Single-domain antibodies and variants thereof against TAB1

Abstract

The present application provides constructs comprising a single-domain antibody (sdAb) moiety that specifically recognizes TAB1. Also provided are methods of making and using these constructs, such as for the treatment of conditions mediated by atypical p38 signaling.

Description

[0001] SINGLE-DOMAIN ANTIBODIES AND VARIANTS THEREOF AGAINST TAB1

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] Priority is hereby claimed to US Provisional Application 63 / 555,736, filed February 20, 2024, which is incorporated herein by reference in its entirety.

[0004] SEQUENCE LISTING

[0005] The instant application contains a Sequence Listing which has been submitted in XML format and is hereby incorporated by reference in its entirety. The XML copy was created on February 19, 2025, is named PCT- -105677.005-2024-041 -02-SEQJJST.xml, and is 35,630 bytes in size.

[0006] FIELD OF THE INVENTION

[0007] The present invention relates to constructs comprising single-domain antibody (sdAb) moieties that specifically recognize TAB1 , and methods of making and using such constructs, such as for the treatment of conditions mediated by atypical p38 signaling.

[0008] BACKGROUND

[0009] In the classical pathway, MKK3 / 6 activates all four p38 isoforms (a / b / d / g); however, there is a growing body of evidence to support MKK3 / 6-independent atypical activation of p38a. Contrary to MKK3 / 6 and Zap70, the scaffolding protein transforming growth factor-p-activated kinasei binding protein-1 (TAB1) binds directly to p38a at two sites, inducing a conformational change, and p38a autophosphorylation, bypassing the requirement for MKK3 / 6- or Zap70-mediated activation. TAB1 -dependent signaling is distinct from Zap70-mediated p38 activation, which is selectively utilized in T-cells. Mutations in the p38 interaction domains of TAB1 (TAB1 KI) block atypical p38 signaling, also termed non-canonical p38 activation or TAB1-dependent p38 activation. The direct TAB1-p38o interaction drives atypical p38-dependent vascular inflammation and edema, ischemic damage, amyloidosis, dermal inflammation, and viral replication. Small molecules have yet to be generated that selective block atypical p38 signaling in cells.

[0010] There is a critical unmet need for effective therapies that can target TAB1 for disrupting TAB1 and p38 interactions and treat conditions mediated by atypical p38 signaling.

[0011] SUMMARY OF THE INVENTION

[0012] One aspect of the invention is directed to an ti-TAB1 constructs comprising single-domain antibody (sdAb) moieties specifically recognizing transforming growth factor (TGF)-beta-activated kinase 1 (MAP3K7) binding protein 1 (TAB1). The anti-TAB1 constructs are capable of disrupting TAB1 and p38 interactions and treating conditions mediated by atypical p38 signaling.

[0013] In some embodiments, the anti-TAB1 construct comprising a single-domain antibody (sdAb) moiety specifically recognizing TAB1 , wherein the sdAb moiety comprises a CDR1, a CDR2, and a CDR3.

[0014] In some embodiments, the CDR1 comprises an amino acid sequence of SGDTWENTGMG (SEQ ID NO: 1 ), SGSTSAFSNMG (SEQ ID NO:2), SGTYTSEIMG (SEQ ID NO:3), or a variant of any of the foregoing comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions.

[0015] In some embodiments, the CDR2 comprises an amino acid sequence of SDFEDMVDYY (SEQ ID NO:4), SGLDDMKHYY (SEQ ID NO:5), SRNFSFKTYY (SEQ ID NO:6), or a variant of any of the foregoing comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions.

[0016] In some embodiments, the CDR3 comprises an amino acid sequence of AMPTMQIRITSDTNSGTWKY (SEQ ID NO:7), AEIELDMHHLELTIHYY (SEQ ID NO:8), AIDLKWKGRLIMTY (SEQ ID NO:9), or a variant of any of the foregoing comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions.

[0017] In some embodiments, the CDR1 comprises an amino acid sequence of SGDTWENTGMG (SEQ ID NON) or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions, the CDR2 comprises an amino acid sequence of SDFEDMVDYY (SEQ ID NO:4) or a variant thereof comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions, and the CDR3 comprises an amino acid sequence of AMPTMQIRITSDTNSGTWKY (SEQ ID NO:7) or a variant thereof comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions.

[0018] In some embodiments, the CDRI comprises an amino acid sequence of SGSTSAFSNMG (SEQ ID NO:2) or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions, the CDR2 comprises an amino acid sequence of SGLDDMKHYY (SEQ ID NO:5) or a variant thereof comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions, and the CDR3 comprises an amino acid sequence of AEIELDMHHLELTIHYY (SEQ ID NO:8) or a variant thereof comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions.

[0019] In some embodiments, the CDR1 comprises an amino acid sequence of SGTYTSEIMG (SEQ ID NO:3) or a variant thereof comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions, the CDR2 comprises an amino acid sequence of SRNFSFKTYY (SEQ ID NO:6) or a variant thereof comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions, and the CDR3 comprises an amino acid sequence of AIDLKWKGRLIMTY (SEQ ID NO:9) or a variant thereof comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions.

[0020] In some embodiments, the sdAb moiety comprises an FR1 , an FR2, an FR3, and an FR4. In some embodiments, the FR1 comprises an amino acid sequence of MAEVQLQASGGGFVQPGGSLRLSCAA (SEQ ID NO:10) or a variant of any of the foregoing comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions.

[0021] In some embodiments, the FR2 comprises an amino acid sequence of WFRQAPGKEREFVSAI (SEQ ID NO: 11) or a variant of any of the foregoing comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions.

[0022] In some embodiments, the FR3 comprises an amino acid sequence of ADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYC (SEQ ID NO: 12) or a variant of any of the foregoing comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions.

[0023] In some embodiments, the FR4 comprises an amino acid sequence of WGQGTQVTVSS (SEQ ID NO: 13) or a variant of any of the foregoing comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions.

[0024] In some embodiments, the sdAb moiety comprises an amino acid sequence having at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity to an sdAb sequence selected from the group consisting of SEQ ID NOs:14, 16, and 18.

[0025] In some embodiments, the anti-TAB1 construct binds an epitope having the amino acid sequence of SAQSTSKTSVTLSLVMPS (SEQ ID NO:22) or PAAGGRVYPVSVPYS (SEQ ID NO:23).

[0026] In some embodiments, the anti-TAB1 construct binds an epitope having PAAGGRVYPVSVPYS (SEQ ID NO:23) and: the CDR1 comprises an amino acid sequence of SGDTWENTGMG (SEQ ID NON) or a variant thereof comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions, the CDR2 comprises an amino acid sequence of SDFEDMVDYY (SEQ ID NO:4) or a variant thereof comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions, and the CDR3 comprises an amino acid sequence of AMPTMQIRITSDTNSGTWKY (SEQ ID NO:7) or a variant thereof comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions; and / or the sdAb moiety comprises an amino acid sequence having at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity to an sdAb sequence of SEQ ID NO: 14; and / or the FR1 comprises an amino acid sequence of MAEVQLQASGGGFVQPGGSLRLSCAA (SEQ ID NON O) or a variant of any of the foregoing comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions, the FR2 comprises an amino acid sequence of WFRQAPGKEREFVSAI (SEQ ID NON 1) or a variant of any of the foregoing comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions, the FR3 comprises an amino acid sequence of ADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYC (SEQ ID NON2) or a variant of any of the foregoing comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions, and the FR4 comprises an amino acid sequence of WGQGTQVTVSS (SEQ ID NO:13) or a variant of any of the foregoing comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions.

[0027] In some embodiments, the anti-TAB1 construct binds an epitope having the amino acid sequence of SAQSTSKTSVTLSLVMPS (SEQ ID NO:22) and: the CDR1 comprises an amino acid sequence of SGSTSAFSNMG (SEQ ID NO:2) or a variant thereof comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions, the CDR2 comprises an amino acid sequence of SGLDDMKHYY (SEQ ID NO:5) or a variant thereof comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions, and the CDR3 comprises an amino acid sequence of AEIELDMHHLELTIHYY (SEQ ID NO:8) or a variant thereof comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions; and / or the sdAb moiety comprises an amino acid sequence having at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity to an sdAb sequence of SEQ ID NO: 16; and / or the FR1 comprises an amino acid sequence of MAEVQLQASGGGFVQPGGSLRLSCAA (SEQ ID NON O) or a variant of any of the foregoing comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions, the FR2 comprises an amino acid sequence of WFRQAPGKEREFVSAI (SEQ ID NON 1) or a variant of any of the foregoing comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions, the FR3 comprises an amino acid sequence of ADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYC (SEQ ID NO:12) or a variant of any of the foregoing comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions, and the FR4 comprises an amino acid sequence of WGQGTQVTVSS (SEQ ID NO:13) or a variant of any of the foregoing comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions.

[0028] In some embodiments, the anti-TAB1 construct binds an epitope having PAAGGRVYPVSVPYS (SEQ ID NO:23) and: the CDR1 comprises an amino acid sequence of SGTYTSEIMG (SEQ ID NO:3) or a variant thereof comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions, the CDR2 comprises an amino acid sequence of SRNFSFKTYY (SEQ ID NO:6) or a variant thereof comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions, and the CDR3 comprises an amino acid sequence of AIDLKWKGRLIMTY (SEQ ID NO:9) or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; and / or the sdAb moiety comprises an amino acid sequence having at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity to an sdAb sequence of SEQ ID NO: 18; and / or the FR1 comprises an amino acid sequence of MAEVQLQASGGGFVQPGGSLRLSCAA (SEQ ID NO: 10) or a variant of any of the foregoing comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions, the FR2 comprises an amino acid sequence of WFRQAPGKEREFVSAI (SEQ ID NON 1) or a variant of any of the foregoing comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions, the FR3 comprises an amino acid sequence of ADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYC (SEQ ID NO: 12) or a variant of any of the foregoing comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions, and the FR4 comprises an amino acid sequence of WGQGTQVTVSS (SEQ ID NO: 13) or a variant of any of the foregoing comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions.

[0029] In some embodiments, the anti-TAB1 construct inhibits binding of p38 to TAB1 .

[0030] In some embodiments, the anti-TAB1 construct inhibits p38 activation.

[0031] Another aspect of the invention is directed to a nucleic acid encoding an anti-TAB1 construct of the invention.

[0032] Another aspect of the invention is directed to a vector comprising a nucleic acid encoding an anti-TAB1 construct of the invention. In some embodiments, the vector is configured for intracellular expression of the anti- TAB1 construct. In some embodiments, the vector is configured for intracellular delivery of the nucleic acid.

[0033] Another aspect of the invention is directed to methods of intracellularly expressing the anti-TAB1 construct of the invention. In some embodiments, the methods comprise delivering a nucleic acid encoding an anti-TAB1 construct of the invention or a vector comprising the nucleic acid into a cell to express the anti-TAB1 construct within the cell.

[0034] Another aspect of the invention is directed to inhibiting p38 activation and / or signaling in a cell. The methods comprise delivering or expressing an anti-TAB1 construct of the invention in the cell in an amount effective to inhibit p38 activation and / or signaling in the cell.

[0035] Another aspect of the invention is directed to methods of treating a condition mediated by atypical p38 signaling in a subject. In some embodiments, the methods comprise administering an anti-TAB1 construct of the invention, a nucleic acid encoding an anti-TAB1 construct of the invention, or a vector comprising a nucleic acid encoding an anti-TAB 1 construct of the invention to the subject in an amount effective to treat the condition.

[0036] In some embodiments, the methods comprise delivering the anti-TAB1 construct or the vector into a cell in the subject.

[0037] In some embodiments, the condition is selected from the group consisting of myocardial ischemia, cardiovascular ischemia and reperfusion, myocardial infarction, cardiomyopathy, amyloidosis, viral infection, viral replication, bacterial infection, parasitic infection (including Toxoplasma gondii), inflammation (e.g., including but not limited to vascular inflammation, dermal inflammation, hepatic and retinal inflammation, neuronal inflammation, viral-induced inflammation, fungal-induced inflammation, bacterial-induced inflammation, and parasite-induced inflammation), cancer, cancer metastasis, cancer inflammation, vascular endothelial dysfunction, leukocyte dysfunction, myeloid cell mobilization and activation, immune system (T-Cell) modulation, neurovascular signaling, traumatic brain injury, edema, pulmonary damage, acute respiratory distress syndrome, acute lung injury, diabetes, diabetes complications (e.g., diabetes-induced neuronal dysregulation, vascular inflammation, vascular edema, hypoxic damage, stroke, and retinopathies), pregnancy complications, retinal damage (e.g., retinal vascular damage, retinal microglial dysfunction, retinal neuronal dysfunction, and photoreceptor dysregulation), retinopathy, oxygen-induced retinopathy, retinopathy of prematurity, age-related macular degeneration, glaucoma, choroidal neovascularization, rheumatoid arthritis, and hepatic damage.

[0038] In some embodiments, the condition is a viral infection.

[0039] In some embodiments, the condition is inflammation.

[0040] In some embodiments, the condition is oxygen-induced retinopathy.

[0041] The objects and advantages of the invention will appear more fully from the following detailed description of the preferred embodiment of the invention made in conjunction with the accompanying drawings.

[0042] BRIEF DESCRIPTION OF THE DRAWINGS

[0043] FIG. 1. Model TAB1-p38 interaction. A. Schematic representation of TABI domains that interact with p38. OS = canonical site, NOS = non-canonical site. B. Model showing the specific amino acid residues of the TAB1 C- terminal tail that interacts with p38.

[0044] FIG. 2. Model representing the screening process for intraTAB (IB) development. A. The synthetic humanized variable heavy domain (VHH) is expressed on bacteria infecting m13 phage. The library represents a complexity of - 3x109. B. Phage selection, using affinity isolation ofVHH expressing phage that binds to full-length human TAB1 . Phage amplified and selection repeated 3x. C. Candidate VHH domains processed through a Yeast two-hybrid screen against the critical TAB1 C-terminus. Twenty candidates interacted with bait protein, representing potential blocking intrabodies, IntraTabs.

[0045] FIG. 3. A. Western blot of biotinylated proteins fixed on streptavidin magnetic beads. Lane 1 : 682 ng of Tab1-biotin without beads; Lane 2: 682 ng of Tab1-biotin on beads (50nM for roundl); lane 3: 200 ng of His- SUMO-TAB1-Halo without beads. Predicted MW of TAB1 -biotin, His-SUMO-tag -130 kDa. B. Yeast-two-hybrid using pB27: LexA DNA-binding domain DBD vector (DBD-bait), and pP9 Gal4 activation domain (AD) vector (AD- prey). pB270 = empty pB27 vector, pP90 = empty pP9 vector. TAB1 = aa304-432 of human TAB1 cloned into pB27. Full-length SMAD and SMURF were used as positive controls. DO-2 = Selective medium without tryptophan and leucine, DO-3 = selective medium without tryptophan, leucine, and histidine. IBi1 = pB27-l Bi 1 intrabody.

[0046] FIG. 4. Intrabody screen in HeLa cells, cotransfected with PAR1 and intrabodies with C-terminal mCherry tag. A. Quantification of initial thrombin-mediated p38 activation. B. Immunoblotting cells stimulated with 10 nM Thrombin (a-Th). Control cells transfected with mCherry only with no intrabody. IBM was detected using RFP antibody. Total and phospho-p38 detected. C. NIH Imaged quantification of 3 independent repeats phosphor-p38 normalized total p38 and represented as the fold increase over the control 0 minute. Each intrabody is shown without (-) or with (+) thrombin treatment (10 nM). Error bars show standard deviation. * = P <0.05, *** = P <0.001. D. Using the samples as in B, probed for TAB1 , Total ERK1 / 2, and phosphor-ERK1 / 2.

[0047] FIG. 5. Atypical p38 activation and HCV. A. MAPK p38 activity in patient liver histology samples + / - HCV infections. B. P38 activity in immune cells isolated from healthy or HCV infected patients. C. Immunoprecipitation of TAB1 from Huh7.5 cells infected with JFH1, induces P38-TAB1 complex formation. D. SB203580 p38 chemical inhibitor, suppresses J399EM replication and egress in Huh7.5 cells. All data from Cheng et al. 2020.1

[0048] FIG. 6. Inhibition of TAB1-p38 block viral replication. A. Huh7.5 cells transfected with three concentrations of intrabody, 50 ng, 100 ng, or 250 ng respectively. After 24 hours, cells infected with HCVcc, and collected for immunoblotting after 24 hours. Cell lysates were probed for p38, TAB1 , HCV core protein, and IB-mCherry, using anti-RFP antibody. Samples compared to 24 hr treatment with P38 inhibitor SB203580. B. Quantification of HCV core protein levels normalized to total p38, displayed as a fold change relative to DMSO treated, HCV infected control (ctrl) cells. N=>2 independent repeats, + / -SD T-test analysis, * = P <0.05, ** = P <0.01 , *** = P <0.001 , **** = P <0.0001.

[0049] FIG. 7. IBI1 colocalizes with endogenous human TAB1 and can immunoisolate endogenous human TAB1 from cells. A. Representative image of HeLa cells co-transfected with pcDNA-IBi1-mCherry co-localized with endogenous TAB1 (Green, Alexa 488) fixed after 24 hours. Yellow in merge panel represents colocalized I BI1 and Tab1. Scale bar = 50 m. B. IBI1 labeled with C-terminal mCherry fusion protein expressed in HeLa cells immunoprecipitated with anti-RFP. Lane 1 = IgG control in the IBi1 expressing cells, Lane2, * = Endogenous TAB1 detected in IP eluate. Total p38 used as loading control for total lysates. FIG. 8. Oxygen Induced Retinopathy. C56BL6 wt and Tabl KI mice post-natal day 7 (P7) mother and pups placed at 75% O2, after 5 days, (P12) mice returned to 21 % O2 for 5 days (P17). A. Representative images from P17 retinal flat mount, stained with isolectin B4 (IB4 red). B. White dotted line inducts avascular region. White puncta indicate neovascular regions. C. Quantified using Image J. Avascular area and neovascular tufts, indicated as % of whole retinal area. n=12 independent mice. Bars shown are for wild type (W) or TablKI (T). One way ANOVA, * = P <0.05, *** = P <0.001.

[0050] FIGS. 9A-9D. IB1 interaction with TAB1 c-terminal tail. FIG. 9A. Schematic of human Tab1 truncation mutants Mutant 1 = M1 , Mutant 2 = M2, Mutant 3 = M3, Mutant 4 = M4. C-t = c-tail linker, NCS = noncanonical p38 binding motif, OS = Canonical p38 binding motif, Link = linker peptide, TAK1-BD = TAK1 binding domain. FIGS. 9B-9D. Representative examples of 3 independent repeats. FIG. 9B. RPF-I Bi1 co-immunoprecipitation of human TAB1. FIG. 9C. RPF-IBi2 co-immunoprecipitation of human TAB1. FIG. 9D. RPF-IBi3 coimmunoprecipitation of human TAB1 . All three intrabodies bind to human TAB1 on the canonical p38 binding motif.

[0051] DETAILED DESCRIPTION OF THE INVENTION

[0052] The present invention provides single-domain antibodies (sdAbs) specifically recognizing transforming growth factor (TGF)-beta activated kinase 1 (MAP3K7) binding protein 1 (TAB1). The anti-TAB1 constructs are capable of disrupting TAB1 and p38 interactions and treating conditions mediated by atypical p38 signaling.

[0053] Single-chain antibodies (sdAbs) are different from conventional 4-chain antibodies by having a single monomeric antibody variable domain, such as heavy chain variable domain (VHH, also abbreviated in the art as VHH), which can exhibit high affinity to an antigen without the aid of a light chain. Camelid VHHs are known as some of the smallest functional antigen-binding fragments with molecular weights of approximately 15 kD.

[0054] The anti-TAB1 constructs described herein can comprise a single-domain antibody (sdAb) moiety that specifically recognizes TAB1 (“anti-TAB1 sdAb”). In some embodiments, the anti-TAB1 construct is an anti-TAB1 sdAb.

[0055] Exemplary sdAbs include, but are not limited to, heavy chain variable domains from heavy-chain only antibodies (e.g., VHH (variable domain of the heavy chain of the heavy chain antibody) in Camelidae or VNAR (Variable domain of the shark New Antigen Receptor) in cartilaginous fish), binding molecules naturally devoid of light chains, single domains (such as VH or VL) derived from conventional 4-chain antibodies, humanized heavychain only antibodies, human single-domain antibodies produced by transgenic mice or rats expressing human heavy chain segments, and engineered domains and single domain scaffolds other than those derived from antibodies. The sdAbs may be derived from any species including, but not limited to mouse, rat, human, camel, llama, lamprey, fish, shark, goat, rabbit, and bovine. Single-domain antibodies contemplated herein also include naturally occurring single-domain antibody molecules from species other than Camelidae and sharks.

[0056] In some embodiments, the sdAb is derived from a naturally occurring single-domain antigen binding molecule known as heavy chain antibody devoid of light chains (also referred herein as “heavy chain-only antibodies”, or “HCAb”). Such single domain molecules are disclosed in WO 94 / 04678 and Hamers-Casterman, C. et al. (1993) Nature 363:446-448, for example. For clarity reasons, the variable domain derived from a heavy chain molecule naturally devoid of light chain is known herein as a VHH to distinguish it from the conventional VH of four chain immunoglobulins. Such a VHH molecule can be derived from antibodies raised in Camelidae species, for example, camel, llama, vicuna, dromedary, alpaca and guanaco. Other species besides Camelidae may produce heavy chain molecules naturally devoid of light chain, and such VHHs are within the scope of the present application.

[0057] In some embodiments, the sdAb is derived from a variable region of the immunoglobulin found in cartilaginous fish. For example, the sdAb can be derived from the immunoglobulin isotype known as Novel Antigen Receptor (NAR) found in the serum of shark. Methods of producing single domain molecules derived from a variable region of NAR (“IgNARs”) are described in WO 03 / 014161 and Streltsov (2005) Protein Sci. 14:2901- 2909.

[0058] In some embodiments, the sdAb is recombinant, CDR-grafted, humanized, camelized, de-immunized and / or in vitro generated (e.g., selected by phage display). In some embodiments, the amino acid sequence of the framework regions may be altered by “camelization" of specific amino acid residues in the framework regions. Camelization refers to the replacement or substitution of one or more amino acid residues in the amino acid sequence of a (naturally occurring) VH domain from a conventional 4-chain antibody by one or more of the amino acid residues that occur at the corresponding position(s) in a VHH domain of a heavy chain antibody. This can be performed in a manner known per se, which will be clear to the skilled person, for example on the basis of the further description herein. Such "camelizing” substitutions are preferably inserted at amino acid positions that form and / or are present at the VH-VL interface, and / or at the so-called Camelidae hallmark residues, as defined herein (see for example WO 94 / 04678, Davies and Riechmann FEES Letters 339: 285-290, 1994; Davies and Riechmann Protein Engineering 9 (6): 531-537, 1996; Riechmann J. Mol. Biol. 259: 957-969, 1996; and Riechmann and Muyldermans J. Immunol. Meth. 231 : 25-38, 1999).

[0059] In some embodiments, the sdAb is a human sdAb produced by transgenic mice or rats expressing human heavy chain segments. See, e g., US20090307787A1, U.S. Pat. No. 8,754,287, US20150289489A1 , US20100122358A1, and W02004049794.

[0060] In some embodiments, naturally occurring VHH domains against a particular antigen or target, can be obtained from (naive or immune) libraries of Camelid VHH sequences. Such methods may or may not involve screening such a library using said antigen or target, or at least one part, fragment, antigenic determinant or epitope thereof using one or more screening techniques known per se. Such libraries and techniques are for example described in WO 99 / 37681 , WO 01 / 90190, WO 03 / 025020 and WO 03 / 035694. Alternatively, improved synthetic or semi-synthetic libraries derived from (naive or immune) VHH libraries may be used, such as VHH libraries obtained from (naive or immune) VHH libraries by techniques such as random mutagenesis and / or CDR shuffling, as for example described in WO 00 / 43507.

[0061] In some embodiments, the sdAbs are generated from conventional four-chain antibodies. See, for example, EP 0 368 684, Ward et al. (Nature 1989 Oct. 12; 341 (6242): 544-6), Holt et al., Trends Biotechnol., 2003, 21 (11):484-490; WO 06 / 030220; and WO 06 / 003388.

[0062] In some embodiments, the anti-TAB1 constructs of the invention comprise a single-domain antibody (sdAb) moiety specifically recognizing TAB1. In some embodiments, the sdAb moiety of the invention comprises a CDR1 , a CDR2, and / or a CDR3. In some embodiments, the sdAb moiety comprises a CDR1 , a CDR2, and a CDR3.

[0063] In some embodiments, the CDR1 comprises an amino acid sequence of SGDTWENTGMG (SEQ ID NO: 1), SGSTSAFSNMG (SEQ ID NO:2), SGTYTSEIMG (SEQ ID NO:3), or a variant of any of the foregoing comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions.

[0064] In some embodiments, the CDR2 comprises an amino acid sequence of SDFEDMVDYY (SEQ ID NO:4), SGLDDMKHYY (SEQ ID NO:5), SRNFSFKTYY (SEQ ID NO:6), or a variant of any of the foregoing comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions.

[0065] In some embodiments, the CDR3 comprises an amino acid sequence of AMPTMQIRITSDTNSGTWKY (SEQ ID NOV), AEIELDMHHLELTIHYY (SEQ ID NO:8), AIDLKWKGRLIMTY (SEQ ID NO:9), or a variant of any of the foregoing comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions. In some embodiments, the CDR1 comprises an amino acid sequence of SGDTWENTGMG (SEQ ID NO:1) or a variant thereof comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions, the CDR2 comprises an amino acid sequence of SDFEDMVDYY (SEQ ID NO:4) or a variant thereof comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions, and the CDR3 comprises an amino acid sequence of AMPTMQIRITSDTNSGTWKY (SEQ ID NO:7) or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions.

[0066] In some embodiments, the CDRI comprises an amino acid sequence of SGSTSAFSNMG (SEQ ID NO:2) or a variant thereof comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions, the CDR2 comprises an amino acid sequence of SGLDDMKHYY (SEQ ID NO:5) or a variant thereof comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions, and the CDR3 comprises an amino acid sequence of AEIELDMHHLELTIHYY (SEQ ID NO:8) or a variant thereof comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions.

[0067] In some embodiments, the CDR1 comprises an amino acid sequence of SGTYTSEIMG (SEQ ID NO:3) or a variant thereof comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions, the CDR2 comprises an amino acid sequence of SRNFSFKTYY (SEQ ID NO:6) or a variant thereof comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions, and the CDR3 comprises an amino acid sequence of AIDLKWKGRLIMTY (SEQ ID NO:9) or a variant thereof comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions.

[0068] In some embodiments, the sdAb moiety comprises an FR1 , an FR2, an FR3, and an FR4.

[0069] In some embodiments, the FR1 comprises an amino acid sequence of MAEVQLQASGGGFVQPGGSLRLSCAA (SEQ ID NQ:10) or a variant of any of the foregoing comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions.

[0070] In some embodiments, the FR2 comprises an amino acid sequence of WFRQAPGKEREFVSAI (SEQ ID NO: 11) or a variant of any of the foregoing comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions.

[0071] In some embodiments, the FR3 comprises an amino acid sequence of ADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYC (SEQ ID NO:12) or a variant of any of the foregoing comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions.

[0072] In some embodiments, the FR4 comprises an amino acid sequence of WGQGTQVTVSS (SEQ ID NO: 13) or a variant of any of the foregoing comprising up to about 3 (such as about any of 1 , 2, or 3) amino acid substitutions.

[0073] In some embodiments, the sdAb moiety comprises an amino acid sequence having at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity to an sdAb sequence selected from the group consisting of SEQ ID NOs:14, 16, and 18.

[0074] SEQ ID NO: 14 is:

[0075] MAEVQLQASGGGFVQPGGSLRLSCAASGDTWENTGMGWFRQAPGKEREFVSAISDFEDMVDYYAD SVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCAMPTMQIRITSDTNSGTWKYWGQGTQVTVSS (SEQ ID NO: 14)

[0076] SEQ ID NO: 14 is the amino acid sequence of the IBi1 sdAb described in the following examples. The CDR1 , CDR2, and CDR3 regions of IBI1 are presented in consecutive order with underlining in SEQ ID NO: 14 above. The FR1 , FR2, FR3, and FR4 regions of IBi1 are presented in consecutive order without underlining in SEQ ID NO: 14 above.

[0077] An exemplary coding sequence of SEQ ID NO: 14 is SEQ ID NO: 15: atggcggaagtgcagctgcaggcttccgggggaggatttgtgcagccgggggggtcattgcgactgagctgcgccgcatccggagatacttgg gagaacacaggtatgggctggtttcgtcaggcccctggcaaggagagagagttcgtttccgccatctccgactttgaagacatggttgactattac gctgacagcgtaaagggaagatttacaattagccgggataactccaaaaacacggtctatctccagatgaacagcctcagggccgaggacac agctacgtattactgtgcaatgcctacaatgcagattcgtattacatcggacacaaactcgggtacatggaagtattggggacaggggacgcag gtaactgtgagtagc (SEQ ID NO:15)

[0078] SEQ ID NO: 16 is:

[0079] MAEVQLQASGGGFVQPGGSLRLSCAASGSTSAFSNMGWFRQAPGKEREFVSAISGLDDMKHYYADS VKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCAEIELDMHHLELTIHYYWGQGTQVTVSS (SEQ ID NO:16)

[0080] SEQ ID NO: 16 is the amino acid sequence of the IBI2 sdAb described in the following examples. The CDR1 , CDR2, and CDR3 regions of IBI2 are presented in consecutive order with underlining in SEQ ID NO: 16 above. The FR1 , FR2, FR3, and FR4 regions of IBI2 are presented in consecutive order without underlining in SEQ ID NO: 16 above.

[0081] An exemplary coding sequence of SEQ ID NO: 16 is SEQ ID NO: 17: atggcggaagtgcagctgcaggcttccgggggaggatttgtgcagccgggggggtcattgcgactgagctgcgccgcatccggatcaacttca gcattttccaacatgggctggtttcgtcaggcccctggcaaggagagagagttcgtttccgccatctcgggtctggacgacatgaagcattactacg ctgacagcgtaaagggaagatttacaattagccgggataactccaaaaacacggtctatctccagatgaacagcctcagggccgaggacaca gctacgtattactgtgccgagattgagctggacatgcaccacctggagctgacaattcactattattggggacaggggacgcaggtaactgtgagt age (SEQ ID NO: 17)

[0082] SEQ ID NO: 18 is:

[0083] MAEVQLQASGGGFVQPGGSLRLSCAASGTYTSEIMGWFRQAPGKEREFVSAISRNFSFKTYYADSVK

[0084] GRFTISRDNSKNTVYLQMNSLRAEDTATYYCAIDLKWKGRLIMTYWGQGTQVTVSS (SEQ ID NO:18)

[0085] SEQ ID NO: 18 is the amino acid sequence of the IBIS sdAb described in the following examples. The CDR1 , CDR2, and CDR3 regions of IBi3 are presented in consecutive order with underlining in SEQ ID NO: 18 above. The FR1 , FR2, FR3, and FR4 regions of IBi3 are presented in consecutive order without underlining in SEQ ID NO: 18 above.

[0086] An exemplary coding sequence of SEQ ID NO: 18 is SEQ ID NO: 19: atggcggaagtgcagctgcaggcttccgggggaggatttgtgcagccgggggggtcattgcgactgagctgcgccgcatccggaacatataca tcggagattatgggctggtttcgtcaggcccctggcaaggagagagagttcgtttccgccatctcccgtaacttttcgtttaagacatattacgctgac agcgtaaagggaagatttacaattagccgggataactccaaaaacacggtctatctccagatgaacagcctcagggccgaggacacagctac gtattactgtgccattgacctgaagtggaagggtcgtctgattatgacatactggggacaggggacgcaggtaactgtgagtagc (SEQ ID NO: 19)

[0087] The anti-TAB1 constructs of the invention preferably bind to a TAB1 protein, such as a human TAB1 protein having the amino acid sequence of SEQ ID NQ:20:

[0088] MAAQRRSLLQSEQQPSWTDDLPLCHLSGVGSASNRSYSADGKGTESHPPEDSWLKFRSENNCFLYG VFNGYDGNRVTNFVAQRLSAELLLGQLNAEHAEADVRRVLLQAFDVVERSFLESIDDALAEKASLQSQL PEGVPQHQLPPQYQKILERLKTLEREISGGAMAWAVLLNNKLYVANVGTNRALLCKSTVDGLQVTQLN VDHTTENEDELFRLSQLGLDAGKIKQVGIICGQESTRRIGDYKVKYGYTDIDLLSAAKSKPIIAEPEIHGA QPLDGVTGFLVLMSEGLYKALEAAHGPGQANQEIAAMIDTEFAKQTSLDAVAQAVVDRVKRIHSDTFAS GGERARFCPRHEDMTLLVRNFGYPLGEMSQPTPSPAPAAGGRVYPVSVPYSSAQSTSKTSVTLSLVM PSQGQMVNGAHSASTLDEATPTLTNQSPTLTLQSTNTHTQSSSSSSDGGLFRSRPAHSLPPGEDGRV

[0089] EPYVDFAEFYRLWSVDHGEQSWTAP (SEQ ID NO:20)

[0090] An exemplary coding sequence for SEQ ID NO:20: is SEQ ID NO:21 : ATGGCGGCGCAGAGGAGGAGCTTGCTGCAGAGTGAGCAGCAGCCAAGCTGGACAGATGACCTGC CTCTCTGCCACCTCTCTGGGGTTGGCTCAGCCTCCAACCGCAGCTACTCTGCTGATGGCAAGGGC ACTGAGAGCCACCCGCCAGAGGACAGCTGGCTCAAGTTCAGGAGTGAGAACAACTGCTTCCTGTA TGGGGTCTTCAACGGCTATGATGGCAACCGAGTGACCAACTTCGTGGCCCAGCGGCTGTCCGCA GAGCTCCTGCTGGGCCAGCTGAATGCCGAGCACGCCGAGGCCGATGTGCGGCGTGTGCTGCTG CAGGCCTTCGATGTGGTGGAGAGGAGCTTCCTGGAGTCCATTGACGACGCCTTGGCTGAGAAGG CAAGCCTCCAGTCGCAATTGCCAGAGGGAGTCCCTCAGCACCAGCTGCCTCCTCAGTATCAGAAG ATCCTTGAGAGACTCAAGACGTTAGAGAGGGAAATTTCGGGAGGGGCCATGGCCGTTGTGGCGG TCCTTCTCAACAACAAGCTCTACGTCGCCAATGTCGGTACAAACCGTGCACTTTTATGCAAATCGA CAGTGGATGGGTTGCAGGTGACACAGCTGAACGTGGACCACACCACAGAGAACGAGGATGAGCT CTTCCGTCTTTCGCAGCTGGGCTTGGATGCTGGAAAGATCAAGCAGGTGGGGATCATCTGTGGGC AGGAGAGCACCCGGCGGATCGGGGATTACAAGGTTAAATATGGCTACACGGACATTGACCTTCTC AGCGCTGCCAAGTCCAAACCAATCATCGCAGAGCCAGAAATCCATGGGGCACAGCCGCTGGATG GGGTGACGGGCTTCTTGGTGCTGATGTCGGAGGGGTTGTACAAGGCCCTAGAGGCAGCCCATGG GCCTGGGCAGGCCAACCAGGAGATTGCTGCGATGATTGACACTGAGTTTGCCAAGCAGACCTCCC TGGACGCAGTGGCCCAGGCCGTCGTGGACCGGGTGAAGCGCATCCACAGCGACACCTTCGCCAG TGGTGGGGAGCGTGCCAGGTTCTGCCCCCGGCACGAGGACATGACCCTGCTAGTGAGGAACTTT GGCTACCCGCTGGGCGAAATGAGCCAGCCCACACCGAGCCCAGCCCCAGCTGCAGGAGGACGA GTGTACCCTGTGTCTGTGCCATACTCCAGCGCCCAGAGCACCAGCAAGACCAGCGTGACCCTCTC CCTTGTCATGCCCTCCCAGGGCCAGATGGTCAACGGGGCTCACAGTGCTTCCACCCTGGACGAA GCCACCCCCACCCTCACCAACCAAAGCCCGACCTTAACCCTGCAGTCCACCAACACGCACACGCA GAGCAGCAGCTCCAGCTCTGACGGAGGCCTCTTCCGCTCCCGGCCCGCCCACTCGCTCCCGCCT GGCGAGGACGGTCGTGTTGAGCCCTATGTGGACTTTGCTGAGTTTTACCGCCTCTGGAGCGTGGA

[0091] CCATGGCGAGCAGAGCGTGGTGACAGCACCGTAG (SEQ ID N0:21)

[0092] In some embodiments, the anti-TAB1 constructs of the invention bind to an epitope having the amino acid sequence of SEQ ID NO:22:

[0093] SAQSTSKTSVTLSLVMPS (SEQ ID NO:22)

[0094] Such binding is exhibited, for example, with IBi2.

[0095] In some embodiments, the anti-TAB1 constructs of the invention bind to an epitope having the amino acid sequence of SEQ ID NO:23: PAAGGRVYPVSVPYS (SEQ ID NO:23)

[0096] Such binding is exhibited, for example, with IBi1 and IBi3.

[0097] The anti-TAB1 constructs of the invention are preferably capable of inhibiting binding of p38 to TAB1. “p38" in this context refers to any isoform of p38 (p38a, p38|3, p38y, p38b). In some versions, the anti-TAB1 constructs of the invention are capable of inhibiting binding of p38o to TAB1 .

[0098] The anti-TAB1 constructs of the invention are preferably capable of inhibiting p38 activation and / or signaling, such as when present within a cell. "p38” in this context refers to any isoform of p38 (p38a, p38p, p38y, p38b). In some versions, the anti-TAB1 constructs of the invention are capable of inhibiting p38a activation and / or signaling. “p38 activation” refers to any activation of p38, whether canonical or non-canonical (atypical), unless the context dictates otherwise. In some versions, the anti-TAB 1 constructs of the invention are capable of inhibiting non-canonical p38 activation and / or signaling, such as non-canonical p38a activation and / or signaling.

[0099] The anti-TAB1 constructs of the invention can present in the form of an intrabody that is present intracellularly within a cell. Any methods can be used to introduce the anti-TAB1 construct intracellularly within a cell. Such methods comprise delivering to a cell an anti-TAB1 construct which may be in any form used by one skilled in the art, for example, a protein, an RNA molecule which is translated, or a DNA vector which is transcribed and translated.

[0100] In instances where a nucleic acid molecule encoding an anti-TAB1 construct is used, techniques known in the art may be used for cloning of the nucleic acid molecule into an expression vector. Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), 1993, Current Protocols in Molecular Biology, John. Wiley & Sons, NY; and Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, N.Y.

[0101] The DNA encoding the an ti-TAB1 construct may be recombinantly engineered into a variety of host vector systems that also provide for replication of the DNA in large scale and contain the necessary elements for directing the transcription of the anti-TAB1 construct. The use of such a vector to transfect target cells in the patient will result in transcription of sufficient amounts of the anti-TAB 1 construct to affect a cellular process, such as atypical p38 activity. For example, a vector can be introduced in vivo such that it is taken up by a cell and directs the transcription of the anti-TAB1 construct. Such a vector can remain episomal or become chromosomally integrated, as long as it can be expressed to produce the desired anti-TAB1 construct. Such vectors can be constructed by recombinant DNA technology methods standard in the art.

[0102] Vectors encoding the anti-TAB1 construct can be plasmid, viral, or others known in the art, used for replication and expression in mammalian cells. Expression of the sequence encoding the anti-TAB1 construct can be regulated by any promoter / enhancer sequences known in the art to act in mammalian, preferably human cells. Such promoters / enhancers can be inducible or constitutive. Such promoters include but are not limited to the SV40 early promoter region (Benoist, C. and Chambon, P. 1981, Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445), the regulatory sequences of the metallothionein gene (Brinster et al., 1982, Nature 296:39-42), the viral CMV promoter, the human p-chorionic gonadotropin-6 promoter (Hollenberg et al., 1994, Mol. Cell. Endocrinology 106:111-119), etc. In one embodiment, cell type specific promoter / enhancer sequences may be used to promote the synthesis of the anti-TAB 1 construct in particular cells or tissue types.

[0103] Vectors for use in the practice of the invention include any eukaryotic expression vectors, including but not limited to viral expression vectors such as those derived from the class of retroviruses, lentiviruses, adenoviruses, or adeno-associated viruses.

[0104] Various delivery systems are known and can be used to transfer compositions of the invention into cells, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the composition, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem 262:4429-4432), construction of a nucleic acid as part of a retroviral, adenoviral, adeno-associated viral or other vector, injection of DNA, electroporation, calcium phosphate mediated transfection, etc.

[0105] In one embodiment, nucleic acids comprising a sequence encoding an anti-TAB1 construct are administered by way of gene delivery and expression into a host cell. In this embodiment of the invention, the nucleic acid mediates an effect by promoting intrabody production. Any of the methods for gene delivery into a host cell available in the art can be used according to the present invention. For general reviews of the methods of gene delivery see Strauss, M. and Barranger, J. A., 1997, Concepts in Gene Therapy, by Walter de Gruyter & Co., Berlin; Goldspiel et al., 1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 33:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62: 191-217; 1993, TIBTECH 11 (5):155-215.

[0106] In a specific embodiment, the nucleic acid encoding the anti-TAB1 construct is directly administered in vivo, under conditions effective for production of an intrabody. This can be accomplished by any of numerous methods known in the art, e.g., by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by infection using a defective or attenuated retroviral or other viral vector (see U.S. Pat. No. 4,980,286), or by direct injection of naked DNA, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, encapsulation in liposomes, microparticles, or microcapsules, or by administering it in linkage to a peptide which is known to enter the nucleus, or by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432). In a specific embodiment, a viral vector that contains sequences encoding the anti-TAB1 construct can be used. For example, a retroviral vector can be utilized that has been modified to delete retroviral sequences that are not necessary for packaging of the viral genome and integration into host cell DNA (see Miller et al., 1993, Meth. Enzymol. 217:581-599). Alternatively, adenoviral or adeno-associated viral vectors can be used for gene delivery to cells or tissues. (See, Kozarsky and Wilson, 1993, Current Opinion in Genetics and Development 3:499- 503 for a review of adenovirus-based gene delivery).

[0107] In some embodiments of the invention, an adeno-associated viral vector may be used to deliver nucleic acid molecules that encode the anti-TAB1 construct The vector is designed so that, depending on the level of expression desired, a promoter and / or enhancer element of choice may be inserted into the vector.

[0108] Another approach to gene delivery into a cell involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection. Usually, the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. The resulting recombinant cells can be delivered to a host by various methods known in the art. In a preferred embodiment, the cell used for gene delivery is autologous to the host cell.

[0109] The anti-TAB1 constructs may be fused or conjugated to a domain or sequence that has translocation activity. For example, the signal peptide of Kaposi fibroblast growth factor (Dell! Bovi, P. et al., 1987, Cell 50:729- 37) contains a hydrophobic sequence (AAVLLPVLLAAP (SEQ ID NO:24)) that functions as a cellular import signal (Shin, I. et al., 2005, Cancer Res. 65:2815-24) and can be fused at the N-terminus of the anti-TAB1 construct. Thus, intrabodies can be produced outside a target cell and then added to a cell culture or administered to a subject.

[0110] Translocation activity has also been identified in amino acids 37-72 (Fawell et al., 1994, Proc. Natl. Acad. Sci. U.S.A. 91 :664-8), amino acids 37-62 (Anderson et al., 1993, Biochem. Biophys. Rex. Commun. 194:876-84) and amino acids 49-58 (having the basic sequence RKKRRQRRR (SEQ ID NO:25)) of HIV-Tat. A longer peptide of HIV-Tat (amino acids 48-60; CGRKICRRQRRRPPQC (SEQ ID NO:26)) can be used for translocation, nuclear localization and trans-activation of cellular genes (Vives et al., 1997, J. Biol. Chem. 272:16010-7). Administration of a fusion protein containing an HIV-Tat translocation sequence and p-galactosidase resulted in delivery of active fusion protein to all tissues of a mouse (Schwarze et al., 1999, Science, 285:1569-72). A 16 amino acid basic peptide from the Drosophila antennapedia homeodomain protein (RQIIKIWFQNRRMKWKIC (SEQ ID NO:27); Derossi, et al., 1994, J. Biol. Chem. 269:10444-50) can also be used to direct intrabodies to the cytoplasm of cells (Theodore, et al., 1995, J. Neurosci. 15:715867).

[0111] It is understood that the anti-TAB1 construct of the invention, where used in a mammal for the purpose of prophylaxis or treatment, will be administered in the form of a composition additionally comprising a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. Pharmaceutically acceptable carriers can further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the binding proteins. The compositions of the injection can, as is well known in the art, be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the mammal.

[0112] The anti-TAB1 construct comprising the anti-TAB 1 sdAb moiety can be of any possible format.

[0113] In some embodiments, the anti-TAB1 construct comprising the anti-TAB1 sdAb moiety may further comprise additional polypeptide sequences, such as one or more antibody moieties, or Fc fragment of immunoglobulin.

[0114] In some embodiments, the additional polypeptide sequences may be a second antibody moiety (such as sdAb, scFv, full-length antibody). The second antibody moiety can recognize the same or a different antigen as the anti-TAB1 sdAb moiety.

[0115] In some embodiments, the additional polypeptide sequences may increase the antibody construct halflife, solubility, or absorption, reduce immunogenicity or toxicity, eliminate or attenuate undesirable side effects, and / or confer other advantageous properties to and / or reduce undesired properties of the an ti-TAB1 construct of the invention, compared to the anti-TAB1 sdAb described herein perse.

[0116] In some embodiments, anti-TAB1 sdAb moiety described herein can be linked to one or more (preferably human) CH2 and / or CH3 domains, optionally via a linker sequence, to increase its half-life in vivo.

[0117] Thus in some embodiments, the anti-TAB1 construct is an HCAb (hereinafter referred to as “anti-TAB1 HCAb”) comprising an anti-TAB1 sdAb moiety described herein fused to an Fc fragment of an immunoglobulin, such as IgA, IgD, IgE, IgG, and IgM. In some embodiments, the anti-TAB1 HCAb comprises an Fc sequence of IgG, such as any of lgG1 , lgG2, lgG3, or lgG4. In some embodiments, the Fc fragment is a human Fc. In some embodiments, the Fc fragment is a human lgG1 Fc. In some embodiments, the anti-TAB1 HCAb is monomeric. In some embodiments, the anti-TAB1 HCAb is dimeric. In some embodiments, the anti-TAB1 sdAb moiety and the Fc fragment are optionally connected by a peptide linker.

[0118] In some embodiments, the anti-TAB1 construct comprises an anti-TAB1 sdAb moiety described herein fused to one or more other antibody moiety (such as an antibody moiety that specifically recognizes TAB1 or another antigen). The one or more other antibody moiety can be of any antibody or antibody fragment format, such as a multispecific sdAb (such as bispecific sdAb), a full-length antibody, a Fab, a Fab', a (Fab')2, an Fv, a single chain Fv (scFv), an scFv-scFv, a minibody, a diabody, or a sdAb.

[0119] In some embodiments, the anti-TAB 1 construct comprises a first anti-TAB 1 sdAb moiety of the invention described herein fused to a second anti-TAB1 sdAb moiety of the invention The first and second anti-TAB1 sdAb moieties can include any anti-TAB 1 sdAb moieties described herein. The first and second anti-TAB1 sdAb moieties can be fused via a peptide linker. In some embodiments, the anti-TAB1 construct comprising an anti-TAB1 sdAb moiety and one or more other antibody moiety is monospecific. In some embodiments, the anti-TAB1 construct comprising an anti-TAB1 sdAb moiety and one or more other antibody moiety is multispecific (such as bispecific). Multispecific molecules are molecules that have binding specificities for at least two different antigens or epitopes (e.g., bispecific antibodies have binding specificities for two antigens or epitopes). Multispecific molecules with more than two valencies and / or specificities are also contemplated. For example, trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60 (1991). It is to be appreciated that one of skill in the art could select appropriate features of individual multispecific molecules described herein to combine with one another to form a multi-specific anti- TAB1 molecule of the invention.

[0120] In some embodiments, the anti-TAB1 construct is multivalent but monospecific, i.e., the anti-TAB1 construct comprises an an ti-TAB1 sdAb moiety described herein and at least a second antibody moiety specifically recognizing the same epitope as the anti-TAB1 sdAb moiety. In some embodiments, the one or more antibody moiety specifically recognizing the same epitope as the anti-TAB1 sdAb moiety described herein may comprise the same CDRs and / or the same VHH amino acid sequence as the anti-TAB 1 sdAb moiety. For example, the anti- TAB1 construct may comprise two or more anti-TAB1 sdAb moieties described herein, wherein the two or more anti-TAB1 sdAb moieties are the same. In some embodiments, the anti-TAB1 sdAb moieties are optionally connected by peptide linker(s).

[0121] In some embodiments, the anti-TAB1 construct is multivalent and multispecific, i.e., the anti-TAB1 construct comprises an an ti-TAB1 sdAb moiety described herein and at least a second antibody moiety specifically recognizing a second antigen other than TAB1 , or a different TAB1 epitope than that recognized by the anti-TAB1 sdAb moiety. In some embodiments, the second antibody moiety is a sdAb.

[0122] In some embodiments, the two or more antibody moieties within the anti-TAB1 construct can be optionally connected by a peptide linker. The length, the degree of flexibility and / or other properties of the peptide linker(s) used in the anti-TAB1 construct may have some influence on properties, including but not limited to the affinity, specificity or avidity for one or more particular antigens or epitopes. For example, longer peptide linkers may be selected to ensure that two adjacent domains do not sterically interfere with one another In some embodiment, a peptide linker comprises flexible residues (such as glycine and serine) so that the adjacent domains are free to move relative to each other. For example, a glycine-serine doublet can be a suitable peptide linker.

[0123] The peptide linker can be of any suitable length. In some embodiments, the peptide linker is at least about any of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100 or more amino acids long. In some embodiments, the peptide linker is no more than about any of 100, 75, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 , 10, 9, 8, 7, 6, 5 or fewer amino acids long. In some embodiments, the length of the peptide linker is any of about 1 amino acid to about 10 amino acids, about 1 amino acid to about 20 amino acids, about 1 amino acid to about 30 amino acids, about 5 amino acids to about 15 amino acids, about 10 amino acids to about 25 amino acids, about 5 amino acids to about 30 amino acids, about 10 amino acids to about 30 amino acids long, about 30 amino acids to about 50 amino acids, about 50 amino acids to about 100 amino acids, or about 1 amino acid to about 100 amino acids.

[0124] The peptide linker may have a naturally occurring sequence, or a non-naturally occurring sequence. For example, a sequence derived from the hinge region of heavy chain only antibodies may be used as the linker. See, for example, WO1996 / 34103. In some embodiments, the peptide linker is a mutated human lgG1 hinge (see SEQ ID NO:445 of US 11 ,673,954). In some embodiments, the peptide linker is a flexible linker. Exemplary flexible linkers include glycine or polymers thereof, glycine-serine polymers (including, for example, GS, GSGGS (SEQ ID NO:28), GGGS (SEQ ID NO:29), and GGGGS (SEQ ID NQ:30), or any number of contiguous repeats of any of the foregoing, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more contiguous repeats, and / or, optionally, up to 10, 15, 20, 25, 30, 35, or more contiguous repeats), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Exemplary peptide linkers can include peptide sequences such as GGGGSGGGS (SEQ ID NO:31 ), GGGGSGGGGSGGGGS (SEQ ID NO:32), GPGGP (SEQ ID NO:33), AALVGPGGQGGGGSGGGGSGGGGSGGGGSGGGGSMA (SEQ ID NO:34), EPKSSDKTHTSPPSP (SEQ ID NO:35), and GPGGQGTGPGGS (SEQ ID NO:36). Other suitable peptide linkers are provided in Klein JS, Jiang S, Galimidi RP, Keeffe JR, Bjorkman PJ. Design and characterization of structured protein linkers with differing flexibilities. Protein Eng Des Sei. 2014 Oct;27(10):325-30.

[0125] I n some embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleic acid sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into and / or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding.

[0126] In some embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the HVRs and FRs Conservative substitutions are shown in Table 1 under the heading of "Preferred substitutions.” More substantial changes are provided in Table 1 under the heading of “exemplary substitutions,” and as further described below in reference to amino acid side chain classes. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity. Table 1. Amino acid substitutions.

[0127] Original Residue Exemplary Substitutions Preferred Substitutions

[0128] Ala (A) Vai; Leu; lie Vai

[0129] Arg (R) Lys; Gin; Asn Lys

[0130] Asn (N) Gin; His; Asp, Lys; Arg Gin

[0131] Asp (D) Glu; Asn Glu

[0132] Cys (C) Ser; Ala Ser

[0133] Gin (Q) Asn; Glu Asn

[0134] Glu (E) Asp; Gin Asp

[0135] Gly (G) Ala Ala

[0136] His (H) Asn; Gin; Lys; Arg Arg

[0137] He (I) Leu; Vai; Met; Ala; Phe; Norleucine Leu

[0138] Leu (L) Norleucine; lie; Vai; Met; Ala; Phe lie

[0139] Lys (K) Arg; Gin; Asn Arg

[0140] Met (M) Leu; Phe; lie Leu

[0141] Phe (F) Trp; Leu; Vai; lie; Ala; Tyr Tyr

[0142] Pro (P) Ala Ala

[0143] Ser (S) Thr Thr

[0144] Thr (T) Vai; Ser Ser

[0145] Trp (W) Tyr; Phe Tyr

[0146] Tyr (Y) Trp; Phe; Thr; Ser Phe

[0147] Vai (V) lie; Leu; Met; Phe; Ala; Norleucine Leu

[0148] Amino acids may be grouped according to common side-chain properties:

[0149] (1) Hydrophobic: Norleucine, Met, Ala, Vai, Leu, lie; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

[0150] (3) Acidic: Asp, Glu;

[0151] (4) Basic: His, Lys, Arg;

[0152] (5) Residues that influence chain orientation: Gly, Pro;

[0153] (6) Aromatic: Trp, Tyr, Phe.

[0154] Non-conservative substitutionswill entail exchanging a member of one of these classes for another class. One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and / or will have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g. binding affinity).

[0155] Alterations (e.g., substitutions) may be made in HVRs, e.g., to improve antibody affinity. Such alterations may be made in HVR “hotspots,” i.e. , residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or SDRs (a- CDRs), with the resulting variant VH or VL being tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., (2001)) In some embodiments of affinity maturation, diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PGR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method to introduce diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.

[0156] In some embodiments, substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such alterations may be outside of HVR “hotspots” or CDRs. In some embodiments of the variant VHH sequences provided above, each HVR either is unaltered, or contains no more than one, two or three amino acid substitutions.

[0157] A useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with antigen is affected. Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions. Alternatively, or additionally, a crystal structure of an antigen-antibody complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.

[0158] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody.

[0159] In some embodiments, an anti-TAB1 construct provided herein may be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available. The moieties suitable for derivatization of the antibody include but are not limited to water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol / propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1 , 3- dioxolane, poly-1 , 3, 6-trioxane, ethylene / maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide / ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer are attached, they can be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.

[0160] Binding specificity of the antibody or antigen-binding domain can be determined experimentally by methods known in the art. Such methods comprise, but are not limited to Western blots, ELISA-, RIA-, ECL-, IRMA-, EIA-, BIAcore-tests and peptide scans.

[0161] In some embodiments, the KdOf the binding between the anti-TAB1 sdAb moiety and TAB1 is about 10-5to about 10-6M, about 10-6M to about 10-7M, about 10-7M to about 10-8M, about 10-8M to about 10-9M, about 10-9M to about 10-10M, about 10_,0M to about 10-11M, about 10-11M to about 10-12M, about 10-5M to about 10-12M, about 10-6M to about 10-12M, about 10-7M to about 10-12M, about 10-8M to about 10-12M, about 10-9M to about 10-12M, about 10-10M to about 10-12M, about 10-5M to about 10-11M, about 10-7M to about 10-11M, about 10-8M to about 10-11M, about 10-9M to about 10-11M, about 10-5M to about 10-10M, about 10-7M to about 10“10M, about 10-8M to about 10-10M, about 10-5M to about 10-9M, about 10-7M to about 10-9M, about 10-5M to about 10-8M , or about 10-6M to about 10-8M . In some embodiments, the Konof the binding between the anti-TAB1 sdAb moiety and TAB1 is about 102M"1s"1to about 104M"1s"1, about 104M"1s-1to about 106M’1s-1, about 106M-1s"1to about 107M’1s"1, about 102M"1s-1to about 107M"1s’1, about 103M’1s-1to about 107M-1s-1, about 104M’1s"1to about 107M’1s"1, about 105M"1s-1to about 107’1s"1, about 103M’1s-1to about 106M“1s_1, or about 104M-1s"1to about 106M-1s’1.

[0162] In some embodiments, the KOffOf the binding between the anti-TAB1 sdAb moiety and TAB1 is about 1 s-1to about 10-2s-1, about 10-2s-1to about 10-4s-1, about 10-4s-1to about 10-5s-1, about 10-6s-1to about 10-6s-1, about 1 s-1to about 10-6s’1, about 10-2s-1to about 10’6s-1, about 10-3s-1to about 10-6s-1, about 10’4s-1to about 10-6s'1, about 10’2s"1to about 10"5s-1, or about 10"3s-1to about 10-5s'1.

[0163] In some embodiments, the anti-TAB1 construct provided herein is a chimeric antibody. Certain chimeric antibodies are described, e.g., in U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81 :6851- 6855 (1984)). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a camelid species, such as llama) and a human constant region. In a further example, a chimeric antibody is a “class switched” antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0164] In some embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non- human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences or are modified to have residues from human antibody sequences. A humanized antibody optionally will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0165] Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are further described, e.g., in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'IAcad. Sci. USA 86: 10029-10033 (1989); U.S. Pat. Nos. 5,821 ,337, 7,527,791 , 6,982,321 , and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing SDR (a-CDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing “resurfacing”); Dall’Acqua et al., Methods 36:43-60 (2005) (describing “FR shuffling”); Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing the “guided selection" approach to FR shuffling); Vincke et al. J Biol Chem. 284(5): 3273-3284 (2009); and Sulea, T. Humanization of Camelid Single-Domain Antibodies. Methods Mol. Biol. 2022, 2446, 299-312.

[0166] Human framework regions that may be used for humanization include but are not limited to: framework regions selected using the “best-fit” method (see, e.g., Sims et al. J. Immunol. 151 :2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151 :2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271 :22611-22618 (1996)).

[0167] In some embodiments, the sdAbs are modified, such as humanized, without diminishing the native affinity of the domain for antigen and while reducing its immunogenicity with respect to a heterologous species. For example, the amino acid residues of the antibody variable domain (VHH) of a llama antibody can be determined, and one or more of the Camelid amino acids, for example, in the framework regions, are replaced by their human counterpart as found in the human consensus sequence, without that polypeptide losing its typical character, i.e. the humanization does not significantly affect the antigen binding capacity of the resulting polypeptide. Humanization of Camelid single-domain antibodies can be obtained by the introduction and mutagenesis of a limited amount of amino acids in a single polypeptide chain. This is in contrast to humanization of scFv, Fab', (Fab')2 and IgG, which requires the introduction of amino acid changes in two chains, the light and the heavy chain and the preservation of the assembly of both chains.

[0168] Single-domain antibodies comprising a VHH domain can be humanized to have human-like sequences. In some embodiments, the FR regions of the VHH domain used herein comprise at least about any one of 50%, 60%, 70%, 80%, 90%, 95% or more of amino acid sequence homology to human VH framework regions. One exemplary class of humanized VHH domains is characterized in that the VHHs carry an amino acid from the group consisting of glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tyrosine, tryptophan, methionine, serine, threonine, asparagine, or glutamine at position 45, such as, for example, L45 and a tryptophan at position 103, according to the Kabat numbering. As such, polypeptides belonging to this class show a high amino acid sequence homology to human VH framework regions and said polypeptides might be administered to a human directly without expectation of an unwanted immune response therefrom, and without the burden of further humanization.

[0169] Another exemplary class of humanized Camelid single-domain antibodies has been described in WO 03 / 035694 and contains hydrophobic FR2 residues typically found in conventional antibodies of human origin or from other species, but compensating this loss in hydrophilicity by the charged arginine residue on position 103 that substitutes the conserved tryptophan residue present in Vnfrom double-chain antibodies. As such, peptides belonging to these two classes show a high amino acid sequence homology to human VH framework regions, and said peptides might be administered to a human directly without expectation of an unwanted immune response therefrom and without the burden of further humanization. Further provided by the present application are pharmaceutical compositions comprising any one of the anti-TAB1 constructs comprising an sdAb specifically recognizing TAB1 as described herein or a nucleic acid configured to express the anti-TAB1 construct.

[0170] The pharmaceutical compositions can optionally include a pharmaceutically acceptable carrier. Pharmaceutical compositions can be prepared by mixing an anti-TAB1 construct or nucleic acid described herein having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions.

[0171] Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers, antioxidants including ascorbic acid, methionine, Vitamin E, sodium metabisulfite; preservatives, isotonicifiers (e.g. sodium chloride), stabilizers, metal complexes (e.g. Zn-protein complexes); chelating agents such as EDTA and / or non-ionic surfactants.

[0172] Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™ or polyethylene glycol (PEG).

[0173] Buffers are used to control the pH in a range which optimizes the therapeutic effectiveness, especially if stability is pH dependent. Buffers are preferably present at concentrations ranging from about 50 mM to about 250 mM. Suitable buffering agents for use in the present application include both organic and inorganic acids and salts thereof. For example, citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate. Additionally, buffers may comprise histidine and trimethylamine salts such as Tris.

[0174] Preservatives are added to retard microbial growth, and are typically present in a range from 0.2%-1 .0% (w / v). The addition of a preservative may, for example, facilitate the production of a multi-use (multiple-dose) formulation. Suitable preservatives for use in the present application include octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium halides (e.g., chloride, bromide, iodide), benzethonium chloride; thimerosal, phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol, 3-pentanol, and m-cresol. Tonicity agents, sometimes known as "stabilizers” are present to adjust or maintain the tonicity of liquid in a composition. When used with large, charged biomolecules such as proteins and antibodies, they are often termed "stabilizers” because they can interact with the charged groups of the amino acid side chains, thereby lessening the potential for inter and intra-molecular interactions. Tonicity agents can be present in any amount between 0.1 % to 25% by weight, preferably 1 % to 5%, taking into account the relative amounts of the other ingredients. Preferred tonicity agents include polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.

[0175] Additional excipients include agents which can serve as one or more of the following: (1) bulking agents, (2) solubility enhancers, (3) stabilizers and (4) and agents preventing denaturation or adherence to the container wall. Such excipients include: polyhydric sugar alcohols (enumerated above); amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclitols (e.g., inositol), polyethylene glycol; sulfur containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, a-monothioglycerol and sodium thio sulfate; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin or other immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides (e.g., xylose, mannose, fructose, glucose; disaccharides (e.g., lactose, maltose, sucrose); trisaccharides such as raffinose; and polysaccharides such as dextrin or dextran.

[0176] Non-ionic surfactants or detergents (also known as “wetting agents”) are present to help solubilize the therapeutic agent as well as to protect the therapeutic protein against agitation-induced aggregation, which also permits the formulation to be exposed to shear surface stress without causing denaturation of the active therapeutic protein or antibody. Non-ionic surfactants are present in a range of about 0.05 mg / ml to about 1 .0 mg / ml, preferably about 0.07 mg / ml to about 0.2 mg / ml.

[0177] Suitable non-ionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), polyoxamers (184, 188, etc.), PLURONIC® polyols, TRITON®, polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.), lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, sucrose fatty acid ester, methyl celluose and carboxymethyl cellulose. Anionic detergents that can be used include sodium lauryl sulfate, dioctyle sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

[0178] In order for the pharmaceutical compositions to be used for in vivo administration, they should be sterile. The pharmaceutical composition may be rendered sterile by filtration through sterile filtration membranes. The pharmaceutical compositions herein generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle. The route of administration is in accordance with known and accepted methods, such as by single or multiple bolus or infusion over a long period of time in a suitable manner, e.g., injection or infusion by subcutaneous, intravenous, intraperitoneal, intramuscular, intra-arterial, intralesional or intraarticular routes, topical administration, inhalation or by sustained release or extended-release means. In some embodiments, the pharmaceutical composition is administered locally.

[0179] The anti-TAB1 constructs, nucleic acids, vectors, or pharmaceutical compositions of the invention (the anti-TAB1 constructs, nucleic acids, vectors, and pharmaceutical compositions collectively being referred to herein as "anti-TAB1 agents”) can be used to treat a variety of conditions. The conditions preferably include conditions mediated by atypical p38 signaling. The methods can comprise administering the anti-TAB1 agents in an amount effective to treat the condition. Such methods preferably comprise delivering the anti-TAB1 construct, the nucleic acid, or the vector into a cell in the subject to thereby provide the anti-TAB 1 construct within the cell of the subject.

[0180] Conditions mediated by atypical p38 signaling are well-known in the art. See, e.g., Burton et al. 2021 (Burton JC, Antoniades W, Okalova J, Roos MM, Grimsey NJ. Atypical p38 Signaling, Activation, and Implications for Disease. Int J Mol Sci. 2021 Apr 17;22(8):4183), Cheng et al. 2020 (Cheng Y, Sun F, Wang L, Gao M, Xie Y, Sun Y, Liu H, Yuan Y, Yi W, Huang Z, Yan H, Peng K, Wu Y, Cao Z. Virus-induced p38 MAPK activation facilitates viral infection. Theranostics. 2020 Oct 30;10(26):12223-12240), Howe et al. 2022 (Howe AYM, Rodrigo C, Cunningham EB, Douglas MW, Dietz J, Grebely J, Popping S, Sfalcin JA, Parczewski M, Sarrazin C, de Salazar A, Fuentes A, Sayan M, Quer J, Kjellin M, Kileng H, Mor O, Lennerstrand J, Fourati S, Di Maio VC, Chulanov V, Pawlotsky JM, Harrigan PR, Ceccherini-Silberstein F, Garcia F; SHARED Collaborators. Characteristics of hepatitis C virus resistance in an international cohort after a decade of direct-acting antivirals. JHEP Rep. 2022 Feb 24; 4(5): 100462), Tanno et al. 2003 (Tanno M, Bass! R, Gorog DA, Saurin AT, Jiang J, Heads RJ, Martin JL, Davis RJ, Flavell RA, Marber MS. Diverse mechanisms of myocardial p38 mitogen-activated protein kinase activation: evidence for MKK-independent activation by a TAB1-associated mechanism contributing to injury during myocardial ischemia. Circ Res. 2003 Aug 8;93(3):254-61), Li et al. 2005 (Li J, Miller EJ, Ninomiya-Tsuji J, Russell RR 3rd, Young LH. AMP-activated protein kinase activates p38 mitogen-activated protein kinase by increasing recruitment of p38 MAPK to TAB1 in the ischemic heart Circ Res. 2005 Oct 28;97(9) :872-9), Fiedler et al. 2006 (Fiedler B, Feil R, Hofmann F, Willenbockel C, Drexler H, Smolensk! A, Lohmann SM, Wollert KC. cGMP- dependent protein kinase type I inhibits TAB1-p38 mitogen-activated protein kinase apoptosis signaling in cardiac myocytes. J Biol Chem. 2006 Oct 27;281 (43):32831 -40), Wang et al. 2013 (Wang Q, Feng J, Wang J, Zhang X, Zhang D, Zhu T, Wang W, Wang X, Jin J, Cao J, Li X, Peng H, Li Y, Shen B, Zhang J. Disruption of TAB1 / p38o interaction using a cell-permeable peptide limits myocardial ischemia / reperfusion injury. Mol Ther. 2013 Sep;21 (9): 1668-77), De Nicola et al 2018 (De Nicola GF, Bass! R, Nichols C, Fernandez-Caggiano M, Golforoush PA, Thapa D, Anderson R, Martin ED, Verma S, Kleinjung J, Laing A, Hutchinson JP, Eaton P, Clark J, Marber MS. The TAB1 -p38a complex aggravates myocardial injury and can be targeted by small molecules. JCI Insight. 2018 Aug 23;3(16):e121144), Lu et al. 2006 (Lu G, Kang YJ, Han J, Herschman HR, Stefani E, Wang Y. TAB-1 modulates intracellular localization of p38 MAP kinase and downstream signaling. J Biol Chem. 2006 Mar 3;281 (9):6087-95), and De Nicola et al. 2013 (DeNicola GF, Martin ED, Chaikuad A, Bass! R, Clark J, Martino L, Verma S, Sicard P, Tata R, Atkinson RA, Knapp S, Conte MR, Marber MS. Mechanism and consequence of the autoactivation of p38a mitogen-activated protein kinase promoted by TAB1. Nat Struct Mol Biol. 2013 Cct;20(10): 1182-90), among others.

[0181] In some embodiments, the condition comprises one or more of myocardial ischemia, cardiovascular ischemia and reperfusion, myocardial infarction, cardiomyopathy, amyloidosis, viral infection, viral replication, bacterial infection, parasitic infection (including Toxoplasma gondii), inflammation (e.g., including but not limited to vascular inflammation, dermal inflammation, hepatic and retinal inflammation, neuronal inflammation, viral-induced inflammation, fungal-induced inflammation, bacterial-induced inflammation, and parasite-induced inflammation), cancer, cancer metastasis, cancer inflammation, vascular endothelial dysfunction, leukocyte dysfunction, myeloid cell mobilization and activation, immune system (T-Cell) modulation, neurovascular signaling, traumatic brain injury, edema, pulmonary damage, acute respiratory distress syndrome, acute lung injury, diabetes, diabetes complications (e.g., diabetes-induced neuronal dysregulation, vascular inflammation, vascular edema, hypoxic damage, stroke, and retinopathies), pregnancy complications, retinal damage (e.g., retinal vascular damage, retinal microglial dysfunction, retinal neuronal dysfunction, and photoreceptor dysregulation), retinopathy, oxygen- induced retinopathy, retinopathy of prematurity, age-related macular degeneration, glaucoma, choroidal neovascularization, rheumatoid arthritis, and hepatic damage.

[0182] In some embodiments, the condition is a viral infection. The viral infection can be an infection of a hepatitis virus (hepatitis A virus, hepatitis B virus, hepatitis C virus), coronavirus (e.g., SARS-CoV-2), influenza virus, herpes simplex virus, varicella-zoster virus, paramyxovirus, human papillomavirus (HPV), measles virus, respiratory syncytial virus, rubella virus, human immunodeficiency virus, rotavirus, norovirus, ebola virus, Junin virus, and / or Zika virus, among others.

[0183] Dosages and desired concentrations of the anti-TAB1 agents may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration is well within the skill of an ordinary artisan. Animal experiments provide reliable guidance for the determination of effective doses for human therapy.

[0184] The anti-TAB1 agents can be administered in accord with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intravenous (i.v.), intra-articular, intrasy novi al, intrathecal, oral, topical, or inhalation routes. A reconstituted formulation can be prepared by dissolving a lyophilized anti-TAB1 construct described herein in a diluent such that the protein is dispersed throughout. Exemplary pharmaceutically acceptable (safe and non-toxic for administration to a human) diluents suitable for use in the present application include, but are not limited to, sterile water, bacteriostatic water for injection (BWFI ), a pH buffered solution (e.g. phosphate- buffered saline), sterile saline solution, Ringer's solution or dextrose solution, or aqueous solutions of salts and / or buffers.

[0185] In some embodiments, the anti-TAB1 agents can be administered to the individual by subcutaneous (i.e. beneath the skin) administration. For such purposes, the anti-TAB1 agents may be injected using a syringe. However, other devices for administration of the anti-TAB1 agents are available such as injection devices; injector pens; auto-injector devices, needleless devices; and subcutaneous patch delivery systems.

[0186] In some embodiments, the anti-TAB 1 agents can be administered to the individual intravenously. In some embodiments, the anti-TAB1 agent is administered to an individual by infusion, such as intravenous infusion. Infusion techniques for immunotherapy are known in the art (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676 (1988)).

[0187] The individual can be an animal, such as a mammal or a human.

[0188] As used herein, “treatment’’ or “treating" is an approach for obtaining beneficial or desired results, including clinical results. For purposes of this invention, beneficial or desired clinical results include but are not limited to one or more of the following: alleviating one or more symptoms resulting from a condition, diminishing the extent of a condition, stabilizing a condition (e.g., avoiding or delaying the worsening of the disease), delaying or slowing the progression of a condition, ameliorating a condition state, decreasing the dose of one or more other medications required to treat the condition, increasing the quality of life, and / or prolonging survival. Also encompassed by “treatment" is a reduction of a pathological consequence of a condition. The methods of the invention contemplate any one or more of these aspects of treatment.

[0189] The term “therapeutically effective amount” used herein refers to an amount of an agent, a combination of agents, or a pharmaceutical composition comprising such agents sufficient to treat a specified disorder, condition, or disease, such as to ameliorate, palliate, lessen, and / or delay one or more of its symptoms.

[0190] The term “antibody” or “antibody moiety” is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multi specific antibodies (e.g., bispecific antibodies), full-length antibodies and antigen-binding fragments thereof, so long as they exhibit the desired antigen-binding activity.

[0191] The term “heavy chain-only antibody” or “HCAb" refers to a functional antibody, which comprises heavy chains, but lacks the light chains usually found in 4-chain antibodies. Camelid animals (such as camels, llamas, or alpacas) are known to produce HCAbs.

[0192] The term “single-domain antibody” or“sdAb” refers to a single antigen-binding polypeptide having three complementary determining regions (CDRs). The sdAb alone is capable of binding to the antigen without pairing with a corresponding CDR-containing polypeptide. In some cases, single-domain antibodies are engineered from camelid HCAbs, and their heavy chain variable domains are referred herein as “VHHs” (variable domain of the heavy chain of the heavy chain antibody). Some VHHs can also be known as nanobodies. Camelid sdAb is one of the smallest known antigen-binding antibody fragments (see, e.g., Hamers-Casterman et al., Nature 363:446-8 (1993); Greenberg et al., Nature 374: 168-73 (1995); Hassanzadeh-Ghassabeh et al., Nanomedicine (Lond), 8: 1013-26 (2013)). A basic VHH has the following structure from the N-terminus to the C-terminus: FR1-CDR1- FR2-CDR2-FR3-CDR3-FR4, in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3.

[0193] In some embodiments, the anti-TAB1 constructs or nucleic acids of the invention are recombinant. “Recombinant” used herein with respect to an amino acid or nucleic acid construct refers to an amino acid or nucleic acid construct having a non-natural amino acid or nucleic acid sequence, respectively. Any anti-TAB1 construct of the invention or nucleic acid encoding same can be recombinant.

[0194] In some embodiments, the anti-TAB1 constructs are isolated. An “isolated” antibody (or construct) is one that has been identified, separated and / or recovered from a component of its production environment (e.g., natural or recombinant). Preferably, the isolated polypeptide is free of association with all other components from its production environment. Contaminant components of its production environment, such as that resulting from recombinant transfected cells, are materials that would typically interfere with research, diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide will be purified: (1) to greater than 95% by weight of antibody as determined by, for example, the Lowry method, and in some embodiments, to greater than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie Blue or, preferably, silver stain.

[0195] The “variable region” or “variable domain” of an antibody refers to the amino-terminal domains of the heavy or light chain of the antibody. The variable domains of the heavy chain and light chain may be referred to as “VH” and “VL”, respectively. These domains are generally the most variable parts of the antibody (relative to other antibodies of the same class) and contain the antigen binding sites. Heavy-chain only antibodies from the Camelid species have a single heavy chain variable region, which is referred to as “VHH”. VHH is thus a special type of VH.

[0196] The term “variable” refers to the fact that certain segments of the variable domains differ extensively in sequence among antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the entire span of the variable domains. Instead, it is concentrated in three segments called complementary determining regions (CDRs) or hypervariable regions (HVRs) both in the light-chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat et al., Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in the binding of antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

[0197] “Humanized” forms of non-human (e.g., llama or camelid) antibodies are antibodies that contain minimal sequence derived from non-human immunoglobulin. In some embodiments, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from an CDR (hereinafter defined) of the recipient are replaced by residues from an CDR of a non-human species (donor antibody) such as mouse, rat, rabbit, camel, llama, alpaca, or non-human primate having the desired specificity, affinity, and / or capacity. In some instances, framework (“FR”) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications can be made to further refine antibody performance, such as binding affinity. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin sequence, and all or substantially all of the FR regions are those of a human immunoglobulin sequence, although the FR regions may include one or more individual FR residue substitutions that improve antibody performance, such as binding affinity, isomerization, immunogenicity, etc. The number of these amino acid substitutions in the FR is typically no more than 6 in the H chain, and in the L chain, no more than 3. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, e.g., Jones et al., Nature 321 :522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, for example, Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1 :105-115 (1998); Harris, Biochem. Soc. Transactions 23: 1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and 7,087,409.

[0198] The term “hypervariable region,” “HVR,” or “HV," when used herein refers to the regions of an antibody variable domain which are hypervariable in sequence and / or form structurally defined loops. Generally, singledomain antibodies comprise three HVRs (or CDRs): HVR1 (or CDR1), HVR2 (or CDR2), and HVR3 (or CDR3). HVR3 (or CDR3) displays the most diversity of the three HVRs, and is believed to play a unique role in conferring fine specificity to antibodies. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996). The term "Complementarity Determining Region” or “CDR” is used to refer to hypervariable regions as defined by the Kabat system. See Kabat et al , Sequences of Proteins of Immunological Interest, 5th Ed Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

[0199] A number of HVR delineations are in use and are encompassed herein. The Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia refers instead to the location of the structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The AbM HVRs represent a compromise between the Kabat HVRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software. The "contact” HVRs are based on an analysis of the available complex crystal structures. The residues from each of these HVRs are noted below in Table 2.

[0200] Table 2. HVR delineations.

[0201] Loop Kabat AbM Chothia Contact

[0202] L1 L24-L34 L24-L34 L26-L32 L30-L36

[0203] L2 L50-L56 L50-L56 L50-L52 L46-L55

[0204] L3 L89-L97 L89-L97 L91-L96 L89-L96

[0205] H1 H31-H35B H26-H35B H26-H32 H30-H35B

[0206] (Kabat Numbering)

[0207] H1 H31-H35 H26-H35 H26-H32 H30-H35

[0208] (Chothia Numbering)

[0209] H2 H50-H65 H50-H58 H53-H55 H47-H58

[0210] H3 H95-H102 H95-H102 H96-H101 H93-H101

[0211] HVRs may comprise “extended HVRs” as follows: 24-36 or 24-34 (L1 ), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in the VLand 26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in the VH. The variable domain residues are numbered according to Kabat et al., supra, for each of these definitions.

[0212] The amino acid residues of a single-domain antibody (such as VHH) can be numbered according to the general numbering for VH domains given by Kabat et al. ("Sequence of proteins of immunological interest”, US Public Health Services, NIH Bethesda, Md., Publication No. 91), as applied to VHH domains from Camelids in the article of Riechmann and Muyldermans, J. Immunol. Methods 2000 Jun. 23; 240 (1-2): 185-195. According to this numbering, FR1 of a VHH comprises the amino acid residues at positions 1-30, CDR1 of a VHH comprises the amino acid residues at positions 31-35, FR2 of a VHH comprises the amino acids at positions 36-49, CDR2 of a VHH comprises the amino acid residues at positions 50-65, FR3 of a VHH comprises the amino acid residues at positions 66-94, CDR3 of a VHH comprises the amino acid residues at positions 95-102, and FR4 of a VHH comprises the amino acid residues at positions 103-113. In this respect, it should be noted that— as is well known in the art for VH domains and for VHH domains— the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (that is, one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering).

[0213] The expression "variable-domain residue-numbering as in Kabat” or "amino-acid-position numbering as in Kabat,” and variations thereof, refers to the numbering system used for heavy-chain variable domains or lightchain variable domains of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or HVR of the variable domain. For example, a heavy-chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat) after heavy-chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence.

[0214] “Framework” or “FR” residues are those variable-domain residues other than the HVR residues as herein defined.

[0215] A “human consensus framework” or “acceptor human framework” is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin V_or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, 5thEd. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Examples include for the VL, the subgroup may be subgroup kappa I, kappa II, kappa III or kappa IV as in Kabat et al., supra. Additionally, for the VH, the subgroup may be subgroup I, subgroup II, or subgroup III as in Kabat et al. Alternatively, a human consensus framework can be derived from the above in which particular residues, such as when a human framework residue is selected based on its homology to the donor framework by aligning the donor framework sequence with a collection of various human framework sequences. An acceptor human framework “derived from” a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain pre-existing amino acid sequence changes. In some embodiments, the number of pre-existing amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less.

[0216] As used herein, the term “specifically binds,” “specifically recognizes,” or is “specific for” refers to measurable and reproducible interactions such as binding between a target and an antigen binding protein (such as a sdAb), which is determinative of the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules. For example, an antigen binding protein (such as a sdAb) that specifically binds a target (which can be an epitope) is an antigen binding protein (such as a sdAb) that binds this target with greater affinity, avidity, more readily, and / or with greater duration than it binds other targets. In some embodiments, the extent of binding of an antigen binding protein (such as a sdAb) to an unrelated target is less than about 10% of the binding of the antigen binding protein (such as sdAb) to the target as measured, e.g., by a radioimmunoassay (RIA). In some embodiments, an antigen binding protein (such as a sdAb) that specifically binds a target has a dissociation constant (Kd) of <10-5M, <10-6M, <10-7M, <10-8M, <10-9M, <10-10M, <10-11M, or <1CH2M. In some embodiments, an antigen binding protein specifically binds an epitope on a protein that is conserved among the protein from different species. In some embodiments, specific binding can include, but does not require exclusive binding.

[0217] The term “specificity” refers to selective recognition of an antigen binding protein (such as a sdAb) for a particular epitope of an antigen. Natural antibodies, for example, are monospecific. The term “multispecific” as used herein denotes that an antigen binding protein has polyepitopic specificity (i.e , is capable of specifically binding to two, three, or more, different epitopes on one biological molecule or is capable of specifically binding to epitopes on two, three, or more, different biological molecules). " Bispecific” as used herein denotes that an antigen binding protein has two different antigen-binding specificities. The term “monospecific” as used herein denotes an antigen binding protein (such as a sdAb) that has one or more binding sites each of which bind the same epitope of the same antigen.

[0218] The term “valent” as used herein denotes the presence of a specified number of binding sites in an antigen binding protein. A natural antibody for example or a full length antibody has two binding sites and is bivalent. As such, the terms “trivalent”, “tetravalent”, “pentavalent” and “hexavalent” denote the presence of two binding site, three binding sites, four binding sites, five binding sites, and six binding sites, respectively, in an antigen binding protein.

[0219] The dissociation constant (KD or Kd) is used as an indicator showing affinity of antibodies to antigens. For example, easy analysis is possible by the Scatchard method using antibodies marked with a variety of marker agents, as well as by using BiacoreX (made by Amersham Biosciences), which is an over-the-counter, measuring kit, or similar kit, according to the user's manual and experiment operation method attached with the kit. The KD value that can be derived using these methods is expressed in units of M (Mols). An antibody or antigen-binding fragment thereof that specifically binds to a target may have a dissociation constant (Kd) of, for example, <10-5M, <10“6M, <1 O’7M, <10-8M, <10-9M, <10-10M, <10’10M, <10’11M, or <10-12M.

[0220] Binding specificity of the antibody or antigen-binding domain can be determined experimentally by methods known in the art. Such methods comprise, but are not limited to Western blots, ELISA-, RIA-, ECL-, IRMA-, EIA-, BIAcore-tests and peptide scans. Half maximal inhibitory concentration (IC50) is a measure of the effectiveness of a substance (such as an antibody) in inhibiting a specific biological or biochemical function. It indicates how much of a particular drug or other substance (inhibitor, such as an antibody) is needed to inhibit a given biological process by half. The values are typically expressed as molar concentration. IC50 is comparable to an ECsofor agonist drug or other substance (such as an antibody). ECso also represents the plasma concentration required for obtaining 50% of a maximum effect in vivo. As used herein, an “IC50” is used to indicate the effective concentration of an antibody (such as an anti-TAB1 sdAb) needed to neutralize 50% of the antigen bioactivity in vitro. ICsoor ECso can be measured by bioassays such as inhibition of ligand binding by FACS analysis (competition binding assay), cell based cytokine release assay, or amplified luminescent proximity homogeneous assay (AlphaLISA).

[0221] “Percent (%) amino acid sequence identity" and “homology” with respect to a peptide, polypeptide or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0222] An “isolated” nucleic acid molecule encoding a construct, antibody, or antigen-binding fragment thereof described herein is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the environment in which it was produced. Preferably, the isolated nucleic acid is free of association with all components associated with the production environment. The isolated nucleic acid molecules encoding the polypeptides and antibodies described herein is in a form other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from nucleic acid encoding the polypeptides and antibodies described herein existing naturally in cells. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0223] The term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0224] The terms “host cell,” “host cell line,” and “host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0225] The term “pharmaceutical formulation” of “pharmaceutical composition” refers to a preparation that is in such form as to permit the biological activity of the active ingredient to be effective, and that contains no additional components that are unacceptably toxic to a subject to which the formulation would be administered. Such formulations are sterile. A “sterile” formulation is aseptic or free from all living microorganisms and their spores.

[0226] It is understood that embodiments of the invention described herein include “consisting” and / or “consisting essentially of' embodiments.

[0227] The term “about X-Y" used herein has the same meaning as “about X to about Y."

[0228] Additional definitions of can be found in US Patent 11 ,673,954, which is incorporated herein by reference. In case of conflicts, the definitions provided explicitly herein control.

[0229] The elements and method steps described herein can be used in any combination whether explicitly described or not.

[0230] All combinations of method steps as used herein can be performed in any order, unless otherwise specified or clearly implied to the contrary by the context in which the referenced combination is made.

[0231] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise.

[0232] Numerical ranges as used herein are intended to include every number and subset of numbers contained within that range, whether specifically disclosed or not. Further, these numerical ranges should be construed as providing support for a claim directed to any number or subset of numbers in that range. For example, a disclosure of from 1 to 10 should be construed as supporting a range of from 2 to 8, from 3 to 7, from 5 to 6, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth.

[0233] All patents, patent publications, and peer-reviewed publications ( / .e., “references”) cited herein are expressly incorporated by reference to the same extent as if each individual reference were specifically and individually indicated as being incorporated by reference. In case of conflict between the present disclosure and the incorporated references, the present disclosure controls.

[0234] It is understood that the invention is not confined to the particular construction and arrangement of parts herein illustrated and described, but embraces such modified forms thereof as come within the scope of the claims.

[0235] EXAMPLES Results

[0236] Atypical p38 Signaling

[0237] The atypical (non-canonical) activation of mitogen-activated protein kinase (MAPK) p38, requires the direct binding of the adaptor protein TAB1 (transforming growth factor (TGF)-beta activated kinase 1 (MAP3K7) binding protein 1 ) to p38 via specific interaction of the TAB1 C-terminal loop peptide and p38. The interaction between TAB1 and p38 occurs via two distinct interaction domains, a canonical and non-canonical pocket on p38 (FIG. 1). In contrast to the broader specificity of the canonical site, the noncanonical binding site is selective for TAB1 interactions.2Our prior studies show that the TAB1-p38 interaction is essential for p38 activation by a family of inflammatory G protein- coupled receptors (GPCRs), including the thrombin receptor (PAR1), Prostaglandin E2 receptors (EP1-4), Histamine receptors (HRH1 -4), and the purinergic receptor (P2Y 1 ).5'8Furthermore, the direct TAB1-p38a interaction drives atypical p38-dependent vascular inflammation and edema,6’8 9ischemic damage,2 3’10amyloidosis,11dermal inflammation,12and viral replication.1We aimed to develop inhibitory nanobodies to bind the TAB1 C-terminal peptides, sterically blocking the TAB1-p38a interaction and thus blocking inflammation and viral replication. Blocking the TAB1-p38a interaction would serve to treat all the disease applications regulated by atypical p38.5

[0238] Nanobodies as Research Tools and Therapeutics

[0239] The small immunologically neutral camelid nanobodies revolutionized the study of GPCRs, allowing the stabilization of the active receptor and enabling the characterization of spatiotemporal GPCR and G-protein signaling dynamics.13Critically, recent studies using nanobodies 37 and 80 (nb37, nb80) in conjunction with subcellular targeted Fluorescence Resonance Energy Transfer (FRET) sensors have elucidated the endosome, endoplasmic reticulum, and Golgi as critical organelles for G protein and ERK1 / 2 signaling by the b-2 adrenergic receptor and the dopamine receptor.14-17However, no current FRET sensors allow for the dissection of spatial p38 signaling. Additionally, atypical p38 signaling is independent of arrestins and indirectly controlled by G proteins, suggesting a functionally distinct mechanism for regulating atypical p38 responses.8Therefore, developing tools to map or block atypical p38 signaling will significantly impact our capacity to address many outstanding questions.

[0240] Camelid VHH or nanobodies are 15 kDa, homologous to human VH domains, and have promising therapeutic potential, with several nanobody-based therapeutics in current phase II and phase III clinical trials, including clinical trials for treating SARS-COv2.18'20Nanobodies can be directly expressed in cells (intrabodies) and are easily labeled with fluorescent tags to trace the endogenous spatiotemporal dynamics of proteins15’21’22or can be used to block protein interactions.23'25Thus, intrabodies can be developed to map atypical p38 activity and sterically block TAB1-p38 interactions. Intrabodies have high solubility and stability in the reducing environment of the cytosol and high expression and thermostability.26 27Nanobodies represent an exciting research tool with high potential for human therapeutics, with the caveat that current targets are localized in the extracellular space and plasma. However, due to their small size, nanobodies can be efficiently coded into AAVs for expression in target cells / tissue, overcoming the potential challenge of therapeutic delivery. Nanobody Screening and Identification

[0241] To screen for TAB1 selective intrabodies, we utilized a phage display library of humanized camelid singledomain antibodies, VHH (NaLi-H1 : Nanobody library humanized 1),28generating lead inhibitors to block atypical p38 signaling (FIG. 2 (A-D)). The recombinant full-length TAB1 protein was expressed using a cell-free system (Synthelis, France) as a His-SUMO-TAB1-Halo construct. The protein was biotinylated on the Halo-Tag with a biotin Halo ligand. TAB1 -Biotin was bound to Streptavidin Magnetic Beads (Dynabeads® M-280 Streptavidin, Life Technologies) with a 50-nM final concentration of biotinylated protein for the first round. His-SUMO-TAB1-Halo-Biotin was bound to Streptavidin Magnetic Beads with a 10-nM final concentration of biotinylated protein for all three rounds. The successful binding of the biotinylated proteins on the streptavidin beads was controlled in an SDS-PAGE / Western Blot using a Streptavidin-HRP conjugate (Thermo Fisher) (FIG. 3 (A)).

[0242] The protein His-SUMO- TAB1 -Halo-Biotin was used in an initial round of synthetic phage display library of humanized llama single domain antibody (NaLi-H1: Nanobody Library Humanized 1) to deplete the library from unspecific binders.

[0243] A fragment containing aa 304-432 of human TAB1 (NM_006116.3) (SEQ ID NO:20) was cloned into pB27 as a C-terminal fusion to LexA (LexA-TAB1 ). The construct was checked by sequencing the insert and used as bait to screen the naive VHH library constructed in pP9.29pB27 derives from the original pBTM 116 plasmid30and pP9 derives from the original pGAD-GH plasmid.29Clones were screened using a mating approach with YHGX13 (Y187 ade2- 101 ::loxP-kanMX-loxP, mata) and L40AGal4 (mata) yeast strains as previously described.31

[0244] The VHH identified from the phage display affinity isolation against TAB 1-Biotin were correlated with positive clones identified in the pP9 yeast two-hybrid-system.

[0245] Full-length SMAD and SMURF were used as bait and prey,32respectively, as positive controls for complementation and grew successfully in both the double (DO-2, medium lacking tryptophan, and leucine, and supplemented with 0.5 mM 3-Aminotriazol) and triple dropout media (DO-3, medium lacking tryptophan, leucine, and histidine and supplemented with 0.5 mM 3-Aminotriazol). As a negative control, TAB1 and the pP9 empty vector (pP90) were paired, growing in DO-2 but inhibited in the DO-3. We show a representative example from our top candidate TAB1 intrabody, IBI1 (see below), when paired with the empty pB27 vector (pB270), which grew in DO-2 but failed to grow in the DO-3 media. Conversely, when paired with TAB1 , IBI1 grew in both DO2 and DO3 media, demonstrating complementation and interaction between bait (TAB1) and pray (IBI1) (FIG. 3 (B)). In total, 48 His+ colonies were selected on DO-3 medium. They correspond to 27 different intrabodies with redundancies from 1 to 10.

[0246] Regulation ofGPCR-lnduced p38 Signaling

[0247] To screen for inhibition of p38 activation, we utilized our previously published assay to assess thrombin- mediated activation of p38 in human fibroblast cells (HeLa).6'8The thrombin receptor protease-activated receptor 1 (PAR1-FLAG) was transfected independently with each of the top 20 N-terminal mCherry-tagged intrabodies. After 24 hours, cells were serum starved (1 mg / ml BSA, 1 m MCaCI2, 10 mM HEPES, In DMEM), stimulated with 10 nM thrombin for 7-minutes, lysed, and processed for immunoblotting. From the initial screen, five intrabodies increased thrombin signaling, four intrabodies had little to no impact, and ten intrabodies decreased p38 activation by thrombin (FIG. 4 (A)). Intrabody 19 was selected as the top candidate (renamed IBM). IBi1 was then carried forward for quantitative analysis in comparison to an intrabody that had no impact of p38 activation and an intrabody that enhanced p38 activity (FIG. 4 (B, compare lanes 2, 4, 6, and 8) (C)). I Bi 1 reduced thrombin-mediated p38 activation by -95%. Conversely, I Bi1 did not impact thrombin-mediated activation of MAPK ERK1 / 2, suggesting selectivity to p38 signaling (FIG. 4 (D)).

[0248] Cell Culture Hepatitis C Virus (HCVcc) Challenge

[0249] HCV robustly activates p38, as seen in patient data sets and primary human hepatocytes infected with HCV (JFH1 )1(FIG. 5 (A, B)). During infection, HCV initiates TAB1-p38 association (FIG. 5 (C)) and autophosphorylation. P38 then directly associates with and phosphorylates HVC core protein regulating core expression. Combined, this suggests that biologies targeting atypical p38 could regulate the replication of all HVC genotypes and be supportive when resistance develops. Deletion of TAB1 or chemical inhibition of p38 (SB203580) can suppress HCV replication (FIG. 5 (D)).33Additionally, p38 inhibition can suppress the replication of many other viruses, including HBV, HSV1 , SARS-cov2, and Junin virus1-34'36.

[0250] To test the capacity of TAB1 -intrabodies to block HCV replication and core antigen production, we infected human hepatocellular carcinoma cells Huh7.5 with a cell culture permissive chimeric virus (HCVcc, FL-J6 / JFH37). We compared p38 inhibition with pre-treatment with SB203580 or I Bi 1 , 1 Bi2 or IBI3.

[0251] HuH7.5 cells were transfected with varying concentrations of IBi1 -3 (50 ng, 100 ng, or 250 ng). After 4 hours, wells were infected with equal volumes of HCVcc. Finally, 4 hours after infection, DMSO or SB203580 were added to control wells. All wells were then collected after 48 hours. SB203580 treatment reduced HCVcc core protein expression by -75% (FIG. 6 (A, compare lane 2 / 3)). Comparably, increasing titrations of intrabodies reduced the expression of HCV core protein for all three of the top intrabody candidates. With the 27% reduction for IBi1 , 50% reduction for I Bi2, and 75% reduction for I Bi3 (FIG. 6 (A, compare lane 2 with lane 6 (Ibi1), lane 9 (IBI2), and lane 12 (IBI3))).

[0252] Intrabody Interaction with Endogenous TAB1

[0253] The two-hybrid screen (FIG. 3 (B)) established that IBi 1 binds to the C-terminus of human TAB1 in yeast. To further validate the interaction between TAB1 and IBM we transfected IBH-mCherry into human cells (HeLa cells). 48 hours after transfection, cells were fixed with 4% PFA permeabilized with 0.01% Triton X-100 and Immunolabeled with anti-Human TAB1 (Cell Signaling Technologies) followed by secondary antibody labeling with anti-rabbit Alexa 488 (Thermo-fisher). Slides were imaged using a Zeiss LSM800 Confocal microscope. IBM displayed a diffuse distribution that colocalized with endogenous TAB1 , seen as yellow in the merged image (FIG. 7 (A)). To confirm that IBI1 can bind to endogenous TAB1 we transfected I Bi1 into Hela Cells and immunoprecipitated the intrabody using anti-RFP (red fluorescent protein). Eluates were then probed for mCherry (IBi1) and endogenous TAB1 (indicated by the *). Endogenous TAB1 was only detected in lane 3 in the presence of IBI1 . (FIG. 7 (B, lane 3)) Oxygen-Induced Retinopathy.

[0254] Atypical p38 signaling is required to drive oxygen-induced retinal damage in mice, a model of retinopathy of prematurity and proliferative diabetic retinopathy in humans.38-49This has applications to glaucoma,50'52choroidal neovascularization (CNV),48-53and neovascular age-related macular degeneration (nAMD).5455Atypical p38 drives ischemia / reperfusion-induced tissue damage and apoptosis mediated by HIFIa ROS, and H2O2 generation.2 4’56 58We predict that atypical p38 signaling drives oxygen-induced retinal damage. Isolectin B4 (IB4) labeling of wt and Tab1KIretina shows a significant reduction in oxygen-induced retinopathy-induced vaso-obliteration and neovascular tufting in Tab1KImice at P17 (FIG. 8).59We predict that TAB1 -intrabodies (e.g., through AAV delivery) will protect the retina from pathological atypical p38 signaling and neovascular responses in oxygen-induced retinopathy.

[0255] Co-Immunoprecipitation of Intrabodies and TAB1 Truncation Mutants

[0256] Using truncation mutation of TAB1 (FIG. 9A), we show that all three intrabodies (IBi1 , IBi2, and IBi3) bind to human TAB1 (FIGS. 9B-9C). The data indicate that IBi2 binds to TAB1 in a conserved 18 amino acid peptide having the sequence of SAQSTSKTSVTLSLVMPS (SEQ ID NO:22) (FIG. 9C), whereas IBi1 and IBi3 bind to a peptide having the sequence of PAAGGRVYPVSVPYS (SEQ ID NO:23) (FIGS. 9B and 9D).

[0257] Conclusions

[0258] Atypical p38 is selectively active by the direct TAB1-p38 interaction. We predicted that TAB1-targeted intracellular nanobodies (intrabodies) would block the TAB1-p38 interaction, selectively inhibiting pathological p38 MAPK signaling.

[0259] Using biotinylated full-length human TAB1 we affinity purified Nanobodies from a phage display library of humanized camelid single-domain antibodies, VHH (NaLi-H1 : Nanobody library humanized 1) (FIG. 2). We next used a truncated TAB1 peptide fragment containing aa 304-432 of human TAB1 , containing the CS and NCS p38 binding domains, but removing the TAK1 binding domain to enhance the likelihood of generating NCS targeting intrabodies and screened the VHH library through a yeast two-hybrid assay, identifying 20 lead candidates. (FIG. 3).

[0260] Our prior studies show that a family of inflammatory GPCRs activate atypical p38 signaling. Atypical p38 signaling is also established to regulate HCV replication and likely regulates multiple other viruses.

[0261] In these examples, we show that IBI1 suppresses GPCR-induced p38 activation, that IBi1 interacts with TAB1 in a yeast-two-hybrid model, colocalizes with endogenous human Tab1 in cells, and immunoisolates endogenous TAB1 , and that when expressed in HuH7.5 cells, IBi1-3 suppresses viral replication as indicated by a loss of HCV core expression.

[0262] Methods

[0263] Reagents and Antibodies

[0264] The following reagents were used: SB203580, was from Sigma-Aldrich. Human alpha thrombin (a-Th) (Enzyme Research Laboratories). The following antibodies were used: mouse IgG antibody, and rabbit anti-RFP (Rockland Immunochemicals), mouse anti-PAR1 WEDE antibody (Beckman Coulter), mouse M2 anti-FLAG and -actin antibodies (Sigma-Aldrich), rabbit anti-TAB1, p38, phospho-p38, p42 / 44 MAPK, phospho-p42 / 44 MAPK, (Cell Signaling Technology), Mouse anti-hepatitis C virus core antigen, C7-50 (Fisher), HRP-conjugated goat-anti-rabbit and goat-anti-mouse antibodies (Bio-Rad Laboratories) and Alexa Fluor 488- and 594-conjugated secondary antibodies (Life Technologies).

[0265] Plasmids

[0266] The FLAG-tagged PAR1 WT pcDNA3.1 was previously characterized.6’60JFH1J6Ctop7(HCVcc) were amplified with max efficiency DH5a E. coli, and purified with a ZYMO maxiprep kit. For RNA transcription, plasmid templates were linearized with Xbal and the 5' overhangs were removed by treatment with mung bean nuclease. Templates were purified with two rounds of phenol-chloroform extraction and ethanol precipitation then resuspended in 20 pL H2O and quantified on the Nanodrop Spectrophotometer. 2 pg of linearized templates were transcribed using the MEGAscript High Yield transcription kit by Ambion and incubated for 3 hours at 37°C. Following synthesis templates were purified with two rounds of phenol-chloroform and RNA were precipitated with 1 volume of isopropanol and 2 pL of glycogen, the mixture was then chilled at -20°C overnight. RNA was then centrifuged at max speed and resuspended in 20 pL of nuclease-free water and quantified. RNA was then aliquoted and stored at -80°C. For the yeast-two-hybrid assay, pB27 is derived from the original pBTM116 plasmid field 3, and pP9 is derived from the original pGAD-GH plasmid.29Both plasmids generated and retained by Hybrigenics, France.

[0267] Cell Lines

[0268] Huh7.5 cells, a human hematoma cell line, was supplemented with 10% FBS and 100 pg / mL of streptomycin / penicillin at 37°C in humidified conditions and 5% CO2. All HCVcc experiments described above were performed using these cells. HeLa cells (Cat. No. Hela CCI2, ATCC Manassa, VA, USA) were used for all experiments as previously described.6 8Dulbecco’s modification of Eagle’s medium (DMEM, Cat. No. 10-013-CV, Corning, Mediatech Inc., Manassas, VA, USA) supplemented with 10% (v / v) fetal bovine serum (FBS; Cat. No. 35-010-CV, Corning, Mediatech, CA, USA) was used for cell maintenance. Cells were incubated at 37 °C in humidified conditions and 5% CO2.

[0269] Cell-Free Protein Synthesis

[0270] The recombinant full-length TAB1 protein was expressed using a cell-free system (Synthelis, France) as a His-Sumo-TAB1-Halo construct. The protein was biotinylated on the Halo-Tag with a biotin Halo ligand (Promega).

[0271] Affinity Purification of VHH 13 Phage

[0272] Affinity purification was carried out by hybrigenics, France using a phage display library of humanized camelid single-domain antibodies, VHH (NaLi-H1 : Nanobody library humanized 1), as previously described.28Briefly, TAB1- Biotin was bound to Streptavidin Magnetic Beads (Dynabeads® M-280 Streptavidin, Life Technologies) with a 50 nM final concentration of biotinylated protein. His-SU MO-TAB 1 -Halo-Biotin was bound to Streptavidin Magnetic Beads with a 10 nM final concentration of biotinylated protein for all three rounds. The successful binding of the biotinylated proteins on the streptavidin beads was controlled in an SDS-PAGE / Western Blot using a Streptavidin-HRP conjugate (Thermo Fischer). Each round used the phage display library.28After affinity isolation, 380,000 VHH were identified and cloned into the pP9 yeast PREY vector

[0273] Yeast Two Hybrid

[0274] A fragment containing aa 304-432 of human TAB1 (NM_006116.3) was cloned into pB27 as a C-terminal fusion to LexA (LexA-TAB1 ). The construct was checked by Sanger sequencing and the insert was used as bait to screen the naive VHH library constructed in pP9.29pB27 derives from the original pBTM 116 plasmid30and pP9 derives from the original pGAD-GH plasmid.29Clones were screened using a mating approach with YHGX13 (Y187 ade2- 101 ::loxP-kanMX-loxP, rnato) and L40AGal4 (mata) yeast strains as previously described.31The VHH identified from the phage display affinity isolation against TAB1 -Biotin were correlated with positive clones identified in the pP9 yeast two-hybrid-system.

[0275] HCV RNA Transfection and HCVcc Growth and Storage

[0276] HCV RNA generation, transfection, and viral storage were adapted from previously described protocols.37’61Briefly, Huh7.5 cells were washed with PBS, trypsinized, resuspended in DMEM supplemented with 10% fetal calf serum (FCS), and centrifuged at 1500 rpm for 10 minutes. Cells were washed once with ice-cold PBS and centrifuged again following the same parameters. Cells were resuspended at a density of 2 x 107cells / mL.10 g of RNA was added to the cell suspension and electroporated using the NeonTM transfection system (Thermo Fisher Scientific). Using 1400 V, 20 ms, 1 pulse. Cells were resuspended in DMEM with 10% FCS before seeding in 6 well plates. Viral supernatants were collected at days 4-7 post-electroporation. Viral supernatants from days 4-7 were pooled, sterile filtered (0.2 pm), and buffered with 1 volume 1 M Hepes. HCVcc stocks were then aliquoted and stored at -80°C. During experiments, Huh7.5 cells were infected with equal volumes of a 1 :20 dilution of HCVcc in a complete medium for 48 hrs.

[0277] Cell Transfections

[0278] Hela cells were transiently transfected with cDNA using linear polyethylenimine (PEI) Polysciences. Briefly, 1mg / ml stock of PEI was mixed with optimum and incubated for 10 minutes, then cDNA was added (1 :6 ratio, cDNA: PEI). This was mixed and incubated for a further 20 minutes. PEI / cDNA / optimum mixture was added to cells seeded 24 h before transfection and at 40% confluency or to cells in suspension. Cells were grown for 24-48 hours before analysis. Huh7.5 cells were seeded in a 24-well plate at 100,000 cells / well. The following day adherent cells were transfected with a master mix containing either 50, 100, or 250 ng intrabody cDNA incubated with Gibco Opti-MEM reduced serum media, P300 (2pL / pg), and Lipofectamine 3000 following Thermo Fisher protocol. HeLa cells were transfected in suspension with polyethyleneimine (PEI) incubated with Gibco Opti-MEM reduced serum media before plating in a 24-well plate coated with human fibronectin and incubated in a humidified incubator for 48 hrs.

[0279] Immunoblotting

[0280] Huh7.5 or HeLa cells were seeded into 24-well plates, transfected as above, and then grown for 48 hours. In some experiments, cells were treated with 10 pM SB203580, a p38o and selective inhibitor. HeLa cells were serum starved overnight at 37 °C before agonist stimulation with 10 nM thrombin. For Huh7.5 infection models, HCVcc was added to the indicated wells at equal volumes and incubated for the indicated time. Cells lysed in 1X Laemmli sample buffer plus 100 mM DTT, boiled for 5 minutes and vortexed, cell lysates resolved by SDS-PAGE and processed for immunoblotting as previously described, with antibody dilutions of 1 : 15,000 for total and phospho-p38, 1 : 4,000 for mRFP, 1 : 1 ,000 for ERK and P-ERK, and 1 : 2,000 for TAB1 and HCV core. Immunoblots were quantified by densitometry using NIH ImageJ software.

[0281] Immunoprecipitation

[0282] As above, HeLa cells were transfected in 6-cm dishes and grown for 48 hrs. Cells were then lysed in Triton lysis buffer containing 50 mM Tris-HCI pH 7.4, 100 mM NaCI, 1 % Triton X-100, 10 mM NaF, 10 mM (3- glycerophosphate, 10 mM NaPP, 10 mg / ml leupeptin, aprotinin, pepstatin, and 20 mM N-ethylmaleimide. Cell lysates were homogenized and cleared by centrifugation, and protein concentrations were determined by bicinchoninic acid assay (BCA). Equivalent amounts of lysates were used for overnight immunoprecipitations using either the anti-RFP or IGG control and Protein-G sepharose (Thermo Fisher). Samples were washed with lysis buffer and eluted with 2X Laemmli sample buffer containing 200 mM dithiothreitol and processed via immunoblotting.

[0283] Fluorescence Microscopy

[0284] HeLa cells were transfected in suspension and then seeded onto 12 mm coverslips coated with 8.25 g / mL of Fibronectin at a density of 70,000 cells / well. 48 hours after, transfected cells were fixed with 4% paraformaldehyde for 5 minutes on ice, quenched with glycine and 1 mM CaCI2+, then permeabilized with 0.1% Triton-X 100 in PBS. Cells were blocked with 1% BSA and 10 mM glycine in PBS for 30 min. Cells were then stained with anti-TAB1 1 :250 overnight at 4°C. The following day the secondary antibodies conjugated with Alexa Flour 488 were added to the cells for one hour at room temperature. The coverslips were then mounted onto glass slides with a mounting medium containing NucBlue. Slides were then imaged with confocal microscopy to visualize co-localization.

[0285] Co-Immunoprecipitation of Intrabodies and TAB1 Truncation Mutants

[0286] T runcation mutations were generated for human T ab 1 through directed mutagenesis, introducing stop codons to remove (1 ) the D-c-terminal Tak1 domain after Pro 432 (Mutant 1), (2) the D-Linker after Ser 413 (Mutant 2), (3) the D-p38 Canonical binding peptide after Ser 395 (Mutant 3), and (4) D-p38 non-canonical binding peptide after Gly 366 (Mutant 4). See FIG. 9A. Huh7.5 cells were transfected with either I Bi1 , 1 BI2, or IBI3 in combination with wt-Tab1 , or Mutant 1 , Mutant 2, Mutant 3, or Mutant 4, using lipofectamine 3000. After 48 co-immunoprecipitations were performed as follows. Cells were lysed in Triton lysis buffer containing 50 mM Tris-HCI pH 7.4, 100 mM NaCI, 1 % Triton X100, 5 mM EDTA, 50 mM NaF, 50 mM b-glycerophosphate, 10 pg / ml leupeptin, aprotinin, trypsin protease inhibitor, pepstatin, 100 pg / ml benzamide, 20 mM W-ethylmaleimide protease inhibitors, and 20 mM N-ethylmaleimide. The lysate was passed through a 21 GA needle and cleared by centrifugation. Protein concentrations were determined by bicinchoninic acid assay, and equivalent amounts of lysates were used for immunoprecipitations using anti-IgG control antibody or anti- RFP (Rockland). Immunoprecipitates were eluted with 2x Laemmli sample buffer containing 200 mM dithiothreitol.

[0287] Data Analysis

[0288] Data were analyzed using Prism 7.0 software (GraphPad Software, La Jolla, CA), and statistical significance was determined using the Student’s t-test and one-way analysis of variance, as indicated.

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Claims

CLAIMSWhat is claimed is:

1. A anti-TAB1 construct comprising a single-domain antibody (sdAb) moiety specifically recognizing TAB1, wherein the sdAb moiety comprises a CDR1 , a CDR2, and a CDR3, wherein: the CDR1 comprises an amino acid sequence of SGDTWENTGMG (SEQ ID NO: 1), SGSTSAFSNMG (SEQ ID NO:2), SGTYTSEIMG (SEQ ID NO:3), or a variant of any of the foregoing comprising up to 3 (such as any of 1 , 2, or 3) amino acid substitutions; the CDR2 comprises an amino acid sequence of SDFEDMVDYY (SEQ ID NO:4), SGLDDMKHYY (SEQ ID NO:5), SRNFSFKTYY (SEQ ID NO:6), or a variant of any of the foregoing comprising up to 3 (such as any of 1 , 2, or 3) amino acid substitutions; and / or the CDR3 comprises an amino acid sequence of AMPTMQIRITSDTNSGTWKY (SEQ ID NO:7), AEIELDMHHLELTIHYY (SEQ ID NO:8), AIDLKWKGRLIMTY (SEQ ID NO:9), or a variant of any of the foregoing comprising up to 3 (such as any of 1 , 2, or 3) amino acid substitutions.

2. The anti-TAB 1 construct of claim 1 , wherein: the CDR1 comprises an amino acid sequence of SGDTWENTGMG (SEQ ID NO: 1), SGSTSAFSNMG (SEQ ID NO:2), SGTYTSEIMG (SEQ ID NO:3), or a variant of any of the foregoing comprising up to 3 (such as any of 1 , 2, or 3) amino acid substitutions; the CDR2 comprises an amino acid sequence of SDFEDMVDYY (SEQ ID NO:4), SGLDDMKHYY (SEQ ID NO:5), SRNFSFKTYY (SEQ ID NO:6), or a variant of any of the foregoing comprising up to 3 (such as any of 1 , 2, or 3) amino acid substitutions; and the CDR3 comprises an amino acid sequence of AMPTMQIRITSDTNSGTWKY (SEQ ID NO:7), AEIELDMHHLELTIHYY (SEQ ID NO:8), AIDLKWKGRLIMTY (SEQ ID NO:9), or a variant of any of the foregoing comprising up to 3 (such as any of 1 , 2, or 3) amino acid substitutions.

3. The anti-TAB 1 construct of claim 1 , wherein: the CDRI comprises an amino acid sequence of SGDTWENTGMG (SEQ ID NO: 1) or a variant thereof comprising up to 3 (such as any of 1 , 2, or 3) amino acid substitutions, the CDR2 comprises an amino acid sequence of SDFEDMVDYY (SEQ ID NO:4) or a variant thereof comprising up to 3 (such as any of 1 , 2, or 3) amino acid substitutions, and the CDR3 comprises an amino acid sequence of AMPTMQIRITSDTNSGTWKY (SEQ ID NO:7) or a variant thereof comprising up to 3 (such as any of 1 , 2, or 3) amino acid substitutions; the CDR1 comprises an amino acid sequence of SGSTSAFSNMG (SEQ ID NO:2) or a variant thereof comprising up to 3 (such as any of 1 , 2, or 3) amino acid substitutions, the CDR2 comprises an amino acid sequence of SGLDDMKHYY (SEQ ID NO:5) or a variant thereof comprising up to 3 (such as any of 1 , 2, or 3) amino acid substitutions, and the CDR3comprises an amino acid sequence of AEIELDMHHLELTIHYY (SEQ ID NO:8) or a variant thereof comprising up to 3 (such as any of 1 , 2, or 3) amino acid substitutions; or the CDR1 comprises an amino acid sequence of SGTYTSEIMG (SEQ ID NO:3) or a variant thereof comprising up to 3 (such as any of 1 , 2, or 3) amino acid substitutions, the CDR2 comprises an amino acid sequence of SRNFSFKTYY (SEQ ID NO:6) or a variant thereof comprising up to 3 (such as any of 1 , 2, or 3) amino acid substitutions, and the CDR3 comprises an amino acid sequence of AIDLKWKGRLIMTY (SEQ ID NO:9) or a variant thereof comprising up to 3 (such as any of 1 , 2, or 3) amino acid substitutions.

4. The anti-TAB 1 construct of claim 1 , wherein: the CDR1 comprises an amino acid sequence of SGDTWENTGMG (SEQ ID NON), the CDR2 comprises an amino acid sequence of SDFEDMVDYY (SEQ ID NON), and the CDR3 comprises an amino acid sequence of AMPTMQIRITSDTNSGTWKY (SEQ ID NO:7); the CDR1 comprises an amino acid sequence of SGSTSAFSNMG (SEQ ID NO:2), the CDR2 comprises an amino acid sequence of SGLDDMKHYY (SEQ ID NO:5), and the CDR3 comprises an amino acid sequence of AEIELDMHHLELTIHYY (SEQ ID NO:8); or the CDR1 comprises an amino acid sequence of SGTYTSEIMG (SEQ ID NO:3), the CDR2 comprises an amino acid sequence of SRNFSFKTYY (SEQ ID NO:6), and the CDR3 comprises an amino acid sequence of AIDLKWKGRLIMTY (SEQ ID NO:9).

5. The anti-TAB1 construct of any prior claim, wherein the sdAb moiety comprises an FR1 , an FR2, an FR3, and an FR4, wherein: the FR1 comprises an amino acid sequence of MAEVQLQASGGGFVQPGGSLRLSCAA (SEQ ID NO: 10) or a variant of any of the foregoing comprising up to 3 (such as any of 1 , 2, or 3) amino acid substitutions; the FR2 comprises an amino acid sequence of WFRQAPGKEREFVSAI (SEQ ID NO: 11) or a variant of any of the foregoing comprising up to 3 (such as any of 1, 2, or 3) amino acid substitutions; the FR3 comprises an amino acid sequence of ADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYC (SEQ ID NO: 12) or a variant of any of the foregoing comprising up to 3 (such as any of 1 , 2, or 3) amino acid substitutions; and / or the FR4 comprises an amino acid sequence of WGQGTQVTVSS (SEQ ID NO:13) or a variant of any of the foregoing comprising up to 3 (such as any of 1, 2, or 3) amino acid substitutions.

6. The anti-TAB1 construct of any prior claim, wherein the sdAb moiety comprises an amino acid sequence having at least 95% sequence identity to an sdAb sequence selected from the group consisting of SEQ ID NOs: 14, 16, and 18.

7. The anti-TAB1 construct of any prior claim, wherein the anti-TAB1 construct binds an epitope having the amino acid sequence of SAQSTSKTSVTLSLVMPS (SEQ ID NO:22) or PAAGGRVYPVSVPYS (SEQ ID NO:23).

8. The anti-TAB1 construct of any prior claim, wherein the anti-TAB1 construct inhibits binding of p38 to TAB1.

9. The anti-TAB 1 construct of any prior claim, wherein the anti-TAB 1 construct inhibits p38 activation.

10. A nucleic acid encoding the anti-TAB1 construct of any prior claim.

11. A vector comprising the nucleic acid of claim 10.

12. The vector of claim 11 , wherein the vector is configured for intracellular expression of the anti-TAB1 construct.

13. The vector of any one of claims 11-12, wherein the vector is configured for intracellular delivery of the nucleic acid.

14. A method of intracellularly expressing the anti-TAB1 construct of any one of claims 1-9, comprising delivering the nucleic acid of claim 10 or the vector of any one of claims 11-13 into a cell to express the anti-TAB1 construct within the cell.

15. A method of treating a condition mediated by atypical p38 signaling in a subject, the method comprising administering the anti-TAB1 construct of any one of claims 1-9, the nucleic acid of claim 10, or the vector of any one of claims 11-13 to the subject in an amount effective to treat the condition.

16. The method of claim 15, wherein the method comprises delivering the anti-TAB1 construct or the vector into a cell in the subject.

17. The method of any one of claims 15-16, wherein the condition is selected from the group consisting of myocardial ischemia, cardiovascular ischemia and reperfusion, myocardial infarction, cardiomyopathy, amyloidosis, viral infection, viral replication, bacterial infection, parasitic infection, inflammation, cancer, cancer metastasis, cancer inflammation, vascular endothelial dysfunction, leukocyte dysfunction, myeloid cell mobilization and activation, immune system modulation, neurovascular signaling, traumatic brain injury, edema, pulmonary damage, acute respiratory distress syndrome, acute lung injury, diabetes, a diabetes complication, a pregnancy complication, retinal damage, retinopathy, oxygen-induced retinopathy, retinopathy of prematurity, age-related macular degeneration, glaucoma, choroidal neovascularization, rheumatoid arthritis, and hepatic damage.

18. The method of any one of claims 15-17, wherein the condition is a viral infection.

19. The method of any one of claims 15-17, wherein the condition is inflammation.

20. The method of any one of claims 15-17, wherein the condition is oxygen-induced retinopathy.

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