: 87); j) QVRLVETGGGLVQAGGSLRLSCAAS (SEQ ID NO: 88); k) QVQLVEAGGGLVQAGGSLRLSCAAS (SEQ ID NO: 73); l) QVQLVETRGGLVQAGGSLRLSCAAS (SEQ ID NO: 89); m) RVQLVETRGGLVQAGGSLRLSCAAS (SEQ ID NO: 90); or n) QVQLVGTGGGLVQAGGSLRLSCAVS (SEQ ID NO: 91). In some embodiments, an anti-FN-EIIIB nanobody comprises a FR1 selected from a) RVQLVETGGGLVQAGGSLRLSCAVS (SEQ ID NO: 66); b) QVQLVETRGGLVQAGGSLRLSCAVS (SEQ ID NO: 72); c) QVQLVETGGGLVQAGGSLRLSCAVS (SEQ ID NO: 71); d) QVQLVETEGGLVQAGGSLRLSCAAS (SEQ ID NO: 70); e) QVQLVETGGGLVQAGGSLRLSCAAS (SEQ ID NO: 64); or f) QVQLVEAGGGLVQAGGSLRLSCAAS (SEQ ID NO: 73). In some embodiments, an anti-FN-EIIIB nanobody comprises a FR1 comprising RVQLVETGGGLVQAGGSLRLSCAVS (SEQ ID NO: 66). In some embodiments, an anti-FN-EIIIB nanobody does not comprise SEQ ID NO: 1.

[0059] In some embodiments, anti-FN-EIIIB nanobodies comprise a FR4 selected from a) WGQGTQVTVSS (SEQ ID NO: 65); b) WGQGIQVTVSS (SEQ ID NO: 69); c) WSQGTQVTVSS (SEQ ID NO: 92); d) WGQRTQVTVSS (SEQ ID NO: 97); e) WGRGAQVTVSS (SEQ ID NO: 93); f) WGQGAQVTVSS (SEQ ID NO: 67); g) WGQGTQVTVFS (SEQ ID NO: 94); h) WGQGTRVTVSS (SEQ ID NO: 95); or i) GGQGTQVTVFS (SEQ ID NO: 96). In some embodiments, anti-FN-EIIIB nanobodies comprise a FR4 selected from a) WGQGAQVTVSS (SEQ ID NO: 67), b) WGQGTQVTVSS (SEQ ID NO: 65), or c) WGQGIQVTVSS (SEQ ID NO: 69). In some embodiments, anti-FN-EIIIB nanobodies comprise a FR4 comprising WGQGAQVTVSS (SEQ ID NO: 67). In some embodiments, the anti-FN-EIIIB nanobody does not comprise SEQ ID NO:1.

[0060] In some embodiments, the anti-FN-EIIIB Nanobody comprises the amino acid sequence of any one of SEQ ID NOs: 2-53. In some embodiments, the anti-FN-EIIIB Nanobody consists essentially of the amino acid sequence of any one of SEQ ID NOs: 2-53. In some embodiments, the anti-FN-EIIIB Nanobody consists of the amino acid sequence of any one of SEQ ID NOs: 2-53. In some embodiments, the anti-FN-EIIIB Nanobody comprises the amino acid sequence of any one of SEQ ID NOs: 2-11. In some embodiments, the anti-FN-EIIIB Nanobody consists essentially of the amino acid sequence of any one of SEQ ID NOs: 2-11. In some embodiments, the anti-FN-EIIIB Nanobody consists of the amino acid sequence of any one of SEQ ID NOs: 2-11. In some embodiments, the anti-FN-EIIIB Nanobody comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the anti-FN-EIIIB Nanobody consists essentially of the amino acid sequence of SEQ ID NO: 3. In some embodiments, the anti-FN-EIIIB Nanobody consists of the amino acid sequence of SEQ ID NO: 3.

[0061] In some embodiments, the term Nanobody refers to analogs, mutants, variants, alleles, homologs and orthologues of the Nanobodies described herein. In general, such analogs may have one or more amino acid residues replaced, deleted and / or added compared to the Nanobodies disclosed herein. Such substitutions, insertions, deletions or additions may be made in one or more of the framework regions and / or in one or more of the CDRs. In some embodiments, substitutions, insertions, deletions or additions may be made in one or more of the framework regions and / or in one or more of the CDRs of any one of SEQ ID NOs: 1-53. In some embodiments, substitutions, insertions, deletions or additions may be made to the CDRs listed in Table 2. In some embodiments, substitutions, insertions, deletions or additions may be made to the framework regions listed in Table 3. In some embodiments, the anti-FN-EIIIB Nanobody does not include SEQ ID NO: 1.

[0062] In some embodiments, anti-FN-EIIIB Nanobodies comprise an amino acid sequence that contains up to 20 amino acid changes (e.g., up to 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid change) compared to the amino acid sequences set forth in SEQ ID NOs: 1-53. In some embodiments, anti-FN-EIIIB Nanobodies do not comprise SEQ ID NO:1.

[0063] In some embodiments, the anti-FN-EIIIB nanobody comprises no more than five (e.g., four, three, two or one) amino acid residue changes in one or more of the CDR regions of one of the exemplified antibodies herein and binds to the same epitope of the antigen with substantially similar affinity (e.g., has a KD value of the same order). In some embodiments, the anti-FN-EIIIB nanobody comprises no more than five (e.g., four, three, two or one) amino acid residue changes in one or more of the framework regions of one of the exemplified antibodies herein and binds to the same epitope of the antigen with substantially similar affinity (e.g., has a KD value of the same order). In one example, the amino acid residue change is a conservative amino acid residue substitution. As used herein, "conservative amino acid substitution" refers to an amino acid substitution that does not change the relative charge or size characteristics of the protein in which the amino acid substitution is made.

[0064] An "analog", as used herein, is a sequence in which each or any of the framework regions and each or any of the complementarity determining regions exhibits at least 80% identity, preferably at least 85% identity, more preferably 90% identity, and even more preferably 95% identity, with the corresponding regions in a reference sequence as measured by BLASTp alignment (i.e. FR1_analog to FR1_ref, CDR1_analog to CDR1_ref, FR2_analog to FR2_ref, CDR2_analog to CDR2_ref, FR3_analog to FR3_ref, CDR3_analog to CDR3_ref, FR4_analog to FR4_ref).

[0065] The substitutions may, for example, be conservative substitutions and / or the amino acid residue may be replaced by another amino acid residue that naturally occurs at the same position in another VHH domain. The deletions and / or substitutions may be designed to remove one or more sites for post-translational modification (e.g., one or more glycosylation sites) or to introduce one or more sites for attachment of a functional group, for example to allow site-specific pegylation.

[0066] In some embodiments, anti-FN-EIIIB Nanobodies comprise an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 1-53. In some embodiments, anti-FN-EIIIB Nanobodies do not comprise SEQ ID NO:1.

[0067] In some embodiments, the anti-FN-EIIIB nanobodies of the present disclosure comprise a GxS linker. In some embodiments, the GxS linker is at the C-terminus.

[0068] Amino acid residues of nanobodies, i.e., on the protein backbone or on the side chains, can be modified. Modifications include the introduction of one or more functional groups, residues or moieties into or on the nanobody. The functional groups can be directly linked to the nanobody or indirectly linked via a linker or spacer. Methods for increasing the half-life and / or reducing the immunogenicity of pharmaceutical proteins include attaching a polymer, such as poly(ethylene glycol) (PEG), or a derivative thereof (e.g., methoxypoly(ethylene glycol), i.e., mPEG). For example, PEG can be attached to a cysteine ​​residue naturally present in the nanobody, or PEG is synthetically added to the end or within the nanobody. In some embodiments, PEG has a molecular weight greater than 5000 and less than 100,000, or greater than 10,000 and less than 200,000. Other modifications include N-linked or O-linked glycosylation, usually as part of co- and / or post-translational modifications, depending on the host cell used to express the Nanobody.

[0069] Suitable biologically active variants of Nanobodies may be fragments, analogs and derivatives of Nanobodies that specifically bind to FN-EIIIB. By "analog" is intended an analog of either a native polypeptide or a fragment of a native polypeptide, including native polypeptide sequences and structures with one or more amino acid substitutions, insertions or deletions. A Nanobody fragment is a peptide that is identical or at least 90% homologous to a less than full-length Nanobody, referred to herein as a portion of a Nanobody. A portion of a Nanobody or antibody represents a full-length Nanobody or antibody polypeptide. A fragment represents a full-length Nanobody if it comprises at least two amino acids (contiguous or non-contiguous) of a Nanobody and binds to FN-EIIIB. In some embodiments, the portion is less than 90% of the entire native Nanobody. In other embodiments, the portion is less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5% of the entire native Nanobody. By "derivative" is intended any suitable modification of the polypeptide of interest, of a fragment of a polypeptide, or of their respective analogs, such as glycosylation, phosphorylation, polymer conjugation (e.g. with polyethylene glycol), or other addition of foreign moieties, provided that the desired biological activity of the Nanobody or antibody is retained. Methods for making polypeptide fragments, analogs and derivatives are generally available in the art.

[0070] Nanobodies can be obtained from selecting from libraries of such VHH domains, e.g., phage libraries or yeast surface display. Phage libraries can be generated by inserting libraries of polynucleotides containing antibody VHH domains from B cells of immunized animals. Diverse phagemid libraries can be engineered to increase and / or modify the specificity of the resulting library polypeptides, and then additional, desirable, molecular properties and the polynucleotides encoding them can be identified. Libraries involve a high diversity level, since one of 32 different codons can be introduced per position, and all 20 amino acids can be introduced. Such libraries theoretically contain 32 different codons for every residue number n. n The synthesis of oligonucleotides with selected nucleotide "degeneracy" at certain positions is known in the art. Phage display libraries are typically 10 9 ~10 10 has a "registration."

[0071] The method of generating a nanobody library generally involves immunizing a camelid (e.g., camel, dromedary, alpaca, vicuna, llama, etc.) with an immunizing material. Lymphocytes can then be isolated from the blood sample, and the lymphocyte RNA can then be used to construct a phage display-based nanobody library, for example using M13 phage. The library can then be screened for novel nanobodies against a diverse set of ECM and ECM-associated antigens involved in disease. The method involves collecting peripheral blood lymphocytes from which nucleic acid can be isolated. The nucleic acid from which the VH domain sequence is derived can be total RNA, or more particularly, mRNA isolated from cells within a sample taken from a host. The expression vector can be any suitable vector used for library construction, preferably a phage or phagemid vector that allows for the selection of target-specific antibody fragments using phage display-based selection methods. Antibody sequences isolated from these libraries can then be used as a scaffold for in vitro affinity maturation to further improve binding to the target antigen.

[0072] In some embodiments, the nanobody is obtained from affinity maturation of an existing nanobody. In some embodiments, the affinity maturation involves yeast surface display. Affinity maturation methods using yeast display are known in the art. See, for example, Chao G, et al. (2006) Nat Protoc 1(2):755-768.

[0073] Further steps may include, for example and without limitation, affinity maturation, expressing and / or modifying the desired amino acid sequence, screening for binding to the desired antigen (ECM epitope) and / or for activity, determining the desired amino acid or nucleotide sequence, introducing one or more humanizing substitutions, formatting into a suitable multivalent and / or multispecific format, screening for certain desired biological and / or physiological properties.

[0074] In some embodiments, the CDRs or other portions of the Nanobodies disclosed herein are incorporated into structures other than Nanobodies. For example, antibodies (e.g., humanized antibodies) comprising one or more of the CDRs of the Nanobodies disclosed herein or analogs thereof can be produced and used in accordance with the present invention. The term "antibody" is used herein in the broadest sense and specifically encompasses intact monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, antibody fragments, and antibody-like molecules, such as scFvs, provided that they exhibit the desired biological activity. Natural antibodies usually refer to heterotetrameric glycoproteins composed of two identical light (L) chains and two identical heavy (H) chains. Each heavy and light chain has regularly spaced interchain disulfide bridges. Each heavy chain has a variable domain (VH) at one end followed by several constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at its other end, with the light chain constant domain aligned with the first constant domain of the heavy chain and the light chain variable domain aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains.

[0075] Nanobodies can be produced by conventional polypeptide production techniques. For example, they can be synthesized using well-known solid-phase synthesis methods (Merrifield (1962) Proc. Soc. Ex. Boil. 21: 412; Merrifield (1963) J. Am. Chem. Soc. 85: 2149; Tam et al. (1983) J. Am. Chem. Soc. 105: 6442), preferably using a commercially available peptide synthesis machine (e.g., from Applied Biosystems, Foster City, Calif.) and following the manufacturer's instructions.

[0076] Alternatively, Nanobodies can be synthesized by recombinant DNA techniques well known to those of skill in the art (Maniatis et al. (1982) Molecular Cloning: a laboratory manual, Cold Spring Harbor Laboratories, NY, 51-54 and 412-430). For example, they can be obtained as DNA expression products after incorporation of a DNA sequence encoding a polypeptide of interest into an expression vector and introduction of these vectors into a suitable prokaryotic or eukaryotic host that will express the polypeptide of interest, and then they can be isolated from the host using techniques well known to those of skill in the art. In some embodiments, the polypeptide of interest is linked to a tag that facilitates its purification. Such tags are well known to those of skill in the art and include, but are not limited to, for example, hexahistidine (6His), glutathione S-transferase (GST), myc tags or influenza virus hemagglutinin (HA). In some embodiments, when a protein is linked to a tag that facilitates its purification, such a protein comprises a sequence between the native sequence and this tag that allows enzymatic cleavage between the protein and this tag.

[0077] Another aspect of the disclosure relates to a nucleic acid comprising a nucleic acid sequence encoding a Nanobody according to the disclosure. In some embodiments, the nucleic acid of the disclosure comprises a nucleic acid sequence encoding a Nanobody comprising the amino acid sequence of any one of SEQ ID NOs: 2-53. In some embodiments, the nucleic acid of the disclosure comprises a nucleic acid sequence encoding a Nanobody consisting essentially of the amino acid sequence of any one of SEQ ID NOs: 2-53. In some embodiments, the nucleic acid of the disclosure comprises a nucleic acid sequence encoding a Nanobody consisting essentially of the amino acid sequence of any one of SEQ ID NOs: 2-53. In some embodiments, the nucleic acid of the disclosure comprises a nucleic acid sequence encoding a Nanobody comprising the amino acid sequence of any one of SEQ ID NOs: 2-11. In some embodiments, the nucleic acid of the disclosure comprises a nucleic acid sequence encoding a Nanobody consisting essentially of ... of the amino acid sequence of any one of SEQ ID NOs: 2-11. In some embodiments, the nucleic acid of the disclosure comprises a nucleic acid sequence encoding a Nanobody comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, the nucleic acid of the disclosure comprises a nucleic acid sequence encoding a Nanobody consisting essentially of the amino acid sequence of SEQ ID NO: 3. In some embodiments, a nucleic acid of the disclosure comprises a nucleic acid sequence encoding a Nanobody consisting of the amino acid sequence of SEQ ID NO:3.

[0078] Typically, said nucleic acid is a DNA or RNA molecule, which can be included in any suitable vector, for example, plasmid, cosmid, episome, artificial chromosome, phage or viral vector.The terms "vector", "cloning vector" and "expression vector" refer to a carrier that can introduce DNA or RNA sequence into host cell, so as to transform the host and promote the expression (e.g., transcription and translation) of the introduced sequence.Therefore, another aspect of the present disclosure relates to a vector that comprises the nucleic acid according to the present invention.

[0079] Such vectors may contain regulatory elements for causing or directing expression of a polypeptide, such as promoters, activators, terminators, etc. Examples of promoters and activators used in expression vectors for animal cells include SV40 early promoter and activator, Moloney murine leukemia virus LTR promoter and activator, immunoglobulin chain promoter and activator, etc.

[0080] Any expression vector for animal cells can be used. Other examples of vectors include replicative plasmids that contain a replication origin or such as pUC, pcDNA, pBR, etc. Other examples of vectors include viral vectors, such as adenovirus, retrovirus, herpes virus, and AAV vectors. Such recombinant viruses can be produced by techniques well known to those skilled in the art, such as by transfection of packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of viral packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses can be found, for example, in applications WO95 / 14785, WO96 / 22378, U.S. Patent Nos. 5,882,887, 6,013,516, 4,861,719, 5,278,056 and WO94 / 19478.

[0081] Another aspect of the present disclosure relates to cells transfected, transduced or transformed with the nucleic acids and / or vectors according to the invention.

[0082] The term "transformation" refers to the introduction of a "foreign" (i.e., exogenous or extracellular) gene or DNA or RNA sequence into a host cell such that the host cell expresses the introduced gene or sequence to produce a substance of interest, typically a protein encoded by the introduced gene or sequence. A host cell that receives and expresses the introduced DNA or RNA has been "transformed."

[0083] The nucleic acids according to the invention can be used to produce the Nanobodies according to the invention in a suitable expression system. The term "expression system" means a host cell and a vector that are compatible under suitable conditions for the expression of a protein encoded by a foreign DNA, e.g., carried by a vector and introduced into the host cell.

[0084] Conventional expression systems include Escherichia coli host cells and plasmid vectors, insect host cells and baculovirus vectors, and mammalian host cells and their vectors. Other examples of host cells include prokaryotic cells (e.g., bacteria) and eukaryotic cells (e.g., yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include Escherichia coli, Kluyveromyces or Saccharomyces yeast, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.), and also primary or established mammalian cell cultures (e.g., those produced from lymphoblasts, fibroblasts, epithelial cells, neuronal cells, adipocytes, etc.). Examples include mouse SP2 / 0-Ag14 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), CHO cells lacking the dihydrofolate reductase gene, and rat YB2 / 3HL.P2.G11.16Ag.20 cells (ATCC CRL1662).

[0085] In particular, the present invention also relates to a method for producing a Nanobody according to the present invention, comprising the steps of (i) culturing a cell containing a nucleic acid or vector of the disclosure under conditions suitable for expressing said Nanobody, and (ii) recovering the expressed Nanobody.

[0086] The Nanobodies according to the invention can be suitably separated from the culture medium by conventional immunoglobulin purification procedures, such as, for example, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, affinity dialysis or chromatography.

[0087] II. Nanobody Conjugates In some embodiments, the anti-FN-EIIIB nanobody of the present disclosure is conjugated with a cargo molecule. The method of the present disclosure includes the induction, validation and deployment of highly specific nanobodies against ECM epitopes that are specifically expressed in cancer, metastasis and other disease conditions. These unique and highly effective nanobodies can be used as conjugated nanobodies to enable highly selective means of delivery (with low systemic background) of imaging probes and other cargo molecules, including but not limited to drugs, toxins, antibodies, cytokines, siRNA, shRNA, nanoparticles, CAR-T cells, CAR-NK cells, CAR-macrophages, etc., to accessible, common, stable extracellular components of diseased tissues. Conjugation techniques and cargo molecules are known in the art, and a brief summary of these is included herein. Alternatively, nanobodies can be attached or associated with nanoparticles, which can further comprise other therapeutic agents, such as drugs, toxins, antibodies, cytokines, siRNA, shRNA, modified mRNA, DNA, oligonucleotides, CRISPR Cas9 reagents, imaging probes and other cargo molecules.

[0088] As used herein, "conjugated" means two entities stably bound to each other by any physiochemical means. It is important that the nature of the attachment is such that it does not substantially impair the effectiveness of either entity. With these parameters in mind, any covalent or non-covalent linkage known to those skilled in the art can be utilized. In some embodiments, covalent linkage is preferred. Non-covalent conjugation includes hydrophobic interactions, ionic interactions, high affinity interactions such as biotin avidin and biotin streptavidin complexation, and other affinity interactions. Such means and methods of attachment are well known to those skilled in the art.

[0089] Cargo molecules can be very easily attached to nanobodies in a site-specific manner for targeted delivery to diseased sites. In some embodiments, the N- or C-terminus of nanobodies can be site-specifically tagged by sortase-mediated reaction ("sortagging") to different cargo molecules. This facilitates targeted delivery of any cargo molecule attached to the nanobody to diseased ECM. ECM remodeling is specific to several diseases, and at least some ECM biomarkers are shared among these diseases. Thus, nanobodies biopanned from these libraries can be applied across a wide range of diseases characterized by ECM remodeling.

[0090] In some embodiments, the nanobody conjugates (eg, nanobody-cytokine fusion proteins) can be recombinantly produced.

[0091] The active agent can be conjugated to the N-terminus or C-terminus or internal amino acid of the nanobody. In some embodiments, the nanobody is directly conjugated to the active agent. If the cargo molecule is directly linked (e.g., by a peptide bond) to an amino acid of the nanobody (i.e., the N-terminus, C-terminus, or internal amino acid), the nanobody is directly conjugated to the cargo molecule. Alternatively, if a linker is used to connect the cargo molecule to the nanobody, the nanobody can be indirectly conjugated to the cargo molecule, or these two components can be indirectly linked to each other by linkage to a common carrier molecule.

[0092] Thus, linker molecules ("linkers") may be used as needed to link Nanobodies to another molecule. Linkers may be peptides consisting of one to several amino acids, or may be non-peptide molecules. Examples of peptide linker molecules useful in the present invention include glycine-rich peptide linkers (see, for example, US Pat. No. 5,908,626), in which more than half of the amino acid residues are glycine. Preferably, such glycine-rich peptide linkers consist of about 20 or fewer amino acids. A specific advantage of Nanobodies is that during the isolation, subcloning and expression of cloned DNA sequences encoding VHH domain Nanobodies, short peptide tags that are recognized by the sortase enzyme are incorporated, thereby allowing easy conjugation with other entities via these tags (Guimares et al.).

[0093] The carrier molecule may, for example, comprise a PEG or TEG molecule. A PEG or TEG carrier modified peptide will be referred to as a PEGylated or TEGylated peptide.

[0094] Linker molecules may also include non-peptide or partially peptide molecules. For example, well-known cross-linking molecules such as glutaraldehyde or EDC (Pierce, Rockford, Illinois) can be used to link nanobodies to other molecules. Bifunctional cross-linking molecules are linker molecules that have two separate reactive sites. For example, one of the reactive sites of the bifunctional linker molecule can react with a functional group on a peptide to form a covalent linkage, and the other reactive site can react with a functional group on another molecule to form a covalent linkage.

[0095] Homobifunctional crosslinker molecules have two reactive sites that are chemically identical. Examples of homobifunctional crosslinker molecules include, but are not limited to, glutaraldehyde; N,N'-bis(3-maleimido-propionyl-2-hydroxy-1,3-propanediol (a sulfhydryl-specific homobifunctional crosslinker); certain N-succinimide esters (e.g., discuccinimyidyl suberate, dithiobis(succinimidyl propionate)); and soluble bissulfonic acids and their salts.

[0096] Preferably, the bifunctional crosslinker molecule is a heterobifunctional linker molecule, which means that the linker has at least two different reactive sites, each of which can be separately linked to a peptide or other molecule. The use of such a heterobifunctional linker allows for chemically separate stepwise addition (directional conjugation) of each reactive site to a selected peptide sequence. Heterobifunctional linker molecules useful in the present invention include, but are not limited to, m-maleimidobenzoyl-N-hydroxysuccinimide ester; m-maleimido-benzoyl sulfosuccinimide ester; γ-maleimidobutyric acid N-hydroxysuccinimide ester; and 3-(2-pyridyl-dithio)propionate N-succinimidyl.

[0097] The linker may be a "cleavable linker" that facilitates the release of the cargo molecule in the cell. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker (Chari et al., Cancer Research 52:127-131 (1992); U.S. Patent No. 5,208,020) can be used.

[0098] The carboxyl-terminal amino acid residues of the Nanobodies described herein can also be modified to block or reduce the reactivity of the free terminal carboxylic acid group, for example to prevent the formation of esters, peptide bonds, and other reactions. Such blocking of the group includes forming an amide of the carboxylic acid group. Other carboxylic acid groups that may be present in the polypeptide can also be blocked, again provided that such blocking does not provoke an undesired immune response or significantly alter the ability of the Nanobody to function specifically.

[0099] Alternatively, nanobodies can be attached to or associated with nanoparticles, which can further comprise other therapeutic agents, such as cargo molecules.

[0100] Examples of suitable cargo molecules include, but are not limited to, molecular imaging probes, drugs, toxins, siRNA, shRNA, antibodies, cytokines, enzymes (e.g., ECM-modifying enzymes), CAR-T cells, radioisotopes, or nanoparticles. In some embodiments, the enzyme is an ECM-modifying enzyme. In some embodiments, the enzyme remodels the tumor microenvironment (e.g., glucose oxidase).

[0101] In some embodiments, the cargo molecule is a molecular imaging probe. Examples of imaging probes include, but are not limited to, radionuclides, fluorophores or other detectable reagents for use in PET, SPECT, NIR, magnetic particle imaging or other imaging modalities.

[0102] In some embodiments, nanobodies can be conjugated to highly radioactive atoms. A variety of radioisotopes are available for the production of radioconjugated nanobodies. Examples include: 211 At, 131 I, 125 I, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P, 212 Pb, and radioactive isotopes of Lu. When a conjugate is used for detection, it is preferably a radioactive atom for scintigraphic examination, e.g. 99m Tc or 123 I, or spin labels for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as 123 I, 131 I, 111 In, 19 F, 13 C. 15 The label may include N, 17O, gadolinium, manganese or iron. The radioactive or other label may be incorporated into the conjugate in a known manner. For example, the conjugated peptide or polypeptide may be biosynthesized or may be synthesized by chemical amino acid synthesis using suitable amino acid precursors, for example, containing fluorine 19 instead of hydrogen. The label may include, for example, 99m Tc or 123 I, 186 Re, 188 Re and 111In can be attached via a cysteine ​​residue in the peptide. Yttrium-90 can be attached via a lysine residue. Iodine-123 can be incorporated using the IODOGEN method (Fraker et al (1978) Biochem. Biophys. Res. Commun. 80: 49-57). Other methods are described in detail in "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989).

[0103] In some embodiments, the nanobodies are conjugated to diagnostic and imaging labels (commonly referred to as in vivo detectable labels), such as, for example, for magnetic resonance imaging (MRI): Gd(DOTA); for nuclear medicine: 201Tl, the gamma ray emitting nuclide 99mTc; for positron emission tomography (PET): positron emitting isotopes, 18F-fluorodeoxyglucose (18FDG), 18F-fluoride, copper 64, gadodiamide, and radioisotopes of Pb(II), e.g. 203Pb, 111In.

[0104] In some embodiments, the nanobody is conjugated to a drug or therapeutic agent. In some embodiments, the drug is an anti-cancer drug. In some embodiments, the cargo molecule is a cytotoxic agent, such as a chemotherapeutic agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant or animal origin, or a fragment thereof, or a small molecule toxin), or a radioisotope (i.e., a radioconjugate), or a CAR-T cell. Other anti-tumor agents that can be conjugated to the nanobodies of the present disclosure include BCNU, streptozotocin, vincristine, 5-fluorouracil and other commercially available, FDA-approved anti-cancer drugs, a family of agents collectively referred to as LL-E33288 conjugates described in U.S. Pat. Nos. 5,053,394, 5,770,710, and esperamicin (U.S. Pat. No. 5,877,296). Enzymatically active toxins and fragments thereof that can be used in conjugates include diphtheria toxin A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and the trichothecenes.

[0105] Conjugates of antibodies and cargo molecules can be made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipomidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bisazide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bisdiazonium derivatives (e.g., bis(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., 2,6-toluene diisocyanate), and diactive fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotides to antibodies. See WO94 / 11026. In some embodiments, drugs are conjugated to nanobodies via cleavable linkers that facilitate the release of cargo molecules inside cells. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers or disulfide-containing linkers (Chari et al., Cancer Research 52:127-131 (1992); U.S. Patent No. 5,208,020) can be used.

[0106] CAR-T cells, as used herein, refer to T cells that have been introduced with chimeric receptors to redirect their specificity to the optimal antigen. Such receptors contain an ectodomain that recognizes antigens regardless of MHC restriction, combined with a cytoplasmic signaling domain. Many different peptides can be introduced into T cells as the ectodomain of chimeric antigen receptors. Examples include nanobodies, monoclonal antibodies, humanized antibodies, chimeric antibodies, human antibodies, or antibody fragments. In some embodiments, the peptide is a nanobody of the present disclosure. In some embodiments, the nanobody is co-expressed with one or more other CAR receptors in these cells.

[0107] In addition to the conventional CAR T cell derived from αβ T cell, other cells can be used to generate CAR cells that are useful according to the present disclosure.For example, CAR can be transferred into different cell types, such as γδ T cell, natural killer cell, natural killer T cell, and myeloid cell, such as macrophage.These cell types have unique characteristics that can be useful to address some of the obstacles of traditional CAR-T cell therapy.

[0108] Nanobodies can also be incorporated into bispecific T cell engagers (BiTEs). BiTEs are a class of artificial bispecific antibodies that link T cells and tumor cells. They can be used as anticancer drugs. BiTE linkage allows cytotoxic T cells to recognize tumor tissue and thus allows the T cell to exert its cytotoxic effect without the need to bind to its specific T cell receptor. This process is therefore comparable to the physiological process that occurs when T cells attack tumor cells, where the T cells produce proteins such as perforin and granzymes that enter the tumor cell and ultimately lead to its apoptosis. The whole process occurs independently of the presence of MHC I or other costimulatory molecules. BiTEs consist of two single chain variable fragments (scFvs) or nanobodies. One scFv / nanobody binds to the T cell via the CD3 receptor, whereas the other scFv / nanobody binds to the ECM. There are several clinical trials involving BiTEs, including MT110, blinaturmomab (MT 103), BAY2010112, MEDI-565, and MEDI-538. Potential clinical applications include the treatment of non-Hodgkin's lymphoma, acute lymphoblastic leukemia, gastrointestinal cancer, lung cancer, melanoma, and acute myeloid leukemia.

[0109] In some embodiments, the nanobody is conjugated to a cytokine, including, but not limited to, IL-2, IL-6, IL-8, IL-10, IL-12, IL-18, TNF, IFN-γ, IFN-β, chemokines, and IFN-α, and variants thereof. Nanobody-cytokine fusions or conjugates are also referred to herein as "immunocytokines."

[0110] In some embodiments, the immunocytokine is a nanobody-cytokine fusion protein having a nanobody that specifically binds to the EIIIB domain of fibronectin fused to a cytokine. Such fusion proteins can include, for example, multivalent and multimeric fusion proteins. A multivalent nanobody fusion is a protein composed of more than one copy of a nanobody attached to a therapeutic agent, such as a cytokine. Multivalent nanobodies can provide increased size and avidity of the therapeutic agent. For example, a multivalent nanobody structure can have at least two nanobodies fused to a cytokine. In some embodiments, a multivalent fusion protein can have 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-15, 2-20, 2-25, 3-30, 2-35, 2-40, 2-45, 2-45, 2-50, 2-60, 2-70, 2-80, 2-90, 2-100, 2-100, 2-20, 2-30 ...30, 2-40, 2-45, 2-45, 2-50, 2-60, 2-70, 2-80, 2-90, 2-100, 2-100 , 3~4, 3~5, 3~6, ​​3~7, 3~8, 3~9, 3~10, 3~15, 3~20, 3~25, 3~30, 3~35, 3~40, 3~45, 3~45, 3~50, 3~60, 3~70, 3~80, 3~90, 3~100, 4~5, 4~6, 4~7, 4~8, 4~9, 4~10, 4~15, 4 ~20, 4~25, 4~30, 4~35, 4~40, 4~45, 4~45, 4~50, 4~60, 4~70, 4~80, 4~90, 4~100, 5~6, 5~7, 5~8, 5~9, 5~10, 5~15, 5~20, 5~25, 5~30, 5~35, 5~40, 5~45, 5~45, 5~50, 5 between 60, 5 and 70, 5 and 80, 5 and 90, 5 and 100, 10 and 100, 10 and 20, 10 and 30, 10 and 40, 10 and 50, 20 and 30, 20 and 40, 20 and 50, 20 and 100, 30 and 40, 30 and 50, 30 and 100, 40 and 50, 40 and 100, or 50 and 100 nanobodies.

[0111] Multivalent fusion proteins consist of multiple copies of the same nanobody. Such structures include NB1 (n=2~50) -cytokine (where NB1 refers to the first nanobody), for example, NB1-NB1-cytokine.

[0112] In some embodiments, the immunocytokine is a multimeric fusion protein. A multimeric fusion protein is composed of multiple copies of a different nanobody (at least two different nanobodies) fused to a therapeutic agent, such as a cytokine. Such structures include NB1 (n=1~50) NB2 (n=1~50) NB3 (n=0~50) NB4 (n=0~50) -cytokine (NB1, NB2, NB3 and NB4 herein refer to the first, second, third and fourth nanobodies), e.g., NB1-NB2-cytokine, NB1-NB2-NB3-cytokine, NB1-NB2-NB3-NB4-cytokine. In some embodiments, the multimeric protein may comprise 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-15, 2-20, 2-25, 3-30, 2-35, 2-40, 2-45, 2-45, 2-50, 2-60, 2-70, 2-80, 2-90, 2-100, 2-100, 2-20, 2-30, 2-40, 2-45, 2-50, 2-60, 2-70, 2-80, 2-90, 2-100, 2-20, 2-30, 2-40 ...50, 2-60, 2-70, 2-80, 2- , 3~4, 3~5, 3~6, ​​3~7, 3~8, 3~9, 3~10, 3~15, 3~20, 3~25, 3~30, 3~35, 3~40, 3~45, 3~45, 3~50, 3~60, 3~70, 3~80, 3~90, 3~100, 4~5, 4~6, 4~7, 4~8, 4~9, 4~10, 4~15, 4~ 20, 4~25, 4~30, 4~35, 4~40, 4~45, 4~45, 4~50, 4~60, 4~70, 4~80, 4~90, 4~100, 5~6, 5~7, 5~8, 5~9, 5~10, 5~15, 5~20, 5~25, 5~30, 5~35, 5~40, 5~45, 5~45, 5~50, 5~6 with 0, 5-70, 5-80, 5-90, 5-100, 10-100, 10-20, 10-30, 10-40, 10-50, 20-30, 20-40, 20-50, 20-100, 30-40, 30-50, 30-100, 40-50, 40-100, or 50-100 different nanobodies.

[0113] In some embodiments, a fusion protein comprises a nanobody with multiple therapeutic agents, such as cytokines, attached to either end. For example, the fusion protein may have a structure such as "cytokine1-NB-cytokine2" or "NB-cytokine1-cytokine2."

[0114] In some embodiments, the nanobody cytokine fusion protein further comprises albumin.

[0115] III.How to use The anti-FN-EIIIB nanobodies of the present disclosure can be used as conjugated nanobodies to enable a highly selective means of delivering imaging probes and other cargo molecules, including but not limited to drugs, toxins, antibodies, cytokines, nanoparticles, CAR-T cells, etc., to accessible, common, stable extracellular compartments of diseased tissues (with low systemic background). Alternatively, the nanobodies can be attached to or associated with nanoparticles, which can further comprise other therapeutic agents, such as drugs, toxins, antibodies, cytokines, nucleic acids, such as siRNA, shRNA, modified mRNA, DNA, oligonucleotides, CRISPR Cas9 reagents, imaging probes and other cargo molecules, etc.

[0116] Thus, in one aspect, the present disclosure provides a method of delivering a cargo molecule to the ECM of a diseased tissue in a subject, the method comprising administering to the subject an effective amount of an anti-FN-EIIIB nanobody of the present disclosure conjugated to a cargo molecule.

[0117] In some embodiments, the methods include determining the presence or absence of the EIIIB domain of fibronectin in the subject, and determining whether the subject has a disease or condition associated with fibronectin.

[0118] In some embodiments, the method includes measuring the presence or absence of the EIIIB domain of fibronectin in tissue samples isolated from the subject at a first time point and a second time point, and determining progression to an advanced stage of the disease or condition based on a change in the presence or absence of the EIIIB domain of fibronectin at the first and second time points, where i) if the EIIIB domain of fibronectin is present at a higher level in the isolated tissue sample from the second time point, the disease or condition has progressed to an advanced stage, and ii) if the EIIIB domain of fibronectin is present at a lower level in the isolated tissue sample from the second time point, the disease or condition has regressed to a less advanced stage.

[0119] In another aspect, the present disclosure provides a method of treating a disease or condition associated with fibronectin, comprising administering to a subject in need thereof an effective amount of an anti-FN-EIIIB nanobody of the present disclosure. In some embodiments, the anti-FN-EIIIB nanobody is conjugated to a cargo molecule (e.g., a therapeutic cargo molecule).

[0120] Several methods based on the application and exploitation of these nanobodies are described herein. Methods are provided for determining the presence of FN-EIIIB from patient samples, and thus for providing evidence as to whether a subject has a particular disease and / or disease progression or regression. The status of a subject can be determined using standard immunological methods, including immunohistochemistry, immunofluorescence, ELISA, Western blot, protein array, non-invasive imaging methods such as immunoPET / CT, and similar methods, using anti-FN-EIIIB nanobodies coupled with cargo molecules. The use of such methods provides diagnostic, prognostic and monitoring information that allows improved management of patient care and treatment.

[0121] Methods are provided for specifically targeting cargo molecules, such as imaging agents (e.g., radionuclides, fluorescent reporters or other detectable reagents for use in PET imaging or other imaging modalities, etc.) to FN-EIIIB within the extracellular matrix of pathological conditions to detect the location, extent and progression of disease, such as primary tumors and / or metastases.

[0122] Methods are also provided for specifically targeting active agents, such as therapeutic agents (e.g., radionuclides, chemotherapeutic drugs, toxins, cytokines, siRNA, shRNA, nanoparticles, CAR-T cells, etc.) to FN-EIIIB in order to concentrate such therapeutic agents in diseased tissues, such as primary tumors and / or metastases, thus improving the therapeutic index. Targeting of therapeutic agents is achieved by attaching them to the anti-FN-EIIIB nanobodies of the present disclosure or by co-formulating them in carriers, such as nanoparticles.

[0123] In some embodiments, FN-EIIIB can be analyzed in isolated tissue samples. As used herein, isolated tissue samples are tissues obtained from tissue biopsy, surgically removed tumors, or any other tumor masses removed from the body using methods known to those skilled in the relevant medical arts. The tissues can be known or suspected to be diseased (i.e., fibronectin-related disease or condition), for example, the tissues can be known or suspected to be cancerous. The phrase "suspected to be cancerous" as used herein refers to cancer tissue samples that are believed to contain cancerous cells by those skilled in the medical arts. Methods for obtaining samples from biopsies include macroscopic distribution of tumor mass, microdissection, laser-based microdissection, or other cell separation methods known in the art. The tissues can also be tissue sections.

[0124] Due to the variability of cell types in diseased tissue biopsies and the variability of the sensitivity of the prediction method used, the sample size required for analysis may be 5 mg, 10 mg, 15 mg, 25 mg, 30 mg, or 50 mg or more of tissue. Alternatively, it may be a portion or section of a tissue sample of biopsy size. The appropriate sample size can be determined based on the composition and condition of the cells of the biopsy, and standard preparation steps for this determination and for the subsequent isolation of the protein used in the present invention are well known to those skilled in the art.

[0125] Alternatively, FN-EIIIB can be analyzed directly in vivo. For example, anti-FN-EIIIB nanobodies can be administered directly to a subject. Nanobodies can be labeled, for example, with fluorescent, luminescent, NIR, MRI, SPECT or PET probes to aid in the visualization of ECM.

[0126] The anti-FN-EIIIB nanobodies of the present disclosure can be used to examine changes in a pathology (e.g., a disease or condition associated with fibronectin) in a subject. A pathology is any physiological condition in a subject caused by under- or overproduction of a biochemical in the subject, invasion of the subject by a foreign organism, i.e., an organism that produces a substance that is toxic to the host, or abnormal proliferation of a part of the subject. For example, pathological conditions include conditions associated with infection, cancer, metastasis, etc., many of which are associated with excessive production and alteration of ECM that can be targeted by the nanobodies of the present disclosure. Changes in the metastatic status of a tumor can be examined over time and / or in response to therapeutic intervention. To accomplish these methods, tissue samples can be isolated from a subject at different time points and / or with or without drug treatment. The different tissue samples can be analyzed for the presence of FN-EIIIB. The difference in FN-EIIIB expression between two or more samples can then be evaluated. The differences can be analyzed qualitatively, using assessment of the different proteins present (e.g., by immunohistochemistry, IHC), or quantitatively, by measuring the level or approximate level of expression of the proteins (e.g., by ELISA). Alternatively, the methods of the invention can be performed in vivo without removing a tissue sample.

[0127] The presence of FN-EIIIB in tissue samples or the level of FN-EIIIB in tissue samples can be evaluated using any known method in the art. Such methodologies are well known. A commonly used method in clinical and pathological evaluation is immunohistochemistry or immunofluorescence, which allows the determination of the presence of a particular protein or set of proteins in tissue samples. Methods of immunohistochemistry and immunofluorescence are well known and widely practiced. When a quantitative evaluation of protein levels is performed, the level of the protein can be compared to either a reference or threshold amount or to the amount found in other samples. For example, when the presence or level of a protein is measured in a primary tumor and its metastasis or in a corresponding normal tissue, they can be compared to provide a relative evaluation of the progression of the tumor or its metastasis. Alternatively, the level can be compared to an amount known (or shown) to be above or below the amount that the tumor normally expresses the protein. The value used for comparison is called the reference or threshold level.

[0128] The actual value of a particular determination of the threshold value may vary with different diseases or conditions (e.g., with different tumors) or may vary under different circumstances, such as the conditions of the assay for determining expression. However, those skilled in the art will be able to identify the exact threshold value based on the situation. For example, threshold values ​​can be easily generated using normal (e.g., non-cancerous) tissue under similar circumstances.

[0129] Reference sample can be any of various biological samples that can be diagnostically evaluated.Examples of reference sample include biological samples from a control population or control samples.Reference sample can be produced by manufacturing and provided to test sample in parallel, for example, reference sample can be provided in diagnostic kit.Suitable reference sample will be clear to those skilled in the art.

[0130] In other embodiments, the expression level of protein in test sample can be determined based on direct comparison with reference level in absolute value.For example, the expression level of protein in reference sample is at least 10%, at least 20%, at least 50%, at least 100%, at least 200%, at least 500%, at least 1000% or more higher.In other embodiments, the expression level of protein in test sample is at least 10%, at least 20%, at least 50%, at least 100%, at least 200%, at least 500%, at least 1000% or more lower than the expression level of protein in reference sample.In further embodiments, the expression level of FN-EIIIB in subject is determined at a first time point and at a second time point later.In addition, the expression level of FN-EIIB can be measured at a later time point, so that the total number of measurements can be 3, 4, 5, 6, 7, 8, 9, 10 or more.The time gap between any two measurements can be only a few days, or it can be longer, for example, weeks, months or years. For example, the time between sample acquisition is 6 months, 8 months, 10 months, 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years, or 5 years. The time between acquisition of the first (or earlier) sample from the subject and acquisition of the subsequent or time-course sample can be sufficient time for a change in disease status to occur. Disease progression or regression can be determined by comparing FN-EIIIB expression levels in isolated tissue samples between two or more time points. For example, the presence or absence of FN-EIIIB or changes in their expression levels between successive time points can indicate disease progression, such as to an advanced stage of disease. In particular, higher expression levels and / or presence of FN-EIIIB in samples taken at later time points can indicate that the disease is progressing to an advanced stage. In some examples, the disease can be cancer, and higher expression levels or wider distribution of FN-EIIB associated with the pathological condition in samples taken at later time points can indicate that metastatic cancer is progressing.Conversely, if the tissue sample isolated from the second time point has a lower FN-EIIIB expression level compared to the tissue sample isolated from the first time point, the disease regresses to a less advanced stage.For example, successively lower expression level measurements may indicate that cancer regresses to a less metastatic state.

[0131] The pathological condition may be any non-physiological condition associated with fibronectin, such as cancer, cardiovascular disease (e.g., atherosclerosis, myocardial infarction), inflammatory disorder, fibrosis, or wound. In some embodiments, the cancer is metastatic cancer.

[0132] The presence / absence or level of a protein or marker can be determined using any of several techniques available to one of skill in the art for protein analysis, such as direct physical measurement (e.g., mass spectrometry), or binding assays (e.g., immunohistochemistry, immunoassays, agglutination assays, and immunochromatography assays). Methods may also include measuring signals resulting from chemical reactions, such as changes in absorbance, changes in fluorescence, production of chemiluminescence or electrochemiluminescence, changes in reflectance, refractive index, or light scattering, accumulation of a detectable label or release from a surface, oxidized or reduced or redox species, current or potential, changes in a magnetic field, and the like. Suitable detection techniques can detect the binding event by measuring the engagement of labeled binding domains, by measuring the labels' photoluminescence (e.g., by measuring fluorescence, time-resolved fluorescence, evanescent wave fluorescence, up-converting phosphor, multiphoton fluorescence, etc.), chemiluminescence, electrochemiluminescence, light scattering, absorbance, radioactivity, magnetic field, enzymatic activity (e.g., by measuring enzymatic activity by an enzymatic reaction that causes a change in absorbance or fluorescence, or causes the emission of chemiluminescence). Alternatively, detection techniques that do not require the use of labels can be used, for example techniques based on measuring the mass of the analyte (e.g., surface acoustic wave measurements), measuring the refractive index (e.g., surface plasmon resonance measurements) or measuring intrinsic luminescence.

[0133] The binding assay for measuring protein epitope level can use solid phase or homogeneous format.Suitable assay methods include sandwich or competitive binding assay.Examples of sandwich immunoassays are described in U.S. Patent No. 4,168,146 to Grubb et al. and U.S. Patent No. 4,366,241 to Tom et al., both of which are incorporated herein by reference.Examples of competitive immunoassays include those disclosed in U.S. Patent No. 4,235,601 to Deutsch et al., U.S. Patent No. 4,442,204 to Liotta, and U.S. Patent No. 5,208,535 to Buechler et al., all of which are incorporated herein by reference.

[0134] Multiple ECM epitopes can be measured using multiplexed assay formats, for example, by using nanobody arrays, multiplexing using spectral discrimination of labels, multiplexing by flow cytometric analysis of binding assays performed with the particles.

[0135] The detection of protein epitopes in a test sample involves conventional methods. Those skilled in the art can detect the presence or absence of a protein using well-known methods. Such methods include a variety of immunoassays. In general, immunoassays involve the binding of an antibody or similar probe to a protein in a sample, such as a tissue section, or the binding of a protein in a sample to a solid support, such as a plastic surface. A detectable antibody (or nanobody) that selectively binds to the protein of interest is added. Detection of the antibody (or nanobody) indicates the presence of the protein. The detectable antibody (or nanobody) can be a labeled antibody or an unlabeled antibody. An unlabeled antibody (or nanobody) can be detected using a labeled secondary antibody that specifically binds to the primary antibody (or nanobody), or an unlabeled secondary antibody that can be detected using labeled protein A, a protein that complexes with the antibody. Various immunoassay procedures are described in Immunoassays for the 80's, A. Voller et al., Eds., University Park, 1981, which is incorporated herein by reference.

[0136] Simple immunoassays, such as dot blots and Western blots, involve the use of a solid support that is contacted with a test sample. Any proteins present in the test sample bind to the solid support, and those proteins can be detected by a specific detectable antibody preparation. The intensity of the signal can be measured to obtain a quantitative measurement, such as by ELISA. Other more complex immunoassays include forward assays for the detection of proteins, in which a primary anti-protein antibody bound to a solid support is contacted with the test sample. After a suitable incubation period, the solid support is washed to remove unbound protein. A separate secondary anti-protein antibody is then added that is specific for a portion of the specific protein that is not recognized by the primary antibody. The secondary antibody is preferably detectable. After a second incubation period that allows the detectable antibody to complex with the specific protein bound to the solid support by the primary antibody, the solid support is washed again to remove unbound detectable antibody. Alternatively, in forward sandwich assays, a detectable tertiary antibody that binds to the secondary antibody is added to the system. Other types of immunometric assays include simultaneous and reverse assays. A simultaneous assay involves a single incubation step in which a primary antibody bound to a solid support, a detectable secondary antibody, and a test sample are added simultaneously. After incubation is completed, the solid support is washed to remove unbound protein. The presence of detectable antibody associated with the solid support is then determined as in a conventional assay. A reverse assay involves the stepwise addition of a solution of detectable antibody to the test sample followed by an incubation period, and the addition of antibody bound to the solid support after an additional incubation period. The solid support is washed in a conventional manner to remove unbound protein / antibody complexes and unreacted detectable antibody.

[0137] Several methods for the detection and quantification of antibodies (or nanobodies) are well known. For example, an antibody can be detectably labeled by linking it to an enzyme and then using it in an enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA), such as a capture ELISA. When the enzyme is then exposed to its substrate, it reacts with the substrate to generate a chemical moiety that can be detected, for example, by spectrophotometric, fluorometric or visual means. Enzymes that can be used to detectably label an antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triosephosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.

[0138] The disclosed methods include methods for in vivo imaging using nanobodies with detectable labels. For example, the in vivo imaging methods may include PET imaging. PET imaging technology allows high quality visualization of physiological processes at the molecular level in real time. Therefore, PET is very useful in clinical diagnostics and drug discovery. Although several technical improvements in PET technology have been developed, including both advances in labeling strategies in the field of tracer development and intelligent design of selective molecular probes capable of visualizing molecular targets involved in physiological and pathophysiological processes, early detection of some tumors and micrometastases remains a challenge.

[0139] A detectable label is a moiety, the presence of which can be directly or indirectly ascertained. In general, detection of a label involves the emission of energy by the label. A label can be directly detected by its ability to emit and / or absorb photons or other atomic particles of a particular wavelength (e.g., radioactivity, luminescence, optical or electron density, etc.). A label can be indirectly detected by its ability to bind to, recruit, and in some cases cleave another moiety that can itself emit or absorb light of a particular wavelength (e.g., epitope tags, such as FLAG epitopes, enzyme tags, such as horseradish peroxidase, etc.). One example of indirect detection is the use of a first enzyme label that cleaves a substrate into a visible product. The label can be of a chemical, peptide, or nucleic acid molecule nature, but is not so limited. The label includes any known label that can be used in imaging techniques, such as PET isotopes, scintigraphy, NMR, etc. Other detectable labels include radioisotopes, such as P32 or H3, luminescent markers, such as fluorescent dyes, optical or electron density markers, or epitope tags, such as FLAG epitopes or HA epitopes, biotin, avidin, and enzyme tags, such as horseradish peroxidase, *-galactosidase, and nanoparticles. Labels can be attached to reagents during or after their synthesis. There are many different labels and labeling methods known to those skilled in the art. Examples of the types of labels that can be used in the present invention include enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, and bioluminescent compounds. Those skilled in the art will know of other suitable labels for the reagents described herein, or will be able to ascertain such labels using routine experimentation. Furthermore, coupling or conjugation of these labels to the reagents of the present invention can be performed using standard techniques common to those skilled in the art. Therapeutic agents can also be delivered by various types of nanoparticles, alone and in combination, and these could be targeted to disease-specific ECM by conjugation to nanoparticles that allow for concentration at disease sites.

[0140] Another labeling technique that can provide greater sensitivity consists of coupling the molecules described herein with low molecular weight haptens. These haptens can then be specifically modified using secondary reactions. For example, it is common to use haptens such as biotin, which reacts with avidin, or dinitrophenol, pyridoxal or fluorescein, which can react with specific anti-hapten antibodies.

[0141] Conjugation of the nanobodies and detectable labels described herein facilitates, among other things, the use of such agents in diagnostic assays. Another category of detectable labels includes diagnostic and imaging labels (commonly referred to as in vivo detectable labels), such as for example, for magnetic resonance imaging (MRI): Gd(DOTA); for nuclear medicine: 201Tl, gamma-ray emitting nuclide 99mTc; for positron emission tomography (PET): positron-emitting isotopes, 18F-fluorodeoxyglucose (18FDG), 18F-fluoride, copper-64, gadodiamide, and radioisotopes of Pb(II), such as 203Pb, 111In, etc.

[0142] The cargo molecule may be a detectable label as described above. Such molecules are useful for the detection and characterization of tumor cells in vitro or in vivo.

[0143] In some embodiments, anti-FN-EIIIB nanobody can be conjugated with cargo molecule that is a drug or therapeutic drug.In some embodiments, anti-FN-EIIIB nanobody is conjugated with anti-cancer drug.Such compound can be used as therapeutic conjugate to treat disease and tumor.

[0144] Therapeutic conjugates include anti-FN-EIIIB nanobodies conjugated with cytotoxic agents, such as chemotherapeutic agents, toxins (e.g., enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof, or small molecule toxins), or radioisotopes (i.e., radioconjugates) or CAR-T cells. Other antitumor agents that can be conjugated with anti-FN-EIIIB nanobodies include BCNU, streptozotocin, vincristine and 5-fluorouracil, a family of agents collectively referred to as LL-E33288 conjugates, described in U.S. Patent Nos. 5,053,394 and 5,770,710, and esperamicin (U.S. Patent No. 5,877,296). Enzymatically active toxins and fragments thereof that can be used in conjugates include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and the trichothecenes.

[0145] Nanobodies can be conjugated with highly radioactive atoms for selective destruction of cells. A variety of radioisotopes are available for the production of radioconjugated antibodies. Examples include 211At, 131I, 125I, 90Y, 186Re, 188Re, 153Sm, 212Bi, 32P, 212Pb, and Lu radioisotopes. When the conjugate is used for detection, it can contain radioactive atoms for scintigraphic examination, such as 99mTc or 123I, or spin labels for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as 123I, 131I, 111In, 19F, 13C, 15N, 17O, gadolinium, manganese, or iron. Radioactive or other labels can be incorporated into the conjugate in a known manner. For example, the binding peptide or polypeptide can be biosynthesized or synthesized by chemical amino acid synthesis using suitable amino acid precursors, for example, containing fluorine-19 instead of hydrogen. Labels, such as 99mTc or 123I, 186Re, 188Re and 111In, can be attached via cysteine ​​residues in the peptide. Yttrium-90 can be attached via lysine residues. Iodine-123 can be incorporated using the IODOGEN method (Fraker et al (1978) Biochem. Biophys. Res. Commun. 80: 49-57). Other methods are described in detail in "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989).

[0146] In other embodiments, selective destruction of cells can be mediated by CAR-T cells having a chimeric antigen receptor with an ectodomain comprising the anti-FN-EIIIB of the present disclosure.

[0147] In addition, nanobody-cytokine conjugates can be used for treatment. Anti-FN-EIIIB can be used to deliver cytokines to diseased (e.g., fibronectin-related disease) tissues (e.g., cancerous tissues), which may have several functions, including enhancing immune response. Cytokines include, but are not limited to, IL-2, IL-6, IL-8, IL-10, IL-12, IL-18, TNF, IFN-γ, IFN-β, chemokines, and IFN-α.

[0148] In one aspect, the present invention provides a method for treating a disease or condition associated with fibronectin (e.g., a disease associated with expression of FN-EIIIB). Examples of disorders or diseases associated with fibronectin epitopes include, but are not limited to, cancer, fibrosis, atheroma, inflammatory disorders, cardiovascular diseases, and aneurysms.

[0149] In one aspect, the present disclosure provides a method for the treatment of cancer. The terms "tumor", "cancer", "cancerous tissue" and "carcinoma" are used interchangeably herein and each refers to uncontrolled cell growth that interferes with the normal function of the body's organs and systems. Cancer, including cancer that migrates from its original location and disseminates to vital organs, may ultimately lead to the death of the subject due to the functional impairment of the affected organ. Cancer can be classified into various categories, including carcinoma, sarcoma and hematopoietic cancer. Carcinoma is a malignant cancer that arises from epithelial cells and includes adenocarcinoma and squamous cell carcinoma.

[0150] As used herein, subject is human, non-human primate, cow, horse, pig, sheep, goat, dog, cat or rodent. In all embodiments, human subject is preferred. In some embodiments, subject is either human suspected of having cancer or diagnosed with cancer. Methods for identifying subjects suspected of having fibronectin-related disease or condition (e.g., cancer) can include physical examination, subject's family history, subject's medical history, biopsy, or some imaging techniques, such as ultrasound, computed tomography, magnetic resonance imaging, magnetic resonance spectroscopy, or positron emission tomography. Methods for diagnosing fibronectin-related disease or condition (e.g., cancer) and clinical features of diagnosis are well known to those skilled in the medical arts.

[0151] Cancer treatments and their dosages, routes of administration and recommended methods of use are known in the art. In some embodiments, the therapeutic compounds of the present invention are formulated into pharmaceutical compositions that further comprise one or more additional anti-cancer agents.

[0152] The nanobodies of the invention are administered to a subject in an effective amount for the treatment of the subject. An "effective amount" is, for example, an amount necessary or sufficient to achieve a desired biological effect. For example, an effective amount is an amount sufficient to prevent or inhibit cancer cell growth or proliferation, or alternatively, to induce apoptosis or induce tumor regression in cancer cells. In some embodiments, an effective amount is an amount useful for reducing the occurrence of metastatic cancer.

[0153] The effective amount of the compound of the present invention in treating a subject may vary depending on the specific compound used, the method of delivery of the compound, and whether it is used alone or in combination. The effective amount for any particular application may vary depending on factors such as the type and / or extent of cancer in the subject, the specific compound administered for treatment, the size of the subject, or the severity of the disorder. Those skilled in the art can empirically determine the effective amount of a particular molecule of the present invention without undue experimentation. By selecting from among various active compounds in conjunction with the teachings provided herein and considering factors such as potency, relative bioavailability, patient weight, severity of adverse side effects, and preferred method of administration, an effective prophylactic or therapeutic treatment regimen that does not itself cause substantial toxicity and is completely effective for treating a particular subject can be designed. The toxicity and effectiveness of the protocol of the present invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine LD50 (the dose lethal to 50% of the population) and ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the LD50 / ED50 ratio. Prophylactic and / or therapeutic agents that exhibit large therapeutic indices are preferred. Prophylactic and / or therapeutic agents that exhibit toxic side effects may be used, but care must be taken to design delivery systems that target such agents to affected tissue sites to minimize potential damage to non-infected cells and thereby reduce side effects. Data obtained from cell culture assays, animal studies, and human studies can be used to formulate dosage ranges of prophylactic and / or therapeutic agents for use in humans. The dosage of such agents is preferably within a range of circulating concentrations that includes the ED50 with little or no toxicity. Dosages can vary within this range depending on the dosage form and route of administration employed. For any agent used in the methods of the present invention, the therapeutically effective dose can be initially estimated from cell culture assays.Dosages can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves half-maximal inhibition of symptoms) determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

[0154] The term "treat", "treated" or "treating" refers to the alleviation of symptoms associated with a condition afflicting a subject, where alleviation is used broadly to refer to at least a reduction in the magnitude of a parameter, e.g., a symptom, associated with the condition being treated, such as a disease or condition associated with fibronectin. In some embodiments, it also encompasses preventing the disease from worsening or slowing the progression of the disease compared to the absence of treatment. Thus, treatment also includes the situation in which a condition or at least symptoms and / or secondary effects associated therewith are completely suppressed, e.g., prevented from occurring, or stopped, e.g., terminated, so that a subject no longer suffers from the condition or at least symptoms that characterize the condition.

[0155] The diagnostic and therapeutic compositions described herein can be administered in combination with other therapeutic agents, and such administration can be simultaneous or sequential. When other therapeutic agents are administered simultaneously, they are administered at the same time, but may be administered in the same formulation, or may be administered in separate formulations. The administration of other therapeutic agents, including chemotherapeutic agents, can also be separated in time, meaning that the therapeutic agent is administered at different times, either before or after the administration of the therapeutic agent described herein. The time gap between the administration of these compounds can be only a few minutes, or it can be longer. When used in combination with the treatment of the present disclosure, the dosage of the known treatment can be reduced in some cases to avoid side effects.

[0156] In some embodiments, the methods of the present disclosure include administering to the subject another cancer treatment (e.g., radiation therapy, chemotherapy, or surgery). Examples of conventional cancer treatments include all-trans retinoic acid, actinomycin D, adriamycin, anastrozole, azacitidine, azathioprine, alkeran, Ara-C, arsenic trioxide (Tricenox), BiCNU, bleomycin, busulfan, CCNU, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, cytoxan, DTIC, daunorubicin, docetaxel, doxifluridine, doxorubicin, 5-fluorouracil, epirubicin, epothilone, etoposide, exemestane, erlotinib, fludarabine, fluorouracil, gemcitabine, hydroxyurea, herceptin, hydrea, ifosfamide, irinotecan, riboflavin ... Examples of such treatments include the treatment of cancer with agents such as rifampin, idarubicin, imatinib, letrozole, lapatinib, leustatin, 6-MP, mithramycin, mitomycin, mitoxantrone, mechlorethamine, megestrol, mercaptopurine, methotrexate, mitoxantrone, navelbine, nitrogen mustard, oxaliplatin, paclitaxel, disodium pamidronate, pemetrexed, rituxan, 6-TG, taxol, topotecan, tamoxifen, taxotere, teniposide, thioguanine, toremifene, trimetrexate, trastuzumab, valrubicin, vinblastine, vincristine, vindesine, vinorelbine, velban, VP-16, and xeloda. Multiple doses of the molecules of the present disclosure are also contemplated. In some cases, when a molecule of the present disclosure is administered together with another therapeutic agent, such as a chemotherapeutic agent, a sub-therapeutic dosage of either or both of the molecules may be used. "Sub-therapeutic dose," as used herein, refers to a dosage that is less than the dosage that would produce a therapeutic result in a subject when administered in the absence of the other agent.

[0157] The pharmaceutical compositions of the present disclosure comprise an effective amount of the anti-FN-EIIIB nanobody of the present disclosure dissolved or dispersed in a pharma- ceutically acceptable carrier. The phrase "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other untoward reactions when administered as required to animals, such as humans. It will further be understood that for administration to animals (e.g., humans), preparations must meet sterility, pyrogenicity, general safety and purity standards as required by the FDA Office of Biological Standards. The compounds are generally suitable for administration to humans. This term requires that the compound or composition is non-toxic and sufficiently pure, such that no further manipulation of the compound or composition is required prior to administration to humans.

[0158] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial, antifungal), isotonicity agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, and similar materials and combinations thereof known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences (1990), which is incorporated herein by reference). Its use in therapeutic or pharmaceutical compositions is contemplated, except where any conventional carrier is incompatible with the active ingredient. The compound may be sterile or non-sterile.

[0159] The compositions described herein can contain different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form and whether it needs to be sterile for routes of administration such as injection.

[0160] The compositions may contain various antioxidants to retard oxidation of one or more components. Additionally, the prevention of the action of microorganisms can be provided by preservatives, such as various antibacterial and antifungal agents, including, but not limited to, parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof.

[0161] The nanobodies of the present disclosure can be formulated in compositions in free base, neutral or salt form. Pharmaceutically acceptable salts include acid addition salts, for example those formed with the free amino groups of the proteinaceous composition, or with inorganic acids such as, for example, hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid or mandelic acid. Salts formed with free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxide, or organic bases such as isopropylamine, trimethylamine, histidine or procaine.

[0162] In embodiments where the composition is in liquid form, the carrier may be a solvent or dispersion medium, including, but not limited to, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), lipid (e.g., triglyceride, vegetable oil, liposome), and combinations thereof. The proper fluidity can be maintained, for example, by the use of a coating agent such as lecithin, by maintaining the required particle size, for example, by dispersion in a carrier such as a liquid polyol or lipid, by the use of a surfactant such as hydroxypropylcellulose, or by a combination of such methods. In some embodiments, the composition includes an isotonic agent, such as sugar, sodium chloride, or a combination thereof.

[0163] In some embodiments, the nanobodies of the present disclosure are administered in a pharma- ceutically acceptable solution, which may typically contain pharma- ceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, adjuvants, and other therapeutic ingredients as appropriate. Pharmaceutically acceptable carriers for nucleic acids, small molecules, peptides, monoclonal antibodies, and antibody fragments are well known to those skilled in the art. As used herein, a pharma- ceutically acceptable carrier means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients.

[0164] Pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. In particular, exemplary pharma-ceutically acceptable carriers for peptides are described in U.S. Pat. No. 5,211,657. Such preparations may typically contain salts, buffers, preservatives, compatible carriers, and other therapeutic agents as necessary. When used in medicine, the salts should be pharma-ceutically acceptable salts, but pharma-ceutically unacceptable salts can be conveniently used to prepare pharma-ceutically acceptable salts thereof, and pharma-ceutically unacceptable salts are not excluded from the scope of the present invention. Such pharmacologically and pharma-ceutically acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, formic acid, malonic acid, succinic acid, and the like. Pharmaceutically acceptable salts can also be prepared as alkali metal or alkaline earth salts, for example, sodium, potassium, or calcium salts. The Nanobodies of the invention may be formulated into preparations in solid, semi-solid, liquid or gaseous form, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, inhalants and injections, as well as in conventional manner for oral, parenteral or surgical administration. The present disclosure also encompasses pharmaceutical compositions formulated for local administration, such as by implant, including those designed for sustained or controlled release.

[0165] Compositions suitable for oral administration can be presented as discrete units, such as capsules, tablets, lozenges, each containing a predetermined amount of the active agent. Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as a syrup, elixir, or an emulsion.

[0166] For oral administration, the composition can be easily formulated by combining the nanobody with a pharma- ceutically acceptable carrier known in the art. Such carriers allow the compounds of the present invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. for oral ingestion by the subject to be treated. In the case of solid excipients, pharmaceutical preparations for oral use can be obtained, if necessary, by grinding the mixture obtained and, if desired, adding suitable auxiliaries, processing the mixture of granules to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers, such as sugars, including lactose, sucrose, mannitol or sorbitol, cellulose preparations, such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, etc., and / or polyvinylpyrrolidone (PVP). If desired, disintegrating agents can be added, such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate. If necessary, the oral preparations can also be formulated in saline or buffers to neutralize internal acidic conditions, or can be administered without any carrier.

[0167] Dragee core is provided with suitable coating.For this purpose, concentrated sugar solution, lacquer solution and suitable organic solvent or solvent mixture can be used, which may contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol and / or titanium dioxide as necessary.Dyes or pigments can also be added to tablet or dragee for identification or to characterize different combinations of active compound doses.

[0168] Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, as well as sealed soft capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. Push-fit capsules may contain the active ingredient in admixture with fillers such as lactose, binders such as starches, and / or lubricants such as talc or magnesium stearate, and optionally stabilizers. In the case of soft capsules, the active compound may be dissolved or suspended in a suitable liquid, such as fatty oils, liquid paraffin, or liquid polyethylene glycol. In addition, stabilizers may be added. Microspheres formulated for oral administration may also be used. Such microspheres are well defined in the art. All formulations for oral administration should be in dosages suitable for such administration. For buccal administration, the composition may take the form of tablets or lozenges formulated in a conventional manner.

[0169] For administration by inhalation, the composition for use according to the present disclosure can be conveniently delivered in the form of aerosol spray presentation from pressurized pack or nebulizer using suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of pressurized aerosol, dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, for example, gelatin, for use in inhaler or insufflator can be formulated, containing a powder mix of compound and suitable powder base, for example, lactose or starch. The technology for preparing aerosol delivery systems is well known to those skilled in the art. In general, such systems should use ingredients that do not significantly impair the biological properties of active agents (see, for example, Sciarra and Cutie, "Aerosols," in Remington's Pharmaceutical Sciences, 18th edition, 1990, pp 1694-1712, which is incorporated by reference). One of ordinary skill in the art can readily determine the various parameters and conditions for generating an aerosol without undue experimentation.

[0170] When it is desired to deliver the compositions to the whole body, they can be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion.The preparations for injection can be provided in unit dosage form, for example, in ampoules or in multi-dose containers with added preservatives.The compositions can take the form of suspension, solution or emulsion in oily or aqueous vehicles, and can contain formulating agents such as suspending agents, stabilizing agents and / or dispersing agents.

[0171] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, sterile aqueous or non-aqueous suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, alcoholic / aqueous emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (e.g., based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases. Lower dosages result from other forms of administration, such as intravenous administration. If the response in a subject is inadequate with the initial dose applied, then higher doses (or effectively higher doses via a different, more localized route of delivery) can be used as tolerated by the patient. Multiple doses per day are contemplated to achieve adequate systemic levels of the compound.

[0172] Both non-biodegradable and biodegradable polymer matrices can be used to deliver the compositions of the present disclosure to a subject. Biodegradable matrices are preferred. Such polymers may be natural or synthetic. Synthetic polymers are preferred. The polymer is selected based on the period of time over which release is desired, generally on the order of a few hours to a year or longer. Typically, release over a period ranging between a few hours to 3-12 months is most desirable. The polymer is optionally in the form of a hydrogel capable of absorbing up to about 90% of its weight in water, and further optionally crosslinked with multivalent ions or other polymers.

[0173] The composition can be administered intravenously, intradermally, intraarterially, intralesionally, intratumorally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctivally, intravesicularly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, locally, topically, by inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, localized irrigation to directly bathe the target cells, by catheter, by lavage, in a cream, in a lipid composition (e.g., liposomes), or by other methods or any combination of the foregoing that would be known to one of skill in the art (see, e.g., Remington's Pharmaceutical Sciences (1990), which is incorporated herein by reference). In some embodiments, the administration is intravenous administration. In some embodiments, the administration is intratumor administration. In some embodiments, the administration is intraperitoneally administration. The compositions of the present invention can be administered directly to a tissue. Direct tissue administration can be accomplished by direct injection.

[0174] The composition can be administered once, or alternatively, in multiple doses.When administered multiple times, the composition can be administered by different routes.For example, the first (or first few) doses can be administered directly to affected tissue, while subsequent doses can be administered systemically.

[0175] IV. Kit The present disclosure also provides a kit that comprises the anti-FN-EIIIB nanobody of the present disclosure.The kit can further comprise an assay diluent, a standard, a control and / or a detectable label.The assay diluent, the standard and / or the control can be optimized for a specific sample.It will be easily known to those skilled in the art that the reagent of the present invention can be easily incorporated into one of the established kit formats that are well known in the art.

[0176] Thus, the agents described herein can be assembled into pharmaceutical or diagnostic or research kits in some embodiments to facilitate their use in therapeutic, diagnostic or research applications. The kits can include one or more containers that contain the components of the present disclosure and instructions for use. "Instructions" typically include written instructions on or accompanying the packaging of the compositions of the present invention. Instructions can also include any oral or electronic instructions provided in any manner. Specifically, such kits can include one or more agents described herein, together with instructions that describe the intended therapeutic application and appropriate administration of these agents. In certain embodiments, the agents in the kits can be in pharmaceutical formulations and dosages suitable for the particular application and method of administration of the agents.

[0177] The kit can be designed to facilitate the use of the method described herein by the physician, and can take many forms.Each of the compositions of the kit can be provided in liquid form (e.g., in solution) or solid form (e.g., dry powder), if applicable.In certain cases, some of the compositions can be configurable or otherwise processable (e.g., into active form), for example, by adding suitable solvents or other species (e.g., water or cell culture medium), which may or may not be provided in the kit.

[0178] The kit may contain any one or more of the components described herein in one or more containers. By way of example, in one embodiment, the kit may include instructions for mixing one or more components of the kit and / or isolating, mixing, and applying to a subject. The kit may include a container that contains the agent described herein. The agent may be prepared aseptically, filled into a syringe, and shipped refrigerated. Alternatively, it may be contained in a vial or other container for storage. A second container may have another agent prepared aseptically. Alternatively, the kit may include an active agent that is shipped premixed in a syringe, vial, tube, or other container.

[0179] The following examples are provided to illustrate specific examples of the practice of the invention and are not intended to limit the scope of the invention. As will be apparent to those skilled in the art, the invention will be applicable to a variety of compositions and methods. EXAMPLES

[0180] Example 1 NJB2-IL-2 immune cytokine We tested whether IL2 fused to the FN-EIIIB-specific nanobody NJB2 could extend the survival of diseased mice. Controls included (1) an active but non-targeting immunocytokine based on NJT6, a nanobody specific for the human tenascin-C protein that does not bind to any protein in mice, and (2) an inactive immunocytokine with a "dead" IL2 tag attached to NJB2 (Figures 1A and 1B). 1 × 10 6 of B16F10 cells were inoculated subcutaneously on day 0. Mice were treated with 100 μg of TA99 (ip) and 1 nmol (32 μg) of nanobody-IL-2 fusion (iv) on the days indicated (Figure 1C). TA99 is an antibody that targets a surface antigen on melanoma cells. Tumor growth and survival were measured. No significant differences were observed between NJB2-IL2 and NJT6-IL2 treated mice (Figure 1D).

[0181] Example 2 Affinity maturation of NJB2 Given that no significant differences were observed between NJB2-IL2 and NJT6-IL2 treated mice, it was tested whether increasing the affinity of NJB2 would result in a better outcome.

[0182] Using a previously described yeast display method (2), the FN-EIIIB-specific nanobody NJB2 was subjected to random mutagenesis followed by several rounds of affinity maturation. Figure 2A shows a schematic of how the nanobody library was expressed as Aga2 fusions on the surface of yeast. Nanobody expression is detected by fluorescent antibodies binding to the epitope tags hemagglutinin (HA) or c-Myc. Yeast are selected for enhanced binding to biotinylated FN-EIIIB as detected by streptavidin Alexa-Fluor 647 (SA-647).

[0183] After two rounds of yeast surface display, 10 clones were selected for affinity characterization using Biolayer Interferometry. A second round of yeast display was performed using clones 1.2C, 1.2G and 1.3J from the first round.

[0184] Nanobodies were subcloned, expressed in E. coli, purified by his-tag purification and then subjected to affinity testing. D The values ​​are shown in Figure 2C. All nanobodies tested had higher affinity than the parental NJB2, and some nanobodies from the second round of yeast display had affinity two orders of magnitude higher than NJB2.

[0185] The sequences of the nanobodies from yeast display are shown in Table 1. In some embodiments, the nanobodies contain a GxS linker at the C-terminus (not shown in Table 1). Various mutations in both the CDR regions and framework residues enhanced affinity (Tables 2 and 3). The positions of the mutations in LMJ1.2C and LMJ2.5I are shown on the predicted structure of NJB2. (Figure 2B)

[0186] In Western blot analysis, the mutants selected by yeast display showed selective binding to FN-EIIIB (Figure 3). Western blot shows that the new biotin-tagged nanobodies detect EIIIB-containing fragments, namely EIIIB-His and FN(7-15)-EIIIB. They do not bind to human or mouse plasma fibronectin, which lacks EIIIB or normal lung ECM. Thus, Western blot shows a significant improvement in affinity while retaining the original specificity. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0187] The CDR regions are underlined. The clones tested are shown in bold. [Table 2-1] [Table 2-2]

[0188] CDR changes relative to the corresponding CDRs of NJB2 are underlined. [Table 3-1] [Table 3-2]

[0189] Changes in framework 1 and 4 regions relative to the corresponding framework regions of NJB2 are underlined.

[0190] Example 3 Testing nanobody-cytokine fusions in tumor models via intravenous administration The efficacy of nanomolar and picomolar concentrations of IL-2 immunocytokine specific for FN-EIIIB was tested in B16F10 and 4T1 tumor models. Nanomolar and picomolar concentrations of IL-2 immunocytokine specific for FN-EIIIB were found to result in a slight delay in B16F10 and 4T1 tumor growth after intravenous administration.

[0191] 1 × 10 6 of B16F10 cells were inoculated subcutaneously (sc) on day 0. Mice were treated with 100 μg of TA99 (ip) and 1 nmol (32 μg) of the following nanobody-IL-2 fusions (iv) on the days indicated: (1) NJB2-IL2 (based on the parent nanobody NJB2), (2) LMJ1.2C-IL2, (3) LMJ2.5I-IL2, and (4) NJT6-IL2 (non-targeting control based on NJT6, a nanobody specific for the human TNC protein) (Figure 4A). NJB2-IL2 extends survival in diseased mice compared to NJT6-IL2 (non-targeting control) (Figure 4B). Although the extension in the B16F10 model was not statistically significant, the trend is promising.

[0192] 0.5 × 10 64T1 cells were inoculated on day 0 and treated (iv) with 1 nmol (32 μg) of the following nanobody-IL-2 fusions on the indicated days: (1) NJB2-IL2 (based on the parent nanobody NJB2), (2) LMJ2.5I-IL2, and (3) NJT6-IL2 (non-targeting control based on NJT6, a nanobody specific for the human TNC protein) (Figure 4C). Tumor growth and survival were assessed. NJB2-IL2 and LMJ2.5I-IL2 prolong survival in disease-bearing mice compared to NJT6-IL2 (non-targeting control) (Figure 4D). In the 4T1 model, this prolongation was statistically significant.

[0193] The sequence of the Nanobody-IL2 fusion tested in Examples 3 and 4 (Nanobody-GGGGGS-mouse IL2-LPETGG-HHHHHH) is provided below. [ka] [ka]

[0194] Example 4 Testing nanobody-cytokine fusions in tumor models by intratumoral administration To avoid the challenges / constraints associated with systemic delivery, we tested whether intratumoral delivery could circumvent this issue and improve survival.

[0195] The efficacy of the nanobody-cytokine fusions was tested by intratumoral administration in the B16F10 solid tumor model. It was found that intratumoral administration of the FN-EIIIB-specific IL-2 immunocytokine enabled high B16F10 cure rates.

[0196] 1 × 10 injected into the right flank of 7–8 week-old mice 6of B16F10 cells were inoculated subcutaneously (sc) on day 0. Mice were treated on the indicated days with 100 μg of TA99 (ip) and 0.4 nmol (12.8 μg) of the following IL-2 fusions (it): (1) NJB2-IL2 (based on the parent nanobody NJB2), (2) LMJ2.5I-IL2, and (3) NJT6-IL2 (non-targeting control based on NJT6, a nanobody specific for the human TNC protein) (Figure 5A). Tumor growth and survival were assessed (Figure 5B).

[0197] Additionally, healed mice were injected with 1 × 10 5 B16F10 cells were re-administered subcutaneously on day 94 and tumor growth was monitored without additional treatment. Re-administration experiments were performed with N=1 for NJT6-IL2, N=6 for NJB2-IL2, and N=7 for LMJ2.5I-IL2. All tumors grew in naive age-matched controls (N=6), but the FN-EIIIB-specific IL-2 immunocytokine showed increased survival (Figure 5C).

[0198] Example 5 Testing IL-12-Based Immunocytokines IL-12-based immunocytokines were also tested. IL-12 immunocytokines specific for NJB2-based FN-EIIIB resulted in mild 4T1 growth retardation after intravenous administration.

[0199] 0.5 × 10 6 4T1 cells were inoculated on day 0 and treated with 12.5 μg of the following nanobody-IL-12 fusions on the indicated days (iv): (1) NJB2-IL12, (2) NJT6-IL2 (Figure 6A). To see if antibodies against PD1 could potentiate the response to IL12, a group was also treated with anti-PD1 to IL12 fusions. Tumor growth and viability were assessed (Figure 6B).

[0200] Mice treated with NJB2-IL12 survived longer than mice treated with NJT6-IL12, and although the trend seems promising, the prolongation was not statistically significant with this dosing scheme.

[0201] The sequence of the IL12-nanobody fusion (IL12p40-linker-IL12p35-linker-nanobody-LPETGG-HHHHHH) is provided below. [ka] [ka] [ka]

[0202] Example 6 Testing IFNα-based immunocytokines IFNα-based immunocytokines were also tested. The following nanobody-IFNα fusions were purified: NJT6-IFNα, NJB2-IFNα, LMJ2.5I-IFNα (Figure 7A). All IFNα fusions were active in vitro as tested by the RAW Lucia macrophage activation assay (Figure 7B).

[0203] The sequence of the nanobody-IFNα fusion (nanobody-linker IFNα-HHHHHH) is provided below. [ka] [ka]

[0204] [ka] [ka]

[0205] Equivalents and Scope Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the embodiments described herein. The scope of the present disclosure is not intended to be limited to the above description, but rather is as set forth in the appended claims.

[0206] Articles such as "a", "an" and "the" may mean at least one more than one, unless otherwise indicated or otherwise clear from the context. A claim or description including "or" between two or more members of a group is considered to stand when one, more than one, or all of the group members are present, unless otherwise indicated or otherwise clear from the context. The disclosure of a group including "or" between two or more group members provides embodiments in which exactly one member of the group is present, embodiments in which more than one member of the group is present, and embodiments in which all members of the group are present. For the sake of brevity, these embodiments are not individually detailed herein, but it should be understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed.

[0207] It should be understood that the present disclosure encompasses all variations, combinations and permutations in which at least one limitation, element, clause or descriptive term from at least one claim or from at least one relevant portion of this specification is introduced into another claim.For example, a claim that is dependent on another claim can be modified to include at least one of the limitations found in any other claim that is dependent on the same base claim.Furthermore, it should be understood that if a claim describes a composition, the method of making or using the composition according to any of the methods of making or using disclosed herein, or according to any method known in the art, if any, is included, unless otherwise indicated or unless a contradiction or inconsistency would be obvious to a person skilled in the art.

[0208] When elements are presented as lists, for example in Markush group format, it is understood that all possible subgroups of the elements are also disclosed, and that any element or subgroup of elements may be removed from the group. It is also noted that the term "comprises" is intended to be an open term, allowing for the inclusion of additional elements or steps. In general, when an embodiment, product, or method is said to include certain elements, features, or steps, it is understood that an embodiment, product, or method consisting of or consisting essentially of such elements, features, or steps is also provided. For the sake of brevity, these embodiments are not individually detailed herein, but it is understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed.

[0209] All transitional phrases, such as "comprising," "including," "holding," "having," "containing," "involving," "holding," "consisting of," and the like, in the above specification, as well as in the claims, are to be construed as open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" are intended to be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures Chapter 2111-03.

[0210] It should also be understood that, unless expressly indicated otherwise, for any method recited in the claims herein that includes more than one step or act, the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are recited.

[0211] Where ranges are given, the endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and / or the understanding of one of ordinary skill in the art, it should be understood that values ​​expressed as ranges can take any particular value within the ranges stated in some embodiments, to the tenth of the unit of the lower limit of that range, unless otherwise clearly dictated by the context. For brevity, the values ​​within each range are not individually set forth herein, but it should be understood that each of these values ​​is provided herein and may be specifically claimed or disclaimed. Furthermore, unless otherwise indicated or otherwise evident from the context and / or the understanding of one of ordinary skill in the art, it should also be understood that values ​​expressed as ranges can take any subranges within the given range, with the endpoints of the subranges being expressed to the same precision as the tenth of the unit of the lower limit of the range.

[0212] In addition, it should be understood that any particular embodiment of the present disclosure may be expressly excluded from any one or more claims. When a range is given, any value within the range may be expressly excluded from any one or more claims. Any embodiment, element, feature, application or aspect of the composition and / or method of the present disclosure may be excluded from any one or more claims. For the sake of brevity, not all of the embodiments in which at least one element, feature, purpose or aspect is excluded are expressly set forth herein.

[0213] All references (e.g., published journal articles, books, etc.), patents, and patent applications disclosed herein are hereby incorporated by reference with respect to the subject matter cited, which in some cases may include the entire document.

Claims

1. A Nanobody that specifically binds to the EIIIB domain of fibronectin, a) a complementarity determining region (CDR) 1 comprising G(S / R / I / N)TFSH(N / S / Y / K)AG(G / S); b) a CDR2 comprising: (G / R)(I / V)(S / R / G / N)S(D / A / Y)GNI(N / S); c) a CDR3 comprising NIRG(S / T)YG(N / S)TYY(S / R)R; and not including SEQ ID NO:

1.

2. a) a CDR1 selected from GSTFSHNAGG (SEQ ID NO:54), GSTFSHSAGG (SEQ ID NO:57), GSTFSHYAGG (SEQ ID NO:107), GSTFSHKAGG (SEQ ID NO:106), GRTFSHNAGG (SEQ ID NO:105), GSTFSHNAGS (SEQ ID NO:104), GITFSHNAGG (SEQ ID NO:103), GRTFSHSAGG (SEQ ID NO:63), and GNTFSHSAGG (SEQ ID NO:102); b) a CDR2 selected from GISSDGNIN (SEQ ID NO:55), GIRSDGNIN (SEQ ID NO:58), GIGSDGNIN (SEQ ID NO:59), GISSAGNIN (SEQ ID NO:60), RISSDGNIN (SEQ ID NO:108), GINSYGNIN (SEQ ID NO:109), GIGSAGNIN (SEQ ID NO:62), GIRSDGNIS (SEQ ID NO:101), and GVRSDGNIN (SEQ ID NO:61); c) a CDR3 selected from NIRGSYGNTYYSR (SEQ ID NO: 56), NIRGTYGNTYYSR (SEQ ID NO: 100), NIRGSYGNTYYRR (SEQ ID NO: 99) and NIRGSYGSTYYSR (SEQ ID NO: 98); The nanobody of claim 1, comprising:

3. (a) GSTFSHSAGG (SEQ ID NO:57), GIRSDGNIN (SEQ ID NO:58), and NIRGSYGNTYYSR (SEQ ID NO:56), respectively; (b) GSTFSHSAGG (SEQ ID NO:57), GIGSDGNIN (SEQ ID NO:59), and NIRGSYGNTYYSR (SEQ ID NO:56), respectively; (c) GSTFSHSAGG (SEQ ID NO:57), GISSAGNIN (SEQ ID NO:60), and NIRGSYGNTYYSR (SEQ ID NO:56), respectively; (d) GSTFSHSAGG (SEQ ID NO:57), GIGSAGNIN (SEQ ID NO:62), and NIRGSYGNTYYSR (SEQ ID NO:56), respectively; (e) GSTFSHSAGG (SEQ ID NO:57), GVRSDGNIN (SEQ ID NO:61), and NIRGSYGNTYYSR (SEQ ID NO:56), respectively; or (f) GRTFSHSAGG (SEQ ID NO: 63), GIRSDGNIN (SEQ ID NO: 58), and NIRGSYGNTYYSR (SEQ ID NO: 56), respectively.

3. The nanobody of claim 1 or 2, comprising a CDR1, a CDR2, and a CDR3 comprising:

4. 4. The nanobody of claim 3, comprising CDR1, CDR2, and CDR3 comprising GSTFSHSAGG (SEQ ID NO: 57), GIRSDGNIN (SEQ ID NO: 58), and NIRGSYGNTYYSR (SEQ ID NO: 56), respectively.

5. a) QVQLVETGGGLVQAGGSLRLSCAAS (SEQ ID NO: 64); b) QVQLVGTGGGLVQAGGSLRLSCAAS (SEQ ID NO: 68); c) RVQLVETGGGLVQAGGSLRLSCAAS (SEQ ID NO: 85); d) QVRLVETGGGLVQAGGSLRLSCAVS (SEQ ID NO: 86); e) RVQLVETGGGLVQAGGSLRLSCAVS (SEQ ID NO:66); f) QVQLVETGGGLVQAGGSLRLSCAVS (SEQ ID NO:71); g) QVQLVETRGGLVQAGGSLRLSCAVS (SEQ ID NO:72); h) QVQLVETEGGLVQAGGSLRLSCAAS (SEQ ID NO: 70); i) RVQLVETEGGLVQAGGSLRLSCAAS (SEQ ID NO:87); j) QVRLVETGGGLVQAGGSLRLSCAAS (SEQ ID NO: 88); k) QVQLVEAGGGLVQAGGSLRLSCAAS (SEQ ID NO: 73); l) QVQLVETRGGLVQAGGSLRLSCAAS (SEQ ID NO:89); m) RVQLVETRGGLVQAGGSLRLSCAAS (SEQ ID NO: 90); or n) QVQLVGTGGGLVQAGGSLRLSCAVS (SEQ ID NO: 91) 5. The nanobody of claim 1 , further comprising a framework 1 comprising:

6. a) RVQLVETGGGLVQAGGSLRLSCAVS (SEQ ID NO: 66); b) QVQLVETRGGLVQAGGSLRLSCAVS (SEQ ID NO:72); c) QVQLVETGGGLVQAGGSLRLSCAVS (SEQ ID NO:71); d) QVQLVETEGGLVQAGGSLRLSCAAS (SEQ ID NO: 70); e) QVQLVETGGGLVQAGGSLRLSCAAS (SEQ ID NO: 64); or f) QVQLVEAGGGLVQAGGSLRLSCAAS (SEQ ID NO: 73) 6. The nanobody of claim 5, comprising a framework 1 comprising:

7. a) WGQGTQVTVSS (SEQ ID NO:65); b) WGQGIQVTVSS (SEQ ID NO:69); c) WSQGTQVTVSS (SEQ ID NO:92); d) WGQRTQVTVSS (SEQ ID NO:97); e) WGRGAQVTVSS (SEQ ID NO:93); f) WGQGAQVTVSS (SEQ ID NO:67); g) WGQGTQVTVFS (SEQ ID NO:94); h) WGQGTRVTVSS (SEQ ID NO:95); or i) GGQGTQVTVFS (SEQ ID NO:96) The nanobody of claim 1 , further comprising a framework 4 comprising:

8. a) WGQGAQVTVSS (SEQ ID NO:67); b) WGQGTQVTVSS (SEQ ID NO:65); or c) WGQGIQVTVSS (SEQ ID NO: 69) The nanobody of claim 7, comprising a framework 4 comprising:

9. 9. The nanobody of any one of claims 1 to 8, comprising the amino acid sequence of any one of SEQ ID NOs: 2 to 53.

10. 10. The nanobody of claim 9, which comprises the amino acid sequence of SEQ ID NO:

3.

11. 11. The nanobody of any one of claims 1 to 10, which is conjugated to a cargo molecule.

12. The nanobody of claim 11 , wherein the cargo molecule is linked to the N-terminus or C-terminus of the nanobody.

13. The nanobody of claim 11, wherein the cargo molecule is a molecular imaging probe, a drug, a toxin, an antibody, a cytokine, an enzyme (e.g. an ECM modifying enzyme), a CAR-T cell, a radioisotope, a protein or a nanoparticle.

14. 13. The nanobody of claim 12, wherein the cargo molecule is a cytokine selected from IL-2, IL-6, IL-8, IL-10, IL-12, IL-15, IL-18, TNF, IFNg, IFNb, IFNa and a chemokine or a mutant thereof.

15. 11. A nanobody according to any one of claims 1 to 10, which is attached to or associated with a carrier.

16. The nanobody according to claim 15, wherein the carrier is a nanoparticle.

17. 17. The nanobody of claim 15 or 16, wherein the carrier further comprises a cargo molecule.

18. 18. The nanobody of claim 17, wherein the cargo molecule is selected from a drug, a toxin, an antibody, a cytokine, a nucleic acid, an siRNA, an shRNA, a modified mRNA, a DNA, a CRISPR Cas9 reagent, an oligonucleotide, and an imaging probe.

19. 20. A pharmaceutical composition comprising a nanobody according to any one of claims 1 to 18 and a pharma- ceutically acceptable carrier.

20. 19. A nucleic acid comprising a nucleic acid sequence encoding a Nanobody according to any one of claims 1 to 18.

21. A vector comprising the nucleic acid of claim 19.

22. 22. A cell comprising the nucleic acid of claim 16 or the vector of claim 21.

23. 23. A method for producing a Nanobody, comprising the steps of: (i) culturing a cell according to claim 22 under conditions suitable for expression of said Nanobody; and (ii) recovering the expressed Nanobody.

24. A method for treating a disease or condition associated with fibronectin, comprising administering to a subject in need thereof an effective amount of a nanobody described in any one of claims 1 to 18 or a pharmaceutical composition described in claim 19.

25. 20. A method comprising administering to a subject having a fibronectin-associated disease or condition an effective amount of a nanobody described in any one of claims 11-14 or 17-18 to deliver the cargo molecule to the extracellular matrix of the diseased tissue.

26. 26. The method of claim 25, comprising determining the presence or absence of the EIIIB domain of fibronectin in a subject and determining whether the subject has a disease or condition associated with fibronectin.

27. 26. The method of claim 25, comprising tracking the progression of the disease or condition associated with fibronectin by measuring the presence or absence of the EIIIB domain of fibronectin over time.

28. measuring the presence or absence of the EIIIB domain of said fibronectin in a tissue sample isolated from a subject at a first time point and at a second time point, and determining progression to an advanced stage of said disease or condition based on a change in the presence or absence of the EIIIB domain of said fibronectin at said first and second time points; i) if the EIIIB domain of fibronectin is present at a higher level in the isolated tissue sample from the second time point, the disease or condition has progressed to an advanced stage; ii) if the EIIIB domain of fibronectin is present at a lower level in the isolated tissue sample from the second time point, the disease or condition has regressed to a less advanced stage; 26. The method of claim 25.

29. 29. The method of any one of claims 24 to 28, wherein the disease or condition is cancer, cardiovascular disease, an inflammatory disorder, or fibrosis.

30. 30. The method of claim 29, wherein the cancer is a metastatic cancer.

31. 31. The method of claim 29 or 30, wherein the cancer is melanoma, breast cancer or pancreatic cancer.

32. 32. The method of any one of claims 24 to 31, wherein the administration is intravenous, intratumoral, subcutaneous, intramuscular, intradermal, intraperitoneal, intranasal, intratracheal, intrathecal, intracranial, intraperitoneal, rectal, nasal or oral administration.

33. A nanobody-cytokine fusion protein comprising a nanobody that specifically binds to the EIIIB domain of fibronectin fused to a cytokine.

34. 11. A Nanobody-cytokine fusion protein comprising a Nanobody according to any one of claims 1 to 10 fused to a cytokine.

35. 35. The fusion protein of claim 33 or 34, wherein the cytokine is selected from IL-2, IL-6, IL-8, IL-10, IL-12, IL-18, TNF, IFN-γ, IFN-β, chemokines, and IFN-α, and variants thereof.

36. 36. The fusion protein of any one of claims 33 to 35, comprising at least two nanobodies fused to said cytokine.

37. The fusion protein of claim 36, comprising 2 to 25 nanobodies fused to said cytokine.

38. The fusion protein of claim 36 or 37, wherein the Nanobody comprises multiple copies of the same Nanobody.

39. The fusion protein of claim 36 or 37, wherein the Nanobody comprises at least two different Nanobodies.

40. 36. The fusion protein of any one of claims 33 to 35, comprising at least two cytokines fused to said nanobody.

41. The fusion protein of claim 40, wherein the at least two cytokines are attached to the distal ends of the Nanobody.

42. 42. The fusion protein of claim 41, wherein the at least two cytokines are the same cytokine.

43. 42. The fusion protein of claim 41, wherein the at least two cytokines are two different cytokines.

44. 44. The fusion protein of any one of claims 33 to 43, further comprising albumin.

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