WO2026006639A1 - Antigen binding molecules targeting transferrin receptor 1 - Google Patents

Abstract

The disclosure provides, in various embodiments, antibodies and antigen binding fragments thereof, and polypeptides that bind transferrin receptor 1 (TFR1, e.g., human TFR1). The disclosure also provides, in various embodiments, polynucleotides encoding antibodies and antigen binding fragments thereof, and polypeptides; vectors and host cells suitable for expressing the antibodies and antigen binding fragments thereof, and polypeptides; and methods for treating diseases or conditions (e.g., TNF-α-associated diseases and/or CNS associated diseases such as neurodegenerative conditions). An antibody, an antigen binding fragment, or a polypeptide disclosed herein may be single-specific, bi-specific, or multi-specific (e.g., targeting multiple drug targets).

Description

5431.1032002 ANTIGEN BINDING MOLECULES TARGETING TRANSFERRIN RECEPTOR 1 RELATED APPLICATIONS _{[0001] This application claims the benefit of U.S. Provisional Application No. 63/664,307,} filed on June 26, 2024 and U.S. Provisional Application No.63/737,271, filed on December 20, 2024. The entire teachings of the above applications are incorporated herein by reference. INCORPORATION BY REFERENCE OF MATERIAL IN XML _{[0002] This application incorporates by reference the Sequence Listing contained in the} following eXtensible Markup Language (XML) file being submitted concurrently herewith: a_{) File name: 54311032002.xml; created June 23, 2025 Bytes 217,754 in size.} GOVERNMENT SUPPORT _{[0003] This invention was made with government support under Grant numbers} R01AG073182 and R41AG080864 from the National Institute on Aging. The government has certain rights in the invention. BACKGROUND _{[0004] Approximately 6 million Americans have Alzheimer’s disease (AD). Without} disease-modifying drugs, this number will triple by 2050. Most AD cases are sporadic with late onset (>65 years old, late onset Alzheimer’s disease or LOAD), and approximately 2% are caused by inherited dominant mutations (onset <65 years old, familial Alzheimer’s disease or FAD). Toxic forms of β-amyloid (Aβ) and tau are believed to cause AD. However, therapies targeting Aβ and/or tau have shown negligible benefits on cognitive decline in patients. There is a critical need to develop therapeutic agents that treat Alzheimer’s disease (AD) by modulating targets other than toxic forms of β-amyloid (Aβ) and tau. Tumor necrosis factor-alpha (TNF-α) plays an important role in late onset Alzheimer’s disease (LOAD). However, use of biologic TNF-α inhibitors in AD therapy is hindered by their limited blood-brain barrier (BBB) penetration. SUMMARY _{[0005] There is a critical need to develop agents that can be used for brain delivery of} biologics such as TNF-α inhibitors. _{[0006] Transferrin receptor 1 (TFR1), also known as TFRC or CD71, is highly expressed on} endothelial cells forming the blood-brain-barrier (BBB), and its transcytosis activity can be - 1 - 4181706.v1 5431.1032002 harnessed to facilitate the delivery of molecules to the central nervous system (CNS). TFR1 exists as a homo-dimeric transmembrane protein, with each monomer capable of binding one molecule of transferrin (TF) to form an iron-TF-TFR1 complex that undergoes endocytosis for cellular uptake, crucial for iron import. Importantly, monovalent interactions with TFR1 favor transcytosis over bivalent interactions, suggesting that TFR1-sdAbs may enhance transcytosis more efficiently than α-TFR1 monoclonal antibodies (MAbs). _{[0007] Provided herein are anti-transferrin receptor 1 (TFR1) antibodies or antigen-binding} fragments thereof, comprising an immunoglobulin heavy chain variable (V_H) domain comprising a heavy chain complementarity-determining region (HCDR) 1, an HCDR2, and an HCDR3 having 100% sequence identity to an HCDR1, an HCDR2, and an HCDR3, respectively, of a V_H domain comprising the amino acid sequence of any one of SEQ ID NOs:190, 176, 99-123, 177- 189, and 191-199. _{[0008] In some embodiments, HCDR1, HCDR2, and HCDR3 of an anti-TFR1 antibody or} antigen-binding fragment thereof are determined by Honneger’s, Kabat, IMGT, or Chothia numbering scheme. _{[0009] In some embodiments: a) an HCDR1 comprises the amino acid sequence of SEQ ID NO:163, an HCDR2} comprises the amino acid sequence of SEQ ID NO:168, and an HCDR3 comprises the amino acid sequence of SEQ ID NO:172; b_{) an HCDR1 comprises the amino acid sequence of SEQ ID NO:161, an HCDR2} comprises the amino acid sequence of SEQ ID NO:166, and an HCDR3 comprises the amino acid sequence of SEQ ID NO:170; c_{) an HCDR1 comprises the amino acid sequence of SEQ ID NO:2, an HCDR2} comprises the amino acid sequence of SEQ ID NO:14, and an HCDR3 comprises the amino acid sequence of SEQ ID NO:26; d_{) an HCDR1 comprises the amino acid sequence of SEQ ID NO:40, an HCDR2} comprises the amino acid sequence of SEQ ID NO:48, and an HCDR3 comprises the amino acid sequence of SEQ ID NO:26; e_{) an HCDR1 comprises the amino acid sequence of SEQ ID NO:62, an HCDR2} comprises the amino acid sequence of SEQ ID NO:74, and an HCDR3 comprises the amino acid sequence of SEQ ID NO:86; - 2 - 4181706.v1 5431.1032002 f_{) an HCDR1 comprises the amino acid sequence of SEQ ID NO:5, an HCDR2} comprises the amino acid sequence of SEQ ID NO:16, and an HCDR3 comprises the amino acid sequence of SEQ ID NO:28; g_{) an HCDR1 comprises the amino acid sequence of SEQ ID NO:41, an HCDR2} comprises the amino acid sequence of SEQ ID NO:51, and an HCDR3 comprises the amino acid sequence of SEQ ID NO:28; or h_{) an HCDR1 comprises the amino acid sequence of SEQ ID NO:65, an HCDR2} comprises the amino acid sequence of SEQ ID NO:76, and an HCDR3 comprises the amino acid sequence of SEQ ID NO:88. _{[0010] In some embodiments: a) an HCDR1 consists of the amino acid sequence of SEQ ID NO:163, an HCDR2} consists of the amino acid sequence of SEQ ID NO:168, and an HCDR3 consists of the amino acid sequence of SEQ ID NO:172; b_{) an HCDR1 consists of the amino acid sequence of SEQ ID NO:161, an HCDR2} consists of the amino acid sequence of SEQ ID NO:166, and an HCDR3 consists of the amino acid sequence of SEQ ID NO:170; c_{) an HCDR1 consists of the amino acid sequence of SEQ ID NO:2, an HCDR2} consists of the amino acid sequence of SEQ ID NO:14, and an HCDR3 consists of the amino acid sequence of SEQ ID NO:26; d_{) an HCDR1 consists of the amino acid sequence of SEQ ID NO:40, an HCDR2} consists of the amino acid sequence of SEQ ID NO:48, and an HCDR3 consists of the amino acid sequence of SEQ ID NO:26; e_{) an HCDR1 consists of the amino acid sequence of SEQ ID NO:62, an HCDR2} consists of the amino acid sequence of SEQ ID NO:74, and an HCDR3 consists of the amino acid sequence of SEQ ID NO:86; f_{) an HCDR1 consists of the amino acid sequence of SEQ ID NO:5, an HCDR2} consists of the amino acid sequence of SEQ ID NO:16, and an HCDR3 consists of the amino acid sequence of SEQ ID NO:28; g_{) an HCDR1 consists of the amino acid sequence of SEQ ID NO:41, an HCDR2} consists of the amino acid sequence of SEQ ID NO:51, and an HCDR3 consists of the amino acid sequence of SEQ ID NO:28; or - 3 - 4181706.v1 5431.1032002 h_{) an HCDR1 consists of the amino acid sequence of SEQ ID NO:65, an HCDR2} consists of the amino acid sequence of SEQ ID NO:76, and an HCDR3 consists of the amino acid sequence of SEQ ID NO:88. _{[0011] In some embodiments, an VH of an anti-TFR1 antibody or antigen-binding fragment} thereof is humanized, contains human framework regions, or both. _{[0012] In some embodiments, an VH domain of an anti-TFR1 antibody or antigen-binding} fragment thereof comprises an amino acid sequence that has at least 70% sequence identity to the amino acid sequence of at least one of SEQ ID NOs:190, 176, 99-123, 177-189, and 191-199. _{[0013] In some embodiments, an VH domain comprises an amino acid sequence that has} 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs:190, 176, 99- 123, 177-189, and 191-199. _{[0014] In some embodiments, an VH domain comprises an amino acid sequence that has at} least 70% sequence identity to the amino acid sequence of SEQ ID NO:190 or SEQ ID NO:176. _{[0015] In some embodiments, an VH domain comprises an amino acid sequence that has} 100% sequence identity to the amino acid sequence of SEQ ID NO:190 or SEQ ID NO:176. _{[0016] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof is} a single-domain antibody (sdAb). _{[0017] Also provided herein are polypeptides comprising an anti-TFR1 antibody or antigen-} binding fragment thereof disclosed herein. _{[0018] In some embodiments, a polypeptide further comprises a therapeutic agent, a} diagnostic agent, or a combination thereof. _{[0019] In some embodiments, a polypeptide further comprises a therapeutic agent. In some} embodiments, a therapeutic agent is an antibody or antigen-binding fragment thereof. In some embodiments, a therapeutic agent is an anti-tumor necrosis factor-alpha (TNF-α) antibody or antigen-binding fragment thereof. _{[0020] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} comprises an immunoglobulin V_H domain comprising a HCDR1, an HCDR2, and an HCDR3 having 100% sequence identity to an HCDR1, an HCDR2, and an HCDR3, respectively, of a V_H domain comprising the amino acid sequence of any one of SEQ ID NOs:225, 223, 224, 226-228, and 132-137. _{[0021] In some embodiments, HCDR1, HCDR2, and HCDR3 of an anti-TNF-α antibody or} antigen-binding fragment thereof are determined by Kabat, IMGT, or Chothia numbering scheme. - 4 - 4181706.v1 5431.1032002 _{[0022] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} comprises a V_H domain comprising: a_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:229, an HCDR2} comprising the amino acid sequence of SEQ ID NO:230, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:231; b_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:138, an HCDR2} comprising the amino acid sequence of SEQ ID NO:139, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:140; c_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:138, an HCDR2} comprising the amino acid sequence of SEQ ID NO:232, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:140; d_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:141, an HCDR2} comprising the amino acid sequence of SEQ ID NO:142, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:143; e_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:144, an HCDR2} comprising the amino acid sequence of SEQ ID NO:145, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:140; or f_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:147, an HCDR2} comprising the amino acid sequence of SEQ ID NO:148, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:149. _{[0023] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} comprises a V_H domain comprising: a_{) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:138, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:139, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:140; b_{) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:141, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:142, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:143; c_{) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:144, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:145, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:140; or - 5 - 4181706.v1 5431.1032002 d_{) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:147, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:148, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:149. _{[0024] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof is} a sdAb. _{[0025] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} comprises a V_H domain comprising an amino acid sequence that has at least 70% sequence identity to the amino acid sequence of at least one of SEQ ID NOs:225, 223, 224, 226-228, and 132-137. _{[0026] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} comprises a V_H domain comprising an amino acid sequence that has 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs:225, 223, 224, 226-228, and 132-137. _{[0027] In some embodiments, a polypeptide comprises: a) one anti-TFR1 sdAb and two anti-TNF-α sdAbs; or b) one anti-TFR1 sdAb and one anti-TNF-α sdAb. [0028] In some embodiments, a polypeptide comprises an amino acid sequence that has at} least 70% sequence identity to the amino acid sequence of at least one of SEQ ID NOs:201, 200, 202-215, 124, and 125. In some embodiments, a polypeptide comprises an amino acid sequence that has at least 70% sequence identity to the amino acid sequence of at least one of SEQ ID NOs:200, 201, 208, and 209. _{[0029] In some embodiments, a polypeptide comprises an amino acid sequence that has} 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs:201, 200, 202- 215, 124, and 125. In some embodiments, a polypeptide comprises an amino acid sequence that has 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs:200, 201, 208, and 209. _{[0030] In some embodiments, a polypeptide further comprises a protein tag. [0031] Also provided herein are polynucleotides encoding an anti-TFR1 antibody or antigen} binding fragment thereof, or a polypeptides disclosed herein. _{[0032] Also provided herein are expression vectors comprising a polynucleotide disclosed} herein. _{[0033] Also provided herein are host cells comprising a polynucleotide or an expression} vector disclosed herein. - 6 - 4181706.v1 5431.1032002 _{[0034] Also provided herein are methods of producing an anti-TFR1 antibody or antigen} binding fragment thereof disclosed herein, comprising expressing the anti-TFR1 antibody or antigen binding fragment thereof, in a host cell disclosed herein and isolating the expressed anti- TFR1 antibody or antigen binding fragment thereof. _{[0035] Also provided herein are methods of producing a polypeptide disclosed herein,} comprising expressing the polypeptide in a host cell disclosed herein and isolating the expressed polypeptide. _{[0036] Also provided herein are compositions comprising an anti-TFR1 antibody or antigen} binding fragment thereof, a polypeptide, a polynucleotide, an expression vector, or a host cell disclosed herein, and a carrier or diluent. In some embodiments, a composition (e.g., a pharmaceutical composition) comprises a pharmaceutically acceptable carrier or diluent. _{[0037] In some embodiments, a composition further comprises at least one additional} therapeutic agent, optionally, the at least one additional therapeutic agent comprises: a_{) an anti-tau antibody, b) an anti-amyloid-beta (Aβ) antibody, c) a BRI2 peptide, d) an amyloid precursor protein (APP)-targeting agent, or} any combination of the foregoing. _{[0038] Also provided herein are kits comprising a composition or a pharmaceutical} composition disclosed herein. _{[0039] Also provided herein are methods of treating a disease in a subject in need thereof,} comprising administering to the subject a therapeutically effective amount of a polypeptide, a polynucleotide, an expression vector, a host cell, or a composition (e.g., a pharmaceutical composition) disclosed herein, thereby treating the disease. _{[0040] Also provided herein are methods of blocking binding of TNF-α to TNFR in a} subject, the methods comprising administering to the subject an effective amount of a polypeptide, a polynucleotide, an expression vector, a host cell, or a composition (e.g., a pharmaceutical composition) disclosed herein, thereby blocking binding of TNF-α to TNFR expressed on the surface of the cell. _{[0041] Also provided herein are methods of treating a TNF-α-associated disease in a subject} in need thereof, comprising administering to the subject an effective amount of a polypeptide, a polynucleotide, an expression vector, a host cell, or a composition (e.g., a pharmaceutical composition) disclosed herein, thereby treating the TNF-α-associated disease. - 7 - 4181706.v1 5431.1032002 _{[0042] In some embodiments, a TNF-α-associated disease is Alzheimer’s disease (AD). In} some embodiments, a TNF-α-associated disease is late onset Alzheimer’s disease (LOAD). In some embodiments, a TNF-α-associated disease is traumatic brain injury (TBI). _{[0043] In some embodiments, a subject is an adult human patient. [0044] In some embodiments, a subject is a pediatric human patient. [0045] In some embodiments, a method further comprises administering to a subject at least} one additional therapeutic agent, optionally wherein the at least one additional therapeutic agent comprises a_{) an anti-serum albumin agent, b) a fusion protein comprising human serum albumin, c) a chemotherapeutic agent, d) an immunosuppressant agent, or} any combination of the foregoing. _{[0046] In some embodiments, a method further comprises administering to a subject} methotrexate. _{[0047] Also provided herein are methods of blocking binding of TNF-α to TNF receptor} (TNFR) expressed on a surface of a cell, the methods comprising contacting the cell with an effective amount of a polypeptide, a polynucleotide, an expression vector, a host cell, or a composition (e.g., a pharmaceutical composition) disclosed herein, thereby blocking binding of TNF-α to TNFR expressed on the surface of the cell. BRIEF DESCRIPTION OF THE DRAWINGS _{[0048] The patent or application file contains at least one drawing executed in color. Copies} of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee. _{[0049] The foregoing will be apparent from the following more particular description of} example embodiments, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments. _{[0050] FIGs. 1A-1B. Camelid single-domain antibodies (sdAbs) targeting human Transferrin} receptor 1 (hTFR1), hTFR1‐sdAbs, bind hTFR1 in a native membrane-displayed conformation. FIG.1A. HEK293 cells transfected with a vector expressing EGFP plus either human TFR1, mouse Tfr1, rat Tfr1, or R. macaque TFR1 were incubated with 400 nM TFR1-sdAb, followed by an anti-His-APC antibody. Twenty-five hTFR1-sdAbs bound hTFR1 (O4B05R3 is shown), - 8 - 4181706.v1 5431.1032002 13 bound R. macaque TFR1, and none bound mouse Tfr1 or rat Tfr1. FIG.1B. Staining of endogenous hTFR1-expressing HL-60 cells.03A01R3 (an sdAb that does not bind hTFR1) + Anti-His-APC staining overlaps with Anti-His-APC alone staining.04B05R3 (an hTFR1‐sdAb) + Anti-His-APC produces a large right-shift of the staining curve indicating binding. _{[0051] FIGs. 2A-2B. Proteins purification and analysis. FIG. 2A. SDS-PAGE Coomassie} staining analysis of 02B02R3. M=MWM; 1: purified 02B02R3 under reducing conditions; 2: purified 02B02R3 under non-reducing conditions. FIG.2B. SEC-HPLC analysis of purified 02B02R3. _{[0052] FIGs. 3A-3I. hTFR1-sdAbs produced by mammalian cells bind to hTFR1 part 1.} HEK293 cells were transfected with a vector expressing hTFR1 (alongside EGFP). Subsequently, cells were treated with each hTFR1-sdAb at a concentration of 400 nanomolar, followed by incubation with an anti-His-APC antibody (R&D Systems, Cat. # IC050A). _{[0053] FIGs. 4A-4J. hTFR1-sdAbs produced by mammalian cells bind to hTFR1 part 2.} FIG.4A shows the specific binding of human Transferrin-FITC (at a concentration of 2.5 nanomolar) to CHEK-ATP089 cells, with no binding observed on HEK293 cells. FIGs.4B1-4J depict the binding of each hTFR1-sdAb at a concentration of 40 nanomolar to CHEK-ATP089 cells. _{[0054] FIGs. 5A-5B. Selection of hTFR1-sdAbs that do not interfere with TF-TFR1} interaction. The top-left panel of FIG.5A demonstrates the dose-dependent competition of unlabeled Transferrin for binding to hTFR1 on the cell surface of CHEK-ATP089 cells in the presence of FITC-Transferrin. The remaining panels of FIGs.5A-5B assess the inhibitory activity of 14 hTFR1-sdAbs from Families A, B, D, G, and I. A sdAb from Family L (which lacks binding to TFR1 on CHEK-ATP089 cells) serves as a negative control. In these experiments, CHEK-ATP089 cells were incubated with the specified proteins for 1 hour on ice before FACS analysis. Human Transferrin-FITC and hTFR1-sdAbs were utilized at a concentration of 2.5 micromolar. _{[0055] FIGs. 6A-6B. Selection of hTFR1-sdAbs that do not interfere with TF uptake} interaction. The panel demonstrates the dose-dependent competition of unlabeled Transferrin pHrodo Red-TF uptake by CHEK-ATP089 cells. The right panel assess the inhibitory activity of representative hTFR1-sdAbs from Families A, B, D, G, and I. A nanobody from Family J (which lacks binding to hTFR1 on CHEK-ATP089 cells) serves as a negative control. _{[0056] FIGs. 7A-7B. Generation of humanized TF and TFR1 rats. FIG. 7A. The rat Tfr1} gene consists of 19 exons, with the ATG start codon located in exon 2 and the TAA stop codon - 9 - 4181706.v1 5431.1032002 in exon 19. In the knock-in (KI) model, the last 32 base pairs (3’) of rat Tfrc gene exon 2, counting from the ATG start codon, were replaced with the human TFR1 CDS (Transcript: 201- ENST00000360110), followed by the rat 3’ untranslated region (3’UTR) and the SV40 polyadenylation sequence. FIG.7B. The rat Tf gene (NM_001013110.1) is comprised of 17 exons, with the ATG start codon located in exon 1 and the TAA stop codon in exon 17. In the KI model, a portion of exon 2 coding sequence (140 base pairs) of the rat Tf gene was substituted with the Human TF CDS (amino acids 20-698, Transcript: 201-ENST00000402696), followed by the rat 3’UTR and the SV40 polyadenylation sequence. Importantly, the murine signal peptide (amino acids 1-19), encoded by exon 1 and the 5’ sequences of rat exon 2, was retained. Consequently, this modified allele results in a TF protein encompassing the rat leader sequence and the human TF protein. _{[0057] FIGs. 8A-8C. Expression pattern of human and rat TF and TFR1 in TF/TFR1} humanized KI rats. FIGs.8A, 8C. Western blots for TF, Tf, TFR1 and Tfr1 were performed utilizing the following antibodies: anti-Human-TF (Cell Signaling Technology, Cat. # 35293), anti-Human-TFR1 (Cell Signaling Technology, Cat. # 13113), anti-rat-Tf (Invitrogen, Cat. # MA529871), anti-rat-Tfr1 (Invitrogen, Cat. # MA170033). FIG.8B. ELISAs for sTFR1 were performed using Kit. #K151P9K-1 (MSD); ELISA for human-TF was performed using Kit ab187391 (ABCAM). _{[0058] FIG. 9. Human TFR1 forms homodimers and chimeric dimers with rat Tfr1. Brain} lysates from the indicated animals were Immunoprecipitated with anti-rat-Tfr1 (Invitrogen, Cat. # MA170033) and Protein-A/G Agarose beads. Western blots were performed using anti- Human-TFR1 (Cell Signaling Technology, Cat. # 13113). _{[0059] FIGs. 10A-10B. Production of TNFI-sdAbs in mammalian cells was performed as for} hTFR1-sdAb. The table shows quantity and purity of TNFI-sdAbs purified from 100 ml culture supernatants. SDS-PAGE Coomassie staining analysis of TNFI-sdAb1 is shown on the right (R = reducing; NR = non-reducing conditions). _{[0060] FIGs. 11A-11B. Production of TNFI-sdAb1-linker-hTFR1- sdAbs in mammalian} cells was performed as for hTFR1-sdAb. The shows quantity and purity of TNFI-sdAbs purified from 100 ml culture supernatants. SDS-PAGE Coomassie staining analysis of TNFI-sdAb1- linker-04B05R3 is shown on the right (R = reducing; NR = non-reducing conditions). _{[0061] FIG. 12. Analysis of brain fraction was performed by Western blot. ** indicates non-} specific bands. - 10 - 4181706.v1 5431.1032002 _{[0062] FIG. 13. Binding of Mutant TfRb-Nabs Produced by Mammalian Cells to Human} TFR1: Family A. HEK293 cells were transfected with a vector expressing human TFR1 alongside EGFP. The cells were then treated with each TfRb-Nab at a concentration of 400 nM, followed by incubation with an anti-His-APC antibody (R&D Systems, Cat. # IC050A). Secondary antibody staining alone (upper-left panel) is shown as a negative control. Binding of the wild-type TfRb-Nab from which the mutants originated is also shown for comparison. _{[0063] FIG. 14. Binding of Mutant TfRb-Nabs Produced by Mammalian Cells to Human} TFR1: Family B. _{[0064] FIG. 15. Binding of Mutant TfRb-Nabs Produced by Mammalian Cells to Human} TFR1: Family D. _{[0065] FIG. 16. Binding of Mutant TfRb-Nabs Produced by Mammalian Cells to Human} TFR1: Family G. _{[0066] FIG. 17. TfRb-Nab mutants with enhanced therapeutic potential determined by} AbNatiV analysis. Changes in Human-ness (H), VHH-ness (VH), and CamSol Intrinsic (S) are presented in the last three columns. _{[0067] FIGs. 18A-18B. Assessment of BBB permeability of optimized TfRb-Nabs. [0068] FIG. 19. The optimization strategy involved two pivotal steps, humanization and} solubility optimization. Humanization, facilitated by Machine Learning algorithm AbNatiV⁴, aimed to enhance the similarity of VHHs to human immune system sequences, minimizing immunogenicity while preserving activity. This process evaluated humanness and VHH- nativeness. A score above 0.8 indicates human-like characteristics and low/no immunogenicity. Solubility Optimization utilized CamSol, a validated sequence-based scoring method, to assess the solubility of nanobodies and the effects of mutations. Additionally, structural resolution of Nanobodies bound to their respective ligands guided optimization of the CDRs through structure-guided mutations. As an additional control step, a deep-learning model named ImmuneBuilder2 was employed to predict structures of wild-type and mutant sequences to predict mutations in frameworks that do not adversely affect binding. Evaluation of mutants optimized TNFI-Nabs led to the selection of two optimized TNFIs (TNFI-^ and TNFI-^) with high humanness and TNFI activity (H=Humanness, V=VH-ness, S=solubility). Following AI- guided optimization, hTFR1 binding assays and in vivo BBB permeability tests, four optimized TfRb-Nab (TfRb-A2, -D1, -D2 and -D3) with high humanness and BBB permeability were selected to produce INNs in combination with either TNFI-α and -β. All 8 INN dimers combinations exhibited TNFI IC₅₀ comparable to TNFI-α or TNFI-β. Notably, the two INN - 11 - 4181706.v1 5431.1032002 trimers INN^β-D3-β and INN^{^-D3-^} with the TfRb-D3 positioned between the two TNFI-Nabs, demonstrated ~10-fold greater TNFI activity than TNFI-α and -β, and ~3-fold better activity than trimers where the TfRb-Nab was positioned at the C-terminus of two TNFI-Nabs. _{[0069] FIGs. 20A-20D. INNβ-D3-β crosses the BBB via hTFR1. (A) INNβ-D3-β was detected} with b-TNF^-based ELISA. Briefly, INNs were captured on Streptavidin plates (MSD, L15SA) coated with 0.25 µg/ml of Biotinylated Trimeric Active hTNFα (b-TNFα). Nabs bound to b- TNFα were detected using anti-VHH goat antibody, followed by anti-Goat SULFO-TAG labeled antibody (MSD) and read using a MESO QuickPlex SQ. INN^β-D3-β in all tissues analyzed. Brain localization -CSF, homogenates (Homo.), parenchymal cells (P.C.) and Vessels- of INN^β-D3-β was hTFR1-dependent (hTFR1). Vasculature and parenchymal components were separated following a fractionation protocol used by Denali’s to evaluate BBB permeability of AVI-based therapeutics¹. S1 and S2 were brain soluble fractions that were discarded during this fractionation protocol. The CSF/Serum ratio varied from 0.123 to 0.26 with an average of 0.17. TfRb-Nabs were consistently present at higher levels in the serum of hTFR1 rats, likely because binding to hTFR1 prolongs their half-life. (B) INN^β-D3-β (green), hTFR1 (red) and CD31 (purple) were stained with anti-His tag, anti-human TFR1 anti-CD31 antibodies, respectively. INN^β-D3-β and hTFR1 were only detected in Tfr1^h/h and not Tfr1^w/w rats. White arrows highlight colocalization (white) in the overlay panel. (C-D) Brain slices were stained with anti-His tag and either the microglia marker anti-IBA1 (red, C) or the astrocytes marker anti-GFAP (red, D). Microglia and astrocytes showed extensive colocalization with INN^β-D3-β (green). White arrows denote colocalization (yellow spots) in the overlay images. _{[0070] FIG. 21. INNβ-D3-β/INNβ-A2 showed no hematotoxicity in rats humanized for both} TFR1 and TF. _{[0071] FIG. 22. INNs crossed the BBB via hTFR1 after SQ injection. [0072] FIG. 23. Heterotrimers are the lead therapeutic candidates. INN trimers, INNβ-D3-β and} INN^{^-D3-^}, with the TfRb-D3 positioned between the two TNFI-Nabs, demonstrated approximately 10-fold greater TNFI activity than TNFI-α or -β alone and ~3-fold higher activity compared to trimers where the TfRb-Nab was positioned at the C-terminus of the two TNFI- Nabs (see FIG.24). Based on these findings, additional trimers, including INN^α-D2-α, INN^β-D2-β, INN^α-D1-α, INN^β-D1-β, INN^α-A2-α, were synthesized. The IC₅₀ values for the newly synthesized INNs are displayed below the INN diagrams. _{[0073] FIGs. 24A-24B. Protein purification and analysis. (A) SDS-PAGE analysis of the} eight heterotrimers stained with Coomassie Brilliant Blue. M: Molecular weight marker; R: - 12 - 4181706.v1 5431.1032002 Purified proteins under reducing conditions; NR: Purified proteins under non-reducing conditions. (B) Size-Exclusion Chromatography-High Performance Liquid Chromatography (SEC-HPLC) analysis of purified INN^α-D3-α, demonstrating a purity of 99%. Comparable purity results were obtained for the other seven heterotrimers. _{[0074] FIGs. 25A-25C. INNβ-D3-β crosses the BBB via hTFR1. INNβ-D3-β was detected with b-} TNF^-based ELISA. Briefly, INNs were captured on Streptavidin plates (MSD, L15SA) coated with 0.25 µg/ml of Biotinylated Trimeric Active hTNFα (b-TNFα). Nabs bound to b-TNFα were detected using anti-VHH goat antibody, followed by anti-Goat SULFO-TAG labeled antibody (MSD) and read using a MESO QuickPlex SQ. INN^β-D3-β in all tissues analyzed. Brain localization -CSF, homogenates (Homo.), parenchymal cells (P.C.) and Vessels- of INN^β-D3-β were hTFR1-dependent (hTFR1). Vasculature and parenchymal components were separated following a fractionation protocol used by Denali’s to evaluate BBB permeability of AVI-based therapeutics¹. S1 and S2 were brain soluble fractions that were discarded during this fractionation protocol. The CSF/Serum ratio varied from 0.123 to 0.26 with an average of 0.17. TfRb-Nabs were consistently present at higher levels in the serum of hTFR1 rats, likely because binding to hTFR1 prolonged their half-life. _{[0075] FIGs. 26A-26C are sequences. [0076] FIGs. 27A-27D. BRI2, APP, and TREM2 processing. Schematic representation of} BRI2 processing in healthy (A) and mutant BRI2 in FDD and FBD (B) by convertase (Conv.), ADAM10, and SPPL2a/b. (C) APP is sequentially processed by β-secretase, releasing the soluble APP ectodomain (sAPPβ) and leaving a membrane-bound C-terminal fragment (C99 or βCTF), which is then cleaved by γ-secretase to produce Aβ and the intracellular domain (ICD) of APP, named AID/AICD. This process is termed the amyloidogenic pathway as it produces Aβ. Alternatively, APP is cleaved by α-secretase, generating sAPPα and αCTF, which is subsequently cleaved by γ-secretase into P3 and the AID. Grey box: mBRI2 binds APP at regions containing the α- and β-secretase cleavage sites, thereby reducing APP cleavage. Additionally, mBRI2 binds to the β-CTF, hindering γ-secretase access and limiting the production of Aβ and AID. (D) Like APP, TREM2 is cleaved by α-secretase, producing a soluble ectodomain (sTREM2) and a membrane-bound C-terminal fragment (TREM2-CTF)^2,3. TREM2-CTF is then further processed by γ-secretase in the transmembrane region. Grey box; BRI2 binds to TREM2 around the α-secretase cleavage site, reducing its processing and regulating TREM2 cleavage. - 13 - 4181706.v1 5431.1032002 _{[0077] FIG. 28A. Depiction of T2-BD1, T2-BD2, BRI2-ECD, BRI2-BRICHOS and BRI2-} T2-BD1 recombinant proteins. APP-BD = APP binding domain of BRI2. TM = transmembrane region. FIG.28B. BRI2/TFRB-D3 fusion protein, ECD-NN, T2BD2-NN and T2BD1-NN. TFRB-D3 will be used as a negative control. All proteins contain a 6xHis-Tag at the COOH- terminus utilized for recombinant protein purification. _{[0078] FIGs. 29A-29D. T2BD1-NN crosses the BBB via human TfR1. (A) T2BD1-NN was} detected with human TfR1-based ELISA. Briefly, T2BD1-NN was captured on Streptavidin plates (MSD, L15SA) coated with 0.25 µg/ml of biotinylated human soluble TfR1 ectodomain. T2BD1-NN bound to biotinylated sTfR1 were detected using anti-VHH goat antibody (specific for VHH-camelid nanobodies FR region), followed by anti-Goat SULFO-TAG labeled antibody (MSD) and read using a MESO QuickPlex SQ. Brain localization of T2BD1-NN was dependent on human TfR1, as T2BD1-NN was only found in the CSF and brain homogenates of Tfr1^h/h rats, but not Tfr1^w/w rats. The CSF/Serum ratio varied from 0.75 to 1.42 with an average of 1.197. T2BD1-NN was present at higher levels in the serum of Tfr1^w/w rats. To determine whether this is due to active uptake of T2BD1-NN by brain tissue via hTfR1 or to reduced serum half-life from general tissue uptake, we will investigate T2BD1-NN uptake across other tissues. (B) NN- T2BD1 (green), hTfR1 (red) and CD31 (purple) were stained with anti-His tag, anti-human TfR1 anti-CD31 antibodies, respectively. T2BD1-NN and hTFR1 were only detected in Tfr1^h/h and not Tfr1^w/w rats (not shown). White arrows highlight colocalization (white) in the overlay panel. (C- D) Brain slices were stained with anti-His tag and either the astrocytes marker anti-GFAP (red, C) or the microglia marker anti-IBA1 (red, D). Microglia and astrocytes showed extensive colocalization with T2BD1-NN (green) in Tfr1^h/h rats. White arrows denote colocalization (yellow spots) in the overlay images. _{[0079] FIG. 30. Inhibition of tumor necrosis factor alpha (TNFα) from rhesus macaque, rat,} and mouse by heterotrimeric nanobodies containing an anti-TNF-α sdAb and NewroBus components. All heterotrimers exhibited potent TNFα-neutralizing activity against rhesus macaque TNFα, with IC₅₀ values in the single-digit picomolar range—comparable to their activity against human TNFα (Table 16). Moderate inhibition was observed against rat TNFα, with half-maximal inhibitor concentration (IC₅₀) values shifted to the double-digit nanomolar range (approximately 10,000-fold reduced potency). In contrast, the heterotrimers displayed minimal or no detectable activity against mouse TNFα. IC₅₀ values are reported as mean ± standard error of the mean (SEM) from quadruplicate measurements and were determined using - 14 - 4181706.v1 5431.1032002 the "Inhibitor vs. Normalized Response" model in GraphPad Prism 10 (GraphPad Software, Boston, MA, USA). _{[0080] FIGs. 31A-31D. INN^-D2-^ and INN^-A2-^ nanobodies bind human TfR1 selectively,} with weak cross-reactivity to monkey but not rodent TfR1 at high concentrations. FIGs.31A- 31B. HEK293T/17 cells ( American Type Culture Collection (ATCC), Manassas, VA, USA; Cat. # CRL-11268) were transiently transfected with bicistronic expression constructs encoding either human TfR1 (hTfR1) or rhesus macaque TfR1 (mkTfR1), each co-expressed with enhanced green fluorescent protein (EGFP) (VectorBuilder Inc., Chicago, IL, USA; plasmids VB211221-1144bue and VB220126-1180vvq, respectively). Transfection was performed using FUGENE® reagent (Promega Corporation, Madison, WI, USA; Cat. # E2311), and EGFP expression enabled gating of transfected cells by fluorescein 5-isothiocyanate (FITC) fluorescence. Cells were stained with either an allophycocyanin (APC)-conjugated anti-human TfR1 antibody (Invitrogen, Waltham, MA, USA; Cat. # 17071942) that cross-reacts with mkTfR1 or an immunoglobulin G (IgG)-APC isotype control (R&D Systems, Minneapolis, MN, USA; Cat. # IC050A/G). As shown in FIG.31A, robust surface staining was observed in FITC⁺ cells expressing either hTfR1 or mkTfR1, confirming surface expression of both receptors. In FIG.31B, HEK293T cells were stained with an APC-conjugated anti-heavy chain variable domain (VHH) secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA; Cat. # 128005230) following incubation with either INN^{^-A2-^}, INN^{^-D2-^}, or phosphate buffered saline (PBS) (control). Both nanobodies strongly stained FITC⁺ hTfR1-expressing cells but showed no staining of mkTfR1-transfected cells, confirming species-specific recognition. APC fluorescence intensity correlated with EGFP expression levels, indicating that nanobody binding scales with receptor abundance. Cells were stained in fluorescence-activated cell sorting (FACS) buffer (PBS + 1% bovine serum albumin (BSA) + 1^mM ethylenediaminetetraacetic acid (EDTA), pH 7.2), and analyzed after a series of washes and staining steps. Cell viability was assessed using propidium iodide (Invitrogen, Cat. # P3566) or acridine orange/propidium iodide (AO/PI) solution (DeNovix Inc., Wilmington, DE, USA; Cat. # CD-AO-PI-7.5). FIG.31C. To further confirm species specificity, enzyme-linked immunosorbent assay (ELISA) plates (Meso Scale Discovery, Rockville, MD , USA; Cat. # L15SA or L45SA) coated with 0.25^µg/mL biotinylated extracellular domains of human, mouse, rat, or Cynomolgus TfR1 (Acro Biosystems, Newark, DE, USA; Cat. #: TFR-H82E3, TFR-M82E3, TFR-R82E3, TFR-C82E3) were incubated with serial 2-fold dilutions of INN^{^-A2-^} or INN^{^-D2-^} (1^nM to 9.765^pM) overnight at 4°C. After washing, bound nanobody was detected with 1^µg/mL goat anti-VHH antibody (Jackson - 15 - 4181706.v1 5431.1032002 ImmunoResearch), followed by 0.5-1^µg/mL SULFO-TAG-labeled anti-goat antibody (Meso Scale Discovery, Cat. # R32AG). Both INN^{^-A2-^} and INN^{^-D2-^} showed strong, dose-dependent binding to human TfR1 only, with no measurable interaction with mouse, rat, or macaque orthologs. FIG.31D. To evaluate potential low-affinity cross-reactivity, ELISAs were repeated using a 1000-fold higher nanobody concentration range (10^µM to 9.765^nM) for INN^{^-A2-^}, INN^{^-D2-^}, and INN^{^-D1-^} against mouse, rat, and monkey TfR1. No binding was observed for rodent TfR1 at any concentration. However, weak binding to monkey TfR1 was detectable at concentrations ≥300^nM, indicating low-affinity cross-reactivity with the macaque ortholog under supraphysiological conditions. _{[0081] FIG. 32. Timeline of injections and blood collections. Rats received subcutaneous} injections on days 0, 5, 8, 12, 15, and 19 (blue circles). Blood samples were collected on day -5 (baseline), 24 hours after the first injection (day 1), and on days 7, 14, and 21 (red squares). This schedule was designed to assess both short- and long-term hematological effects. _{[0082] FIGs. 33A-33C. Hematological toxicity assessment of central nervous system (CNS)-} targeted nanobodies. Rats were assigned to three groups: vehicle (PBS; 3 males, 2 females), INN^{^-A2-^} (4 males, 3 females), and INN^{^-D1-^} (4 males, 3 females). Complete blood counts analysis included leukocyte subsets, red blood cell indices, hemoglobin, hematocrit, and platelet parameters. The study was designed to identify potential hematological toxicity of repeated nanobody dosing, including hematopoietic suppression. Statistical analysis was performed using two-way analysis of variance (ANOVA) to assess effects of treatment and time. No statistically significant differences were observed in any hematological parameter at any time point, indicating that INN^{^-A2-^} and INN^{^-D1-^} were well tolerated. _{[0083] FIGs. 34A-34C. IHC data related to the samples shown in FIGs. 25A-25C. The} methods are similar to those used in FIGs.20B-20D. DETAILED DESCRIPTION _{[0084] A description of example embodiments follows. [0085] Several aspects of the disclosure are described below, with reference to examples for} illustrative purposes only. Numerous specific details, relationships, and methods are set forth to provide a full understanding of the disclosure. One having ordinary skill in the relevant art will readily recognize that the disclosure can be practiced without one or more of the specific details and/or practiced with other materials (e.g., reagents, cells, animals), techniques, and/or procedures. Many techniques and procedures described herein are well understood and commonly employed by those skilled in the art using conventional methodology. - 16 - 4181706.v1 5431.1032002 Definitions _{[0086] Unless otherwise defined, all terms of art, notations and other scientific terms or} terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this disclosure pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. It will be further understood that terms, such as those defined in commonly-used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and/or as otherwise defined herein. _{[0087] The terminology used herein is for the purpose of describing particular embodiments} only and is not intended to be limiting. _{[0088] When introducing elements disclosed herein, the articles “a,” “an,” “the,” and “said”} are intended to mean that there are one or more of the elements. Further, the one or more elements may be the same or different. For example, unless the context clearly indicates otherwise, “a polypeptide” includes a single polypeptide, and two or more polypeptides. _{[0089] Throughout this specification and the claims which follow, unless the context} requires otherwise, the term “comprise,” and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of, e.g., a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integer or step. When used herein, the term “comprising” can be substituted with the term “containing” or “including.” _{[0090] As used herein, the term “consisting of” excludes any element, step, or ingredient not} specified in the claim element. _{[0091] When used herein, the term “consisting essentially of” does not exclude materials or} steps that do not materially affect the basic and novel characteristics of the claim. _{[0092] Also provided herein are corresponding embodiments for each and every embodiment} featuring the term “comprising,” “containing,” “including,” or “having,” wherein those terms are replaced by the term “consisting of” or “consisting essentially of.” _{[0093] As used herein, the conjunctive term “and/or” between multiple recited elements is} understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and/or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one - 17 - 4181706.v1 5431.1032002 of these options is understood to fall within the meaning, and, therefore, satisfy the requirement of the term “and/or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and, therefore, satisfy the requirement of the term “and/or.” _{[0094] It should be understood that for all numerical bounds describing some parameter in} this application, such as “about,” “at least,” “less than,” “fewer than,” and “more than,” the description also necessarily encompasses any range bounded by the recited values. Accordingly, for example, the description “at least 1, 2, 3, 4, or 5” also describes, inter alia, the ranges 1-2, 1- 3, 1-4, 1-5, 2-3, 2-4, 2-5, 3-4, 3-5, and 4-5, et cetera. _{[0095] When a list is presented, unless stated otherwise, it is to be understood that each} individual element of that list, and every combination of that list, is a separate embodiment. For example, a list of embodiments presented as “A, B, or C” is to be interpreted as including the embodiments, “A,” “B,” “C,” “A or B,” “A or C,” “B or C,” or “A, B, or C.” _{[0096] As used herein, the term “about” means within an acceptable error range for a} particular value, as determined by one of ordinary skill in the art. Typically, an acceptable error range for a particular value depends, at least in part, on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within an acceptable standard deviation, per the practice in the art. Alternatively, “about” can mean a range of ± 20%, e.g., ± 10%, ± 5% or ± 1% of a given value. It is to be understood that the term “about” can precede any particular value specified herein, except for particular values used in the Exemplification. When “about” precedes a range, as in “1-20”, the term “about” should be read as applying to both given values of the range, such that “about 1-20” means about 1 to about 20. _{[0097] As used herein, the term “polypeptide” refers to a polymer of at least two amino acids} covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). A polypeptide can comprise any suitable L-and/or D-amino acid, for example, common ^-amino acids (e.g., alanine, glycine, valine), non-^-amino acids (e.g., ^-alanine, 4-aminobutyric acid, 6-aminocaproic acid, sarcosine, statine), and unusual amino acids (e.g., citrulline, homocitruline, homoserine, norleucine, norvaline, ornithine). The amino, carboxyl, and/or other functional groups on a polypeptide can be free (e.g., unmodified) or protected with a suitable protecting group. Suitable protecting groups for amino and carboxyl groups, and methods for adding or removing protecting groups are known in the art and are disclosed in, for example, Green and Wuts, “Protecting Groups in Organic Synthesis,” John Wiley and Sons, 1991. The functional groups of a polypeptide can also be derivatized (e.g., - 18 - 4181706.v1 5431.1032002 alkylated) or labeled (e.g., with a detectable label, such as a fluorogen or a hapten) using methods known in the art. A polypeptide can comprise one or more modifications (e.g., amino acid linkers, acylation, acetylation, amidation, methylation, terminal modifiers (e.g., cyclizing modifications), N-methyl-^-amino group substitution), if desired. In addition, a polypeptide can be an analog of a known and/or naturally-occurring peptide, for example, a peptide analog having conservative amino acid residue substitution(s). _{[0098] As used herein, a “polynucleotide” is defined as a plurality of nucleotides and/or} nucleotide analogs linked together in a single molecule. In some embodiments, a polynucleotide disclosed herein comprises deoxyribonucleotides. In some embodiments, the polynucleotide comprises ribonucleotides. Non-limiting examples of polynucleotides include single-, double- or multi-stranded DNA or RNA, DNA-RNA hybrids (e.g., each “T” position may be independently substituted by a “U” or vice versa), or a polymer comprising purine and pyrimidine bases, or other natural, chemically, or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of the polynucleotide can comprise sugars and phosphate groups, modified or substituted sugar or phosphate groups, a polymer of synthetic subunits such as phosphoramidates, or a combination thereof. _{[0099] As used herein, the term “sequence identity” refers to the extent to which two} nucleotide sequences have the same residues at the same positions when the sequences are aligned to achieve a maximal level of identity, expressed as a percentage. For sequence alignment and comparison, typically one sequence is designated as a reference sequence, to which test sequences are compared. Sequence identity between reference and test sequences is expressed as a percentage of positions across the entire length of the reference sequence where the reference and test sequences share the same nucleotide or amino acid upon alignment of the reference and test sequences to achieve a maximal level of identity. As an example, two sequences are considered to have 70% sequence identity when, upon alignment to achieve a maximal level of identity, the test sequence has the same nucleotide residue at 70% of the same positions over the entire length of the reference sequence. _{[00100] Alignment of sequences for comparison to achieve maximal levels of identity can be} readily performed by a person of ordinary skill in the art using an appropriate alignment method or algorithm. In some instances, alignment can include introduced gaps to provide for the maximal level of identity. Examples include the local homology algorithm of Smith & Waterman, Adv. Appl. Math.2:482 (1981), the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol.48:443 (1970), the search for similarity method of Pearson & Lipman, - 19 - 4181706.v1 5431.1032002 Proc. Nat’l. Acad. Sci. USA 85:2444 (1988), computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), and visual inspection (see generally Ausubel et al., Current Protocols in Molecular Biology). _{[00101] When using a sequence comparison algorithm, test and reference sequences are input} into a computer, subsequent coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. A commonly used tool for determining percent sequence identity is Protein Basic Local Alignment Search Tool (BLASTP) available through National Center for Biotechnology Information, National Library of Medicine, of the United States National Institutes of Health. (Altschul et al., 1990). _{[00102] As used herein, the term “antibody” refers to an immunoglobulin molecule capable of} specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable domain of the immunoglobulin molecule. As used herein, the term “antibody” refers to a full-length antibody. In some embodiments, an antibody is a modified and/or engineered antibody; non-limiting examples of modified and/or engineered antibodies include chimeric antibodies, humanized antibodies, multiparatopic antibodies, bispecific antibodies, and multispecific antibodies. _{[00103] As used herein, a “humanized antibody” is an antibody in which the antigen binding} sites are derived from non-human species and the framework regions are derived from human immunoglobulin sequences. _{[00104] As used herein, a “human antibody” is an antibody having heavy and light chain} variable regions in which the framework and the antigen binding sites are derived from sequences of human origin. _{[00105] As used herein, the term “antibody mimetic” refers to polypeptides capable of} mimicking an antibody’s ability to bind an antigen, but structurally differ from native antibody structures. Examples of antibody mimetics include, but not limited to, Adnectins, Affibodies, Affilins, Affimers, Affitins, Alphabodies, Anticalins, Avimers, DARPins, Fynomers, Kunitz domain peptides, monobodies, nanoCLAMPs, and Versabodies. _{[00106] As used herein, the term “single-domain antibody (sdAb)” or “NANOBODY®” are} used interchangeably to refer to an immunoglobulin molecule consisting of a single monomeric - 20 - 4181706.v1 5431.1032002 variable antibody domain. The single-domain antibodies identified herein as anti-TNF-α sdAb 1- 3 are also referred to herein as “TNF-α-Nab 1-3” and “TNFI-Nab1-3.” _{[00107] As used herein, the term “antigen-binding fragment” refers to a portion of an} immunoglobulin molecule (e.g., antibody) that retains the antigen binding properties (e.g., of a corresponding full-length antibody). Non-limiting examples of antigen-binding fragments include a V_H region, a single-domain antibody (sdAb), a V_L region, a Fab fragment, a F(ab’)₂ fragment, a Fd fragment, a Fv fragment, and a domain antibody (dAb) consisting of one V_H domain or one V_L domain, etc. V_H and V_L domains may be linked together via a synthetic linker to form various types of single-chain antibody designs in which the V_H/V_L domains pair intramolecularly, or intermolecularly in those cases when the V_H and V_L domains are expressed by separate chains, to form a monovalent antigen binding site, such as single chain Fv (scFv) or diabody. In some embodiments, an antigen-binding fragment is Fab, F(ab’)₂, Fab’, scFv, or Fv. In some embodiments, antigen-binding fragment is a scFv. _{[00108] As used herein, a “complementarity determining region (CDR)” encompasses CDRs} defined by any art-recognized method for identifying the CDR residues on an antibody. See, e.g., Kabat, E.A., et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No.91-3242, Chothia et al., (1989) Nature 342:877, Chothia, C. et al., (1987) J. Mol. Biol.196:901-917, Al-lazikani et al., (1997) J. Molec. Biol.273:927-948, and Almagro, J. Mol. Recognit.17:132-143 (2004). See also hgmp.mrc.ac.uk and bioinf.org.uk/abs. Two antibodies are determined to have the same CDR as one another with respect to an HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and/or LCDR3, when the identity of that CDR is determined for both antibodies using the same method. _{[00109] The extent of the framework region and the CDRs of an antibody can be identified} using one of several suitable methodologies that are well known in the art, for example, by the Kabat definition, the Chothia definition, the AbM definition, and/or the contact definition. Publicly and/or commercially available tools for identifying framework and/or CDR regions include, IgBlast (accessible at www.ncbi.nlm.nih.gov/igblast/), Scaligner (available from drugdesigntech at www.scaligner.com/), IMGT rules and/or tools (see, for example, www.imgt.org/IMGTScientificChart/Nomenclature/IMGT-FRCDRdefinition.html, also accessible at www.imgt.org/), Chothia Canonical Assignment (accessible at www.bioinf.org.uk/abs/chothia.html), Antigen receptor Numbering And Receptor Classification (ANARCI, accessible at opig.stats.ox.ac.uk/webapps/newsabdab/sabpred/anarci/), or the Paratome web server (accessible at www.ofranlab.org/paratome/ see Vered Kunik, et al, Nucleic - 21 - 4181706.v1 5431.1032002 Acids Research, Volume 40, Issue W1, 1 July 2012, Pages W521-W524). Also see, e.g., Dondelinger et al., Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface/Residue Definition, Front Immunol.9:2278 (2018), Polonelli et al., Antibody complementarity-determining regions (CDRs) can display differential antimicrobial, antiviral and antitumor activities, PLoS One.3(6):e2371 (2008). _{[00110] As used herein, the term “KD,” also referred to as “binding constant,” “equilibrium} dissociation constant” or “affinity constant,” is a measure of the extent of a reversible association between two molecular species (e.g., antibody and target protein) and includes both the actual binding affinity as well as the apparent binding affinity. Binding affinity can be determined using methods known in the art including, for example, by measurement of surface plasmon resonance, e.g., using a Biolayer interferometry (Octet, ForteBio) or a surface plasmon resonance (Biacore) system and assay. A reference that compares various surface technologies for measuring binding affinity and kinetics is Yang, D., Singh, A., Wu, H., & Kroe-Barrett, R., Comparison of biosensor platforms in the evaluation of high affinity antibody-antigen binding kinetics, Analytical Biochemistry 508: 78-96 (2016), the contents of which are incorporated by reference herein in their entirety. _{[00111] The term “expression vector” refers to a replicable nucleic acid from which one or} more proteins can be expressed when the expression vector is transformed into a suitable expression host cell. _{[00112] As used herein, the term “promoter” refers to a region of DNA to which RNA} polymerase binds and initiates the transcription of a gene. _{[00113] As used herein, the term “operably linked” means that the nucleic acid is positioned} in the recombinant polynucleotide, e.g., vector, in such a way that enables expression of the nucleic acid under control of the element (e.g., promoter) to which it is linked. _{[00114] As used herein, the term “selectable marker element” is an element that confers a trait} suitable for artificial selection. Selectable marker elements can be negative or positive selection markers. _{[00115] As used herein, the term “expression host cell” refers to a cell useful for receiving,} maintaining, reproducing and/or amplifying a vector. _{[00116] The phrase “pharmaceutically acceptable” means that the substance or composition} the phrase modifies is, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, and are commensurate with a reasonable benefit/risk ratio. - 22 - 4181706.v1 5431.1032002 _{[00117] As used herein, the term “pharmaceutically acceptable salt” refers to those salts} which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of mammals without undue toxicity, irritation, allergic response, and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, the relevant teachings of which are incorporated herein by reference in their entirety. Pharmaceutically acceptable salts of the agents/compounds described herein include salts derived from suitable inorganic and organic acids, and suitable inorganic and organic bases. _{[00118] Examples of salts derived from suitable acids include salts of an amino group formed} with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art, such as ion exchange. Other pharmaceutically acceptable salts derived from suitable acids include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, cinnamate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, glutarate, glycolate, hemisulfate, heptanoate, hexanoate, hydroiodide, hydroxybenzoate, 2-hydroxy-ethanesulfonate, hydroxymaleate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 2-phenoxybenzoate, phenylacetate, 3-phenylpropionate, phosphate, pivalate, propionate, pyruvate, salicylate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. _{[00119] Either the mono-, di- or tri-acid salts can be formed, and such salts can exist in either} a hydrated, solvated or substantially anhydrous form. _{[00120] Salts derived from appropriate bases include salts derived from inorganic bases, such} as alkali metal, alkaline earth metal, and ammonium bases, and salts derived from aliphatic, alicyclic or aromatic organic amines, such as methylamine, trimethylamine and picoline, or N⁺((C₁-C₄)alkyl)₄ salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, barium and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and - 23 - 4181706.v1 5431.1032002 amine cations formed using counterions such as halide, hydroxide, carboxyl, sulfate, phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate. _{[00121] A “pharmaceutical composition” refers to a formulation of one or more therapeutic} agents and a medium generally accepted in the art for delivery of a biologically active agent to subjects, e.g., humans. In some embodiments, a pharmaceutical composition may include one or more pharmaceutically acceptable excipients, diluents, or carriers. In some embodiments, a pharmaceutical composition suitable for use in methods disclosed herein further comprises one or more pharmaceutically acceptable carriers. _{[00122] “Pharmaceutically acceptable carrier, diluent, or excipient” includes any adjuvant,} carrier, excipient, glidant, sweetening agent, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals. _{[00123] “Pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical} composition, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative. In some embodiments, the carrier may be a diluent, adjuvant, excipient, or vehicle with which the agent (e.g., polynucleotide) is administered. Such vehicles may be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. For example, 0.4% saline and 0.3% glycine can be used. These solutions are sterile and generally free of particulate matter. They may be sterilized by conventional, well-known sterilization techniques (e.g., filtration). Compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, stabilizing, thickening, lubricating, and coloring agents, etc. The concentration of the agent in such pharmaceutical formulation may vary widely, i.e., from less than about 0.5%, to at least about 1%, or to as much as 15% or 20%, 25%, 30%, 35%, 40%, 45% or 50% by weight. The concentration will be selected primarily based on required dose, fluid volumes, viscosities, etc., according to the mode of administration. Suitable vehicles and formulations, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in Remington: The Science and Practice of Pharmacy, 21^st Edition, Troy, D.B. ed., Lipincott Williams and Wilkins, Philadelphia, PA 2006, Part 5, Pharmaceutical Manufacturing: 691-1092 (e.g., pages 958-89). - 24 - 4181706.v1 5431.1032002 _{[00124] Non-limiting examples of pharmaceutically acceptable carriers are solvents,} dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible, such as salts, buffers, antioxidants, saccharides, aqueous or non-aqueous carriers, preservatives, wetting agents, surfactants or emulsifying agents, or combinations thereof. _{[00125] Non-limiting examples of buffers are acetic acid, citric acid, formic acid, succinic} acid, phosphoric acid, carbonic acid, malic acid, aspartic acid, histidine, boric acid, Tris buffers, HEPPSO, and HEPES. _{[00126] Non-limiting examples of antioxidants are ascorbic acid, methionine, cysteine} hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, lecithin, citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, and tartaric acid. _{[00127] Non-limiting examples of amino acids are histidine, isoleucine, methionine, glycine,} arginine, lysine, L-leucine, tri-leucine, alanine, glutamic acid, L-threonine, and 2-phenylamine. _{[00128] Non-limiting examples of surfactants are polysorbates (e.g., polysorbate-20 or} polysorbate-80), polyoxamers (e.g., poloxamer 188), Triton, sodium octyl glycoside, lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine, lauryl-, myristyl-, linoleyl- or stearyl-sarcosine, linoleyl-, myristyl-, or cetyl-betaine, lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g., lauroamidopropyl), myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine, sodium methyl cocoyl-, or disodium methyl oleyl-taurate, and the MONAQUA™ series (Mona Industries, Inc., Paterson, N.J.), polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g., PLURONICS™, PF68, etc.). _{[00129] Non-limiting examples of preservatives are phenol, m-cresol, p-cresol, o-cresol,} chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride, alkylparaben (methyl, ethyl, propyl, butyl, and the like), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, and thimerosal, or mixtures thereof. _{[00130] Non-limiting examples of saccharides are monosaccharides, disaccharides,} trisaccharides, polysaccharides, sugar alcohols, reducing sugars, nonreducing sugars such as glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerin, dextran, erythritol, glycerol, arabitol, sylitol, sorbitol, mannitol, mellibiose, melezitose, raffinose, mannotriose, stachyose, maltose, lactulose, maltulose, glucitol, maltitol, lactitol, or iso-maltulose. - 25 - 4181706.v1 5431.1032002 _{[00131] Non-limiting examples of salts are acid addition salts and base addition salts. Acid} addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous, and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium, and the like, as well as from nontoxic organic amines, such as N,N’-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, and the like. In some embodiments, the salt is sodium chloride (NaCl). _{[00132] The term “subject” or “patient” refers to an animal (e.g., a mammal such as a human),} diagnosed with or suspected of having an inflammation-associated disease or condition (e.g., Alzheimer’s disease), or one at risk of developing such conditions. Diagnosis may be performed by any method or technique known in the art. One skilled in the art will understand that a subject to be treated according to the present disclosure may have been subjected to standard tests or may have been identified, without examination, as one at risk due to the presence of one or more risk factors associated with the disease or condition. _{[00133] “Treating” or “treatment,” as used herein, refers to taking steps to deliver a therapy to} a subject, such as a mammal, in need thereof (e.g., as by administering to a mammal one or more therapeutic agents). “Treating” or “treatment” includes inhibiting the disease or condition (e.g., as by slowing or stopping its progression or causing regression of the disease or condition) and relieving the symptoms resulting from the disease or condition. _{[00134] The term “treating,” or “treatment” refers to the medical management of a subject} with the intent to improve, ameliorate, stabilize (i.e., not worsen), prevent, or cure a disease, pathological condition, or disorder—such as the particular indications exemplified herein. This term includes active treatment (treatment directed to improve the disease, pathological condition, or disorder), causal treatment (treatment directed to the cause of the associated disease, pathological condition, or disorder), palliative treatment (treatment designed for the relief of symptoms), preventative treatment (treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder), and supportive treatment (treatment employed to supplement another therapy). Treatment also includes diminishment of the extent of the disease or condition, preventing spread of the disease or condition, delay or slowing the progress of the disease or condition, amelioration or palliation - 26 - 4181706.v1 5431.1032002 of the disease or condition, and remission (whether partial or total), whether detectable or undetectable. “Ameliorating” or “palliating” a disease or condition means that the extent and/or undesirable clinical manifestations of the disease, disorder, or condition are lessened and/or time course of the progression is slowed or lengthened, as compared to the extent or time course in the absence of treatment. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder, as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented. _{[00135] As used herein, the term “a TNF-α associated disorder” refers to a disease,} pathological condition, or disorder that is associated with, results from, and/or occurs in response to, an elevated level of TNF-α. In some embodiments, an elevated level of TNF-α is an episodic elevated level of TNF-α activity (e.g., a local and/or systemic increase in TNF activity). In some embodiments, an elevated level of TNF-α is a chronic elevated level of TNF-α activity (e.g., a local and/or systemic increase in TNF activity). In some embodiments, a TNF-α associated disorder is a local disorder where TNF-α is a primary mediator. In some embodiments, a TNF-α associated disorder is a systemic disorder where TNF-α is a primary mediator. _{[00136] Compositions (e.g., antibodies or antigen binding fragments thereof, polynucleotides)} described herein may be prepared in accordance with standard procedures and are administered at dosages that are selected to reduce, prevent, or eliminate, or to slow or halt progression of, a condition being treated (see, e.g., Remington’s Pharmaceutical Sciences, Mack Publishing Company, Easton, PA, and Goodman and Gilman’s The Pharmaceutical Basis of Therapeutics, McGraw-Hill, New York, N.Y., the contents of which are incorporated herein by reference, for a general description of methods for administering various agents for human therapy). _{[00137] “Administering” or “administration,” as used herein, refers to providing a compound,} composition, or pharmaceutically acceptable salt thereof described herein to a subject in need of treatment or prevention. Administering can be performed, for example, once, a plurality of times, and/or over one or more extended periods. Administration includes both direct administration (including self-administration), and indirect administration (including an act of prescribing a drug or directing a subject to consume an agent). For example, as used herein, one (e.g., a physician) who instructs a subject (e.g., a human patient) to self-administer an agent (e.g., a drug), or to have an agent administered by another and/or who provides a patient with a prescription for a drug is administering an agent to a subject. - 27 - 4181706.v1 5431.1032002 _{[00138] “A therapeutically effective amount,” “an effective amount” or “an effective dosage”} refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result (e.g., treatment, healing, inhibition or amelioration of physiological response or condition, etc.). A full therapeutic effect does not necessarily occur by administration of one dose and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations. A therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of a mammal (e.g., a human patient), mode of administration, and the ability of a therapeutic or a combination of therapeutics to elicit a desired response. _{[00139] An effective amount of an agent to be administered can be determined by a clinician} of ordinary skill using the guidance provided herein and other methods known in the art. Relevant factors include the given agent, the pharmaceutical formulation, the route of administration, the type of disease or disorder, the identity of the subject (e.g., age, sex, weight) or host being treated, and the like. For example, suitable dosages can be from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.01 mg/kg to about 1 mg/kg body weight per treatment. Determining the dosage for a particular agent, subject and disease is well within the abilities of one of skill in the art. Preferably, the dosage does not cause or produces minimal adverse side effects. _{[00140] Desired response or desired results include effects at the cellular level, tissue level, or} clinical results. As such, “a therapeutically effective amount” or synonym thereto depends upon the context in which it is being applied. For example, in some embodiments it is an amount of the composition sufficient to achieve a treatment response as compared to the response obtained without administration of the composition. In other embodiments, it is an amount that results in a beneficial or desired result in a subject as compared to a control. As defined herein, a therapeutically effective amount of a composition (e.g., a pharmaceutical composition) disclosed herein may be readily determined by one of ordinary skill by routine methods known in the art. Dosage regimen and route of administration may be adjusted to provide the optimum therapeutic response. Anti-Transferrin Receptor 1 (TFR1) Antibodies or Antigen-Binding Fragments Thereof _{[00141] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof is} a full-length (e.g., whole, intact) antibody. In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof is an antigen-binding fragment comprising (e.g., consisting of) a V_H region, a single-domain antibody (sdAb), a Fab fragment, a F(ab’)₂ fragment, a Fd - 28 - 4181706.v1 5431.1032002 fragment, a Fv fragment, or a domain antibody (dAb). In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof is a sdAb, Fab, F(ab’)₂, Fab’, scFv, or Fv. In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof is a sdAb. _{[00142] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof is} an isolated antibody. In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof is an isolated antigen-binding fragment of an antibody. In some embodiments, an anti- TFR1 antibody or antigen-binding fragment thereof is an isolated sdAb. _{[00143] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof is} recombinantly produced. In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof is synthetically produced. _{[00144] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof is} produced recombinantly or synthetically, using routine methods and reagents that are well known in the art. For example, an antibody or antigen-binding fragment thereof can be produced recombinantly in a suitable host cell (e.g., bacteria) according to methods known in the art. See, e.g., Current Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992; and Molecular Cloning: a Laboratory Manual, 2nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press. For example, a nucleic acid molecule comprising a nucleotide sequence encoding an anti-TFR1 antibody or antigen-binding fragment thereof can be introduced and expressed in a suitable host cell (e.g., E. coli), and the expressed anti-TFR1 antibody or antigen-binding fragment thereof can be isolated/purified from the host cell (e.g., in inclusion bodies) using routine methods and readily available reagents. For example, DNA fragments coding for different protein sequences (e.g., a light-responsive domain, a heterologous peptide component) can be ligated together in-frame in accordance with conventional techniques. In another embodiment, an antibody or antigen-binding fragment thereof can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of nucleic acid fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive nucleic acid fragments that can subsequently be annealed and re-amplified to generate a chimeric nucleic acid sequence (see e.g., Ausubel et al., Current Protocols in Molecular Biology, 1992). _{[00145] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} binds (e.g., specifically binds) human transferrin receptor 1 (hTFR1, e.g., SEQ ID NO:1). _{[00146] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} has spontaneous BBB permeability. In some embodiments, an anti-TFR1 antibody or antigen- - 29 - 4181706.v1 5431.1032002 binding fragment thereof has an isoelectric point (pI) of 9-10 (e.g., 9.1-10, 9.1-9.9, 9.2-9.9, 9.2- 9.8, 9.3-9.8, 9.3-9.7, 9.4-9.7, 9.4-9.6, or 9.5-9.6). In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof has an isoelectric point (pI) of about 9.5. Complementarity-Determining Regions Complementarity-Determining Regions of Heavy Chain Variable Domain (V_H) _{[00147] In some embodiments, an anti-TFR1 antibody or antigen- thereof} comprises an immunoglobulin heavy chain variable (V_H) domain a chain complementarity-determining region (HCDR) 1, an HCDR2, and an HCDR3 having at least 80% sequence identity (e.g., having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to an HCDR1, an HCDR2, and an HCDR3, respectively, of a V_H domain comprising the amino acid sequence of any one of SEQ ID NOs:190, 176, 99-123, 177-189, and 191-199. _{[00148] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises a V_H domain comprising an HCDR1, an HCDR2, and an HCDR3 having 100% sequence identity to an HCDR1, an HCDR2, and an HCDR3, respectively, of a V_H domain comprising the amino acid sequence of any one of SEQ ID NOs:190, 176, 99-123, 177-189, and 191-199. _{[00149] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises a V_H domain comprising an HCDR1, an HCDR2, and an HCDR3 having at least 80% sequence identity (e.g., having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to an HCDR1, an HCDR2, and an HCDR3, respectively, of a V_H domain comprising the amino acid sequence of any one of SEQ ID NOs:176-190. _{[00150] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises a V_H domain comprising an HCDR1, an HCDR2, and an HCDR3 having 100% sequence identity to an HCDR1, an HCDR2, and an HCDR3, respectively, of a V_H domain comprising the amino acid sequence of any one of SEQ ID NOs:176-190. _{[00151] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises a V_H domain comprising an HCDR1, an HCDR2, and an HCDR3 having at least 80% sequence identity (e.g., having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to an HCDR1, an HCDR2, and an HCDR3, respectively, of a V_H domain comprising the amino acid sequence of SEQ ID NO:176. - 30 - 4181706.v1 5431.1032002 _{[00152] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises a V_H domain comprising an HCDR1, an HCDR2, and an HCDR3 having 100% to an HCDR1, an HCDR2, and an HCDR3, respectively, of a V_H domain comprising the amino acid sequence of SEQ ID NO:176. _{[00153] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises a V_H domain comprising an HCDR1, an HCDR2, and an HCDR3 having at least 80% sequence identity (e.g., having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to an HCDR1, an HCDR2, and an HCDR3, respectively, of a V_H domain comprising the amino acid sequence of SEQ ID NO:190. _{[00154] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises a V_H domain comprising an HCDR1, an HCDR2, and an HCDR3 having 100% to an HCDR1, an HCDR2, and an HCDR3, respectively, of a V_H domain comprising the amino acid sequence of SEQ ID NO:190. _{[00155] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises a V_H domain comprising an HCDR1, an HCDR2, and an HCDR3 having at least 80% sequence identity (e.g., having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to an HCDR1, an HCDR2, and an HCDR3, respectively, of a V_H domain comprising the amino acid sequence of any one of SEQ ID NOs:99-123. _{[00156] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises a V_H domain comprising an HCDR1, an HCDR2, and an HCDR3 having 100% sequence identity to an HCDR1, an HCDR2, and an HCDR3, respectively, of a V_H domain comprising the amino acid sequence of any one of SEQ ID NOs:99-123. _{[00157] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises a V_H domain comprising an HCDR1, an HCDR2, and an HCDR3 having at least 80% sequence identity (e.g., having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to an HCDR1, an HCDR2, and an HCDR3, respectively, of a V_H domain comprising the amino acid sequence of any one of SEQ ID NOs:99-107, 110-112, and 118-120. _{[00158] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises a V_H domain comprising an HCDR1, an HCDR2, and an HCDR3 having 100% - 31 - 4181706.v1 5431.1032002 sequence identity to an HCDR1, an HCDR2, and an HCDR3, respectively, of a V_H domain comprising the amino acid sequence of any one of SEQ ID NOs:99-107, 110-112, and 118-120. _{[00159] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises a V_H domain comprising an HCDR1, an HCDR2, and an HCDR3 having at least 80% sequence identity to an HCDR1, an HCDR2, and an HCDR3, respectively, of a V_H domain comprising the amino acid sequence of any one of SEQ ID NOs:99-105, 107, 110-112, and 118- 120. _{[00160] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises a V_H domain comprising an HCDR1, an HCDR2, and an HCDR3 having 100% sequence identity to an HCDR1, an HCDR2, and an HCDR3, respectively, of a V_H domain comprising the amino acid sequence of any one of SEQ ID NOs:99-105, 107, 110-112, and 118- 120. Complementarity-Determining Regions _{[00161] In some embodiments, HCDR1, HCDR2, and HCDR3 of an anti-TFR1 antibody or} antigen-binding fragment thereof are determined by the Honneger’s, Chothia, Kabat, or IMGT numbering scheme. Honneger’s Numbering Scheme _{[00162] In some embodiments, HCDR1, HCDR2, and HCDR3 of an anti-TFR1 antibody or} antigen-binding fragment thereof are determined by the Honneger’s numbering scheme. _{[00163] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 having at least 80% sequence identity (e.g., having at least 81%, 82%,} 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of SEQ ID NO:163, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:168, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:172; b_{) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:161, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:166, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:170; - 32 - 4181706.v1 5431.1032002 c_{) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:162, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:167, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:171; d_{) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:164, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:169, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:173; or e_{) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:165, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:169, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:174. _{[00164] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:163, an HCDR2} comprising the amino acid sequence of SEQ ID NO:168, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:172; b_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:161, an HCDR2} comprising the amino acid sequence of SEQ ID NO:166, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:170; c_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:162, an HCDR2} comprising the amino acid sequence of SEQ ID NO:167, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:171; d_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:164, an HCDR2} comprising the amino acid sequence of SEQ ID NO:169, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:173; or e_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:165, an HCDR2} comprising the amino acid sequence of SEQ ID NO:169, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:174. - 33 - 4181706.v1 5431.1032002 _{[00165] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:163, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:168, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:172; b_{) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:161, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:166, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:170; c_{) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:162, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:167, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:171; d_{) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:164, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:169, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:173; or e_{) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:165, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:169, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:174. _{[00166] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 having at least 80% sequence identity (e.g., having at least 81%, 82%,} 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of SEQ ID NO:161, b_{) an HCDR2 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:166, and c_{) an HCDR3 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:170. _{[00167] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:161, b) an HCDR2 comprising the amino acid sequence of SEQ ID NO:166, and c) an HCDR3 comprising the amino acid sequence of SEQ ID NO:170.} - 34 - 4181706.v1 5431.1032002 _{[00168] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:161, b) an HCDR2 consisting of the amino acid sequence of SEQ ID NO:166, and c) an HCDR3 c consisting of the amino acid sequence of SEQ ID NO:170. [00169] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 having at least 80% sequence identity (e.g., having at least 81%, 82%,} 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of SEQ ID NO:163, b_{) an HCDR2 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:168, and c_{) an HCDR3 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:172. _{[00170] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:163, b) an HCDR2 comprising the amino acid sequence of SEQ ID NO:168, and c) an HCDR3 comprising the amino acid sequence of SEQ ID NO:172. [00171] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:163, b) an HCDR2 consisting of the amino acid sequence of SEQ ID NO:168, and c) an HCDR3 consisting of the amino acid sequence of SEQ ID NO:172.} Chothia Numbering Scheme _{[00172] In some embodiments, HCDR1, HCDR2, and HCDR3 of an anti-TFR1 antibody or} antigen-binding fragment thereof are determined by the Chothia numbering scheme. _{[00173] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 having at least 80% sequence identity (e.g., having at least 81%, 82%,} 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of SEQ ID - 35 - 4181706.v1 5431.1032002 NO:2, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:14, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:26; _{b) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:3, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:15, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:27; _{c) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:4, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:15, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:28; _{d) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:5, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:16, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:28; _{e) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:6, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:17, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:29; _{f) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:7, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:18, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:30; _{g) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:8, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:19, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:31; _{h) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:9, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:19, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:32; _{i) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:10, an HCDR2 having at least 80% sequence identity to the amino - 36 - 4181706.v1 5431.1032002 acid sequence of SEQ ID NO:20, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:33; _{j) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:10, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:21, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:34; _{k) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:10, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:22, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:34; _{l) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:11, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:22, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:35; _{m) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:11, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:22, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:34; _{n) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:12, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:23, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:36; _{o) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:12, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:23, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:37; _{p) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:13, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:24, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:38; or _{q) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:13, an HCDR2 having at least 80% sequence identity to the amino - 37 - 4181706.v1 5431.1032002 acid sequence of SEQ ID NO:25, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:39. _{[00174] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:2, an HCDR2} comprising the amino acid sequence of SEQ ID NO:14, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:26; b_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:3, an HCDR2} comprising the amino acid sequence of SEQ ID NO:15, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:27; c_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:4, an HCDR2} comprising the amino acid sequence of SEQ ID NO:15, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:28; d_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:5, an HCDR2} comprising the amino acid sequence of SEQ ID NO:16, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:28; e_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:6, an HCDR2} comprising the amino acid sequence of SEQ ID NO:17, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:29; f_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:7, an HCDR2} comprising the amino acid sequence of SEQ ID NO:18, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:30; g_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:8, an HCDR2} comprising the amino acid sequence of SEQ ID NO:19, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:31; h_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:9, an HCDR2} comprising the amino acid sequence of SEQ ID NO:19, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:32; i_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:10, an HCDR2} comprising the amino acid sequence of SEQ ID NO:20, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:33; - 38 - 4181706.v1 5431.1032002 j_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:10, an HCDR2} comprising the amino acid sequence of SEQ ID NO:21, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:34; k_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:10, an HCDR2} comprising the amino acid sequence of SEQ ID NO:22, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:34; l_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:11, an HCDR2} comprising the amino acid sequence of SEQ ID NO:22, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:35; m_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:11, an HCDR2} comprising the amino acid sequence of SEQ ID NO:22, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:34; n_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:12, an HCDR2} comprising the amino acid sequence of SEQ ID NO:23, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:36; o_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:12, an HCDR2} comprising the amino acid sequence of SEQ ID NO:23, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:37; p_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:13, an HCDR2} comprising the amino acid sequence of SEQ ID NO:24, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:38; or q_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:13, an HCDR2} comprising the amino acid sequence of SEQ ID NO:25, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:39. _{[00175] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:2, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:14, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:26; b_{) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:3, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:15, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:27; - 39 - 4181706.v1 5431.1032002 _{c) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:4, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:15, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:28; _{d) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:5, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:16, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:28; _{e) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:6, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:17, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:29; _{f) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:7, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:18, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:30; _{g) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:8, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:19, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:31; _{h) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:9, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:19, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:32; _{i) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:10, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:20, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:33; _{j) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:10, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:21, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:34; _{k) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:10, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:22, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:34; _{l) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:11, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:22, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:35; _{m) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:11, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:22, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:34; - 40 - 4181706.v1 5431.1032002 n_{) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:12, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:23, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:36; o_{) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:12, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:23, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:37; p_{) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:13, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:24, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:38; or q_{) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:13, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:25, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:39. _{[00176] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 having at least 80% sequence identity (e.g., having at least 81%, 82%,} 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of SEQ ID NO:2, b_{) an HCDR2 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:14, and c_{) an HCDR3 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:26. _{[00177] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:2, b) an HCDR2 comprising the amino acid sequence of SEQ ID NO:14, and c) an HCDR3 comprising the amino acid sequence of SEQ ID NO:26. [00178] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:2, b) an HCDR2 consisting of the amino acid sequence of SEQ ID NO:14, and c) an HCDR3 c consisting of the amino acid sequence of SEQ ID NO:26.} - 41 - 4181706.v1 5431.1032002 _{[00179] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 having at least 80% sequence identity (e.g., having at least 81%, 82%,} 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of SEQ ID NO:5, b_{) an HCDR2 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:16, and c_{) an HCDR3 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:28. _{[00180] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:5, b) an HCDR2 comprising the amino acid sequence of SEQ ID NO:16, and c) an HCDR3 comprising the amino acid sequence of SEQ ID NO:28. [00181] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:5, b) an HCDR2 consisting of the amino acid sequence of SEQ ID NO:16, and c) an HCDR3 consisting of the amino acid sequence of SEQ ID NO:28.} Kabat Numbering Scheme _{[00182] In some embodiments, HCDR1, HCDR2, and HCDR3 of an anti-TFR1 antibody or} antigen-binding fragment thereof are determined by the Kabat numbering scheme. _{[00183] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 having at least 80% sequence identity (e.g., having at least 81%, 82%,} 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of SEQ ID NO:40, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:48, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:26; b_{) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:40, an HCDR2 having at least 80% sequence identity to the amino - 42 - 4181706.v1 5431.1032002 acid sequence of SEQ ID NO:216, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:26; _{c) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:41, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:49, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:27; _{d) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:217, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:49, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:27; _{e) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:41, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:50, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:28; _{f) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:41, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:51, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:28; _{g) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:42, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:52, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:29; _{h) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:43, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:53, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:30; _{i) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:44, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:54, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:31; _{j) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:44, an HCDR2 having at least 80% sequence identity to the amino - 43 - 4181706.v1 5431.1032002 acid sequence of SEQ ID NO:218, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:31; _{k) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:44, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:54, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:32; _{l) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:44, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:219, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:32; _{m) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:45, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:55, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:33; _{n) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:45, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:56, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:34; _{o) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:45, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:57, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:34; _{p) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:45, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:57, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:35; _{q) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:45, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:57, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:34; _{r) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:46, an HCDR2 having at least 80% sequence identity to the amino - 44 - 4181706.v1 5431.1032002 acid sequence of SEQ ID NO:58, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:36; s_{) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:46, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:59, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:37; t_{) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:47, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:60, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:38; or u_{) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:47, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:61, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:39. _{[00184] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:40, an HCDR2} comprising the amino acid sequence of SEQ ID NO:48, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:26; b_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:40, an HCDR2} comprising the amino acid sequence of SEQ ID NO:216, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:26; c_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:41, an HCDR2} comprising the amino acid sequence of SEQ ID NO:49, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:27; d_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:217, an HCDR2} comprising the amino acid sequence of SEQ ID NO:49, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:27; e_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:41, an HCDR2} comprising the amino acid sequence of SEQ ID NO:50, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:28; - 45 - 4181706.v1 5431.1032002 _{f) an HCDR1 comprising the amino acid sequence of SEQ ID NO:41, an HCDR2} comprising the amino acid sequence of SEQ ID NO:51, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:28; _{g) an HCDR1 comprising the amino acid sequence of SEQ ID NO:42, an HCDR2} comprising the amino acid sequence of SEQ ID NO:52, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:29; _{h) an HCDR1 comprising the amino acid sequence of SEQ ID NO:43, an HCDR2} comprising the amino acid sequence of SEQ ID NO:53, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:30; _{i) an HCDR1 comprising the amino acid sequence of SEQ ID NO:44, an HCDR2} comprising the amino acid sequence of SEQ ID NO:54, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:31; _{j) an HCDR1 comprising the amino acid sequence of SEQ ID NO:44, an HCDR2} comprising the amino acid sequence of SEQ ID NO:218, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:31; _{k) an HCDR1 comprising the amino acid sequence of SEQ ID NO:44, an HCDR2} comprising the amino acid sequence of SEQ ID NO:54, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:32; _{l) an HCDR1 comprising the amino acid sequence of SEQ ID NO:44, an HCDR2} comprising the amino acid sequence of SEQ ID NO:219, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:32; _{m) an HCDR1 comprising the amino acid sequence of SEQ ID NO:45, an HCDR2} comprising the amino acid sequence of SEQ ID NO:55, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:33; _{n) an HCDR1 comprising the amino acid sequence of SEQ ID NO:45, an HCDR2} comprising the amino acid sequence of SEQ ID NO:56, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:34; _{o) an HCDR1 comprising the amino acid sequence of SEQ ID NO:45, an HCDR2} comprising the amino acid sequence of SEQ ID NO:57, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:34; _{p) an HCDR1 comprising the amino acid sequence of SEQ ID NO:45, an HCDR2} comprising the amino acid sequence of SEQ ID NO:57, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:35; - 46 - 4181706.v1 5431.1032002 q_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:45, an HCDR2} comprising the amino acid sequence of SEQ ID NO:57, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:34; r_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:46, an HCDR2} comprising the amino acid sequence of SEQ ID NO:58, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:36; s_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:46, an HCDR2} comprising the amino acid sequence of SEQ ID NO:59, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:37; t_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:47, an HCDR2} comprising the amino acid sequence of SEQ ID NO:60, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:38; or u_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:47, an HCDR2} comprising the amino acid sequence of SEQ ID NO:61, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:39. _{[00185] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:40, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:48, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:26; b_{) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:40, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:216, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:26; c_{) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:41, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:49, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:27; d_{) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:217, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:49, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:27; e_{) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:41, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:50, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:28; - 47 - 4181706.v1 5431.1032002 _{f) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:41, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:51, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:28; _{g) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:42, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:52, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:29; _{h) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:43, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:53, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:30; _{i) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:44, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:54, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:31; _{j) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:44, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:218, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:31; _{k) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:44, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:54, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:32; _{l) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:44, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:219, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:32; _{m) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:45, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:55, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:33; _{n) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:45, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:56, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:34; _{o) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:45, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:57, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:34; _{p) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:45, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:57, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:35; - 48 - 4181706.v1 5431.1032002 q_{) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:45, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:57, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:34; r_{) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:46, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:58, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:36; s_{) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:46, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:59, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:37; t_{) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:47, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:60, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:38; or u_{) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:47, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:61, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:39. _{[00186] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 having at least 80% sequence identity (e.g., having at least 81%, 82%,} 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of SEQ ID NO:40, b_{) an HCDR2 having at least 80% sequence identity (e.g., having at least 81%, 82%,} 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of SEQ ID NO:48, and c_{) an HCDR3 having at least 80% sequence identity (e.g., having at least 81%, 82%,} 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of SEQ ID NO:26. _{[00187] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:40, b) an HCDR2 comprising the amino acid sequence of SEQ ID NO:48, and} - 49 - 4181706.v1 5431.1032002 c_{) an HCDR3 comprising the amino acid sequence of SEQ ID NO:26. [00188] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:40, b) an HCDR2 consisting of the amino acid sequence of SEQ ID NO:48, and c) an HCDR3 consisting of the amino acid sequence of SEQ ID NO:26. [00189] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 having at least 80% sequence identity (e.g., having at least 81%, 82%,} 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of SEQ ID NO:41, b_{) an HCDR2 having at least 80% sequence identity (e.g., having at least 81%, 82%,} 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of SEQ ID NO:51, and c_{) an HCDR3 having at least 80% sequence identity (e.g., having at least 81%, 82%,} 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of SEQ ID NO:28. _{[00190] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:41, b) an HCDR2 comprising the amino acid sequence of SEQ ID NO:51, and c) an HCDR3 comprising the amino acid sequence of SEQ ID NO:28. [00191] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:41, b) an HCDR2 consisting of the amino acid sequence of SEQ ID NO:51, and c) an HCDR3 consisting of the amino acid sequence of SEQ ID NO:28.} IMGT Numbering Scheme _{[00192] In some embodiments, HCDR1, HCDR2, and HCDR3 of an anti-TFR1 antibody or} antigen-binding fragment thereof are determined by the IMGT numbering scheme. - 50 - 4181706.v1 5431.1032002 _{[00193] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 having at least 80% sequence identity (e.g., having at least 81%, 82%,} 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of SEQ ID NO:62, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:74, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:86; b_{) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:62, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:74, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:220; c_{) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:63, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:75, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:87; d_{) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:63, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:75, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:221; e_{) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:64, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:75, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:88; f_{) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:65, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:76, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:88; g_{) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:65, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:76, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:222; - 51 - 4181706.v1 5431.1032002 _{h) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:66, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:77, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:89; _{i) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:67, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:78, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:90; _{j) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:68, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:79, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:91; _{k) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:69, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:79, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:92; _{l) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:70, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:80, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:93; _{m) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:70, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:81, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:94; _{n) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:70, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:82, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:94; _{o) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:71, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:82, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:95; - 52 - 4181706.v1 5431.1032002 p_{) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:71, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:82, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:94; q_{) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:72, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:83, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:95; r_{) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:72, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:83, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:96; s_{) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:73, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:84, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:97; or t_{) an HCDR1 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:73, an HCDR2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:85, and an HCDR3 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:98. _{[00194] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:62, an HCDR2} comprising the amino acid sequence of SEQ ID NO:74, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:86; b_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:62, an HCDR2} comprising the amino acid sequence of SEQ ID NO:74, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:220; c_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:63, an HCDR2} comprising the amino acid sequence of SEQ ID NO:75, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:87; - 53 - 4181706.v1 5431.1032002 _{d) an HCDR1 comprising the amino acid sequence of SEQ ID NO:63, an HCDR2} comprising the amino acid sequence of SEQ ID NO:75, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:221; _{e) an HCDR1 comprising the amino acid sequence of SEQ ID NO:64, an HCDR2} comprising the amino acid sequence of SEQ ID NO:75, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:88; _{f) an HCDR1 comprising the amino acid sequence of SEQ ID NO:65, an HCDR2} comprising the amino acid sequence of SEQ ID NO:76, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:88; _{g) an HCDR1 comprising the amino acid sequence of SEQ ID NO:65, an HCDR2} comprising the amino acid sequence of SEQ ID NO:76, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:222; _{h) an HCDR1 comprising the amino acid sequence of SEQ ID NO:66, an HCDR2} comprising the amino acid sequence of SEQ ID NO:77, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:89; _{i) an HCDR1 comprising the amino acid sequence of SEQ ID NO:67, an HCDR2} comprising the amino acid sequence of SEQ ID NO:78, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:90; _{j) an HCDR1 comprising the amino acid sequence of SEQ ID NO:68, an HCDR2} comprising the amino acid sequence of SEQ ID NO:79, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:91; _{k) an HCDR1 comprising the amino acid sequence of SEQ ID NO:69, an HCDR2} comprising the amino acid sequence of SEQ ID NO:79, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:92; _{l) an HCDR1 comprising the amino acid sequence of SEQ ID NO:70, an HCDR2} comprising the amino acid sequence of SEQ ID NO:80, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:93; _{m) an HCDR1 comprising the amino acid sequence of SEQ ID NO:70, an HCDR2} comprising the amino acid sequence of SEQ ID NO:81, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:94; _{n) an HCDR1 comprising the amino acid sequence of SEQ ID NO:70, an HCDR2} comprising the amino acid sequence of SEQ ID NO:82, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:94; - 54 - 4181706.v1 5431.1032002 o_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:71, an HCDR2} comprising the amino acid sequence of SEQ ID NO:82, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:95; p_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:71, an HCDR2} comprising the amino acid sequence of SEQ ID NO:82, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:94; q_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:72, an HCDR2} comprising the amino acid sequence of SEQ ID NO:83, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:95; r_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:72, an HCDR2} comprising the amino acid sequence of SEQ ID NO:83, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:96; s_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:73, an HCDR2} comprising the amino acid sequence of SEQ ID NO:84, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:97; or t_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:73, an HCDR2} comprising the amino acid sequence of SEQ ID NO:85, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:98. _{[00195] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:62, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:74, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:86; b_{) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:62, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:74, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:220; c_{) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:63, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:75, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:87; d_{) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:63, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:75, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:221; - 55 - 4181706.v1 5431.1032002 _{e) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:64, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:75, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:88; _{f) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:65, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:76, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:88; _{g) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:65, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:76, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:222; _{h) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:66, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:77, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:89; _{i) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:67, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:78, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:90; _{j) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:68, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:79, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:91; _{k) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:69, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:79, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:92; _{l) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:70, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:80, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:93; _{m) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:70, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:81, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:94; _{n) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:70, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:82, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:94; _{o) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:71, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:82, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:95; - 56 - 4181706.v1 5431.1032002 p_{) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:71, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:82, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:94; q_{) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:72, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:83, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:95; r_{) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:72, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:83, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:96; s_{) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:73, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:84, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:97; or t_{) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:73, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:85, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:98. _{[00196] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 having at least 80% sequence identity (e.g., having at least 81%, 82%,} 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of SEQ ID NO:62, b_{) an HCDR2 having at least 80% sequence identity (e.g., having at least 81%, 82%,} 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of SEQ ID NO:74, and c_{) an HCDR3 having at least 80% sequence identity (e.g., having at least 81%, 82%,} 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of SEQ ID NO:86. _{[00197] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:62, b) an HCDR2 comprising the amino acid sequence of SEQ ID NO:74, and} - 57 - 4181706.v1 5431.1032002 c_{) an HCDR3 comprising the amino acid sequence of SEQ ID NO:86. [00198] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:62, b) an HCDR2 consisting of the amino acid sequence of SEQ ID NO:74, and c) an HCDR3 consisting of the amino acid sequence of SEQ ID NO:86. [00199] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 having at least 80% sequence identity (e.g., having at least 81%, 82%,} 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of SEQ ID NO:65, b_{) an HCDR2 having at least 80% sequence identity (e.g., having at least 81%, 82%,} 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of SEQ ID NO:76, and c_{) an HCDR3 having at least 80% sequence identity (e.g., having at least 81%, 82%,} 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of SEQ ID NO:88. _{[00200] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:65, b) an HCDR2 comprising the amino acid sequence of SEQ ID NO:76, and c) an HCDR3 comprising the amino acid sequence of SEQ ID NO:88. [00201] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:65, b) an HCDR2 consisting of the amino acid sequence of SEQ ID NO:76, and c) an HCDR3 consisting of the amino acid sequence of SEQ ID NO:88.} Heavy Chain Variable Domain (V_H) _{[00202] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises an immunoglobulin heavy chain variable domain (V_H). In some embodiments, an anti- - 58 - 4181706.v1 5431.1032002 TFR1 antibody or antigen-binding fragment thereof comprises a mammalian V_H domain. In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof comprises a Camelidae (e.g., llama, camel, or alpaca) V_H domain. In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof comprises a humanized V_H domain (e.g., at least about: 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% humanized). In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof comprises human framework regions. _{[00203] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises a V_H domain having at least 70% sequence identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity) to the amino acid sequence of at least one of SEQ ID NOs:99-123 and 176-199. _{[00204] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises a V_H domain having at least 70% sequence identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity) to the amino acid sequence of at least one of SEQ ID NOs:99-123. _{[00205] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises a V_H domain having at least 70% sequence identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity) to the amino acid sequence of at least one of SEQ ID NOs:176-199. _{[00206] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises a V_H domain having at least 70% sequence identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity) to the amino acid sequence of SEQ ID NO:176. _{[00207] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises a V_H domain having at least 70% sequence identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity) to the amino acid sequence of SEQ ID NO:190. _{[00208] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises a V_H domain comprising at least one amino acid substitution relative to the amino acid sequence of at least one of SEQ ID NOs:99-123 and 176-199. For example, the number of amino acid substitutions can be at least about: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, or about: 1-20, 1-19, 2-19, 2-18, 2-17, 3-17, 3-16, 4-16, 4-15, 5-15, 5-14, 6-14, 6-13, 7-13, 7-12, 8-12, 8-11 or 9-11. In some embodiments, an anti-TFR1 antibody or antigen-binding - 59 - 4181706.v1 5431.1032002 fragment thereof comprises a V_H that comprises about 1-10 amino acid substitutions, relative to the amino acid sequence of at least one of SEQ ID NOs:99-123 and 176-199. _{[00209] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises a V_H domain comprising at least one amino acid substitution relative to the amino acid sequence of at least one of SEQ ID NOs:99-123. For example, the number of amino acid substitutions can be at least about: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, or about: 1-20, 1-19, 2-19, 2-18, 2-17, 3-17, 3-16, 4-16, 4-15, 5-15, 5-14, 6-14, 6-13, 7-13, 7- 12, 8-12, 8-11 or 9-11. In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof comprises a V_H that comprises about 1-10 amino acid substitutions, relative to the amino acid sequence of at least one of SEQ ID NOs:99-123. _{[00210] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises a V_H domain comprising at least one amino acid substitution relative to the amino acid sequence of at least one of SEQ ID NOs:176-199. For example, the number of amino acid substitutions can be at least about: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, or about: 1-20, 1-19, 2-19, 2-18, 2-17, 3-17, 3-16, 4-16, 4-15, 5-15, 5-14, 6-14, 6-13, 7-13, 7- 12, 8-12, 8-11 or 9-11. In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof comprises a V_H that comprises about 1-10 amino acid substitutions, relative to the amino acid sequence of at least one of SEQ ID NOs:176-199. _{[00211] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises a V_H domain comprising at least one amino acid substitution relative to the amino acid sequence of SEQ ID NO:176. For example, the number of amino acid substitutions can be at least about: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, or about: 1-20, 1-19, 2-19, 2-18, 2-17, 3-17, 3-16, 4-16, 4-15, 5-15, 5-14, 6-14, 6-13, 7-13, 7-12, 8-12, 8-11 or 9-11. In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof comprises a V_H that comprises about 1-10 amino acid substitutions, relative to the amino acid sequence of SEQ ID NO:176. _{[00212] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises a V_H domain comprising at least one amino acid substitution relative to the amino acid sequence of SEQ ID NO:190. For example, the number of amino acid substitutions can be at least about: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, or about: 1-20, 1-19, 2-19, 2-18, 2-17, 3-17, 3-16, 4-16, 4-15, 5-15, 5-14, 6-14, 6-13, 7-13, 7-12, 8-12, 8-11 or 9-11. In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof comprises a - 60 - 4181706.v1 5431.1032002 V_H that comprises about 1-10 amino acid substitutions, relative to the amino acid sequence of SEQ ID NO:190. _{[00213] In some embodiments, an amino acid substitution is a conservative substitution. The} term “a conservative amino acid substitution” or “a conservative substitution” refers to an amino acid substitution having a value of 0 or greater in BLOSUM62. _{[00214] In some embodiments, an amino acid substitution is a highly conservative} substitution. The term “a highly conservative amino acid substitution” or “a highly conservative substitution” refers to an amino acid substitution having a value of at least 1 (e.g., at least 2) in BLOSUM62. _{[00215] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises a V_H having 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs:99-123 and 176-199. In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof comprises a V_H comprising (e.g., consisting of) the amino acid sequence of any one of SEQ ID NOs:99-123 and 176-199. _{[00216] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises a V_H having 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs:99-123. In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof comprises a V_H comprising (e.g., consisting of) the amino acid sequence of any one of SEQ ID NOs:99-123. _{[00217] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises a V_H having 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs:176-199. In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof comprises a V_H comprising (e.g., consisting of) the amino acid sequence of any one of SEQ ID NOs:176-199. _{[00218] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises a V_H having 100% sequence identity to the amino acid sequence of SEQ ID NO:176. In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof comprises a V_H comprising (e.g., consisting of) the amino acid sequence of SEQ ID NO:176. _{[00219] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} comprises a V_H having 100% sequence identity to the amino acid sequence of SEQ ID NO:190. In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof comprises a V_H comprising (e.g., consisting of) the amino acid sequence of SEQ ID NO:190. - 61 - 4181706.v1 5431.1032002 Conjugates _{[00220] In some embodiments, an anti-TFR1 antibody or antigen-binding fragment thereof} (e.g., a hTFR1-sdAb) is linked to an agent (e.g., a therapeutic to be transported across the blood- brain barrier) via a linker. _{[00221] In some embodiments, a linkage is enzymatically cleavable (e.g., by an enzyme} present in the central nervous system). Non-limiting examples of enzymatically cleavable linkers include acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers, and disulfide-containing linkers. _{[00222] An agent may be linked to any region of an anti-TFR1 antibody or antigen-binding} fragment thereof herein (e.g., a hTFR1-sdAb), so long as the agent does not interfere with binding of an anti-TFR1 antibody or antigen-binding fragment thereof to the transferrin receptor. In some embodiments, an agent is linked to the N-terminal region of an anti-TFR1 antibody or antigen-binding fragment thereof (e.g., a hTFR1-sdAb). In some embodiments, an agent is linked to the C-terminal region of an anti-TFR1 antibody or antigen-binding fragment thereof. _{[00223] In some embodiments, an agent is a therapeutic agent, for example, a peptide, a DNA} molecule, an RNA molecule, or a chemical moiety. In some embodiments, an agent is a cytotoxic agent. In some embodiments, an agent is a drug for a neurological disorder. _{[00224] In some embodiments, an agent is a diagnostic agent (e.g., an imaging agent). [00225] In some embodiments, an agent is less than 1,000 Da (e.g., less than: 900, 800, 750,} 600, 500, 400, 300, or 250 Da). _{[00226] In some embodiments, a conjugate is generated using a chemical cross-linking} reagent. In some embodiments, a chemical cross-linking reagent is a heterobifunctional cross- linker. Non-limiting examples of heterobifunctional cross-linkers include EDC (l-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride), IT (iminothiolane), LC-SPDP (succinimidyl 6-[3-(2- pyridyldithio)propionate]hexanoate), MBS (m-maleimidobenzoyl-N- hydroxysuccinimide ester), NHS (N-hydroxysuccinimide), SIAB (N-succinimidyl (4-iodoacetyl) aminobenzoate), SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-l- carboxylate), SMPB (succinimidyl 4-(p- maleimidophenyl)butyrate), SMPT (4-succinimidyloxycarbonyl-a- methyl-a-(2-pyridyldithio)-toluene), SPDP (N-succinimidyl 3-(2-pyridyldithio)propionate), and sulfo-NHS (N- hydroxysulfosuccinimide). In some embodiments, a heterobifunctional cross- linker is a derivative bifunctional imidoester (e.g., dimethyl adipimidate HCl), an active ester (e.g., disuccinimidyl suberate), an aldehyde (e.g., glutaraldehyde), a bis-azido compound (e.g., bis(pazidobenzoyl)hexanediamine), a bis-diazonium derivative (e.g., bis-(p-diazoniobenzoyl)- - 62 - 4181706.v1 5431.1032002 ethylenediamine), a diisocyanates (e.g., toluene-2,6-diisocyanate), or a bis-active fluorine compound (e.g., 1,5-difluoro-2,4-dinitrobenzene). _{[00227] In some embodiments, a cross-linking agent having a N-hydroxysuccinimide moiety} is obtained as a N-hydroxysulfosuccinimide analog. In some embodiments, a cross-linking agent having a disulfide bridge within the linking chain is synthesized as an alkyl derivative (e.g., to reduce the amount of linker cleavage in vivo). _{[00228] In some embodiments, a chemical cross-linking reagent is a homobifunctional cross-} linker. Non-limiting examples of homobifunctional cross-linkers include DSS (Disuccinimidyl subcrate), BMH (bismaleimidohexane) and DMP (dimethylpimelimidate.2HCl). _{[00229] In some embodiments, a chemical cross-linking reagent is a photoreactive cross-} linker. Non-limiting examples of photoreactive cross-linkers include BASED (bis-[B-(4- azidosalicylamido)ethyl]disulfide) and SANPAH (N- succinimidyl-6(4’-azido-2’- nitrophenylamino)hexanoate). _{[00230] In some embodiments, a linker is a peptide linker. In some embodiments, a peptide} linker is configured to allow for rotation between an agent and an anti-TFR1 antibody or antigen- binding fragment thereof (e.g., a hTFR1-sdAb) relative to each other. In some embodiments, a peptide linker is configured to allow for resistant to digestion by one or more proteases. In some embodiments, a linker is a flexible linker (containing amino acids such as Gly, Asn, Ser, Thr, Ala, and/or Gly-Ser repeats). In some embodiments, a peptide linker is at least 10 amino acids in length, for example, at least: 12, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length. In some embodiments, a peptide linker is at least 25 amino acids in length. In some embodiments, a peptide linker is about 10-70 amino acids in length, for example, 12-70, 12-65, 15-65, 15-60, 20- 60, 20-55, 25-55, 25-50, 30-50, 30-45, 35-45, or 35-40 amino acids in length. Polypeptides _{[00231] The disclosure also provides, among other things, a polypeptide comprising at least} one anti-TFR1 antibody or antigen-binding fragment thereof disclosed herein. _{[00232] In some embodiments, a polypeptide is recombinantly produced. In some} embodiments, a polypeptide is synthetically produced. _{[00233] In some embodiments, a polypeptide has spontaneous BBB permeability. In some} embodiments, a polypeptide has an isoelectric point (pI) of 9-10 (e.g., 9.1-10, 9.1-9.9, 9.2-9.9, 9.2-9.8, 9.3-9.8, 9.3-9.7, 9.4-9.7, 9.4-9.6, or 9.5-9.6). In some embodiments, a polypeptide has an isoelectric point (pI) of about 9.5. - 63 - 4181706.v1 5431.1032002 _{[00234] In some embodiments, a polypeptide comprises an isolated antibody. In some} embodiments, a polypeptide comprises an isolated antigen-binding fragment of an antibody. _{[00235] In some embodiments, a polypeptide comprises a bispecific antibody or an antigen-} binding fragment thereof. In some embodiments, a polypeptide comprises a multispecific antibody or an antigen-binding fragment thereof. _{[00236] In some embodiments, a polypeptide comprises an isolated sdAb. [00237] In some embodiments, a polypeptide comprises a monomeric sdAb. [00238] In some embodiments, a polypeptide comprises a bispecific sdAb. In some} embodiments, a bispecific sdAb is a bispecific monomeric sdAb. See, e.g., Hanke et al., A bispecific monomeric nanobody induces spike trimer dimers and neutralizes SARS-CoV-2 in vivo, Nat Commun.13(1):155 (2022). In some embodiments, a bispecific sdAb is a bispecific dimeric sdAb. See, e.g., Biesemann et al., Additive efficacy of a bispecific anti-TNF/IL-6 nanobody compound in translational models of rheumatoid arthritis, Sci Transl Med. 15(681):eabq4419 (2023), the contents of which are incorporated herein by reference. In some embodiments, a bispecific sdAb is a bispecific multimeric (e.g., trimeric) sdAb. See, e.g., PCT application PCT/EP2006/004678 (WO2006122786), the contents of which are incorporated herein by reference. Additional information on bispecific or multispecific sdAbs and preparation thereof can be found in, e.g., Conrath et al., Camel single-domain antibodies as modular building units in bispecific and bivalent antibody constructs, J Biol Chem.276(10):7346-50 (2001), Muyldermans, Single domain camel antibodies: current status, J Biotechnol.74(4):277- 302 (2001), and PCT applications PCT/EP1996/001725 (WO1996034103) and PCT/EP1998/006991 (WO1999023221), the contents of which are incorporated herein by reference. _{[00239] In some embodiments, a polypeptide comprises a multispecific sdAb. [00240] In some embodiments, a polypeptide comprises a dimeric sdAb. In some} embodiments, a polypeptide comprises a multimeric (e.g., trimeric) sdAb. _{[00241] In some embodiments, a polypeptide comprises one anti-TFR1 sdAb. [00242] In some embodiments, a polypeptide comprises two anti-TFR1 sdAbs. In some} embodiments, a polypeptide comprises two identical anti-TFR1 sdAbs. In some embodiments, a polypeptide comprises two non-identical anti-TFR1 sdAbs. _{[00243] In some embodiments, a polypeptide further comprises a therapeutic agent, a} diagnostic agent, a tag (e.g., a protein tag), or a combination thereof. In some embodiments, a polypeptide further comprises a diagnostic agent. In some embodiments, a polypeptide further - 64 - 4181706.v1 5431.1032002 comprises a tag (e.g., a protein tag). In some embodiments, a polypeptide further comprises a therapeutic agent. _{[00244] In some embodiments, a polypeptide comprises: a) an anti-TFR1sdAb, and b) a therapeutic agent. [00245] In some embodiments, a polypeptide comprises the following elements arranged in} the following sequence: a_{) an anti-TFR1 sdAb, and b) a therapeutic agent. [00246] In some embodiments, a polypeptide comprises the following elements arranged in} the following sequence: a_{) a therapeutic agent, and b) an anti-TFR1 sdAb. [00247] In some embodiments, a polypeptide comprises the following elements arranged in} the following sequence: a_{) a first therapeutic agent, b) an anti-TFR1 sdAb, and c) a second therapeutic agent. [00248] In some embodiments, a second therapeutic agent is identical to a first therapeutic} agent. In some embodiments, a second therapeutic agent differs from a first therapeutic agent. _{[00249] In some embodiments, a therapeutic agent comprises an antibody or antigen-binding} fragment thereof. In some embodiments, a therapeutic agent comprises a sdAb. _{[00250] In some embodiments, a polypeptide comprises two sdAbs connected by a linker} sequence (e.g., GGGGSGGGS (SEQ ID NO:130)). _{[00251] In some embodiments, a polypeptide comprises three sdAbs connected by linker} sequences (e.g., GGGGSGGGS (SEQ ID NO:130)). Anti-Tumor Necrosis Factor-Alpha (TNF-α) Antibodies or Antigen-Binding Fragments Thereof _{[00252] In some embodiments, a therapeutic agent comprises an anti-tumor necrosis factor-} alpha (TNF-α) antibody or antigen-binding fragment thereof. _{[00253] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof is} a full-length (e.g., whole, intact) antibody. In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof is an antigen-binding fragment comprising (e.g., consisting of) a V_H region, a single-domain antibody (sdAb), a Fab fragment, a F(ab’)₂ fragment, a Fd - 65 - 4181706.v1 5431.1032002 fragment, a Fv fragment, or a domain antibody (dAb). In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof is a sdAb, Fab, F(ab’)₂, Fab’, scFv, or Fv. In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof is a sdAb. _{[00254] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof is} an isolated antibody. In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof is an isolated antigen-binding fragment of an antibody. In some embodiments, an anti- TNF-α antibody or antigen-binding fragment thereof is an isolated sdAb. _{[00255] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof is} recombinantly produced. In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof is synthetically produced. _{[00256] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof is} produced recombinantly or synthetically, using routine methods and reagents that are well known in the art. For example, an antibody or antigen-binding fragment thereof can be produced recombinantly in a suitable host cell (e.g., bacteria) according to methods known in the art. See, e.g., Current Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992; and Molecular Cloning: a Laboratory Manual, 2nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press. For example, a nucleic acid molecule comprising a nucleotide sequence encoding an anti-TNF-α antibody or antigen-binding fragment thereof can be introduced and expressed in a suitable host cell (e.g., E. coli), and the expressed anti-TNF-α antibody or antigen-binding fragment thereof can be isolated/purified from the host cell (e.g., in inclusion bodies) using routine methods and readily available reagents. For example, DNA fragments coding for different protein sequences (e.g., a light-responsive domain, a heterologous peptide component) can be ligated together in-frame in accordance with conventional techniques. In another embodiment, an antibody or antigen-binding fragment thereof can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of nucleic acid fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive nucleic acid fragments that can subsequently be annealed and re-amplified to generate a chimeric nucleic acid sequence (see e.g., Ausubel et al., Current Protocols in Molecular Biology, 1992). _{[00257] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} binds (e.g., specifically binds) human TNF-α. [00258] - 66 - 4181706.v1 5431.1032002 Complementarity-Determining Regions _{[00259] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} comprises an immunoglobulin heavy chain variable (V_H) domain comprising a heavy chain complementarity-determining region (HCDR) 1, an HCDR2, and an HCDR3 having at least 80% sequence identity (e.g., having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to an HCDR1, an HCDR2, and an HCDR3, respectively, of a V_H domain comprising the amino acid sequence of any one of SEQ ID NOs:225, 223, 224, 226-228, and 132-137. _{[00260] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} comprises a V_H domain comprising an HCDR1, an HCDR2, and an HCDR3 having 100% sequence identity to an HCDR1, an HCDR2, and an HCDR3, respectively, of a V_H domain comprising the amino acid sequence of any one of SEQ ID NOs:225, 223, 224, 226-228, and 132-137. _{[00261] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} comprises a V_H domain comprising an HCDR1, an HCDR2, and an HCDR3 having at least 80% sequence identity (e.g., having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to an HCDR1, an HCDR2, and an HCDR3, respectively, of a V_H domain comprising the amino acid sequence of any one of SEQ ID NOs:132-137. _{[00262] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} comprises a V_H domain comprising an HCDR1, an HCDR2, and an HCDR3 having 100% sequence identity to an HCDR1, an HCDR2, and an HCDR3, respectively, of a V_H domain comprising the amino acid sequence of any one of SEQ ID NOs:132-137. _{[00263] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} comprises a V_H domain comprising an HCDR1, an HCDR2, and an HCDR3 having at least 80% sequence identity (e.g., having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to an HCDR1, an HCDR2, and an HCDR3, respectively, of a V_H domain comprising the amino acid sequence of any one of SEQ ID NOs:135-137. _{[00264] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} comprises a V_H domain comprising an HCDR1, an HCDR2, and an HCDR3 having 100% sequence identity to an HCDR1, an HCDR2, and an HCDR3, respectively, of a V_H domain comprising the amino acid sequence of any one of SEQ ID NOs:135-137. - 67 - 4181706.v1 5431.1032002 _{[00265] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} comprises a V_H domain comprising an HCDR1, an HCDR2, and an HCDR3 having at least 80% sequence identity (e.g., having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to an HCDR1, an HCDR2, and an HCDR3, respectively, of a V_H domain comprising the amino acid sequence of any one of SEQ ID NOs:223-228. _{[00266] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} comprises a V_H domain comprising an HCDR1, an HCDR2, and an HCDR3 having 100% sequence identity to an HCDR1, an HCDR2, and an HCDR3, respectively, of a V_H domain comprising the amino acid sequence of any one of SEQ ID NOs:223-228. _{[00267] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} comprises a V_H domain comprising an HCDR1, an HCDR2, and an HCDR3 having at least 80% sequence identity (e.g., having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to an HCDR1, an HCDR2, and an HCDR3, respectively, of a V_H domain comprising the amino acid sequence of SEQ ID NO:225. _{[00268] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} comprises a V_H domain comprising an HCDR1, an HCDR2, and an HCDR3 having 100% to an HCDR1, an HCDR2, and an HCDR3, respectively, of a V_H domain comprising the amino acid sequence of SEQ ID NO:225. _{[00269] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} comprises a V_H domain comprising an HCDR1, an HCDR2, and an HCDR3 having at least 80% sequence identity (e.g., having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to an HCDR1, an HCDR2, and an HCDR3, respectively, of a V_H domain comprising the amino acid sequence of SEQ ID NO:223. _{[00270] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} comprises a V_H domain comprising an HCDR1, an HCDR2, and an HCDR3 having 100% to an HCDR1, an HCDR2, and an HCDR3, respectively, of a V_H domain comprising the amino acid sequence of SEQ ID NO:223. - 68 - 4181706.v1 5431.1032002 _{[00271] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 having at least 80% sequence identity (e.g., having at least 81%, 82%,} 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of SEQ ID NO:229, b_{) an HCDR2 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:230, and c_{) an HCDR3 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:231. _{[00272] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 having at least 80% sequence identity (e.g., having at least 81%, 82%,} 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of SEQ ID NO:138, b_{) an HCDR2 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:139 or 232, and c_{) an HCDR3 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:140. _{[00273] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 having at least 80% sequence identity (e.g., having at least 81%, 82%,} 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of SEQ ID NO:138, b_{) an HCDR2 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:139, and c_{) an HCDR3 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:140. - 69 - 4181706.v1 5431.1032002 _{[00274] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 having at least 80% sequence identity (e.g., having at least 81%, 82%,} 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of SEQ ID NO:138, b_{) an HCDR2 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:232, and c_{) an HCDR3 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:140. _{[00275] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 having at least 80% sequence identity (e.g., having at least 81%, 82%,} 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of SEQ ID NO:141, b_{) an HCDR2 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:142, and c_{) an HCDR3 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:143. _{[00276] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 having at least 80% sequence identity (e.g., having at least 81%, 82%,} 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of SEQ ID NO:144, b_{) an HCDR2 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:145, and c_{) an HCDR3 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:140. - 70 - 4181706.v1 5431.1032002 _{[00277] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 having at least 80% sequence identity (e.g., having at least 81%, 82%,} 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of SEQ ID NO:147, b_{) an HCDR2 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:148, and c_{) an HCDR3 having at least 80% sequence identity to the amino acid sequence of} SEQ ID NO:149. _{[00278] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} comprises a V_H domain comprising: a_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:229, an HCDR2} comprising the amino acid sequence of SEQ ID NO:230, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:231; b_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:138, an HCDR2} comprising the amino acid sequence of SEQ ID NO:232, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:140; c_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:138, an HCDR2} comprising the amino acid sequence of SEQ ID NO:139, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:140; d_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:141, an HCDR2} comprising the amino acid sequence of SEQ ID NO:142, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:143; e_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:144, an HCDR2} comprising the amino acid sequence of SEQ ID NO:145, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:140; or f_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:147, an HCDR2} comprising the amino acid sequence of SEQ ID NO:148, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:149. _{[00279] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:229,} - 71 - 4181706.v1 5431.1032002 b_{) an HCDR2 comprising the amino acid sequence of SEQ ID NO:230, and c) an HCDR3 comprising the amino acid sequence of SEQ ID NO:231. [00280] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:138, b) an HCDR2 comprising the amino acid sequence of SEQ ID NO:139 or 232, and c) an HCDR3 comprising the amino acid sequence of SEQ ID NO:140. [00281] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:138, b) an HCDR2 comprising the amino acid sequence of SEQ ID NO:139, and c) an HCDR3 comprising the amino acid sequence of SEQ ID NO:140. [00282] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:138, b) an HCDR2 comprising the amino acid sequence of SEQ ID NO:232, and c) an HCDR3 comprising the amino acid sequence of SEQ ID NO:140. [00283] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:141, b) an HCDR2 comprising the amino acid sequence of SEQ ID NO:142, and c) an HCDR3 comprising the amino acid sequence of SEQ ID NO:143. [00284] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:144, b) an HCDR2 comprising the amino acid sequence of SEQ ID NO:145, and c) an HCDR3 comprising the amino acid sequence of SEQ ID NO:140. [00285] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} comprises: a_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:147, b) an HCDR2 comprising the amino acid sequence of SEQ ID NO:148, and c) an HCDR3 comprising the amino acid sequence of SEQ ID NO:149.} - 72 - 4181706.v1 5431.1032002 Heavy Chain Variable Domain (V_H) _{[00286] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} comprises a V_H. In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof comprises a mammalian V_H domain. In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof comprises a Camelidae (e.g., llama, camel, or alpaca) V_H domain. In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof comprises a humanized V_H domain (e.g., at least about: 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% humanized). In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof comprises human framework regions. _{[00287] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} comprises a V_H domain having at least 70% sequence identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity) to the amino acid sequence of at least one of SEQ ID NOs:225, 223, 224, 226-228, and 132-137. _{[00288] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} comprises a V_H domain having at least 70% sequence identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity) to the amino acid sequence of at least one of SEQ ID NOs:132-137. _{[00289] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} comprises a V_H domain having at least 70% sequence identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity) to the amino acid sequence of at least one of SEQ ID NOs:223-228. _{[00290] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} comprises a V_H domain having at least 70% sequence identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity) to the amino acid sequence of SEQ ID NO:225. _{[00291] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} comprises a V_H domain having at least 70% sequence identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity) to the amino acid sequence of SEQ ID NO:223. _{[00292] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} comprises a V_H domain comprising at least one amino acid substitution relative to the amino acid sequence of at least one of SEQ ID NOs:225, 223, 224, 226-228, and 132-137. For example, the number of amino acid substitutions can be at least about: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, or about: 1-20, 1-19, 2-19, 2-18, 2-17, 3-17, 3-16, 4-16, 4-15, 5-15, 5- - 73 - 4181706.v1 5431.1032002 14, 6-14, 6-13, 7-13, 7-12, 8-12, 8-11 or 9-11. In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof comprises a V_H that comprises about 1-10 amino acid substitutions, relative to the amino acid sequence of at least one of SEQ ID NOs:225, 223, 224, 226-228, and 132-137. _{[00293] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} comprises a V_H domain comprising at least one amino acid substitution relative to the amino acid sequence of at least one of SEQ ID NOs:132-137. For example, the number of amino acid substitutions can be at least about: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, or about: 1-20, 1-19, 2-19, 2-18, 2-17, 3-17, 3-16, 4-16, 4-15, 5-15, 5-14, 6-14, 6-13, 7-13, 7- 12, 8-12, 8-11 or 9-11. In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof comprises a V_H that comprises about 1-10 amino acid substitutions, relative to the amino acid sequence of at least one of SEQ ID NOs:132-137. _{[00294] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} comprises a V_H domain comprising at least one amino acid substitution relative to the amino acid sequence of at least one of SEQ ID NOs:223-228. For example, the number of amino acid substitutions can be at least about: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, or about: 1-20, 1-19, 2-19, 2-18, 2-17, 3-17, 3-16, 4-16, 4-15, 5-15, 5-14, 6-14, 6-13, 7-13, 7- 12, 8-12, 8-11 or 9-11. In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof comprises a V_H that comprises about 1-10 amino acid substitutions, relative to the amino acid sequence of at least one of SEQ ID NOs:223-228. _{[00295] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} comprises a V_H domain comprising at least one amino acid substitution relative to the amino acid sequence of SEQ ID NO:225. For example, the number of amino acid substitutions can be at least about: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, or about: 1-20, 1-19, 2-19, 2-18, 2-17, 3-17, 3-16, 4-16, 4-15, 5-15, 5-14, 6-14, 6-13, 7-13, 7-12, 8-12, 8-11 or 9-11. In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof comprises a V_H that comprises about 1-10 amino acid substitutions, relative to the amino acid sequence of SEQ ID NO:225. _{[00296] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} comprises a V_H domain comprising at least one amino acid substitution relative to the amino acid sequence of SEQ ID NO:223. For example, the number of amino acid substitutions can be at least about: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, or about: 1-20, 1-19, 2-19, 2-18, 2-17, 3-17, 3-16, 4-16, 4-15, 5-15, 5-14, 6-14, 6-13, 7-13, 7-12, 8-12, 8-11 or 9-11. - 74 - 4181706.v1 5431.1032002 In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof comprises a V_H that comprises about 1-10 amino acid substitutions, relative to the amino acid sequence of SEQ ID NO:223. _{[00297] In some embodiments, an amino acid substitution is a conservative substitution. The} term “a conservative amino acid substitution” or “a conservative substitution” refers to an amino acid substitution having a value of 0 or greater in BLOSUM62. _{[00298] In some embodiments, an amino acid substitution is a highly conservative} substitution. The term “a highly conservative amino acid substitution” or “a highly conservative substitution” refers to an amino acid substitution having a value of at least 1 (e.g., at least 2) in BLOSUM62. _{[00299] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} comprises a V_H having 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs:225, 223, 224, 226-228, and 132-137. In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof comprises a V_H comprising (e.g., consisting of) the amino acid sequence of any one of SEQ ID NOs:225, 223, 224, 226-228, and 132-137. _{[00300] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} comprises a V_H having 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs:132-137. In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof comprises a V_H comprising (e.g., consisting of) the amino acid sequence of any one of SEQ ID NOs:132-137. _{[00301] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} comprises a V_H having 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs:223-228. In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof comprises a V_H comprising (e.g., consisting of) the amino acid sequence of any one of SEQ ID NOs:223-228. _{[00302] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} comprises a V_H having 100% sequence identity to the amino acid sequence of SEQ ID NO:225. In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof comprises a V_H comprising (e.g., consisting of) the amino acid sequence of SEQ ID NO:225. _{[00303] In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof} comprises a V_H having 100% sequence identity to the amino acid sequence of SEQ ID NO:223. In some embodiments, an anti-TNF-α antibody or antigen-binding fragment thereof comprises a V_H comprising (e.g., consisting of) the amino acid sequence of SEQ ID NO:223. - 75 - 4181706.v1 5431.1032002 _{[00304] In some embodiments, a polypeptide comprises: a) one anti-TFR1 sdAb and one anti-TNF-α sdAb; or b) one anti-TFR1 sdAb and two anti-TNF-α sdAbs. [00305] In some embodiments, a polypeptide comprises an amino acid sequence that has at} least 70% sequence identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity) to the amino acid sequence of at least one of SEQ ID NOs:201, 200, 202-215, 124, and 125. _{[00306] In some embodiments, a polypeptide comprises an amino acid sequence that has} 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs:201, 200, 202- 215, 124, and 125. _{[00307] In some embodiments, a polypeptide comprises one anti-TFR1 sdAb and two anti-} TNF-α sdAbs. _{[00308] In some embodiments, a polypeptide comprises an amino acid sequence that has at} least 70% sequence identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity) to the amino acid sequence of at least one of SEQ ID NOs:201, 200, 202-215. _{[00309] In some embodiments, a polypeptide comprises an amino acid sequence that has} 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs:201, 200, 202- 215. _{[00310] In some embodiments, a polypeptide comprises an amino acid sequence that has at} least 70% sequence identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity) to the amino acid sequence of at least one of SEQ ID NOs:200, 201, 208, and 209. _{[00311] In some embodiments, a polypeptide comprises an amino acid sequence that has} 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs:200, 201, 208, and 209. _{[00312] In some embodiments, a polypeptide comprises an amino acid sequence that has at} least 70% sequence identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity) to the amino acid sequence of SEQ ID NO:200 or 201. _{[00313] In some embodiments, a polypeptide comprises an amino acid sequence that has} 100% sequence identity to the amino acid sequence of SEQ ID NO:200 or 201. - 76 - 4181706.v1 5431.1032002 _{[00314] In some embodiments, a polypeptide comprises an amino acid sequence that has at} least 70% sequence identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity) to the amino acid sequence of SEQ ID NO:200. _{[00315] In some embodiments, a polypeptide comprises an amino acid sequence that has} 100% sequence identity to the amino acid sequence of SEQ ID NO:200. _{[00316] In some embodiments, a polypeptide comprises an amino acid sequence that has at} least 70% sequence identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity) to the amino acid sequence of SEQ ID NO:201. _{[00317] In some embodiments, a polypeptide comprises an amino acid sequence that has} 100% sequence identity to the amino acid sequence of SEQ ID NO:201. _{[00318] In some embodiments, a polypeptide comprises an amino acid sequence that has at} least 70% sequence identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity) to the amino acid sequence of SEQ ID NO:208 or 209. _{[00319] In some embodiments, a polypeptide comprises an amino acid sequence that has} 100% sequence identity to the amino acid sequence of SEQ ID NO:208 or 209. _{[00320] In some embodiments, a polypeptide comprises an amino acid sequence that has at} least 70% sequence identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity) to the amino acid sequence of SEQ ID NO:208. _{[00321] In some embodiments, a polypeptide comprises an amino acid sequence that has} 100% sequence identity to the amino acid sequence of SEQ ID NO:208. _{[00322] In some embodiments, a polypeptide comprises an amino acid sequence that has at} least 70% sequence identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity) to the amino acid sequence of SEQ ID NO:209. _{[00323] In some embodiments, a polypeptide comprises an amino acid sequence that has} 100% sequence identity to the amino acid sequence of SEQ ID NO:209. _{[00324] In some embodiments, a polypeptide comprises one anti-TFR1 sdAb and one anti-} TNF-α sdAb. - 77 - 4181706.v1 5431.1032002 _{[00325] In some embodiments, a polypeptide comprises an amino acid sequence that has at} least 70% sequence identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity) to the amino acid sequence of SEQ ID NO:124 or 125. _{[00326] In some embodiments, a polypeptide comprises an amino acid sequence that has} 100% sequence identity to the amino acid sequence of SEQ ID NO:124 or 125. Tags _{[00327] In some embodiments, a polypeptide further comprises a protein tag (e.g., for} detecting and/or purifying an antibody or antigen-binding fragment thereof). In some embodiments, a protein tag comprises a polyhistidine-tag. _{[00328] Non-limiting examples of protein tags include His-tag (e.g., 6xHis-tag), Strep-tag,} Avi-tag, and Flag-tag. In some embodiments, a protein tag (e.g., 6xHis-tag (SEQ ID NO:126)) is at the C-terminal end of a polypeptide. In some embodiments, a protein tag (e.g., 6xHis-tag (SEQ ID NO:126)) is at the N-terminal end of a polypeptide. _{[00329] 6xHis-tag: HHHHHH (SEQ ID NO:126) [00330] Strep-tag: WSHPQFEK (SEQ ID NO:127) [00331] Avi-tag: GLNDIFEAQKIEWHE (SEQ ID NO:128) [00332] 3X Flag-tag: DYKDHDGDYKDHDIDYKDDDDK (SEQ ID NO:129)} Modifications _{[00333] In some embodiments, a polypeptide is modified, e.g., conjugated to a heterologous} moiety. The term “conjugated” refers to attached, via a covalent or noncovalent interaction. Conjugation can employ any of suitable linking agents; non-limiting examples include peptide linkers, compound linkers, and chemical cross-linking agents. _{[00334] In some embodiments, a heterologous moiety comprises a marker (e.g., a fluorescent} or radioactive marker), a molecule that stabilizes the antibody or the antigen-binding fragment thereof, a molecule that targets the antibody or the antigen-binding fragment thereof (e.g., to a particular tissue or cell, to facilitate crossing the blood brain barrier), a therapeutic agent, a diagnostic agent, or any combination thereof. _{[00335] In some embodiments, a heterologous moiety is selected from polyethylene glycol} (PEG), hexadecanoic acid, hydrogels, nanoparticles, multimerization domains and carrier peptides. In some embodiments, a nanoparticle is a lipid nanoparticle. In some embodiments, a nanoparticle is a polymer nanoparticle. In some embodiments, a polymer is an amphiphilic - 78 - 4181706.v1 5431.1032002 polymer. In some embodiments, a polymer is a hydrophobic or hydrophilic polymer. Non- limiting examples of polymers include poly(lactic acid)-poly(ethylene glycol), poly(lactic-co- glycolic acid)-poly(ethylene glycol), poly(lactic-co-glycolic acid) (PLGA), poly(lactic-co- glycolic acid)-d-α-tocopheryl polyethylene glycol succinate, poly(lactic-co-glycolic acid)- ethylene oxide fumarate, poly(glycolic acid)-poly(ethylene glycol), polycaprolactone- poly(ethylene glycol), or any salts thereof. In some embodiments, a polymer nanoparticle comprises PLGA. _{[00336] In some embodiments, a heterologous moiety facilitates transport of a polypeptide} across the blood brain barrier (BBB), for example, by increasing lipid solubility of the polypeptide, increasing intra- and/or intercellular vesicular transport, or increasing receptor- mediated transcytosis (RMT). See, e.g., Bellettato & Scarpa, Possible strategies to cross the blood-brain barrier, Ital J Pediatr.44(Suppl 2):131 (2018), Ceña & Játiva, Nanoparticle crossing of blood-brain barrier: a road to new therapeutic approaches to central nervous system diseases, Nanomedicine (Lond).13(13):1513-16 (2018), Pulgar, Transcytosis to Cross the Blood Brain Barrier, New Advancements and Challenges, Front Neurosci.12:1019 (2019), and Wu et al., The blood-brain barrier: structure, regulation, and drug delivery, Signal Transduct Target Ther.8(1):217 (2023), the contents of which are incorporated by reference herein in their entirety. _{[00337] In some embodiments, a heterologous moiety facilitates transport of a polypeptide} across the BBB by increasing lipid solubility of the polypeptide. In some embodiments, a heterologous moiety is or comprises a lipid group or functional group (e.g., to be added to the polar ends of a polypeptide). _{[00338] In some embodiments, a heterologous moiety facilitates transport of a polypeptide} across the BBB by increasing intra- and/or intercellular vesicular transport. In some embodiments, a heterologous moiety is or comprises lipid- and polymer-based nanoparticles (NPs) (e.g., g7-NP). _{[00339] In some embodiments, a heterologous moiety facilitates transport of a polypeptide} across the BBB by increasing RMT. In some embodiments, a heterologous moiety is or comprises a ligand (e.g., an endogenous ligand or an antibody or antigen-binding fragment) that binds an exofacial epitope on a BBB receptor. Non-limiting examples of receptor systems capable of mediating RMT cargo delivery through the BBB include insulin receptor (IR), and low density lipoprotein receptor (LDLR). - 79 - 4181706.v1 5431.1032002 _{[00340] In some embodiments, a polypeptide is linked to a second polypeptide. The term} “linked” means attached, via a covalent or noncovalent interaction. Conjugation can employ a suitable linking agent. Non-limiting examples include peptide linkers, compound linkers, and chemical cross-linking agents. In some embodiments, a linker is a disulfide bond. _{[00341] In some embodiments, a polypeptide is produced recombinantly or synthetically,} using routine methods and reagents that are well known in the art. For example, a polypeptide can be produced recombinantly in a suitable host cell (e.g., bacteria) according to methods known in the art. See, e.g., Current Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992; and Molecular Cloning: a Laboratory Manual, 2nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press. For example, a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide can be introduced and expressed in a suitable host cell (e.g., E. coli), and the expressed polypeptide can be isolated/purified from the host cell (e.g., in inclusion bodies) using routine methods and readily available reagents. For example, DNA fragments coding for different protein sequences (e.g., a light-responsive domain, a heterologous peptide component) can be ligated together in-frame in accordance with conventional techniques. In another embodiment, a polypeptide can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of nucleic acid fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive nucleic acid fragments that can subsequently be annealed and re-amplified to generate a chimeric nucleic acid sequence (see e.g., Ausubel et al., Current Protocols in Molecular Biology, 1992). Nucleic Acids & Vectors _{[00342] The disclosure also provides, among other things, a polynucleotide (e.g., DNA, RNA,} or an analog of either, e.g., optionally including one or more modified nucleotides) encoding any one or more of the antibodies or antigen-binding fragments thereof, or polypeptides disclosed herein. In some embodiments, a polynucleotide is a DNA. In some embodiments, a polynucleotide is an RNA. In some embodiments, a polynucleotide is linear (e.g., a linear DNA or a linear RNA). In some embodiments, a polynucleotide is circular (e.g., a circular DNA or a circular RNA). In some embodiments, a polynucleotide comprises a nucleotide sequence that is codon-optimized for a chosen cell (e.g., a host cell). _{[00343] In some embodiments, an antibody or antigen-binding fragment thereof, or} polypeptide is encoded by a single polynucleotide. In some embodiments, an antibody or antigen-binding fragment thereof, or polypeptide is encoded by multiple polynucleotides. - 80 - 4181706.v1 5431.1032002 _{[00344] In some embodiments, a polynucleotide encodes a sdAb. In some embodiments, a} polynucleotide encodes a monomeric sdAb. In some embodiments, a polynucleotide encodes a dimeric sdAb. In some embodiments, a polynucleotide encodes a multimeric (e.g., trimeric) sdAb. _{[00345] The disclosure also provides, among other things, a vector (e.g., an expression vector,} including a viral-delivery vector) comprising any one or more of the polynucleotides disclosed herein. _{[00346] In some embodiments, a vector (e.g., expression vector) further comprises an} expression control polynucleotide sequence operably linked to the polynucleotide, and/or a polynucleotide sequence encoding a selectable marker. In some embodiments, an expression control polynucleotide sequence comprises a promoter sequence and/or an enhancer sequence. In some embodiments, an expression control polynucleotide sequence comprises an inducible promoter sequence. Host Cells and Methods of Production _{[00347] The disclosure also provides, among other things, a host cell (e.g., a recombinant cell)} comprising any one or more of the antibodies or antigen binding fragments thereof, polypeptides, polynucleotides, and/or expression vectors disclosed herein. _{[00348] Non-limiting examples of host cells (e.g., recombinant cells) include mammalian} cells such as hybridoma cells, Chinese hamster ovary (CHO) cells, CV-1 Origin defective SV-40 (COS) cells, human embryonic kidney (HEK), yeast cells such as Pichia pastoris cells, and bacterial cells such as E. coli, including DH5α competent cells. _{[00349] The disclosure also provides, among other things, a method of producing any one or} more of the antibodies or antigen binding fragments thereof, or polypeptides disclosed herein, comprising expressing the antibodies or antigen binding fragments thereof, or polypeptides in a host cell disclosed herein and isolating the expressed antibodies or antigen binding fragments thereof, or polypeptides. Compositions & Kits _{[00350] The disclosure also provides, among other things, a composition comprising any one} or more of the anti-TFR1 antibodies or antigen-binding fragments thereof, polypeptides, polynucleotides, vectors, or host cells (e.g., recombinant cells) disclosed herein, and a carrier or diluent (e.g., a pharmaceutically acceptable carrier or diluent). In some embodiments, a composition is a pharmaceutical composition. - 81 - 4181706.v1 5431.1032002 _{[00351] In some embodiments, a composition (e.g., pharmaceutical composition) further} comprises at least one additional therapeutic agent. _{[00352] In some embodiments, a composition (e.g., pharmaceutical composition) further} comprises: a_{) an anti-tau antibody, b) an anti-amyloid-beta (Aβ) antibody, c) a BRI2 peptide, d) an amyloid precursor protein (APP)-targeting agent, or} any combination of the foregoing. _{[00353] In some embodiments, a composition (e.g., pharmaceutical composition) further} comprises pharmaceutically acceptable carriers, excipients, stabilizers, diluents or tonifiers (Remington’s Pharmaceutical Sciences 16^th edition, Osol, A. Ed. (1980)). Suitable pharmaceutically acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed. Non-limiting examples of pharmaceutically acceptable carriers, excipients, stabilizers, diluents or tonifiers include buffers (e.g., L-histidine), antioxidants (e.g., ascorbic acid or methionine), preservatives, proteins (e.g., serum albumin, gelatin or immunoglobulins), hydrophilic polymers, amino acids, carbohydrates (e.g., monosaccharides, disaccharides, glucose, mannose or dextrins), chelating agents (e.g., EDTA), sugars (e.g., sucrose), salt-forming counter-ions (e.g., sodium), metal complexes (e.g., Zn-protein complexes), non-ionic surfactants (e.g., Tween), PLURONICS™ and polyethylene glycol (PEG). _{[00354] In some embodiments, a composition comprises an antibody or an antigen-binding} fragment thereof and any one of a) to e): a_{) L-arginine hydrochloride, sodium chloride, and sucrose, b) mannitol, sucrose, and tromethamine, c) sucrose, polysorbate 80, monobasic sodium phosphate (monohydrate), and dibasic} sodium phosphate (dihydrate), d_{) mannitol and polysorbate 80, e) citric acid monohydrate, dibasic sodium phosphate dihydrate, mannitol,} monobasic sodium phosphate dihydrate, polysorbate 80, sodium chloride, and sodium citrate. _{[00355] In some embodiments, a composition (e.g., a pharmaceutical composition) is} formulated for a suitable administration schedule and route. Non-limiting examples of - 82 - 4181706.v1 5431.1032002 administration routes include intra-cerebroventricular, intranasal, intraperitoneal, intrathecal, intravenous, oral, peri-spinal, and subcutaneous. In some embodiments, a composition (e.g., a pharmaceutical composition) is stored in the form of an aqueous solution or a dried formulation (e.g., lyophilized). In some embodiments, a composition is formulated to be administered by infusion (e.g., intravenous infusion). In other embodiments a composition is formulated for subcutaneous administration. _{[00356] In some embodiments, a composition is provided in a dosage form, e.g., in a prefilled} syringe, prefilled pen, or autoinjector. _{[00357] In some embodiments, a pharmaceutical composition comprises from about 5 mg to} about 300 mg of an antibody or an antigen-binding fragment thereof disclosed herein, for example, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 80 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 10-300 mg, about 10-250 mg, about 15-250 mg, about 15-200 mg, about 20-200 mg, about 20-150 mg, about 30-150 mg, about 30-100 mg, about 40-100 mg, about 40-80 mg, about 50-80 mg, or about 50-60 mg. _{[00358] In some embodiments, a composition is formulated to be administered with at least} one additional therapeutic agent as a combination therapy. In some embodiments, at least one additional therapeutic agent comprises an anti-serum albumin agent, such as an anti-serum albumin sdAb. In some embodiments, at least one additional therapeutic agent comprises methotrexate. _{[00359] The disclosure also provides, among other things, kits comprising a container and} optionally an instruction for use, wherein the container comprises any one or more of the compositions (e.g., pharmaceutical compositions) disclosed herein. Methods of Use _{[00360] The disclosure also provides, among other things, a method of treating a disease in a} subject in need thereof, comprising administering to the subject a therapeutically effective amount of any one or more of the polypeptides, polynucleotides, expression vectors, host cells, or compositions (e.g., pharmaceutical compositions) disclosed herein, thereby treating the disease. _{[00361] In some embodiments, a disease is a brain disease, for example, a brain tumor, brain} malformation, a cerebellar disorder, concussion, or a neurodegenerative disease. _{[00362] In some embodiments, a disease is a neurodegenerative disease. Non-limiting} examples of neurodegenerative diseases include Alzheimer’s disease (AD), amyotrophic lateral - 83 - 4181706.v1 5431.1032002 sclerosis (ALS), Creutzfeldt-Jakob disease, dementia with Lewy bodies (LBD), frontotemporal dementia (FTD), Huntington’s disease (HD), mixed dementia, multiple sclerosis (MS), multiple system atrophy (MSA), Parkinson’s disease (PD), prion diseases, spinocerebellar ataxias (SCAs), tauopathies, and vascular dementia. _{[00363] In some embodiments, a neurodegenerative disease is a late onset disease. [00364] In some embodiments, a neurodegenerative disease affects basal ganglia, thalamus,} red nucleus, locus coeruleus, parahippocampal gyrus, or a combination thereof. In some embodiments, a neurodegenerative disease affects basal ganglia and/or thalamus. _{[00365] In some embodiments, a disease is a neurological disorder. Non-limiting examples of} neurological disorders include, amyloidosis, Angelman syndrome, a behavioral disorder, bulbar palsy, a cancer (e.g., a CD20-positive cancer with brain metastases, a Her2-positive cancer with brain metastases), a central nervous system (CNS) inflammation, cystic fibrosis, an eye disease disorder, hepatolenticular degeneration, ischemia, Lafora disease, Lesch-Nyhan syndrome, Liddle syndrome, a lysosomal storage disease, stroke, Menkes syndrome, muscular dystrophy, a neurodegenerative disease (e.g., Alexander’s disease, Alper's disease, Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), ataxia telangiectasia, Batten disease, Canavan disease, Cockayne syndrome, corticobasal degeneration, Creutzfeldt-Jakob disease, dementia (e.g., dementia with Lewy bodies (LBD), frontotemporal dementia, HIV-related dementia, mixed dementia, vascular dementia), Friedreich's ataxia, frontotemporal lobar degeneration, Huntington’s disease, Kennedy disease, microbial diseases, motor neuron diseases, multiple sclerosis, multiple system atrophy, muscle atrophy spinal cord, Parkinson’s disease, primary lateral sclerosis, prion diseases (e.g., bovine spongiform encephalopathy, scrapie, kuru, Gerstmann-Straussler-Scheinker disease, chronic wasting disease, and fatal familial insomnia), progressive supranuclear palsy, Refsum’s disease, spinocerebellar ataxias (SCAs), tauopathies (e.g., episodic age-related tauopathy), Tourette’s syndrome, transverse myelitis), neuronal ceroid lipofuscinosis, a neuropathic disorder, Paget’s disease, Pick’s disease, progressive supranuclear palsy, Rett syndrome, a seizure disorder, spinal muscular atrophy, traumatic brain injury, Unverricht-Lundborg syndrome, and viral diseases. _{[00366] Non-limiting examples of neuropathic disorders include pain caused by injury to a} tissue, cancer-related pain, neuropathic pain (pain caused by abnormalities in the nerves, spinal cord, or brain), and psychogenic pain, headache, migraine, neuropathy, and associated symptoms (e.g., vertigo or nausea). In some embodiment, a neuropathic disorder is a chronic pain (e.g., nociceptive pain). - 84 - 4181706.v1 5431.1032002 _{[00367] Non-limiting examples of amyloidosis include Alzheimer’s disease (AD),} amyotrophic lateral sclerosis (ALS), cerebral amyloid angiopathy, Creutzfeldt-Jakob disease, dementia with Lewy bodies (LBD), Down’s syndrome, eye diseases related to amyloid beta deposition (e.g., macular degeneration, optic neuropathy related to drusen and cataracts), hereditary cerebral hemorrhage with amyloidosis (Dutch type), HIV-related dementia, Huntington’s disease, inclusion body myositis (IBM), mild cognitive impairment (MCI), multiple sclerosis, Parkinson-Guam dementia complex, Parkinson’s disease, secondary amyloidosis, progressive supranuclear palsy, senile amyloidosis, and transmissible spongiform encephalopathy. _{[00368] Non-limiting examples of CNS cancers include acoustic neuroma, astrocytoma,} chondroma, CNS metastases from peripheral tumors (e.g., CD20- positive cancers or HER2- positive cancers), extradural tumors, ganglioglioma, glioblastoma multiforme, glioma, intradural tumors, intramedullary tumors, medulloblastomas, meningioma, neuroblastoma, neurofibroma, oligodendroglioma, and schwannoma. _{[00369] Non-limiting examples of eye disorders (e.g., diseases) include aniridia, Argyll} Robertson’s sign, disorders of the ciliary body (e.g., arc eye, corneal abrasion, corneal ulcer, Fuchs’ dystrophy, iritis, keratitis, keratoconjunctivitis sicca, keratoconus, niphablepsia, scleritis, superficial punctate keratitis, Thygeson’s corneal neovascularization, and uveitis), choroidal and retinal disorders (e.g., retinal detachment, retinoschisis, hypertensive retinopathy, diabetic retinopathy, retinopathy , retinopathy of prematurity, age-related macular degeneration, macular degeneration (wet or dry), epiretinal membrane, retinitis pigmentosa and macular edema), color blindness, conjunctivitis, cornea, eye muscle disorders/binocular movement/accommodation/refraction (e.g., strabismus, ophthalmoparesis, progressive external ophthalmoplegia, convergent strabismus, divergent strabismus, hyperopia, myopia, astigmatism, anisometropia, presbyopia, and ophthalmoplegia), iris, lens disorders (e.g., cataracts), keratomycosis, and sclera, glaucoma, floaters, optic nerve and visual pathway disorders (e.g., Leber hereditary optic neuropathy and drusen optic disc), visual disturbances and blindness (e.g., amblyopia, Lever’s congenital amaurosis, scotoma, achromatopsia, nocturnal amblyopia, blindness, onchocerciasis and microphthalmia/coloboma), and xerophthalmia. _{[00370] Non-limiting examples of viral or microbial CNS infections include infections with} bacteria (e.g., Neisseria sp., Streptococcus sp., Pseudomonas sp. , Proteus sp., E. coli, S. aureus, Pneumococcus sp., Meningococcus sp., Haemophilus sp., and Mycobacterium tuberculosis), infections with viruses (e.g., influenza, HIV, poliovirus, rubella), and infections with other - 85 - 4181706.v1 5431.1032002 microorganisms (e.g., fungi (e.g., yeasts, Cryptococcus neoformans), parasites (e.g., Toxoplasma gondii) or amoebas), resulting in CNS pathophysiologies (e.g., meningitis, encephalitis, myelitis, vasculitis, and abscess). In some embodiments, a CNS pathophysiology is acute. In some embodiments, a CNS pathophysiology is chronic. _{[00371] Non-limiting examples of CNS ischemia include aneurysm, focal cerebral ischemia,} global cerebral ischemia, and stroke (e.g., subarachnoid hemorrhage and intracerebral hemorrhage). _{[00372] Non-limiting examples of seizures include abdominal epilepsy, akinetic seizures,} atonic seizures, autonomic seizures, catamenial epilepsy, complex partial seizures, Dravet syndrome, emotional seizures, epilepsy (e.g., absence seizures, atonic seizures, benign rolandic epilepsy, infantile absence seizure, and clonic seizures), febrile seizures, focal seizures, frontal lobe epilepsy, gelastic seizures, generalized onset seizures, infantile spasms, Jacksonian progression, juvenile myoclonic epilepsy, juvenile absence epilepsy, Lafora disease, Landau- Kleffner syndrome, Lennox-Gastaut syndrome, limbic epilepsy, massive bilateral myoclonus, mild seizures, mitochondrial disorders, motor seizures, multifocal seizures, myoclonic seizures, nocturnal seizures, Ohtahara syndrome, partial-onset seizures, photosensitive seizures, progressive myoclonic epilepsies, psychogenic seizures (pseudoseizures), psychomotor seizures, Rasmussen syndrome, reflex epilepsy, secondary generalized seizures, sensory seizures, simple partial seizures, status epileptic, Sylvan seizures, temporal lobe epilepsy, tonic-clonic seizures, tonic seizures, visual reflex seizures, West syndrome, and withdrawal seizures. _{[00373] Non-limiting examples of behavioral disorders include behavioral and developmental} disorders (e.g., autism , Rett syndrome, Asperger syndrome), eating disorders (e.g., anorexia or bulimia), mood disorders (e.g., depression, depression with suicide attempt, anxiety, chronic affective disorders, phobias, panic attacks, obsessive-compulsive disorder, attention deficit hyperactivity disorder (ADHD), attention deficit disorder (ADD), chronic fatigue syndrome, agoraphobia, post-traumatic stress disorder, bipolar disorder), personality disorders, psychosis, psychotic disorders (e.g., schizophrenia, delusional disorder), and sleep disorders (e.g., insomnia, parasomnias, night terrors, circadian rhythm sleep disorders and narcolepsy). _{[00374] Non-limiting examples of lysosomal storage diseases include Canavan} leukodystrophy, Fabry disease, Farber’s disease, Gaucher disease, glycogenosis, GM1 gangliosidosis, Krabbe disease, metachromatic leukodystrophy, mucopolysaccharidoses (types I, II, III, IV, V, VI, and VII), neuronal ceroid lipofuscinosis, Niemann-Pick disease, Pompe disease, and Tay-Sachs disease. - 86 - 4181706.v1 5431.1032002 _{[00375] In some embodiments, a subject is a mammal. In some embodiments, a subject is a} mammal selected from the group consisting of a dog, a cat, a mouse, a rat, a hamster, a guinea pig, a horse, a pig, a sheep, a cow, a chimpanzee, a macaque, a cynomolgus, and a human. In some embodiments, a subject is a primate. In some embodiments, a subject is a human. _{[00376] In some embodiments, a subject (e.g., a human patient) is 2 years or older. In some} embodiments, a subject (e.g., a human patient) is 4 years or older. In some embodiments, a subject (e.g., a human patient) is 5 years or older. In some embodiments, a subject (e.g., a human patient) is 6 years or older. In some embodiments, a subject (e.g., a human patient) is 12 years or older. _{[00377] In some embodiments, a subject is a pediatric human patient. In some embodiments, a} subject is 2 to 17 years of age, for example, 4 to 17 years of age, 5 to 17 years of age, 6 to 17 years of age, or 12 to 17 years of age. In some embodiments, a subject is 2 to 11 years of age, for example, 4 to 11 years of age, 5 to 11 years of age, or 6 to 11 years of age. _{[00378] In some embodiments, a subject is an adult human patient. In some embodiments, a} subject is 18 years of age or older, for example, about: 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or 80 years of age or older. In some embodiments, a subject is about: 18-80, 20-80, 20-75, 25- 75, 25-70, 30-70, 30-65, 35-65, 35-60, 40-60, 40-55, 45-55 years of age. In some embodiments, a subject is about 18-65 years of age. In some embodiments, a subject is 65 years of age or older. _{[00379] The disclosure also provides, among other things, a method of blocking binding of} TNF-α to TNF receptor (TNFR) expressed on a surface of a cell, the method comprising contacting the cell with an effective amount of any one or more of the polypeptides, polynucleotides, expression vectors, host cells, or compositions (e.g., pharmaceutical compositions) disclosed herein, thereby blocking binding of TNF-α to TNFR expressed on the surface of the cell. _{[00380] The disclosure also provides, among other things, a method of blocking binding of} TNF-α to TNFR in a subject, the method comprising administering to the subject an effective amount of any one or more of the polypeptides, polynucleotides, expression vectors, host cells, or compositions (e.g., pharmaceutical compositions) disclosed herein, thereby blocking the binding of TNF-α to TNFR in the subject. _{[00381] The disclosure also provides, among other things, a method of treating a TNF-α-} associated disease in a subject in need thereof, comprising administering to the subject an effective amount of any one or more of the polypeptides, polynucleotides, expression vectors, - 87 - 4181706.v1 5431.1032002 host cells, or compositions (e.g., pharmaceutical compositions) disclosed herein, thereby treating the TNF-α-associated disease. _{[00382] The disclosure also provides, among other things, a method of reducing inflammation} in a subject in need thereof, comprising administering to the subject an effective amount of any one or more of the polypeptides, polynucleotides, expression vectors, host cells, or compositions (e.g., pharmaceutical compositions) disclosed herein, thereby reducing inflammation in the subject. _{[00383] In some embodiments, a TNF-α-associated disease is Alzheimer’s disease (AD). In} some embodiments, a TNF-α-associated disease is late onset Alzheimer’s disease (LOAD). In some embodiments, a TNF-α-associated disease is an inflammatory disease. In some embodiments, a TNF-α-associated disease is an autoimmune disease. In some embodiments, a TNF-α-associated disease is rheumatoid arthritis (RA) (e.g., moderately to severely active RA), juvenile idiopathic arthritis (JIA) (e.g., polyarticular JIA), psoriatic arthritis (PsA), ankylosing spondylitis (AS), plaque psoriasis (PsO or Ps, e.g., moderate to severe chronic plaque psoriasis), Crohn’s disease (CD), pediatric Crohn’s disease, ulcerative colitis (UC), pediatric ulcerative colitis, hidradenitis suppurativa (HS), or uveitis (UV). EMBODIMENTS _{1. An antibody or an antigen-binding fragment thereof, comprising an immunoglobulin} heavy chain variable (V_H) domain comprising a heavy chain complementarity- determining region (HCDR) 1, an HCDR2, and an HCDR3 having 100% sequence identity to an HCDR1, an HCDR2, and an HCDR3, respectively, of a V_H domain comprising the amino acid sequence of any one of SEQ ID NOs:99-123. _{2. An antibody or an antigen-binding fragment thereof, comprising an immunoglobulin} heavy chain variable (V_H) domain comprising a heavy chain complementarity- determining region (HCDR) 1, an HCDR2, and an HCDR3 having 100% sequence identity to an HCDR1, an HCDR2, and an HCDR3, respectively, of a V_H domain comprising the amino acid sequence of any one of SEQ ID NOs:99-105, 107, 110-112, and 118-120. _{3. The antibody or the antigen-binding fragment thereof of Embodiment 1, wherein:} - 88 - 4181706.v1 5431.1032002 _{a) the HCDR1 comprises the amino acid sequence of SEQ ID NO:2, the HCDR2} comprises the amino acid sequence of SEQ ID NO:14, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:26; _{b) the HCDR1 comprises the amino acid sequence of SEQ ID NO:3, the HCDR2} comprises the amino acid sequence of SEQ ID NO:15, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:27; _{c) the HCDR1 comprises the amino acid sequence of SEQ ID NO:4, the HCDR2} comprises the amino acid sequence of SEQ ID NO:15, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:28; _{d) the HCDR1 comprises the amino acid sequence of SEQ ID NO:5, the HCDR2} comprises the amino acid sequence of SEQ ID NO:16, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:28; _{e) the HCDR1 comprises the amino acid sequence of SEQ ID NO:6, the HCDR2} comprises the amino acid sequence of SEQ ID NO:17, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:29; _{f) the HCDR1 comprises the amino acid sequence of SEQ ID NO:7, the HCDR2} comprises the amino acid sequence of SEQ ID NO:18, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:30; _{g) the HCDR1 comprises the amino acid sequence of SEQ ID NO:8, the HCDR2} comprises the amino acid sequence of SEQ ID NO:19, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:31; _{h) the HCDR1 comprises the amino acid sequence of SEQ ID NO:9, the HCDR2} comprises the amino acid sequence of SEQ ID NO:19, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:32; _{i) the HCDR1 comprises the amino acid sequence of SEQ ID NO:10, the HCDR2} comprises the amino acid sequence of SEQ ID NO:20, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:33; _{j) the HCDR1 comprises the amino acid sequence of SEQ ID NO:10, the HCDR2} comprises the amino acid sequence of SEQ ID NO:21, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:34; _{k) the HCDR1 comprises the amino acid sequence of SEQ ID NO:10, the HCDR2} comprises the amino acid sequence of SEQ ID NO:22, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:34; - 89 - 4181706.v1 5431.1032002 l_{) the HCDR1 comprises the amino acid sequence of SEQ ID NO:11, the HCDR2} comprises the amino acid sequence of SEQ ID NO:22, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:35; m_{) the HCDR1 comprises the amino acid sequence of SEQ ID NO:11, the HCDR2} comprises the amino acid sequence of SEQ ID NO:22, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:34; n_{) the HCDR1 comprises the amino acid sequence of SEQ ID NO:12, the HCDR2} comprises the amino acid sequence of SEQ ID NO:23, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:36; o_{) the HCDR1 comprises the amino acid sequence of SEQ ID NO:12, the HCDR2} comprises the amino acid sequence of SEQ ID NO:23, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:37; p_{) the HCDR1 comprises the amino acid sequence of SEQ ID NO:13, the HCDR2} comprises the amino acid sequence of SEQ ID NO:24, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:38; or q_{) the HCDR1 comprises the amino acid sequence of SEQ ID NO:13, the HCDR2} comprises the amino acid sequence of SEQ ID NO:25, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:39. _{4. The antibody or the antigen-binding fragment thereof of Embodiment 1 or 3, wherein: a) the HCDR1 consists of the amino acid sequence of SEQ ID NO:2, the HCDR2} consists of the amino acid sequence of SEQ ID NO:14, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:26; b_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:3, the HCDR2} consists of the amino acid sequence of SEQ ID NO:15, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:27; c_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:4, the HCDR2} consists of the amino acid sequence of SEQ ID NO:15, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:28; d_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:5, the HCDR2} consists of the amino acid sequence of SEQ ID NO:16, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:28; - 90 - 4181706.v1 5431.1032002 _{e) the HCDR1 consists of the amino acid sequence of SEQ ID NO:6, the HCDR2} consists of the amino acid sequence of SEQ ID NO:17, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:29; _{f) the HCDR1 consists of the amino acid sequence of SEQ ID NO:7, the HCDR2} consists of the amino acid sequence of SEQ ID NO:18, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:30; _{g) the HCDR1 consists of the amino acid sequence of SEQ ID NO:8, the HCDR2} consists of the amino acid sequence of SEQ ID NO:19, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:31; _{h) the HCDR1 consists of the amino acid sequence of SEQ ID NO:9, the HCDR2} consists of the amino acid sequence of SEQ ID NO:19, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:32; _{i) the HCDR1 consists of the amino acid sequence of SEQ ID NO:10, the HCDR2} consists of the amino acid sequence of SEQ ID NO:20, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:33; _{j) the HCDR1 consists of the amino acid sequence of SEQ ID NO:10, the HCDR2} consists of the amino acid sequence of SEQ ID NO:21, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:34; _{k) the HCDR1 consists of the amino acid sequence of SEQ ID NO:10, the HCDR2} consists of the amino acid sequence of SEQ ID NO:22, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:34; _{l) the HCDR1 consists of the amino acid sequence of SEQ ID NO:11, the HCDR2} consists of the amino acid sequence of SEQ ID NO:22, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:35; _{m) the HCDR1 consists of the amino acid sequence of SEQ ID NO:11, the HCDR2} consists of the amino acid sequence of SEQ ID NO:22, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:34; _{n) the HCDR1 consists of the amino acid sequence of SEQ ID NO:12, the HCDR2} consists of the amino acid sequence of SEQ ID NO:23, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:36; _{o) the HCDR1 consists of the amino acid sequence of SEQ ID NO:12, the HCDR2} consists of the amino acid sequence of SEQ ID NO:23, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:37; - 91 - 4181706.v1 5431.1032002 p_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:13, the HCDR2} consists of the amino acid sequence of SEQ ID NO:24, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:38; or q_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:13, the HCDR2} consists of the amino acid sequence of SEQ ID NO:25, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:39. _{5. The antibody or the antigen-binding fragment thereof of any one of Embodiments 1-3,} wherein: a_{) the HCDR1 comprises the amino acid sequence of SEQ ID NO:2, the HCDR2} comprises the amino acid sequence of SEQ ID NO:14, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:26; b_{) the HCDR1 comprises the amino acid sequence of SEQ ID NO:3, the HCDR2} comprises the amino acid sequence of SEQ ID NO:15, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:27; c_{) the HCDR1 comprises the amino acid sequence of SEQ ID NO:5, the HCDR2} comprises the amino acid sequence of SEQ ID NO:16, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:28; d_{) the HCDR1 comprises the amino acid sequence of SEQ ID NO:8, the HCDR2} comprises the amino acid sequence of SEQ ID NO:19, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:31; e_{) the HCDR1 comprises the amino acid sequence of SEQ ID NO:9, the HCDR2} comprises the amino acid sequence of SEQ ID NO:19, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:32; f_{) the HCDR1 comprises the amino acid sequence of SEQ ID NO:12, the HCDR2} comprises the amino acid sequence of SEQ ID NO:23, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:36; or g_{) the HCDR1 comprises the amino acid sequence of SEQ ID NO:12, the HCDR2} comprises the amino acid sequence of SEQ ID NO:23, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:37. _{6. The antibody or the antigen-binding fragment thereof of any one of Embodiments 1-5,} wherein: - 92 - 4181706.v1 5431.1032002 a_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:2, the HCDR2} consists of the amino acid sequence of SEQ ID NO:14, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:26; b_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:3, the HCDR2} consists of the amino acid sequence of SEQ ID NO:15, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:27; c_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:5, the HCDR2} consists of the amino acid sequence of SEQ ID NO:16, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:28; d_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:8, the HCDR2} consists of the amino acid sequence of SEQ ID NO:19, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:31; e_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:9, the HCDR2} consists of the amino acid sequence of SEQ ID NO:19, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:32; f_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:12, the HCDR2} consists of the amino acid sequence of SEQ ID NO:23, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:36; or g_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:12, the HCDR2} consists of the amino acid sequence of SEQ ID NO:23, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:37. _{7. The antibody or the antigen-binding fragment thereof of Embodiment 1, wherein: a) the HCDR1 comprises the amino acid sequence of SEQ ID NO:40, the HCDR2} comprises the amino acid sequence of SEQ ID NO:48, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:26; b_{) the HCDR1 comprises the amino acid sequence of SEQ ID NO:41, the HCDR2} comprises the amino acid sequence of SEQ ID NO:49, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:27; c_{) the HCDR1 comprises the amino acid sequence of SEQ ID NO:41, the HCDR2} comprises the amino acid sequence of SEQ ID NO:50, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:28; - 93 - 4181706.v1 5431.1032002 _{d) the HCDR1 comprises the amino acid sequence of SEQ ID NO:41, the HCDR2} comprises the amino acid sequence of SEQ ID NO:51, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:28; _{e) the HCDR1 comprises the amino acid sequence of SEQ ID NO:42, the HCDR2} comprises the amino acid sequence of SEQ ID NO:52, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:29; _{f) the HCDR1 comprises the amino acid sequence of SEQ ID NO:43, the HCDR2} comprises the amino acid sequence of SEQ ID NO:53, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:30; _{g) the HCDR1 comprises the amino acid sequence of SEQ ID NO:44, the HCDR2} comprises the amino acid sequence of SEQ ID NO:54, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:31; _{h) the HCDR1 comprises the amino acid sequence of SEQ ID NO:44, the HCDR2} comprises the amino acid sequence of SEQ ID NO:54, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:32; _{i) the HCDR1 comprises the amino acid sequence of SEQ ID NO:45, the HCDR2} comprises the amino acid sequence of SEQ ID NO:55, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:33; _{j) the HCDR1 comprises the amino acid sequence of SEQ ID NO:45, the HCDR2} comprises the amino acid sequence of SEQ ID NO:56, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:34; _{k) the HCDR1 comprises the amino acid sequence of SEQ ID NO:45, the HCDR2} comprises the amino acid sequence of SEQ ID NO:57, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:34; _{l) the HCDR1 comprises the amino acid sequence of SEQ ID NO:45, the HCDR2} comprises the amino acid sequence of SEQ ID NO:57, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:35; _{m) the HCDR1 comprises the amino acid sequence of SEQ ID NO:45, the HCDR2} comprises the amino acid sequence of SEQ ID NO:57, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:34; _{n) the HCDR1 comprises the amino acid sequence of SEQ ID NO:46, the HCDR2} comprises the amino acid sequence of SEQ ID NO:58, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:36; - 94 - 4181706.v1 5431.1032002 o_{) the HCDR1 comprises the amino acid sequence of SEQ ID NO:46, the HCDR2} comprises the amino acid sequence of SEQ ID NO:59, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:37; p_{) the HCDR1 comprises the amino acid sequence of SEQ ID NO:47, the HCDR2} comprises the amino acid sequence of SEQ ID NO:60, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:38; or q_{) the HCDR1 comprises the amino acid sequence of SEQ ID NO:47, the HCDR2} comprises the amino acid sequence of SEQ ID NO:61, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:39. _{8. The antibody or the antigen-binding fragment thereof of Embodiment 1 or 7, wherein: a) the HCDR1 consists of the amino acid sequence of SEQ ID NO:40, the HCDR2} consists of the amino acid sequence of SEQ ID NO:48, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:26; b_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:41, the HCDR2} consists of the amino acid sequence of SEQ ID NO:49, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:27; c_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:41, the HCDR2} consists of the amino acid sequence of SEQ ID NO:50, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:28; d_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:41, the HCDR2} consists of the amino acid sequence of SEQ ID NO:51, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:28; e_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:42, the HCDR2} consists of the amino acid sequence of SEQ ID NO:52, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:29; f_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:43, the HCDR2} consists of the amino acid sequence of SEQ ID NO:53, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:30; g_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:44, the HCDR2} consists of the amino acid sequence of SEQ ID NO:54, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:31; - 95 - 4181706.v1 5431.1032002 h_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:44, the HCDR2} consists of the amino acid sequence of SEQ ID NO:54, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:32; i_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:45, the HCDR2} consists of the amino acid sequence of SEQ ID NO:55, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:33; j_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:45, the HCDR2} consists of the amino acid sequence of SEQ ID NO:56, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:34; k_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:45, the HCDR2} consists of the amino acid sequence of SEQ ID NO:57, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:34; l_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:45, the HCDR2} consists of the amino acid sequence of SEQ ID NO:57, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:35; m_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:45, the HCDR2} consists of the amino acid sequence of SEQ ID NO:57, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:34; n_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:46, the HCDR2} consists of the amino acid sequence of SEQ ID NO:58, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:36; o_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:46, the HCDR2} consists of the amino acid sequence of SEQ ID NO:59, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:37; p_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:47, the HCDR2} consists of the amino acid sequence of SEQ ID NO:60, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:38; or q_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:47, the HCDR2} consists of the amino acid sequence of SEQ ID NO:61, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:39. _{9. The antibody or the antigen-binding fragment thereof of any one of Embodiments 1, 2,} and 7, wherein: - 96 - 4181706.v1 5431.1032002 a_{) the HCDR1 comprises the amino acid sequence of SEQ ID NO:40, the HCDR2} comprises the amino acid sequence of SEQ ID NO:48, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:26; b_{) the HCDR1 comprises the amino acid sequence of SEQ ID NO:41, the HCDR2} comprises the amino acid sequence of SEQ ID NO:49, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:27; c_{) the HCDR1 comprises the amino acid sequence of SEQ ID NO:41, the HCDR2} comprises the amino acid sequence of SEQ ID NO:51, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:28; d_{) the HCDR1 comprises the amino acid sequence of SEQ ID NO:44, the HCDR2} comprises the amino acid sequence of SEQ ID NO:54, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:31; e_{) the HCDR1 comprises the amino acid sequence of SEQ ID NO:44, the HCDR2} comprises the amino acid sequence of SEQ ID NO:54, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:32; f_{) the HCDR1 comprises the amino acid sequence of SEQ ID NO:46, the HCDR2} comprises the amino acid sequence of SEQ ID NO:58, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:36; or g_{) the HCDR1 comprises the amino acid sequence of SEQ ID NO:46, the HCDR2} comprises the amino acid sequence of SEQ ID NO:59, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:37. _{10. The antibody or the antigen-binding fragment thereof of any one of Embodiments 1, 2,} and 7-9, wherein: a_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:40, the HCDR2} consists of the amino acid sequence of SEQ ID NO:48, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:26; b_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:41, the HCDR2} consists of the amino acid sequence of SEQ ID NO:49, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:27; c_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:41, the HCDR2} consists of the amino acid sequence of SEQ ID NO:51, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:28; - 97 - 4181706.v1 5431.1032002 d_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:44, the HCDR2} consists of the amino acid sequence of SEQ ID NO:54, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:31; e_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:44, the HCDR2} consists of the amino acid sequence of SEQ ID NO:54, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:32; f_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:46, the HCDR2} consists of the amino acid sequence of SEQ ID NO:58, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:36; or g_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:46, the HCDR2} consists of the amino acid sequence of SEQ ID NO:59, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:37. _{11. The antibody or the antigen-binding fragment thereof of Embodiment 1, wherein: a) the HCDR1 comprises the amino acid sequence of SEQ ID NO:62, the HCDR2} comprises the amino acid sequence of SEQ ID NO:74, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:86; b_{) the HCDR1 comprises the amino acid sequence of SEQ ID NO:63, the HCDR2} comprises the amino acid sequence of SEQ ID NO:75, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:87; c_{) the HCDR1 comprises the amino acid sequence of SEQ ID NO:64, the HCDR2} comprises the amino acid sequence of SEQ ID NO:75, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:88; d_{) the HCDR1 comprises the amino acid sequence of SEQ ID NO:65, the HCDR2} comprises the amino acid sequence of SEQ ID NO:76, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:88; e_{) the HCDR1 comprises the amino acid sequence of SEQ ID NO:66, the HCDR2} comprises the amino acid sequence of SEQ ID NO:77, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:89; f_{) the HCDR1 comprises the amino acid sequence of SEQ ID NO:67, the HCDR2} comprises the amino acid sequence of SEQ ID NO:78, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:90; - 98 - 4181706.v1 5431.1032002 _{g) the HCDR1 comprises the amino acid sequence of SEQ ID NO:68, the HCDR2} comprises the amino acid sequence of SEQ ID NO:79, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:91; _{h) the HCDR1 comprises the amino acid sequence of SEQ ID NO:69, the HCDR2} comprises the amino acid sequence of SEQ ID NO:79, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:92; _{i) the HCDR1 comprises the amino acid sequence of SEQ ID NO:70, the HCDR2} comprises the amino acid sequence of SEQ ID NO:80, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:93; _{j) the HCDR1 comprises the amino acid sequence of SEQ ID NO:70, the HCDR2} comprises the amino acid sequence of SEQ ID NO:81, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:94; _{k) the HCDR1 comprises the amino acid sequence of SEQ ID NO:70, the HCDR2} comprises the amino acid sequence of SEQ ID NO:82, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:94; _{l) the HCDR1 comprises the amino acid sequence of SEQ ID NO:71, the HCDR2} comprises the amino acid sequence of SEQ ID NO:82, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:95; _{m) the HCDR1 comprises the amino acid sequence of SEQ ID NO:71, the HCDR2} comprises the amino acid sequence of SEQ ID NO:82, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:94; _{n) the HCDR1 comprises the amino acid sequence of SEQ ID NO:72, the HCDR2} comprises the amino acid sequence of SEQ ID NO:83, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:95; _{o) the HCDR1 comprises the amino acid sequence of SEQ ID NO:72, the HCDR2} comprises the amino acid sequence of SEQ ID NO:83, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:96; _{p) the HCDR1 comprises the amino acid sequence of SEQ ID NO:73, the HCDR2} comprises the amino acid sequence of SEQ ID NO:84, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:97; or _{q) the HCDR1 comprises the amino acid sequence of SEQ ID NO:73, the HCDR2} comprises the amino acid sequence of SEQ ID NO:85, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:98. - 99 - 4181706.v1 5431.1032002 _{12. The antibody or the antigen-binding fragment thereof of Embodiment 1 or 11, wherein: a) the HCDR1 consists of the amino acid sequence of SEQ ID NO:62, the HCDR2} consists of the amino acid sequence of SEQ ID NO:74, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:86; _{b) the HCDR1 consists of the amino acid sequence of SEQ ID NO:63, the HCDR2} consists of the amino acid sequence of SEQ ID NO:75, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:87; _{c) the HCDR1 consists of the amino acid sequence of SEQ ID NO:64, the HCDR2} consists of the amino acid sequence of SEQ ID NO:75, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:88; _{d) the HCDR1 consists of the amino acid sequence of SEQ ID NO:65, the HCDR2} consists of the amino acid sequence of SEQ ID NO:76, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:88; _{e) the HCDR1 consists of the amino acid sequence of SEQ ID NO:66, the HCDR2} consists of the amino acid sequence of SEQ ID NO:77, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:89; _{f) the HCDR1 consists of the amino acid sequence of SEQ ID NO:67, the HCDR2} consists of the amino acid sequence of SEQ ID NO:78, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:90; _{g) the HCDR1 consists of the amino acid sequence of SEQ ID NO:68, the HCDR2} consists of the amino acid sequence of SEQ ID NO:79, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:91; _{h) the HCDR1 consists of the amino acid sequence of SEQ ID NO:69, the HCDR2} consists of the amino acid sequence of SEQ ID NO:79, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:92; _{i) the HCDR1 consists of the amino acid sequence of SEQ ID NO:70, the HCDR2} consists of the amino acid sequence of SEQ ID NO:80, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:93; _{j) the HCDR1 consists of the amino acid sequence of SEQ ID NO:70, the HCDR2} consists of the amino acid sequence of SEQ ID NO:81, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:94; - 100 - 4181706.v1 5431.1032002 k_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:70, the HCDR2} consists of the amino acid sequence of SEQ ID NO:82, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:94; l_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:71, the HCDR2} consists of the amino acid sequence of SEQ ID NO:82, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:95; m_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:71, the HCDR2} consists of the amino acid sequence of SEQ ID NO:82, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:94; n_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:72, the HCDR2} consists of the amino acid sequence of SEQ ID NO:83, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:95; o_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:72, the HCDR2} consists of the amino acid sequence of SEQ ID NO:83, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:96; p_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:73, the HCDR2} consists of the amino acid sequence of SEQ ID NO:84, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:97; or q_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:73, the HCDR2} consists of the amino acid sequence of SEQ ID NO:85, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:98. _{13. The antibody or the antigen-binding fragment thereof of any one of Embodiments 1, 2,} and 11, wherein: a_{) the HCDR1 comprises the amino acid sequence of SEQ ID NO:62, the HCDR2} comprises the amino acid sequence of SEQ ID NO:74, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:86; b_{) the HCDR1 comprises the amino acid sequence of SEQ ID NO:63, the HCDR2} comprises the amino acid sequence of SEQ ID NO:75, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:87; c_{) the HCDR1 comprises the amino acid sequence of SEQ ID NO:65, the HCDR2} comprises the amino acid sequence of SEQ ID NO:76, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:88; - 101 - 4181706.v1 5431.1032002 d_{) the HCDR1 comprises the amino acid sequence of SEQ ID NO:68, the HCDR2} comprises the amino acid sequence of SEQ ID NO:79, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:91; e_{) the HCDR1 comprises the amino acid sequence of SEQ ID NO:69, the HCDR2} comprises the amino acid sequence of SEQ ID NO:79, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:92; f_{) the HCDR1 comprises the amino acid sequence of SEQ ID NO:72, the HCDR2} comprises the amino acid sequence of SEQ ID NO:83, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:95; or g_{) the HCDR1 comprises the amino acid sequence of SEQ ID NO:72, the HCDR2} comprises the amino acid sequence of SEQ ID NO:83, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:96. _{14. The antibody or the antigen-binding fragment thereof of any one of Embodiments 1, 2,} and 11-13, wherein: a_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:62, the HCDR2} consists of the amino acid sequence of SEQ ID NO:74, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:86; b_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:63, the HCDR2} consists of the amino acid sequence of SEQ ID NO:75, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:87; c_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:65, the HCDR2} consists of the amino acid sequence of SEQ ID NO:76, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:88; d_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:68, the HCDR2} consists of the amino acid sequence of SEQ ID NO:79, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:91; e_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:69, the HCDR2} consists of the amino acid sequence of SEQ ID NO:79, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:92; f_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:72, the HCDR2} consists of the amino acid sequence of SEQ ID NO:83, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:95; or - 102 - 4181706.v1 5431.1032002 g_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:72, the HCDR2} consists of the amino acid sequence of SEQ ID NO:83, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:96. _{15. The antibody or the antigen-binding fragment thereof of any one of Embodiments 1-14,} wherein the V_H domain comprises an amino acid sequence that has at least 70% sequence identity to the amino acid sequence of at least one of SEQ ID NOs:99-123. _{16. The antibody or the antigen-binding fragment thereof of any one of Embodiments 1-15,} wherein the V_H domain comprises an amino acid sequence that has 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs:99-123. _{17. The antibody or the antigen-binding fragment thereof of any one of Embodiments 1-15,} wherein the V_H domain comprises an amino acid sequence that has at least 70% sequence identity to the amino acid sequence of at least one of SEQ ID NOs:99-105, 107, 110-112, and 118-120. _{18. The antibody or the antigen-binding fragment thereof of any one of Embodiments 1-17,} wherein the V_H domain comprises an amino acid sequence that has 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs:99-105, 107, 110-112, and 118-120. _{19. The antibody or the antigen-binding fragment thereof of any one of Embodiments 1-18,} wherein the antigen-binding fragment is a single-domain antibody (sdAb). _{20. The antibody or the antigen-binding fragment thereof of any one of Embodiments 1-19,} wherein the antibody or the antigen-binding fragment thereof binds human transferrin receptor 1 (hTFR1). _{21. The antibody or the antigen-binding fragment thereof of any one of Embodiments 1-20,} wherein the antibody is bispecific or multispecific. _{22. A polypeptide comprising the antibody or the antigen-binding fragment thereof of any} one of Embodiments 1-21. _{23. The polypeptide of Embodiment 22, further comprising an antibody or an antigen-} binding fragment thereof that binds tumor necrosis factor-alpha (TNF-α). - 103 - 4181706.v1 5431.1032002 _{24. The polypeptide of Embodiment 23, wherein the antibody or the antigen-binding} fragment thereof that binds TNF-α comprises an immunoglobulin heavy chain variable (V_H) domain comprising a heavy chain complementarity-determining region (HCDR) 1, an HCDR2, and an HCDR3 having 100% sequence identity to an HCDR1, an HCDR2, and an HCDR3, respectively, of a V_H domain comprising the amino acid sequence of any one of SEQ ID NOs:132-137. _{25. The polypeptide of Embodiment 23 or 24, wherein the antibody or the antigen-binding} fragment thereof of that binds TNF-α comprises a V_H domain comprising: a_{) an HCDR1 comprises the amino acid sequence of SEQ ID NO:138, b) an HCDR2 comprises the amino acid sequence of SEQ ID NO:139, and c) an HCDR3 comprises the amino acid sequence of SEQ ID NO:140. 26. The polypeptide of any one of Embodiments 23-25, wherein the antibody or the antigen-} binding fragment thereof of that binds TNF-α comprises a V_H domain comprising: a_{) an HCDR1 consists of the amino acid sequence of SEQ ID NO:138, b) an HCDR2 consists of the amino acid sequence of SEQ ID NO:139, and c) an HCDR3 consists of the amino acid sequence of SEQ ID NO:140. 27. The polypeptide of any one of Embodiments 23-26, wherein the antibody or the antigen-} binding fragment thereof of that binds TNF-α comprise a V_H domain comprising four V_H framework regions (V_H FR1-4) and three heavy chain complementarity determining regions (HCDR1-3) in the following N-terminal to C-terminal order: V_H FR1-HCDR1- V_H FR2-HCDR2-V_H FR3-HCDR3-V_H FR4, wherein: a_{) the VH FR1 comprises an amino acid sequence having at least 80% sequence} identity to the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO:10) or QVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO:151), b_{) the VH FR2 comprises an amino acid sequence having at least 80% sequence} identity to the amino acid sequence of WMYWVRQAPGKE (SEQ ID NO:152), c_{) the VH FR3 comprises an amino acid sequence having at least 80% sequence} identity to the amino acid sequence of DFVKGRFTVSRDNAKNTLYLQMDSLRPEDTAVYS (SEQ ID NO:158), DFVKGRFTVSRDNAKNTLYLQMNSLEPEDTAVYS (SEQ ID NO:159) or DFVKGRFTVSRDNAENTLYLQMNSLEPEDTAVYS (SEQ ID NO:160), or - 104 - 4181706.v1 5431.1032002 d_{) the VH FR4 comprises an amino acid sequence having at least 80% sequence} identity to the amino acid sequence of LRGQGTQVTVSS (SEQ ID NO:156) or LRGQGTQVTVSSGPGGQ (SEQ ID NO:157), or any combination of a) to d). _{28. The polypeptide of any one of Embodiments 23-27, wherein the antibody or the antigen-} binding fragment thereof of that binds TNF-α comprise a V_H domain comprising an amino acid sequence that has at least 70% sequence identity to the amino acid sequence of at least one of SEQ ID NOs:132-137. _{29. The polypeptide of any one of Embodiments 23-28, wherein the antibody or the antigen-} binding fragment thereof of that binds TNF-α comprise a V_H domain comprising an amino acid sequence that has 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs:132-137. _{30. The polypeptide of any one of Embodiments 23-29, wherein the antibody or the antigen-} binding fragment thereof of that binds TNF-α is a single-domain antibody (sdAb). _{31. The polypeptide of any one of Embodiments 23-30, further comprising: a) a protein tag, b) a therapeutic agent, optionally, wherein the therapeutic agent comprises a} polypeptide and/or a small molecule, c_{) a diagnostic agent, or} any combination of the foregoing. _{32. A polynucleotide encoding the antibody or antigen binding fragment thereof of any one} of Embodiments 1-21, or the polypeptide of any one of Embodiments 22-31. _{33. An expression vector comprising the polynucleotide of Embodiment 32. 34. A host cell comprising the polynucleotide of Embodiment 32, or the expression vector of} Embodiment 33. _{35. A method of producing the antibody or the antigen binding fragment thereof of any one} of Embodiments 1-21, comprising expressing the antibody or the antigen binding fragment thereof, in the host cell of Embodiment 34 and isolating the expressed antibody or the antigen binding fragment thereof. - 105 - 4181706.v1 5431.1032002 _{36. A method of producing the polypeptide of any one of Embodiments 22-31, comprising} expressing the polypeptide in the host cell of Embodiment 34 and isolating the expressed polypeptide. _{37. A composition comprising the antibody or the antigen binding fragment thereof of any} one of Embodiments 1-21, the polypeptide of any one of Embodiments 22-31, the polynucleotide of Embodiment 32, the expression vector of Embodiment 33, or the host cell of Embodiment 34. _{38. The composition of Embodiment 37, further comprising at least one additional} therapeutic agent, optionally, wherein the at least one additional therapeutic agent comprises an anti-tau antibody, an anti-amyloid-beta (Aβ) antibody, a BRI2 peptide, and/or an amyloid precursor protein (APP)-targeting agent. _{39. A pharmaceutical composition comprising the composition of Embodiment 37 or 38, and} a pharmaceutically acceptable carrier or diluent. _{40. A kit comprising the composition of Embodiment 37 or 38, or the pharmaceutical} composition of Embodiment 39. _{41. A method of treating a disease in a subject in need thereof, comprising administering to} the subject a therapeutically effective amount of the antibody or the antigen binding fragment thereof of any one of Embodiments 1-21, the polypeptide of any one of Embodiments 22-31, the composition of Embodiment 37 or 38, or the pharmaceutical composition of Embodiment 39, thereby treating the disease. _{42. A method of blocking binding of TNF-α to TNF receptor (TNFR) expressed on a surface} of a cell, the method comprising contacting the cell with an effective amount of the antibody or the antigen binding fragment thereof of any one of Embodiments 1-21, the polypeptide of any one of Embodiments 22-31, the composition of Embodiment 37 or 38, or the pharmaceutical composition of Embodiment 39, thereby blocking binding of TNF- α to TNFR expressed on the surface of the cell. _{43. A method of blocking binding of TNF-α to TNFR in a subject, the method comprising} administering to the subject an effective amount of the antibody or the antigen binding fragment thereof of any one of Embodiments 1-21, the polypeptide of any one of - 106 - 4181706.v1 5431.1032002 Embodiments 22-31, the composition of Embodiment 37 or 38, or the pharmaceutical composition of Embodiment 39, thereby blocking binding of TNF-α to TNFR expressed in the subject. _{44. A method of treating a TNF-α-associated disease in a subject in need thereof, comprising} administering to the subject an effective amount of the antibody or the antigen binding fragment thereof of any one of Embodiments 1-21, the polypeptide of any one of Embodiments 22-31, the composition of Embodiment 37 or 38, or the pharmaceutical composition of Embodiment 39, thereby treating the TNF-α-associated disease. _{45. The method of Embodiment 44, wherein the TNF-α-associated disease is Alzheimer’s} disease (AD). _{46. The method of Embodiment 44 or 45, wherein the TNF-α-associated disease is late onset} Alzheimer’s disease (LOAD). _{47. The method of Embodiment 44 or 45, wherein the TNF-α-associated disease is traumatic} brain injury (TBI). _{48. The method of any one of Embodiments 41-47, wherein the subject is an adult human} patient. _{49. The method of any one of Embodiments 41-47, wherein the subject is a pediatric human} patient. _{50. The method of any one of Embodiments 42-49, further comprising administering to the} subject at least one additional therapeutic agent. _{51. The method of Embodiment 50, wherein the at least one additional therapeutic agent} comprises an anti-serum albumin agent and/or a fusion protein comprising human serum albumin. _{52. The method of Embodiment 50, wherein the at least one additional therapeutic agent} comprises at least one chemotherapeutic agent and/or at least one immunosuppressant agent. _{53. The method of Embodiment 50, wherein the at least one additional therapeutic agent} comprises methotrexate. - 107 - 4181706.v1 5431.1032002 The method of any one of Embodiments 50-53, wherein the at least one additional therapeutic agent is formulated in nanoparticles, hydrogel depots, or microarray patches. EXEMPLIFICATION _{[00384] Camelids produce heavy-chain-only antibodies (Hamers-Casterman et al.), which} consist of the antigen-binding variable domain (VHH) followed by constant domains CH2/CH3. These single-domain antibodies (sdAbs, Nabs) typically range from 12 to 14 kDa and lack an Fc region. Notably, certain sdAbs possess the ability to spontaneously traverse the blood-brain barrier (BBB) ( Li et al. (2012), Li et al. (2016)). _{[00385] TFR1, also known as TFRC or CD71, is highly expressed on endothelial cells} forming the blood-brain-barrier (BBB), and its transcytosis activity can be harnessed to facilitate the delivery of molecules to the central nervous system (CNS) (Xiao & Gan, Sehlin et al., Johnsen et al., Pardridge et al., Lee et al.). TFR1 exists as a homo-dimeric transmembrane protein, with each monomer capable of binding one molecule of transferrin (TF) to form an iron- TF-TFR1 complex that undergoes endocytosis for cellular uptake, crucial for iron import. Importantly, monovalent interactions with TFR1 favor transcytosis over bivalent interactions (Lee et al.), suggesting that sdAbs that bind TFR1 may enhance transcytosis more efficiently than monoclonal antibodies (mAbs) that bind TFR1. Without being bound by theory, sdAbs targeting human cell-membrane TFR1 (hTFR1-sdAbs) hold promise for circumventing the restricted BBB penetration that impedes the utilization of numerous drugs, including biologics, for CNS disorders. Example 1. Identification of Novel Single-Domain Antibodies that Bind Human TFR1. _{[00386] Example 1 reports identification of 25 novel single-domain antibodies (sdAbs) that} bind human TFR1 (hTFR1). One llama and one alpaca were immunized with hTFR1 extracellular domain (Sino Biological, HPLC-11020-H07H). A total of 470 variable heavy domain of heavy chain (VHH) domain clones were isolated from peripheral blood mononuclear cells (PBMCs), and 106 unique VHH sequences were identified. _{[00387] To evaluate antigen binding, HEK293 cells, which have low levels of endogenous} TFR1, were transfected with a vector expressing hTFR1 and EGFP.25 sdAbs (AB-1 to AB-25) were found to bind cell-membrane hTFR1 but not mouse or rat Tfr1 (FIG.1A). Anti-hTFR1- APC, Anti-mTfr1-APC and Anti-rTfr1-APC antibodies (Thermo-Fisher, 17-0719-42, 17-0711- 82, 17-0710-82) were used as positive controls (not shown), isotype control-APC and Anti-His- APC alone are negative controls (not shown). Among the 25 sdAbs, 13 were found to bind - 108 - 4181706.v1 5431.1032002 Rhesus (R.) macaque TFR1 (FIG.1A). The hTFR1-sdAbs also bound to endogenous cell-surface TFR1 of HL-60 cells (FIG.1B). HL-60 is a human promyeloblast line expressing hTFR1 levels comparable to endothelial cells. Accordingly, the results demonstrated that the hTFR1-sdAbs bind endogenous hTFR1 in a native membrane-displayed conformation. Example 2. Categorizing the hTFR1-sdAbs Based on CDR Sequences. _{[00388] The complementarity-determining regions (CDRs) were analyzed following three} established methods: Chothia, Kabat, and the International ImMunoGeneTics Information System (IMGT). Chothia is a computational approach developed by G. Chothia and A.M. Lesk for identifying the CDRs. It relies on structural analysis of antibody-antigen complexes to define the regions of the antibody that are most likely to interact with antigens. This method considers the three-dimensional structure of antibodies to predict CDRs. The Kabat method, named after E.A. Kabat, is another computational approach commonly used for CDR analysis. This method relies on empirical rules derived from a large dataset of antibody sequences to identify CDRs. It considers sequence characteristics such as amino acid composition, length, and position within the antibody chain to predict CDRs. IMGT is a comprehensive database and analysis tool for immunoglobulin and T cell receptor sequences. IMGT provides a standardized and curated framework for the annotation and analysis of antibody sequences, including the identification of CDRs. IMGT incorporates both sequence-based and structural-based approaches to CDR analysis, combining empirical rules with information on antibody structure and function. Based on similarities in CDR sequences, the hTFR1-sdAbs were grouped into 10 families (Tables 1-5). - 109 - 4181706.v1 5431.1032002 Table 1. hTFR1-sdAbs F_{amily Name Code} - 110 - 4181706.v1 5431.1032002 Table 2. HCDR1 Sequences as Determined by Chothia Numbering H_{CDR1 SEQ ID HCDR2 SEQ ID HCDR3 SEQ ID} - 111 - 4181706.v1 5431.1032002 Table 3. HCDR1 Sequences as Determined by Kabat Numbering H_{CDR1 SEQ ID HCDR2 SEQ ID HCDR3 SEQ ID} - 112 - 4181706.v1 5431.1032002 Table 4. HCDR1 Sequences as Determined by ImMunoGeneTics (IMGT) Numbering H_{CDR1 SEQ ID HCDR2 SEQ ID HCDR3 SEQ ID} - 113 - 4181706.v1 5431.1032002 Table 5. V_H Sequences V_H SEQ ID - 114 - 4181706.v1 5431.1032002 EVQLVESGGGFVQAGGSLRLSCAGPQSIFSSRAMGWFRQAPGKEREFVAAIMWSGDSTE AB 20 YADSVKGRFTISRDNAKNTVYLQMNSLEPEDTAVYYCALGQSPWLGGGSNFVSWGQGTQ NO:118 Example 3. Production of hTFR1-sdAbs in CHO-S Cells. _{[00391] Chinese Hamster Ovary (CHO) cells are mammalian cells that can produce complex} proteins with proper folding, post-translational modifications, and biological activity like human proteins. This is crucial for many therapeutic proteins. Proteins produced in CHO cells are more likely to be recognized as “self” by the human immune system, reducing the risk of immune responses in patients. CHO cells often provide a more suitable environment for the correct folding and assembly of complex proteins, ensuring proper functionality. CHO cell-based expression systems have a long history of use in the production of biopharmaceuticals and have gained regulatory approval for numerous therapeutic proteins. In contrast, proteins expressed in Bacteria (e.g., Escherichia coli), may lack proper post-translational modifications, which can be critical for the functionality and safety of therapeutic proteins. In addition, Bacterial expression systems, especially E. coli, may produce endotoxins, which can pose challenges for downstream purification and may require additional steps to ensure product safety. In summary, CHO cells are generally preferred for producing therapeutic proteins that require proper folding, post- translational modifications, and mammalian-like functionality. Thus, whether hTFR1-sdAbs can be efficiently produced in CHO-S cells, was tested. _{[00392] Each cDNA coding a unique hTFR1-sdAb identified in Example 1 was subcloned} into pcDNA3.4. A sequence encoding a signal peptide (MGWSCIILFLVATATGVHS (SEQ ID NO:131), Ig heavy chain Mus musculus P01750) was added at the 5’-end of the sdAb coding sequences to enable secretion of the hTFR1-sdAbs. A sequence encoding 6xHis was added to the 3’-end (His-Tag) to enable purification of secreted hTFR1-sdAbs. Each construct was transfected - 115 - 4181706.v1 5431.1032002 into CHO-S cells, and the TFR1-sdAbs were purified by a single step via the His-Tag. Table 6 lists the quantity and purity of the hTFR1-sdAbs purified from 100 ml culture supernatants. As shown in FIGs.2A-2B (representative data from one sdAb) and Table 6, hTFR1-sdAbs were efficiently produced in CHO-S cells. Table 6. Production of hTFR1-sdAbs in Mammalian Cells. N_{ame Family} Conc. Purity SDS-PAGE Purity SEC-HPLC Endotoxin Level Total l (%) (%) (EU ) ( ) . . _{[00393] The biological activities of the hTFR1-sdAbs produced in CHO-S cells were} evaluated. In the first set of experiments, bindings between mammalian cell produced hTFR1- sdAbs to hTFR1 were assessed in HEK293 cells transfected with a plasmid expressing hTFR1 and EGFP. As illustrated in FIGs.3A-3I, EGFP-positive (transfected) cells were effectively stained with the hTFR1-sdAbs of Families A, B, D, G, and I. In contrast, TFR1-positive HEK293 cells were not stained with the hTFR1-sdAbs of Families E, F, J, and L. - 116 - 4181706.v1 5431.1032002 _{[00394] In the second set of experiments, bindings between mammalian cell produced} hTFR1-sdAbs to hTFR1 were assessed in CHEK-ATP089, a HEK293/human transferrin receptor stable cell line purchased from ACROBiosystems (Newark, DE). To confirm hTFR1 expression in CHEK-ATP089 cells, both HEK293 and CHEK-ATP089 cells with human Transferrin-FITC were stained (FIGs.4A-4J). The staining pattern revealed strong affinity of Transferrin-FITC to CHEK-ATP089 cells, but not to HEK293 cells, confirming hTFR1 expression in this cell line. _{[00395] It is worth noting that Transferrin-FITC was employed at a concentration of 2.5} micromolar in these staining assays, approximately one-tenth of the concentration of Transferrin typically found in human serum. The results were consistent with those from transiently transfected HEK293 cells. The hTFR1-sdAbs of Families A, B, D, G, and I exhibited strong staining of the CHEK-ATP089 cells, while those of Families E, F, J, and L did not (FIGs.4A- 4J). _{[00396] The reasons why the hTFR1-sdAbs of Families E, F, J, and L produced in bacteria} bind to hTFR1 while those produced in mammalian cells do not, is currently under investigation. The hTFR1-sdAb of Family C, 01E01R3, has not been produced in mammalian cells. However, successful binding is predicted based on the CDR sequences. Example 5. Effects of hTFR1-sdAbs on TF Binding to TFR1 & TF Uptake. _{[00397] TF is the major ferric iron transport protein, which binds ferric (Fe3+) ions. The iron} binding affinity of transferrin is pH dependent. In a neutral pH environment, TF (apotransferrin) binds iron with high affinity to form iron-bound TF (holotransferrin). In an acidic pH environment, binding affinity of transferrin to iron decreases, and iron is dissociated from holotransferrin and released into the environment. The importance of holotransferrin is to sequester Fe³⁺ ions in a relatively nonreactive and inert state to ensure normal free iron homeostasis in the body. _{[00398] Holotransferrin delivers iron to cells by binding to transferrin receptor (TFR1 or} TFR2). Neutral pH at the cell surface promotes binding of holotransferrin to TFR1/TFR2. The receptor-ligand complex enters the cell through receptor-mediated endocytosis and is internalized into endosomes. Relatively lower endosomal pH results in the release of iron. The receptor-ligand complex is recycled to the cell surface, where apotransferrin dissociates from TFR1/TFR2 (Kawabata (2019)). - 117 - 4181706.v1 5431.1032002 _{[00399] Interference with these processes might lead to unintended toxic effects in patients} treated with therapeutics based on the hTFR1-sdAbs. Therefore, whether the hTFR1-sdAbs disrupt: (1) binding of TF to TFR1, and (2) cellular uptake of TF, were investigated. Example 5A. Select hTFR1-sdAbs Do Not Interfere with TF Binding to TFR1. _{[00400] First, effects of the hTFR1-sdAbs on TF-hTFR1 binding was investigated using} CHEK-ATP089 cells, where unlabeled TF competes for binding of TF-FITC to TFR1. Competitive bindings were observable across TF/TF-FITC ratios of 3 and 1, but not 0.3, demonstrating a dose-dependent relationship (FIGs.5A-5B). _{[00401] Next, whether the hTFR1-sdAbs diminish binding of TF-FITC to CHEK-ATP089} cells was examined at a 1:1 TF-FITC/hTFR1-sdAb ratio (with both at a concentration of 2.5 micromolar). At this ratio, all three hTFR1-sdAbs of Family I reduced binding of TF-FITC to CHEK-ATP089 cells. Conversely, none of the remaining 11 hTFR1-sdAbs (5 of Family A, 2 of Family B, 1 of Family D, and 3 of Family G) interfered with TF-TFR1 interaction (FIGs.5A- 5B). Example 5B. Select hTFR1-sdAbs Do Not Interfere with TF Uptake. _{[00402] Effects of the hTFR1-sdAbs on TF uptake was investigated using CHEK-ATP089} cells, where uptake of pHrodo Red-TF was monitored using the Incucyte in vivo imaging system. The emission of pHrodo Red-TF is inversely related to pH, i.e., its intensity increases within the cellular organelles where pH decreases. _{[00403] First, whether TF competes for uptake of pHrodo Red-TF was investigated using} CHEK-ATP089 cells. The results indicate that TF inhibits pHrodo Red-TF uptake in a dose- dependent manner (FIG.6A). Notably, even at a pHrodo Red-TF/TF ratio of 10.42 (312.5 nanomolar pHrodo Red-TF vs.30 nanomolar TF), competition for uptake remained highly significant. These findings validated the system, highlighting potentially adverse effects of hTFR1-sdAbs in competing with TF for hTFR1 binding. _{[00404] Next, effects of the hTFR1-sdAbs on pHrodo Red-TF uptake was determined. In line} with the binding competition data depicted in FIGs.5A-5B, 01B01R3, a member of Family I, significantly attenuated pHrodo Red-TF uptake (FIG.6B). It is worth noting that this inhibitory effect is less pronounced than that observed with a 30 nanomolar concentration of TF. Importantly, members of Families A, B, D, and G did not exhibit this inhibitory effect. Additionally, a member of Family J, which showed no binding to hTFR1, also had no impact pHrodo Red-TF uptake. - 118 - 4181706.v1 5431.1032002 _{[00405] Results of Examples 4 and 5 suggest that members of Families A, B, D, and G are the} most promising candidates for further development. Nonetheless, the potential utility of members of Families J, E, F, C, L, and I cannot be entirely dismissed, as those sdAbs may provide alternative avenues for drug development. Assessing In Vivo BBB Permeability. Example 6. Generation & Validation of Human-TFR1 and TF Knock-in Rats. _{[00406] The blood-brain barrier (BBB) permeability of the hTFR1-sdAbs relies on TFR1-} mediated transcytosis. Since the hTFR1-sdAbs do not cross react with rodent Tfr1, rodents are unsuitable for in vivo assessment of permeability of the hTFR1-sdAbs. This challenge extends to anti-hTFR1 monoclonal antibodies (mAbs), which are also species-specific. To evaluate whether targeting therapeutic agents to TFR1 enhances BBB permeability in preclinical studies, therapeutics were conjugated with anti-rodent Tfr1 mAbs. However, these mAbs do not bind hTFR1 and are unsuitable for human therapy. This proof-of-concept approach is inadequate for evaluating the hTFR1-sdAbs. While some of the hTFR1-sdAbs bind to Rhesus Macaque TFR1, conducting in vivo BBB permeability testing in Rhesus macaques poses ethical and economic challenges. Therefore, for in vivo BBB permeability assessment, a rat model that expresses hTFR1 and hTF mRNAs and proteins was produced by modifying the rat Tfr1 and Tf genes. _{[00407] Given the considerable sizes of the rat and human genes involved, human TFR1 and} TF coding sequences were inserted into exon 2 of the corresponding rat genes (FIGs.7A-7B). The knock-in rats express the type II human TFR1 protein and a fully human TF protein. Regarding the later, while the transcript includes the rat leader sequence, the leader sequence is removed during protein synthesis. _{[00408] The knock-in rat model was validated using Western blots of human TFR1 and} human TF. As shown in FIG.8A, human TF and TFR1 were detected in the protein lysates from the lung, liver, brain, duodenum, and kidney of double heterozygous (Tf^h/w:Tfr1^h/w) male and female rats, but not in wild-type rats (Tf^w/w:Tfr1^w/w). _{[00409] The protein expression data was further validated using: (1) a rat with two humanized} Tf alleles and two wild-type Tfr1 alleles (Tf^h/h:Tfr1^w/w), and (2) a rat with two wild-type Tf alleles and two humanized Tfr1 alleles (Tf^w/w:Tfr1^h/h). As shown in FIG.8C, in lysates prepared from tissues including the pancreas, spleen, and heart, hTFR1 expression was exclusively detected in the Tf^w/w:Tfr1^h/h rat, and hTF expression was exclusively detected in the in the Tf^h/h:Tfr1^w/w rat. Importantly, the pattern and level of hTFR1 expression in the Tf^w/w:Tfr1^h/h rat mirrored those of rTfr1 expression in the Tf^h/h:Tfr1^w/w rat; and the pattern and level of hTF expression in the - 119 - 4181706.v1 5431.1032002 Tf^h/h:Tfr1^w/w rat mirrored those of rTf expression in the Tf^w/w:Tfr1^h/h rat. It is important to note that although the anti-hTFR1 antibody is highly specific for hTFR1, the anti-rTfr1 antibody exhibits some degree of cross-reactivity with hTFR1. _{[00410] The knock-in rat model was also validated using ELISA of human TFR1 and human} TF. Erythroblasts are the primary source of serum soluble TFR1 (sTFR1), which is a truncated monomer lacking its first 100 amino acids circulating in complex with TF. As shown in FIG.8B, human sTFR1 and human TF were detected in the serum of double heterozygous (Tf^h/w:Tfr1^h/w) rats, but not in wild-type rats (Tf^w/w:Tfr1^w/w). _{[00411] Functional hTFR1 is displayed on cell membranes as a ~190 kDa homo-dimeric} receptor comprising two identical subunits linked by two disulfide bridges (Kawabata (2019)). To investigate potential dimerization of hTFR1, brain lysates from Tfr1^w/w, Tfr1^h/w, and Tfr1^h/h rats were subjected to immunoprecipitation using the anti-rat Tfr1 antibody. Both total lysates and immunoprecipitated proteins were analyzed via Western blotting under reducing or non- reducing conditions, employing an anti-hTFR1 antibody. Analysis of the total lysates showed that under reducing conditions, hTFR1 was detected as a monomeric protein of approximately 95 kDa. Under non-reducing conditions, hTFR1 was detected as a dimeric protein of approximately 190 kDa, consistent with dimerization via disulfide bridges (FIG.9). _{[00412] The levels of monomeric and dimeric hTFR1 were significantly higher in the Tfr1h/h} lysates, compared to the Tfr1^h/w lysates, and hTFR1 was not detected in the Tfr1^w/w samples (FIG.9). Efficient immunoprecipitation of human TFR1 by the anti-rat Tfr1 antibody was observed in the Tfr1^h/w samples. Cross-reactivity of anti-rTfr1 was again indicated by small amounts of hTFR1 precipitated from the Tfr1^h/h samples. However, despite higher hTFR1 levels in the Tfr1^h/h lysates, the amounts of human TFR1 immunoprecipitated by the anti-rTfr1 antibody were notably smaller in the Tfr1^h/h lysates, compared to the Tfr1^h/w lysates. The results suggest that hTFR1 forms chimeric dimers with rTfr1 in Tfr1^h/w rats. _{[00413] These data strongly supports the functionality of hTFR1 and hTF in the novel knock-} in rat model. The successful generation of homozygous humanized TFR1 and TF rats further reinforces this conclusion. Indeed, homozygous null Tfr1 mice exhibit an embryonic lethal phenotype, failing to progress beyond embryonic day 12.5. Hypotransferrinemic (Tf^hpx/hpx) mice have a severe deficiency in serum transferrin as the result of a spontaneous to the murine Tf locus. Hypotransferrinemic mice are born alive, but die from severe anemia before weaning, unless they are treated with exogenous Tf or red blood cell transfusions (Trenor et al. (2000)). The ability to obtain homozygous humanized TFR1 and TF KI rats, as well as double - 120 - 4181706.v1 5431.1032002 homozygous rats, which are fertile, underscores the fidelity of the model in mimicking Tfr1/Tf functions. Thus, the knock-in rat model can be used for testing whether an hTFR1-sdAb can cross the BBB in vivo. Example 7. In Vivo Brain-Blood Barrier Permeability Assays. _{[00414] The initial brain-blood barrier (BBB) permeability screening was performed on} knock-in rats with various genotypes, sexes, and ages obtained during the expansion and crossing of Tf and Tfr1 knock-in rat lines. The rats were intravenously injected with 14 anti- hTFR1-sdAbs at indicated concentrations (Table 7). After a specified post-injection time, blood was collected, and the rats were perfused with PBS to clear blood from brain tissues. Brain homogenates were then fractionated into soluble and precipitant fractions by centrifugation at 100,000 G for one hour. The presence of the anti-hTFR1 sdAbs was determined enzyme-linked immunosorbent assay (ELISA). Streptavidin plates (Meso Scale Diagnostics (MSD), Rockville, MD, L15SA) were coated with 0.25 ^g/ml of biotinylated hTFR1 protein (ACROBiosystems, Newark, DE, TFR-H82E5). The samples were incubated with the coated-wells, and sdAbs bound to hTFR1 were detected by 1 ^g/ml of goat anti-alpaca IgG, VHH domain (Jackson ImmunoResearch Laboratories, Inc, West Grove, PA, 128-005-230), followed by 1 ^g/ml of donkey SULFO-TAG labeled anti-goat antibody (MSD, R32AG). ELISA were read on a MESO QuickPlex SQ instrument. Table 7. Human TFR1-Based ELISA of hTFR1-sdAbs in Rat Brain Samples. G_enotype Age Tim _{Concentration [pM] of sdAb il l k} e* - 121 - 4181706.v1 5431.1032002 3_{4.4 h/w h/w m 114 16-18 88.5 635.1 120.4 301.7 325 h/w h/h m 39 16-18 4148 6244 3700 3811 [00415] In Table 7, human TFR1-based ELISA reveals the presence of hTFR1-sdAbs in brain} tissues of rats expressing human TFR1, whereas such Nabs are absent in rats solely expressing rat Tfr1. Columns 1, 2, and 3 indicate the name, family, and amount of the hTFR1-sdAbs injected, respectively. Columns 4, 5, 6, and 7 indicate the genotype at the Tf and Tfr1 gene loci, sex, and age of the animals injected, respectively. Column 8 indicates the time of tissue harvesting after injection. Columns 9, 10, 11, and 12 indicate the concentration of TFR1b-Nab found in serum, brain homogenates, brain S100, and P100 fractions, respectively. _{[00416] Specificity of the ELISA was further demonstrated using 100 nM of hTNFI^-sdAb1,} an sdAb specific for human TNF^, with a reading similar to that of vehicle alone (data not shown). The data in Table 7 show that: (1) all tested hTFR1-sdAbs were detected in the brain homogenates, S100, and P100 fractions of rats expressing hTFR1; and (2) the hTFR1-sdAbs were not detected in brain homogenates, S100, and P100 fractions of rats expressing rat Tfr1 only. These findings strongly suggest that the hTFR1-sdAbs can localize to the brain in a human TFR1-dependend manner. _{[00417] Next, BBB permeabilities of three hTNFIα-sdAbs (hTNFIα-sdAb1, hTNFα-sdAb2,} and hTNFα-sdAb3) were analyzed using ELISA. Levels of the hTNFIα-sdAbs were quantified using plates coated with 0.25 ^g/ml of biotinylated human TNF^ protein active trimer (Acro- Biosystems, TNA-H82E3). As shown in Table 8, none of the sdAbs was detectable 12 hours post-injection in any sample analyzed, suggesting that the hTNFIα-sdAbs are short-lived and do not cross the BBB. Production of the hTNFIα-sdAbs in CHO-S are shown in FIG.10A. - 122 - 4181706.v1 5431.1032002 Table 8. Human TNFα-Based ELISA of hTNFIα-sdAbs in Rat Brain Samples. N_{anobody nmol/Kg Tf Tfr1 sex Age in days} Time Concentration [pM] of sdAb h _{[00418] The possibility that the expression of hTFR1α may compromise the integrity of the} BBB was investigated by co-injecting hTNFIα-sdAb1 and 04B05R3, an hTFR1-sdAb. Serum and brain samples were harvested 4-6 hours post-injection to maximize the probability of detecting hTNFIα-sdAb1 in the serum. While 04B05R3, the hTFR1-sdAb, was detected in the brains of rats expressing hTFR1 (Table 9, hTFR1-based ELISA), hTNFIα-sdAb1 was only detected in the sera but not in the brains of all analyzed rats (Table 9, hTNF^-based ELISA). The data indicate that BBB permeability in the knock-in rats is not altered by the expression of hTFR1. Table 9. Assessment of Blood-Brain Barrier Integrity in Rats Expressing Human TFR1. (40 nmol/kg TNFI-Nab1 + 40 nmol/kg 04B05R3) Concentration [pM] of sdAb Genotype _{[00419] Next, whether linking hTNFIα-sdAb1 to an hTFR1-sdAb could promote the brain} localization of hTNFIα-sdAbs was investigated using 04B05R3 and 05D01R3. The sequences of these two heterodimers are: - 123 - 4181706.v1 5431.1032002 _{[00420] TNFIα-sdAb1-linker-05D01R3-His-tag: [00421] EVQLVESGGGLVQPGGSLRLSCAASGFAFNDHWMYWVRQAPGKELEWVSE} INTNGLITKYADFVKGRFTVSRDNAKNTLYLQMDSLRPEDTAVYSCSRNQIGTLRGQGT QVTVSSGGGGSGGGSQVQLVESGGGLVQAGGSLRLSCAASGRTFNNYVMGWFRGKEREFV ASISLRGGSPSYADSVKDRFFISTANAKNTVSLQMNSLKPGDTADYYCAASGLTNTGGYYFRRP DQYPYWGQGTLVTVSSHHHHHH (SEQ ID NO:124) _{[00422] TNFIα-sdAb1-linker-04B05R3-His-tag: [00423] EVQLVESGGGLVQPGGSLRLSCAASGFAFNDHWMYWVRQAPGKELEWVSE} INTNGLITKYADFVKGRFTVSRDNAKNTLYLQMDSLRPEDTAVYSCSRNQIGTLRGQGT QVTVSSGGGGSGGGQLQLVESGGGLVQAGGSLRLSCAVSGIAFAGRGMSWYRQAPGKQRE WVAGITGGGHANYAESVKGRFTISRDNAENTGHLQMTSLKAEDTGVYYCWVATNYWGKGTQ VTVSSHHHHHH (SEQ ID NO:125) _{[00424] As shown in Table 10, when linked to either hTFR1-sdAb, hTNFIα-sdAb1 was} detected in and only in the brains of rats expressing hTFR1. The data suggests that the hTFR1- sdAbs, when coupled with hTNFIα-sdAb1 in a heterodimeric configuration, promote CNS accumulation of hTNFIα-sdAb1. Accordingly, the hTFR1-sdAbs can be used to facilitate brain localization of proteins such as hTNFIα-sdAbs. Table 10. Shuttle Activity of hTFR1-sdAbs Across BBB in Rats Expressing Human TFR1. SdAb _Genotype Ti _{Concentration [pM] of hTNFIα-sdAb Age} me _{[00425] Because the rats underwent perfusion with PBS for approximately 10 minutes before} the brains were removed for analysis, there remained a possibility that the hTFR1-sdAbs detected in the brain fractions were originated from intra-vessel hTFR1-sdAbs. The following measures were implemented to minimize this possibility: (1) the tissues were harvest occurred 48 hours after injection, when serum levels of the hTFR1-sdAbs were anticipated to be lower; and (2) cerebrospinal fluid (CSF), which presumably closely mirrored the composition of brain - 124 - 4181706.v1 5431.1032002 parenchymal interstitial fluids, were collected following perfusion. As shown in Table 11. Human TNF^-based ELISA reveals presence of hTNFIα-sdAb1 in brain tissues of rats injected with hTNFIα-sdAb1-linker-04B05R3 and hTNFIα-sdAb1-linker-05D01R3. hTFR1-sdAbs 04B05R3 and 04F01R3 were identified in the CSF of all three Tfr1^h/w rats but were absent in the wild-type rat. Conversely, while hTNFIα-sdAb1-linker-04B05R3 was detectable in the sera of all four rats, it was not detectable in the CSF of the wild-type rat. Accordingly, the two hTFR1- sdAbs can transport hTNFIα-sdAb1 to the brain of rats expressing human TFR1, but not rats solely expressing rat Tfr1. Table 11. Serum and CSF Concentrations of Human hTFR1-sdAb1. s_{dAb Genotype Sex Age Concentration [pM] of Time (Hours) hTNF1α-sdAb [00426] The ability of hTFR1α-sdAbs to cross the BBB in the brain was further evaluated} using a protocol previously used for evaluating BBB passage of AVI-based therapeutics (Kariolis, 2020). This protocol divides the tissue into two fractions: one containing the CNS vasculature (vasculature fraction) and the other containing vascular-depleted parenchymal cells (parenchymal fraction). The ability of hTNFIα-sdAb1-linker-04B05R3 heterodimer to cross the BBB in the brain was examined. _{[00427] The fractionation protocol was validated by conducting a series of Western blot} analyses on brain homogenates, vasculature fractions, and parenchymal fractions. As reported (Kariolis, 2020), the vasculature endothelial cell marker Glut1 was found to be highly enriched in the vasculature fractions and depleted in the parenchymal fractions (FIG.12). Conversely, Gapdh showed enrichment in the parenchymal fraction, indicating higher cellularity compared to total homogenates and vasculature fractions, which contain extracellular components as well. Based on the relative intensity of neuronal (Vamp2, NmdaR2b), microglial (Iba1), astrocytic (Eaat2), and oligodendrocyte (Mbp) markers, the parenchymal fraction appears to be enriched in - 125 - 4181706.v1 5431.1032002 microglia. Human TFR1 was detected in all three brain fractions from Tfr1^h/w rats (but not in the Tfr1^w/w rat), exhibiting enrichment in the vasculature fraction. This enrichment aligns with expectations, as endothelial cells lining the brain’s vessels express very high levels of TFR1. _{[00428] ELISAs analysis of CSF, serum and brain fractions demonstrate that TNFI-Nab1-} linker-04B05R3 is present in the serum, CSF, brain homogenates, vasculature fraction, and parenchymal fraction of all three rats expressing human TFR1 (Table 12). Conversely, the rat expressing solely rat Tfr1 exhibited TNFI-Nab1-linker-04B05R3 solely in the serum. Table 12. ELISA of hTNFIα-sdAb1-linker-04B05R3 (40 nM/kg) in Rats. Concentration [pM] In _{[00429] Two noteworthy observations emerged. First, the levels of hTNFIα-sdAb1-linker-} 04B05R3, determined by both the TFR1- and TNF-based ELISAs, yielded similar results. Second, upon normalization for protein content in the brain fractions, the parenchymal fraction displayed the highest levels of hTNFIα-sdAb1-linker-04B05R3 compared to total homogenates and the vasculature fraction. The presence of hTNFIα-sdAb1-linker-04B05R3 in the parenchymal fraction suggests uptake by CNS cells via their TFR1 (and potentially membrane TNFα). _{[00430] hTFR1-sdAbs and hTNFIα-hTFR1-sdAb heterodimers are present in the CSF and} parenchymal fraction of the CNS, indicating that CNS accumulation involves transcytosis through the BBB from the bloodstream to the CNS tissue. _{[00431] Finally, as evidenced by the following evidence, humanized TF and TFR1 rats exhibit} functionality: a_{) The expression pattern of human TF and TFR1 is similar to the expression pattern} of rat Tf and Tfr1. b_{) The presence of human TF and soluble TFR1 (sTFR1) in the serum. c) The ability of TFR1 to form both homodimers and chimeric dimers with rat Tfr1.} - 126 - 4181706.v1 5431.1032002 d_{) The viability and fertility of single and double humanized TF and TFR1} homozygous rats. e_{) The capacity of human TFR1 to facilitate the transcytosis of TFR1-Nabs.} Example 8 Humanization and Developability Optimization of TfRb-Nabs _{[00432] The optimization strategy for TfRb-Nabs was like that used for TNF1-Nabs. Based on} the most favorable starting points for humanness, VHH-nativeness, and CamSol solubility, TfRb-Nabs 05F02, 04F01, and 04G05 were selected for TfRb-Nabs Families A, B, and D, respectively. For Family G, 03E01 and 05D01 were chosen, as this family presents a single amino acid variation in both CDR1 and CDR3 (Tables 13A-13B). Table 13A. AbNatiV Assessment of TfRb-Nabs. I_{D Sequence H V S} - 127 - 4181706.v1 5431.1032002 _{[00433] Table 13A. The amino acids that differ among members of the same family are} shown in bold. The CDR regions (as defined by the AHo numbering) are underlined. Key metrics include Human-ness (H), VHH-ness (VH), and CamSol Intrinsic (S). Table 13B. HCDR1 Sequences as Determined by Honneger’s Numbering H_{CDR1 SEQ ID HCDR2 SEQ ID HCDR3 SEQ ID} A AbNatiV analysis. (Mutations are shown in bold and underlined characters.) I_{D Family A} EVQLVESGGGFVQPGGSLRLSCAVSGIAFAGRGMSWYRQAPGKQREWVAGI - 128 - 4181706.v1 5431.1032002 EVQLVESGGGLVQPGGSLRLSCAVSGIAFAGRGMSWVRQAPGKGREWVAG exhaustive 7 ITGGGHANYAESVKGRFTISRDNAKNTGYLQMTSLKAEDTGVYYCAVATN - 129 - 4181706.v1 5431.1032002 EVQLVESGGGLVQPGGSLRLSCTASGIDASSRAMAWHRQPPGKGREWVAAI enhanced v2 HRGGTLNYADSVKGRFTISRDNSKNTVYLQMDSLKPEDTAVYYCAVPTNY Table 14B TfRb-Nab mutants with enhanced therapeutic potential determined by AbNatiV analysis. (Changes in Human-ness (H), VHH-ness (VH), and CamSol Intrinsic (S)) I_{D H V S} - 130 - 4181706.v1 5431.1032002 Family D _{[00434] Tables 14A-14B show TfRb-Nab mutants with enhanced therapeutic potential} determined by AbNatiV analysis. Optimization mutations have been confined to the frameworks. We produced these proteins in CHO-S cells and evaluated them for binding to TFR1 on the cell surface. EGFP-positive (transfected) cells were effectively stained with the indicated TfRb-Nab mutants (FIGs.13-16). _{[00435] FIG. 17 summarizes the 13 optimized mutants that showed binding to cell-membrane} TFR1. The mutants highlighted in black exhibit the best features for drug development, including solubility and humanness. Consequently, their BBB permeability was assessed in vivo using our Tfr1 humanized rats. As shown in FIGs.18A-18B, all eight TfRb-Nabs were enriched in the CSF of Tfr1^h/w rats compared to Tfr1^w/w animals. Example 9. Generate TNFI/TfRb-Nabs with therapeutic potential, targeting TNFα in the CNS with minimized immunogenicity and iron uptake interference. _{[00436] Based on these findings, we proceeded to create heterodimers using all eight} optimized TfRb-Nabs in combination with either TNFI-α or TNFI-β. For initial testing, we adopted a configuration like that used for exploratory heterodimers, positioning the TNFI-Nab at the N-terminus relative to the TfRb-Nab. A slightly longer linker was utilized to maximize flexibility and provide a high degree of freedom between the two Nabs. Family G nanobodies were excluded (FIG.17). _{[00437] To further optimize, we selected TfRb-D3 to explore trimeric configurations,} incorporating TfRb-D3 and two TNFI-Nabs. Heterodimers containing TfRb-B1, TfRb-B2, TfRb- A1, and TfRb-A3 were eliminated due to reduced BBB permeability (data not shown). The - 131 - 4181706.v1 5431.1032002 remaining eight dimers and the four pilot trimers were then tested for TNFI activity. For simplicity, we refer to humanized heterodimers and heterotrimers as INN. _{[00438] Notably, the two heterotrimer configurations with TfRb-D3 positioned between two} TNFI-Nabs (INN^α-D3-α and INN^β-D3-β), demonstrated approximately 10-fold greater TNFI activity than TNFI-α or -β alone and showed ~3-fold improved activity compared to trimers where the TfRb-Nab was positioned at the C-terminus of the TNFI-Nabs (INN^α-α-D3 and INN^β-β-D3, FIG.19). Example.10. In vivo BBB permeability _{[00439] Rats were intravenously (IV) injected with 1µl of a 40^M INNs solution in PBS per} gram of rat body weight. After the designated time points, blood was collected, and rats were perfused with PBS to remove blood from tissues prior to harvesting. We collected CSF, brain, kidney, heart, duodenum, liver, and lung tissues. Brains were homogenized, and homogenates were fractionated into vasculature and parenchymal components with a protocol used by Denali’s to evaluate BBB permeability of AVI-based therapeutics¹. All Nabs concentrations are shown in picomolar (pM). These trimers bound hTFR1 (not shown) and crossed the BBB in vivo. One representative experiment in shown FIG.20A. _{[00440] Forty-eight hours post-injection, INNβ-D3-β was detected in the CSF, which reflects} the CNS’ interstitial fluid (ISF), brain homogenates, parenchymal fractions (indicating intracellular content), and brain vessel fractions. BBB crossing was observed only in rats expressing hTFR1. Of note, the CSF/Serum ratio was on average 0.17, ranging from 0.123 to 0.26. Thus, INN^β-D3-β efficiently crosses the BBB via hTFR1, reaching the CSF/ISF and intracellular compartments. INN^β-D3-β was found in other tissues, with heart and kidney showing a potential hTFR1-dependent distribution. While the concentrations of nanobodies in the CSF and serum reflected their actual levels in the body, for brain lysates, brain fractions, and other tissues, we report the picomolarity normalized to a solution with 1 mg/mL of protein content. This normalization accounted for variations in sample preparation, tissue size, and total protein content, providing a more accurate and biologically relevant comparison of nanobody concentrations across different tissues. _{[00441] Fixed brain slices from one hemibrain were stained with anti-His tag antibodies} (green) to detect the His-tagged INN^β-D3-β, alongside with either anti-hTFR1 (red), anti-GFAP (red, for astrocytes), anti-Iba1 (red, for microglia), or anti-NeuN (red, for neurons). The results (FIGs.20B-20D) show that: - 132 - 4181706.v1 5431.1032002 _{[00442] a. INNβ-D3-β and hTFR1 were detected in the brains of rats expressing hTFR1 (Tfr1h/h)} but not in those expressing rTfr1 (Tfr1^w/w). _{[00443] b. INNβ-D3-β showed significant overlap with hTFR1 in brain vessels, as confirmed by} co-staining with the endothelial cell marker CD31. _{[00444] c. INNβ-D3-β colocalized with IBA1+ microglia and GFAP+ astrocytes, as evidenced} by the comparison of His tag panels with IBA1 and GFAP panels (FIGs.20C-20D). Yellow staining observed in some microglia and astrocytes indicates proximity between INN^β-D3-β and IBA1 or GFAP, respectively (overlay panels, FIGs.20C-20D). _{[00445] d. No obvious colocalization of INNβ-D3-β with NeuN+ neurons were observed (not} shown). _{[00446] In all, these findings demonstrate that hTFR1 was physiologically expressed at high} levels in brain vessels and that INN^β-D3-β effectively crossed the BBB via hTFR1, reaching the CSF/ISF and microglia/astrocytes intracellular compartments. Example 11. In vivo hematotoxicity _{[00447] TF transports Fe3+ ions. Its Fe3+ binding affinity varies with pH: under neutral} conditions, TF binds iron tightly and interacts with TFR1/TFR2, facilitating endocytosis. Lower endosomal pH leads to iron release. The receptor-ligand complex is recycled to the cell surface, where TF dissociates from TFR1/TFR2⁵. Interfering with TF-TFR1 interaction and/or uptake can have toxic effects, especially anemia. Homozygous null Tfr1 mice display an embryonic lethal phenotype, while hypo-transferrinemic mice suffer severe anemia⁶. Although TFR1-Nabs from Families D and A did not interfere with TF binding or uptake by CHEK-ATP089 cells (not shown), we assessed the potential hematotoxicity of INNs in vivo. A complete blood count (CBC), conducted by the IRVS core at Rutgers, was performed on ~4 months-old rats humanized for both TF and TFR1, and administered either INN^β-D3-β, INN^β-A2 or PBS. Despite being a heterodimer, INN^β-A2 was included in the study to assess potential hematotoxicity for heterotrimers incorporating TFR1-A2. This is because hematotoxicity, particularly anemia, is potentially influenced by TFR1 binding. The experimental design is detailed in Table 15. The first CBC (Day -3) established baseline values. Subsequent CBCs (Day 1, Day 17 after three injections, and Day 24 after four injections) monitored for acute and long-term hematotoxic effects. All values remained within normal physiological ranges for rats, indicating that the humanization of Tf and Tfr1 genes has preserved normal blood cell functions. Neither INN^{β-D3- β} nor INN^β-A2 caused significant changes in CBC parameters, including anemia indicators (RBC, HGB, HCT, FIG.21), compared to PBS controls. Given that these tests were performed in rats - 133 - 4181706.v1 5431.1032002 expressing human TF and TFR1, the findings are expected to closely reflect the INNs’ effects in humans, especially regarding holo-TF-TFR1 interactions and iron uptake. This supports the likelihood of low hematotoxicity in humans, particularly given that the therapeutic dosage of INNs may be significantly lower than those tested in these experiments. Table 15. Hematotoxicity assessment. D _{-3 D0 D1 D7 D14 D17 D21 D24 [00448] Table 15. Rats were injected IV on the indicated days (inj.) with either PBS (G1),} INN^β-D3-β (G2), or INN^β-A2 (G3) (1 µL of a 40 µM INN solution in PBS per gram of rat body weight). Complete blood counts (CBC) were performed at several time points: 3 days before the first injection (D-3), 24 hours after the first injection (D1), on Day 17 following three injections, and on Day 24 after four injections. The CBC measurements included white blood cells (WBC), neutrophils (NEU), lymphocytes (LYM), monocytes (MONO), eosinophils (EOS), basophils (BAS), as well as their percentages (NEU %, LYM %, MONO %, EOS %, BAS %), red blood cells (RBC), hemoglobin concentration (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW %), platelet count (PLT), and mean platelet volume (MPV). Example 12. Subcutaneous delivery route _{[00449] The tests established proof-of-concept that INNs can enter the CNS via TFR1-} mediated transcytosis through brain endothelial cells, with IV injection serving as a direct method to validate this. Next, we evaluated the SQ route, which offers several advantages: Ease of Use: SQ injections are simple for patients to administer themselves, enhancing compliance and convenience. Examples include Adalimumab (Humira), Etanercept (Enbrel), and Golimumab (Simponi), which are all SQ-administered, allowing patients to manage their treatment at home using prefilled syringes or autoinjectors. Gradual Absorption: SQ administration allows INN to be absorbed slowly and steadily, maintaining therapeutic levels over an extended period. This is ideal for biologics, reducing dosing frequency. Resource Efficiency: SQ administration minimizes the need for specialized clinical infrastructure and professional time, lowering healthcare costs and increasing accessibility. This is in contrast to IV-administeration of biologics, which require more resource-intensive setups. Minimized Discomfort: SQ injections are less invasive and cause less discomfort compared to IV infusions, - 134 - 4181706.v1 5431.1032002 which are more cumbersome and time-consuming. Cost Efficiency: SQ administration reduces overall treatment costs by eliminating frequent clinic visits and lowering the burden on healthcare resources. In summary, SQ administration of INN offers practical benefits in patient convenience, cost-effectiveness, and ease of use. The success of SQ biologics such as Humira, Enbrel, and Simponi underscores these advantages. _{[00450] We administered INNβ-D3-β (3 males and 3 females of the Tfr1h/h genotype), IINNβ-D1} or INN^β-A2 (to Tfr1^h/h and Tfr1^w/w rats, 2 males and 2 females for each genotype) via SC injection (1 µL of a 40 µM INN solution in PBS per gram of rat body weight). INN levels were measured 72 hours post-injection. Serum concentration of INN^β-D3-β were 191.617 ± 28.69 pM, and CSF concentrations were 27.783 ± 3.867 pM, resulting in a CSF/serum ratio of 0.1476 ± 0.0201. INN^β-D3-β was also found in other tissues with a patter similar to that shown in FIGs.20A-20D (data not shown). For INN^β-A2 and INN^β-D1, the data were summarized in FIG.22. In hTFR1 rats, the CSF/serum ratios were 0.14 for INN^β-A2 and 0.37 for INN^β-D1, with average CSF levels of 18.8 pM for INN^β-A2 and 5.5 pM for INN^β-D1. In summary, INN^β-D3-β and INN^β-A2 exhibited higher total INN levels, while INN^β-D1 demonstrated superior BBB permeability. Once again, only the heart (and possibly the kidney) displayed a distribution pattern dependent on hTFR1. These results confirmed that INNs efficiently cross the BBB when administered subcutaneously in hTFR1-expressing rats, indicating that SQ is a viable route for human therapy, effectively delivering INNs across the BBB. The significant levels of INNs detected in both CSF and serum 3 days post-injection indicate that INNs have a stability significantly greater than the few hours typically reported for nanobodies. Furthermore, the consistently higher serum levels of nanobodies containing a TfRb-Nab unit in humanized TFR1 rats compared to WT rats underscore the extended half-life of these nanobodies, attributable to the presence of the ligand. Example 13. Heterotrimeric INN lead candidates _{[00451] Based on the data, heterotrimers were further developed. The eight INNs listed in} FIG.23 (INN^α-D3-α, INN^β-D3-β, plus the new INN^α-D2-α, INN^β-D2-β, INN^α-D1-α, INN^β-D1-β, INN^α-A2-α and INN^β-A2-β) were synthesized. To ensure the highest purity, these proteins underwent a two- step purification process using AmMag™ Ni Magnetic Beads followed by a Chromdex 200pg 320ml column. Employing this methodology resulted in INN preparations with 99% purity (FIGs.24A-24B). Their TNFI activities were evaluated (FIG.23), and a comparison of IC₅₀ values for the new preparations of INN^α-D3-α and INN^β-D3-β with the original batches (FIGs.20A- 20D) showed consistent and reproducible results across preparations. - 135 - 4181706.v1 5431.1032002 Example 14. In Vivo BBB Permeability Testing of Humanized INN Heterotrimerics _{[00452] Nomenclature [00453] The humanized TNFI-Nabs and TfRb-Nabs, as well as the trimers, disclosed herein} have the following designations: Table 16. Humanized antibody designations. B_{iochemical Name Composition of the trimer} ^^{-A2-^} ^ ^ _{[00454] We administered INN^-D3-^, INN^-D3-^, INN^-A2-^, INN^-A2-^, INN^-D1-^, INN^-D1-^,} INN^{^-D2-^}, or INN^{^-D2-^} (1 µL of a 17.3 µM solution in PBS per gram of rat body weight) SQ injection to Tfr1^h/h rats (2 males and 2 females for each NN). Nanobody levels were measured 24 hours post-injection, and the data are summarized in FIGs.25A-25C. _{[00455] Serum Levels: Ranged from 390 pM (INN^-A2-^) to 50.2 pM (INN^-D3-^). [00456] CSF Levels: Ranged from 60 pM (INN^-A2-^) to 18.3 pM (INN^-D3-^). [00457] CSF/Serum Ratios: Varied from 0.14 (INN^-A2-^ and INN^-D1-^) to 0.4 (INN^-D3-^). [00458] The tissue distributions of these 8 heterotrimers were consistent with that observed} for INN^{^-D3-^} (FIGs.20A-20D) and two heterodimers (FIGs.25A-25C). Overall, these results confirmed that these INN heterotrimers effectively cross the BBB following subcutaneous administration in hTFR1-expressing rats. This indicates that SQ administration is a viable and efficient route for delivering these therapeutics across the BBB in a human therapeutic context. Example 15. NewroBus: In Vivo Capability for Transporting Diverse Proteins Across the Blood- Brain Barrier _{[00459] BRI2 is a type II transmembrane protein cleaved by convertases in the trans-Golgi} network, producing the C-terminal Bri2-23 peptide and membrane-anchored mBRI2. mBRI2 is further processed by ADAM10 into the BRI2-BRICHOS ectodomain and BRI2-NTF, which is then cleaved by SPPL2a/b to release the BRI2 intracellular domain⁷ (FIG.27A). Mutations in ITM2B, the gene encoding BRI2, are responsible for rare ADRDs. Two of these mutations - 136 - 4181706.v1 5431.1032002 cause FBD and FDD^8,9, with two additional mutations identified more recently^10,11. FBD and FDD show an AD-like pathology (amyloid plaques, neurofibrillary tangles, neuroinflammation, and neurodegeneration). The FBD mutation at the stop codon causes a stop codon readthrough, producing a mutant BRI2 protein with an extended C-terminal tail of 11 additional amino acids. In FDD, a 10-nucleotide insertion just upstream of the stop codon causes a frameshift, similarly producing a mutant BRI2 protein with an 11-amino-acid C-terminal extension. These C-terminal extensions are identical in length but of different amino acid sequences. Convertase cleavage generates longer amyloidogenic peptides of 34 amino acids, known as ABri (in FBD) and ADan (in FDD), along with mBRI2 (FIG.27B), which are the main components of plaques in FBD and FDD patients. FBD and FDD align with the amyloid cascade hypothesis, with other key differences: while Aβ is produced in all people, primarily by neurons, ABri and ADan are only produced in mutation carriers, mainly by microglia¹². _{[00460] We became interested in BRI2 after an unbiased genetic screen revealed that mBRI2} interacts with APP and down-regulates its processing^13-16 (FIG.27C). The evidence that the BRI2-BRICHOS domain binds Aβ, reducing its oligomerization further supports the BRI2-AD connection^17-19. To study the function of BRI2 and the pathogenesis of FDD and FBD, we developed both pan and cell-type-specific Itm2b-KO mice, as well as FDD and FBD KI models in mice and rats. In KI models the pathogenic mutations were introduced into the endogenous rodent genes. The key advantage of a KI model is that pathogenic mutations are expressed at physiological levels in the appropriate cell types and developmental stages, both in the CNS and peripheral tissues, providing an accurate model of the disease’s pathophysiology. The KI method avoids several problems inherent to the transgenic approach, which overexpresses mutant mini- genes under neuron-specific promoters^20,21. A major concern is that transgenic models can turn a partial LOF mutation into an artificial gain-of-function. If the FDD mutation leads to reduced BRI2 function in humans, a transgenic model—showing increased mBRI2 levels²²— would represent the exact opposite of the disease’s underlying mechanism, making the KI model not just different, but essential. _{[00461] Using these models, we have made several key observations: [00462] 1) FDD and FBD KI rodents show synaptic plasticity and L&M deficits without brain} pathology^23-25, similar to those observed in hetero and homozygous Itm2b-KO mice^23,26-28. _{[00463] 2) The FDD/FBD mutations reduce maturation of mutant BRI2, lowering mBRI2} level^26,27. _{[00464] 3) APP haploinsufficiency prevents these deficits in FDD/FBD KI mice29,30.} - 137 - 4181706.v1 5431.1032002 _{[00465] 4) MoBA, a BRI2-derived peptide that binds APP at the β-secretase cleavage site and} inhibits APP cleavage by β-secretase, rescues L&M deficits in FDD KI mice^31-33. _{[00466] 5) Itm2b deletion in excitatory neurons (EN) affects both pre- and post-synaptic} transmission²⁸. _{[00467] 6) ITM2B is expressed in all CNS cell types, with the highest levels in microglia31,} which explains why ADan and ABri are primarily produced by microglia. _{[00468] 7) Sc-RNA-seq unveiled Trem2/Bri2-dependent microglia clusters, underscoring} their functional interaction³¹. _{[00469] 8) BRI2 binds to TREM2, a microglia-specific protein associated with increased risk} for AD/ADRDs^34-45, and negatively modulates TREM2 processing in a manner similar to its effects on APP processing³¹ (FIG.27D). _{[00470] 9) Pan- and microglia-specific deletion of Itm2b reduces phagocytic activity and} alters the cytokine production profile in activated microglia³¹. These last three points indicate that BRI2 plays a cell-autonomous role in microglia. Finally, FDD patient brains show reduced mBRI2 and increased APP processing¹³. _{[00471] These data suggest that two mechanisms, both due to elongated C-terminal sequences} in the mutant proteins, may underlie FDD/FBD pathogenesis: (1) amyloid-driven, from ADan and ABri, and (2) LOF from reduced mBRI2. If BRI2 LOF is pathogenically relevant, understanding its CNS function could help identify therapeutic targets and develop treatments for FDD, FBD, and potentially AD. _{[00472] The region of APP between the β- and α-secretase cleavage sites (amino acids 593–} 625 of APP-695) mediates binding to BRI2^15,16, while BRI2 amino acids 74-102 are necessary and sufficient for its interaction with APP and inhibition of APP cleavage⁴⁶. Using a peptide- scanning approach, we identified a BRI2 peptide (amino acids 84-93: DDVYYCGIKY (SEQ ID NO:233)) that binds to APP and reduces β-cleavage of APP without affecting BACE1 activity on other substrates⁴⁶. Through alanine scanning mutagenesis, we enhanced its potency by substituting the second residue with alanine (DAVYYCGIKY (SEQ ID NO:234)) , leading to the development of a stronger inhibitor known as MoBA (Modulator of β-cleavage of APP). These findings open therapeutic opportunities for AD by targeting APP processing via BACE1 without inhibiting BACE1 activity, which has shown toxicity in clinical trials. MoBA restored LTP and NOR deficits in mFDD⁴⁶, highlighting its therapeutic promise. _{[00473] We identified two TREM2-binding domains in BRI231, one between amino acids 81} and 131 (T2-BD1) and the other encompassing the BRICHOS domain (T2-BD2) (FIG.28A). - 138 - 4181706.v1 5431.1032002 The interaction of BRI2 with TREM2 inhibits α-cleavage of TREM2³¹. T2-BD1 overlaps with the APP-binding domain of BRI2. Treatment of mouse Itm2b-KO PMM microglia with a 2^M concentration of recombinant BRI2-ECD, which contains both T2-BD1 and T2-BD2, reduced the Trem2-CTF/Trem2 ratio, decreased sTrem2 levels, and Syk phosphorylation³¹, suggesting that BRI2-ECD compensates for Bri2 functions in Trem2 processing and signaling. _{[00474] The evidence above supports investigating BRI2-derived polypeptides’ impact on} APP and TREM2 processing and their therapeutic potential. However, CNS-targeting biologics face limited CNS delivery due to the blood-brain barrier (BBB). To address this, we have used the humanized anti-TfR1 nanobody TFRB-D3, that achieves high CNS penetration without detectable peripheral toxicity, such as anemia. To test this technology for delivering BRI2-based biologics to the CNS, we produced an BRI2T2-BD1/TFRB-D3 fusion protein, termed T2BD1- NN (FIG.28B), and assessed its CNS accessibility using humanized TfR1 rats. Rats were intravenously injected with 1µl of a 40^M T2BD1-NN solution in PBS per gram of rat body weight. As controls, WT rats were also injected. After 24 hours, blood was collected, and rats were perfused with PBS to remove blood from tissues prior to harvesting. We collected CSF and brains. T2BD1-NN was found in the CSF, which reflects the CNS’ interstitial fluid (ISF), and brain homogenates only in rats expressing hTfR1 (FIG.29A). Remarkably, the CSF/serum ratio was 1.197. To further test whether T2BD1-NN penetrated the brain, fixed brain slices from one hemibrain of injected rats were stained with anti-His tag antibodies (green) to detect the His- tagged T2BD1-NN, alongside with either anti-hTfR1 (red), anti-GFAP (astrocytes), anti-Iba1 (microglia) or anti-NeuN (neurons). T2BD1-NN was detected in the brains of rats expressing hTfR1 (FIG.29B) but not in those expressing rTfr1 (not shown). T2BD1-NN showed significant overlap with hTfR1 in brain vessels, as confirmed by co- staining with the endothelial cell marker CD31 (FIG.29B). T2BD1-NN colocalized with GFAP+ astrocytes and Iba1+ microglia (FIGs.29C-29D). No obvious colocalization of T2BD1-NN with neurons were observed (not shown). These findings demonstrate that T2BD1-NN crossed the BBB via hTfR1, supporting the rationale for fusing BRI2-derived biologics with TFRB-D3. _{[00475] These data further underscore the versatility of NewroBus (TFRB-D3) in facilitating} the penetration of biologics across the BBB. _{[00476] NewroBus: Expanding the Potential for CNS Drug Delivery Across the Blood-Brain} Barrier _{[00477] The NewroBus platform represents a transformative approach to delivering biologics} and other traditionally BBB-impermeable therapeutics directly to the central nervous system - 139 - 4181706.v1 5431.1032002 (CNS). By leveraging a humanized nanobody that binds to the transferrin receptor 1 (TFR1) for transcytosis across the BBB, NewroBus efficiently bypasses the physiological barriers that limit CNS drug delivery. This innovation opens the door to a wide array of therapeutic possibilities for treating neurodegenerative diseases and other CNS disorders characterized by limited treatment options. _{[00478] Key Advantages of NewroBus for CNS Drug Delivery (Non-limiting examples) [00479] Versatile Transport Mechanism: [00480] NewroBus can deliver biologics, small molecules, and gene therapies that would} otherwise be unable to cross the BBB. _{[00481] Its modular design allows for the attachment of various therapeutic agents, providing} a flexible platform for targeting multiple CNS diseases as well as multiple targets in one disease simultaneously. _{[00482] Multi-Pathway Targeting: [00483] The platform can carry single or multiple therapeutic agents, enabling simultaneous} targeting of one, two, or multiple pathogenic pathways. _{[00484] This capability allows for combination therapies, such as delivering: [00485] A TNFα inhibitor and a neuroprotective agent simultaneously for Alzheimer’s} disease. _{[00486] A monoclonal antibody against amyloid-β or tau aggregates paired with an anti-} inflammatory agent. _{[00487] Bi-Functional and Multi-Functional Approaches: [00488] NewroBus supports the development of bi-functional antibodies or heterotrimers that} target different disease mechanisms within the CNS. _{[00489] For example: [00490] Bi-functional antibodies targeting both amyloid plaques and TNFα. [00491] Heterotrimers combining two TNFα inhibitors with a TFR1-targeting nanobody for} efficient BBB transport and therapeutic action. _{[00492] Enhanced Clinical Applicability: [00493] NewroBus offers a pathway for subcutaneous (SC) administration, improving ease of} use, patient compliance, and therapeutic accessibility compared to invasive administration routes like intracerebroventricular (ICV). _{[00494] SC administration enables gradual absorption and potentially sustained CNS drug} levels. - 140 - 4181706.v1 5431.1032002 _{[00495] Increased CNS Localization: [00496] Based on preclinical in vivo data, NewroBus significantly enhances the CNS delivery} of biologics. For instance: _{[00497] When fused to Lecanemab (an anti-amyloid-β antibody), NewroBus is predicted to} achieve a CSF/Serum ratio of 0.2-0.5, a dramatic improvement over the typical ratio of <0.01 for unconjugated antibodies. _{[00498] Broad Spectrum of Therapeutic Applications [00499] The following are potential therapeutic areas where NewroBus could revolutionize} CNS drug delivery: _{[00500] Neurodegenerative Diseases: [00501] Alzheimer's Disease: Delivery of anti-amyloid, anti-tau, and TNFα inhibitors. [00502] Parkinson’s Disease: Transport of neuroprotective agents targeting α-synuclein or} mitochondrial dysfunction. _{[00503] Huntington’s Disease: Delivery of RNA-based therapies or gene-modifying agents. [00504] Neuroinflammatory Disorders: [00505] Multiple Sclerosis: Transport of biologics that modulate the immune response or} promote remyelination. _{[00506] Neuromyelitis Optica: Delivery of monoclonal antibodies targeting pathogenic} autoantibodies. _{[00507] Brain Tumors: [00508] Delivery of chemotherapeutics, targeted antibodies, or immunotherapies directly to} the tumor site within the brain. _{[00509] CNS Genetic Disorders: [00510] Lysosomal Storage Diseases: Transport of enzyme replacement therapies or gene} therapy vectors. _{[00511] Spinal Muscular Atrophy (SMA): Delivery of antisense oligonucleotides or gene-} editing tools. _{[00512] Psychiatric and Cognitive Disorders: [00513] Targeting pathways involved in depression, schizophrenia, or cognitive dysfunction} by delivering neuropeptides, growth factors, or neuromodulatory agents. - 141 - 4181706.v1 5431.1032002 Example 16. INN heterotrimers potently inhibit Rhesus TNFα but show reduced or no activity against rodent TNFα. _{[00514] To evaluate species cross-reactivity of TNFα inhibition, we tested eight NN-800} heterotrimers (e.g., comprising a TfRb-Nab positioned between two TNFI-Nabs) for their ability to neutralize recombinant TNFα from rhesus macaque, rat, and mouse. _{[00515] TNFα-induced cytotoxicity was measured using WEHI 13VAR cells. Cells were} harvested, and viability and cell count were determined using acridine orange/propidium iodide (AO/PI) staining on a DeNovix CELLDROP® cell counter. The cell suspension was adjusted to 30,000 cells per well, and 90^µL was seeded into 96-well plates containing Roswell Park Memorial Institute (RPMI) 1640 medium with 10% fetal bovine serum (FBS) and 1^µg/mL Actinomycin-D. _{[00516] Nanobodies were pre-mixed with recombinant TNFα (final concentration:} 0.25^ng/mL) and incubated at room temperature for 30 minutes prior to cell treatment. The following recombinant TNFα trimers were used: human TNFα (Acro Biosystems, Cat. # TNA- H5228), mouse TNFα (Acro Biosystems, Cat. # TNA-M82E9), rat TNFα (R&D Systems, Cat. # 510-RT-010/C), and rhesus/cynomolgus TNFα (Thermo Fisher, Waltham, MA, USA; Cat. #A42631). Serial dilutions of nanobodies were performed to generate dose–response curves. After incubation, 10^µL of the TNFα–nanobody mixture was added to each well of WEHI 13VAR cells. _{[00517] For Caspase-3/7 activity assays, the Caspase-3/7 dye (final concentration: 0.5^µM)} was added directly to the TNFα–nanobody mixture before adding to the cells. Plates were incubated and monitored in real-time using the INCUCYTE® live-cell analysis system under standard culture conditions (37°C, 5% CO₂). To quantify TNFα-induced apoptosis, background Caspase-3/7 activity from wells treated with Actinomycin-D alone was subtracted from the signal in wells treated with Actinomycin-D plus TNFα. TNFα inhibition by nanobodies was calculated as the percentage reduction in Caspase-3/7 activity relative to the maximal activity induced by Actinomycin-D plus TNFα, which was set to 100%. _{[00518] TNFα-induced cytotoxicity was measured using WEHI-164 cells (a TNFα-sensitive} fibrosarcoma cell line) in a caspase-3/7 activity assay. Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS and seeded in 96-well plates. Recombinant TNFα (R&D Systems) was pre-incubated with serial dilutions of each nanobody for 30 minutes at 37°C, followed by addition to WEHI cells. After 16–18 hours of incubation, caspase-3/7 activity was quantified using a luminescent substrate (CASPASE-GLO® 3/7 Assay, Promega), and IC₅₀ values were calculated based on the resulting luminescence signal. - 142 - 4181706.v1 5431.1032002 _{[00519] As shown in FIG. 30, all eight INN heterotrimers potently inhibited rhesus macaque} TNFα, with IC₅₀ values in the low single-digit picomolar range—comparable to their previously measured potency against human TNFα. These results support strong conservation of neutralizing epitopes between human and nonhuman primate TNFα. _{[00520] In contrast, inhibition of rat TNFα was markedly less potent, with IC₅₀ values in the} 20–60^nM range, indicating an approximately 10,000-fold reduction in activity. Against mouse TNFα, INN heterotrimers showed minimal or no detectable activity. The results against rodent TNFα species are consistent with our previous findings during the characterization of TNFI-^ and TNFI-^⁵⁹. _{[00521] Notably, this cross-species assay was conducted approximately 6.5 months after the} initial production of the nanobodies. Two nanobody samples, INN^{^-A2-^} and NN^{^-A2-^} underwent a single freeze–thaw cycle, while the other six were tested from original frozen stocks stored at – 80°C. All samples maintained high activity against rhesus TNFα, indicating excellent long-term stability of the INN heterotrimers. _{[00522] Collectively, these results confirm that the INN platform is highly effective against} primate TNFα but ineffective against endogenous rodent TNFα, highlighting the necessity of humanized rodent models for preclinical efficacy evaluation. Example 17. INN^{^-A2-^} and INN^{^-D2-^} bind human TfR1 with high specificity and exhibit low- affinity interaction with monkey TfR1 only at high concentrations. _{[00523] To determine the species specificity of the heterotrimeric nanobodies INN^-A2-^ and} INN^β-D2-β, we used two complementary approaches: flow cytometry (FACS) in transfected cells and ELISA with recombinant proteins. HEK293T/17 cells were transiently transfected with bicistronic constructs expressing either human or rhesus macaque TfR1 along with EGFP to allow identification of transfected cells. Staining with an APC-conjugated anti-human TfR1 antibody—which cross-reacts with macaque TfR1—confirmed robust surface expression of both receptors in EGFP⁺ cells (FIG.31A). _{[00524] Staining with INN^-A2-^ or INN^-D2-^, followed by an APC-conjugated anti-VHH} secondary antibody, revealed strong signal in hTfR1-transfected (FITC⁺) cells but no detectable staining in cells expressing mkTfR1 (FIG.31B). Signal intensity correlated with FITC fluorescence, indicating that nanobody binding scales with receptor expression. These data demonstrate that both nanobodies are highly specific for human TfR1 and do not bind the macaque ortholog. - 143 - 4181706.v1 5431.1032002 Table 17. Summary of IC₅₀ values. INN^{^-D2-^} INN^{^-D2-^} INN^{^-D3-^} INN^{^-D3-^} Table 17 (continued). Summary of IC₅₀ values. INN^{^-A2-^} INN^{^-A2-^} INN^{^-D1-^} INN^{^-D1-^} _{[00525] Table 17. Summary of IC₅₀ values (and corresponding ranges) from the experiment} shown in FIG.30, along with previously determined IC₅₀ values for human TNFα. _{[00526] To extend these findings, we performed ELISAs using recombinant biotinylated} extracellular domains of human, mouse, rat, and Cynomolgus macaque TfR1. INN^{^-A2-^} and INN^{^-D2-^} were applied in 2-fold serial dilutions (1^nM to 9.765^pM). Both nanobodies bound human TfR1 in a concentration-dependent manner, but showed no detectable interaction with mouse, rat, or macaque TfR1 at any concentration tested (FIG.31C). _{[00527] To test for low-affinity cross-reactivity, we repeated the ELISA with a 1000-fold} higher nanobody concentration range (10^µM to 9.765^nM) for INN^{^-A2-^}, INN^{^-D2-^}, and INN^{^-D1-^}. No interaction with rodent TfR1 was observed at any concentration tested. However, beginning at approximately 300^nM, weak binding to monkey TfR1 became detectable for all three nanobodies (FIG.31D). These findings confirm the high specificity of INN^{^-A2-^} and - 144 - 4181706.v1 5431.1032002 INN^{^-D2-^} for human TfR1, with minimal off-target binding to monkey TfR1 only at supraphysiological concentrations, and no detectable interaction with rodent TfR1 even at high doses. Example 18. In Vivo Toxicity Assessment of Nanobodies. _{[00528] To evaluate the in vivo toxicity of transferrin receptor 1 (TfR1)-targeting nanobody} conjugates, 60-day-old rats were randomized into three treatment groups receiving either (1) TNFI-β−TfR1b-D1−TNFI-β, (2) TNFI-β−TfR1b-A2−TNFI-β, or (3) PBS vehicle control. These bivalent, bispecific nanobody constructs are designed to bind TfR1 and cross the blood-brain barrier (BBB) through receptor-mediated transcytosis, allowing TNFα to be delivered into the brain from the periphery for targeted immune modulation. _{[00529] All nanobody solutions were prepared at a concentration of 30^µM in PBS and} administered subcutaneously into the dorsal interscapular region at a dose of 1^μL per gram of body weight. Injections were performed using a MONOJECT® tuberculin syringe (CardinalHealth, Dublin, OH, USA) with a 25G needle. The subcutaneous route was chosen to allow slow systemic absorption and avoid first-pass metabolism, while also being easy to administer repeatedly. Dosing was conducted on days 0, 5, 8, 12, 15, and 19. This schedule was chosen to mimic repeated dosing and monitor both short-term and longer-term effects. A pretest assessment was conducted on day -5 to establish baseline hematological parameters. Blood was collected from the tail vein 24 hours after the first injection and again on days 7, 14, and 21 (FIG.32). _{[00530] Tail vein collection was carried out using the ONGUARD® blood collection set} (25G, Medline, Northfield, IL, USA). Approximately 200^μL of blood was obtained per animal during the morning hours to minimize circadian variability. The collected blood was immediately transferred into MINICOLLECT® ethylenediaminetetraacetic acid dipotassium salt dihydrate (K2EDTA) tubes (Greiner Bio-One, Monroe, NC, USA) to prevent coagulation and maintained at 4°C until analysis. Complete blood count (CBC) parameters were measured using the Heska ELEMENT HT5® CBC Analyzer (Vermont, VIC, Australia). The following hematological parameters were assessed: white blood cell count (WBC, 10³/μL), neutrophil count (NEU, 10³/μL), lymphocyte count (LYM, 10³/μL), monocyte count (MONO, 10³/μL), eosinophil count (EOS, 10³/μL), basophil count (BAS, 10³/μL), neutrophil percentage (NEU%), lymphocyte percentage (LYM%), monocyte percentage (MONO%), eosinophil percentage (EOS%), basophil percentage (BAS%), red blood cell count (RBC, 10⁶/μL), hemoglobin (HGB, g/dL), hematocrit (HCT, %), mean corpuscular volume (MCV, fL), mean corpuscular - 145 - 4181706.v1 5431.1032002 hemoglobin (MCH, pg), mean corpuscular hemoglobin concentration (MCHC, g/dL), red cell distribution width percentage (RDW%), platelet count (PLT, 10³/μL), and mean platelet volume (MPV, fL). This experimental framework was specifically designed to assess potential hematological toxicity of nanobodies, including off-target immune activation, hematopoietic suppression, or systemic cytokine-related effects. By using repeated dosing and serial blood sampling, we aimed to detect early and cumulative signs of toxicity and assess whether these constructs are well tolerated in vivo for CNS-targeted applications. _{[00531] As shown in FIGs. 33A-33C, none of the values were significantly different from the} PBS control in animals treated with either of the two heterotrimers. Without being bound by any theory, if iron import via TfR1/Tf is impaired, iron availability for erythropoiesis becomes limited; this leads to iron-restricted erythropoiesis, most commonly seen in iron-deficiency anemia. Each value would typically change as follows: _{[00532] Values that would decrease: a) Hemoglobin (HGB, g/dL)– ↓. Less iron means less hemoglobin synthesis. b) Hematocrit (HCT, %)– ↓. Fewer and smaller RBCs result in a lower proportion of} blood volume occupied by RBCs. c_{) Red Blood Cell Count (RBC, 106/μL)– ↓ or normal. Often decreased but can} remain in the low-normal range initially. d_{) Mean Corpuscular Volume (MCV, fL)– ↓. Microcytic anemia: RBCs are smaller} due to impaired hemoglobin synthesis. e_{) Mean Corpuscular Hemoglobin (MCH, pg)– ↓. Less hemoglobin per cell due to} iron deficiency. f_{) Mean Corpuscular Hemoglobin Concentration (MCHC, g/dL)– ↓. or normal Less} hemoglobin in smaller cells; MCHC can be slightly decreased but often normal. _{[00533] Values that would increase: a) Red Cell Distribution Width (RDW%)– ↑. Increased variability in RBC size due} to a mix of old normal-sized cells and new microcytic ones. Table 18. Summary Table. P_{arameter Change with impaired TfR1/Tf iron import Reason} - 146 - 4181706.v1 5431.1032002 M_{CHC ↓ or normal Lower hemoglobin concentration} Example 19. Immunogenicity. _{[00534] Immunogenicity remains a major challenge for protein therapeutics. Immune} responses against biologics can reduce efficacy and, in some cases, cause severe adverse events. CD4⁺ T cell–mediated responses—initiated by dendritic cell (DC) presentation of major histocompatibility complex (MHC) class II–bound peptides—can drive the formation of neutralizing anti-drug antibodies (ADA) and immune complex–mediated inflammation. Thus, minimizing T cell epitopes is critical for reducing immunogenicity To this end, we employed AI- guided mutagenesis to humanize all nanobody components, followed by experimental validation to ensure functional integrity. To confirm low immunogenicity of our constructs, we evaluated the immunogenicity of eight heterotrimeric constructs. As shown in Table 16, these molecules were designed with TNFI-Nbs TNFI-^ or TNFI-^ positioned at both termini of one of four BBB- permeable NewroBus nanobodies (TfRb-D1 [D1], TfRb-D2 [D2], TfRb-D3 [D3], and TfRb-A2 [A2]). This configuration preserves BBB transcytosis while enhancing TNFα-neutralizing potency by up to 20-fold compared to heterodimers (lower IC₅₀; see Table 17), offering a promising therapeutic profile. Immunogenicity was assessed using ex vivo assays with monocyte-derived DCs and CD4⁺ T cells from a cohort of 15 human donors selected based on high-resolution human leukocyte antigen (HLA) typing. This cohort captures the distribution and frequency of MHC class II alleles representative of both European/North American and global populations. This design ensures broad relevance of the immunogenicity findings and reflects allele coverage sufficient to predict potential T cell responses across diverse patient populations. Three complementary methods were used: _{[00535] MHC-Associated Peptide Proteomics (MAPPs, EPISCREEN®): Monocyte-} derived dendritic cells were pulsed with the test proteins, and MHC class II–bound peptides were then isolated from the cell surface and identified using liquid chromatography–tandem mass spectrometry (LC-MS/MS). This approach captures the naturally processed and presented epitopes most relevant to in vivo antigen presentation, allowing direct identification of nanobody-derived peptides likely to trigger T cell responses. A total of 40 unique peptides (Table 19) were identified and further analyzed using iTope-AI. Notably, none of the identified peptides included sequences from the linker regions used to assemble the nanobody multimers, indicating that the chosen linkers are unlikely to contribute to immunogenicity. - 147 - 4181706.v1 5431.1032002 _{[00536] iTope-AI: This in silico machine learning platform predicts peptide–HLA binding} affinities across 46 human MHC class II alleles, enabling high-throughput evaluation of potential immunogenic regions. The predictions help guide rational de-immunization by highlighting sequences most likely to be presented across diverse patient populations, thus refining design decisions before empirical testing. _{[00537] T Cell Epitope Mapping (TCEM): The 40 MAPPs-identified peptides were} synthesized and presented by autologous dendritic cells to matched CD4⁺ T cells from the same donors. T cell activation, measured by interleukin-2 (IL-2) and interferon gamma (IFNγ) release, provided functional validation of immunogenicity. As summarized in Table 19, 42.5% of peptides (17/40) elicited no detectable response, while 37.5% showed responses in only one donor (6.7% response rate). Another 17.5% of peptides had weak responses in two donors (13.3%), and only one peptide—peptide 30—elicited a response in three donors (20%). Importantly, all positive responses were weak in magnitude. _{[00538] Among the heterotrimers, INN^-D3-^ and INN^-D3-^—both containing peptide 30—had} the highest average response rates (Table 20). Similarly, TfR1b-D3 (D3), which also includes peptide 30, was the most immunogenic TfR1b monomer (Table 21). TNFI-α showed slightly higher average immunogenicity than TNFI-β (Table 21). In contrast, INN^{^-D1-^} and INN^{^-D2-^} exhibited the lowest immunogenicity, with most peptide responses limited to a single donor (Table 20). _{[00539] In this same experiment, all tested nanobodies exhibited potential immunogenicity} that was lower than—or, at worst, comparable to—that of anti-human epidermal growth factor receptor 2 (HER2) monoclonal antibody trastuzumab (HERCEPTIN®). Trastuzumab is a widely used therapeutic monoclonal antibody with a well-established clinical safety record and is commonly used as a benchmark for low immunogenicity in biologic drug development. This comparison reinforces the favorable immunogenicity profile of our nanobody-based constructs. Table 19. Rate of positive responses observed to each of the 4015-mer peptides. % _{Response Rate Peptide % of peptides responding at this rate 0% 1 7 9 12 15 17 19 21 24 26} 425% - 148 - 4181706.v1 5431.1032002 Table 20. Heterotrimer ranking based on percentage of positive responses observed in the EPISCREEN® TCEM assay (n=15). P_{rotein name Number of} Average Contains P tid P t f t i k Table 21. Monomers ranking based on percentage of positive responses observed in the EPISCREEN® TCEM assay (n=15). P_{rotein name Number of Peptides} Average Percentage of Contains at di P iti t i k tid ? Table 22. Hematotoxicity assessment. D _{-3 D0 D1 D7 D14 D17 D21 D24 CBC I CBC I I CBC I CBC [00540] Table 22. Hematotoxicity assessment. Rats were injected intravenously (IV) on the} indicated days with either PBS (3 males and 4 females) or INN^{^-D3-^} (3 males and 5 females) at a dose of 1^µL of a 40^µM heterotrimer solution in PBS per gram of body weight. CBCs were performed at multiple time points: 3 days prior to the first injection (D–3), 24 hours after the first injection (D1), on Day 17 following three injections, and on Day 24 after four injections. 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Tamayev, R., Matsuda, S., Arancio, O. & D'Adamio, L. β- but not γ-secretase proteolysis of} APP causes synaptic and memory deficits in a mouse model of dementia. EMBO Mol Med 4, 171-179 (2012). https://doi.org:10.1002/emmm.201100195 - 154 - 4181706.v1 5431.1032002 _{48. Hamers-Casterman et al., Naturally occurring antibodies devoid of light chains, Nature} 363(6428):446-48 (1993). _{49. Li et al., Cell-penetrating anti-GFAP VHH and corresponding fluorescent fusion protein} VHH-GFP spontaneously cross the blood-brain barrier and specifically recognize astrocytes: application to brain imaging, FASEB J.26(10):3969-79 (2012). _{50. Li et al., A versatile tool for in vivo imaging of extracellular and intracellular brain targets, J} Control Release 243:1-10 (2016). _{51. Xiao & Gan, Receptor-mediated endocytosis and brain delivery of therapeutic biologics, Int} J Cell Biol.2013:703545 (2013). _{52. Sehlin et al., Brain delivery of biologics using a cross-species reactive transferrin receptor 1} VNAR shuttle, FASEB J.34(10):13272-83 (2020). _{53. Johnsen et al., Targeting the transferrin receptor for brain drug delivery, Prog Neurobiol.} 181:101665 (2019). _{54. Pardridge et al., Selective transport of an anti-transferrin receptor antibody through the} blood-brain barrier in vivo, J Pharmacol Exp Ther.259(1):66-70 (1991). _{55. Lee et al., Targeting rat anti-mouse transferrin receptor monoclonal antibodies through} blood-brain barrier in mouse, J Pharmacol Exp Ther.292(3):1048-52 (2000). _{56. Kawabata, Transferrin and transferrin receptors update, Free Radic Biol Med. 133:46-54} (2019). _{57. Trenor et al., The molecular defect in hypotransferrinemic mice, Blood 96(3):1113-18} (2000). _{58. Kariolis et al., Brain delivery of therapeutic proteins using an Fc fragment blood-brain} barrier transport vehicle in mice and monkeys, Sci Transl Med.12(545):eaay1359 (2020). _{59. Yin et al., Development of potent humanized TNFalpha inhibitory nanobodies for} therapeutic applications in TNFalpha-mediated diseases, MAbs 17, 2498164 (2025). _{[00541] The teachings of all patents, published applications, and references cited herein are} incorporated by reference in their entirety. _{[00542] While example embodiments have been particularly shown and described, it will be} understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the embodiments encompassed by the appended claims. - 155 - 4181706.v1

Claims

5431.1032002 CLAIMS What is claimed is: _{1. An anti-transferrin receptor 1 (TFR1) antibody or antigen-binding fragment thereof,} comprising an immunoglobulin heavy chain variable (V_H) domain comprising a heavy chain complementarity-determining region (HCDR) 1, an HCDR2, and an HCDR3 having 100% sequence identity to an HCDR1, an HCDR2, and an HCDR3, respectively, of a V_H domain comprising the amino acid sequence of any one of SEQ ID NOs:190, 176, 99-123, 177-189, and 191-199. _{2. The anti-TFR1 antibody or antigen-binding fragment thereof of claim 1, wherein the} HCDR1, HCDR2, and HCDR3 are determined by Honneger’s, Chothia, Kabat, or IMGT numbering scheme. _{3. The anti-TFR1 antibody or antigen-binding fragment thereof of claim 1 or 2, wherein: a) the HCDR1 comprises the amino acid sequence of SEQ ID NO:163, the HCDR2} comprises the amino acid sequence of SEQ ID NO:168, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:172; b_{) the HCDR1 comprises the amino acid sequence of SEQ ID NO:161, the HCDR2} comprises the amino acid sequence of SEQ ID NO:166, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:170; c_{) the HCDR1 comprises the amino acid sequence of SEQ ID NO:2, the HCDR2} comprises the amino acid sequence of SEQ ID NO:14, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:26; d_{) the HCDR1 comprises the amino acid sequence of SEQ ID NO:40, the HCDR2} comprises the amino acid sequence of SEQ ID NO:48, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:26; e_{) the HCDR1 comprises the amino acid sequence of SEQ ID NO:62, the HCDR2} comprises the amino acid sequence of SEQ ID NO:74, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:86; f_{) the HCDR1 comprises the amino acid sequence of SEQ ID NO:5, the HCDR2} comprises the amino acid sequence of SEQ ID NO:16, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:28; - 156 - 4181706.v1

5431.1032002 g_{) the HCDR1 comprises the amino acid sequence of SEQ ID NO:41, the HCDR2} comprises the amino acid sequence of SEQ ID NO:51, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:28; or h_{) the HCDR1 comprises the amino acid sequence of SEQ ID NO:65, the HCDR2} comprises the amino acid sequence of SEQ ID NO:76, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:88. _{4. The anti-TFR1 antibody or antigen-binding fragment thereof of any one of claims 1-3,} wherein: a_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:163, the HCDR2} consists of the amino acid sequence of SEQ ID NO:168, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:172; b_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:161, the HCDR2} consists of the amino acid sequence of SEQ ID NO:166, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:170; c_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:2, the HCDR2} consists of the amino acid sequence of SEQ ID NO:14, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:26; d_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:40, the HCDR2} consists of the amino acid sequence of SEQ ID NO:48, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:26; e_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:62, the HCDR2} consists of the amino acid sequence of SEQ ID NO:74, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:86; f_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:5, the HCDR2} consists of the amino acid sequence of SEQ ID NO:16, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:28; g_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:41, the HCDR2} consists of the amino acid sequence of SEQ ID NO:51, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:28; or h_{) the HCDR1 consists of the amino acid sequence of SEQ ID NO:65, the HCDR2} consists of the amino acid sequence of SEQ ID NO:76, and the HCDR3 consists of the amino acid sequence of SEQ ID NO:88. - 157 - 4181706.v1

5431.1032002 _{5. The anti-TFR1 antibody or antigen-binding fragment thereof of any one of claims 1-4,} wherein the V_H is humanized, contains human framework regions, or both. _{6. The anti-TFR1 antibody or antigen-binding fragment thereof of any one of claims 1, 2,} and 5, wherein the V_H domain comprises an amino acid sequence that has at least 70% sequence identity to the amino acid sequence of at least one of SEQ ID NOs:190, 176, 99-123, 177-189, and 191-199. _{7. The anti-TFR1 antibody or antigen-binding fragment thereof of any one of claims 1, 2, 5,} and 6, wherein the V_H domain comprises an amino acid sequence that has 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs:190, 176, 99- 123, 177-189, and 191-199. _{8. The anti-TFR1 antibody or antigen-binding fragment thereof of any one of claims 1-7,} wherein the V_H domain comprises an amino acid sequence that has at least 70% sequence identity to the amino acid sequence of SEQ ID NO:190 or SEQ ID NO:176. _{9. The anti-TFR1 antibody or antigen-binding fragment thereof of any one of claims 1-8,} wherein the V_H domain comprises an amino acid sequence that has 100% sequence identity to the amino acid sequence of SEQ ID NO:190 or SEQ ID NO:176. _{10. The anti-TFR1 antibody or antigen-binding fragment thereof of any one of claims 1-9,} wherein the anti-TFR1 antibody or antigen-binding fragment thereof is a single-domain antibody (sdAb). _{11. A polypeptide comprising the anti-TFR1 antibody or antigen-binding fragment thereof of} any one of claims 1-10. _{12. The polypeptide of claim 11, further comprising a therapeutic agent, a diagnostic agent,} or a combination thereof. _{13. The polypeptide of claim 12, wherein the therapeutic agent is an antibody or antigen-} binding fragment thereof. _{14. The polypeptide of claim 12 or 13, wherein the therapeutic agent is an anti-tumor} necrosis factor-alpha (TNF-α) antibody or antigen-binding fragment thereof. - 158 - 4181706.v1

5431.1032002 _{15. The polypeptide of claim 14, wherein the anti-TNF-α antibody or antigen-binding} fragment thereof comprises a V_H domain comprising an HCDR1, an HCDR2, and an HCDR3 having 100% sequence identity to an HCDR1, an HCDR2, and an HCDR3, respectively, of a V_H domain comprising the amino acid sequence of any one of SEQ ID NOs:225, 223, 224, 226-228, and 132-137. _{16. The polypeptide of claim 15, wherein the HCDR1, HCDR2, and HCDR3 of the anti-} TNF-α antibody or antigen-binding fragment thereof are determined by Kabat, IMGT, Chothia, or Honneger’s numbering scheme. _{17. The polypeptide of claim 15 or 16, wherein the anti-TNF-α antibody or antigen-binding} fragment thereof comprises a V_H domain comprising: a_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:229, an HCDR2} comprising the amino acid sequence of SEQ ID NO:230, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:231; b_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:138, an HCDR2} comprising the amino acid sequence of SEQ ID NO:232, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:140; c_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:138, an HCDR2} comprising the amino acid sequence of SEQ ID NO:139, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:140; d_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:141, an HCDR2} comprising the amino acid sequence of SEQ ID NO:142, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:143; e_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:144, an HCDR2} comprising the amino acid sequence of SEQ ID NO:145, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:140; or f_{) an HCDR1 comprising the amino acid sequence of SEQ ID NO:147, an HCDR2} comprising the amino acid sequence of SEQ ID NO:148, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:149. _{18. The polypeptide of any one of claims 15-17, wherein the anti-TNF-α antibody or antigen-} binding fragment thereof comprises a V_H domain comprising: - 159 - 4181706.v1

5431.1032002 a_{) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:138, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:139, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:140; b_{) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:141, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:142, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:143; c_{) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:144, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:145, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:140; or d_{) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:147, an HCDR2} consisting of the amino acid sequence of SEQ ID NO:148, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:149. _{19. The polypeptide of any one of claims 14-18, wherein the anti-TNF-α antibody or antigen-} binding fragment thereof is a sdAb. _{20. The polypeptide of any one of claims 14-19, wherein the anti-TNF-α antibody or antigen-} binding fragment thereof comprises a V_H domain comprising an amino acid sequence that has at least 70% sequence identity to the amino acid sequence of at least one of SEQ ID NOs:225, 223, 224, 226-228, and 132-137. _{21. The polypeptide of any one of claims 14-20, wherein the anti-TNF-α antibody or antigen-} binding fragment thereof comprises a V_H domain comprising an amino acid sequence that has 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs:225, 223, 224, 226-228, and 132-137. _{22. The polypeptide of any one of claims 14-21, comprising: a) one anti-TFR1 sdAb and two anti-TNF-α sdAbs; or b) one anti-TFR1 sdAb and one anti-TNF-α sdAb. 23. The polypeptide of any one of claims 14-22, comprising an amino acid sequence that has} at least 70% sequence identity to the amino acid sequence of at least one of SEQ ID NOs:201, 200, 202-215, 124, and 125. - 160 - 4181706.v1

5431.1032002 _{24. The polypeptide of any one of claims 14-23, comprising an amino acid sequence that has} 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs:201, 200, 202-215, 124, and 125. _{25. The polypeptide of any one of claims 11-24, further comprising a protein tag. 26. A polynucleotide encoding the anti-TFR1 antibody or antigen binding fragment thereof} of any one of claims 1-10, or the polypeptide of any one of claims 11-25. _{27. An expression vector comprising the polynucleotide of claim 26. 28. A host cell comprising the polynucleotide of claim 26, or the expression vector of claim} 27. _{29. A method of producing the anti-TFR1 antibody or antigen binding fragment thereof of} any one of claims 1-10, comprising expressing the anti-TFR1 antibody or antigen binding fragment thereof in the host cell of claim 28 and isolating the expressed anti-TFR1 antibody or antigen binding fragment thereof. _{30. A method of producing the polypeptide of any one of claims 11-25, comprising} expressing the polypeptide in the host cell of claim 28 and isolating the expressed polypeptide. _{31. A composition comprising the anti-TFR1 antibody or antigen binding fragment thereof of} any one of claims 1-10, the polypeptide of any one of claims 11-25, the polynucleotide of claim 26, the expression vector of claim 27, or the host cell of claim 28, and a carrier or diluent, optionally wherein the carrier or diluent is a pharmaceutically acceptable carrier or diluent. _{32. The composition of claim 31, further comprising at least one additional therapeutic agent,} optionally, wherein the at least one additional therapeutic agent comprises: a_{) an anti-tau antibody, b) an anti-amyloid-beta (Aβ) antibody, c) a BRI2 peptide, d) an amyloid precursor protein (APP)-targeting agent, or} any combination of the foregoing. - 161 - 4181706.v1

5431.1032002 _{33. A kit comprising the composition of claim 31 or 32. 34. A method of treating a disease in a subject in need thereof, comprising administering to} the subject a therapeutically effective amount of the polypeptide of any one of claims 11- 25, the polynucleotide of claim 26, the expression vector of claim 27, the host cell of claim 28, or the composition of claim 31 or 32, thereby treating the disease. _{35. A method of blocking binding of TNF-α to TNFR in a subject, comprising administering} to the subject an effective amount of the polypeptide of any one of claims 11-25, the polynucleotide of claim 26, the expression vector of claim 27, the host cell of claim 28, or the composition of claim 31 or 32, thereby blocking binding of TNF-α to TNFR expressed in the subject. _{36. A method of treating a TNF-α-associated disease in a subject in need thereof, comprising} administering to the subject an effective amount of the polypeptide of any one of claims 11-25, the polynucleotide of claim 26, the expression vector of claim 27, the host cell of claim 28, or the composition of claim 31 or 32, thereby treating the TNF-α-associated disease. _{37. The method of claim 36, wherein the TNF-α-associated disease is Alzheimer’s disease} (AD). _{38. The method of claim 36 or 37, wherein the TNF-α-associated disease is late onset} Alzheimer’s disease (LOAD). _{39. The method of claim 36, wherein the TNF-α-associated disease is traumatic brain injury} (TBI). _{40. The method of any one of claims 34-39, wherein the subject is an adult human patient. 41. The method of any one of claims 34-39, wherein the subject is a pediatric human patient. 42. The method of any one of claims 34-41, further comprising administering to the subject: a) an anti-serum albumin agent, b) a fusion protein comprising human serum albumin, c) a chemotherapeutic agent, d) an immunosuppressant agent, or} - 162 - 4181706.v1

5431.1032002 any combination of the foregoing. _{43. The method of any one of claims 34-42, further comprising administering to the subject} methotrexate. _{44. A method of blocking binding of TNF-α to TNF receptor (TNFR) expressed on a surface} of a cell, the method comprising contacting the cell with an effective amount of the polypeptide of any one of claims 11-25, the polynucleotide of claim 26, the expression vector of claim 27, the host cell of claim 28, or the composition of claim 31 or 32, thereby blocking binding of TNF-α to TNFR expressed on the surface of the cell. - 163 - 4181706.v1