WO2025090840A1

WO2025090840A1 - Antigen binding molecules targeting tnf-alpha

Info

Publication number: WO2025090840A1
Application number: PCT/US2024/052920
Authority: WO
Inventors: Luciano D'adamio; Tao Yin
Original assignee: Rutgers State University of New Jersey
Current assignee: Rutgers State University of New Jersey
Priority date: 2023-10-27
Filing date: 2024-10-25
Publication date: 2025-05-01
Anticipated expiration: 2026-04-27

Abstract

The disclosure provides, in various embodiments, antibodies and antigen binding fragments thereof, and polypeptides that bind tumor necrosis factor-alpha (TNF-α). 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 TNF-α-associated diseases or conditions.

Description

Antigen Binding Molecules Targeting TNF-alpha

RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application No. 63/593,885, filed on October 27, 2023. The entire teachings of the above application 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: 54311028001. xml; created October 23, 2024, 42,882 Bytes in size.

GOVERNMENT SUPPORT

[0003] This invention was made with government support under grant nos. R01 AG073182- 03 and R41 AG080864-01 awarded by the National Institutes of Health. 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 P-amyloid (AP) and tau are believed to cause AD. However, therapies targeting Ap and/or tau have shown negligible benefits on cognitive decline in patients.

SUMMARY

[0005] There is a critical need to develop therapeutic agents that treat Alzheimer’s disease (AD) by modulating targets other than toxic forms of P-amyloid (AP) and tau. Tumor necrosis factor-alpha (TNF-a) plays an important role in late onset Alzheimer’s disease (LOAD). The disclosure provided herein is based, in part, on the discovery that antibodies and antigen-binding fragments thereof disclosed herein specifically bind to human TNF-a and demonstrate various additional beneficial properties.

[0006] The disclosure provides, among other things, an antibody or an antigen-binding fragment thereof comprising an immunoglobulin heavy chain variable domain (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:40-45 and 4-6.

[0007] In some embodiments, an antibody or an antigen-binding fragment thereof comprises a V_H domain comprising: a) an HCDR1 comprising (e.g., consisting of) the amino acid sequence of SEQ ID NO: 1, b) an HCDR2 comprising (e.g., consisting of) the amino acid sequence of SEQ ID NO:46 or 2, and c) an HCDR3 comprising (e.g., consisting of) the amino acid sequence of SEQ ID NO:3.

[0008] In some embodiments, an antibody or an antigen-binding fragment thereof comprises a V_H domain comprising: a) an HCDR1 comprising (e.g., consisting of) the amino acid sequence of SEQ ID NO:29, b) an HCDR2 comprising (e.g., consisting of) the amino acid sequence of SEQ ID NO:30, and c) an HCDR3 comprising (e.g., consisting of) the amino acid sequence of SEQ ID NO:31.

[0009] In some embodiments, an antibody or an antigen-binding fragment thereof comprises a V_H domain comprising: a) an HCDR1 comprising (e.g., consisting of) the amino acid sequence of SEQ ID NO:32, b) an HCDR2 comprising (e.g., consisting of) the amino acid sequence of SEQ ID NO:33, and c) an HCDR3 comprising (e.g., consisting of) the amino acid sequence of SEQ ID NO:3.

[0010] In some embodiments, an antibody or an antigen-binding fragment thereof comprises a V_H domain comprising: a) an HCDR1 comprising (e.g., consisting of) the amino acid sequence of SEQ ID NO:34, b) an HCDR2 comprising (e.g., consisting of) the amino acid sequence of SEQ ID NO:35, and c) an HCDR3 comprising (e.g., consisting of) the amino acid sequence of SEQ ID NO:36.

[0011] In some embodiments, a V_H domain comprises (e.g., consists of) an amino acid sequence that has at least 70% sequence identity to the amino acid sequence of at least one of SEQ ID NOs:40-45 and 4-6. In some embodiments, a V_H domain comprises (e.g., consists of) an amino acid sequence that has at least 70% sequence identity to the amino acid sequence of at least one of SEQ ID NOs:40, 42, and 4-6.

[0012] In some embodiments, a V_H domain comprises (e.g., consists of) an amino acid sequence that has 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs:40-45 and 4-6. In some embodiments, a V_H domain comprises (e.g., consists of) an amino acid sequence that has 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs:40, 42, and 4-6.

[0013] In some embodiments, an antigen-binding fragment is a single-domain antibody (sdAb).

[0014] In some embodiments, an antibody or an antigen-binding fragment thereof binds tumor necrosis factor-alpha (TNF-a).

[0015] The disclosure also provides, among other things, a polypeptide comprising any one or more of the antibodies or antigen binding fragments thereof disclosed herein.

[0016] In some embodiments, a polypeptide comprises a single antibody or antigen binding fragment thereof disclosed herein.

[0017] In some embodiments, a polypeptide comprises two or more of the antibodies or antigen binding fragments thereof disclosed herein.

[0018] In some embodiments, a polypeptide further comprises a protein tag, a heterologous moiety, a therapeutic agent, a diagnostic agent, or any combination of the foregoing.

[0019] The disclosure also provides, among other things, a polynucleotide or vector (e.g., expression vector) encoding any one or more of the antibodies or antigen binding fragments thereof, or polypeptides disclosed herein.

[0020] The disclosure also provides, among other things, a vector (e.g., expression vector) comprising any one or more of the polynucleotides disclosed herein. [0021] The disclosure also provides, among other things, a host cell comprising any one or more of the antibodies or antigen binding fragments thereof, polypeptides, polynucleotides, and/or vectors (e.g., expression vectors) disclosed herein.

[0022] 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 one or more antibodies or antigen binding fragments thereof, or polypeptides in a host cell disclosed herein and isolating the expressed one or more antibodies, antigen binding fragments thereof, or polypeptides.

[0023] The disclosure also provides, among other things, a composition (e.g., a pharmaceutical composition) comprising any one or more of the antibodies or antigen-binding fragments thereof, polypeptides, polynucleotides, vectors, or host cells disclosed herein.

[0024] 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.

[0025] The disclosure also provides, among other things, a method of blocking binding of TNF-a 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 antibodies or antigen binding fragments thereof, polypeptides, or compositions (e.g., pharmaceutical compositions) disclosed herein, thereby blocking binding of TNF-a to TNFR expressed on the surface of the cell.

[0026] The disclosure also provides, among other things, a method of blocking binding of TNF-a to TNFR in a subject, the method comprising administering to the subject an effective amount of any one or more of the antibodies or antigen binding fragments thereof, polypeptides, or compositions (e.g., pharmaceutical compositions) disclosed herein, thereby blocking the binding of TNF-a to TNFR in the subject.

[0027] The disclosure also provides, among other things, a method of treating a TNF-a- associated disease in a subject in need thereof, comprising administering to the subject an effective amount of any one or more of the antibodies or antigen binding fragments thereof, polypeptides, or compositions (e.g., pharmaceutical compositions) disclosed herein, thereby treating the TNF-a-associated disease.

[0028] 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 antibodies or antigen binding fragments thereof, polypeptides, or compositions (e.g., pharmaceutical compositions) disclosed herein, thereby reducing inflammation in the subject.

[0029] In some embodiments, a pharmaceutical composition comprises a pharmaceutically acceptable carrier and any one or more of the antibodies or antigen binding fragments thereof disclosed herein.

[0030] In some embodiments, a pharmaceutical composition comprises a pharmaceutically acceptable carrier and any one or more of the polypeptides disclosed herein.

[0031] In some embodiments, an antibody or antigen binding fragment thereof comprises e.g., consists of) a single-domain antibody (sdAb).

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] 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.

[0033] 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.

[0034] FIGs. 1A-1D. Tumor necrosis factor-alpha (TNF-a) and Alzheimer’s disease (AD) pathogenesis. FIG. 1A. Glia cells set physiological excitatory/inhibitory balance and long-term potentiation (LTP) via TNF-a. FIG. IB. TREM2 expression is restricted to microglia; thus, microglia expressing the p.R47H variant (Trem2^7?47H-microglia) are likely the source of supraphy si ologi cal TNF-a. Also, 7 /'c/7?2^/'⁷ '' ''-microglia may promote TNF-a production by other cell types, such as astrocytes. Supraphy si ologi cal TNF-a impairs LTP and increases the excitatory/inhibitory balance by enhancing glutamate and reducing y-aminobutyric acid (GABA) transmission. Exocytosis of a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMP AR) and endocytosis of GABA_A receptor (GABA_AR) at post-synaptic termini could be one mechanism by which supraphy si ologi cal TNF-a increases the excitatory/inhibitory balance. These dysfunctions may enhance glutamate exci totoxi city and neuronal cells death, culminating in cognitive deficits, pathology, and neurodegeneration decades later. FIG. 1C. Resetting TNF-a activity at physiological levels normalizes excitatory/inhibitory balance and LTP and could prevent neurodegeneration. FIG. ID. Increased excitatory transmission may cause activitydependent increase in Ap levels. Supraphysiological TNF-a also promotes amyloid pathology by favoring Ap production over clearance. [0035] FIGs. 2A-2D. Identification of TNF-a inhibitor single-domain antibodies (sdAbs)l-3 (TNFI-Nabl-3). FIG. 2A. SDS-PAGE showing purity of TNFI-Nabl-3 produced in bacteria.

One inactive anti-TNF-a-Nab (01E03R3) is also shown. FIG. 2B. TNFI-Nabl reduced cytotoxicity of human soluble TNF-a (sTNF-a). FIGs. 2C-2D. TNFI-Nabl binds membrane- tethered human mature TNF-a.

[0036] FIG. 3. TNFI-Nabs have low predicted immunogenicity. Total Scores and Hotspot Max of TNFI-Nabl-3 were compared to those of the indicated therapeutic antibodies or nonhuman proteins.

[0037] FIG. 4. TNFI-Nab ELISA.

[0038] FIG. 5. Production of TNFI-Nabs in mammalian cells.

[0039] FIG. 6. TNF inhibitory activities of mutant TNFI-Nabs.

[0040] FIG. 7. Comparative kinetic analysis of kinetic parameters for TNFa against immobilized nanobodies. Two-dimensional iso-affinity kinetic plot of rate constants is shown for interactions characterized on sensors with immobilized nanobodies. Diagonal lines depict equilibrium binding constants and are shown to help with the visualization of the affinity distribution. Each circle represents one set of kinetic values determined based on a 1 : 1 binding model.

DETAILED DESCRIPTION

[0041] A description of example embodiments follows.

[0042] 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.

Definitions

[0043] 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.

[0044] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0045] 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.

[0046] 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.” [0047] As used herein, the term “consisting of’ excludes any element, step, or ingredient not specified in the claim element.

[0048] 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.

[0049] 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.”

[0050] 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 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.”

[0051] 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.

[0052] 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 ” [0053] 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. 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. [0054] 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 a-amino acids (e.g., alanine, glycine, valine), non-a-amino acids (e.g., P-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., 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), A-methyl-a-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). [0055] 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.

[0056] 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.

[0057] 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, Proc. Nat ’I. 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).

[0058] 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 etal., 1990).

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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 variable antibody domain. The single-domain antibodies identified herein as anti-TNF-a sdAb 1- 3 are also referred to herein as “TNF-a-Nab 1-3” and “TNFI-Nabl-3.”

[0064] 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.

[0065] 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.

[0066] 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-FRCDRdefmition.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 Acids Research, Volume 40, Issue Wl, 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).

[0067] As used herein, the term “K_D,” 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.

[0068] 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.

[0069] As used herein, the term “promoter” refers to a region of DNA to which RNA polymerase binds and initiates the transcription of a gene.

[0070] 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.

[0071] 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.

[0072] As used herein, the term “expression host cell” refers to a cell useful for receiving, maintaining, reproducing and/or amplifying a vector.

[0073] 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.

[0074] 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.

[0075] 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, dodecyl sulfate, 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, phenyl acetate, 3 -phenylpropionate, phosphate, pivalate, propionate, pyruvate, salicylate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like.

[0076] 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.

[0077] 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⁺((Ci-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 amine cations formed using counterions such as halide, hydroxide, carboxyl, sulfate, phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate.

[0078] 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. [0079] “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.

[0080] “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, z.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).

[0081] 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.

[0082] 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. [0083] 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.

[0084] 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.

[0085] Non-limiting examples of surfactants are polysorbates (e.g., polysorbate-20 or polysorbate-80), poly oxamers (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, 1 auroamidopropyl-, 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.).

[0086] 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.

[0087] 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.

[0088] 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).

[0089] 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.

[0090] “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.

[0091] The term “treating,” or “treatment” refers to the medical management of a subject with the intent to improve, ameliorate, stabilize (z.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 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. [0092] As used herein, the term “a TNF-a 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-a. In some embodiments, an elevated level of TNF-a is an episodic elevated level of TNF-a activity (e.g., a local and/or systemic increase in TNF activity). In some embodiments, an elevated level of TNF-a is a chronic elevated level of TNF-a activity (e.g., a local and/or systemic increase in TNF activity). In some embodiments, a TNF-a associated disorder is a local disorder where TNF-a is a primary mediator. In some embodiments, a TNF-a associated disorder is a systemic disorder where TNF-a is a primary mediator.

[0093] 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).

[0094] “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.

[0095] 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. [0096] 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.

[0097] 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.

Antibodies or Antigen-Binding Fragments Thereof

[0098] In some embodiments, an antibody or an antigen-binding fragment thereof is a full- length (e.g., whole, intact) antibody. In some embodiments, an antibody or an 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 fragment, a Fv fragment, or a domain antibody (dAb). In some embodiments, an antibody or an antigen-binding fragment thereof is a sdAb, Fab, F(ab’)₂, Fab’, scFv, or Fv. In some embodiments, an antibody or an antigen-binding fragment thereof is a sdAb.

Complementarity -Determining Regions of Heavy Chain Variable Domain (VH) [0099] In some embodiments, an antibody or an antigen-binding fragment thereof (e.g., a sdAb) comprises an immunoglobulin heavy chain variable domain (V_H). In some embodiments, an antibody or an antigen-binding fragment thereof (e.g., a sdAb) comprises a mammalian V_H domain. In some embodiments, an antibody or an antigen-binding fragment thereof (e.g., a sdAb) comprises a Camelidae e.g., llama, camel, or alpaca) V_H domain.

[00100] In some embodiments, an antibody or an antigen-binding fragment thereof (e.g., a sdAb) comprises a humanized V_H domain (e.g., at least about: 10%, 20%, 30%, 40%, 50%, 60%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% humanized). In some embodiments, a V_H domain is at least 75% humanized. In some embodiments, a V_H domain is at least 78% humanized. In some embodiments, a V_H domain is at least 80% humanized. In some embodiments, a V_H domain is at least 81% humanized. In some embodiments, a V_H domain is at least 82% humanized.

[00101] In some embodiments, an antibody or an antigen-binding fragment thereof (e.g., a sdAb) comprises human framework regions.

[00102] In some embodiments, an antibody or an antigen-binding fragment thereof (e.g., a sdAb) comprises a V_H domain comprising a heavy chain complementarity-determining region (HCDR) 1, an HCDR2, and an HCDR3 having at least about 80% sequence identity (e.g., having at least about: 82%, 85%, 88%, 90%, 92%, 95%, 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:40-45 and 4-6.

[00103] In some embodiments, an antibody or an antigen-binding fragment thereof (e.g., a sdAb) comprises a V_H domain comprising a heavy chain complementarity-determining region (HCDR) 1, an HCDR2, and an HCDR3 having at least about 80% sequence identity (e.g., having at least about: 82%, 85%, 88%, 90%, 92%, 95%, 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:40, 42, and 4-6.

[00104] In some embodiments, an antibody or an antigen-binding fragment thereof (e.g., a sdAb) 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:40-45 and 4-6.

[00105] In some embodiments, an antibody or an antigen-binding fragment thereof (e.g., a sdAb) 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:40, 42, and 4-6. [00106] EVQLVESGGGLVQPGGSLRLSCAASGF AFNDHWMYWVRQAPGKELEWVSE INTNGLITKYADFVKGRFTVSRDNAKNTLYLQMDSLRPEDTAVYSCSRNQIGTLRGQGT QVTVSS (SEQ ID NO:4) TNFI-Nabla

[00107] QVQLVESGGGLVQPGGSLRLSCAASGF AFNDHWMYWVRQAPGKELEWVSE INTNGLITKYADFVKGRFTVSRDNAENTLYLQMNSLEPEDTAVYSCSRNQIGTLRGQGT QVTVSS (SEQ ID NO: 5) TNFI-Nab2a

[00108] QVQLVESGGGLVQPGGSLRLSCAASGF AFNDHWMYWVRQAPGKELEWVSE INTNGLITKYADFVKGRFTVSRDNAKNTLYLQMNSLEPEDTAVYSCSRNQIGTLRGQGT QVTVSS (SEQ ID NO: 6) TNFI-Nab3a

[00109] EVQLVESGGGLVQPGGSLRLSCAASGF AFNDHWMYWVRQAPGKELEWVSE INTNGLITKYADFVKGRFTVSRDNAKNTLYLQMDSLRPEDTAVYSCSRNQIGTLRGQGT QVTVSSGPGGQ (SEQ ID NO:7) TNFI-Nablb

[00110] Q VQLVESGGGLVQPGGSLRLSCAASGF AFNDHWMYWVRQAPGKELEWVSE INTNGLITKYADFVKGRFTVSRDNAENTLYLQMNSLEPEDTAVYSCSRNQIGTLRGQGT QVTVSSGPGGQ (SEQ ID NO: 8) TNFI-Nab2b

[00111] Q VQLVESGGGLVQPGGSLRLSCAASGF AFNDHWMYWVRQAPGKELEWVSE INTNGLITKYADFVKGRFTVSRDNAKNTLYLQMNSLEPEDTAVYSCSRNQIGTLRGQGT QVTVSSGPGGQ (SEQ ID NOV) TNFI-Nab3b

[00112] EVQLVESGGGLVQPGGSLRLSC AASGF AFNDHWMYWVRQAPGKGLEWVSE INTNGLITKYADSVKGRFTVSRDNAKNTLYLQMDSLRPEDTAVYSCSRNQIGTLRGQGT QVTVSS (SEQ ID NO:40) TNFLNabl Ml

[00113] EVQLVESGGGLVQPGGSLRLSC AASGF AFNDHWMYWVRQAPGKGLEWVSE INTNGLITKYADSVKGRFTVSRDNAKNTLYLQMDSLRPEDTAVYSCSRNQIGTLWGQGT QVTVSS (SEQ ID NO:41) TNFI-Nabl_M2

[00114] EVQLVESGGGLVQPGGSLRLSC AASGF AFNDHWMYWVRQAPGKGLEWVSE INTNGLITKYADSVKGRFTVSRDNSKNTLYLQMDSLRPEDTAVYSCSRNQIGTLRGQGT LVTVSS (SEQ ID NO:42) TNFI-Nabl_M3

[00115] EVQLVESGGGLVQPGGSLRLSCKASGF AFNDHWMYWVRQAPGKGLEWVSE INTNGLITKYADSVKGRFTVSRDNSKNTLYLQMDSLRPEDTAVYSCSRNQIGTLWGQGT LVTVSS (SEQ ID NO:43) TNFI-Nabl_M4

[00116] EVQLVESGGGLVQPGGSLRLSC AASGF AFNDHWMYWVRQAPGKGLEWVSE INTNGLITKYADSVKGRFTVSRDNAKNTLYLQMDSLRPEDTAVYSCSRNQIGTLRGQGT LVTVSS (SEQ ID NO:44) TNFI-Nabl_M5 [00117] EVQLVESGGGLVQPGGSLRLSC AASGF AFNDHWMYWVRQAPGKGLEWVSE INTNGLITKYADSVKGRFTVSRDNAKNTLYLQMDSLRPEDTAVYSCSRNQIGTLWGQGT LVTVSS (SEQ ID NO:45) TNFI-Nabl_M6

[00118] In some embodiments, an antibody or an antigen-binding fragment thereof (e.g., a sdAb) comprises a V_H domain comprising an HCDR1, an HCDR2, and an HCDR3 having at least about 80% sequence identity (e.g., having at least about: 82%, 85%, 88%, 90%, 92%, 95%, 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:40.

[00119] In some embodiments, an antibody or an antigen-binding fragment thereof (e.g., a sdAb) 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 SEQ ID NO:40.

[00120] In some embodiments, an antibody or an antigen-binding fragment thereof (e.g., a sdAb) comprises a V_H domain comprising an HCDR1, an HCDR2, and an HCDR3 having at least about 80% sequence identity (e.g., having at least about: 82%, 85%, 88%, 90%, 92%, 95%, 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:42.

[00121] In some embodiments, an antibody or an antigen-binding fragment thereof (e.g., a sdAb) 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 SEQ ID NO:42.

[00122] In some embodiments, an antibody or an antigen-binding fragment thereof (e.g., a sdAb) comprises a V_H domain comprising an HCDR1, an HCDR2, and an HCDR3 having at least about 80% sequence identity (e.g., having at least about: 82%, 85%, 88%, 90%, 92%, 95%, 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:4.

[00123] In some embodiments, an antibody or an antigen-binding fragment thereof (e.g., a sdAb) 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 SEQ ID NO:4.

[00124] In some embodiments, an antibody or an antigen-binding fragment thereof (e.g., a sdAb) comprises a V_H domain comprising an HCDR1, an HCDR2, and an HCDR3 having at least about 80% sequence identity (e.g., having at least about: 82%, 85%, 88%, 90%, 92%, 95%, 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:5.

[00125] In some embodiments, an antibody or an antigen-binding fragment thereof (e.g., a sdAb) 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 SEQ ID NO:5.

[00126] In some embodiments, an antibody or an antigen-binding fragment thereof (e.g., a sdAb) comprises a V_H domain comprising an HCDR1, an HCDR2, and an HCDR3 having at least about 80% sequence identity (e.g., having at least about: 82%, 85%, 88%, 90%, 92%, 95%, 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:6.

[00127] In some embodiments, an antibody or an antigen-binding fragment thereof (e.g., a sdAb) 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 SEQ ID NO:6.

Complementarity -Determining Regions

[00128] Kabat numbering

[00129] In some embodiments, an antibody or an antigen-binding fragment thereof (e.g., a sdAb) comprises a V_H domain comprising: a) a heavy chain complementarity-determining region (HCDR) 1 having at least about 80% sequence identity (e.g., having at least about: 82%, 85%, 88%, 90%, 92%, 95%, 98% or 99% sequence identity) to the amino acid sequence of SEQ ID NO: 1, b) an HCDR2 having at least about 80% sequence identity (e.g., having at least about: 82%, 85%, 88%, 90%, 92%, 95%, 98% or 99% sequence identity) to the amino acid sequence of SEQ ID NO:46 or SEQ ID NO:2, and c) an HCDR3 having at least about 80% sequence identity (e.g., having at least about: 82%, 85%, 88%, 90%, 92%, 95%, 98% or 99% sequence identity) to the amino acid sequence of SEQ ID NO:3.

[00130] HCDR1 : DHWMY (SEQ ID NO: 1)

[00131] HCDR2: EINTNGLITKYADFVKG (SEQ ID NO:2) or EINTNGLITKYADSVKG (SEQ ID NO:46)

[00132] HCDR3 : NQIGTL (SEQ ID NO:3) [00133] The sequences identified as SEQ ID NO: 1, SEQ ID NOs:2 and 46, and SEQ ID NO:3 correspond to HCDR1, HCDR2, and HCDR3 sequences, respectively, as determined by Kabat numbering.

[00134] In some embodiments, an antibody or an antigen-binding fragment thereof (e.g., a sdAb) comprises a V_H domain comprising: a) an HCDR1 having at least about 80% sequence identity (e.g., having at least about: 82%, 85%, 88%, 90%, 92%, 95%, 98% or 99% sequence identity) to the amino acid sequence of SEQ ID NO: 1, b) an HCDR2 having at least about 80% sequence identity (e.g., having at least about: 82%, 85%, 88%, 90%, 92%, 95%, 98% or 99% sequence identity) to the amino acid sequence of SEQ ID NO:46, and c) an HCDR3 having at least about 80% sequence identity (e.g., having at least about: 82%, 85%, 88%, 90%, 92%, 95%, 98% or 99% sequence identity) to the amino acid sequence of SEQ ID NO:3.

[00135] In some embodiments, an antibody or an antigen-binding fragment thereof (e.g., a sdAb) comprises a V_H domain comprising: a) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, b) an HCDR2 comprising the amino acid sequence of SEQ ID NO:46, and c) an HCDR3 comprising the amino acid sequence of SEQ ID NO:3.

[00136] In some embodiments, an antibody or an antigen-binding fragment thereof (e.g., a sdAb) comprises a V_H domain comprising: a) an HCDR1 consisting of the amino acid sequence of SEQ ID NO: 1, b) an HCDR2 consisting of the amino acid sequence of SEQ ID NO:46, and c) an HCDR3 consisting of the amino acid sequence of SEQ ID NO:3.

[00137] In some embodiments, an antibody or an antigen-binding fragment thereof (e.g., a sdAb) comprises a V_H domain comprising: a) an HCDR1 having at least about 80% sequence identity (e.g., having at least about: 82%, 85%, 88%, 90%, 92%, 95%, 98% or 99% sequence identity) to the amino acid sequence of SEQ ID NO: 1, b) an HCDR2 having at least about 80% sequence identity (e.g., having at least about: 82%, 85%, 88%, 90%, 92%, 95%, 98% or 99% sequence identity) to the amino acid sequence of SEQ ID NO:2, and c) an HCDR3 having at least about 80% sequence identity (e.g., having at least about: 82%, 85%, 88%, 90%, 92%, 95%, 98% or 99% sequence identity) to the amino acid sequence of SEQ ID NO:3.

[00138] In some embodiments, an antibody or an antigen-binding fragment thereof (e.g., a sdAb) comprises a V_H domain comprising: a) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, b) an HCDR2 comprising the amino acid sequence of SEQ ID NO:2, and c) an HCDR3 comprising the amino acid sequence of SEQ ID NO:3.

[00139] In some embodiments, an antibody or an antigen-binding fragment thereof (e.g., a sdAb) comprises a V_H domain comprising: a) an HCDR1 consisting of the amino acid sequence of SEQ ID NO: 1, b) an HCDR2 consisting of the amino acid sequence of SEQ ID NO:2, and c) an HCDR3 consisting of the amino acid sequence of SEQ ID NO:3.

[00140] IMGT numbering

[00141] In some embodiments, an antibody or an antigen-binding fragment thereof (e.g., a sdAb) comprises a V_H domain comprising: a) an HCDR1 having at least about 80% sequence identity (e.g., having at least about: 82%, 85%, 88%, 90%, 92%, 95%, 98% or 99% sequence identity) to the amino acid sequence of SEQ ID NO:29, b) an HCDR2 having at least about 80% sequence identity (e.g., having at least about: 82%, 85%, 88%, 90%, 92%, 95%, 98% or 99% sequence identity) to the amino acid sequence of SEQ ID NO: 30, and c) an HCDR3 having at least about 80% sequence identity (e.g., having at least about: 82%, 85%, 88%, 90%, 92%, 95%, 98% or 99% sequence identity) to the amino acid sequence of SEQ ID NO: 31.

[00142] HCDR1 : GFAFNDHW (SEQ ID NO:29)

[00143] HCDR2: INTNGLIT (SEQ ID NO:30)

[00144] HCDR3 : SRNQIGTL (SEQ ID NO:31)

[00145] The sequences identified as SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31 correspond to HCDR1, HCDR2, and HCDR3 sequences, respectively, as determined by IMGT numbering. [00146] In some embodiments, an antibody or an antigen-binding fragment thereof (e.g., a sdAb) comprises a V_H domain comprising: a) an HCDR1 comprising the amino acid sequence of SEQ ID NO:29, b) an HCDR2 comprising the amino acid sequence of SEQ ID NO:30, and c) an HCDR3 comprising the amino acid sequence of SEQ ID NO:31.

[00147] In some embodiments, an antibody or an antigen-binding fragment thereof (e.g., a sdAb) comprises a V_H domain comprising: a) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:29, b) an HCDR2 consisting of the amino acid sequence of SEQ ID NO:30, and c) an HCDR3 consisting of the amino acid sequence of SEQ ID NO:31.

[00148] Chothia numbering

[00149] In some embodiments, an antibody or an antigen-binding fragment thereof (e.g., a sdAb) comprises a V_H domain comprising: a) an HCDR1 having at least about 80% sequence identity (e.g., having at least about: 82%, 85%, 88%, 90%, 92%, 95%, 98% or 99% sequence identity) to the amino acid sequence of SEQ ID NO:32, b) an HCDR2 having at least about 80% sequence identity (e.g., having at least about: 82%, 85%, 88%, 90%, 92%, 95%, 98% or 99% sequence identity) to the amino acid sequence of SEQ ID NO:33, and c) an HCDR3 having at least about 80% sequence identity (e.g., having at least about: 82%, 85%, 88%, 90%, 92%, 95%, 98% or 99% sequence identity) to the amino acid sequence of SEQ ID NO:3.

[00150] HCDR1 : GFAFNDH (SEQ ID NO:32)

[00151] HCDR2: NTNGLI (SEQ ID NO:33)

[00152] HCDR3 : NQIGTL (SEQ ID NO:3)

[00153] The sequences identified as SEQ ID NO:32, SEQ ID NO:33, and SEQ ID NO:3 correspond to HCDR1, HCDR2, and HCDR3 sequences, respectively, as determined by Chothia numbering.

[00154] In some embodiments, an antibody or an antigen-binding fragment thereof (e.g., a sdAb) comprises a V_H domain comprising: a) an HCDR1 comprising the amino acid sequence of SEQ ID NO:32, b) an HCDR2 comprising the amino acid sequence of SEQ ID NO:33, and c) an HCDR3 comprising the amino acid sequence of SEQ ID NO:3. [00155] In some embodiments, an antibody or an antigen-binding fragment thereof (e.g., a sdAb) comprises a V_H domain comprising: a) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:32, b) an HCDR2 consisting of the amino acid sequence of SEQ ID NO:33, and c) an HCDR3 consisting of the amino acid sequence of SEQ ID NO:3.

[00156] In some embodiments, an antibody or an antigen-binding fragment thereof (e.g., a sdAb) comprises a V_H domain comprising: a) an HCDR1 having at least about 80% sequence identity (e.g., having at least about: 82%, 85%, 88%, 90%, 92%, 95%, 98% or 99% sequence identity) to the amino acid sequence of SEQ ID NO:34, b) an HCDR2 having at least about 80% sequence identity (e.g., having at least about: 82%, 85%, 88%, 90%, 92%, 95%, 98% or 99% sequence identity) to the amino acid sequence of SEQ ID NO: 35, and c) an HCDR3 having at least about 80% sequence identity (e.g., having at least about: 82%, 85%, 88%, 90%, 92%, 95%, 98% or 99% sequence identity) to the amino acid sequence of SEQ ID NO: 36.

[00157] HCDR1 : SGFAFNDH (SEQ ID NO:34)

[00158] HCDR2: LEWVSEINTNGLITKYA (SEQ ID NO:35)

[00159] HCDR3 : CSRNQIGT (SEQ ID NO:36)

[00160] The sequences identified as SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36 correspond to HCDR1, HCDR2, and HCDR3 sequences, respectively, as determined by Chothia numbering.

[00161] In some embodiments, an antibody or an antigen-binding fragment thereof (e.g., a sdAb) comprises a V_H domain comprising: a) an HCDR1 comprising the amino acid sequence of SEQ ID NO:34, b) an HCDR2 comprising the amino acid sequence of SEQ ID NO:35, and c) an HCDR3 comprising the amino acid sequence of SEQ ID NO:36.

[00162] In some embodiments, an antibody or an antigen-binding fragment thereof (e.g., a sdAb) comprises a V_H domain comprising: a) an HCDR1 consisting of the amino acid sequence of SEQ ID NO:34, b) an HCDR2 consisting of the amino acid sequence of SEQ ID NO:35, and c) an HCDR3 consisting of the amino acid sequence of SEQ ID NO:36. Framework Regions of Heavy Chain Variable Domain VH

[00163] In some embodiments, a V_H domain of an antibody or an antigen-binding fragment thereof comprises 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 V_H FR1 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 10) or QVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 11), b) the V_H FR2 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of WMYWVRQAPGKE (SEQ ID NO: 12), c) the V_H FR3 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of KNTLYLQMDSLR (SEQ ID NO: 18), KNTLYLQMNSLE (SEQ ID NO: 19) or ENTLYLQMNSLE (SEQ ID NO:20), d) the V_H FR4 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of LRGQGTQVTVSS (SEQ ID NO: 16) or LRGQGTQVTVSSGPGGQ (SEQ ID NO: 17), or any combination of a) to d).

[00164] In some embodiments: a) the V_H FR1 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 10) or QVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 11), b) the V_H FR2 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of WMYWVRQAPGKE (SEQ ID NO: 12), c) the V_H FR3 having at least 80% sequence identity e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of KNTLYLQMDSLR (SEQ ID NO: 18), KNTLYLQMNSLE (SEQ ID NO: 19) or ENTLYLQMNSLE (SEQ ID NO:20), and d) the V_H FR4 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of LRGQGTQVTVSS (SEQ ID NO: 16) or LRGQGTQVTVSSGPGGQ (SEQ ID NO: 17).

[00165] In some embodiments: a) the V_H FR1 comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 10) or QVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 11), b) the V_H FR2 comprises the amino acid sequence of WMYWVRQAPGKE (SEQ ID NO: 12), c) the V_H FR3 comprises the amino acid sequence of KNTLYLQMDSLR (SEQ ID NO: 18), KNTLYLQMNSLE (SEQ ID NO: 19) or ENTLYLQMNSLE (SEQ ID NO:20), or d) the V_H FR4 comprises the amino acid sequence of LRGQGTQVTVSS (SEQ ID NO: 16) or LRGQGTQVTVSSGPGGQ (SEQ ID NO: 17), or any combination of a) to d).

[00166] In some embodiments: a) the V_H FR1 comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 10) or QVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 11), b) the V_H FR2 comprises the amino acid sequence of WMYWVRQAPGKE (SEQ ID NO: 12), c) the V_H FR3 comprises the amino acid sequence of KNTLYLQMDSLR (SEQ ID NO: 18), KNTLYLQMNSLE (SEQ ID NO: 19) or ENTLYLQMNSLE (SEQ ID NO:20), and d) the V_H FR4 comprises the amino acid sequence of LRGQGTQVTVSS (SEQ ID NO: 16) or LRGQGTQVTVSSGPGGQ (SEQ ID NO: 17).

[00167] In some embodiments, a V_H domain of an antibody or an antigen-binding fragment thereof comprises 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 V_H FR1 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 10), b) the V_H FR2 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of WMYWVRQAPGKE (SEQ ID NO: 12), c) the V_H FR3 having at least 80% sequence identity e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of KNTLYLQMDSLR (SEQ ID NO: 18), d) the V_H FR4 having at least 80% sequence identity e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of LRGQGTQVTVSS (SEQ ID NO: 16) or LRGQGTQVTVSSGPGGQ (SEQ ID NO: 17), or any combination of a) to d).

[00168] In some embodiments: a) the V_H FR1 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 10), b) the V_H FR2 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of WMYWVRQAPGKE (SEQ ID NO: 12), c) the V_H FR3 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of KNTLYLQMDSLR (SEQ ID NO: 18), and d) the V_H FR4 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of LRGQGTQVTVSS (SEQ ID NO: 16) or LRGQGTQVTVSSGPGGQ (SEQ ID NO: 17).

[00169] In some embodiments: a) the V_H FR1 comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 10), b) the V_H FR2 comprises the amino acid sequence of WMYWVRQAPGKE (SEQ ID NO: 12), c) the V_H FR3 comprises the amino acid sequence of KNTLYLQMDSLR (SEQ ID NO: 18), d) the V_H FR4 comprises the amino acid sequence of LRGQGTQVTVSS (SEQ ID NO: 16) or LRGQGTQVTVSSGPGGQ (SEQ ID NO: 17), or any combination of a) to d).

[00170] In some embodiments: a) the V_H FR1 comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 10), b) the V_H FR2 comprises the amino acid sequence of WMYWVRQAPGKE (SEQ ID NO: 12), c) the V_H FR3 comprises the amino acid sequence of KNTLYLQMDSLR (SEQ ID NO: 18), and d) the V_H FR4 comprises the amino acid sequence of LRGQGTQVTVSS (SEQ ID NO: 16) or LRGQGTQVTVSSGPGGQ (SEQ ID NO: 17).

[00171] In some embodiments, a V_H domain of an antibody or an antigen-binding fragment thereof comprises 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 V_H FR1 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of QVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 11), b) the V_H FR2 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of WMYWVRQAPGKE (SEQ ID NO: 12), c) the V_H FR3 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of KNTLYLQMNSLE (SEQ ID NO: 19), d) the V_H FR4 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of LRGQGTQVTVSS (SEQ ID NO: 16) or LRGQGTQVTVSSGPGGQ (SEQ ID NO: 17), or any combination of a) to d).

[00172] In some embodiments: a) the V_H FR1 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of QVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 11), b) the V_H FR2 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of WMYWVRQAPGKE (SEQ ID NO: 12), c) the V_H FR3 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of KNTLYLQMNSLE (SEQ ID NO: 19), and d) the V_H FR4 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of LRGQGTQVTVSS (SEQ ID NO: 16) or LRGQGTQVTVSSGPGGQ (SEQ ID NO: 17). [00173] In some embodiments: a) the V_H FR1 comprises the amino acid sequence of QVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 11), b) the V_H FR2 comprises the amino acid sequence of WMYWVRQAPGKE (SEQ ID NO: 12), c) the V_H FR3 comprises the amino acid sequence of KNTLYLQMNSLE (SEQ ID NO: 19), d) the V_H FR4 comprises the amino acid sequence of LRGQGTQVTVSS (SEQ ID NO: 16) or LRGQGTQVTVSSGPGGQ (SEQ ID NO: 17), or any combination of a) to d).

[00174] In some embodiments: a) the V_H FR1 comprises the amino acid sequence of QVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 11), b) the V_H FR2 comprises the amino acid sequence of WMYWVRQAPGKE (SEQ ID NO: 12), c) the V_H FR3 comprises the amino acid sequence of KNTLYLQMNSLE (SEQ ID NO: 19), and d) the V_H FR4 comprises the amino acid sequence of LRGQGTQVTVSS (SEQ ID NO: 16) or LRGQGTQVTVSSGPGGQ (SEQ ID NO: 17).

[00175] In some embodiments, a V_H domain of an antibody or an antigen-binding fragment thereof comprises 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 V_H FR1 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of QVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 11), b) the V_H FR2 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of WMYWVRQAPGKE (SEQ ID NO: 12), c) the V_H FR3 having at least 80% sequence identity e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of ENTLYLQMNSLE (SEQ ID NO:20), d) the V_H FR4 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of LRGQGTQVTVSS (SEQ ID NO: 16) or LRGQGTQVTVSSGPGGQ (SEQ ID NO: 17), or any combination of a) to d).

[00176] In some embodiments: a) the V_H FR1 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of QVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 11), b) the V_H FR2 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of WMYWVRQAPGKE (SEQ ID NO: 12), c) the V_H FR3 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of ENTLYLQMNSLE (SEQ ID NO:20), and d) the V_H FR4 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of LRGQGTQVTVSS (SEQ ID NO: 16) or LRGQGTQVTVSSGPGGQ (SEQ ID NO: 17).

[00177] In some embodiments: a) the V_H FR1 comprises the amino acid sequence of QVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 11), b) the V_H FR2 comprises the amino acid sequence of WMYWVRQAPGKE (SEQ ID NO: 12), c) the V_H FR3 comprises the amino acid sequence of ENTLYLQMNSLE (SEQ ID NO:20), d) the V_H FR4 comprises the amino acid sequence of LRGQGTQVTVSS (SEQ ID NO: 16) or LRGQGTQVTVSSGPGGQ (SEQ ID NO: 17), or any combination of a) to d).

[00178] In some embodiments: a) the V_H FR1 comprises the amino acid sequence of QVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 11), b) the V_H FR2 comprises the amino acid sequence of WMYWVRQAPGKE (SEQ ID NO: 12), c) the V_H FR3 comprises the amino acid sequence of ENTLYLQMNSLE (SEQ ID NO:20), and d) the V_H FR4 comprises the amino acid sequence of LRGQGTQVTVSS (SEQ ID NO: 16) or LRGQGTQVTVSSGPGGQ (SEQ ID NO: 17).

[00179] In some embodiments, a V_H domain of an antibody or an antigen-binding fragment thereof comprises 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 V_H FR1 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 10) or QVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 11), b) the V_H FR2 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of WMYWVRQAPGKE (SEQ ID NO: 12), c) the V_H FR3 having at least 80% sequence identity e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of DFVKGRFTVSRDNAKNTLYLQMDSLRPEDTAVYS (SEQ ID NO: 13), DFVKGRFTVSRDNAKNTLYLQMNSLEPEDTAVYS (SEQ ID NO: 14) or DFVKGRFTVSRDNAENTLYLQMNSLEPEDTAVYS (SEQ ID NO: 15), or d) the V_H FR4 having at least 80% sequence identity e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of LRGQGTQVTVSS (SEQ ID NO: 16) or LRGQGTQVTVSSGPGGQ (SEQ ID NO: 17), or any combination of a) to d).

[00180] In some embodiments: a) the V_H FR1 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 10) or QVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 11), b) the V_H FR2 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of WMYWVRQAPGKE (SEQ ID NO: 12), c) the V_H FR3 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of DFVKGRFTVSRDNAKNTLYLQMDSLRPEDTAVYS (SEQ ID NO: 13), DFVKGRFTVSRDNAKNTLYLQMNSLEPEDTAVYS (SEQ ID NO: 14) or DFVKGRFTVSRDNAENTLYLQMNSLEPEDTAVYS (SEQ ID NO: 15), and d) the V_H FR4 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of LRGQGTQVTVSS (SEQ ID NO: 16) or LRGQGTQVTVSSGPGGQ (SEQ ID NO: 17).

[00181] In some embodiments: a) the V_H FR1 comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 10) or QVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 11), b) the V_H FR2 comprises the amino acid sequence of WMYWVRQAPGKE (SEQ ID NO: 12), c) the V_H FR3 comprises the amino acid sequence of DFVKGRFTVSRDNAKNTLYLQMDSLRPEDTAVYS (SEQ ID NO: 13), DFVKGRFTVSRDNAKNTLYLQMNSLEPEDTAVYS (SEQ ID NO: 14) or DFVKGRFTVSRDNAENTLYLQMNSLEPEDTAVYS (SEQ ID NO: 15), d) the V_H FR4 comprises the amino acid sequence of LRGQGTQVTVSS (SEQ ID NO: 16) or LRGQGTQVTVSSGPGGQ (SEQ ID NO: 17), or any combination of a) to d).

[00182] In some embodiments: a) the V_H FR1 comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 10) or QVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 11), b) the V_H FR2 comprises the amino acid sequence of WMYWVRQAPGKE (SEQ ID NO: 12), c) the V_H FR3 comprises the amino acid sequence of DFVKGRFTVSRDNAKNTLYLQMDSLRPEDTAVYS (SEQ ID NO: 13), DFVKGRFTVSRDNAKNTLYLQMNSLEPEDTAVYS (SEQ ID NO: 14) or DFVKGRFTVSRDNAENTLYLQMNSLEPEDTAVYS (SEQ ID NO: 15), and d) the V_H FR4 comprises the amino acid sequence of LRGQGTQVTVSS (SEQ ID NO: 16) or LRGQGTQVTVSSGPGGQ (SEQ ID NO: 17).

[00183] In some embodiments: a) the V_H FR1 consists of the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 10) or QVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 11), b) the V_H FR2 consists of the amino acid sequence of WMYWVRQAPGKE (SEQ ID NO: 12), c) the V_H FR3 consists of the amino acid sequence of DFVKGRFTVSRDNAKNTLYLQMDSLRPEDTAVYS (SEQ ID NO: 13), DFVKGRFTVSRDNAKNTLYLQMNSLEPEDTAVYS (SEQ ID NO: 14) or DFVKGRFTVSRDNAENTLYLQMNSLEPEDTAVYS (SEQ ID NO: 15), d) the V_H FR4 consists of the amino acid sequence of LRGQGTQVTVSS (SEQ ID NO: 16) or LRGQGTQVTVSSGPGGQ (SEQ ID NO: 17), or any combination of a) to d).

[00184] In some embodiments: a) the V_H FR1 consists of the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 10) or QVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 11), b) the V_H FR2 consists of the amino acid sequence of WMYWVRQAPGKE (SEQ ID NO: 12), c) the V_H FR3 consists of the amino acid sequence of DFVKGRFTVSRDNAKNTLYLQMDSLRPEDTAVYS (SEQ ID NO: 13), DFVKGRFTVSRDNAKNTLYLQMNSLEPEDTAVYS (SEQ ID NO: 14) or DFVKGRFTVSRDNAENTLYLQMNSLEPEDTAVYS (SEQ ID NO: 15), and d) the V_H FR4 consists of the amino acid sequence of LRGQGTQVTVSS (SEQ ID NO: 16) or LRGQGTQVTVSSGPGGQ (SEQ ID NO: 17).

[00185] In some embodiments, a V_H domain of an antibody or an antigen-binding fragment thereof comprises 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 V_H FR1 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 10), b) the V_H FR2 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of WMYWVRQAPGKE (SEQ ID NO: 12), c) the V_H FR3 having at least 80% sequence identity e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of DFVKGRFTVSRDNAKNTLYLQMDSLRPEDTAVYS (SEQ ID NO: 13), d) the V_H FR4 having at least 80% sequence identity e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of LRGQGTQVTVSS (SEQ ID NO: 16) or LRGQGTQVTVSSGPGGQ (SEQ ID NO: 17), or any combination of a) to d).

[00186] In some embodiments: a) the V_H FR1 having at least 80% sequence identity e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 10), b) the V_H FR2 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of WMYWVRQAPGKE (SEQ ID NO: 12), c) the V_H FR3 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of DFVKGRFTVSRDNAKNTLYLQMDSLRPEDTAVYS (SEQ ID NO: 13), and d) the V_H FR4 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of LRGQGTQVTVSS (SEQ ID NO: 16) or LRGQGTQVTVSSGPGGQ (SEQ ID NO: 17).

[00187] In some embodiments: a) the V_H FR1 comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 10), b) the V_H FR2 comprises the amino acid sequence of WMYWVRQAPGKE (SEQ ID NO: 12), c) the V_H FR3 comprises the amino acid sequence of DFVKGRFTVSRDNAKNTLYLQMDSLRPEDTAVYS (SEQ ID NO: 13), d) the V_H FR4 comprises the amino acid sequence of LRGQGTQVTVSS (SEQ ID NO: 16) or LRGQGTQVTVSSGPGGQ (SEQ ID NO: 17), or any combination of a) to d).

[00188] In some embodiments: a) the V_H FR1 comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 10), b) the V_H FR2 comprises the amino acid sequence of WMYWVRQAPGKE (SEQ ID NO: 12), c) the V_H FR3 comprises the amino acid sequence of DFVKGRFTVSRDNAKNTLYLQMDSLRPEDTAVYS (SEQ ID NO: 13), and d) the V_H FR4 comprises the amino acid sequence of LRGQGTQVTVSS (SEQ ID NO: 16) or LRGQGTQVTVSSGPGGQ (SEQ ID NO: 17). [00189] In some embodiments: a) the V_H FR1 consists of the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 10), b) the V_H FR2 consists of the amino acid sequence of WMYWVRQAPGKE (SEQ ID NO: 12), c) the V_H FR3 consists of the amino acid sequence of DFVKGRFTVSRDNAKNTLYLQMDSLRPEDTAVYS (SEQ ID NO: 13), d) the V_H FR4 consists of the amino acid sequence of LRGQGTQVTVSS (SEQ ID NO: 16) or LRGQGTQVTVSSGPGGQ (SEQ ID NO: 17), or any combination of a) to d).

[00190] In some embodiments: a) the V_H FR1 consists of the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 10), b) the V_H FR2 consists of the amino acid sequence of WMYWVRQAPGKE (SEQ ID NO: 12), c) the V_H FR3 consists of the amino acid sequence of DFVKGRFTVSRDNAKNTLYLQMDSLRPEDTAVYS (SEQ ID NO: 13), and d) the V_H FR4 consists of the amino acid sequence of LRGQGTQVTVSS (SEQ ID NO: 16) or LRGQGTQVTVSSGPGGQ (SEQ ID NO: 17).

[00191] In some embodiments, a V_H domain of an antibody or an antigen-binding fragment thereof comprises 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 V_H FR1 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of QVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 11), b) the V_H FR2 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of WMYWVRQAPGKE (SEQ ID NO: 12), c) the V_H FR3 having at least 80% sequence identity e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of DFVKGRFTVSRDNAKNTLYLQMNSLEPEDTAVYS (SEQ ID NO: 14), d) the V_H FR4 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of LRGQGTQVTVSS (SEQ ID NO: 16) or LRGQGTQVTVSSGPGGQ (SEQ ID NO: 17), or any combination of a) to d).

[00192] In some embodiments: a) the V_H FR1 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of QVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 11), b) the V_H FR2 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of WMYWVRQAPGKE (SEQ ID NO: 12), c) the V_H FR3 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of DFVKGRFTVSRDNAKNTLYLQMNSLEPEDTAVYS (SEQ ID NO: 14), and d) the V_H FR4 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of LRGQGTQVTVSS (SEQ ID NO: 16) or LRGQGTQVTVSSGPGGQ (SEQ ID NO: 17).

[00193] In some embodiments: a) the V_H FR1 comprises the amino acid sequence of QVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 11), b) the V_H FR2 comprises the amino acid sequence of WMYWVRQAPGKE (SEQ ID NO: 12), c) the V_H FR3 comprises the amino acid sequence of DFVKGRFTVSRDNAKNTLYLQMNSLEPEDTAVYS (SEQ ID NO: 14), d) the V_H FR4 comprises the amino acid sequence of LRGQGTQVTVSS (SEQ ID NO: 16) or LRGQGTQVTVSSGPGGQ (SEQ ID NO: 17), or any combination of a) to d).

[00194] In some embodiments: a) the V_H FR1 comprises the amino acid sequence of QVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 11), b) the V_H FR2 comprises the amino acid sequence of WMYWVRQAPGKE (SEQ ID NO: 12), c) the V_H FR3 comprises the amino acid sequence of DFVKGRFTVSRDNAKNTLYLQMNSLEPEDTAVYS (SEQ ID NO: 14), and d) the V_H FR4 comprises the amino acid sequence of LRGQGTQVTVSS (SEQ ID NO: 16) or LRGQGTQVTVSSGPGGQ (SEQ ID NO: 17).

[00195] In some embodiments: a) the V_H FR1 consists of the amino acid sequence of QVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 11), b) the V_H FR2 consists of the amino acid sequence of WMYWVRQAPGKE (SEQ ID NO: 12), c) the V_H FR3 consists of the amino acid sequence of DFVKGRFTVSRDNAKNTLYLQMNSLEPEDTAVYS (SEQ ID NO: 14), d) the V_H FR4 consists of the amino acid sequence of LRGQGTQVTVSS (SEQ ID NO: 16) or LRGQGTQVTVSSGPGGQ (SEQ ID NO: 17), or any combination of a) to d).

[00196] In some embodiments: a) the V_H FR1 consists of the amino acid sequence of QVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 11), b) the V_H FR2 consists of the amino acid sequence of WMYWVRQAPGKE (SEQ ID NO: 12), c) the V_H FR3 consists of the amino acid sequence of DFVKGRFTVSRDNAKNTLYLQMNSLEPEDTAVYS (SEQ ID NO: 14), and d) the V_H FR4 consists of the amino acid sequence of LRGQGTQVTVSS (SEQ ID NO: 16) or LRGQGTQVTVSSGPGGQ (SEQ ID NO: 17).

[00197] In some embodiments, a V_H domain of an antibody or an antigen-binding fragment thereof comprises 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 V_H FR1 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of QVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 11), b) the V_H FR2 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of WMYWVRQAPGKE (SEQ ID NO: 12), c) the V_H FR3 having at least 80% sequence identity e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of DFVKGRFTVSRDNAENTLYLQMNSLEPEDTAVYS (SEQ ID NO: 15), d) the V_H FR4 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of LRGQGTQVTVSS (SEQ ID NO: 16) or LRGQGTQVTVSSGPGGQ (SEQ ID NO: 17), or any combination of a) to d).

[00198] In some embodiments: a) the V_H FR1 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of QVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 11), b) the V_H FR2 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of WMYWVRQAPGKE (SEQ ID NO: 12), c) the V_H FR3 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of DFVKGRFTVSRDNAENTLYLQMNSLEPEDTAVYS (SEQ ID NO: 15), and d) the V_H FR4 having at least 80% sequence identity (e.g., having at least 85% sequence identity, having at least 90% sequence identity, or having at least 95% sequence identity) to the amino acid sequence of LRGQGTQVTVSS (SEQ ID NO: 16) or LRGQGTQVTVSSGPGGQ (SEQ ID NO: 17).

[00199] In some embodiments: a) the V_H FR1 comprises the amino acid sequence of QVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 11), b) the V_H FR2 comprises the amino acid sequence of WMYWVRQAPGKE (SEQ ID NO: 12), c) the V_H FR3 comprises the amino acid sequence of

DFVKGRFTVSRDNAENTLYLQMNSLEPEDTAVYS (SEQ ID NO: 15), d) the V_H FR4 comprises the amino acid sequence of LRGQGTQVTVSS (SEQ ID NO: 16) or LRGQGTQVTVSSGPGGQ (SEQ ID NO: 17), or any combination of a) to d).

[00200] In some embodiments: a) the V_H FR1 comprises the amino acid sequence of QVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 11), b) the V_H FR2 comprises the amino acid sequence of WMYWVRQAPGKE (SEQ ID NO: 12), c) the V_H FR3 comprises the amino acid sequence of

DFVKGRFTVSRDNAENTLYLQMNSLEPEDTAVYS (SEQ ID NO: 15), and d) the V_H FR4 comprises the amino acid sequence of LRGQGTQVTVSS (SEQ ID NO: 16) or LRGQGTQVTVSSGPGGQ (SEQ ID NO: 17).

[00201] In some embodiments: a) the V_H FR1 consists of the amino acid sequence of QVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 11), b) the V_H FR2 consists of the amino acid sequence of WMYWVRQAPGKE (SEQ ID NO: 12), c) the V_H FR3 consists of the amino acid sequence of

DFVKGRFTVSRDNAENTLYLQMNSLEPEDTAVYS (SEQ ID NO: 15), d) the V_H FR4 consists of the amino acid sequence of LRGQGTQVTVSS (SEQ ID NO: 16) or LRGQGTQVTVSSGPGGQ (SEQ ID NO: 17), or any combination of a) to d). [00202] In some embodiments: a) the V_H FR1 consists of the amino acid sequence of QVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO: 11), b) the V_H FR2 consists of the amino acid sequence of WMYWVRQAPGKE (SEQ ID NO: 12), c) the V_H FR3 consists of the amino acid sequence of DFVKGRFTVSRDNAENTLYLQMNSLEPEDTAVYS (SEQ ID NO: 15), and d) the V_H FR4 consists of the amino acid sequence of LRGQGTQVTVSS (SEQ ID NO: 16) or LRGQGTQVTVSSGPGGQ (SEQ ID NO: 17).

Heavy Chain Variable Domains (V_a)

[00203] In some embodiments, an antibody or an 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:40-45 and 4-6.

[00204] In some embodiments, an antibody or an antigen-binding fragment thereof comprises a V_H comprising at least one amino acid substitution relative to the amino acid sequence of at least one of SEQ ID NOs:40-45 and 4-6. 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 antibody or an 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:40-45 and 4-6.

[00205] 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.

[00206] 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.

[00207] In some embodiments, an antibody or an antigen-binding fragment thereof comprises a V_H having 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs:40- 45 and 4-6. In some embodiments, an antibody or an antigen-binding fragment thereof comprises a V_H comprising (e.g., consisting of) the amino acid sequence of any one of SEQ ID NOs:40-45 and 4-6.

[00208] In some embodiments, an antibody or an 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:40, 42, and 4-6.

[00209] In some embodiments, an antibody or an antigen-binding fragment thereof comprises a V_H comprising at least one amino acid substitution relative to the amino acid sequence of at least one of SEQ ID NOs:40, 42, and 4-6. 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 antibody or an 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:40, 42, and 4-6.

[00210] In some embodiments, an amino acid substitution is a conservative substitution.

[00211] In some embodiments, an amino acid substitution is a highly conservative substitution.

[00212] In some embodiments, an antibody or an antigen-binding fragment thereof comprises a V_H having 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs:40, 42, and 4-6. In some embodiments, an antibody or an antigen-binding fragment thereof comprises a V_H comprising (e.g., consisting of) the amino acid sequence of any one of SEQ ID NOs:40, 42, and 4-6.

[00213] In some embodiments, an antibody or an 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:40.

[00214] In some embodiments, an antibody or an antigen-binding fragment thereof comprises a V_H comprising at least one amino acid substitution relative to the amino acid sequence of SEQ ID NO:40. 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 antibody or an 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:40. In some embodiments, an amino acid substitution is a conservative substitution. In some embodiments, an amino acid substitution is a highly conservative substitution.

[00215] In some embodiments, an antibody or an antigen-binding fragment thereof comprises a V_H having 100% sequence identity to the amino acid sequence of SEQ ID NO:40. In some embodiments, an antibody or an antigen-binding fragment thereof comprises a V_H comprising (e.g., consisting of) the amino acid sequence of SEQ ID NO:40.

[00216] In some embodiments, an antibody or an 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:42.

[00217] In some embodiments, an antibody or an antigen-binding fragment thereof comprises a V_H comprising at least one amino acid substitution relative to the amino acid sequence of SEQ ID NO:42. 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 antibody or an 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:42. In some embodiments, an amino acid substitution is a conservative substitution. In some embodiments, an amino acid substitution is a highly conservative substitution.

[00218] In some embodiments, an antibody or an antigen-binding fragment thereof comprises a V_H having 100% sequence identity to the amino acid sequence of SEQ ID NO:42. In some embodiments, an antibody or an antigen-binding fragment thereof comprises a V_H comprising (e.g., consisting of) the amino acid sequence of SEQ ID NO:42.

[00219] In some embodiments, an antibody or an 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:4.

[00220] In some embodiments, an antibody or an antigen-binding fragment thereof comprises a V_H comprising at least one amino acid substitution relative to the amino acid sequence of SEQ ID NO:4. 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 antibody or an 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:4. In some embodiments, an amino acid substitution is a conservative substitution. In some embodiments, an amino acid substitution is a highly conservative substitution.

[00221] In some embodiments, an antibody or an antigen-binding fragment thereof comprises a V_H having 100% sequence identity to the amino acid sequence of SEQ ID NO:4. In some embodiments, an antibody or an antigen-binding fragment thereof comprises a V_H comprising (e.g., consisting of) the amino acid sequence of SEQ ID NO:4.

[00222] In some embodiments, an antibody or an 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: 5.

[00223] In some embodiments, an antibody or an antigen-binding fragment thereof comprises a V_H comprising at least one amino acid substitution relative to the amino acid sequence of SEQ ID NO:5. 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 antibody or an 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:5. In some embodiments, an amino acid substitution is a conservative substitution. In some embodiments, an amino acid substitution is a highly conservative substitution.

[00224] In some embodiments, an antibody or an antigen-binding fragment thereof comprises a V_H having 100% sequence identity to the amino acid sequence of SEQ ID NO: 5. In some embodiments, an antibody or an antigen-binding fragment thereof comprises a V_H comprising (e.g., consisting of) the amino acid sequence of SEQ ID NO:5.

[00225] In some embodiments, an antibody or an 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:6.

[00226] In some embodiments, an antibody or an antigen-binding fragment thereof comprises a V_H comprising at least one amino acid substitution relative to the amino acid sequence of SEQ ID NO:6. 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 antibody or an 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:6. In some embodiments, an amino acid substitution is a conservative substitution. In some embodiments, an amino acid substitution is a highly conservative substitution.

[00227] In some embodiments, an antibody or an antigen-binding fragment thereof comprises a V_H having 100% sequence identity to the amino acid sequence of SEQ ID NO:6. In some embodiments, an antibody or an antigen-binding fragment thereof comprises a V_H comprising (e.g., consisting of) the amino acid sequence of SEQ ID NO:6.

Polypeptides

[00228] The disclosure also provides, among other things, a polypeptide comprising at least one antibody or antigen-binding fragment thereof (e.g., a sdAb) disclosed herein.

[00229] In some embodiments, a polypeptide comprises a single antibody or antigen-binding fragment thereof (e.g., a sdAb). In some embodiments, a polypeptide comprises two or more antibodies or antigen-binding fragments thereof (e.g., sdAbs). In some embodiments, a polypeptide comprises two antibodies or antigen-binding fragments thereof (e.g., sdAbs).

[00230] In some embodiments, a polypeptide comprises two sdAbs. In some embodiments, a polypeptide comprising two sdAbs has a lower IC₅₀ than each sdAb. In some embodiments, a polypeptide comprising two sdAbs has a longer serum half-life than each sdAb.

[00231] In some embodiments, a polypeptide comprises an amino acid sequence 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:40-45 and 4-6.

[00232] In some embodiments, a polypeptide comprises an amino acid sequence comprising at least one amino acid substitution relative to the amino acid sequence of at least one of SEQ ID NOs:40-45 and 4-6. 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, a polypeptide comprises an amino acid sequence comprising about 1-10 amino acid substitutions, relative to the amino acid sequence of at least one of SEQ ID NOs:40-45 and 4-6. In some embodiments, an amino acid substitution is a conservative substitution. In some embodiments, an amino acid substitution is a highly conservative substitution.

[00233] In some embodiments, a polypeptide comprises an amino acid sequence having 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs:40-45 and 4-6. In some embodiments, a polypeptide comprises the amino acid sequence of any one of SEQ ID NOs:40- 45 and 4-6.

[00234] In some embodiments, a polypeptide comprises an amino acid sequence 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:40, 42, and 4-6.

[00235] In some embodiments, a polypeptide comprises an amino acid sequence comprising at least one amino acid substitution relative to the amino acid sequence of at least one of SEQ ID NOs:40, 42, and 4-6. 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, a polypeptide comprises an amino acid sequence comprising about 1-10 amino acid substitutions, relative to the amino acid sequence of at least one of SEQ ID NOs:40, 42, and 4-6. In some embodiments, an amino acid substitution is a conservative substitution. In some embodiments, an amino acid substitution is a highly conservative substitution.

[00236] In some embodiments, a polypeptide comprises an amino acid sequence having 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs:40, 42, and 4-6. In some embodiments, a polypeptide comprises the amino acid sequence of any one of SEQ ID NOs:40, 42, and 4-6.

[00237] In some embodiments, a polypeptide comprises an amino acid sequence 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:40.

[00238] In some embodiments, a polypeptide comprises an amino acid sequence comprising at least one amino acid substitution relative to the amino acid sequence of SEQ ID NO:40. 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, a polypeptide comprises an amino acid sequence comprising about 1-10 amino acid substitutions, relative to the amino acid sequence of SEQ ID NO:40. In some embodiments, an amino acid substitution is a conservative substitution. In some embodiments, an amino acid substitution is a highly conservative substitution. [00239] In some embodiments, a polypeptide comprises an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO:40. In some embodiments, a polypeptide comprises the amino acid sequence of SEQ ID NO:40.

[00240] In some embodiments, a polypeptide comprises an amino acid sequence 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:42.

[00241] In some embodiments, a polypeptide comprises an amino acid sequence comprising at least one amino acid substitution relative to the amino acid sequence of SEQ ID NO:42. 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, a polypeptide comprises an amino acid sequence comprising about 1-10 amino acid substitutions, relative to the amino acid sequence of SEQ ID NO:42. In some embodiments, an amino acid substitution is a conservative substitution. In some embodiments, an amino acid substitution is a highly conservative substitution.

[00242] In some embodiments, a polypeptide comprises an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO:42. In some embodiments, a polypeptide comprises the amino acid sequence of SEQ ID NO:42.

[00243] In some embodiments, a polypeptide comprises an amino acid sequence 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:4.

[00244] In some embodiments, a polypeptide comprises an amino acid sequence comprising at least one amino acid substitution relative to the amino acid sequence of SEQ ID NO:4. 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, a polypeptide comprises an amino acid sequence comprising about 1-10 amino acid substitutions, relative to the amino acid sequence of SEQ ID NO:4. In some embodiments, an amino acid substitution is a conservative substitution. In some embodiments, an amino acid substitution is a highly conservative substitution. [00245] In some embodiments, a polypeptide comprises an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO:4. In some embodiments, a polypeptide comprises the amino acid sequence of SEQ ID NO:4.

[00246] In some embodiments, a polypeptide comprises an amino acid sequence 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:5.

[00247] In some embodiments, a polypeptide comprises an amino acid sequence comprising at least one amino acid substitution relative to the amino acid sequence of SEQ ID NO:5. 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, a polypeptide comprises an amino acid sequence comprising about 1-10 amino acid substitutions, relative to the amino acid sequence of SEQ ID NO:5. In some embodiments, an amino acid substitution is a conservative substitution. In some embodiments, an amino acid substitution is a highly conservative substitution.

[00248] In some embodiments, a polypeptide comprises an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO: 5. In some embodiments, a polypeptide comprises the amino acid sequence of SEQ ID NO:5.

[00249] In some embodiments, a polypeptide comprises an amino acid sequence 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:6.

[00250] In some embodiments, a polypeptide comprises an amino acid sequence comprising at least one amino acid substitution relative to the amino acid sequence of SEQ ID NO:6. 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, a polypeptide comprises an amino acid sequence comprising about 1-10 amino acid substitutions, relative to the amino acid sequence of SEQ ID NO:6. In some embodiments, an amino acid substitution is a conservative substitution. In some embodiments, an amino acid substitution is a highly conservative substitution. [00251] In some embodiments, a polypeptide comprises an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO:6. In some embodiments, a polypeptide comprises the amino acid sequence of SEQ ID NO:6.

[00252] In some embodiments, a polypeptide comprises at least one antibody or antigenbinding fragment thereof (e.g., a sdAb) and a protein tag. In some embodiments, a protein tag comprises a polyhistidine-tag.

[00253] In some embodiments, a polypeptide comprises, an antibody or an antigen-binding fragment thereof (e.g., a sdAb) and a protein tag (e.g., a His-tag, a Strep-tag, an Avi-tag, or a Flag-tag). In some embodiments, a protein tag (e.g., a 6xHis-tag (SEQ ID NO:21)) is at the C- terminal end of the polypeptide.

[00254] 6xHis-tag: HHHHHH (SEQ ID NO:21)

[00255] Strep-tag: WSHPQFEK (SEQ ID NO:37)

[00256] Avi-tag: GLNDIFEAQKIEWHE (SEQ ID NO:38)

[00257] 3X Flag-tag: DYKDHDGDYKDHDIDYKDDDDK (SEQ ID NO:39)

[00258] In some embodiments, a polypeptide 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:22-27 (e.g., at least one of SEQ ID NOs:25-27).

[00259] EVQLVESGGGLVQPGGSLRLSCAASGFAFNDHWMYWVRQAPGKELEWVSE INTNGLITKYADFVKGRFTVSRDNAKNTLYLQMDSLRPEDTAVYSCSRNQIGTLRGQGT QVTVSSHHHHHH (SEQ ID NO:22) TNFI-Nabla-6xHis

[00260] QVQLVESGGGLVQPGGSLRLSCAASGFAFNDHWMYWVRQAPGKELEWVSE INTNGLITKYADFVKGRFTVSRDNAENTLYLQMNSLEPEDTAVYSCSRNQIGTLRGQGT QVTVSSHHHHHH (SEQ ID NO:23) TNFI-Nab2a-6xHis

[00261] QVQLVESGGGLVQPGGSLRLSCAASGFAFNDHWMYWVRQAPGKELEWVSE INTNGLITKYADFVKGRFTVSRDNAKNTLYLQMNSLEPEDTAVYSCSRNQIGTLRGQGT QVTVSSHHHHHH (SEQ ID NO:24) TNFI-Nab3a-6xHis

[00262] EVQLVESGGGLVQPGGSLRLSCAASGFAFNDHWMYWVRQAPGKELEWVSE INTNGLITKYADFVKGRFTVSRDNAKNTLYLQMDSLRPEDTAVYSCSRNQIGTLRGQGT QVTVSSGPGGQHHHHHH (SEQ ID NO:25) TNFI-Nablb-6xHis

[00263] QVQLVESGGGLVQPGGSLRLSCAASGFAFNDHWMYWVRQAPGKELEWVSE INTNGLITKYADFVKGRFTVSRDNAENTLYLQMNSLEPEDTAVYSCSRNQIGTLRGQGT QVTVSSGPGGQHHHHHH (SEQ ID NO:26) TNFI-Nab2b-6xHis [00264] QVQLVESGGGLVQPGGSLRLSCAASGFAFNDHWMYWVRQAPGKELEWVSE INTNGLITKYADFVKGRFTVSRDNAKNTLYLQMNSLEPEDTAVYSCSRNQIGTLRGQGT QVTVSSGPGGQHHHHHH (SEQ ID NO:27) TNFI-Nab3b-6xHis

[00265] The disclosure also provides, among other things, a polypeptide 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:22-27 (e.g, at least one of SEQ ID NOs:25-27).

[00266] In some embodiments, a polypeptide comprises at least one amino acid substitution relative to the amino acid sequence of at least one of SEQ ID NOs:22-27 (e.g., at least one of SEQ ID NOs:25-27). 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, a polypeptide comprises about 1-10 amino acid substitutions, relative to the amino acid sequence of at least one of SEQ ID NOs:22-27 (e.g., at least one of SEQ ID NOs:25-27). In some embodiments, an amino acid substitution is a conservative substitution. In some embodiments, an amino acid substitution is a highly conservative substitution.

[00267] In some embodiments, a polypeptide has 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs:22-27 (e.g., any one of SEQ ID NOs:25-27). In some embodiments, a polypeptide comprises (e.g, consists of) the amino acid sequence of any one of SEQ ID NOs:22-27 (e.g., any one of SEQ ID NOs:25-27).

[00268] In some embodiments, a polypeptide 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:22.

[00269] In some embodiments, a polypeptide comprises at least one amino acid substitution relative to the amino acid sequence of SEQ ID NO:22. 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, a polypeptide comprises about 1-10 amino acid substitutions, relative to the amino acid sequence of SEQ ID NO:22. In some embodiments, an amino acid substitution is a conservative substitution. In some embodiments, an amino acid substitution is a highly conservative substitution. [00270] In some embodiments, a polypeptide has 100% sequence identity to the amino acid sequence of SEQ ID NO:22. In some embodiments, a polypeptide comprises (e.g., consists of) the amino acid sequence of SEQ ID NO:22.

[00271] In some embodiments, a polypeptide 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:23.

[00272] In some embodiments, a polypeptide comprises at least one amino acid substitution relative to the amino acid sequence of SEQ ID NO:23. 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, a polypeptide comprises about 1-10 amino acid substitutions, relative to the amino acid sequence of SEQ ID NO:23. In some embodiments, an amino acid substitution is a conservative substitution. In some embodiments, an amino acid substitution is a highly conservative substitution.

[00273] In some embodiments, a polypeptide has 100% sequence identity to the amino acid sequence of SEQ ID NO:23. In some embodiments, a polypeptide comprises (e.g., consists of) the amino acid sequence of SEQ ID NO:23.

[00274] In some embodiments, a polypeptide 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:24.

[00275] In some embodiments, a polypeptide comprises at least one amino acid substitution relative to the amino acid sequence of SEQ ID NO:24. 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, a polypeptide comprises about 1-10 amino acid substitutions, relative to the amino acid sequence of SEQ ID NO:24. In some embodiments, an amino acid substitution is a conservative substitution. In some embodiments, an amino acid substitution is a highly conservative substitution.

[00276] In some embodiments, a polypeptide has 100% sequence identity to the amino acid sequence of SEQ ID NO:24. In some embodiments, a polypeptide comprises (e.g., consists of) the amino acid sequence of SEQ ID NO:24. [00277] In some embodiments, a polypeptide 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:25.

[00278] In some embodiments, a polypeptide comprises at least one amino acid substitution relative to the amino acid sequence of SEQ ID NO:25. 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, a polypeptide comprises about 1-10 amino acid substitutions, relative to the amino acid sequence of SEQ ID NO:25. In some embodiments, an amino acid substitution is a conservative substitution. In some embodiments, an amino acid substitution is a highly conservative substitution.

[00279] In some embodiments, a polypeptide has 100% sequence identity to the amino acid sequence of SEQ ID NO:25. In some embodiments, a polypeptide comprises (e.g., consists of) the amino acid sequence of SEQ ID NO:25.

[00280] In some embodiments, a polypeptide 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:26.

[00281] In some embodiments, a polypeptide comprises at least one amino acid substitution relative to the amino acid sequence of SEQ ID NO:26. 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, a polypeptide comprises about 1-10 amino acid substitutions, relative to the amino acid sequence of SEQ ID NO:26. In some embodiments, an amino acid substitution is a conservative substitution. In some embodiments, an amino acid substitution is a highly conservative substitution.

[00282] In some embodiments, a polypeptide has 100% sequence identity to the amino acid sequence of SEQ ID NO:26. In some embodiments, a polypeptide comprises (e.g., consists of) the amino acid sequence of SEQ ID NO:26.

[00283] In some embodiments, a polypeptide 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:27.

[00284] In some embodiments, a polypeptide comprises at least one amino acid substitution relative to the amino acid sequence of SEQ ID NO:27. 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, a polypeptide comprises about 1-10 amino acid substitutions, relative to the amino acid sequence of SEQ ID NO:27. In some embodiments, an amino acid substitution is a conservative substitution. In some embodiments, an amino acid substitution is a highly conservative substitution.

[00285] In some embodiments, a polypeptide has 100% sequence identity to the amino acid sequence of SEQ ID NO:27. In some embodiments, a polypeptide comprises (e.g., consists of) the amino acid sequence of SEQ ID NO:27.

[00286] In some embodiments, an antibody or an antigen-binding fragment thereof is a bispecific antibody or an antigen-binding fragment thereof. In some embodiments, an antibody or an antigen-binding fragment thereof is a bispecific single-domain antibody (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 PCTZEP2006/004678 (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/EP 1996/001725 (WO 1996034103) and PCT/EP1998/006991 (WO1999023221), the contents of which are incorporated herein by reference.

[00287] In some embodiments, an antibody or an antigen-binding fragment thereof is a multispecific antibody or an antigen-binding fragment thereof. In some embodiments, an antibody or an antigen-binding fragment thereof is a multispecific sdAb.

[00288] In some embodiments, an antibody or an antigen-binding fragment thereof is a monomeric sdAb. In some embodiments, an antibody or an antigen-binding fragment thereof comprises a dimeric sdAb. In some embodiments, an antibody or an antigen-binding fragment thereof comprises a multimeric (e.g., trimeric) sdAb. In some embodiments, two sdAbs are connected by a linker sequence (e.g., GGGGSGGGS (SEQ ID NO:28)).

[00289] In some embodiments, an antibody or an antigen-binding fragment thereof is an isolated antibody. In some embodiments, an antibody or an antigen-binding fragment thereof is an isolated antigen-binding fragment of an antibody. In some embodiments, an antibody or an antigen-binding fragment thereof is an isolated sdAb.

[00290] In some embodiments, an antibody or an antigen-binding fragment thereof is recombinantly produced. In some embodiments, an antibody or an antigen-binding fragment thereof is synthetically produced.

[00291] In some embodiments, an antibody or an antigen-binding fragment thereof binds human TNF-a. In some embodiments, an antibody or an antigen-binding fragment thereof binds human TNF-a and human serum albumin (HSA).

[00292] In some embodiments, an antibody or an antigen-binding fragment thereof has spontaneous BBB permeability. In some embodiments, an antibody or an antigen-binding fragment thereof has an isoelectric point (pl) 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 antibody or an antigenbinding fragment thereof has an isoelectric point (pl) of about 9.5.

[00293] In some embodiments, an antibody or an antigen-binding fragment thereof, or 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.

[00294] In some embodiments, a conjugated antibody or a conjugated antigen-binding fragment thereof has spontaneous BBB permeability. In some embodiments, a conjugated antibody or a conjugated antigen-binding fragment thereof has an isoelectric point (pl) 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 antibody or an antigen-binding fragment thereof has an isoelectric point (pl) of about 9.5.

[00295] 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. [00296] 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 polymer. In some embodiments, a polymer is a hydrophobic or hydrophilic polymer. Nonlimiting 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-a-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.

[00297] In some embodiments, a heterologous moiety facilitates transport of the antibody or the antigen-binding fragment thereof across the blood brain barrier (BBB), for example, by increasing lipid solubility of the antibody or the antigen-binding fragment thereof, 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), Cena & Jativa, 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.

[00298] In some embodiments, a heterologous moiety facilitates transport of the antibody or the antigen-binding fragment thereof across the BBB by increasing lipid solubility of the antibody or the antigen-binding fragment thereof. 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 an antibody or an antigen-binding fragment thereof).

[00299] In some embodiments, a heterologous moiety facilitates transport of the antibody or the antigen-binding fragment thereof 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).

[00300] In some embodiments, a heterologous moiety facilitates transport of the antibody or the antigen-binding fragment thereof 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), low density lipoprotein receptor (LDLR), and transferrin receptor (TfR).

[00301] 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.

[00302] In some embodiments, an antibody or antigen-binding fragment thereof, or a polypeptide 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, or 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 an antibody or antigenbinding fragment thereof, or a polypeptide can be introduced and expressed in a suitable host cell (e.g., E. colt), and the expressed antibody or antigen-binding fragment thereof, or 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 inframe in accordance with conventional techniques. In another embodiment, an antibody or antigen-binding fragment thereof, or 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

[00303] 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).

[00304] 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.

[00305] 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.

[00306] 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.

[00307] 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

[00308] 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.

[00309] 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 DH5a competent cells. In some embodiments, a host cell is a CHO cell.

[00310] 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

[00311] The disclosure also provides, among other things, a composition comprising any one or more of the antibodies or antigen-binding fragments thereof, polypeptides, polynucleotides, vectors, or host cells (e.g., recombinant cells) disclosed herein. In some embodiments, a composition is a pharmaceutical composition.

[00312] 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).

[00313] 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.

[00314] In some embodiments, a composition (e.g., a pharmaceutical composition) is formulated for a suitable administration schedule and route. Non-limiting examples of 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.

[00315] In some embodiments, a composition is provided in a dosage form, e.g., in a prefilled syringe, prefilled pen, or autoinjector.

[00316] 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.

[00317] 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.

[00318] 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

[00319] The disclosure also provides, among other things, a method of blocking binding of TNF-a 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 antibodies or antigen binding fragments thereof, polypeptides, or compositions (e.g., pharmaceutical compositions) disclosed herein, thereby blocking binding of TNF-a to TNFR expressed on the surface of the cell.

[00320] The disclosure also provides, among other things, a method of blocking binding of TNF-a to TNFR in a subject, the method comprising administering to the subject an effective amount of any one or more of the antibodies or antigen binding fragments thereof, polypeptides, or compositions (e.g., pharmaceutical compositions) disclosed herein, thereby blocking the binding of TNF-a to TNFR in the subject. [00321] The disclosure also provides, among other things, a method of treating a TNF-a- associated disease in a subject in need thereof, comprising administering to the subject an effective amount of any one or more of the antibodies or antigen binding fragments thereof, polypeptides, or compositions (e.g., pharmaceutical compositions) disclosed herein, thereby treating the TNF-a-associated disease.

[00322] 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 antibodies or antigen binding fragments thereof, polypeptides, or compositions (e.g., pharmaceutical compositions) disclosed herein, thereby reducing inflammation in the subject.

[00323] In some embodiments, a pharmaceutical composition comprises a pharmaceutically acceptable carrier and any one or more of the antibodies or antigen binding fragments thereof disclosed herein.

[00324] In some embodiments, a TNF-a-associated disease is Alzheimer’s disease (AD). In some embodiments, a TNF-a-associated disease is late onset Alzheimer’s disease (LOAD). In some embodiments, a TNF-a-associated disease is an inflammatory disease. In some embodiments, a TNF-a-associated disease is an autoimmune disease. In some embodiments, a TNF-a-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).

[00325] 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.

[00326] 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.

[00327] 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.

[00328] 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.

Embodiments

1. An antibody or an antigen-binding fragment thereof, comprising an immunoglobulin heavy chain variable (V_H) domain comprising a heavy chain complementaritydetermining 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:4-9.

2. The antibody or the antigen-binding fragment thereof of Embodiment 1, wherein: a) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, b) the HCDR2 comprises the amino acid sequence of SEQ ID NO:2, and c) the HCDR3 comprises the amino acid sequence of SEQ ID NO:3.

3. The antibody or the antigen-binding fragment thereof of Embodiment 1, wherein: a) the HCDR1 consists of the amino acid sequence of SEQ ID NO: 1, b) the HCDR2 consists of the amino acid sequence of SEQ ID NO:2, and c) the HCDR3 consists of the amino acid sequence of SEQ ID NO:3.

4. The antibody or the antigen-binding fragment thereof of any one of claims 1-3, wherein the V_H domain of the antibody or the antigen-binding fragment thereof comprises 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: VH FR1-HCDR1- V_H FR2-HCDR2-V_H FR3-HCDR3-V_H FR4, wherein: a) the V_H 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: 11), b) the V_H FR2 comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of WMYWVRQAPGKE (SEQ ID NO: 12), c) the V_H FR3 comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of DFVKGRFTVSRDNAKNTLYLQMDSLRPEDTAVYS (SEQ ID NO: 13), DFVKGRFTVSRDNAKNTLYLQMNSLEPEDTAVYS (SEQ ID NO: 14) or DFVKGRFTVSRDNAENTLYLQMNSLEPEDTAVYS (SEQ ID NO: 15), or d) the V_H FR4 comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of LRGQGTQVTVSS (SEQ ID NO: 16) or LRGQGTQVTVSSGPGGQ (SEQ ID NO: 17), or any combination of a) to d). The antibody or the antigen-binding fragment thereof of any one of claims 1-4, 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:4-9. The antibody or the antigen-binding fragment thereof of any one of claims 1-5, 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:4-9. The antibody or the antigen-binding fragment thereof of any one of claims 1-6, wherein the V_H domain comprises the amino acid sequence of SEQ ID NO:4. The antibody or the antigen-binding fragment thereof of any one of claims 1-6, wherein the V_H domain comprises the amino acid sequence of SEQ ID NO:5. The antibody or the antigen-binding fragment thereof of any one of claims 1-6, wherein the V_H domain comprises the amino acid sequence of SEQ ID NO:6. The antibody or the antigen-binding fragment thereof of any one of claims 1-6, wherein the V_H domain comprises the amino acid sequence of SEQ ID NO:7. The antibody or the antigen-binding fragment thereof of any one of claims 1-6, wherein the V_H domain comprises the amino acid sequence of SEQ ID NO:8. The antibody or the antigen-binding fragment thereof of any one of claims 1-6, wherein the V_H domain comprises the amino acid sequence of SEQ ID NO:9. The antibody or the antigen-binding fragment thereof of any one of claims 1-12, wherein the antigen-binding fragment is a single-domain antibody (sdAb). The antibody or the antigen-binding fragment thereof of any one of claims 1-13, wherein the antibody or the antigen-binding fragment thereof binds tumor necrosis factor-alpha (TNF-a). The antibody or the antigen-binding fragment thereof of any one of claims 1-14, wherein the antibody is bispecific or multispecific. A polypeptide comprising the antibody or the antigen-binding fragment thereof of any one of claims 1-15, and optionally: a) a protein tag for detecting and/or purifying the antibody or the antigen-binding fragment thereof, b) a heterologous moiety that targets the antibody or the antigen-binding fragment thereof to a particular tissue or cell, and/or facilitates transport of the antibody or the antigen-binding fragment thereof across the blood brain barrier (BBB), c) a therapeutic agent, d) a diagnostic agent, or any combination of a) to d). A polynucleotide encoding the antibody or antigen binding fragment thereof of any one of claims 1-15, or the polypeptide of Embodiment 16. An expression vector comprising the polynucleotide of Embodiment 17. A host cell comprising the polynucleotide of Embodiment 17, or the expression vector of Embodiment 18. A method of producing the antibody or the antigen binding fragment thereof of any one of claims 1-15, comprising expressing the antibody or the antigen binding fragment thereof, in the host cell of Embodiment 19 and isolating the expressed antibody or the antigen binding fragment thereof. A composition comprising the antibody or the antigen binding fragment thereof of any one of claims 1-15, the polypeptide of Embodiment 16, the polynucleotide of Embodiment 17, the expression vector of Embodiment 18, or host cell of Embodiment 19. The composition of Embodiment 21, further comprising at least one additional therapeutic agent. A pharmaceutical composition comprising the composition of Embodiment 21 or 22, and a pharmaceutically acceptable carrier or diluent. A kit comprising the composition of Embodiment 21 or 22, or the pharmaceutical composition of Embodiment 23. A method of blocking binding of TNF-a 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 claims 1-15, the polypeptide of Embodiment 16, the composition of Embodiment 21 or 22, or the pharmaceutical composition of Embodiment 23, thereby blocking binding of TNF-a to TNFR expressed on the surface of the cell. A method of blocking binding of TNF-a 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 claims 1-15, the polypeptide of Embodiment 16, the composition of Embodiment 21 or 22, or the pharmaceutical composition of Embodiment 23, thereby blocking binding of TNF-a to TNFR expressed in the subject. A method of treating a TNF-a-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 claims 1-15, the polypeptide of Embodiment 16, the composition of Embodiment 21 or 22, or the pharmaceutical composition of Embodiment 23, thereby treating the TNF-a-associated disease. The method of Embodiment 27, wherein the TNF-a-associated disease is Alzheimer’s disease (AD). The method of Embodiment 27 or 28, wherein the TNF-a-associated disease is late onset Alzheimer’s disease (LOAD). 30. The method of Embodiment 27, wherein the TNF-a-associated disease is an inflammatory disease or an autoimmune disease.

31. The method of Embodiment 30, wherein the TNF-a-associated disease is rheumatoid arthritis (RA), juvenile idiopathic arthritis (JIA), psoriatic arthritis (PsA), ankylosing spondylitis (AS), plaque psoriasis (PsO), Crohn’s disease (CD), pediatric Crohn’s disease, ulcerative colitis (UC), pediatric ulcerative colitis, hidradenitis suppurativa (HS), or uveitis (UV).

32. The method of any one of claims 26-31, wherein the subject is an adult human patient.

33. The method of any one of claims 26-31, wherein the subject is a pediatric human patient.

34. The method of any one of claims 26-33, further comprising administering to the subject at least one additional therapeutic agent.

35. The method of Embodiment 34, wherein the at least one additional therapeutic agent comprises an anti-serum albumin agent.

36. The method of Embodiment 34, wherein the at least one additional therapeutic agent comprises methotrexate.

EXEMPLIFICA TION

[00329] Tumor necrosis factor-alpha (TNF-a) and late onset Alzheimer’s disease (LOAD). [00330] A pathogenic role for neuroinflammation in late onset Alzheimer’s disease (LOAD) has emerged (Akiyama et al. (2000), Tarkowski etal., J Neurol Neurosurg Psychiatry (2003)).

Variants of TREM2, whose CNS expression is restricted to microglia (Schmid et al. (2002)), increase the risk of LOAD (Guerreiro et al. (2013)), consolidating the neuroinflammation-to- LOAD link. TNF-a, a trimeric transmembrane protein (m TNF-a) that is cleaved into a soluble trimer (sTNF-a) by TACE (Tang et al. (1996)), is a major neuroinflammatory cytokine (Janelsins et al. (2008), Janelsins et al. (2005), Lieberman et al. (1989)).

[00331] TNF-a binds two receptors. TNFR1 is ubiquitously expressed, is activated by both sTNF-a and mTNF-a and triggers divergent effects including cell proliferation and death.

TNFR2 is expressed by immune and endothelial cells (Sedger et al. (2014)), is activated primarily by mTNF-a, and triggers inflammatory and pro-survival pathways (McCoy & Tansey (2008)). TNFR2 can also enhance TNFR1 -mediated cytotoxicity (Weiss et al. (1997)). [00332] Several lines of data support a role of TNF-a in LOAD. First, TNF-a is elevated in blood, cerebrospinal fluid (CSF), and the central nervous system (CNS) of AD patients (Tarkowski et al., J Neurol Neurosurg Psychiatry (2003), Fillit et al. (1991), Tarkowski et al., Brain Res Bull. (2003), Tarkowski et al., Dement Geriatr Cogn Disord. (2003), Alvarez et al. (2007)). Second, TNF-a, TNFR1 and TNFR2 polymorphisms are associated with LOAD (Perry etal., Am J Med Genet. (2001), Perry etal., Neurobiol Aging (2001)). Third, TNF-a levels are elevated in transgenic FAD mouse models, increasing Ap production (Sly et al. (2001), Billings et al. (2005), Yamamoto et al. (2007), Liao et al. (2004)), and reducing clearance of Ap (Hickman et al. (2008)). Fourth, ablation of TNFR1 expression reduced Ap levels, plaques, and cognitive deficits (He et al. (2007)).

[00333] Rats carrying the p.R47H pathogenic TREM2 variant in the rat Trem2 gene (Trem2R47H rats) (Tambini & D’Adamio Sci Rep. (2020)) have supraphy si ologi cal levels of sTNF-a in the CSF and CNS, without changes in Ap levels (Tambini & D’Adamio Sci Rep. 10( 1 ) :4122 (2020), Ren et al. (2020), Ren et al. (2021)). TNF-a regulates surface levels of AMPA and GABA receptors promoting exocytosis of AMP AR and endocytosis of GAB AAR, thus augmenting excitatory synaptic strength, and decreasing inhibitory synaptic strength (Stellwagen et al. (2005), Ogoshi et al. (2005), Beattie et al. (2002), Stellwagen & Malenka (2006)). In accord with these data, Trem2^R47H rats show increased glutamatergic transmission, decreased GABAergic transmission and impaired LTP, a long-lasting form of synaptic plasticity that is a cellular basis for learning and memory (Tambini & D’Adamio Sci Rep. 10( 1 ):4122 (2020), Ren et al. (2020), Ren et al. (2021)). Reducing TNF-a activity with a TNF inhibitor monoclonal antibody (TNFI Mab) rapidly normalizes these alterations (Tambini & D’Adamio Sci Rep. (2020), Ren et al. (2020), Ren et al. (2021)).

[00334] This supraphysiological TNF-a-to-synaptic plasticity alterations nexus is the core of a pathogenic hypothesis (FIGs. 1A-1C). Noteworthy, genes linked to familial dementia (APP, PSEN1 and ITM2b), play a physiological role in glutamatergic transmission, which is altered by pathogenic mutations (Del Prete et al. (2014), Groemer et al. (2011), Yao et al. J Neurosci.

(2019), Lundgren et al. (2015), Kohli et al. (2012), Norstrom et al. (2010), Fanutza et al. (2015), Tambini et al. (2019), Wu et al. (2013), Xia et al. (2015), Matsuda et al. (2005), Fotinopoulou et al. (2005), Matsuda et al. (2008), Matsuda et al. (2011), Yao et al. Sci Rep. (2019), Tamayev et al., J Neurosci. (2010), Tamayev et al. Proc Natl Acad Sci U S A. (2010)), hinting that enhanced excitation may be a pathogenic pathway shared by LOAD and FAD. These early deficits may initiate a cascade, such as potentiation of glutamate neuronal excitotoxicity, leading to neurodegeneration decades later. Increased excitation also augments Ap brain levels (Cirrito et al. (2008), Cirrito et al. (2005)). Thus, supraphysiological TNF-a could trigger dementia via distinct mechanisms (FIG. ID), which support the disease-modifying potential of TNFIs in AD. [00335] Based on this conceptually innovative link, it is hypothesized that synaptic alterations can be corrected by lowering CNS TNF-a activity to physiological levels. The most potent FDA approved TNFI used to treat peripheral inflammatory conditions are biologic drugs, such as etanercept, an approximately 125 kDa fusion protein, consisting of a TNFR2 domain coupled to the Fc portion of human IgGl (Tracey et al. (2008)), and anti-TNF-a monoclonal antibodies (MAbs), such as adalimumab, infliximab, golimumab, and certolizumab (Cheng et al. (2014)). Intra-cerebroventricular injection of TNFIs reduced cognitive deficits in mice (McAlpine et al. (2009), Shi et al. Brain Res. (2011), Medeiros et al. (2007), Kim et al. (2016)). Remarkably, peri-spinal and intrathecal administration of etanercept and infliximab improved cognition in LOAD patients (Tobinick et al. (2006), Shi etal., J Am Geriatr Soc. (2011)). However, biologic TNFIs have limited blood-brain barrier (BBB) penetration (Boado et al. (2010), Etanercept in Alzheimer disease: A randomized, placebo-controlled, double-blind, phase 2 trial, Neurology 85(23):2084 (2015)), because of the large size and the presence the Fc-receptor mediated efflux to the blood (Cooper et al. (2013)). To overcome this issue, camelid single-domain antibody (sdAbs, Nabs) against human TNF-a were generated. Camelids produce heavy-chain-only antibodies (Hamers-Casterman et al. (1993)), composed of the antigen-binding variable domain (VHH) followed by constant domains CH2/CH3. Nabs, are approximately 12-14 kDa and lack an Fc region. Some Nabs can spontaneously cross the BBB (Li et al. (2012), Li et al. (2016)). Thus, Nabs neutralizing TNF-a activity (TNFLNab) may act as TNFI in the CNS.

[00336] Nabs have several advantages over monoclonal antibodies. First, unlike conventional MAbs, which form a flat shaped paratope, VHH domain form paratopes with a convex shape that enables the binding to molecular clefts, such as receptor binding sites, inaccessible to MAbs. Second, Nabs are stable over a large pH range. Third, Nabs have low immunogenicity, which can be further reduced via humanization/deimmunization. Fourth, Nabs are more suitable for oral delivery. Fifth, half-life of a Nab can be prolonged by targeting to Albumin. Sixth, Nabs may have spontaneous BBB permeability.

Example 1. Identifying Novel TNF-a Inhibitor Single-Domain Antibodies with High Efficacy [00337] Example 1 identified three novel TNF-a inhibitor single-domain antibodies (sdAbs) with high efficacy in inhibiting human TNF-a. One llama and one alpaca were immunized with active trimeric human sTNF-a (Aero Biosystems, Newark, DE, TNA-H5228). SdAb (Nab) sera titers were determined by ELISA on antigen-coated plates. Peripheral blood mononuclear cells (PBMCs) isolated from high-titer samples were used for cDNA synthesis of VHH domain genes, which were cloned into pADL-20c phagemid vector and transformed into TGI cells. Specific clones were enriched by panning on antigens-coated plates, and 470 individual clones were expressed in E. coli. His-tag Nabs were recovered in the periplasmic fraction by osmotic shock and were tested for binding to antigen by ELISA.

[00338] Eighty -five unique anti-TNF-a-Nab VHH sequences were identified. Anti-TNF-a-

Nabs were produced in bacteria, purified (FIG. 2A), and tested for their abilities to inhibit cytotoxicity elicited by active trimeric human sTNF-a. WEHI-13VAR cells, a mouse cell line for measuring rodent and human TNF-a at high sensitivity, were incubated with 0.25 ng/mL active trimeric human soluble TNF-a (sTNF-a) (plus 1 pg/mL Actinomycin-D, Sigma- Aldrich, St. Louis, MO) for 24 hours, alone or with 2-fold dilutions (from 25 nM to 0.76294 pM) of anti- TNF-a-Nab 1. Cytotoxicity was determined using Counting Kit-8 (CCK-8, Dojindo Laboratories Co., Ltd., Kumamoto, Japan) and comparing viability to cells treated with Actinomycin-D only. TNFI-Nabl results are shown as mean+/- standard deviation (SD) of triplicates and analyzed by inhibitor versus (vs.) response GraphPad Prism 9 software. TNFLNabs do not inhibit cytotoxicity of biotinylated mouse TNF-a Protein (His, AVITAG™, active trimer, Aero Biosystems, Newark, DE, TNA-M82E9) or rat recombinant TNF-a (Sino Biological Inc., Wayne, PA, 80045-RNAE), even when used at 100 nM. Three anti-TNF-a-Nabs reduced human sTNF-a cytotoxicity with an IC₅₀ in the pM range: TNFI-Nabl (13.06 kDa) IC₅₀ = 78 to 116 pM; TNFI-Nab2 (13.03 kDa) IC₅₀ = 136 to 223 pM; TNFI-Nab3 (13.04 kDa) IC₅₀ = 179 to 269 pM (Table 1, FIG. 2B). The IC₅₀ values of monovalent TNFI-Nabl, TNFI-Nab2, and TNFI-Nab3 (referenced together as TNFI-Nabl -3) are comparable to that of bivalent TNFI MAbs used in human therapy (Infliximab, IC50 = 67 to 134 pM; Adalimumab, ICso = 100 to 200 pM).

Table 1. TNFI Activity and TNFa Affinity

[00339] Surface plasmon resonance (SPR) showed that TNFI-Nab2 exhibited the slowest dissociation rate, resulting in the highest affinity, while TNFI-Nabl’ s higher affinity over TNFI- Nab3 was due to the fastest association rate. [00340] LTP deficits of Trem2^R47H rats were reversed by a neutralizing antibody to rat TNF-a at a concentration of approximately 0.5% of the IC₅₀ (Tambini & D’ Adamio Sci Rep. (2020), Ren et al. (2020), Ren et al. (2021)). See, e.g., Ren et al. (2020) Figure 7E. LTP impairment in Trem2^R47H/R47H rats (RH/RH) was rescued by an anti-TNF-a antibody at 0.5% of the IC₅₀. Wildtype (w/w + Isotype vs. RH/RH + Isotype: p<0.0001. w/w + anti-TNF-a vs. RH/RH + Isotype: p<0.0001. RH/RH + anti-TNF-a vs. RH/RH + Isotype: p<0.0001. Accordingly, based on a pathogenic model (FIGs. 1A-1D), a therapeutically effective concentration of one of TNFI- Nabl-3 in the CNS is predicted to be approximately 350-600 fM. Like TNFLMAbs used in human therapy, TNFLNabl-3 do not neutralize rodent TNF-a (FIG. 2B).

[00341] Fluorescence activated cell sorting (FACS) analysis was performed. HEK293 cells were transfected with a vector expressing human TNF-a plus EGFP. An allophycocyanin (APC) anti-human TNF-a antibody (BioLegend, San Diego, CA, 502912) and an APC anti-mouse/rat TNF-a antibody (BioLegend, 506107) were used as positive controls (not shown). For sdAb binding, transfected cells were incubated with 40 nM anti-TNF-a-Nabl, followed by an anti-His- APC antibody (R&D Systems, Minneapolis, MN, IC050A). Anti-His-APC alone was used as a negative control. The FACS analysis showed that TNFLNabl-3 bind membrane-tethered human mature TNF-a (FIGs. 2C-2D), indicating that they also inhibit the function of human mature TNF-a.

[00342] In sum, the experiments described in this Example 1 identified three novel TNF-a inhibitor sdAbs that inhibit human TNF-a with an IC₅₀ in the pM range.

Example 2, Predicting Human Immunogenicity /wv hi-Silico Modelling

[00343] Efficacy of protein therapeutics can be tampered by immune responses. Anti-drug antibodies (ADA) can impact the pharmacokinetics, pharmacodynamics, and activity of protein therapeutics. Immunoreactions can also cause life-threatening complications. Hence, European Medicines Agency (EMA) and Food and Drug Administration (FDA) stress the importance of predicting human immunogenicity before clinical trials. Example 2 predicts immunogenicity of the newly identified TNF-a inhibitor sdAbs in human.

[00344] Methods of predicting immunogenicity use animal models, in-silico human immunogenicity modelling, and/or ex vivo assays with human immune cells. Because of species-specific differences in antigen processing and MHC class II and T cell receptor repertoires, animal models are poor predictors of immunogenicity in humans. Thus, to predict immunogenicity risks in humans more accurately before clinical trials, in-silico human immunogenicity modelling was performed to be combined with ex vivo assays. [00345] The in-silico immunogenicity modelling employed combinatory iTope & TCED proprietary technologies (Abzena, San Diego, CA). iTope- Al is an in-silico immunogenicity risk assessment and deimmunization platform based on Augmented Intelligence, using a state-of-the-art Machine Learning algorithm to predict peptide binding specificities across 46 major HLA-DR, DP and DQ isotypes, which represent the most common HLA alleles found world-wide with no weighting attributed to those found most prevalently in any ethnic population.

[00346] The location of key binding residues was achieved by the in-silico generation of 9- mer peptides that overlap by eight amino acids spanning sequences of the TNFI-Nabs. Individual peptides were given a binding score from 0 to 3 for each allotype, and those scores were added together for all HLA-DR, DP, DQ alleles to provide an overall risk score (“Position Risk Score”). A peptide was considered a weak, medium, or strong promiscuous MHC class II binder when its Position Risk Score reached 1-2, 3-5, or >6. “Total Score” of the test protein was calculated by adding the Position Risk Scores obtained for all individual peptides. The highest Position Risk Score was provided to inform the presence of strong binders (“Hotspot Max”). Peptides identical to sequences from the human proteome were excluded from the analysis since germline sequences were unlikely to have immunogenic potential due to T cell tolerance.

[00347] TCED is a database of known T cell epitopes identified in studies using EPISCREEN® T cell epitope mapping assays especially of antibody V regions (Bryson et al. Prediction of immunogenicity of therapeutic proteins: validity of computational tools, BioDrugs 24(1): 1-8 (2010)). Peptides with Position Risk Scores of “>0” were interrogated against the TCED database to identify any high sequence homology with >10,000 peptides from unrelated proteins and antibodies that stimulated T cell responses in previous ex vivo EPISCREEN® studies (T cell epitopes). The iTope- Al algorithm was able to accurately predict 95% of peptide binding core motifs previously identified by 3D structure from X-ray crystallography. Known HLA-DR, DP and DQ promiscuous binders were also predicted to bind the expected isotypes, demonstrating iTope-AI specificity.

[00348] In-silico immunogenicity analysis of TNFI-Nabl -3 indicated a low human immunogenicity risk. Compared to biologies used in human therapies, the Total Score for TNFI-Nabl, 2, and 3 were 72, 62, and 59, respectively, and the Hotspot Max score for TNFI-Nabl, 2, and 3 were 20, 13, and 12, respectively. These values are comparable to those of the least immunogenic human MAbs (FIG. 3). Also, the C-terminal 6-His tag did not affect the Total Score for these sequences and does not cause an immunogenicity risk. [00349] In view of the above data and analysis, it is predicted that the three novel TNF-a inhibitor sdAbs have low or no immunogenicity in humans.

Example 3, Producing TNF-a Inhibitor SdAbs in Mammalian Cells

[00350] Nab production in mammalian cells could favor physiological folding. Furthermore, endotoxin contamination of bacteria preps may trigger production of cytotoxic factors by WEHI- 13 VAR cells used in TNF -bioassays, including murine TNF-a, causing underestimation of TNFI activity. Accordingly, TNFI-Nabl-3 will be produced in HEK293 cells. Furthermore, binding affinity (K_D), on-rate (k_on), and off-rate (k_og-) of TNFI-Nabl-3/TNF-a interactions will be determined using surface plasmon resonance (SPR) (Tamayev et al. (2009)).

Example 4, Determining Human Immunogenicity Using Ex Vivo Assays

[00351] In-silico analysis may overestimate the immunogenicity of TNFI-Nabl-3 because it does not account for the following factors: (z) protein processing by antigen presenting cells (APCs) (not all the peptides analyzed in-silico are formed and presented by APCs in vivo) (zz) recognition by the T cell receptor (the T cell receptor repertoire); and (zzz) T cell tolerance to the peptide. Accordingly, it is critical to combine the in-silico prediction with ex vivo assays with human immune cells.

[00352] Immunogenicity of TNFI-Nabl-3 produced in mammalian cells will be tested using the EPISCREEN® time course T cell assay, a test used extensively across the industry to test immunogenicity of protein therapeutics during preclinical early lead selection. Using CD8⁺ T cell depleted PBMCs, this assay measures CD4⁺ T cell responses, the primary drivers of an immune cascade leading to ADA formation. The CD8⁺ T cell depleted PBMCs will be isolated from 50 donors, with >80% DRB1 allotypes coverage of the world population and containing APCs and CD4⁺ T cells at physiological ratios.

[00353] T cell proliferation and IL-2 secretion, two markers of T cell activation, will be measured. A strong correlation between these markers will enable the detection of CD4⁺ effector T cell activation and discrimination between T cell effector and regulatory responses. Significant immunogenicity will be determined through statistical assessment of the dataset using Student’s Ltest. Details on magnitude of T cell response will be determined based on stimulation index normalization against background control. Since inhibition of TNF-a activity interferes with APC’s differentiation, rodent TNF-a, which is not inhibited by TNFI-Nabs will be supplied.

[00354] The experiments will include at least 6 technical replicates. Data will be analyzed using GraphPad Prism 9 software. All experiments and analyses will be blinded. Example 5, Reducing Human Immunogenicity

[00355] If a TNFI-Nab shows immunogenicity, EPISCREEN® MHC-Associated Peptide Proteomics (MAPPs) will be used to identify Nab-derived peptides that are processed and presented on MHC Class II by APCs. Identified peptides will be run through an EPISCREEN® T Cell Epitope Mapping Assay to identify epitopes that activate human CD4⁺ T cells. In silico guided deimmunization of these sequences will be performed. Mutagenized Nabs will be tested for TNFI activity, to avoid detrimental substitutions.

Example 6, Determining Intrinsic In Vivo Blood-Brain Barrier Permeability

[00356] The intrinsic in vivo blood-brain barrier (BBB) permeability of TNFI-Nab 1-3 will be tested for in rats. In the three test groups, TNFI-Nabl, 2, or 3 (4 mg/kg=250 nM/kg) plus 0.5% Evans Blue will be injected via the lateral tail vein. In a control group, vehicle plus 0.5% Evans Blue will be injected. CSF and serum will be collected approximately 6 and 12 hours after injection as previously reported (Ren et al. (2020)). Six rats will be used for each time point, and a total of 48 rats will be used.

[00357] To quantify active TNFI-Nab 1-3 that crossed the BBB, TNFI-Nab 1-3 will be captured by active trimeric human sTNF-a and detected with an anti-alpaca VHH domain antibody. An ELISA assay detecting concentrations of active TNFI-Nabs in the sub-pM range (as illustrated in FIG. 4), significantly below the predicted therapeutic dose, will be used. Evans Blue (molecular weight of 960) will be quantified using a spectrophotometric method. If the BBB integrity is not compromised, Evans Blue should not be present in the CSF. Accordingly, detection of a TNFI-Nab in the CSF, and concurrently an absence of Evan Blue in the CSF, will demonstrate that the TNFI-Nab has intrinsic BBB permeability. The degree of intrinsic BBB permeability will be estimated by analyzing the serum level of a TNFI-Nab and calculating the CSF/serum ratio.

[00358] In FIG. 4, streptavidin coated plates (Meso Scale Diagnostics, LLC (MSD), Rockville, MD, L15SA) were coated with 0.25 pg/ml of Biotinylated Human TNF-a protein active trimer (ACROBiosystems, Newark, DE, TNAH82E3). The plates were incubated with the indicated amounts of Nabs. Nabs bound to Human TNF-a were detected by 1 pg/ml of goat antialpaca IgG, VHH domain (Jackson ImmunoResearch Labs, West Grove, PA, 128-005-230), followed by 1 pg/ml of SULFO-TAG labeled anti-goat donkey antibody (MSD R32AG). The ELISA plates were read on MESO QuickPlex SQ 120. The ELISA was specific because 100 nM solution of TfRb-Nabl, a negative control antibody, read like vehicle alone. [00359] Association between TNFI and rare inflammatory central and peripheral neuroinflammatory syndromes has been reported (Gelfand & Yazdany (2020)). Also, TNFI can lower the ability of the immune system to fight infections causing infections and tumor development. These adverse events depend on the extent of TNF-a inhibition. Pathogenic changes in synaptic transmission and LTP caused by supraphysiological TNF-a are corrected by a TNFI MAb at a concentration of approximately 0.5% of the IC₅₀. The therapeutic serum concentration of TNFI biologies used in peripheral inflammatory disease is around approximately 50% of the IC₅₀ (for example, the effective Adalimumab concentrations for psoriasis is approximately 30%-60% of the IC₅₀ (Menting et al. (2015)). Thus, TNFI-Nab with a CSF/serum concentration ratio of approximately 0.2/0.6 could prove therapeutically effective in AD at serum concentrations approximately 2.5%/0.8% of the IC₅₀, reducing the probability of adverse events reported for TNFI currently used in human therapy. Still, potential toxicity of TNFI-Nab will be addressed in future preclinical and clinical studies.

[00360] Alternative routes of sdAb administration (subcutaneous, intraperitoneal, intramuscular) will be tested. Brain interstitial fluid (ISF) is contiguous with the CSF. However, TNFI-Nab levels in the ISF in various brain regions (e.g., frontal cortex, hippocampus) will be tested using micro dialysis probes implanted stereo-tactically in these brain regions, as previously described (Tamayev et al. Mol Neurodegener. (2012), Tamayev et al. EMBO Mol Med. (2012), Tamayev & D’Adamio Mol Neurodegener. (2012)).

[00361] The experiments will include at least 6 technical replicates. Data will be analyzed using GraphPad Prism 9 software. All experiments and analyses will be blinded.

Example 7, Evaluating TNFI-Nabs in Rodent AD Models

[00362] If TNFI-Nabs show intrinsic BBB permeability, humanized TNF-a rodents will be used to determine if hTNF-a, which binds TNFI-Nabl-3, modifies BBB permeability and/or TNFI-Nabl-3 detection. Because TNFI-Nabl-3 do not neutralize rodent TNF-a, existing rodent AD models cannot be used. The rat TNF-a gene will be replaced with the human TNF-a gene to generate humanized TNF-a rats. For preclinical studies, rat disease models expressing hTNF-a will be generated by crossing these rats to FAD, LOAD or AD related dementias rat models (Tambini & D’Adamio, Sci Rep. (2020), Ren et al. (2020), Ren et al. (2021), Tambini et al. (2019), Yin et al. (2021), Tambini & D’Adamio, J Biol Chem. (2020), Tambini et al. (2020)). Similarly, mouse AD models expressing hTNF-a Human can be created using a humanized TNF-a mouse model (GenOway, Lyon, France). Example 8, Producing TNFI-Nabs in Chinese Hamster Ovary (CHO) Cells

[00363] CHO cells are mammalian cells capable of producing complex proteins with proper folding, post-translational modifications, and biological activities akin to human 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. This environment ensures proper folding and assembly of complex proteins, crucial for functionality. CHO cell-based expression systems have a long history of producing biopharmaceuticals and have regulatory approval for numerous therapeutic proteins. In contrast, proteins expressed in bacteria (e.g., E. coif) may lack necessary post-translational modifications, potentially compromising functionality and safety. Moreover, bacterial expression systems can produce endotoxins, posing challenges for purification and requiring additional safety steps.

[00364] cDNAs coding for the TNFI-Nabs were clones into pcDNA3.4. A coding sequence for signal peptide MGWSCIILFLVATATGVHS (SEQ ID N0:x) was added to the 5’-end of the nanobody coding sequences to allow secretion of the TNFI-Nabs. A sequence coding for 6xHis (His-Tag) was added to the 3’-end to allow purification of secreted TNFI-Nabs. The constructs were transfected into CHO-S cells, and the TNFI-Nabs were purified by a single step via the His- Tag. As shown in Table 2 and FIG. 5, TNFI-Nabs were efficiently produced in CHO-S cells.

Table 2. TNFI-Nabs Purified from 100 ml Culture Supernatants

Example 9, Optimization Mutagenesis Utilizing AbNatiV

[00365] The Machine Learning algorithm AbNatiV (Ramon et al., (2024)) aims to enhance VHHs’ similarity to human immune system sequences, minimizing immunogenicity while preserving nanobody physio-integrity. The optimization strategy for TNFI-Nabs involves two steps: Humanization and Solubility Optimization.

[00366] Humanization

[00367] This step, facilitated by AbNatiV, enhances VHHs’ similarity to human sequences, minimizing immunogenicity while preserving physio-integrity. The process evaluates humanness and VHH-nativeness, crucial in predicting immunogenicity. A sequence score above 0.8 typically indicates human-like characteristics, while a score below 0.8 suggests non-human attributes, potentially leading to immunogenicity. Two mutational sampling strategies were followed: (i) Enhanced Sampling: Iteratively explores the mutational space to generate a single humanized sequence, and (ii) Exhaustive Sampling: Assesses all mutation combinations within the mutational space and selects the best sequences, returning variants with the highest VH- humanness for each beneficial mutation.

[00368] Solubility Optimization

[00369] Utilizes CamSol, a validated sequence-based scoring method, to assess nanobodies’ solubility and the effects of mutations. Structural resolution of nanobodies bound to their respective ligands guides CDR optimization through structure-guided mutations. A deeplearning model, ImmuneBuilder2 (Abanades et al. (2023)), predicts structures of wild-type and mutant sequences, with RMSD analysis ensuring framework mutations do not adversely affect binding by altering CDR conformation.

[00370] Based on favorable starting points for humanness, VHH-nativeness, and CamSol solubility, TNFI-Nabl was selected as the representative of the TNFI-Nab Family (Table 3).

Table 3.

[00371] TNFI-Nabl Mutants

[00372] WEHI-13VAR cells were incubated for 10 hours with 0.25 ng/mL active trimeric human sTNFa (plus 1 pg/mL Actinomycin-D, Sigma), either alone or with 2-fold serial dilutions (ranging from 10,000 to 12.2 pM) of TNFI-Nabs. Apoptosis was determined using the IQUE® Caspase 3/7 Reagent Kit (Sartorius, Gottingen, Germany), which includes a dye that fluoresces when cleaved by activated caspases 3 and 7, key players in the apoptotic pathway. An IC₅₀ value represents the concentration of TNFI-Nabl or a mutant thereof required to inhibit 50% of the Caspase 3/7 activity induced by TNFa.

[00373] Table 5 shows TNFI-Nabl mutants (TNFI-Nab 1_M1 -6) with enhanced therapeutic potential determined by AbNatiV analysis. Optimization mutations were confined to the frameworks. Structural insights into TNFI-Nabs bound to TNFa were essential for fine-tuning CDRs through structure-guided mutations.

Table 4.

[00374] The mutant Nabs were produced in CHO-S cells and evaluated for TNFI activity

While TNFI-Nabl_M2, TNFI-Nabl_M4, TNFI-Nabl_M5, and TNFI-Nabl_M6 mutants exhibited reduced IC₅₀ against TNFa-induced caspase-3/7 activation, TNFI-Nabl_Ml and TNFI-Nabl_M3 mutants showed lower IC₅₀ values (FIG. 6 and Tables 4&5). Specifically, TNFI-Nabl has an IC₅₀ of 133.5 pM, TNFI-Nabl_Ml has an IC₅₀ of 48.84 pM, and TNFI-

Nabl_M3 has an IC₅₀ of 64.31 pM. TNFI-Nabl_Ml has a lower IC₅₀, and TNFI-Nabl_M3 has higher humanness (0.826 vs. 0.809) and solubility (1.047 vs. 0.975).

Table 5. TNFI Activity of Mutant TNFI-Nabs

Example 10. Kinetic Analysis of Nanobody Interaction with TNFa

[00375] Kinetic Analysis of Nanobody Interaction with TNFa was performed on a Biacore IK SPR system using Carboxyl CM5 sensors. Purity of TNFI-Nabl_Ml, TNFI-Nab_M3, and human TNFa were greater than 90%. The nanobodies were used as the ligands, and TNFa was used as the analyte. Multi-cycle kinetics using a 1 : 1 binding model was used for determining the kinetic parameters.

[00376] Immobilization Optimization [00377] Screening of immobilization conditions was performed on CM5 Carboxyl sensors. Conditions tested included a range of ligand concentrations to eliminate the possibility of the steric hindrance and ligand crowding, immobilization buffer optimization to account for isoelectric points of each ligand, as well as a range of flow rates and cycles.

[00378] Optimal ligand concentration of 2.5 pg/mL was determined to provide the highest level of specific binding with minimal steric hindrance. A range of pH conditions for acetate buffer from 4.0-5.5 were assayed for preconcentration effect of the nanobodies based on their isoelectric points. Optimal immobilization setups were devised based on the mildest buffer conditions exhibiting the most ideal preconcentration effect. Those conditions were used for the remainder of the study.

[00379] Each immobilization setup provided sufficient levels of ligand immobilization based on CM5 sensor guidelines, aiming for the absolute response maximum (R_max, i.e., maximum analyte response (for a given ligand density), expressed in response units (Rus)) being at 20-40 RUs for each setup.

[00380] In particular, R_max for a given binding system is governed by the availability of the binding sites as well as the molecular weight/size of binding partners. Note that theoretical R_max is rarely reached in actual binding experiments due to a variety of factors including < 100% ligand activity or sub-optimal ligand orientation, steric effects, non-specific binding, and such. [00381] Low R_max can be an issue if signal to noise ratio is reduced to a point where it affects confidence in fitting analysis. Given the size ratio of ligand to analyte in this experiment (13.353 kDa for the nanobodies and 63 kDa for TNFa), the theoretical Rmax was determined to be 34.44 RUs for TNFI-Nabl_Ml and 12.74 RUs for TNFI-Nabl_M3 ligands, based on the average ligand capture.

[00382] Achieved analyte responses on Biacore IK were within a standard range based on each respective R_max and sufficient to perform kinetic fitting.

[00383] Assay Development: Buffer Conditions

[00384] Optimal conditions were determined based on minimal non-specific binding (NSB) to the Reference and Active Channel surface, best signal to noise ratio, and low bulk shift in the mid-nanomolar range for each analyte, lx PBS running buffer supplemented with 3 mM EDTA and 0.05% v/v Surfactant P20 was determined to provide optimal binding and was used for full- kinetic analysis.

[00385] Assay Development: System Setup [00386] Biacore IK system priming was performed with the running buffer twice prior to the start of immobilization. Sensitivity calibration for the Active and Reference Channel was verified with a 2% glycerol solution prepared in the in the running buffer according to manufacturer’s specifications. Ligand was immobilized in the Active Channel at the concentration of 2.5 pg/mL via the standard amine coupling chemistry. A mixture of 100 mM EDC and 100 mM NHS was used to covalently couple the primary amine groups of each nanobody ligand to the free carboxyl groups on the gold surface of sensors at the flow rate of 20 pL/min. Blocking of the sensor surface in Active and Reference Channels was performed with 1 M ethanolamine quenching solution.

[00387] Assay Development: Validation of Binding and Kinetic Models

[00388] Binding of the TNFa analyte was validated in picomolar range. The analyte exhibited binding patterns to each ligand following a standard 1 : 1 binding model where both association and the dissociation processes are single exponentials. A bivalent analyte binding model was also explored; however, the 1 :1 model was found to be most appropriate, as validated by fit curves (indicated by green checkmarks set by the Biacore Insights analysis software), residuals (z.e., the difference between the fit and the data), and the Chi² analysis of kinetic fits.

[00389] Kinetic Analysis

[00390] For each test, analytes were diluted into the running buffer at the highest concentration to be injected and then further diluted in a 3 -fold series to the lowest concentration to be injected; a total of 9 serial dilutions were prepared. Analyte was then injected following buffer blank(s), in the order of lowest to highest concentration. Data was exported into Biacore Insights analysis software for kinetic fitting.

[00391] Multi-cycle Kinetics

[00392] Analyte responses were found to be specific and dose-dependent, as determined by the analysis of residuals; comparison of unprocessed, raw sensorgrams for the Active Channel vs. the Reference Channel; and standard errors. Kinetic binding analysis resulted in good fits using the 1 : 1 Langmuir binding model, as opposed to the bivalent analyte binding model. In each case ka and kd parameters were set to Global to provide the most stringent criteria.

[00393] Validations of the fitting curves for each set were performed by the analysis of the residuals and Chi². Data residuals were found to meet the good-fit criteria, as they were found to be below 10% of each individual response and showing a random scattering of data points that do not have a significant impact on the sampling readout. Chi² values for all 1 : 1 fits were validated to be below 10% Rmax cutoff, confirming that under the optimized conditions binding responses primarily follow the 1 : 1 model.

[00394] Multi-cycle kinetic analysis of two independent tests for each nanobody interacting with TNFa resulted in calculated K_D values of 167 pM for TNFI-Nabl_Ml and 405 pM for TNFI-Nabl_M3. TNFa was therefore found to have comparable high affinity towards both nanobodies (Table 6).

Table 6. Kinetics of TNFa Analytes Binding to Immobilized Nanobodies.

[00395] Iso-affinity Analysis

[00396] Comparative kinetic analysis was performed by Iso-affinity plotting, comparing both nanobody-TNFa interactions characterized in this study (FIG. 7). The iso-affinity plot, which assesses the relationship between the association rate (y-axis) and the dissociation rate (x-axis) for each interaction, clarifies the input of each parameter on the overall kinetic analysis. The faster association of TNFa to TNFI-Nabl_Ml ligand is the most meaningful difference in kinetics between the two interactions, and the major contributor to the slightly greater affinity of this pair (Table 6).

[00397] Conclusions

[00398] Binding of TNFa to the TNFI-Nabl_Ml and TNFI-Nabl_M3 ligands was characterized by multi-cycle kinetic analysis. Kinetics parameters were determined using a 1 : 1 binding model. The equilibrium constant was determined for each interaction. Overall, TNFa was found to have the highest affinity for TNFI-Nabl_Ml, though it also has a comparably high affinity for TNFI-Nabl_M3 that is within the same order of magnitude.

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[00399] The teachings of all patents, published applications, and references cited herein are incorporated by reference in their entirety.

[00400] 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.

Claims

CLAIMS What is claimed is:

1. An antibody or an antigen-binding fragment thereof, comprising an immunoglobulin heavy chain variable (V_H) domain comprising a heavy chain complementaritydetermining 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:40, 42, and 4-6.

2. The antibody or the antigen-binding fragment thereof of claim 1, wherein: a) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, b) the HCDR2 comprises the amino acid sequence of SEQ ID NO:46 or SEQ ID NO:2, and c) the HCDR3 comprises the amino acid sequence of SEQ ID NO:3.

3. The antibody or the antigen-binding fragment thereof of claim 1, wherein: a) the HCDR1 comprises the amino acid sequence of SEQ ID NO:29, b) the HCDR2 comprises the amino acid sequence of SEQ ID NO:30, and c) the HCDR3 comprises the amino acid sequence of SEQ ID NO:31.

4. The antibody or the antigen-binding fragment thereof of claim 1, wherein: a) the HCDR1 comprises the amino acid sequence of SEQ ID NO:32, b) the HCDR2 comprises the amino acid sequence of SEQ ID NO:33, and c) the HCDR3 comprises the amino acid sequence of SEQ ID NO:3.

5. The antibody or the antigen-binding fragment thereof of claim 1, wherein: a) the HCDR1 comprises the amino acid sequence of SEQ ID NO:34, b) the HCDR2 comprises the amino acid sequence of SEQ ID NO:35, and c) the HCDR3 comprises the amino acid sequence of SEQ ID NO:36.

6. The antibody or the antigen-binding fragment thereof of any one of claims 1-5, 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:40, 42, and 4-6.

7. The antibody or the antigen-binding fragment thereof of any one of claims 1-6, 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:40, 42, and 4-6.

8. The antibody or the antigen-binding fragment thereof of any one of claims 1-7, wherein the antigen-binding fragment is a single-domain antibody (sdAb).

9. The antibody or the antigen-binding fragment thereof of any one of claims 1-8, wherein the antibody or the antigen-binding fragment thereof binds tumor necrosis factor-alpha (TNF-a).

10. A polypeptide comprising the antibody or the antigen-binding fragment thereof of any one of claims 1-9.

11. The polypeptide of claim 10, further comprising a protein tag, a heterologous moiety, a therapeutic agent, a diagnostic agent, or any combination of the foregoing.

12. A polynucleotide or an expression vector encoding the antibody or antigen binding fragment thereof of any one of claims 1-9, or the polypeptide of claim 10 or 11.

13. A host cell comprising the polynucleotide or expression vector of claim 12.

14. A composition comprising the antibody or the antigen binding fragment thereof of any one of claims 1-9, the polypeptide of claim 10 or 11, the polynucleotide or expression vector of claim 12, or host cell of claim 13.

15. A pharmaceutical composition comprising the composition of claim 14 and a pharmaceutically acceptable carrier or diluent.

16. A kit comprising the composition of claim 14, or the pharmaceutical composition of claim 15.

17. A method of blocking binding of TNF-a 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 claims 1-9, the polypeptide of claim 10 or 11, the composition of claim 14, or the pharmaceutical composition of claim 15, thereby blocking binding of TNF-a to TNFR expressed on the surface of the cell.

18. A method of blocking binding of TNF-a 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 claims 1-9, the polypeptide of claim 10 or 11, the composition of claim 14, or the pharmaceutical composition of claim 15, thereby blocking binding of TNF-a to TNFR expressed in the subject.

19. A method of treating a TNF-a-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 claims 1-9, the polypeptide of claim 10 or 11, the composition of claim 14, or the pharmaceutical composition of claim 15, thereby treating the TNF-a-associated disease.

20. The method of claim 19, wherein the TNF-a-associated disease is Alzheimer’s disease (AD), late onset Alzheimer’s disease (LOAD), an inflammatory disease, an autoimmune disease, rheumatoid arthritis (RA), juvenile idiopathic arthritis (JIA), psoriatic arthritis (PsA), ankylosing spondylitis (AS), plaque psoriasis (PsO), Crohn’s disease (CD), pediatric Crohn’s disease, ulcerative colitis (UC), pediatric ulcerative colitis, hi dradenitis suppurativa (HS), or uveitis (UV).