Combination of novel Anti-fibroblast activation protein alpha (FAP) nanobody (VHH) technology with actinium-225

Abstract

An anti-fibroblast activation protein alpha (FAP) polypeptide comprising at least one single-domain antibody directed against FAP. A pharmaceutical composition comprising an anti-FAP single-domain antibody conjugated to a radioisotope at a ratio that maximizes specific activity but does not compromise affinity. A method of reducing a tumor is a subject in need thereof, comprising administering the pharmaceutical composition to a subject.

Description

COMBINATION OF NOVEL ANTI-FIBROBLAST ACTIVATION PROTEINALPHA (FAP) NANOBODY (VHH) TECHNOLOGY WITH ACTINIUM-225REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit to and priority to US Application Serial Number 63 / 702,491, filed on October 2, 2024, which is hereby incorporated by this reference in its entirety.FIELD

[0002] This invention relates generally to a single-domain antibody directed against FAP and methods of using the same.BACKGROUND

[0003] Glioblastoma (GBM) is the most common primary adult brain cancer in the United States (Society, A. C. Cancer Facts & Figures 2023. 1-80 (2023)). Its infiltrative pattern and tumor heterogeneity make treating this form of brain cancer extremely difficult (Agosti, E, Biology (Basel) 12 (2023); Duerinck, J., Front Immunol 14, 1183641 (2023); Shi, T., Cancers (Basel) 15 (2023)). Current standard of care (SOC) involves maximum safe surgical resection, temozolomide therapy, and ionizing radiation. However, this aggressive treatment regimen severely limits patient quality of life (QOL). Despite this approach, GBM has a median survival of only 14-16 months and 5-year overall survival (OS) < 10% (Ostrom, Q.T., Curr Neural Neurosci Rep 11, 329-335 (2011); Ostrom, Q.T., Neuro Oneal 16, 896-913 (2014); Ostrom, Q.T., Neuro Oneal 21, vl-vlOO (2019); Ostrom, Q.T., Neura Oneal 23, iiil- iii 105 (2021); Ostrom, Q.T., Francis, S. S., Curr Neural Neurosci Rep 21, 68 (2021); Ostrom, Q.T., Neura Oneal 15 Suppl 2, iil-56 (2013); Ostrom, Q.T., Neura Oncol 17 Suppl 4, ivl-iv62 (2015); Ostrom, Q.T., Gittleman, H., Neura Oneal 16 Suppl 4, ivl-63 (2014); Ostrom, Q.T., Neuro Oneal 19, vl-v88 (2017); Ostrom, Q.T., Gittleman, Cancer Treat Res 163, 1-14 (2015)) Thus, novel, innovative strategies are needed to enhance clinical outcomes, improve patient QOL, improve mortality rates for patients.

[0004] Fibroblast activation protein alpha (FAP) is a serine protease that is expressed at basal levels in benign tissues. FAP is considered a bona fide marker of pathology since it has low / no expression in healthy tissues but is overexpressed in a variety of pathologies, including cancer, fibrosis, arthritis, and cardiovascular disease. FAP is prominently expressed on the cell surface of neuroepithelial cancer cells and on- 1 -061685.109PCTtumor associated fibroblasts (Lo, A., Cancer Res 75, 2800-2810 (2015); Cremasco, V., Cancer Immunol Res 6, 1472-1485 (2018); Mentlein, R., Biol Chem 392, 199-207 (2011)). Several publications have described FAP overexpression in GBM tumors including high-grade gliomas with a mesenchymal subtype (Mentlein, R., 2011; Balaziova, E., Cancers (Basel) 13 (2021); Busek, P., Tumour Biol 37, 13961-13971 (2016); Busek, P., Histochem Cell Biol 143, 497-504 (2015); Busek, P., Front Biosci (Landmark Ed) 23, 1933-1968 (2018); Matrasova, I., Biomed Pap Med Fae Univ Palacky 0 / omouc Czech Repub 161, 252-260 (2017); Simkova, A., Biochim Biophys Acta Proteins Proteom 1868, 140409 (2020); Zubal, M., Pathology 10.1016 / j.pathol.2023.05.003 (2023)). Furthermore, additional studies indicated that FAP expression correlates with extracellular matrix remodeling, glioma cell invasion, and inflammation, suggesting a link between FAP expression and the aggressive tissue remodeling, necrosis, and inflammatory infiltrates observed in these tumors. Finally, FAP expression was observed on a variety of stromal cell populations within GBM tumors, suggesting that targeting FAP for imaging and therapy may provide a comprehensive treatment approach that simultaneously targets tumor cells and the pro- tumorigenic microenvironment of this cancer. Despite this unique expression profile, designing effective diagnostic and therapeutic agents that effectively target this biomarker remains elusive.

[0005] Single variable domain on a heavy chain (VHH) antibodies, also referred to as Nanobodies®, are a class of immunoglobulins that consist of only one heavy chain with a single variable domain (Dekempeneer, Y., Expert Opin Biol Ther 16, 1035-1047 (2016); D'Huyvetter, M., Expert Opin Drug De / iv 11, 1939-1954 (2014); Barakat, S., Free Radie Biol Med 182, 260-275 (2022); Arezumand, R., Front Immunol 8, 1746 (2017); Alirahimi, E., Biochim Biophys Acta Gen Subj 1862, 2955-2965 (2018); AI- Baradie, R. S., Hum Antibodies 28, 259-272 (2020)). VHH antibodies retain high affinity and specificity for their target antigens, with low off-target accumulation. Furthermore, unlike conventional antibodies, they can tolerate environmental conditions associated with225Ac (ti / 2 = 10 d; Eomax = 6-8 MeV) radiochemistry, including high temperatures, elevated pressures, and non-physiological pH levels. Finally, their small size and ability to penetrate the blood-brain barrier (BBB) make them ideal ligands for delivering systemic alpha particle (a) radiotherapy to GBM tumors while reducing any off-target effects to normal tissues (Dekempeneer, Y., 2016; Barakat, S., 2022;- 1 -061685.109PCTAlirahimi, E., 2018; Vaneycken, I., Curr Opin Biotechnol 22, 877-881 (2011); Rodak, M., Mol Cancer Ther 21, 1835-1845 (2022); Pruszynski, M., Mol Pharm 15, 1457- 1466 (2018); Lecocq, Q., Theranostics 9, 7772-7791 (2019); Bathula, N. V., Cancer Biother Radiopharm 2021, 36, 109-122; Awad, R. M., Ini Rev Cell Mol Biol 369, 143- 199 (2022); Kunikowska, J., Front Med (Lausanne) 9, 1085245 (2022)).

[0006] Provided herein are novel single-domain antibodies (sdAbs) and methods of making thereof. The invention describes the structural characterization of the interaction between novel sdAbs and FAP using cryo-electron microscopy. The reconstructions were determined to a resolution of 2.7 A and contained two distinct populations: one sdAb bound and two sdAb molecules bound to the FAP dimer. In both cases, the sdAbs bound a unique epitope that was distinct from the active site of the enzyme. Furthermore, this invention provides the rational mutation of specific residues within the complementarity-determining region 3 (CDR3) loop to computationally enhance affinity and selectivity of the sdAbs molecule for FAP. Also provided is a novel anti-fibroblast activation protein alpha (FAP) nanobody (VHH) technology utilizing actinium-225 (225 Ac) to create a unique platform for delivering targeted alpha particle therapy (TAT) to glioblastoma tumors. Additionally, a novel platform is provided herein, where Zr-89 and Ac-225 can be used as a theranostic pair or alone to diagnose and treat GBM, respectively.SUMMARY

[0007] In accordance with the purpose(s) of this invention, as embodied and broadly described herein, this invention, in one aspect, relates to an anti-fibroblast activation protein alpha (FAP) polypeptide comprising at least one single-domain antibody directed against FAP wherein the at least one single-domain antibody comprises at least 95% sequence identity to nucleic acid sequences of SEQ ID NO: 1, 6, 11, or the combination thereof or at least 95% sequence identity to amino acid sequences of SEQ ID NO: 2, 7, 12, or the combination thereof..

[0008] In one embodiment, the single-domain antibody comprises: a) a variable heavy domain of heavy chain (VHH) sequence having at least 95% sequence identity to amino acid sequences of SEQ ID NO: 2, and wherein the VHH comprises a complementarity determining regions (CDR), CDR1 having at least 95% sequence identity to amino acid sequences of SEQ ID NO: 3, CDR2 having at least 95% sequence identity to amino acid sequences of SEQ ID NO: 4, and CDR3 having at least 95%- 3 -061685.109PCTsequence identity to amino acid sequences of SEQ ID NO: 5; b) a variable heavy domain of heavy chain (VHH) sequence having at least 95% sequence identity to amino acid sequences of SEQ ID NO: 7, and wherein the VHH comprises a complementarity determining regions (CDR), CDR1 having at least 95% sequence identity to amino acid sequences of SEQ ID NO: 8, CDR2 having at least 95% sequence identity to amino acid sequences of SEQ ID NO: 9, and CDR3 having at least 95% sequence identity to amino acid sequences of SEQ ID NO: 10; or c) a variable heavy domain of heavy chain (VHH) sequence having at least 95% sequence identity to amino acid sequences of SEQ ID NO: 12, and wherein the VHH comprises a complementarity determining regions (CDR), CDR1 having at least 95% sequence identity to amino acid sequences of SEQ ID NO: 3, CDR2 having at least 95% sequence identity to amino acid sequences of SEQ ID NO: 4, and CDR3 having at least 95% sequence identity to amino acid sequences of SEQ ID NO: 13.

[0009] In another embodiment, the VHH sequence comprising at least 95% sequence identity to amino acid sequences of SEQ ID NO: 2 binds to the extracellular surface of FAP by making important contacts between a CDR3 loop of VHH’ s CDR3 and a unique FAP epitope, that is distinct from a FAP active site.

[0010] In another embodiment, amino acids VI 07 and SI 09 of the VHH’s CDR3 are mutated to computationally increase the affinity and stability of the single-domain antibody. In particular embodiments, amino acid SI 09 is mutated to S109R, forming two new hydrogen bonds and a salt bridge, which positively increases affinity and stability.

[0011] In other embodiments, the unique FAP epitope is FAP’s FR2 region. In another embodiment, the CDR3 loop interacts with the extracellular surface of FAP at FAP Y274. In additional embodiments, FAP has multiple FAP loops, and FAP Y274 has multiple interactions with the CDR3 loop and sits within a pocket formed by the CDR3 loop. In yet another embodiment, the CDR3 loop interacts with one FAP loop at a relatively uncharged region and a second FAP loop at a positively charged region. In particular embodiments, FAP and CDR3 loop multiple interactions have residue pairs having hydrogen bond interactions comprising Y274:P108, Y274:W47, E325:S109, and D326:V107. In yet another embodiment, there is a pi-stacking interaction between Y27EF110.- 4 -061685.109PCT

[0012] In one embodiment, the number of single-domain antibodies directed against FAP is at least two, wherein the at least one single-domain antibody is a Camelidae VHH or a humanized Camelidae VHH. In another embodiment, at least one single-domain antibody is a homologous sequence, a functional portion, or a functional portion of a homologous sequence of the full-length single-domain antibody. In another embodiment, the anti-FAP polypeptide is a homologous sequence, a functional portion, or a functional portion of a homologous sequence of the full length anti-FAP polypeptide.

[0013] In another aspect, the invention relates to a pharmaceutical composition comprising an anti-FAP single-domain antibody as described above and a radioisotope, wherein the radioisotope is conjugated to the anti-FAP single-domain antibody at a ratio that maximizes specific activity but does not compromise affinity, wherein the composition is delivered to a tumor of a subject in need thereof, and wherein the composition is a theranostic agent used to treat or diagnose a disease characterized by overexpression of fibroblast activation protein (FAP).

[0014] In one embodiment, the radioisotope is selected from a group of radionuclides consisting of "mTc,67Ga,68Ga,66Ga,47Sc,51Cr,UC ’167Im,141Ce,i nIn,212Pb,134Ce,133Ce,133La,134La,149Tb,152Tb,155Tb,94Tc, "Tc,43Sc,44Sc,117Sn,52Mn,53Mn,54Mn,55Mn,13N,150,223Fr,227Th,229Th,228Th,226Ra,224Ra,219Rn,215Po,212Po,216Po,206Po,211Pb,2O7T1,2O8T1,211PO,107Ag,109Ag,195mPt,103Pd,223Ra,82Rb’89Sr,85Sr,90Sr,45Ti,44Ti,73As,119Sb,55Fe,59Fe,22Na,48V,63Ni,65Zn,109Cd,153Gd,148Gd,194Hg,26Al,32Si,68Ge,73As,77Br, or147Pm.

[0015] In another embodiment, the anti-FAP single-domain antibody is conjugated to225Ac or89Zr, and a chelating group, and wherein Zr-89 and Ac-225 can be used as a theranostic pair or alone to diagnose and treat the disease characterized by overexpression of fibroblast activation protein (FAP).

[0016] In yet another aspect, the invention relates to a method of reducing a tumor is a subject in need thereof comprising administering the pharmaceutical composition described above to the subject. In one embodiment, the disease characterized by overexpression of fibroblast activation protein (FAP) is selected from the group- 5 -061685.109PCTconsisting of cancer, chronic inflammation, atherosclerosis, fibrosis, tissue remodeling or keloid disorder. In another embodiment, the cancer is a solid tumor selected from the group consisting of glioblastoma multiforme (GBM), breast cancer, pancreatic cancer, small intestine cancer, colon cancer, rectal cancer, lung cancer, head and neck cancer, ovarian cancer, hepatocellular carcinoma, esophageal cancer, hypopharynx cancer, nasopharynx cancer, larynx cancer, myeloma cells, bladder cancer, cholangiocellular carcinoma, clear cell renal carcinoma, mesothelioma, neuroendocrine tumor, oncogenic osteomalacia, sarcoma, and carcinoma of unknown primary (CUP). In other embodiments, the subject has a neurological disorder.

[0017] In another embodiment, the radioisotope is conjugated to the anti-FAP single-domain antibody to create a targeted alpha particle therapy (TAT) that selectively delivers alpha particle radiation to tumors. The pharmaceutical composition targets a FAP biomarker found the tumors to selectively deliver alpha particle radiation to GBM tumors. In another embodiment, the composition is administered intravenously.

[0018] Additional advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.BRIEF DESCRIPTIONS OF THE DRAWINGS

[0019] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate (one) several embodiment(s) of the invention and together with the description, serve to explain the principles of the invention.

[0020] Figures 1A-1C show mass photometry analysis of FAP with SUMO-13 complexes. FIG. 1A shows the mass distribution of 15 nM FAP alone. The molecular weight (MW) observed by MP for FAP is 198 ± 8.3 kDa, which agrees well with the predicted MW of the dimeric FAP (170 kDa) with glycosylation. FIG. IB shows a sensorgram from bio-layer interferometry (BLI) data showing binding of FAP with increasing concentrations of SUMO-13. Data is representative of triplicate measurements. FIG. 1C shows the mass distribution of 15 nM BS3 cross-linked FAP- 6 -061685.109PCTwith SUMO-13 in 1 :5 molar ratio. The MWs observed are 57 ± 7.8 kDa, 200 ± 10.2 kDa, 227 ± 9.5 kDa, and 253 ± 8.8 kDa, which corresponds to the expected MWs of two SUMO-13 (54 kDa), glycosylated FAP alone (198 kDa, panel 1A), and FAP with one SUMO-13 (225 kDa) or 2 SUMO-13 (252 kDa) molecules bound, respectively.

[0021] Figures 2A-2B are graphs showing the binding affinity of FAP-MBP-I3 by BLI. FIG. 2A shows kinetic fits (red lines) for 1 : 1 model from FAP-MBP-I3 binding data (green lines). R2= 0.98 X2= 0.01. FIG. 2B shows steady state analysis of FAP - MBP-I3 binding data. R2= 0.998 Rmax= 0.2403 ± 0.004. Kd= 1.80 ± 0.09 pM.

[0022] Figures 3A-3C show the cryo-EM data processing workflow. FIG. Figure 3A illustrates the processing workflow employed in cryoSPARC to obtain reconstructions of one and two SUMO-13 molecules bound to FAP. FIG. 3B shows the particle view distribution for one SUMO-13 bound to FAP (Cl symmetry). FIG. 3C shows the particle view distribution for two SUMO-13 bound to FAP (C2 symmetry).

[0023] Figures 4A-4F show the cryo-EM structures of FAP-I3 complexes. FIG. 4A shows FAP-I3 reconstruction with the local resolution map. FIG. 4B shows the FAP-I3 model. FIG. 4C shows the FSC curve for the 3D reconstruction of the cryo-EM map of FAP-I3. The overall resolution is 2.7 A based on the FSC value of 0.143. FIG. 4D shows FAP-(I3)2 reconstruction with the local resolution map. FIG. 4E shows the FAP-(I3)2 model. FIG. 4F FSC curve for the 3D reconstruction of the cryo-EM map of FAP-(I3)2. The overall resolution is 2.7 A based on the FSC value of 0.143. Cartoon models in FIG. 4B and FIG. 4E shows the FAP dimer in two shades of blue and 13 in green. Yellow sticks represent sites of glycosylation on FAP.

[0024] Figures 5A-5D show interactions of FAP with 13. FIG. 5 A is an overview of 13 bound to FAP, showing various VHH regions typically involved in the paratope. Blue and red spheres correspond to the first and last residues modeled for 13. FIG. 5B shows residues involved in specific interactions at the FAP-I3 interface. FIG. Figure 5C shows an electrostatic surface map of 13 and residues from FAP involved in epitope formation, represented in blue sticks. FIG. 5D is a surface representation of FAP, highlighting the 13 epitope region and its location relative to the active site.

[0025] Figures 6A-6D show in silico affinity maturation of 13. FIG. 6A shows an electrostatic surface map of FAP and some residues of 13 near the surface in green sticks. Residues chosen for mutation are in cyan. FIG. 6B shows a VI 07 mutation change in affinity and stability results compared with the original 13 sequence. FIG. 6C- 7 -061685.109PCTshows a S109 mutation change in affinity and stability results compared with the original 13 sequence. FIG. 6D shows a comparison for the number of hydrogen bonds (HB), salt bridges (SB), and pi stacking interactions present at the interfaces of 13 and the V107R and S109R mutants.

[0026] Figures 7A-7D show FAP epitope comparison with DPP4. FIG. 7A shows the overall structure-based alignment of FAP with DPP4. FIG. Figure 7B highlights the FAP epitope region, showing conformational differences in DPP4. FIG. 7C shows a structure-based sequence alignment of the FAP epitope region with DPP4. Red asterisks indicate two key residue differences. FIG. 7D shows BLI binding data showing MBP-I3 (10 uM) specifically interacts with FAP and not DPP4.

[0027] Figures 8A-8D show a map and model overlay. FIG. Figure 8A shows a cartoon model of FAP-I3 overlaid with the final reconstruction map, illustrating various orientations. FIG. 8B shows a map-to-model FSC for FAP-I3. FIG. 8C shows a cartoon model of FAP + 2 SUMO-13 overlaid with the final reconstruction map showing different orientations. FIG. 8D shows a map-to-model FSC for FAP + 2 SUMO-13.

[0028] Figures 9A-9B show the overall alignment of FAP with other DPPs and comparison of the 13 epitope region of FAP to structurally aligned regions in DPPs. PDB codes: FAP (1Z68), DPP4 (2ONC), DPP8 (6EOO), DPP9 (7A3F).

[0029] Figures 10A-10F show that FAP is expressed at basal levels in normal bone marrow across species. Whole bone marrow isolates were rinsed, RBC lysed and cytospun onto prepared slides. Multiplexed, multispectral analysis was performed using anti-FAP, anti-a-SMA, anti-CD45, and DAPI. Non-human primate, pig, rat, rabbit, and mouse and the human cancer U87cell line (FAP+ control) were surveyed.

[0030] Figure 11 shows that FAP+ cells are present at extremely low densities in the normal bone marrow of mammals. Multiple images from each species' bone marrow (n = 10) were imported into inForm, spectrally unmixed and segmented to detect FAP (red), a-SMA (green), CD45 (yellow) and DAPI (blue). Both FAP+ / aSMA+, were coregistered and the total FAP+ / aSMA+ cells were quantified and normalized over 300 cells.

[0031] Figure 12 is a cryo-EM map of the I3:FAP protein complex. The structure was determined at a resolution of 2.7 A using cryo-EM. The 13 VHH binds to FAP in a 2: 1 ratio.- 8 -061685.109PCT

[0032] Figures 13A-13B show mass photometry (MP) data of MBP-Iowa5 with FAP.

[0033] Figures 14A-14D show BLI data of MBP-Iowa28 with FAP.

[0034] Figure 15 is an illustration of the generation of the three distinct anti -FAP nanobodies, 1-3, 1-5 and 1-28.

[0035] Figures 16A-16F are FACS data showing the analysis of novel phage display-derived nanobodies binding to U87MG cells.

[0036] Figures 17A and 17B show the analysis of flow cytometry data for 128 (Fig. 16D), which demonstrates improved cell binding of FAP+ U87mg cells compared to 13.

[0037] Figures 18A-18G show the analysis of Iowa 28 dimer nanobodies binding on U87MG cells using FACS (Iowa 28 dimer titration).

[0038] Figures 19A-19B show a conjugation scheme of MBP-I-3 with PCTA-Bn- NCS (FIG. 19A) and a radiolabeling scheme of PCTA-MBP-I-3 with zirconium-89 or actinium-225 (FIG. 19B).

[0039] Figures 20A-20B show the radiochemical purity of89Zr-PCTA-MBP-I-3 by radio-TLC. Figures 20C-20D show the radiochemical purity of89Zr-PCTA-MBP- 1-3 by radio-HPLC. Stationary Phase: ITLC-SG. Mobile phase: 50 mM EDTA, pH 5.0.

[0040] Figures 21A-21B show in vitro serum stability analysis by radio-TLC. Radio-ITLC of89Zr-PCTA-MBP-I-3 solution in human serum at 37 °C after 0 h (FIG. 21A) and 7 days (FIG. 21B). Figures 21C-21D show in vitro serum stability analysis by radio-HPLC. Radio-HPLC of89Zr-PCTA-MBP-I-3 solution in human serum at 37 °C after 0 h (FIG. 21C) and 7 days (FIG. 2 ID). Stationary phase: ITLC-SG. Mobile phase: 50mM EDTA, pH 5.0.

[0041] Figures 22A-22B show radiochemical purity of225Ac-PCTA-MBP-I-3 by Radio-TLC. Quality control by radio-TLC: ITLC-SG of225Ac(NO3)3 (FIG. 22A), and225AC-PCTA-MBP-I-3 (FIG. 22B). Figures 22C-22D show radiochemical purity of225AC-PCTA-MBP-I-3 by Radio-HPLC. Quality control by radio-HPLC: UV-HPLC chromatogram (220 nm) of nonradioactive PCTA-MBP-I-3 (FIG. 22C) compared with radio-HPLC chromatogram of225Ac-PCTA-MBP-I-3 (FIG. 22D). Stationary phase: ITLC-SA. Mobile phase: 50mM DTP A, pH 7.0.- 9 -061685.109PCT

[0042] Figures 23A-23B show in vitro serum stability study225Ac-PCTA-MBP- I-3( Radio-TLC). Radio-ITLC of225Ac-PCTA-MBP-I-3 solution in human serum at 37 °C after 0 h (FIG. 23A) and 7 days (FIG. 23B). Figures 23C-23D show an in vitro serum stability study of225Ac-PCTA-MBP-I-3 (Radio-HPLC). Radio-HPLC of225Ac- PCTA-MBP-I-3 solution in human serum at 37 °C after 0 h (FIG. 23C) and 7 days (FIG. 23D). Stationary phase: ITLC-SA. Mobile phase: 50mM DTP A, pH 7.0.DETAILED DESCRIPTION

[0043] The present invention may be understood more readily by reference to the following detailed description of preferred embodiments of the invention and the Examples included therein and to the Drawings and their previous and following description.I. Definitions

[0044] To facilitate an understanding of the principles and features of the various embodiments of the disclosure, various illustrative embodiments are explained herein. Although exemplary embodiments of the disclosure are explained in detail, it is to be understood that other embodiments are contemplated. Accordingly, it is not intended that the disclosure is limited in its scope to the details of construction and arrangement of components set forth in the description or examples. The disclosure is capable of other embodiments and of being practiced or carried out in various ways.

[0045] In view of the teachings of the present Specification and the Examples, one of ordinary skill in the art can apply conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant polynucleotides, as taught, for example, by the following standard texts: Cellular and Molecular Immunology, Ninth Edition, A. K. Abbas., et al., Elsevier (2017), ISBN 978-0323479783; Cancer Immunotherapy Principles and Practice, First Edition, L. H. Butterfield, et al., Demos Medical (2017), ISBN 978-1620700976; Janeway's Immunobiology, Ninth Edition, Kenneth Murphy, Garland Science (2016), ISBN 978-0815345053; Clinical Immunology and Serology: ALaboratory Perspective, Fourth Edition, C. Dorresteyn Stevens, et al., F. A. Davis Company (2016), ISBN 978- 0803644663; Antibodies: A Laboratory Manual, Second edition, E. A. Greenfield, Cold Spring Harbor Laboratory Press (2014), ISBN 978-1-936113-81-1; Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, Seventh Edition, R. I. Freshney, Wiley-Blackwell (2016), ISBN 978-1118873656; Transgenic Animal- 10 -061685.109PCTTechnology, Third Edition: A Laboratory Handbook, C. A. Pinkert, Elsevier (2014), ISBN 978-0124104907; The Laboratory Mouse, Second Edition, H. Hedrich, Academic Press (2012), ISBN 978-0123820082; Manipulating the Mouse Embryo: A Laboratory Manual, Fourth Edition, R. Behringer, et al., Cold Spring Harbor Laboratory Press (2013), ISBN 978-1936113019; PCR 2: A Practical Approach, M. J. McPherson, et al., IRL Press (1995), ISBN 978-0199634248; Methods in Molecular Biology (Series), J. M. Walker, ISSN 1064-3745, Humana Press; RNA: A Laboratory Manual, D. C. Rio, et al., Cold Spring Harbor Laboratory Press (2010), ISBN 978- 0879698911; Methods in Enzymology (Series), Academic Press; Molecular Cloning: A Laboratory Manual (Fourth Edition), M. R. Green, et al., Cold Spring Harbor Laboratory Press (2012), ISBN 978-1605500560; Bioconjugate Techniques, Third Edition, G. T. Hermanson, Academic Press (2013), ISBN 978-0123822390; Methods in Plant Biochemistry and Molecular Biology, W. V. Dashek, CRC Press (1997), ISBN 978-0849394805; Plant Cell Culture Protocols (Methods in Molecular Biology), V. M. Loyola-Vargas, et al., Humana Press (2012), ISBN 978-1617798177; Plant Transformation Technologies, C. N. Stewart, et al., Wiley-Blackwell (2011), ISBN 978-0813821955; Recombinant Proteins from Plants (Methods in Biotechnology), C. Cunningham, et al., Humana Press (2010), ISBN 978-1617370212; Plant Genomics: Methods and Protocols (Methods in Molecular Biology), W. Busch, Humana Press (2017), ISBN 978-1493970018; Plant Biotechnology: Methods in Tissue Culture and Gene Transfer, R. Keshavachandran, et al., Orient Blackswan (2008), ISBN 978- 8173716164.

[0046] In describing the exemplary embodiments, specific terminology will be resorted to for the sake of clarity. As used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, a reference to a component is also intended to include the composition of a plurality of components. References to a composition containing “a” constituent is intended to include other constituents in addition to the one named.

[0047] Ranges may be expressed herein as from “about” or “approximately” or “substantially” one particular value and / or to “about” or “approximately” or “substantially” another particular value. When such a range is expressed, other- 11 -061685.109PCTexemplary embodiments include from the one particular value and / or to the other particular value.

[0048] The term “immunoglobulin” refers to a class of structurally related proteins generally comprising two pairs of polypeptide chains: one pair of light (L) chains and one pair of heavy (H) chains. In an “intact immunoglobulin,” all four of these chains are interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized. See, e.g., Paul, Fundamental Immunology 7th ed., Ch. 5 (2013) Lippincott Williams & Wilkins, Philadelphia, Pa. Briefly, each heavy chain typically comprises a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region typically comprises three domains, abbreviated CHI, CH2, and CH3. Each light chain typically comprises a light chain variable region (VL) and a light chain constant region. The light chain constant region typically comprises one domain, abbreviated CL.

[0049] The term “antigen-binding protein” (ABP) refers to a protein comprising one or more antigen-binding domains that specifically bind to an antigen or epitope. In some embodiments, the antigen-binding domain binds the antigen or epitope with specificity and affinity similar to that of naturally occurring antibodies. In some embodiments, the ABP comprises an antibody or a nanobody. In some embodiments, the ABP consists of an antibody or a nanobody. In some embodiments, the ABP consists essentially of an antibody or a nanobody. In some embodiments, the ABP comprises an alternative scaffold. In some embodiments, the ABP consists of an alternative scaffold. In some embodiments, the ABP consists essentially of an alternative scaffold. In some embodiments, the ABP comprises an antibody or a nanobody fragment. In some embodiments, the ABP consists of an antibody or a nanobody fragment. In some embodiments, the ABP consists essentially of an antibody or a nanobody fragment.

[0050] The term “antibody” is used herein in its broadest sense and includes certain types of immunoglobulin molecules comprising one or more antigen-binding domains that specifically bind to an antigen or epitope. An antibody specifically includes intact antibodies (e.g., intact immunoglobulins), antibody fragments, and multi-specific antibodies. An antibody is one type of ABP.

[0051] The term “alternative scaffold” refers to a molecule in which one or more regions may be diversified to produce one or more antigen-binding domains that- 12 -061685.109PCTspecifically bind to an antigen or epitope. In some embodiments, the antigen-binding domain binds the antigen or epitope with specificity and affinity similar to that of an antibody. Exemplary alternative scaffolds include those derived from fibronectin (e.g., Adnectins™), the P-sandwich (e.g., iMab), lipocalin (e.g., Anticalins®), EETI- n / AGRP, BPTI / LACI-D1 / ITI-D2 (e.g., Kunitz domains), thioredoxin peptide aptamers, protein A (e.g., Affibody®), ankyrin repeats (e.g., DARPins), gamma-B- crystallin / ubiquitin (e.g., Affilins), CTLD3 (e.g., Tetranectins), Fynomers, and (LDLR- A module) (e.g., Avimers). Additional information on alternative scaffolds is provided in Binz et al., Nat Biotechnol. 2005 Oct;23(10): 1257-68; Skerra, , Curr Opin Biotechnol. 2007 Aug;18(4):295-304; and Silacci., J. Biol. Chem., 2014, 289: 14392- 14398; each of which is incorporated by reference in its entirety. An alternative scaffold is one type of ABP.

[0052] The term “antigen-binding domain” means the portion of an ABP that is capable of specifically binding to an antigen or epitope. One example of an antigenbinding domain is an antigen-binding domain formed by a VH-VL dimer of an antibody.

[0053] The terms “full length antibody,” “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a naturally occurring antibody structure and having heavy chains that comprise an Fc region. For example, when used to refer to an IgG molecule, a “full length antibody” is an antibody that comprises two heavy chains and two light chains.

[0054] The term “Fc region” means the C-terminal region of an immunoglobulin heavy chain that, in naturally occurring antibodies, interacts with Fc receptors and certain proteins of the complement system. The structures of the Fc regions of various immunoglobulins, and the glycosylation sites contained therein, are known in the art. See Schroeder and Cavacini, J. Allergy Clin. Immunol., 2010, 125:S41-52, incorporated by reference in its entirety. The Fc region may be a naturally occurring Fc region, or an Fc region modified as described in the art or elsewhere in this disclosure.

[0055] The VH and VL regions may be further subdivided into regions of hypervariability (“hypervariable regions (HVRs);” also called “complementarity determining regions” (CDRs)) interspersed with regions that are more conserved. The more conserved regions are referred to as framework regions (FRs). Each VH and VL generally comprises three CDRs and four FRs, arranged in the following order (from N-terminus to C-terminus): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The CDRs are- 13 -061685.109PCTinvolved in antigen binding, and influence antigen specificity and binding affinity of the antibody. See Kabat et al., Sequences of Proteins of Immunological Interest 5th ed. (1991) Public Health Service, National Institutes of Health, Bethesda, Md., incorporated by reference in its entirety.

[0056] The light chain from any vertebrate species can be assigned to one of two types, called kappa (K) and lambda (X), based on the sequence of its constant domain.

[0057] The heavy chain from any vertebrate species can be assigned to one of five different classes (or isotypes): IgA, IgD, IgE, IgG, and IgM. These classes are also designated a, 5, a, y, and p, respectively. The IgG and IgA classes are further divided into subclasses based on differences in sequence and function. Humans express the following subclasses: IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2.

[0058] The amino acid sequence boundaries of a CDR can be determined by one of skill in the art using any of a number of known numbering schemes, including those described by Kabat et al., supra (“Kabat” numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 (“Chothia” numbering scheme); MacCallum et al., 1996, J. Mol. Biol. 262:732-745 (“Contact” numbering scheme); Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 (“IMGT” numbering scheme); and Honegge and Pliickthun, J. Mol. Biol., 2001, 309:657-70 (“AHo” numbering scheme); each of which is incorporated by reference in its entirety.

[0059] An “antibody fragment” comprises a portion of an intact antibody, such as the antigen-binding or variable region of an intact antibody. Antibody fragments include, for example, Fv fragments, Fab fragments, F(ab')2 fragments, Fab' fragments, scFv (sFv) fragments, and scFv-Fc fragments.

[0060] “Fv” fragments comprise a non-covalently-linked dimer of one heavy chain variable domain and one light chain variable domain.

[0061] “Fab” fragments comprise, in addition to the heavy and light chain variable domains, the constant domain of the light chain and the first constant domain (CHI) of the heavy chain. Fab fragments may be generated, for example, by recombinant methods or by papain digestion of a full-length antibody.

[0062] “F(ab')2” fragments contain two Fab' fragments joined, near the hinge region, by disulfide bonds. F(ab')2 fragments may be generated, for example, by recombinant methods or by pepsin digestion of an intact antibody. The F(ab') fragments can be dissociated, for example, by treatment with 1 -mercaptoethanol.- 14 -061685.109PCT

[0063] “Single-chain Fv” or “sFv” or “scFv” antibody fragments comprise a VH domain and a VL domain in a single polypeptide chain. A peptide linker generally links the VH and VL. See Pliickthun A. (1994). Any suitable linker may be used. In some embodiments, the linker is a (GGGGS)n (SEQ ID NO: 7). In some embodiments, n=l, 2, 3, 4, 5, or 6. See Antibodies from Escherichia coli. In Rosenberg M. & Moore G. P. (Eds.), The Pharmacology of Monoclonal Antibodies vol. 113 (pp. 269-315). Springer- Verlag, New York, incorporated by reference in its entirety.

[0064] scFv-Fc” fragments comprise an scFv attached to an Fc domain. For example, an Fc domain may be attached to the C-terminal of the scFv. The Fc domain may follow the VH or VL, depending on the orientation of the variable domains in the scFv (i.e., VH-VL or VL-VH). Any suitable Fc domain known in the art or described herein may be used. In some cases, the Fc domain comprises an IgG4 Fc domain.

[0065] The term “single-domain antibody” refers to a molecule in which one variable domain of an antibody specifically binds to an antigen without the presence of the other variable domain. Single-domain antibodies, and fragments thereof, are described in Arabi Ghahroudi et al., FEBS Letters, 1998, 414:521-526 and Muyldermans et al., Trends in Biochem. Sci., 2001, 26:230-245, each of which is incorporated by reference in its entirety. Single-domain antibodies are also known as sdAbs or nanobodies.

[0066] The term “monoclonal antibody” refers to an antibody from a population of substantially homogeneous antibodies. A population of substantially homogeneous antibodies comprises antibodies that are substantially similar and that bind the same epitope(s), except for variants that may usually arise during production of the monoclonal antibody. Such variants are generally present in only minor amounts. A monoclonal antibody is typically obtained by a process that includes the selection of a single antibody from a plurality of antibodies. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, yeast clones, bacterial clones, or other recombinant DNA clones. The selected antibody can be further altered, for example, to improve affinity for the target (“affinity maturation”), to humanize the antibody, to improve its production in cell culture, and / or to reduce its immunogenicity in a subject.- 15 -061685.109PCT

[0067] The term “chimeric antibody” refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0068] “Humanized” forms of non-human antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. A humanized antibody is generally a human antibody (recipient antibody) in which residues from one or more CDRs are replaced by residues from one or more CDRs of a non-human antibody (donor antibody). The donor antibody can be any suitable non-human antibody, such as those from a mouse, rat, rabbit, chicken, or non-human primate, that has the desired specificity, affinity, or biological effect. In some instances, selected framework region residues of the recipient antibody are replaced by the corresponding framework region residues from the donor antibody. Humanized antibodies may also comprise residues that are not found in either the recipient antibody or the donor antibody. Such modifications may be made to refine antibody function further. For further details, see Jones et \., Nature, 1986, 321 :522-525; Riechmann et al., Nature, 1988, 332:323-329; and Presta, Curr. Op. Struct. Biol., 1992, 2:593-596, each of which is incorporated by reference in its entirety.

[0069] A “human antibody” possesses an amino acid sequence corresponding to that of an antibody produced by a human or a human cell, or derived from a non-human source that utilizes a human antibody repertoire or human antibody-encoding sequences (e.g., obtained from human sources or designed de novo). Human antibodies specifically exclude humanized antibodies.

[0070] An “isolated ABP” or “isolated nucleic acid” is an ABP or nucleic acid that has been separated and / or recovered from a component of its natural environment. Components of the natural environment may include enzymes, hormones, and other proteinaceous or nonproteinaceous materials. In some embodiments, an isolated ABP is purified to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence, for example by use of a spinning cup sequenator. In some embodiments, an isolated ABP is purified to homogeneity by gel electrophoresis (e.g., SDS-PAGE) under either reducing or non-reducing conditions, with detection using Coomassie blue or silver stain. In some embodiments, an isolated ABP may include an ABP in situ within recombinant cells, since at least one component of the ABP's natural environment is not present. In some aspects, an isolated ABP or isolated nucleic acid is- 16 -061685.109PCTprepared by at least one purification step. In some embodiments, an isolated ABP or isolated nucleic acid is purified to at least 80%, 85%, 90%, 95%, or 99% by weight. In some embodiments, an isolated ABP or isolated nucleic acid is purified to at least 80%, 85%, 90%, 95%, or 99% by volume. In some embodiments, an isolated ABP or isolated nucleic acid is provided as a solution comprising at least 85%, 90%, 95%, 98%, 99% to 100% ABP or nucleic acid by weight. In some embodiments, an isolated ABP or isolated nucleic acid is provided as a solution comprising at least 85%, 90%, 95%, 98%, 99% to 100% ABP or nucleic acid by volume.

[0071] “Affinity” refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an ABP) and its binding partner (e.g., an antigen or epitope). Unless indicated otherwise, as used herein, “affinity” refers to intrinsic binding affinity, which reflects a 1 : 1 interaction between members of a binding pair (e.g., ABP and antigen or epitope). The affinity of a molecule X for its partner Y can be represented by the dissociation equilibrium constant (KD). The kinetic components that contribute to the dissociation equilibrium constant are described in more detail below. Affinity can be measured by common methods known in the art, including those described herein, such as surface plasmon resonance (SPR) technology (e.g., BIACORE®) or biolayer interferometry (e.g., Sartorius®).

[0072] Concerning the binding of an ABP to a target molecule, the terms “bind,” “specific binding,” “specifically binds to,” “specific for,” “selectively binds,” and “selective for” a particular antigen (e.g., a polypeptide target) or an epitope on a particular antigen mean binding that is measurably different from a non-specific or non- selective interaction (e.g., with a non-target molecule). Specific binding can be measured, for example, by measuring binding to a target molecule and comparing it to binding to a non-target molecule. Specific binding can also be determined by competition with a control molecule that mimics the epitope recognized on the target molecule. In that case, specific binding is indicated if the binding of the ABP to the target molecule is competitively inhibited by the control molecule.

[0073] The term “epitope” means a portion of an antigen that specifically binds to an ABP. Epitopes frequently consist of surface-accessible amino acid residues and / or sugar side chains and may have specific three dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter may be lost- 17 -061685.109PCTin the presence of denaturing solvents. An epitope may comprise amino acid residues that are directly involved in the binding, and other amino acid residues, which are not directly involved in the binding.

[0074] Percent “identity” between a polypeptide sequence and a reference sequence, is defined as the percentage of amino acid residues in the polypeptide sequence that are identical to the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA, or MUSCLE software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0075] A “conservative substitution” or a “conservative amino acid substitution,” refers to the substitution an amino acid with a chemically or functionally similar amino acid. Conservative substitution tables providing similar amino acids are well known in the art.

[0076] A single-domain antibody of the present invention is directed against FAP or a closely related family member.

[0077] FAP is a principal target according to the invention. According to the invention, as discussed below, a polypeptide construct may further comprise singledomain antibodies directed against other targets. A single-domain antibody directed against a target means a single-domain antibody that is capable of binding to said target with an affinity of better than 105M. As used herein, targets may also be fragments of said targets. Thus, a target is also a fragment of said target, capable of eliciting an immune response. A target is also a fragment of said target, capable of binding to a single-domain antibody raised against the full-length target.

[0078] A “fragment” as used herein refers to less than 100% of the sequence (e.g., 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% etc.), but comprising 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids. A fragment is of sufficient length such that the interaction of interest is maintained with an affinity of 1 x 105M or better.- 18 -061685.109PCT

[0079] A fragment as used herein also refers to optional insertions, deletions, and substitutions of one or more amino acids which do not substantially alter the ability of the target to bind to a single-domain antibody raised against the wild-type target. The number of amino acid insertions deletions or substitutions is preferably up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 amino acids.

[0080] As used herein, a “homologous sequence” of the present invention may comprise additions, deletions, or substitutions of one or more amino acids, which do not substantially alter the functional characteristics of the polypeptides of the invention. For the anti-FAP polypeptides, the number of amino acid deletions or substitutions is preferably up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 amino acids.

[0081] A homologous sequence according to the present invention may be a sequence modified by the addition, deletion, or substitution of amino acids, said modification not substantially altering the functional characteristics compared with the unmodified polypeptide.

[0082] A homologous sequence according to the present invention may be a sequence that exists in other Camelidae species, such as, for example, camel, dromedary, llama, vicuna, alpaca, and guanaco. The homologous sequence according to the present invention may be a sequence that exists in cartilaginous fishes, such as but not restricted to sharks, rays, skates, ghost sharks, ratfish, elephantfish, and rabbitfish.

[0083] Where homologous sequence indicates sequence identity, it means a sequence that presents a high sequence identity (more than 70%, 75%, 80%, 85%, 90%, 95% or 98% sequence identity) with the parent sequence and is preferably characterized by similar properties of the parent sequence, namely affinity, said identity calculated using known methods.

[0084] Alternatively, a homologous sequence may also be any amino acid sequence resulting from allowed substitutions at any number of positions of the parent sequence according to the formula below:- 19 -061685.109PCTSer substituted by Ser, Thr, Gly, and Asn;Arg substituted by one of Arg, His, Gin, Lys, and Glu;Leu substituted by one of Leu, He, Phe, Tyr, Met, and Vai;Pro substituted by one of Pro, Gly, Ala, and Thr;Thr substituted by one of Thr, Pro, Ser, Ala, Gly, His, and Gin;Ala substituted by one of Ala, Gly, Thr, and Pro;Vai substituted by one of Vai, Met, Tyr, Phe, He, and Leu;Gly substituted by one of Gly, Ala, Thr, Pro, and Ser;He substituted by one of He, Met, Tyr, Phe, Vai, and Leu;Phe substituted by one of Phe, Trp, Met, Tyr, He, Vai, and Leu;Tyr substituted by one of Tyr, Trp, Met, Phe, He, Vai, and Leu;His substituted by one of His, Glu, Lys, Gin, Thr, and Arg;Gin substituted by one of Gin, Glu, Lys, Asn, His, Thr, and Arg;Asn substituted by one of Asn, Glu, Asp, Gin, and Ser;Lys substituted by one of Lys, Glu, Gin, His, and Arg;Asp substituted by one of Asp, Glu, and Asn;Glu substituted by one of Glu, Asp, Lys, Asn, Gin, His, and Arg;Met substituted by one of Met, Phe, He, Vai, Leu, and Tyr.

[0085] A homologous sequence may also be any amino acid sequence resulting from allowed substitutions at any number of positions with non-natural amino acids. The non-natural amino acid may be selected from at least 2-amino-3-(4- azidophenyl)propanoic acid (pAF), 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid (pAMF), 2-amino-3-(5-(azidomethyl)pyridin-2-yl)propanoic acid, 2-amino-3-(4- (azidomethyl)pyridin-2-yl)propanoic acid, 2-amino-3-(6-(azidomethyl)pyri din-3 - yl)propanoic acid, 2-amino-5-azidopentanoic acid, and 2-amino-3-(4- (azidomethyl)phenyl)propanoic acid, or any combination thereof.

[0086] A homologous nucleotide sequence according to the present invention may refer to nucleotide sequences of more than 50, 100, 200, 300, 400, 500, 600, 800, or 1000 nucleotides able to hybridize to the reverse-complement of the nucleotide sequence capable of encoding the patent sequence, under stringent hybridization conditions (such as the ones described by Sambrook et al., Molecular Cloning, Laboratory Manuel, Cold Spring, Harbor Laboratory press, New York.- 20 -061685.109PCT

[0087] As used herein, a “functional portion” refers to a sequence of a singledomain antibody that is of sufficient size such that the interaction of interest is maintained with affinity of 1 x I O5M or better.

[0088] As used herein, a functional portion as it refers to the polypeptide sequence of an anti-FAP polypeptide refers to less than 100% of the sequence (e.g., 99%, 90%, 80%, 70%, 60% 50% etc.), but comprising 5 or more amino acids or 15 or more nucleotides.

[0089] As used herein “SUMO” refers to small ubiquitin-like modifiers which can be attached by sumoylation-dependent ubiquitin ligases, such as the sumoylation- dependent E3 ligases RanBP2, Pc2 and members of the PIAS family. In humans and mice, the SUMO family consists of three members, SUMO-1, SUMO-2, and SUMO- 3, which are encoded by separate genes. SUMO conjugation requires sequential El- dependent activation, E2-dependent conjugation, and E3-dependent ligation steps. The human SUMO El enzyme comprises a heterodimer of the SAE1 and SAE2 proteins and forms a thioester bond with glycine 97 of SUMO-1. Subsequently, SUMO-1 is transferred by transesterification to the SUMO-specific E2-conjugating enzyme, Ubc9. Ubc9 can directly conjugate the carboxy-terminal glycine of SUMO to lysines in target proteins that are situated in the consensus motif yKxE / D, where y stands for valine, leucine, isoleucine, methionine, or phenylalanine, and x stands for any amino acid. SUMO can be removed by desumoylation proteins or SUMO proteases, such as SuPrl, SENP1 (sentrin / SUMO-specific protease), or ULPs (ubiquitin-like protein-specific proteases).

[0090] As used herein, “MBP” refers to maltose binding protein. The joining of a polymer of interest with another molecule of interest, such as a bioactive molecule and the like, is carried out using known methods, such as chemical synthetic methods using the chemical characteristics of the modified branched polymer and of the molecule to be bound thereto. Thus, the modified branched polymer can contain, for example, amine groups that can be used as the reactive site to which a molecule of interest can be bound through covalent linkages. Alternatively, the joining may occur through the mere mixing of the polymer and the molecule to be bound, forming non- covalent linkages between them. The linking of another entity to the polymer of interest can also be achieved through a combination of both. For example, a polymer of interest can be covalently linked to a bioactive material, followed by physical adsorption of a- 21 -061685.109PCTreporter particle through non-covalent linkages to form a bioactive material-polymer- reporter particle conjugate, which can be readily used for bioassays.

[0091] As used herein, the terms “immunologic,” “immunological,” or “immune” response are the development of a beneficial humoral (antibody-mediated) and / or a cellular (mediated by antigen-specific T cells or their secretion products) response directed against a peptide in a recipient patient. Such a response can be an active response induced by the administration of an immunogen or a passive response induced by the administration of an antibody or primed T-cells. A cellular immune response is elicited by the presentation of polypeptide epitopes in association with Class I or Class II MHC molecules to activate antigen-specific CD4+T helper cells and / or CD8+cytotoxic T cells. The response may also involve activation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglia cells, eosinophils, activation or recruitment of neutrophils or other components of innate immunity. The presence of a cell-mediated immunological response can be determined by proliferation assays (CD4+T cells) or CTL (cytotoxic T lymphocyte) assays. The relative contributions of humoral and cellular responses to the protective or therapeutic effect of an immunogen can be distinguished by separately isolating antibodies and T- cells from an immunized syngeneic animal and measuring the protective or therapeutic effect in a second subj ect.

[0092] An “immunogenic agent” or “immunogen” is capable of inducing an immunological response against itself on administration to a mammal, optionally in conjunction with an adjuvant.

[0093] As used herein, a molecule is said to be able to “immunospecifically bind” a second molecule if such binding exhibits the specificity and affinity of an antibody to its cognate antigen. Antibodies are said to be capable of immunospecifically binding to a target region or conformation (“epitope”) of an antigen if such binding involves the antigen recognition site of the immunoglobulin molecule. An antibody that immunospecifically binds to a particular antigen may bind to other antigens with lower affinity if the other antigen has some sequence or conformational similarity that is recognized by the antigen recognition site as determined by, e.g., immunoassays, BIACORE® assays, or other assays known in the art, but would not bind to a totally unrelated antigen. In some embodiments, however, antibodies (and their antigenbinding fragments) will not cross-react with other antigens. Antibodies may also bind- 22 -061685.109PCTto other molecules in a manner that is not immunospecific, such as to FcR receptors, through binding domains in other regions / domains of the molecule that do not involve the antigen recognition site, such as the Fc region.

[0094] As used herein, “immunosuppressive” refers to the expression of a non- endogenous polypeptide that has the effect of alleviating the patient host's immune response against the donor's immune cells.

[0095] As used herein, the term “prophylactic agent” refers to an agent that can be used in the prevention of a disorder or disease before the detection of any symptoms of such disorder or disease. A “prophylactically effective” amount is the amount of prophylactic agent sufficient to mediate such protection. A prophylactically effective amount may also refer to the amount of the prophylactic agent that provides a prophylactic benefit in the prevention of disease.

[0096] As used herein, the term “cancer” refers to a neoplasm or tumor resulting from abnormal, uncontrolled growth of cells. The term “cancer” refers to a disease involving cells that have the potential to metastasize to distal sites and exhibit phenotypic traits that differ from those of non-cancer cells, for example, formation of colonies in a three-dimensional substrate such as soft agar or the formation of tubular networks or web-like matrices in a three-dimensional basement membrane or extracellular matrix preparation. Non-cancer cells do not form colonies in soft agar and form distinct sphere-like structures in three-dimensional basement membrane or extracellular matrix preparations.II. COMPOSITIONSA. Single-domain Antibodies

[0097] The present invention relates to polypeptide constructs comprising one or more single-domain antibodies directed to one or more target molecule(s), each in a suitable dosage form, either directly or as part of a composition containing an ingredient that facilitates delivery. The invention further relates to polypeptide constructs comprising anti-FAP single-domain antibodies.

[0098] Single-domain antibodies (sdAbs or VHH) are antibodies whose complementary determining regions are part of a single-domain polypeptide. Singledomain antibodies were first detected in the sera of Camelidae, are a class of immunoglobulins that lack light chains and consist of only one heavy chain with a- 23 -061685.109PCTsingle variable domain (AI-Baradie, R. S., 2020; Bathula, N. V., 2021; Schumacher, D.; Angew Chem Int Ed Engl 57, 2314-2333 (2018), Angew Chem Int Ed Engl 57, 2314-2333 (2018)). Examples include, but are not limited to, heavy-chain antibodies, antibodies naturally devoid of light chains, single-domain antibodies derived from conventional four-chain antibodies, engineered antibodies, and single-domain scaffolds other than those derived from antibodies. Single-domain antibodies may be any of the art, or any future single-domain antibodies. Single-domain antibodies may be derived from any species, including, but not limited to, mouse, human, camel, llama, goat, rabbit, bovine, and shark.

[0099] According to one aspect of the invention, a single-domain antibody as used herein is a naturally occurring single-domain antibody known as heavy chain antibody devoid of light chains. Such single-domain antibodies are disclosed in WO 9404678, for example. For clarity reasons, this variable domain derived from a heavy chain antibody naturally devoid of light chain is known herein as a VHH or nanobody to distinguish it from the conventional VH of four-chain immunoglobulins. Such a VHH molecule can be derived from antibodies raised in Camelidae species, for example, in camels, llamas, dromedaries, alpacas, and guanacos. Other species besides Camelidae may produce heavy chain antibodies naturally devoid of light chain; such VHHs are within the scope of the invention.

[0100] VHH molecules are about 10x smaller than IgG molecules. They are single polypeptides and very stable, resisting extreme pH and temperature conditions. Moreover, they are resistant to the action of proteases which is not the case for conventional antibodies. Furthermore, in vitro expression of VHHs produces high yield, properly folded functional VHHs. In addition, antibodies generated in Camelids will recognize epitopes other than those recognized by antibodies generated in vitro through the use of antibody libraries or via immunization of mammals other than Camelids (WO 9749805). Since VHHs are known to bind into ‘unusual’ or non-conventional epitopes such as cavities (WO 97 / 49805), the affinity of such VHHs to circulating albumin may be increased.

[0101] When compared to conventional heavy chains (VH) of regular IgG molecules, their three complementarity determining regions (CDRs) are enlarged to provide a greater surface area for antigen interactions making them well-suited for binding restricted sites such as cavities or sterically hindered epitopes. Moreover, they- 24 -061685.109PCTcontain additional hydrophilic amino acids within the conserved framework region. VHH proteins retain high affinity and specificity for their target antigens, with low off- target accumulation. Compared to the stability exhibited by a conventional antibody, they are unexpectedly robust due to their high refolding capacity, recovering from chemical denaturation with minimal damage to functionality. Furthermore, unlike conventional antibodies, they can tolerate environmental conditions associated with radiochemistry including high temperatures, elevated pressures and non-physiological pHs. Additionally, nanobodies are relatively simple and inexpensive to produce on the milligram scale in a laboratory setting since they lack post-translational modifications and can be synthesized in microbial systems. As a result, the last three decades have witnessed significant growth in research related to these molecules, with their use as diagnostic and therapeutic agents for various pathologies. As of 2020, there were over 15 clinical trials involving sdAbs (Shi, T., 2023; Wen, P. Y., 2008).B. Fibroblast Activation Protein Alpha

[0102] Another embodiment of the present invention is an anti-fibroblast activation protein alpha (FAP or seprase) consisting of a sequence corresponding to that of a Camelidae VHH directed towards FAP or a closely related family member. The invention also relates to a homologous sequence, a function portion, or a functional portion of a homologous sequence of said polypeptide. The invention also relates to nucleic acids capable of encoding said polypeptides. A single-domain antibody of the present invention is directed against FAP or a closely related family member.

[0103] The dipeptidyl peptidase (DPP) family of proteins is a metalloproteases that cleave the N-terminal dipeptide from peptides with Pro or Ala in the penultimate position; the family’s substrates include growth factors, chemokines, neuropeptides, vasoactive peptides, and extracellular matrix molecules such as collagen. This family has seven family members, including DPP4, DPP8, DPP9, DPPII, prolyl carboxypeptidase (PRCP), prolyl oligopeptidase (PREP), and fibroblast activation protein alpha (FAP or seprase) (Ostrom, Q. T., 2011; Ostrom, Q.T., Bauchet, L., 2014). Of these family members significant research activity has revolved around FAP, which is a 170 kDa type II transmembrane serine protease since it is unique among this enzyme family because of its endopeptidase activity and substrate selectivity (Ostrom, Q.T., Cioffi, G., 2021; Ostrom, Q.T., Cioffi, G., 2019; Ostrom, Q. T., Francis, S. S. 2021). Moreover, unlike other members of this protein family, FAP exhibits a unique- 25 -061685.109PCTexpression profile and is considered a robust biomarker of pathology since it demonstrates negligible expression in normal adult tissues, but is prominently expressed in a variety of pathologies, including cancer, arthritis, atherosclerosis, and fibrosis. Several reports describe strategies to target FAP expression for imaging and therapy using peptides, antibodies, antibody fragments, nanoparticles, and small molecules have appeared in the literature (Ostrom, Q.T., Gittleman, H., 2013; Ostrom, Q.T., Gittleman, H., 2015; Ostrom, Q.T., Gittleman, H., 2014; Ostrom, Q.T., Gittleman,H., 2017; Ostrom, Q.T., Gittleman, H., 2015; Lo, A., 2015; Cremasco, V., 2018; Mentlein, R., 2011). Recently, single-domain antibodies targeting FAP have been described as potential theranostic agents. For example, Xu et al. identified two novel anti-FAP VHH proteins engineered to contain the Fc fragment of IgG4 (Xu, J., Cancer Biother Radiopharm 2022; Balaziova, E., 2021). These recombinant proteins were radiolabeled with zirconium-89 (89Zr: t / 2= 78.4 h, P+: 22.8 %, Ep+max = 901 keV; EC: 77%, EY= 909 keV) and lutetium-177 (177Lu3+: P' - emitter: ti / 2 = 6.7d; E p'max = 0.497 MeV) (Busek, P., 2016; Busek, P., 2015). Ex vivo biodistribution analysis of the89Zr- agent revealed good tumor uptake at later time points, while therapy studies with the177Lu-agent demonstrated tumor growth control without significant animal toxicity. Recently, a study by Dekempeneer et al. revealed additional single-domain anti-FAP antibodies with KD values in the nano-to-picomolar range (Dekempeneer, Y., J Nucl Med 2023, 64, 1941-1948). These VHH molecules were radiolabeled with several PET, SPECT, and therapeutic radioisotopes (Busek, P., 2016; Busek, P., 2018; Matrasova,I., 2017). These agents exhibited specific accumulation in human FAP+tumors, while being excreted rapidly in most cases. The proteins radiolabeled with actinium-225 (225AC3+: a++- emitter: ti / 2 = 10 d; Eamax = 6-8 MeV) demonstrated kidney retention but provided tumor growth control in FAP+tumor-bearing mice. Collectively, these publications demonstrate the potential of anti-FAP single-domain antibodies in the development of theranostics. However, despite interesting data reported, neither group of authors specifically described the binding epitope of the reported VHH molecules. Unfortunately, this lack of structural information hinders further development from a rational drug design perspective.

[0104] FAP is a principal target according to the invention. According to the invention, as discussed below, a polypeptide construct may further comprise singledomain antibodies directed against other targets, such as other members of the DPP- 26 -061685.109PCTfamily of proteins. A single-domain antibody directed against a target means a singledomain antibody that is capable of binding to said target with an affinity of better than I O5M.

[0105] The present invention further relates to anti-FAP single-domain proteins, wherein a single-domain antibody is a VHH belonging to a class having human-like sequences.

[0106] Any of the anti-FAP VHHs disclosed herein may be of the traditional class or of a class of human-like Camelidae antibodies. Said antibodies may be directed against whole FAP or a fragment thereof, or a fragment of a homologous sequence thereof. These polypeptides include the full-length Camelidae antibodies, namely Fc and VHH domains.

[0107] Another embodiment of the present invention is a multivalent anti-FAP polypeptide as disclosed herein, comprising at least two single-domain antibodies directed against FAP. Such multivalent anti-FAP polypeptides have the advantage of unusually high functional affinity for the target, displaying inhibitory properties that are significantly higher than expected compared to their monovalent counterparts.

[0108] A multivalent anti-FAP polypeptide as used herein refers to a polypeptide comprising two or more anti-FAP polypeptides that have been covalently linked. The anti-FAP polypeptides may be identical in sequence or different in sequence, but they are directed against the same target or antigen. Depending on the number of anti-FAP polypeptides linked, a multivalent anti- FAP polypeptide may be bivalent (2 anti- FAP polypeptides), trivalent (3 anti- FAP polypeptides), tetravalent (4 anti- FAP polypeptides) or have a higher valency molecules.

[0109] According to one aspect of the present invention, the anti-FAP polypeptides are linked to each other directly, without the use of a linker. According to another aspect of the present invention, the anti-FAP polypeptides are linked to each other via a peptide linker sequence. Such a linker sequence may be a naturally occurring sequence or a non-naturally occurring sequence. The linker sequence is expected to be non-immunogenic in the subject to which the anti-FAP polypeptides are administered. The linker sequence may provide sufficient flexibility to the multivalent anti-FAP polypeptide, while being resistant to proteolytic degradation. A non-limiting example of a linker sequence can be derived from the hinge region of VHHs described in WO 96 / 34103.- 27 -061685.109PCT

[0110] It is an aspect of the invention that the multivalent anti-FAP polypeptides disclosed above may be used instead of or as well as the single unit anti-FAP polypeptides in the therapies and methods of delivery as mentioned herein.

[0111] The single-domain antibodies may be joined to form any of the anti-FAP polypeptides disclosed herein comprising more than one single-domain antibody using methods known in the art or any future method. They may be joined non-covalently (e.g. using streptavidin / biotin combination, antibody / tag combination) or covalently. They may be fused by chemical cross-linking by reacting amino acid residues with an organic derivatizing agent, such as described by Blattler et al, Biochemistry 24,1517- 1524; EP294703. Alternatively, the single-domain antibody may be fused genetically at the DNA level, i.e., an anti-FAP polypeptide formed that encodes the complete polypeptide comprising one or more anti-FAP single-domain antibodies. A method for producing a bivalent or multivalent anti-FAP polypeptide is disclosed in PCT patent application WO 96 / 34103. One way of joining VHH antibodies is via the genetic route by linking a VHH antibody coding sequence either directly or via a peptide linker. For example, the C-terminal end of the VHH antibody may be linked to the N-terminal end of the next single-domain antibody.

[0112] This linking mode can be extended in order to link additional singledomain antibodies for the construction and production of tri-, tetra-, etc. functional constructs.

[0113] The polypeptide disclosed herein may be made by an artisan of ordinary skill in the art according to methods known in the art or any future method. For example, VHHs may be obtained using methods known in the art, such as by immunizing a camel and obtaining hybridomas therefrom, or by cloning a library of single-domain antibodies using molecular biology techniques known in the art and subsequent selection by ELISA with individual clones of unselected libraries, or by using phage display or methods for in silico design.

[0114] According to an aspect of the invention, an anti-FAP polypeptide may be a homologous sequence of a full-length anti-FAP polypeptide. According to another aspect of the invention, an anti-FAP polypeptide may be a functional portion of a full- length anti-FAP polypeptide. According to another aspect of the invention, an anti-FAP polypeptide may be a homologous sequence of a full-length anti-FAP polypeptide. According to another aspect of the invention, an anti-FAP polypeptide may be a- 28 -061685.109PCTfunctional portion of a homologous sequence of a full-length anti-FAP polypeptide. According to an aspect of the invention, an anti-FAP polypeptide may comprise a sequence of an anti-FAP polypeptide.

[0115] According to an aspect of the invention, a single-domain antibody used to form an anti-FAP polypeptide may be a complete single-domain antibody (e.g., a VHH) or a homologous sequence thereof. According to another aspect of the invention, a single-domain antibody used to form an anti-FAP polypeptide may be a functional portion of a complete single-domain antibody. According to another aspect of the invention, a single-domain antibody used to form an anti-FAP polypeptide may be a homologous sequence of a complete single-domain antibody. According to another aspect of the invention, a single-domain antibody used to form an anti-FAP polypeptide may be a functional portion of a homologous sequence of a complete single-domain antibody.

[0116] A portion as it refers to the polypeptide of an anti-FAP polypeptide, refers to less than 100% of the sequence (e.g., 99%, 90%, 80%, 70%, 60% 50% etc.), but comprising 5 or more amino acids or 15 or more nucleotides.III. METHODS OF MAKINGA. Preparing modified polypeptides

[0117] One embodiment of the present invention relates to a method for preparing modified polypeptides by determining the amino acid residues of the antibody variable domain (VHH) which may be modified without diminishing the native affinity of the domain for antigen and while reducing its immunogenicity with respect to a heterologous species; the use of VHHs having modifications at the identified residues which are helpful for administration to heterologous species; and to the VHH so modified. More specifically, the invention relates to the preparation of modified VHHs, which are modified for administration to humans, the resulting VHHs themselves, and the use of such “humanized” VHHs in the treatment of diseases in humans. By humanized is meant mutated so that immunogenicity upon administration in human patients is minor or nonexistent. Humanizing a polypeptide, according to the present invention, comprises a step of replacing one or more of the Camelidae amino acids by their human counterpart as found in the human consensus sequence, without that polypeptide losing its typical character, i.e., the humanization does not significantly affect the antigen binding capacity of the resulting polypeptide. Such methods are- 29 -061685.109PCTknown by the skilled artisan. Humanization of Camelidae single-domain antibodies requires the introduction and mutagenesis of a limited number of amino acids in a single polypeptide chain. This is in contrast to the humanization of scFv, Fab', (Fab')2, and IgG, which requires the introduction of amino acid changes in both the light and heavy chains, while preserving the assembly of these chains.B. Characterize the interaction of the single-domain antibodies

[0118] The methods provided herein describe the utilization of cryo-EM, mass photometry (MP), and biolayer interferometry (BLI) to characterize the interaction of single-domain antibodies with the serine protease, fibroblast activation protein alpha. To our knowledge, this is the first report to describe the structure of FAP in complex with a nanobody, using any method, and the binding interaction between FAP and an anti-FAP VHH, as determined by cryo-EM.

[0119] In the prior art, the primary tools for determining protein structure are the x-ray diffraction (XRD) and the Nuclear Magnetic Resonance (NMR) techniques. Nevertheless, the structures of only a small number of macromolecular complexes and proteins have been successfully determined by using XRD, comprising only a tiny portion of the protein data bank. In fact, not all proteins can be crystallized for XRD measurements. Crystallization of proteins remains a major hurdle, and to date, 99.5% of proteins are difficult to form as crystals. Moreover, the crystallization process for every type of protein is highly challenging, and it may misrepresent the native form of the protein. On the other hand, the conventional NMR technique is only suitable for the observation of small molecules and proteins and is quite difficult for larger macromolecular complexes and proteins.1. Cryo-electron microscopy

[0120] Cryo-electron microscopy (cryo-EM) is a structure determination technique that enables visualization of large and / or dynamic macromolecules (Simkova, A., 2020; Zubal, M., 2023; Dekempeneer, Y., 2016; DHuyvetter, M., 2014; Barakat, S., 2022; Arezumand, R., 2017; Alirahimi, E., 2018; AI-Baradie, R. S., 2020; Vaneycken, I., 2011). As a complementary technique to X-ray crystallography and NMR, cryo-EM has become an important tool in the drug discovery process and a valuable asset for structural biologists who wish to interrogate the structure and function of large protein complexes at near atomic resolution (Rodak, M., 2022). While still evolving, cryo-EM methodologies have become robust enough to confidently- 30 -061685.109PCTmodel amino acid side chains and ligands into the density maps. These improvements continue to transform the drug discovery process, enabling faster identification of lead molecules.

[0121] Cryo-EM can be used to observe protein structure and does not require crystallization. Rather, the biological specimens are prepared by rapid sample freezing in liquid ethane, forming amorphous ice around the specimens. In recent years, the resolutions of resulting 3D reconstructions have been steadily increasing. With the current methodology, near-atomic resolutions can be routinely obtained. As hardware and software enhancements continue, the pharmaceutical industry will further adopt this technique to understand how drugs bind to challenging protein targets. Recent advancements in grid preparation have reduced sample requirements and increased sample throughput, but further development remains (Hirst IJ, Biochem Soc Trans. Jun 26;52(3): 1529-1537 (2024)).2. Mass photometry

[0122] In recent years, interferometric scattering (iSCAT) microscopy, most notably mass photometry (MP), has been developed as a powerful analytical technology for single-molecule detection, offering a simple and cost-efficient alternative to assays such as ELISA. iSCAT microscopy provides information about the relative distribution of particles of different masses in solution, without the requirement to add a label, and has been described previously for the detection of purified single proteins (Cole et al., ACS Photonics. 2017 Feb 15;4(2):211-2167, and W02018 / 011591), and for the detection of lipoproteins and determination of concentrations of a molecule in solution (WO2019 / 110977). Mass photometry in particular has been described in Young et al., 2018 and Li et al., 2020.

[0123] Interferometric scattering mass spectrometry (iSCAMS), also known as mass photometry (MP), is a method for detecting and measuring the mass of single objects and the complexes they form in solution. MP detects single molecules by their light scattering as they bind non-specifically or specifically to a surface. Each binding event leads to a change in refractive index at the surface / solution interface, which effectively alters the local light scattering and can be detected with high accuracy by taking advantage of optimized interference between scattered and reflected light. The magnitude of the signal change can be converted into a molecular mass, with approximately 2% mass accuracy and up to 20 kDa mass resolution, by calibration with- 31 -061685.109PCTmolecules of known mass. The scattering signal is thus directly proportional to the molecule's mass, making it possible to weigh single molecules with light. However, such a technique cannot be be applied to complex solutions where one molecule may be present in low abundance. Thus, although the molecule of low abundance may bind non-specifically to the surface and permit a detection of mass, there may be too many confounding components present to make an accurate determination of mass and, therefore, identity of the molecule. This makes the identification of said molecule complicated and the determination of concentration difficult. The application of this technique to low-abundance biomarkers in samples has been limited.

[0124] Mass photometry is unique in its capability for accurate mass measurement of single molecules in solution, in their native state, and without the need for labels.3. Biolayer interferometry (BLI)

[0125] The term “binding” as used herein refers to the binding of an antigenbinding molecule, such as an antibody, to a predetermined antigen or target.

[0126] The skilled reader will be familiar with the concept of affinity and the equilibrium dissociation constant KD. The dissociation constant KD can be measured by BLI. KD values may be determined by biolayer interferometry (BLI) in an Octet RED96 instrument using the antigen-binding molecule, e.g., the antibody, as the immobilized ligand and the antigen as the analyte.

[0127] Biolayer interferometry utilizes an optical analytical technique that analyzes the interference pattern of light reflected from a layer of immobilized protein on a biosensor tip and an internal reference layer. Changes in the number of molecules bound to the biosensor tip cause shifts in the interference pattern that can be measured in real-time. A non-limiting exemplary device for biolayer interferometry is the Sartorius Octet® RED96 system. See, e.g., Abdiche et al., 2008, Anal. Biochem. B. 3TB. 209-277.C. Alpha Particle Radiation Delivered Systemically with anti -FAP VHH Proteins

[0128] Reducing the mortality rate from GBM requires new strategies for diagnosis and treatment. We posit that GBM can be treated with alpha particle radiation that is delivered systemically with anti-FAP VHH proteins. Provided herein are three novel VHH proteins that bind FAP. The three novel VHH protein interactions with the FAP protein have been characterized using molecular photometry and cry-electron- 32 -061685.109PCTmicroscopy. A novel chelation strategy involving the macrocycle PCTA demonstrates that it facilitates225Ac radiochemistry when compared to the macrocycle DOTA.1 . Radionuclides

[0129] Radiochemistry can be completed under mild conditions and in a shorter time frame without compromising radiopharmaceutical stability. Digital autoradiography, in addition to traditional biodistribution studies, is used to characterize the VHH pharmacokinetics, dosimetry, and its interaction with GBM tumors and organs of excretion. Provided herein are embodiments where radiochemistry is completed with the use of a radionuclide.

[0130] In one embodiment, the radionuclide is suitable for PET imaging. In one embodiment, the radionuclide is selected from the group consisting of isotopes: "mTc,67Ga,68Ga,66Ga,47Sc,51Cr,UC,167Tm,141Ce,mIn,123I,124I,125I,131I,18F,nC,15N,168Yb,175Yb,140La,90Y,88Y,86Y,153Sm,166Ho,165Dy,166Dy,62Cu,64Cu,67Cu,60Cu,61Cu,211At,97RU,103RU,186Re,188Re,203Pb,211Bi,212Bi,213Bi,207Bi,76Br,89Zr,225Ac, ,105Rh,109Pd,117mSn,149Pm,161Tb,177Lu,198Au,199Au,212Pb,134Ce,133Ce,133La,134La,149Tb,152Tb,155Tb,94Tc, "Tc,43Sc,44Sc,117Sn,52Mn,53Mn,54Mn,55Mn,13N,150,223Fr,227Th,229Th,228I,226Ra,224Ra,219Rn,215Po,212Po,216Po,206Po,211Pb, 207T1,2O8T1,21 1PO,107Ag,109Ag,195mPt,103Pd,223Ra,82Rb’89Sr,85Sr,90Sr,45Ti,44Ti,73As,119Sb,55Fe,59Fe,22Na,48V,63Ni,65Zn,109Cd,153Gd,148Gd,194Hg,26Al,32Si,68Ge,73As,77Br, or147Pm. In one embodiment, the radionuclide is selected from the group consisting of18F,64Cu,68Ga, and89Zr isotopes. In one embodiment, the radionuclide is64Cu. In one embodiment, the radionuclide is89Zr.

[0131] In one embodiment, the radionuclide is suitable for SPECT imaging. In one embodiment, the radionuclide is selected from the group consisting of isotopes:67Ga,n iIn,123I,125I,131I, and99Tc. In one embodiment, the radionuclide is selected from the group consisting of isotopes: "Tc,mIn, and123I.

[0132] In one embodiment, the radionuclide is suitable for targeted radionuclide therapy (alpha, beta-emitters, or auger). In one embodiment, the radionuclide is selected from the group consisting of isotopes:67Cu,177Lu,89Sr,H 1In,90Y,117Sn,131I,153Sm,166HO,186Re,188Re,211At,212Pb,212Bi,213Bi,223Ra,224Ra,225Ac, and227Th. In one embodiment, the radionuclide is selected from the group consisting of the isotopes67Cu,90Y,177Lu,H 1In,211At,212Pb,225Ac, and227Th.- 33 -061685.109PCT

[0133] In a particular embodiment, the alpha particle-emitting radionuclide225Ac is combined with a novel anti-FAP VHH radionuclide to provide an innovative and highly effective GBM treatment strategy.2. Chelating Groups

[0134] Provided herein is a novel radiochemistry strategy that utilizes the bifunctional chelator PCTA over DOTA is employed.

[0135] In one embodiment, the chelating group is selected from the group consisting of: DOTA, TCMC, DTPA, 1B4M-DTPA, CHX=A”-DTPA, NOTA, NODAGA, NODASA, NETA, TETA, CB-TE2A, H2dedpa, H2octapa, H2CHXdedpa, H2CXHoctapa, HYNIC, EDD / HYNIC-TOC, Sar bicyclic chelators T3, 4BCPP, N(NOEt)2isomers, HBED-CC, PCTA-NCS, MANOTA, THP, DFO, DFO*, and DFOcyclo*. In one embodiment, the chelating group is DOTA.3. Linking Groups

[0136] The nature of the linking group is not critical, provided the final conjugate has the requisite properties for its intended use (e.g., as an imaging agent or as a therapeutic agent.

[0137] The linker can vary in length and atom composition, and for example, can be branched, non-branched, cyclic, or a combination thereof. The linker may also modulate the properties of the targeted conjugate, such as but not limited to solubility, stability, and aggregation.

[0138] In one embodiment, the linker comprises about 3-5000 atoms. In one embodiment, the linker comprises about 3-4000 atoms. In one embodiment, the linker comprises about 3-2000 atoms. In one embodiment, the linker comprises about 3-1000 atoms. In one embodiment, the linker comprises about 3-750 atoms. In one embodiment, the linker comprises about 3-500 atoms. In one embodiment, the linker comprises about 3-250 atoms. In one embodiment, the linker comprises about 3-100 atoms. In one embodiment, the linker comprises about 3-50atoms. In one embodiment, the linker comprises about 3-25 atoms.

[0139] In one embodiment, the linker comprises about 10-5000 atoms. In one embodiment, the linker comprises about 10-4000 atoms. In one embodiment, the linker comprises about 10-2000 atoms. In one embodiment, the linker comprises about 10- 1000 atoms. In one embodiment, the linker comprises about 10-750 atoms. In one embodiment, the linker comprises about 10-500 atoms. In one embodiment, the linker- 34 -061685.109PCTcomprises about 10-250 atoms. In one embodiment, the linker comprises about 10-100 atoms. In one embodiment, the linker comprises about 10-50atoms. In one embodiment, the linker comprises about 10-25 atoms.

[0140] In one embodiment, the linker comprises atoms selected from H, C, N, S, and O.

[0141] In one embodiment, the linker comprises atoms selected from H, C, N, S, P, and O.

[0142] In one embodiment, the linker comprises a branched or unbranched, saturated or unsaturated, hydrocarbon chain, having from about 1 to 1000 (or 1-750, 1- 500, 1-250, 1-100, 1-50, 1-25, 1-10, 1-5, 5-1000, 5-750, 5-500, 5-250, 5-100, 5-50, 5- 25, 5-10 or 2-5 carbon atoms) wherein one or more of the carbon atoms is optionally replaced independently by -O-, -S, -N(Ra)-, 3-7 membered heterocycle, 5-6-membered heteroaryl or carbocycle and wherein each chain, 3-7 membered heterocycle, 5-6- membered heteroaryl or carbocycle is optionally and independently substituted with one or more (e.g. 1, 2, 3, 4, 5 or more) substituents selected from (Ci-Ce)alkyl, (Ci- Ce)alkoxy, (C3-Ce)cycloalkyl, (Ci-Ce)alkanoyl, (Ci-Ce)alkanoyloxy, (Ci- Ce)alkoxycarbonyl, (Ci-Ce)alkylthio, azido, cyano, nitro, halo, -N(Ra)2, hydroxy, oxo (=0), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy, wherein each Rais independently H or (Ci-Ce)alkyl. In one embodiment the linker comprises a branched or unbranched, saturated or unsaturated, hydrocarbon chain, having from about 1 to 1000 (or 1-750, 1-500, 1-250, 1-100, 1-50, 1-25, 1-10, 1-5, 5-1000, 5-750, 5-500, 5- 250, 5-100, 5-50, 5-25, 5-10 or 2-5 carbon atoms) wherein one or more of the carbon atoms is optionally replaced independently by -O-, -S, -N(Ra).,

[0143] In one embodiment, the linker comprises one or more amino acids or peptides. In one embodiment, the linker comprises at least one amino acid. In one embodiment, the linker comprises at least two amino acids. In one embodiment, the linker comprises at least three amino acids.

[0144] In one embodiment, the linker comprises 10-100 atoms selected from H, C, N, S, and O.

[0145] In one embodiment, the linker comprises 10-100 atoms selected from H, C, N, S, and O, including one or more amino acids.

[0146] In one embodiment, the linker comprises 10-50 atoms selected from H, C, N, S, and O.- 35 -061685.109PCT

[0147] In one embodiment, the linker comprises 10-50 atoms selected from H, C, N, S, and O, including one or more amino acids.

[0148] In one embodiment, the linker comprises:

[0149] In one embodiment, the linker is:

[0150] Processes for preparing conjugates of the invention are also provided as further embodiments of the invention.4.225Ac-PCTA-conjugates

[0151] Data provided herein indicate that225Ac-PCTA-conjugates can be generated more readily under milder radiochemistry conditions, with improved specific activity and better long-term stability. Three anti-FAP VHH molecules, 1-3, 1-5, and 1-28, that are selective for FAP were developed, as described herein. Furthermore, using 1-3, the VHH binding site on FAP has been characterized, demonstrating that two VHH molecules can bind to the FAP protein. Theoretically, this would allow greater delivery of225Ac to the tumor. The low molecular weight nanobodies are less expensive to produce than monoclonal antibodies (mAbs), and the robust nature of VHH proteins facilitates225Ac radiochemistry, creating a radiotherapeutic with higher specific activity than can be achieved with mAbs. The short biological half-life of the VHH protein, combined with FAP's restricted expression profile in healthy tissues, is expected to improve dosimetry, allowing for the delivery of therapeutic doses of alpha particles while minimizing damage to healthy neural parenchyma.

[0152] In recent publications by the inventors, it was demonstrated that properly targeted,225Ac-radiopharmaceuticals can reduce tumor burden and exhibit excellent dosimetry profiles in vivo (Tafreshi, N.K., JNuclMed 60, 1124-1133 (2019); Tafreshi, N.K., ACS Pharmacol Transl Sci 4, 953-965 (2021); Pandya, D.N., Theranostics 6, 698- 709 (2016); Tichacek, C.J., Mol Pharm 17, 4180-4188 (2020)). Studies by the inventors also suggest that225Ac-PCTA-VHH would be a potent, targeted radionuclide therapy for GBM. However, it is unknown if the VHHs will retain their FAP+ cell - 36 -061685.109PCTselectivity and internalization properties in the presence of the conjugated225Ac-PCTA complex. Furthermore, it is unknown how the biodistribution, dosimetry and therapeutic efficacy will be affected by225Ac-PCTA chelates conjugated to the VHH. Therefore, the studies presented herein aim to investigate the selectivity and internalization properties of FAP+ cells in the presence of the conjugated225Ac-PCTA complex. The studies also evaluate the biodistribution, dosimetry, and therapeutic potential of225Ac-PCTA-VHH using mouse models of GBM.IV. METHODS OF USE

[0153] The disclosed compositions thereof can be used to modulate an immune response in a subject in need thereof. One embodiment provides a method of treating a disease characterized by the overexpression of FAP in a subject in need thereof, comprising administering a pharmaceutical composition comprising one or more anti- FAP single-domain antibodies to the subject.

[0154] Methods of inducing or enhancing an immune response in a subject are provided. Typically, the methods include administering to a subject an effective amount of one or more of the disclosed compositions thereof to immunospecifically reduce or block fibrosis associated with FAP.

[0155] In one embodiment, the methods include administering to a subject an effective amount of one or more of the disclosed compositions, wherein the disease characterized by overexpression of fibroblast activation protein (FAP) is selected from the group consisting of cancer, chronic inflammation, atherosclerosis, arthritis, fibrosis, tissue remodeling, or keloid disorder.

[0156] In another embodiment, the methods include administering to a subject an effective amount of one or more of the disclosed compositions, wherein the cancer is a solid tumor selected from the group consisting of glioblastoma multiforme (GBM), breast cancer, pancreatic cancer, small intestine cancer, colon cancer, rectal cancer, lung cancer, head and neck cancer, ovarian cancer, hepatocellular carcinoma, esophageal cancer, hypopharynx cancer, nasopharynx cancer, larynx cancer, myeloma cells, bladder cancer, cholangiocellular carcinoma, clear cell renal carcinoma, neuroendocrine tumor, oncogenic osteomalacia, sarcoma, mesothelioma, and carcinoma of unknown primary (CUP).- 37 -061685.109PCT

[0157] In one particular embodiment, the subject has glioblastoma multiforme. The immune response can be, for example, inducing, promoting, or enhancing T cell activation, secretion of cytokines by immune cells, and T-cell proliferation. The disclosed anti-FAP single-domain antibodies or antigen-binding fragments thereof can be administered to a subject in need thereof in an effective amount to overcome T-cell suppression. Overcoming T cell suppression can be determined by measuring T cell function using known techniques.

[0158] The methods can be used in vivo or ex vivo to induce, promote, or enhance a stimulating immune response.

[0159] In some embodiments, the anti-FAP single-domain antibody or antigen binding fragment thereof, or nucleic acid encoding the anti-FAP single-domain antibody or antigen binding fragment thereof, is administered directly to the subject. In some embodiments, the anti-FAP single-domain antibody or antigen-binding fragment thereof is contacted with cells (e.g., immune cells) ex vivo, and the treated cells are administered to the subject (e.g., adoptive transfer). The anti-FAP single-domain antibody or antigen-binding fragment thereof can enable a more robust immune response.A. Subjects to be Treated1. Fibrotic Disorders

[0160] Progressive scarring (fibrosis) is a pathological feature of many chronic inflammatory diseases and is an important cause of morbidity and mortality worldwide. Fibrosis is characterized by the accumulation of excess extracellular matrix components (e.g., collagen, fibronectin) that form fibrous connective tissue in and around an inflamed or damaged tissue. Fibrosis may cause overgrowth, hardening, and / or scarring that disrupts the architecture of the underlying organ or tissue. While controlled tissue remodeling and scarring are part of the normal wound healing process promoted by transdifferentiation of fibroblasts into myofibroblasts, excessive and persistent scarring due to severe or repetitive injury or dysregulated wound healing (e.g., persistence of myofibroblasts) can eventually result in permanent scarring, organ dysfunction and failure, and even death.

[0161] Fibrotic changes can occur in various vascular disorders (e.g., peripheral vascular disease, cardiac disease, cerebral disease) and in all major tissue and organ- 38 -061685.109PCTsystems (e.g., lungs, liver, kidneys, heart, skin). Fibrotic disorders include a wide range of clinical presentations, including multisystemic disorders, such as systemic sclerosis, multifocal fibrosclerosis, and organ-specific disorders, such as pulmonary, cardiac, liver, and kidney fibrosis (Rosenbloom et al., Ann Intern Med. 2010 Feb 2; 152(3): 159- 66; Wynn, Nat Rev Immunol. 2004 Aug;4(8):583-94). While the etiology and causative mechanisms of individual fibrotic disorders may vary (e.g., ischemic event, exposure to a chemical, radiation, or infectious agent) and are poorly understood, they all share the common feature of abnormal and excessive deposition of extracellular matrix in affected tissues (Wynn and Ramalingam, Nat Med. 2012 Jul 6;18(7): 1028-40).

[0162] In certain embodiments, a fibrotic disorder or disease is associated with the persistent presence of myofibroblasts in and around fibrotic foci or lesions. Excessive and persistent fibrosis can progressively remodel and destroy normal tissue, which may lead to dysfunction and failure of affected organs, and ultimately death. A fibrotic disorder may affect any tissue in the body and is generally initiated by an injury and the transdifferentiation of fibroblasts into myofibroblasts. As used herein, “transdifferentiation” refers to the direct conversion of one cell type into another. It is to be understood that fibrosis alone, triggered by normal wound healing processes that have not progressed to a pathogenic state is not considered a fibrotic disorder or disease of this disclosure. A “fibrotic lesion” or “fibrotic plaque” refers to a focal area of fibrosis.

[0163] Non-limiting examples of fibrotic disorders or fibrotic diseases include pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, liver fibrosis (e.g., cirrhosis), cardiac fibrosis, endomyocardial fibrosis, vascular fibrosis (e.g., atherosclerosis, stenosis, restenosis), atrial fibrosis, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, progressive massive fibrosis (e.g., lungs), chronic kidney disease, nephrogenic systemic fibrosis, Crohn's disease, hypertrophic scarring, keloid, scleroderma, systemic sclerosis (e.g., skin, lungs), athrofibrosis (e.g., knee, shoulder, other joints), Peyronie's disease, Dupuytren's contracture, adhesive capsulitis, organ transplant associated fibrosis, ischemia associated fibrosis, or the like.2. Glioblastoma Multiforme (GBM)

[0164] Provided herein are methods for targeting fibroblast activation protein alpha (FAP) - a unique biomarker found on glioblastoma multiforme (GBM) cells with- 39 -061685.109PCTVHH proteins to create a novel targeted alpha particle therapy (TAT) that selectively delivers alpha particle radiation to GBM tumors.

[0165] Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor in adults with an annual incidence of approximately 14,000 newly diagnosed cases per year in the U.S (Wen, P. Y., N Engl J Med 359, 492-507 (2008); Ostrom, Q. T., 2021; Ostrom, Q.T., 2014; Ostrom, Q. T., 2022; Miller, B. W ., Nucl Instrum Methods Phys Res A 767, 146-152 (2014)). Standard of care (SOC) is maximal safe surgical resection followed by combined temozolomide (TMZ) and radiation (RT) and then adjuvant TMZ post-RT. Despite this aggressive regimen, the median time to local recurrence or progression in the irradiated field is seven months from diagnosis (Stupp, R., Lancet Oneal 10, 459-466 (2009); Brandes, A. A., J Clin Oneal 27, 1275- 1279 (2009); Gebhardt, B. J., Radiat Oneal 9, 130 (2014)). Due to the propensity for recurrence and progression, prognosis is poor with a median overall survival (OS) of only 14-16 months (Stupp, R., 2009; Gilbert, M. R., N Engl J Med 370, 699-708 (2014)).

[0166] FAP has low to undetectable expression in most normal adult tissues, but is highly upregulated in several cancers, including almost all carcinomas. In tumors, various mesenchymal cells express FAP, including mesenchymal stem cells (MSCs), CAFs, sarcoma, and melanoma cells (Xiao, Z., et al., Nat Commun. 2023 Aug 22;14(1):5110; Sahai, E., Nat Rev Cancer 20, 174-186 (2020); Pure, E., Expert Opin Ther Targets 2009, 13, 967-973; Jacob, M., Curr Mol Med 2012, 12, 1220-1243; Barrett, R. L., £life 9 (2020); Barrett, R., Curr Opin Immunol 64, 80-87 (2020)). With respect to neuroepithelial cancers, Mentlein and colleagues, using quantitative reverse transcriptase PCR (RT-PCR) and immunohistochemistry, determined that FAP expression was elevated in several glioma subtypes(Mentlein, R., 2011). Moreover, the data revealed that FAP enabled glioma cell invasion through brain tissue, suggesting its role in tumor cell infiltration and facilitating the degradation of the brain parenchyma. Additionally, while examining human tumor samples and tumor cell lines to understand the relevance of FAP expression, Busek and colleagues discovered elevated levels of FAP protein in most human high-grade gliomas with a mesenchymal subtype and in several glioma tumor cells (Balaziova, E., 2021; Matrasova, I., 2017; Busek, P., Pancreatology 16, 829-838 (2016); Balaziova, E., Mol Cell Biochem 354, 283-289 (2011)). Furthermore, their studies indicated that FAP expression correlated with- 40 -061685.109PCTextracellular matrix remodeling and inflammation. This suggests a link between FAP expression and the aggressive tissue remodeling, necrosis, and inflammatory infiltrates observed in glioma tumors. Finally, they also found FAP expression on a variety of stromal cell populations within these tumors. Thus, targeting FAP may provide a comprehensive treatment strategy that simultaneously targets tumor cells and the pro- tumorigenic microenvironment of GBM.

[0167] Nanobodies (or VHH), which were first discovered in camelids, are a class of immunoglobulins that consist of only one heavy chain with a single variable domain (Dekempeneer, Y., 2016; D'Huyvetter, M., 2014; Barakat, S., 2022; Arezumand, R., 2017; Alirahimi, E., 2018; AI-Baradie, R.S., 2020). Nanobodies retain high affinity and specificity for their target antigens, with low off-target accumulation. They're unexpectedly robust due to their high refolding capacity, recovering from chemical denaturation with minimal damage to functionality. Furthermore, unlike conventional antibodies, they can tolerate environmental conditions associated with radiochemistry including high temperatures, elevated pressures, non-physiological pHs (3.0-9.0), and even the strongest of chemical denaturants (2-3M guanidinium chloride, 6-8M urea). From a manufacturing standpoint, nanobodies are simple and inexpensive to produce. Lacking post-translational modifications, nanobodies can be synthesized in microbial systems. Finally, their small size and ability to penetrate the BBB make them ideal ligands to deliver alpha particle radiotherapy to GBM tumors.B. Methods of Treatment

[0168] The present invention includes methods of inducing an immune response in a subject to a fibrotic disease by administering a composition as described herein to the subject. The immune response may include a humoral immune response and / or a cellular immune response. The immune response may enhance an innate and / or adaptive immune response.

[0169] The disclosed composition can be used in gene therapy and / or therapeutic approaches for the treatment of disease, which involve the increase or decrease of a nucleotide sequence of interest in a host cell. In these embodiments, the expressible heterologous nucleotide sequence may be derived from a mammalian genome. In other embodiments, the heterologous nucleotide sequence encodes a secreted protein.- 41 -061685.109PCT

[0170] In other embodiments, the expressible heterologous nucleotide sequence responds to positive selection stimuli. In other embodiments, the expressible heterologous nucleotide sequence also responds to negative selection stimuli. In further embodiments, it may be useful for the polynucleotide sequences to further comprise a reporter gene. For example, the reporter gene can be a luciferase or green fluorescent protein.

[0171] It is contemplated that when used to treat various diseases, the compositions and methods of the disclosure can be combined with other therapeutic agents suitable for the same or similar diseases. Additionally, two or more embodiments of the disclosure may also be co-administered to generate additive or synergistic effects. When co-administered with a second therapeutic agent, the embodiment of the disclosure and the second therapeutic agent may be simultaneously or sequentially (in any order). Suitable therapeutically effective dosages for each agent may be lowered due to the additive action or synergy.

[0172] As a non-limiting example, the disclosure can be combined with other therapies that prevent fibrosis. In some embodiments, the compositions and methods disclosed herein are useful for enhancing the efficacy of anti-FAP single-domain antibodies or antigen-binding fragments thereof directed against fibrotic diseases or conditions. The compositions and methods of the disclosure can be administered to a subject either simultaneously with or before (e.g., 1-30 days before) a reagent (including but not limited to small molecules, antibodies, or cellular reagents) that acts to elicit an immune response (e.g., to treat cancer or an infection). The compositions and methods of the disclosure can also be administered in combination with a secondary antibody directed at a pathogenic antigen or allergen.

[0173] The pharmaceutical compositions of the invention can be readily employed in a variety of therapeutic or prophylactic applications, such as treating fibrotic diseases and conditions or eliciting an immune response to fibrosis in a subject. In various embodiments, the compositions can be used for treating or preventing fibrosis caused by a pathogen. Therapeutic and prophylactic applications of compositions derived from the other immunogens described herein can be similarly performed. Depending on the specific subject and conditions, pharmaceutical compositions of the invention can be administered to subjects by a variety of- 42 -061685.109PCTadministration modes known to the person of ordinary skill in the art, for example, topical, oral, intranasal, intramuscular, subcutaneous, intravenous, intra-arterial, intraarticular, intraperitoneal, or parenteral routes. In some aspects, administration is to a mucosal surface. When administered by injection, the immunogenic composition or vaccine may be administered parenterally. Parenteral administration includes, for example, administration by intravenous, subcutaneous, intramuscular, or intraperitoneal injection.

[0174] The description exemplifies illustrative embodiments. Throughout the application, guidance is provided through lists of examples that can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.

[0175] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

[0176] The following examples illustrate the present invention. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.EXAMPLESExample 1 : Characterization of 13 binding to FAP

[0177] Materials and Methods

[0178] Reagents and equipment. Unless noted, chemicals and materials were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA) or ThermoFisher Scientific, Inc. Solutions were prepared using ultrapure water (18 MW-cm resistivity). Protein purification was accomplished using an NGC FPLC system (Bio-Rad, Hercules, CA). Human recombinant fibroblast activation protein alpha and dipeptidyl peptidase IV (DPP4) were purchased from Biolegend (San Diego, CA). The singledomain antibodies Iowa 3 (13), Iowa 5 (15), and Iowa 28 (128) were isolated from a naive camelid library and purchased from Neoclone Biotechnologies, International (Madison, WI).

[0179] VHH sequences: The sequences coding for 13, 15, and 128 were synthesized at Integrated DNA Technologies (Coralville, IA).- 43 -061685.109PCT

[0180] The nucleotide sequence for 13 is:GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAACCTGGGGGTTCTCTGAGACTCTCCTGTGCAGCCTCGGGATTCACCTTCAGTAGCTATGCTATGAGCTGGGTCCGCCAGGCTCCAGGAAAGGGGCTCGAGTGGGTCTCAGCTATTAATAGTGGTGGTGGTAGCACAAGCTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTGCAAAAGCCCGGACTGGGTGGTCCCTAGCAGTTCCTAGTTTTGGTTCCTGGGGCCAGGGGACCC AGGTCACCGTCTCCTCA (SEQ ID NO: 1).

[0181] The amino acid sequence for 13 is:EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINSGGGSTSYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAKARTGWSLAVPSFGSWGQGTQVTVSS (SEQ ID NO: 2) with

[0182] CDR1 : SSYAMS (SEQ ID NO: 3),

[0183] CDR2: WVSAINSGGGSTS (SEQ ID NO: 4),

[0184] CDR3 : AKARTGWSLAVPSFGS (SEQ ID NO: 5).

[0185] The nucleotide sequence for 15 is:GAGGTCCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGGGAGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAACATCATCAACATCAATCGCATGGACTGGTACCGCCAGGCGCCAGGGAAGGATCGCGAGTTGGTCGCACGTATTACTGGTAGTGGTAGTACAAATTATGCTGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTTTCTGCAAATGAACAGCCTAAAACCTGAGGACACGGCCGTCTATACGTGTAATGGGTCCTTACTCATGACTGCGATGGGGGGATTTCTCGAACTTCGGGGCCACGGTACCCTGGTCA CCGTCTCCTCA (SEQ ID NO:6).

[0186] The amino acid sequence for 15 is:EVQLVESGGGLVQAGESLRLACVGSGISFRNFAIGWYRQAPGKQRELVVSISSIGVTNYGDSVKGRFTISRDNAKNTVSLQMNSLKPEDTAVYYCNAAGTDWRTLARRDYWGQGTQVTVSS (SEQ ID NO: 7) with

[0187] CDR1 : RNFAIG (SEQ ID NO: 8),

[0188] CDR2: LVVSISSIGVTN (SEQ ID NO: 9),

[0189] CDR3 : NAAGTDWRTLARRDY (SEQ ID NO: 10)- 44 -061685.109PCT

[0190] The nucleotide sequence for 128 is: GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAACCTGGGGGTTC TCTGAGACTCTCCTGTGCAGCCTCGGGATTCACCTTCAGTAGCTATGCTAT GAGCTGGGTCCGCCAGGCTCCAGGAAAGGGGCTCGAGTGGGTCTCAGCTA TTAATAGTGGTGGTGGTAGCACAAGCTATGCAGACTCCGTGAAGGGCCGA TTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAA CAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTGCTTCTGCTTCTTC TTCTGATTATTATGATTATTATTATGCTTATTATTGGGGCCAGGGGACCCA GGTCACCGTCTCCTCA (SEQ ID NO: 11).

[0191] The amino acid sequence for 128 is:

[0192] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKG LEWVSAINSGGGSTSYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYY CASASSSDYYDYYYAYYWGQGTQVTVSS (SEQ ID NO: 12) with

[0193] CDR1 : SSYAMS (SEQ ID NO: 3),

[0194] CDR2: WVSAINSGGGSTS (SEQ ID NO: 4),

[0195] CDR3: ASASSSDYYDYYYAYY (SEQ ID NO: 13).

[0196] Preparation of the single-domain antibody, 13. The coding sequence of 13, along with an N-terminal FLAG tag, was cloned into the pRham™ N-His SUMO Kan vector (Lucigen) following the manufacturer’s recommendations. This expression construct facilitated the expression of 13 fused to an N-terminal 6X His-SUMO-FLAG tag, which is referred to as SUMO-13. The SUMO-13 construct was transformed into Shuffle T7 Escherichia coli strain (New England Biolabs) for expression, which allows for the cytoplasmic disulfide bond formation. The transformed cells were selected on LB agar plates containing 30 pg / ml kanamycin and cells were grown at 30°C. A single colony was then inoculated in 25 ml LB media supplemented with 30 pg / ml kanamycin, 0.2% (w / v) rhamnose and 0.075% (w / v) glucose and the culture was grown at 30°C at 220 rpm for overnight. The cells were harvested by spinning them at 4,000 rpm for 10 minutes at room temperature. The cell pellet was resuspended in 4 ml of Tris-buffered saline (TBS; 50 mM Tris, 150 mM NaCl pH 8) containing 0.1 mg / ml lysozyme. The cell suspension was incubated on ice for 20 minutes, and cells were lysed by sonicating them at 70% amplitude with twenty pulses of 10 seconds each, followed by a 40-second gap. The cell lysate was centrifuged at 13,000 rpm at 4°C, and the supernatant was collected. The supernatant was then incubated with 500 pl Ni-NTA resin, pre-- 45 -061685.109PCTequilibrated in TBS, for 30 minutes at 4°C. The resin bound to the recombinant sdAb was then packed in a Poly-Prep chromatography column (Bio-Rad, Hercules, CA, USA) and washed with 10 column volumes of TBS, followed by washing with 10 column volumes each of TBS containing 10 mM and 20 mM imidazole. Stepwise elution of the His-tagged sdAb was performed with two column volumes each of TBS containing 50-, 100-, 200- and 300- mM imidazole. Chromatography was performed in a gravitational flow mode with a flow rate of ~0.5 mL / min. Fractions were analyzed for the presence of the sdAb on 12% SDS-PAGE and visualized after Coomassie blue staining. The major fraction was subjected to size exclusion chromatography using a Superdex 75 column (Cytiva) on an NGC FPLC system (Bio-Rad) in 100 mM PBS pH 7.2 buffer. The peak fractions were collected and concentrated using an Amicon ultra centrifugal filter with a 3 kDa MWCO (MilliporeSigma).

[0197] The coding sequence of 13 was also cloned into a custom pMAL-c5X vector (New England Biolabs) which allows for sdAb expression with an N-terminal MBP-fusion protein cleavable with TEV protease and a C-terminal 6X His-tag. The protein was expressed in Shuffle T7 Escherichia coli strain (New England Biolabs) for expression. The transformed cells were selected on LB agar plates containing 100 pg / ml ampicillin and cells were grown at 30°C. A single colony was then inoculated in 100 ml LB media supplemented with 100 pg / ml ampicillin and the culture was grown at 30°C at 220 rpm for overnight. Two flasks containing 1 L LB media each containing 100 ug / mL ampicillin was inoculated with 10 mL of overnight grown culture and the culture was grown at 30°C till OD600 reached 0.5-0.6. The culture was then induced by adding 0.5 mM IPTG and the culture was grown overnight at 18°C. The cells were harvested at 5,000 rpm for 30 minutes at 4 °C. The cell pellet was resuspended in buffer (50 mM Na-phosphate, 300 mM NaCl, 10% glycerol and 5 mM Imidazole, pH 7.5) with EDTA-free protease inhibitor cocktail (Roche), 0.1 mg / mL lysozyme and DNase I. The cell suspension was incubated on ice for 20 minutes and cells were lysed by sonication. The cell lysate was centrifuged at 35,000 rpm for 60 minutes at 4°C and the supernatant was collected. The protein was purified using a 5mL Ni-NTA column with a 50 mL gradient of 5 to 300 mM imidazole for elution. The pooled elution fraction was subjected to size exclusion chromatography on a Superdex 200 column (Cytiva) using an NGC FPLC system (Bio-Rad) in lx Dulbecco's phosphate-buffered saline (DPBS) pH 7.2 buffer. The peak fractions were collected and concentrated using an Amicon- 46 -061685.109PCTultra centrifugal filter with a 3 kDa MWCO (MilliporeSigma). The sdAbs were quantitated by measuring their absorbance at 280 nm on a nanodrop instrument (Thermo Scientific). The proteins were stored on ice for subsequent experiments.

[0198] Cross-linking of FAP with SUMO-13. Human FAP (2 pM, Biolegend) was mixed with 5-fold molar excess of SUMO-13 sdAb in the binding buffer (100 mM PBS pH 7.2) and incubated at room temperature overnight. The complexes were then incubated with 1 mM bis(sulfosuccinimidyl)suberate (BS3) cross-linker (Pierce) for 30 minutes at room temperature and then the free cross-linker was quenched by adding 50 mM Tris-HCl pH 8 buffer.

[0199] Mass Photometry . Mass photometry experiments were performed on a Refeyn TwoMP mass photometer (Refeyn Ltd, Oxford, UK). Microscope coverslips (24 mm x 50 mm, Thorlabs Inc.) were cleaned by serial rinsing with Milli-Q water and HPLC-grade isopropanol (Sigma Aldrich) followed by drying with a filtered air stream. Silicon gaskets (Grace Bio-Labs) to hold the sample drops were cleaned in the same procedure immediately prior to measurement. All mass photometry measurements were performed at room temperature using DPBS without calcium and magnesium (ThermoFisher). The instrument was calibrated using a protein standard mixture: P- amylase (Sigma-Aldrich, 56, 112 and 224 kDa), and thyroglobulin (Sigma-Aldrich, 670 kDa). Before each measurement, 15 pL of DPBS buffer was placed in the well to find focus. The focus position was searched and locked using the default droplet-dilution autofocus function after which 5 pL of protein was added to make final concentration at 15 nM and pipetted up and down to briefly mix before movie acquisition was promptly started. Movies were acquired for 60 s (3000 frames) using AcquireMP (version 2.3.0; Refeyn Ltd) using standard settings. All movies were processed and analyzed using DiscoverMP (version 2.3.0; Refeyn Ltd).

[0200] Biolayer interferometry (BLI). Affinity determination measurements were performed on the Octet RED96 (Sartorius). All assays were per- formed using streptavidin (SA) coated biosensors (Sartorius) in kinetics buffer (PBS pH 7.4, 0.5 mg / mL BSA, and 0.01% (v / v) Tween-20) at 25°C. Biosensors were equilibrated for 30 min prior to beginning the assay. Assay step order and corresponding times were as follows: equilibration (30 s), loading (90 s), baseline (30 s), association (10 s), and dissociation (15 s). Biotinylated FAP and DPP4 (2 pg / mL, AcroBiosystems) were loaded onto SA sensors to a response of 0.5 nm. Association measurements were- 47 -061685.109PCTperformed using a dilution series of MBP-I3 from 0.25 to 10 pM. Baseline drift was corrected by subtracting the response of a ligand-loaded sensor in kinetics buffer. Data analysis was performed with Octet Data Analysis 11.1 software using a global fit 1 : 1 model to determine affinity and kinetic parameters. Affinity and kinetic data reported are representative of three independent experiments.

[0201] Results

[0202] To analyze the complex and determine the stoichiometry of MBP-I3 and SUMO-13 binding to FAP, mass photometry (MP) was employed. The FAP protein was confirmed to be a dimer (198 kDa) at the low nM concentrations used for mass photometry (Fig. 1A), matching the previously determined structure. Detection of the FAP complex with SUMO-13 by MP required a cross-linking reagent due to the weak affinity and fast dissociation rate (see methods). Complex formation was confirmed and contained a heterogenous mix of populations including FAP alone, FAP + SUMO-13, and FAP + 2 SUMO-13 (Fig. 1C). This verified one 13 binding site per FAP monomer. Characterization of affinity and binding kinetics were done using biolayer interferometry (BLI). A site-specific biotinylated version of FAP and an MBP-I3 construct were used for BLI. The MBP-I3 construct was chosen for its larger size, and therefore greater response in BLI. The biotinylated FAP was immobilized on streptavidin coated biosensors and dipped into a range of MBP-I3 concentrations to measure association and then into buffer wells to measure dissociation (Fig. IB). The affinity (Kd) was determined to be 2.0 ± 0.3 pM by fitting to the kinetic data and 2.7 ± 0.1 pM by steady state analysis (Fig. 2A-2B). Additionally, the kinetic rates for association (kon) and dissociation (koir) were 1.8 (± 0.3) *105M^s’1and 3.6 (± 0.2) *10’1s’1, respectively.Example 2: FAP-I3 complex structure

[0203] Materials and Methods

[0204] Cryo-EM sample preparation and data acquisition. The cross-linked FAP with SUMO-13 sample (0.4 mg / mL) was vitrified on QuantiFoil Rl.2 / 1.3 300 mesh copper grids (SPT Labtech) in 20 mM Tris-HCl pH 8.0 and 150 mM NaCl using a Vitrobot Mark IV (ThermoFisher). Grids were glow discharged for 60 s, -15 mA on a PELCO easiGlow (Ted Pella) system. Sample (3 pL) was applied to grids in the Vitrobot chamber (4 °C and 95% humidity) and blotted for three seconds with -5 blotting force before plunge-freezing in liquid ethane. Data were collected on a Titan- 48 -061685.109PCTKrios G3 microscope (300 kV) using SerialEM with a K3 direct electron detector (Gatan) as shown in Table 1. A total of 5,450 movies were collected at a pixel size of 0.43 A / pixel (super-resolution mode) with a dose of ~65 electrons / A2, exposure time of 2.43 seconds, 45 frames, and a defocus range of -0.5 to -2.5 pm.

[0205] Table 1. Cryo-EM data collection parameters and model refinement statistics.- 49 -061685.109PCT

[0206] Cryo-EM image processing and 3D reconstruction. Movies were subject to patch motion correction and patch CTF estimation in cryoSPARC45. Initial particle picks were performed on a subset of data using the blob picker followed by two- dimensional (2D) classification to generate 2D templates for template-based picking on the full dataset in cryoSPARC Live. Particles (2,293,048) were extracted using a 300- pixel box size fourier cropped to 150 pixels (1.72 A / pixel) and cleaned with multiple rounds of 2D classification. The volume from streaming refinement in cryoSPARC Live was fed into heterogeneous refinement in cryoSPARC using three classes to isolate classes of FAP + SUMO-13 and FAP + 2 SUMO-13. Two classes from this Hetero refinement job were selected to re-extract separate particle stacks for FAP + SUMO-13 (696,327) and FAP + 2 SUMO-13 (356,520). Particles were extracted using a 300-pixel box size (0.86 A / pixel) and used separately for non-uniform refinement in either Cl or C2 symmetry. A 3D classification job was used to further clean each particle stack, which resulted in final particle numbers of 272,711 for FAP + SUMO- 13 (Cl symmetry) and 238,246 for FAP + 2 SUMO-13 (C2 symmetry). Processing was initially performed in cryoSPARC version 3.1 and completed in version 3.3.1. Final maps were post-processed using DeepEMhancer and used for visualization.

[0207] Model building and refinement. An initial model for complex of FAP with SUMO-13 sdAb was generated by the crystal structure (PDB 1Z68) and Alphafold46. The model was initially docked into the density map using Fit in Map in Chimera47. Manual model building was performed in Coot (Emsley, P., Acta Crystallogr D Biol Crystallogr 2010, 66, 486-501) and refinement using real-space refinement in Phenix Liebschner, D., Acta Crystallogr D Struct Biol 2019, 75, 861-877). Figures were - 50 -061685.109PCTgenerated in Chimera and PyMOL. Software used for data processing, model building, and refinement, except for cryoSPARC, was curated by SBGrid (Morin, A., Elife 2013, 2, e01456).

[0208] Results

[0209] To determine the epitope for 13, the structure of SUMO-13 bound to FAP was determined by cryo-EM. Structure determination used cross-linked sample as it resulted in far greater complex compared to uncross-linked sample (data not shown). Like the dual populations observed in the MP data, particles with both one and two 13 molecules bound to FAP were isolated during the cryo-EM data processing workflow (Fig. 3A). The reconstructions for both FAP 13 complexes were determined to 2.7 A for one 13 bound in Cl symmetry and two 13 molecules bound in C2 symmetry (Fig. 3B-3C). Local resolution maps and final structural models for each complex are shown in Figures 4A-4F. Due to the flexible portion between the SUMO and 13 regions of the fusion protein, the SUMO region is not observed in the reconstruction. The local resolution throughout most of the FAP core region is ~2.6 A and ~3.0 A at the FAP-I3 interface. Both FAP molecules and the 13 molecules from each complex overlay well with very subtle differences (C-alpha RMSDs, FAP dimer 0.141, 13, 0.199). 13 interacts with FAP through its CDR3 loop and FR2 region, instead of the more typical interaction with all three CDR loops (Fig. 5 A). Specific residues involved in the FAP -13 interaction can be seen in Fig. 5B. FAP Y274 has multiple interactions with 13 and sits within a pocket formed by 13 (Fig. 5B,5C). The electrostatic surface map of 13 shows that the one loop from FAP engages a relatively uncharged region while the second FAP loop interacts with a positively charged region (Fig. 5C). The epitope footprint on the surface of FAP is shown in Fig. 5D. The following FAP:I3 residue pairs have a hydrogen bond interaction; Y274:P108, Y274:W47, E325:S109, and D326:V107. Additionally, there is a p-stacking interaction between Y271 :F110.Example 3 : In silico rational design to enhance 13

[0210] Materials and Methods

[0211] In silico rational design. The cryo-EM structure of FAP-I3 was imported into the Bioluminate package (Schrodinger Release 2023-3) and passed through the Protein Preparation Wizard. The FAP-I3 complex interface was manually inspected to identify rational mutations that could both enhance and disrupt the complex. Positions VI 07 and SI 09 in 13 were chosen as sites that could create additional interactions with- 51 -061685.109PCTFAP. Next, the Residue Scanning Module in Bioluminate was used to introduce mutations into 13. Stability and affinity calculations were performed optimizing for the affinity, and backbone minimization was used with a cutoff of 5 A. Interface interactions were determined using the Protein Interaction Analysis Module.

[0212] Results

[0213] Since the affinity of 13 for FAP was weak compared to typical sdAbs, rational mutations were chosen to improve the affinity. The sites VI 07 and SI 09 were identified on 13 that could potentially benefit from having a mutation with positive electrostatic potential or an aromatic residue (Fig. 6A). In silico mutations were chosen for VI 07 and SI 09 that could potentially increase and decrease (as a control) the FAP- 13 interaction (Fig. 6B, 6C). The calculated changes in affinity (dAffinity) and stability (dStability) from the original sequence for both sites showed approximate trends expected with various mutations. The Arg mutations were projected to provide the largest increase in both affinity and stability. The comparison of hydrogen bonds, salt bridges, and p-stacking interactions at the interfaces shows S109R to form 2 new hydrogen bonds and a salt bridge, which positively increases affinity and stability (Fig. 6D).Example 4: Comparison of FAP epitope to homologous DPP4 protein

[0214] Results

[0215] FAP belongs to the dipeptidyl peptidase (DPP) family and shares 52% sequence identity (71% similarity) with DPP4. Both FAP and DPP4 are dimeric and share high overall structural homology (Fig. 7A). Comparison of the FAP 13 epitope region to the same region in DPP4 shows distinct differences in orientation of the crucial FAP loop containing Y274 (Fig. 7B; Fig. 8A-8D). The equivalent loop in DPP4 (residues 275-283) protrudes less from the overall protein. Analysis of the sequences from this epitope region reveals large differences in residue composition in addition to the structural conformation (Fig. 7C). The lack of MBP-I3 binding to DPP4 was tested and confirmed by BLI (Fig. 7D).

[0216] The ability to identify an antibody binding region or epitope of a protein considered to be an important biomarker of disease has important implications for disease diagnosis, vaccine development, and elucidating disease mechanisms (Humanna Press, 2018; Brooks, B. D.; Drug Discov Today 2014, 19, 1040-1044; Ladner, R. C., Biotechnol Genet Eng Rev 2007, 24, 1-30; Volk, A. L., Set Rep 2016, 6,- 52 -061685.109PCT31365; Zhang, F. F., Int Immunopharmacol 2022, 772, 109237). Additionally, the characterization of an antigen-binding region enables the characterization of therapeutic antibodies and has important intellectual property implications. Epitope mapping is the determination of which amino acid sequences and three-dimensional interactions directly contribute to the affinity between an antibody or its derivatives and a specific antigen. Furthermore, epitope mapping allows investigators to study how the binding of specific epitopes may alter protein function.

[0217] The invention demonstrates that the single-domain antibody 13, which has not been previously disclosed within the Structural Antibody Database (SAbDab), binds to the extracellular surface of FAP by making important contacts through its CDR3 (SEQ ID NO: 5) at a unique epitope that is distinct from the active site. These unexpected results may have a profound effect on how this molecule may be utilized for diagnostic or therapeutic purposes since the role of FAP has been shown to be context and disease dependent (Pure, E., 2009; Pure, E., Oncogene 2018, 37, 4343- 4357; Juillerat-Jeanneret, L., Expert Opin Ther Targets 2017, 27, 977-991) the expression of FAP may be beneficial under some circumstances such as pulmonary fibrosis while detrimental in other circumstances such as cancer41. While antigen binding without enzyme inhibition still needs to be confirmed, being able to target the protein without inhibiting its function may be a viable path to the successful development of a new class of diagnostic and therapeutic molecules for various disease states.

[0218] Also, given the current data supporting the cell -surface heteroprotein complex formation that occurs between FAP, dipeptidyl peptidase IV (DDP4), matrix metalloproteinases (MMPs), integrins and uPAR, understanding how 13 binds to FAP may facilitate the rational design of bi-specific ligands that target FAP and additional partners at the cell surface, and offer another strategy for improved development of agents with diagnostic or therapeutic value (Fitzgerald, A. A., Cancer Metastasis Rev 2020, 39, 783-803). A structural alignment comparison of FAP with DPP4, DPP8, and DPP9 shows the 13 footprint region differs for these S9 family members (Fig. 9A-9B). This is an important factor of the 13 epitope as these proteins all share sequence and structural similarity. Finally, since sdAbs are often used to stabilize larger proteins, the use of 13 may have utility as a molecular chaperone when studying FAP and unexplored aspects of its biology that cannot be elucidated using current structural and molecular- 53 -061685.109PCTbiology techniques (de Marco, A, Microb Cell Fact 2011, 10, 44). Such areas include the true expression pattern of FAP on different cell types, more rigorously distinguishing the enzymatic and non-enzymatic roles of FAP in healthy and diseased tissues and understanding the mechano-signaling aspects of FAP.

[0219] Finally, this work represents a powerful example of how cryo-EM can be applied to the drug discovery process and the rational design of sdAbs. Furthermore, it is expected to play an ever more important role now that the de novo design of sdAbs is on the horizon. Recently, Bennett et al. described a computational strategy to accurately design sdAbs and tested this methodology by creating sdAbs specific for influenza hemagglutinin (Bennett, N.R., bioRxiv 2024, 1-30). In their report the authors demonstrated that the computationally derived sdAbs were nearly identical in CDR conformation and overall binding to the binding models developed using conventional molecular biology and elucidated using cryo-EM. Thus, as these techniques become more sophisticated and routine due to technological advancements in machine learning, the more tedious and time-consuming way of generating sdAbs, which involve animal immunization and library screening, will become secondary options; these artificial intelligence (Al) tools are expected to form the foundation of a new period in the rational design of antibodies and their derivatives.

[0220] As provided herein, the invention describes a unique epitope on the FAP protein that is occupied by the single-domain antibody, 13. This work also reveals the important residues necessary for the FAP-I3 interaction and rationalizes why 13 selectively binds to FAP and not its closest S9 family member DPP4. Considering the current research interest in FAP as a therapeutic target in a variety of disorders including cancer, fibrosis, arthritis and cardiovascular disease, this work will assist investigators in developing theranostic agents to assess and mitigate these disease states.Example 5: Demonstrate the Therapeutic Efficacy of225Ac-PCTA-VHH I / / Vitro.

[0221] Materials and Methods

[0222] Cell Lines and Animal Models: The human GBM cell lines U87mg (FAP+) and U373 (FAP-) are obtained from ATCC (Manassas, VA). FAP receptor expression are validated biannually by Western blot and FACS, using the anti-FAP F19 mAb. Balb / c mice are used in MTD studies. Balb / c nu / nu mice (n=6; Charles River; male and female in equal ratios) bearing one U87mg (FAP+) and one U373 (FAP-) xenograft tumors in contralateral flanks are used for biodistribution and therapy studies.- 54 -061685.109PCTTumor volume is evaluated twice weekly using calipers (volume = 0.52 X [width]2X [length]) until they arelOO mm3and then used in subsequent studies. The total number of mice used for this study is 433.

[0223] Chemistry, Radiochemistry, and in vitro Serum Stability: The macrocycle PCTA-NCS are obtained from Macrocyclics, Dallas, TX.225Ac(NO3)3 is obtained from Oak Ridge National Laboratories (Oak Ridge, TN) and is used as the225Ac source for all radiosynthesis and as a radiochemical standard (Tafreshi, N. K., 2019; Tafreshi, N. K., 2021; Pandya, D. N., 2016) PCTA-NCS are conjugated to each VHH using NCS coupling chemistry. Further, we will use our previously established protocols to prepare the225AC-PCTA-VHHS and the La-PCTA-VHHs, which will be used as nonradioactive standards (Tafreshi, N. K., 2019; Tafreshi, N. K., 2021; Pandya, D. N., 2016). All conjugates will be purified using a Waters size exclusion chromatography system. The number of La-PCTA complexes conjugated to the VHH will be determined by mass spectrometry. We and others have demonstrated that La is a useful nonradioactive surrogate for225Ac and can be used in in vitro studies to evaluate affinity (Tafreshi, N. K., 2019; Tafreshi, N. K., 2021; Pandya, D. N., 2016). We will use the chemistry conditions needed to prepare La-PCTA- VHH as a starting point for the preparation of225Ac-PCTA-VHH, which we will use for cell uptake and in vivo studies. Once radiochemistry conditions have been optimized for225Ac incorporation, the research team will focus on specific activity (As) optimization. Finally, in vitro stability will be determined by incubating the225Ac-PCTA-VHHs with 500 pL of human serum (at 37°C). Samples (n = 3 samples / time point) will be analyzed for 10 days using a Bioscan AR2000 radio-TLC scanner or radio- HPLC, fraction collection and gamma counting. Any peaks not corresponding to225Ac(NO3)3 (standard) or the intact radiopharmaceutical after secular equilibrium will be considered products of instability (Tafreshi, N. K., 2019; Tafreshi, N. K., 2021; Pandya, D. N., 2016).

[0224] In vitro Assays: Affinity (Kd) Determination, Internalization and Cell Proliferation: The affinity of La-PCTA-VHHs for recombinant FAP will be evaluated using surface plasmon resonance spectroscopy as previously described by us (see support letter).42For cell proliferation assays,225Ac-PCTA-VHHs will be incubated with cells seeded in 96 well plates (10,000 cells / well) for 2 h (37°C). After incubation, the cells will be washed thrice and provided fresh media. An MTT assay will be performed using a Vybrant® MTT cell proliferation kit {Thermo Fisher, Inc. Waltham,- 55 -061685.109PCTMA). Additionally, y-H2AX foci, which are considered biomarkers for double stranded DNA breaks, will be imaged using confocal fluorescence microscopy and an Oxi Select DNA Double Strand Break Kit (CellBio Labs, Inc, San Diego, CA) according to kit instructions (Tafreshi, N. K., 2019; Tafreshi, N. K., 2021; Bozon-Petitprin, A., Eur J Nuc / Med Mol Imaging 42, 252-263 (2015); Zhu, C., Biomateria / s 130, 67-75 (2017); Zhu, C., Mol Cancer Ther 15, 106-113 (2016)).

[0225] For internalization studies,225Ac-PCTA-VHH will be added to cells (IxlO6cells / 500pL) in microfuge tubes to achieve a final concentration of 4 nM (Wadas, T. J., Chem Rev 2010, 110, 2858-2902). Tubes will be incubated at 37°C with rotation. To block specific binding at each time point, tubes containing a 500 pL cell suspension (IxlO6cells) will be incubated for 5 minutes at room temperature with excess La-PCTA-VHHs. At each time point, surface-bound fractions will be collected by acid extraction of cells. Internalized radioactivity will be collected by lysing the cells in 0.5% SOS. Radioactivity in each fraction will be counted in a gamma counter. Total protein concentration in the cell lysate will be determined using a standard BCA protein assay (Pierce Biotechnology). Internalized and surface-bound fractions will be expressed as counts per minute per mg protein.

[0226] To further validate specificity, FAP+ (U87mg) cells or FAP- (U373) cells will be cultured in multi -well microscope slides (LabTek) and incubated with each VHH that is conjugated to Alexafluor-647 (Life Technologies, Inc., Grand Island, NY). Cells will be imaged using an Olympus FvlOOO confocal microscope (Waltham, MA). Any detected fluorescence within the cell will be interpreted as FAP- mediated internalization. The cell lines will also be co-incubated with an Alexafluor 488 (Life Technologies, Inc.) anti-CD105 antibody to delineate the cell membrane, and antibodies to markers of early (e.g., anti-EEAl), and late (e.g., anti-RAB7), endosomes. Coregistration of images will distinguish surface binding and cell internalization.

[0227] Results

[0228] Figures 10A-10F agree with the scientific consensus that characterizes FAP as a robust GBM biomarker that may be exploited for TAT Delivery. Three novel VHH proteins that bind FAP were developed, 1-3, 1-5, and 1-28, and their interaction with the FAP protein was characterized using molecular photometry and cryo-electron microscopy (Figures 1A-1C and 12).225Ac-PCTA-conjugates can be generated more readily under milder radiochemistry conditions, with improved specific activity and- 56 -061685.109PCTbetter long-term stability (Table 2). Using 1-3, characterization of the VHH binding site on FAP and demonstration that two VHH molecules can bind to the FAP protein indicates that the method presented herein would allow greater delivery of225Ac to the tumor (Figures 1A-1C and 12). This data suggest that225Ac-PCTA-VHH would be a potent, targeted radionuclide therapy for GBM. However, it is unknown if the VHHs will retain their FAP+ cell selectivity and internalization properties in the presence of the conjugated225Ac-PCTA complex.

[0229] Table 2. Data Summary Comparison of PCTA and DOTA in225AcRadiochemistryExample 6: Demonstrate the Therapeutic Efficacy of225Ac-PCTA- VHH In Vivo.

[0230] Materials and Methods

[0231] Biodistribution: Animals (n = 6 / time point) with one U87mg and one U373 tumor on contralateral flanks will be injected with225Ac-PCTA-VHH (0.07 MBq (3 pCi)) and then sacrificed at 1, 4, 24, 48, 72, 144 and 240 h p.i. Organs of interest, including both tumors will be removed, weighed, and counted on a gamma counter. Percent injected dose / gram (%ID / g) will be counted and compared to a weighed, counted standard. To ensure rigor, all samples will be measured in a gamma counter 24- 57 -061685.109PCTh after collection to ensure secular equilibrium exists between Ac and the daughter decay products (Tafreshi, N. K., 2019; Tafreshi, N. K., 2021; Tichacek, C. J., Mol Pharm 17, 4180-4188 (2020); Tichacek, C. J., Molecules 24 (2019)).

[0232] Digital Autoradiography and Histology: Animals will be injected with a225AC-PCTA-VHH (0.56 MBq (15 pCi)) and sacrificed at 1, 24, 72 and 240 h p.i. Tumors and tissues will be harvested and embedded in optimum cutting temperature (OCT) compound (Miles, Inc., Elkhart, IN). Frozen sections (4-6 pm) will be cut for autoradiography and developed using an iQID camera system (Qscint, Inc.) according to known procedures and a Typhoon 9210 Variable Mode Imager (Molecular Devices, Sunnyvale, CA) (Miller, W. H., Cancer Biother Radiopharm 20, 436-449 (2005); Miller, B. W ., 2014; Miller, B. W ., Med Phys 42, 4094-4105 (2015); Miller, B. W ., Appl Radiat / sot 166, 109348 (2020); Miller, B. W ., Semin Nucl Med 48, 367-376 (2018)) Signal intensities will be quantified using a set of autoradiography standards (GE Healthcare) and published procedures (Gambhir, S.S., J Nucl Med 39, 2003-2011 (1998)). Serial sections will also be cut, fixed in ice-cold acetone, and stained with hematoxylin and eosin (H&E). FAP expression will be probed using anti-FAP mAbs (Abeam, Inc.). Autoradiography and histology results will be compared to document antigen binding by the radiotracers.

[0233] Dosimetry: To assess radiation retained in tumors and risk of damage to normal organs, dosimetry will be determined using biodistribution data and digital autoradiography (Song, H., 2009) . We will apply a multi-compartment model of radioactivity distribution using the acquired organ data and the generalized internal dosimetry schema of the MIRO Committee for alpha-particle emitters (P, H., 2009; Sgouros, G., 2010; Bolch, W. E., 2009). According to MIRD #21, the absorbed dose D(rs,t) is the mean energy imparted to target tissue rr per unit tissue mass, definedwhere A(rs , t) is the time-dependent activity of225Ac and its daughter products in source tissue rs, and S(rr+-rs, t) is the radionuclide-specific quantity representing the mean absorbed dose rate to rr at time t after administration per unit activity present in rs.225Ac and its daughter products are mainly alpha emitters and we will estimate the alpha radioactivity of isotopes in tissue samples by measuring the gamma emissions of the decay products using gamma spectrometry and known conversion factors from gamma- 58 -061685.109PCTray abundance to alpha-particle activity (for 100 alpha decays of225Ac there is 1 90 keV gamma ray emission) (Radchenko, V ., J Chromatogr A 1380, 55-63 (2015); Schopper, H., Excited Nuclear States for Ac-225 (Actinium). (Springer Materials, 2013); Sansonetti, J., Journal of Physical and Chemical Reference Data. 36, 497-498 (2007)). We will estimate the alpha activity and absorbed dose from225Ac and its decay products221Fr(11.4 gammas of 218.1 keV per 100 alphas) and213Bi (25.9 gammas of 440.5 keV per 100 alpha decays) (Radchenko, V., 2015; Schopper, H., 2013; Sansonetti, J., 2007) using the measured gamma spectra of225Ac, and221Fr and213Bi decay products in tissue samples using a modified Wizard gamma Counter (Perkin Elmer, Inc.) with a Nal scintillation detector, with a peak resolution of 7% at 81 keV (Radchenko, V., 2015; Schopper, H., 2013; Sansonetti, J., 2007).

[0234] Therapy Studies: Maximum tolerated dose studies (MTD) will be conducted using eight cohorts of normal BALB / c mice (Tafreshi, N. K., 2019; Tafreshi, N. K., 2021; Pandya, D. N., 2016; Tafreshi, N. K., Kil, H., 2021). After a single intravenous injection of 225Ac-PCTA-VHH or saline, cohorts (n = 5 mice / cohort) will be weighed thrice weekly, and monitored for 110 days for signs of distressed behavior. At the end of that period, animals will be euthanized, and serum will be collected. Blood urea nitrogen (BUN) and creatinine content in serum will be analyzed using commercial ELISA kits (Biotrend Chemikalien Gmbh, Germany). Heart; lung; liver; kidney; spleen, pancreas, stomach, small intestine, large intestine, muscle; and bone will be harvested, fixed in 10% formalin, embedded in paraffin, sectioned (4-6 pm thickness), stained with hematoxylin and eosin, and examined. Weight, serum, and pathology data will be correlated to determine the MTD.

[0235] For therapy studies, U87mg and U373 tumors will be formed on the contralateral flanks of nude mice as described herein and then randomly divided into cohorts (n = 16 / cohort). Each cohort will receive a single intravenous injection of either saline, a225Ac-PCTA-VHH (non-targeting) or225Ac-PCTA-VHH. Animals will be monitored daily for signs of distressed behavior, and tumor growth will be evaluated thrice weekly. Cohorts will be followed until clinical endpoints are achieved as previously described (Tafreshi, N. K., 2019; Tafreshi, N. K., 2021; Pandya, D. N., 2016; Tafreshi, N. K., Kil, H., 2021). Tissue samples will be stained with anti-FAP, anti-Ki- 67, anti -caspase-3 and anti-LC3B mAbs (all Sigma Aldrich, St. Louis, MO.) to probe FAP expression, cell proliferation, apoptosis and autophagy, respectively.- 59 -061685.109PCT

[0236] Statistical Analysis: There are two statistical designs. For longitudinal biodistribution studies a repeated cross-sectional design will be used since at each time point mice must be sacrificed to assess the outcomes of interest. At each time point 6 animals bearing bilateral FAP+ / - tumors will be sacrificed to allow enough data to be collected to be able to assess the biodistributions in each organ of interest. With this sample size, a 95% confidence interval at any time point will have a distance from the mean value to the confidence limits equal to 0.836 standard deviations. Since there are 7-time points (per agent), a pooled estimate of biodistribution data will include 42 total mice (per agent) and thus be able to measure the 95% confidence interval with a distance from the mean value to the confidence limit equal to 0.268 standard deviations.

[0237] For MTD studies, a conservative approach to comparing groups is to use a 2-sample t-test at the last time point of assessment. With n = 5 mice per group there is 80% power to detect a 1.5 standard deviation difference between groups with alpha=0.05 (2-sided). As stated above, this is a conservative calculation since the repeated measurements will add precision and reduce variability in outcome measures and allow smaller differences to be detected between groups.

[0238] For therapy studies, this model will consider rate of tumor growth differed by group over time. A conservative power calculation would be to perform a paired t- test comparing tumor volume at a fixed point in time. With n = 16 mice / cohort, there is 80% power to detect a difference in tumor volume equal to 1.16 standard deviations in magnitude with alpha=0.05 (2-sided test). This calculation is conservative since it does not account for the repeated tumor volume assessments that will be made over time. When these additional tumor volume measures are used this will allow smaller differences to be detected since the repeated measures will add precision to the tumor volume change measures.

[0239] Results

[0240] Given our positive preliminary data and our published experience with225Ac radiochemistry we do not anticipate challenges in completing the synthesis of the225AC-PCTA-VHHS (Tafreshi, N. K., 2019; Tafreshi, N. K., 2021; Pandya, D. N., 2016; Tafreshi, N. K., Kil, H., 2021; Miller, W. H., 2005; Ostrom, Q.T., Sattiraju, A., Mol Cancer Ther 16, 2191-2200 (2017); Sattiraju, A., Solingapuram Sai, K. K., OncotargetS, 42997-43007 (2017)). In vitro, we would expect the Kavalues of the native VHH and those conjugated with La-PCTA to be similar. However, if Ka- 60 -061685.109PCTdecreases by 10-fold or more, our studies will not proceed. Rather, we will optimize the number of chelates per VHH or introduce an amino acid linker between the PCTA ligand and the VHH. Our goal will be to establish a ratio that maximizes specific activity but does not compromise affinity. Such methods have been used successfully to optimize the performance of similar radiopharmaceuticals in vitro (Li, M., Cancer Res 55, 5726s-5728s (1995); Kukis, D. L., Cancer Res 55, 878-884 (1995)).

[0241] In vivo, the anti-FAP VHH should dictate the biodistribution of the radiopharmaceutical. Animals bearing U87mg tumors should demonstrate elevated levels of radioactivity while U373 tumors should demonstrate less radioactivity retention. Due to the size {~15KDa) of the VHHs, renal clearance is expected. Further, the localization of daughter products {213Bi and221Fr) within the kidney may lead to elevated levels of radioactivity in kidney tissue resulting in unwanted cytotoxicity. Several strategies can be used to minimize daughter product (213Bi and221Fr)-induced kidney toxicity if observed in vivo (Jaggi, J. S., IntJ Radiat Oneal Biol Phys 64, 1503- 1512 (2006); Jaggi, J. S., J Am Soc Nephrol 16, 2677- 2689 (2005); Jaggi, J. S., Kappel, B. J., Cancer Res 65, 4888-4895 (2005); Antczak, C., Bioconjug Chem 17, 1551-1560 (2006)). These include amino acid co-infusion to block reabsorption; angiotensin 1 blockade; chelation therapy to sequester225Ac daughter products; and diuresis using spironolactone, furosemide or chlorothiazide. If we observe significantly more radioactivity in the kidneys of mice injected with225Ac-PCTA-VHH, we will explore the use of these strategies to reduce radiation damage (Song, H, 2009). Finally, the research team has published experience with performing autoradiography and sequential histological studies on tissue (Kunikowska, J., 2022). Thus, we anticipate no problems in performing this task or using the accumulated data to determine dosimetry. We will evaluate tissue dosimetry using a multi-compartment model of radioactivity distribution and the MIRD #21 schema. However, if the team encounters difficulties determining tissue dosimetry using this method it will reevaluate tissue dosimetry using micro-dosimetry methods that have been published previously (Hobbs, R. F., Phys Med Biol 57, 4403-4424 (2012); Hobbs, R. F., Med Phys 36, 904-907 (2009); Hobbs, R. F., Med Phys 38, 2892-2903 (2011), Hobbs, R. F., Radiat Res 10.1667 / RR1343.1 (2013). Hobbs, R. F., Radiat Res 181, 90-98 (2014); Baechler, S., Med Phys 39, 6118-6128 (2012); Keane, F. M., FEBS Open Bio 4, 43-54 (2013)).- 61 -061685.109PCT

[0242] Figure 16 describes the binding of 128 with FAP+ U87mg cells (Fig. 16D and 16F) using flow cytometry. Figures 16A-C and 16E are negative controls that demonstrate the specificity of 128 for FAP.

[0243] Figure 17 A describes the flow cytometry peak shift describing the binding of 128 to FAP+ cells. Figure 17B describes the quantified results of % labeled cells from flow cytometry experiments. 128 and 13 bind to cells whereas all other agents (non-stained, buffer and 13 -negative control) do not bind to FAP+ cells.

[0244] Figure 18 describes the binding of a dimeric form of 128 with FAP+ U87mg cells. Similar to 128 (Fig. 18F), the 128 dimer (Fig. 18E.) binds to FAP+ cells in contrast to unstained cells and buffer controls (Fig 18A-B). Fig. 18G quantifies the percentage of labeled cells by 128 and 128 dimeric species.Example 7: Alpha Particle Radiation Delivered Systemically with anti -FAP VHH Proteins.

[0245] A conjugation scheme of MBP-I-3 with PCTA-Bn-NCS is shown in FIG. 19A. The conjugation of MBP-I-3 with PCTA-BN-NCS occurred using standard NCS coupling chemistry. The VHH-conjugate was obtained in high yield and purity. A radiolabeling scheme of PCTA-MBP-I-3 with zirconium-89 or actinium-225 is shown in FIG. 19B. Either reaction can be achieved under mild conditions. Radiochemical purity and specific activity are excellent and consistent with literature. Zirconium-89 is a diagnostic PET radionuclide. Using it demonstrates that the PCTA-MBP-I-3 can be radiolabeled wit PET or SPECT radioisotopes for clinical imaging. Actinium-225 is an alpha particle emitting radionuclide. Using it demonstrates that PCTA-MBP-I-3 can be radiolabeled with alpha particle, beta minus or auger emitting radionuclides that are used in radiotherapy applications.89

[0246] Quality control was performed by radio-TLC: ITLC-SG of ZrCl4(FIG. 8920A), and Zr-PCTA-MBP-I-3 (FIG. 20B). In this analysis system, un-complexed Zr- 89 will move to the solvent front, while any Zr-89 that is bound to the PCTA-MBP-I-3 will remain at the origin. Based upon this data, the radiochemical purity of89Zr-PCTA- MBP-I-3 is greater than 99%.

[0247] Figures 20C-20D show quality control by radio-HPLC: UV-HPLC chromatogram (220 nm) of nonradioactive PCTA-MBP-I-3 (FIG. 20C) compared with radio-HPLC chromatogram of89Zr-PCTA-MBP-I-3 (FIG. 20D). Based upon UV- 62 -061685.109PCTanalysis at 220 nm, PCTA-MBP-I-3 has a retention time of 20.2 min (FIG. 20C). Based upon radio-HPLC analysis,89Zr-PCTA-MBP-I-3 has a similar retention time (FIG. 20D). In this analysis system, un-complexed Zr-89 will elute at a time point that is greater than 30 minutes. Based upon this data, the radiochemical purity of89Zr-PCTA- MBP-I-3 is greater than 99% and corroborates the radio-TLC data.

[0248] Table 3 is a summary of the data from FIG. 20A-20D. Based upon radio- TLC and radio-HPLC analysis, the radiochemical yield, radiochemical purity and specific activity are excellent and in agreement with published literature.

[0249] Table 3: Summary of radiochemical data derived for89Zr-PCTA-MBP-I- 3.Example 8: In vitro serum stability studies of89Zr-PCTA-MBP-I-3.

[0250] In vitro serum stability was carried out by adding 50 pL of89Zr-PCTA- MBP-3 (105 pCi) to 450 pL of human serum (Table 4). The solutions were incubated at 37 °C for 7 days and were analysed at suitable time points by Radio-TLC and Radio- HPLC. The radio-TLC and radio-HPLC data are in good agreement. Based upon this analysis the radiopharmaceutical remains greater that 95% stable in human serum after 7 days. This suggests that the radiopharmaceutical will remain stable upon in vivo injection.

[0251] Table 4: In vitro serum stability study of89Zr-PCTA-MBP-I-3.- 63 -061685.109PCT

[0252] In vitro serum stability was carried out by adding 50 pL of89Zr-PCTA- MBP-3 (105 pCi) to 450 pL of human serum. The solutions were incubated at 37 °C for 7 days and were analysed at suitable time points by radio-TLC. In this analysis system, any un-complexed Zr-89 will migrate to the solvent front. FIG. 21 A was performed at time = 0, the radiopharmaceutical is greater than 99% intact. FIG. 2 IB was performed at time = 7 days, approximately 4.5 % Zr-89 is observed at the solvent front.

[0253] In vitro serum stability was carried out by adding 50 pL of89Zr-PCTA- MBP-3 (105 pCi) to 450 pL of human serum. The solutions were incubated at 37 °C for 7 days and were analysed at suitable time points by radio-HPLC. In this analysis system, any un-complexed Zr-89 will elute after the radiopharmaceutical. FIG. 21C was performed at time = 0, the radiopharmaceutical is greater than 99% intact. FIG. 21D was performed at time = 7 days, approximately 4.8 % Zr-89 is observed in the reaction mixture.Example 9: Quality control of225Ac-PCTA-MBP-I-3

[0254] Figures 22A-22B show Radio-TLC of225Ac-PCTA-MBP-I-3. In this analysis system, un-complexed Ac-225 will move to the solvent front, while any Ac- 225 that is bound to the PCTA-MBP-I-3 will remain at the origin. Based upon this data, the radiochemical purity of225Ac-PCTA-MBP-I-3 is greater than 99%.

[0255] Figures 22C-22D show Radio-HPLC of225Ac-PCTA-MBP-I-3. Based upon UV analysis at 220 nm, PCTA-MBP-I-3 has a retention time of 20.2 min (FIG. 22C). Based upon radio-HPLC analysis,225Ac-PCTA-MBP-I-3 has a similar retention time (FIG. 22D). In this analysis system, un-complexed Ac-225 will elute at a time point greater than 30 minutes. Based upon this data, the radiochemical purity of225Ac- PCTA-MBP-I-3 is greater than 99% and corroborates the radio-TLC data.

[0256] Table 5 is a summary of radiochemical data derived for225Ac-PCTA-MBP- I-3 (FIG. 22A-22D). Based upon radio-TLC and radio-HPLC analysis, the- 64 -061685.109PCTradiochemical yield, radiochemical purity and specific activity are excellent and in agreement with published literature.

[0257] Table 5: Summary of radiochemical data derived for225Ac-PCTA-MBP-I-3.

[0258] In vitro serum stability was carried out by adding 50 pL of225Ac-PCTA- MBP-I-3 (65 pCi) to 450 pL of human serum (Table 6). The solutions were incubated at 37 °C for 7 days and were analysed at suitable time points by radio-TLC and radio- HPLC. The radio-TLC and radio-HPLC data are in good agreement. Based upon this analysis the radiopharmaceutical remains greater that 95% stable in human serum after 7 days. This suggests that the radiopharmaceutical will remain stable upon in vivo injection.

[0259] Table 6: In vitro serum stability analysis of225Ac-PCTA-MBP-I-3.Example 10: In vitro serum stability studies of225Ac-PCTA-MBP-I-3.

[0260] Figures 23 A-23B show Radio-TLC analysis of225Ac-PCTA-MBP-I-3 in serum. In this analysis system, un-complexed Ac-225 will move to the solvent front,- 65 -061685.109PCTwhile any Ac-225 that is bound to the PCTA-MBP-I-3 will remain at the origin. Based upon this data, the radiochemical purity of225Ac-PCTA-MBP-I-3 is greater than 95%.

[0261] The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.- 66 -061685.109PCT

Claims

What is claimed is:

1. An anti-fibroblast activation protein alpha (FAP) polypeptide comprising at least one single-domain antibody directed against FAP wherein the at least one singledomain antibody comprises at least 95% sequence identity to nucleic acid sequences of SEQ ID NO: 1, 6, 11, or the combination thereof or at least 95% sequence identity to amino acid sequences of SEQ ID NO: 2, 7, 12, or the combination thereof.

2. The anti -FAP polypeptide of claim 1, wherein the single-domain antibody comprises: a) a variable heavy domain of heavy chain (VHH) sequence having at least 95% sequence identity to amino acid sequences of SEQ ID NO: 2, and wherein the VHH comprises a complementarity determining regions (CDR), CDR1 having at least 95% sequence identity to amino acid sequences of SEQ ID NO: 3, CDR2 having at least 95% sequence identity to amino acid sequences of SEQ ID NO: 4, and CDR3 having at least 95% sequence identity to amino acid sequences of SEQ ID NO: 5; b) a variable heavy domain of heavy chain (VHH) sequence having at least 95% sequence identity to amino acid sequences of SEQ ID NO: 7, and wherein the VHH comprises a complementarity determining regions (CDR), CDR1 having at least 95% sequence identity to amino acid sequences of SEQ ID NO: 8, CDR2 having at least 95% sequence identity to amino acid sequences of SEQ ID NO: 9, and CDR3 having at least 95% sequence identity to amino acid sequences of SEQ ID NO: 10; or c) a variable heavy domain of heavy chain (VHH) sequence having at least 95% sequence identity to amino acid sequences of SEQ ID NO: 12, and wherein the VHH comprises a complementarity determining regions (CDR), CDR1 having at least 95% sequence identity to amino acid sequences of SEQ ID NO: 3, CDR2 having at least 95% sequence identity to amino acid sequences of SEQ ID NO: 4, and CDR3 having at least 95% sequence identity to amino acid sequences of SEQ ID NO: 13.- 67 -061685.109PCT3. The anti-FAP polypeptide of claim 2, wherein the VHH sequence comprising at least 95% sequence identity to amino acid sequences of SEQ ID NO: 2 binds to the to the extracellular surface of FAP by making important contacts between a CDR3 loop of VHH’ s CDR3 and a unique FAP epitope, that is distinct from a FAP active site, wherein amino acids VI 07 and SI 09 of the VHH’s CDR3 are mutated to computationally increase the affinity and stability of the single-domain antibody.

4. The anti-FAP polypeptide of claim 3, wherein amino acid S109 is mutated to S109R to form 2 new hydrogen bonds and a salt bridge, which positively increases affinity and stability.

5. The anti-FAP polypeptide of claim 3, wherein the unique FAP epitope is FAP’s FR2 region.

6. The anti-FAP polypeptide of claim 3, wherein the CDR3 loop interacts with the extracellular surface of FAP at FAP Y274, wherein FAP has multiple FAP loops and FAP Y274 has multiple interactions with the CDR3 loop and sits within a pocket formed by the CDR3 loop, and wherein the CDR3 loop interacts with one FAP loop at a relatively uncharged region and a second FAP loop at a positively charged region.

7. The anti-FAP polypeptide of claim 6, wherein FAP and CDR3 loop multiple interactions have residue pairs having hydrogen bond interactions comprising Y274:P108, Y274:W47, E325:S109, and D326:V107.

8. The anti-FAP polypeptide of claim 6, wherein there is a p-stacking interaction between Y27EF110.

9. The anti-FAP polypeptide of claim 1, wherein the number of single-domain antibodies directed against FAP is at least two, wherein said at least one single-domain antibody is a homologous sequence, a functional portion, or a functional portion of a homologous sequence of the full-length single-domain antibody..- 68 -061685.109PCT10. The anti -FAP polypeptide of claim 1, wherein the at least one single-domain antibody is a Camelidae VHH or a humanized Camelidae VHH.

11. The anti-FAP polypeptide of claim 1, wherein the anti-FAP polypeptide is a homologous sequence, a functional portion, or a functional portion of a homologous sequence of the full length anti-FAP polypeptide.

12. A pharmaceutical composition comprising the anti-FAP single-domain antibody of claim 1 and a radioisotope, wherein the radioisotope is conjugated to the anti-FAP single-domain antibody at a ratio that maximizes specific activity but does not compromise affinity, wherein the composition is delivered to a tumor of a subject in need thereof, and wherein the composition is a theranostic agent used to treat or diagnose a disease characterized by overexpression of fibroblast activation protein (FAP).

13. The pharmaceutical composition of claim 12, wherein the radioisotope is selected from a group of radionuclides consisting of227Th, "mc,67Ga,68Ga,66Ga,47Sc,51Cr,167Tm,141Ce,n iIn,123I,124I,125I,131I,18F,nC,15N,168Yb,175Yb,140La,90Y,88Y,86Y,153Sm,166HO,165Dy,166Dy,62Cu,64Cu,67Cu,60Cu,61Cu,211At,97Ru,103Ru,186Re,188Re,203Pb,211Bi,212Bi,213Bi,207Bi,76Br,89Zr,225Ac,105Rh,109Pd,117mSn,149Pm,161Tb,177LU,198AU,199AU,212Pb,134Ce,133Ce,133La,134La,149Tb,152Tb,155Tb,94TC, "TC,43SC,44SC,117Sn,52Mn,53Mn,54Mn,55Mn,13N,150,223Fr,227Th,229Th,228Th,226Ra,224Ra,219Rn,215Po,212Po,216Po,206Po,211Pb,2O7T1,2O8T1,211Po,107Ag,109Ag,195mPt,103Pd,223Ra,82Rb, 89Sr,85Sr,90Sr,45Ti,44Ti,73As,119Sb,55Fe,59Fe,22Na,48V,63Ni,65Zn,109Cd,153Gd,148Gd,194Hg,26Al,32Si,68Ge,73As,77Br, and147Pm.

14. The pharmaceutical composition of claim 12, wherein the anti-FAP singledomain antibody is conjugated to225Ac or89Zr, and a chelating group, and wherein Zr- 89 and Ac-225 can be used as a theranostic pair or alone to diagnose and treat the disease characterized by overexpression of fibroblast activation protein (FAP).- 69 -061685.109PCT15. A method of treating a disease characterized by overexpression of fibroblast activation protein (FAP) in a subject in need thereof, comprising administering the pharmaceutical composition of claim 12 to the subject.

16. The method of claim 15, wherein the disease characterized by overexpression of fibroblast activation protein (FAP) is selected from the group consisting of cancer, chronic inflammation, atherosclerosis, fibrosis, tissue remodeling or keloid disorder.

17. The method of claim 16, wherein the cancer is a solid tumor selected from the group consisting of glioblastoma multiforme (GBM), breast cancer, pancreatic cancer, small intestine cancer, colon cancer, rectal cancer, lung cancer, head and neck cancer, ovarian cancer, hepatocellular carcinoma, esophageal cancer, hypopharynx cancer, nasopharynx cancer, larynx cancer, myeloma cells, bladder cancer, cholangiocellular carcinoma, clear cell renal carcinoma, mesothelioma, neuroendocrine tumor, oncogenic osteomalacia, sarcoma, and carcinoma of unknown primary (CUP).

18. The method of claim 15, wherein the radioisotope is conjugated to the anti -FAP single-domain antibody to create a novel targeted alpha particle therapy (TAT) that selectively delivers alpha particle radiation to the tumors.

19. The method of claim 15, wherein the pharmaceutical composition targets a FAP biomarker found in tumors to selectively deliver alpha particle radiation to the tumors.

20. The method of claim 15, wherein the composition is administered intravenously.- 70 -061685.109PCT

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