CROSS REFERENCE TO RELATED APPLICATIONS

[001] This application claims benefit to U.S. Provisional Application No. 63/357,535, filed June 30, 2022, which is incorporated by reference herein in its entirety.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[002] The instant application contains a Sequence Listing, which has been submitted electronically in .xml format. The contents of the electronic sequence listing (00958 l_antiTNF.xml; Size: 207,636 bytes; and Date of Creation: June 24, 2023) is herein incorporated by reference in its entirety.

INCORPORATION BY REFERENCE

[003] All publications, patents, and patent applications cited herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicates to be incorporated by reference. In the event of a conflict between a term herein and a term in an incorporated reference, the term herein controls.

BACKGROUND

[004] Tumor Necrosis Factor-alpha (TNF-alpha or TNF-oc) is a homo-trimeric pro- inflammatory cytokine that is released by and interacts with cells of the immune system. TNF- alpha has been shown to be up-regulated in a number of chronic human diseases, such as rheumatoid arthritis, Crohn’s disease, ulcerative colitis, and multiple sclerosis. Antibodies to TNF-alpha have been approved for therapeutic uses, such as (i) infliximab (Remicade®), a chimeric IgG anti- human monoclonal; (ii) adalimumab (Humira®), a fully human monoclonal antibody (mAb); (iii) golimumab (Simponi®), a fully human mAb; and (iv) certolizumab (Cimzia®), a PEGylated Fab fragment. See, e.g., Monaco, C., et al., Int’l Immunol. 2015, 27(l):55-62. Additionally, US 2020/0079844 describes improved Nanobodies™, a singledomain antibody (sdAb) against TNF-alpha, for prophylactic, therapeutic, or diagnostic purposes.

[005] Genetically modified microorganisms (e.g., bacteria) have been used to deliver therapeutic molecules (e.g., cytokines and monoclonal antibodies) to mucosal tissues. See, e.g., Steidler, L., et al., Nat. Biotechnol. 2003, 21(7): 785-789; and Robert S. and Steidler L., Microb. Cell Fact. 2014, 13 Suppl. 1: Si l.

[006] However, there is still a need in the art for genetically modified bacterial strains that are stable and suitable for clinical usage and which constitutively or inducibly express a TNF- alpha antibody and one or more bioactive polypeptides. Such bacterial strains can be expected to more effectively deliver the TNF-alpha antibody to a subject’s mucosal tissues. The present disclosure addresses these needs.

SUMMARY

[007] The current disclosure provides antibody polypeptides comprising a single variable domain that specifically binds a human Tumor Necrosis Factor-alpha (TNF-a) comprising the amino acid sequence of SEQ ID NO:1.

[008] In one aspect, the single variable domain comprises: (a) a complementary determining region 1 (CDR1) sequence having up to one amino acid substitution in the amino acid sequence of Ile-Tyr-Trp-Met-Thr (SEQ ID NO:3), (b) a complementary determining region 2 (CDR2) sequence having up to one amino acid substitution in the amino acid sequence of Glu- Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg-Arg-Tyr-Ala-Asp-Ser-Val-Glu- Gly (SEQ ID NO: 4), and (cl) a complementary determining region 3 (CDR3) sequence having from one to three amino acid substitutions in the amino acid sequence of Lys-Asp-Phe-Gly-Gly-Gln-Ile (SEQ ID NO: 5), or (c2) a complementary determining region 3 (CDR3) sequence is a CDR3+1 sequence having from one to three amino acid substitutions in the amino acid sequence of Lys-Asp-Phe-Gly-Gly- Gln-Ile-Lys (SEQ ID NO: 8).

[009] In some examples according to any of the above embodiments, the CDR1 sequence is Ile-Tyr-Trp-Met-Thr (SEQ ID NOG), and (b) the CDR2 sequence is Glu-Ile-Asn-Thr-Asn- Gly-Leu-Ile-Arg-Arg-Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NOG).

[0010] In certain examples, the CDR1 sequence is Ile-Tyr-Trp-Met-Thr (SEQ ID NOG), the CDR2 sequence is the CDR2 sequence is Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg-Arg-Tyr-Ala- Asp-Ser-Val-Glu-Gly (SEQ ID NOG), and the CDR3 sequence is Xi- X2--X3-Gly-Gly-Gln-Ile (SEQ ID NO:97). In certain examples, the CDR1 sequence is Ile-Tyr-Trp-Met-Thr (SEQ ID NOG), the CDR2 sequence is the CDR2 sequence is Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg- Arg-Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NOG), and the CDR3 sequence is the CDR3+1 Xi- X2--X3-Gly-Gly-Gln-Ile-Lys (SEQ ID NO:98), wherein Xi is Lys, Leu, or Gin; X2 is Asp or Asn; and X3 is Phe, Pro, Thr, Arg, Gin, or Glu.

[0011] In aspects of the antibody polypeptide, the third amino acid residue in the CDR3 or CDR3+1 sequence is Pro. In some examples, the CDR3 sequence is KDPGGQI (SEQ ID NO:27; TNA019), . QDPGGQI (SEQ ID NO: 21; TNA030), LDPGGQI (SEQ ID NO: 15; TNA028), QNPGGQI (SEQ ID NO:.11; TNAO33), or KNPGGQI (SEQ ID NO:13; TNA031) In some examples, the CDR3+1 sequence is KDPGGQIK (SEQ ID NO:96; TNA019). In some examples, the CDR33+1 sequence is KDPGGQIK (SEQ ID NO: 96; TNA019), QDPGGQIK (SEQ ID NO: 93; TNA030), LDPGGQIK (SEQ ID NO: 90; TNA028), QNPGGQIK (SEQ ID NO:88; TNAO33), or KNPGGQIK (SEQ ID NO:89; TNA031).

[0012] In aspects of the antibody polypeptide, the second amino acid residue in the CDR3 or CDR3+1 sequence is asparagine (Asn). In some examples, the CDR3 sequence is LNTGGQI (SEQ ID NO:25; TNA037), LNRGGQI (SEQ ID NO:23; TNAO38), LNQGGQI (SEQ ID NO: 19; TNA036), LNEGGQI (SEQ ID NO: 17; TNA035), LNPGGQI (SEQ ID NO:9; TNA032), QNPGGQI (SEQ ID NO:11; TNAO33), or KNPGGQI (SEQ ID NO:13; TNA031). In some examples, the CDR3+1 sequence is LNTGGQIK (SEQ ID NO:95; TNA037), LNRGGQIK (SEQ ID NO:94; TNAO38), LNQGGQIK (SEQ ID NO:92; TNA036), LNEGGQIK (SEQ ID NO:91; TNA035), LNPGGQIK (SEQ ID NO:87; TNA032), QNPGGQIK (SEQ ID NO:88; TNAO33), or KNPGGQIK (SEQ ID NO:89; TNA031).

[0013] In aspects of the antibody polypeptide, the second amino acid residue in the CDR3 or CDR3+1 sequence is aspartic acid (Asp). In some examples, the CDR3 sequence is KDPGGQI (SEQ ID NO: 27; TNA019), QDPGGQI (SEQ ID NO: 21; TNA030), or LDPGGQI (SEQ ID NO: 15; TNA028). In some examples, the CDR3+1 sequence is KDPGGQIK (SEQ ID NO: 96; TNA019), QDPGGQIK (SEQ ID NO: 93; TNA030), or LDPGGQIK (SEQ ID NO: 90; TNA028).

[0014] In aspects of the antibody polypeptide, the first amino acid residue in the CDR3 or CDR3+1 sequence is leucine (Leu). In some examples, the CDR3 sequence is LNTGGQI (SEQ ID NO:25; TNA037), LNRGGQI (SEQ ID NO:23; TNAO38), LNQGGQI (SEQ ID NO: 19; TNA036), LNEGGQI (SEQ ID NO: 17; TNA035), LDPGGQI (SEQ ID NO: 15; TNA028), or LNPGGQI (SEQ ID NO:9; TNA032). In some examples, the CDR3+1 sequence is LNTGGQIK (SEQ ID NO:95; TNA037), LNRGGQIK (SEQ ID NO:94; TNAO38), LNQGGQIK (SEQ ID NO:92; TNA036), LNEGGQIK (SEQ ID N0:91; TNA035), LDPGGQIK (SEQ ID NO: 90;

TNA028), or LNPGGQIK (SEQ ID NO:87; TNA032).

[0015] In one aspect of the antibody polypeptide, the first amino acid residue of the CDR3 or CDR3+1 sequence is glutamine (Gin). In some examples, the CDR3 sequence is QDPGGQI (SEQ ID NO:21; TNA030) or QNPGGQI (SEQ ID NO:11; TNAO33). In some examples, the CDR3+1 sequence is QDPGGQIK (SEQ ID NO:93; TNA030) or QNPGGQIK (SEQ ID NO:88; TNAO33).

[0016] In some examples of the antibody polypeptide, the CDR1, CDR2, and CDR3 sequences are (a) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile- Arg-Arg-Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and LNPGGQI (SEQ ID NO:9; TNA032); (b) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg- Arg-Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and QNPGGQI (SEQ ID NO: 11; TNAO33); (c) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg- Arg-Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and KNPGGQI (SEQ ID NO: 13; TNA031); (d) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg- Arg-Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and LDPGGQI (SEQ ID NO: 15; TNA028); (e) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg- Arg-Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and LNEGGQI (SEQ ID NO: 17; TNA035); (f) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg- Arg-Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and LNQGGQI (SEQ ID NO: 19; TNA036); (g) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg- Arg-Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and QDPGGQI (SEQ ID NO:21; TNA030); (h) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg- Arg-Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and LNRGGQI (SEQ ID NO:23; TNAO38); (i) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg- Arg-Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and LNTGGQI (SEQ ID NO:25; TNA037); or (j) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg- Arg-Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and KDPGGQI (SEQ ID NO:27; TNA019).

[0017] In other examples of the antibody polypeptide, the CDR1, CDR2, and CDR3+1 sequences are (a) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile- Arg-Arg-Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and LNPGGQIK (SEQ ID NO:87; TNA032); (b) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg- Arg-Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and QNPGGQIK (SEQ ID NO:88; TNAO33); (c) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg- Arg-Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and KNPGGQIK (SEQ ID NO:89; TNA031); (d) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg- Arg-Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and LDPGGQIK (SEQ ID NO:90; TNA028); (e) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg- Arg-Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and LNEGGQIK (SEQ ID NO:91; TNA035); (f) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg- Arg-Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and LNQGGQIK (SEQ ID NO:92; TNA036); (g) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg- Arg-Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and QDPGGQIK (SEQ ID NO:93; TNA030); (h) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg- Arg-Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and LNRGGQIK (SEQ ID NO:94; TNAO38); (i) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg- Arg-Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and LNTGGQIK (SEQ ID NO:95; TNA037); or (j) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg- Arg-Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and KDPGGQIK (SEQ ID NO:96; TNA019).

[0018] In some examples of the antibody polypeptide, the single variable domain comprises (a) a framework 1 (FR1) sequence having at least 80% sequence identity to QVQLVESGGGLVQPGGSLTLSCAASGFDFG (SEQ ID NO:33); (b) a framework 2 (FR2) sequence having at least 80% sequence identity to WVRQTPGKGLEWVS (SEQ ID NO:34); (c) a framework 3 (FR3) sequence having at least 80% sequence identity to RFTVSRDNAKNMMYLQMNSLASEDTAVYYCA (SEQ ID NO:35), and (d) a framework 4 (FR4) sequence having at least 80% sequence identity to KGQGTQVTVSS (SEQ ID NO:36) for CDR3 or a framework 4 (FR4) sequence having at least 80% sequence identity to GQGTQVTVSS (SEQ ID NO:99) for CDR3+1. In certain examples of the antibody polypeptide, the single variable domain comprises (a) a framework 1 (FR1) sequence having at least 90% sequence identity to QVQLVESGGGLVQPGGSLTLSCAASGFDFG (SEQ ID NO:33); (b) a framework 2 (FR2) sequence having at least 90% sequence identity to WVRQTPGKGLEWVS (SEQ ID NO:34); (c) a framework 3 (FR3) sequence having at least 90% sequence identity to RFTVSRDNAKNMMYLQMNSLASEDTAVYYCA (SEQ ID NO:35), and (d) a framework 4 (FR4) sequence having at least 90% sequence identity to KGQGTQVTVSS (SEQ ID NO:36) for CDR3 or a framework 4 (FR4) sequence having at least 80% sequence identity to GQGTQVTVSS (SEQ ID NO:99) for CDR3+1.

[0019] In some examples, the single variable domain can be humanized. In some examples, the single variable domain further comprises a hinge sequence at the carboxy terminus (C- terminus. In some examples, the hinge sequence can be EPKTPKPQ (SEQ ID NO: 6), EPKTPKPQPQPQ (SEQ ID NO:75) or AHHSEDPS (SEQ ID NO:83).

[0020] The present disclosure also provides a polynucleic acid encoding the antibody polypeptide comprising a single variable domain that specifically binds a human Tumor Necrosis Factor- alpha (TNF-a). In some examples, the polynucleic acid further encodes a secretion leader sequence fused in frame to the antibody polypeptide. In certain examples, the secretion leader is an endogenous L. lactis secretion leader sequence or variants thereof. In some examples, the polynucleic acid further comprises an hllA promoter (P/z/ZA) driving expression of the antibody polypeptide or the antibody polypeptide fused with the secretion leader.

[0021] Further provided is a polycistronic expression unit comprising, in 5’ to 3’ order (a) a promoter endogenous to a Gram-positive bacterium; (b) a gene endogenous to the Gram-positive bacterium; (c) an intergenic region active in the Gram-positive bacterium; and (d) a polynucleic acid encoding antibody polypeptide as described above. In certain examples, the endogenous gene is: eno, gapB, usp45, rplS, pyk, rpmB, pdhD, sodA, or tufA. In certain examples of the polycistronic expression unit, the endogenous promoter is a native promoter of the endogenous gene. In certain examples, the intergenic region is the intergenic region preceding rplW, rplP, rpmD, rplB, rpsG, rpsE, rplN, rplM, rplE, or rplF.

[0022] Also provided is a vector comprising any of the polynucleic acids or polycistronic expression unit described above and herein.

[0023] In yet another aspect, the present disclosure provides a Gram-positive bacterium comprising the polynucleic acid. In some examples, the Gram-positive bacterium is a lactic acid bacterium (LAB), a Bifidobacterium, or a Staphylococcus. In an example, the Gram-positive bacterium is L. lactis. A pharmaceutical composition comprising the Gram-positive bacterium is also provided.

[0024] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising an isolated antibody, or antigen binding portion thereof comprising a single variable domain that specifically binds a human Tumor Necrosis Factor-alpha (TNF-a) as described herein, and optionally a pharmaceutically acceptable carrier.

[0025] In a further aspect, the present disclosure also provides therapeutic methods for treating an inflammatory condition or a pathological condition in a patient in need thereof. The method comprises administering to the patient a therapeutically effective amount of the disclosed Gram-positive bacterium or a pharmaceutical composition comprising the bacterium. In other embodiments, the method comprises administering to the patient a therapeutically effective amount of an isolated antibody, or antigen binding portion thereof of the disclosure or a pharmaceutical composition comprising the an isolated antibody, or antigen binding portion thereof. In some embodiments, the inflammatory condition is mucosal inflammation, skin inflammation, inflammatory bowel disease (IBD), Crohn’s disease (CD), ulcerative colitis (UC), psoriasis, irritable bowel syndrome (IBS), oral mucositis (OM), recurrent aphthous stomatitis (RAS), mTOR inhibitor associated stomatitis (mlAS), graft-versus-host disease (GVHD), oral pemphigus vulgaris (OPV), oral lichen planus (OLP), mucous membrane pemphigoid (MMP), vulvar lichen planus (VLP), vulvar lichen sclerosus (VLS), vulvar lichen simplex (VLSi), vulvar pemphigus vulgaris (VPV), Lipschiitz ulcer, vulvodynia, granulomatosis with polyangiitis (GPA), alopecia, lung inflammation, ocular inflammation, or inflammatory pain. In some embodiments, the pathological condition is myalgic encephalomyelitis, chronic fatigue syndrome (CFS), depression, or Parkinson’s disease (PD).

[0026] The present disclosure further provides a microorganism as described herein (e.g., an LAB in accordance with any of the above embodiments), a composition as described herein, or a pharmaceutical composition as described herein, for use in the treatment of an inflammatory condition or a pathological condition.

[0027] The present disclosure further provides a microorganism as described herein (e.g., an LAB in accordance with any of the above embodiments), a composition as described herein, or a pharmaceutical composition as described herein, for use in the preparation of a medicament, e.g., for the treatment of a disease, e.g., an inflammatory condition or a pathological condition.

[0028] The current disclosure further provides kits containing (1) a microorganism e.g., an LAB) according to any of the embodiments disclosed herein, a composition containing a microorganism (e.g., LAB) according to any of the embodiments described herein, a pharmaceutical composition containing a microorganism (e.g., LAB) according to any of the embodiments described herein, or a unit dosage form containing a microorganism (e.g., LAB) according to any of the embodiments described herein; and (2) instructions for administering the microorganism (e.g., LAB), the composition, the pharmaceutical composition, or the unit dosage form to a mammal, e.g., a human e.g., a human patient).

BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 depicts a schematic overview of the VHHTNAOOlh expression module from pAGX2701. The primers used to amplify the insert are indicated by oAGX codes oAGX0169 and oAGX5534 and yield a 1041 base pair (bp) PCR fragment.

[0030] Figures 2A and 2B collectively depict the sequence of the pAGX2701 expression module (SEQ ID NO: 159), amplified with oAGX0169 and oAGX5534 , as shown in Figure 1. The CDRs of VHHTNAOOlh (SEQ ID NO:84) are labeled and are underlined. The expression module is under the control of the constitutive promoter of the HU-like DNA-binding protein gene (PhllA; Gene ID: 4797353). Open reading frames are indicated with amino acids in single letter code. SS21 (SEQ ID NO:41) is the signal peptide sequence (also referred to as secretion signal sequence and secretion leader sequence herein) fromps356 endolysin (Gene ID: 4798697; UniProtKB A2RJJ4; amino acids 1-26). The hinge sequence (SEQ ID NO:6) is indicated. Em’ (SEQ ID NO: 160) = incomplete erythromycin gene.

[0031] Figures 2C and 2D collectively depict the sequence of the pAGX2702 expression module (SEQ ID NO:161), amplified with oAGX0169 and oAGX5534, as shown in Figure 1. The CDRs of VHHTNA002h (SEQ ID NO:85) are labeled and are underlined. The expression module is under the control of the constitutive promoter of the HU-like DNA-binding protein gene (PhllA; Gene ID: 4797353) (not shown). Open reading frames are indicated with amino acids in single letter code. SS21 (SEQ ID NO:41) is the signal peptide sequence (also referred to as secretion signal sequence and secretion leader sequence herein) from ps356 endolysin (Gene ID: 4798697; UniProtKB A2RJJ4; amino acids 1-26). The hinge sequence (SEQ ID NO:75) is indicated. Em‘ (SEQ ID NO: 162)= incomplete erythromycin gene.

[0032] Figures 2E and 2F collectively depict the sequence of the pAGX2703 expression module (SEQ ID NO: 163), amplified with oAGX0169 and oAGX5534, as shown in Figure 1. The CDRs of VHHTNA003h (SEQ ID NO:86 are labeled and are underlined. The expression module is under the control of the constitutive promoter of the HU-like DNA-binding protein gene (PhllA; Gene ID: 4797353) (not shown). Open reading frames are indicated with amino acids in single letter code. SS09 (SEQ ID NO:45) is the signal peptide sequence (also referred [0033] to as secretion signal sequence and secretion leader sequence) from gamma- glutamyl-diamino acid-endopeptidase-(Gene ID: 4798983; UniProtKB A2RLK0). The hinge sequence (SEQ ID NO:75) is indicated. Em‘ (SEQ ID NO: 164) = incomplete erythromycin gene.

[0034] Figure 3 depicts partial amino acid sequences of VHHTNAOOlh (amino acids 89- 123 of SEQ ID NO:7), VHHTNA002h (amino acids 89-137 of SEQ ID NO:71), and VHHTNA003h (amino acids 89-136 of SEQ ID NO:76). Identical sequences are boxed, the hinge region is indicated in bold, and CDR3 is underlined.

[0035] Figure 4 depicts a western blot of indicated VHH configurations with the CDR3 exchanged. Equivalents of 1 ml of culture supernatant of L. lactis strains carrying pTINX, pAGX2701, pAGX2906, or pAGX2907 were analyzed. Recombinant sdAbs (single domain antibodies) were detected with goat anti-llama IgG, followed by IRDye 800CW donkey antigoat IgG. Fluorescent signals were visualized by Odyssey CLx. Expected molecular weights of recombinant VHH are indicated in the left column MG1363[pAGX2701] serves as the positive, and MG1363[pTlNX] as the negative control. Reference molecular weight markers of 15 kDa and 25 kDa are indicated by arrowheads.

[0036] Figures 5A-5F collectively depict absorbance data obtained from a screening ELISA for hTNF-a binding from individual colonies of random mutagenesis of a position of CDR3+1 (indicates CDR3 and including the C-terminal flanking amino acid, which is lysine for this example; the “+1” is the N-terminal amino acid of the FR4 and not an additional inserted amino acid) (KDFGGQIK; SEQ ID NO:8) of VHHTNAOOlh. The data are the absorbance read at 450 nm for measuring (A450). Absorbance was read at 595 nm as reference. Well Al on each 96 well plate is the positive control. Well A2 on each 96 well plate is the negative control. 0.1% casein was added to A3 on each 96 well plate. High binding clones are stippled (A450 sample > average A450 of positive control x 1.2 = 0.422). Intermediate binding clones are bolded (A450 sample between A450 of positive control and 0.422). Figure 5A depicts data for clones having KI randomly mutated (SEQ ID NO: 100). Figure 5B depicts data for clones having D2 randomly mutated (SEQ ID NO: 105). Figure 5C depicts data for clones having F3 randomly mutated (SEQ ID NO: 107). Figure 5D depicts data for clones having Q6 randomly mutated (SEQ ID NO: 108). Figure 5E depicts data for clones having 17 randomly mutated (SEQ ID NO: 110). Figure 5F depicts data for clones having K8 randomly mutated (SEQ ID NO: 113). [0037] Figure 6 depicts an overview of all the identified amino acids that increase hTNF-oc binding and their corresponding codons. The original amino acid for each position is indicated in the column “pAGX2701.” As defined in Figures 5A-5F, high hTNF-oc binders and intermediate binders are indicated in the table header as “High” and “Medium,” respectively.

[0038] Figures 7A-7E collectively depict an overview of hTNF-oc binding of the identified single mutants of VHHTNAOOlh. In each graph, the positive control (red) is MG1363[pAGX2701] with CDR3+1 = KDFGGQIK (SEQ ID NO:8), and the negative control (olive green) is MG1363[pTlNX]. In Figures 7A-7D, the graph header indicates the CDR3+1 with the mutated amino acid X (respectively, SEQ ID NO: 100; SEQ ID NO: 107; SEQ ID NO: 108; SEQ ID NO: 110). The amino acid change and corresponding codon are detailed in the legend. In Figure 7E, the respective CDR3+1 is indicated in the legend, and the mutated amino acid is underlined (Sequences in legend, from top to bottom, SEQ ID NO:8; SEQ ID NO: 106; SEQ ID NO: 124). Figure 7A depicts data for seven clones having KI mutated (pAGX2908d). Figure 7B depicts data for clones having F3 mutated (pAGX2910d). Figure 7C depicts data for clones having Q6 mutated (pAGX2911d). Figure 7D depicts data for clones having 17 mutated (pAGX2912d). Figure 7E depicts data for a clone having D2 mutated (pAGX2909d) and a clone having K8 mutated (pAGX2913d).

[0039] Figure 8 depicts a western blot of all ten single mutants of single domain antibody (sdAb) VHHTNAOOlh (KDFGGQIK (pAGX2701; SEQ ID NO:8). Sequences in the right column, from top to bottom, SEQ ID NO:8; SEQ ID NO: 104; SEQ ID NO: 101 ; SEQ ID NO: 102; SEQ ID NO:103; SEQ ID NO:106; SEQ ID NO:96; SEQ ID NO:109; SEQ ID NO:111; SEQ ID NO: 112; SEQ ID NO: 114. The sdAbs are indicated by plasmid name (pTINX or pAGXNNNN). Expected molecular weights of recombinant VHH are indicated in the left and right columns. VHHTNAOOlh (original wild type CDR3+1; pAGX2701) serves as a positive control and [pTINX] as a negative control. Reference molecular weight markers (MWM) of 15 kDa and 25 kDa are indicated by arrowheads.

[0040] Figures 9A and 9B depict an overview binding to hTNF-a of 10 selected single mutants. For each mutant, the amino acid changed in the CDR3+1 is underlined. pAGX2701 serves as a positive control (VHHTNAOOlh; original wild-type CDR3+1) and pTINX as a negative control. A450: absorbance at 450 nm. Figure 9A depicts binding data for sdAbs mutated at position 1, 2, or 3 of CDR3+1 of VHHTNAOOlh. Sequences in legend, from top to bottom, SEQ ID NO:8; SEQ ID NO:102; SEQ ID NO:103; SEQ ID NO:104; SEQ ID NO:101; SEQ ID NO: 106; SEQ ID NO:96 . Figure 9B depicts binding data for sdAbs mutated at position 6, 7, or 8 of CDR3+1 of VHHTNAOOlh. Sequences in legend, from top to bottom, SEQ ID NO:8; SEQ ID NO: 109; SEQ ID NO: 111 ; SEQ ID NO: 112; SEQ ID NO: 114.

[0041] Figure 10 depicts the expression of crude culture supernatants of selected single, double and triple mutants. pTINX serves as the negative control, and pAGX2701 (original CDR3+1, wild type = wt) is used as the positive control. The amino acid sequence by single letter code is indicated in the column after the plasmid name. For each mutant, the amino acid changes in CDR3+1 are underlined. Expected sizes in kDa are indicated in the left column. Right panel: protein gel stained with Imperial Protein Stain. Markl2 unstained standard was used as Molecular Weight Marker (MWM). 14.4 kDa and 55.4 kDa reference markers are indicated by arrowheads. Left panel: western blot. Reference molecular weight markers of 15 kDa and 25 kDa are indicated by arrowheads. Sequences, from top to bottom, SEQ ID NO:8; SEQ ID NO:102; SEQ ID NO:103; SEQ ID NO:106; SEQ ID NO:96; SEQ ID NO:112; SEQ ID NO: 114; SEQ ID NO: 115; SEQ ID NO: 116; SEQ ID NO:93; SEQ ID NO:89; SEQID NO: 117; SEQ ID NO:87; SEQ ID NO:88.

[0042] Figure 11 depicts binding to hTNF-a of expression samples from mutants derived from pAGX2933. A 1/2 dilution series of culture supernatant (BRPMIE) was analyzed for binding to hTNF-a. Bound single domain antibodies were detected with MonoRab™ rabbit anti-camelid VHH IgG, followed by goat anti-rabbit IgG-HRP and TMB solution for detection. The reaction was stopped by adding 0.5 M HC1, and the absorbance was read at 450 nm for measuring and 595 nm as reference. For each mutant, the amino acid changes in CDR3+1 are underlined. MG1363[pAGX2701] serves as positive control (original CDR3+1, wt) and MG1363[pTlNX] as negative control. Sequences in legend, from top to bottom, SEQ ID NO:8; SEQ ID NO: 102; SEQ ID NO: 106; SEQ ID NO:96; SEQ ID NO:93; SEQ ID NO:89; SEQ ID NO:116; SEQ ID NO:88.

[0043] Figure 12 depicts hTNF-a neutralization of mutants derived from pAGX2933. A 1/3 dilution series of culture supernatant (BRPMIE), mixed with an equal volume of 10 lU/ml hTNF a and 2 pg/ml Actinomycin D, was added to a suspension of L929 cells. Untreated cells and cells treated with hTNF a and Actinomycin D served as controls. CellTiter 96® Aqueous One Solution Reagent was added after 20 to 24 h incubation. The absorbance was read at 490 nm for measuring and 700 nm as reference to determine the amount of cell death. The result is expressed as “% neutralization” versus hTNF-a controls. For each mutant, the amino acid change in CDR3+1 is underlined. MG1363[pAGX2701] serves as positive control (original CDR3+1, wt). MG1363[pTlNX] serves as a negative control. Sequences in legend, from top to bottom, SEQ ID NO:8; SEQ ID NO: 102; SEQ ID NO: 106; SEQ ID NO:96; SEQ ID NO: 116; SEQ ID NO:93; SEQ ID NO:89; SEQ ID NO:88.

[0044] Figure 13 depicts binding to hTNF-a of expression samples from mutants derived from pAGX2934. A 1/2 dilution series of culture supernatant (BRPMIE) was analyzed for binding to hTNF-a. Bound single domain antibodies were detected with MonoRab™ rabbit anti-camelid VHH IgG, followed by goat anti-rabbit IgG-HRP and TMB solution for detection. The reaction was stopped by adding 0.5 M HC1, and the absorbance was read at 450 nm for measuring and 595 nm as reference. For each mutant, the amino acid changes in CDR3+1 are underlined. MG1363[pAGX2701] serves as positive control (original CDR3+1, wt) and MG1363[pTlNX] as negative control. indicates these data are from a later expression study. Sequences in legend, from top to bottom, SEQ ID NO:8; SEQ ID NO: 103; SEQ ID NO: 105; SEQ ID NO:96; SEQ ID NO:90; SEQ ID NO:89; SEQ ID NO:115; SEQ ID NO:87.

[0045] Figure 14 depicts hTNF-a neutralization data of mutants derived from pAGX2934. A 1/3 dilution series of culture supernatant (BRPMIE), mixed with an equal volume of 10 lU/ml hTNF-a and 2 pg/ml Actinomycin D, was added to a suspension of L929 cells. Untreated cells and cells treated with hTNF-a and Actinomycin D served as controls. CellTiter 96® Aqueous One Solution Reagent was added after 20 to 24 h incubation, and the absorbance was read at 490 nm for measuring and 700 nm as a reference to determine the amount of cell death. The result is expressed as “% neutralization” versus hTNF-a controls. For each mutant, the amino acid change in CDR3+1 is underlined. MG1363[pAGX2701] serves as positive control (original CDR3+1, wt) and MG1363[pTlNX] as negative control. indicates that these data are from a later expression study, while “2” indicates a combination of dilutions from two assays. Sequences in legend, from top to bottom, SEQ ID NO:8; SEQ ID NO: 103; SEQ ID NO: 106; SEQ ID NO:96; SEQ ID NO:90; SEQ ID NO:89; SEQ ID NO: 115; SEQ ID NO:87.

[0046] Figure 15 depicts data for hTNF-a binding of expression samples from single and double mutants (second half of CDR3+1). A 1/2 dilution series of culture supernatant (BRPMIE) was analyzed for binding to hTNF-a. Bound single domain antibodies were detected with MonoRab™ rabbit anti-camelid VHH IgG, followed by goat anti-rabbit IgG-HRP and TMB solution for detection. The reaction was stopped by adding 0.5 M HC1, and the absorbance was read at 450 run for measuring and 595 nm as reference. For each mutant, the amino acid changes in CDR3+1 are underlined. MG1363[pAGX2701] serves as the positive control (original CDR3+1, wt) and MG1363[pTlNX] as the negative control. Sequences in legend, from top to bottom, SEQ ID NO:8; SEQ ID NO: 112; SEQ ID NO: 114; SEQ ID NO: 117; SEQ ID NO: 115; SEQ ID NO:89.

[0047] Figure 16 depicts hTNF-a neutralization data of single and double mutants in the second half of CDR3+1. A 1/3 dilution series of culture supernatant (BRPMIE), mixed with an equal volume of 10 lU/ml hTNF-a and 2 pg/ml Actinomycin D, was added to a suspension of L929 cells. Untreated cells and cells treated with hTNF-a and Actinomycin D served as controls. After 20 to 24 h incubation CellTiter 96® Aqueous One Solution Reagent was added, and the absorbance was read at 490 nm for measuring and 700 nm as a reference to determine the amount of cell death. The result is expressed as % neutralization versus hTNF-a controls. For each mutant, the amino acid change in CDR3+1 is underlined. MG1363[pAGX2701] serves as the positive control (original CDR3+1, wt), and MG1363[pTlNX] serves as the negative control. Sequences in legend, from top to bottom, SEQ ID NO:8; SEQ ID NO: 112; SEQ ID NO: 114; SEQ ID NO: 117; SEQ ID NO: 115; SEQ ID NO:89.

[0048] Figures 17A-17B depict absorbance data from screening ELISA for hTNF-a binding of double mutants. Figure 17A: LXFGGQIK (SEQ ID NO: 118). Figure 17B: QXFGGQIK (SEQ ID NO: 119). There were two positive controls. Positive control 1, related to the single mutant that was used for generating the new mutations (well A4 on each 96 well plate); LDFGGQIK (SEQ ID NO: 103) in plate LXFGGQIK and QDFGGQIK (SEQ ID NO: 102) in plate QXFGGQIK). KDFGGQIK (wt; MG1363[pAGX2701]; SEQ ID NO:8) was used as positive control 2 (well Al on each 96 well plate). MG1363[pTlNX] was used as negative control (well A2 on each 96 well plate). 0.1% casein was added to A3 on each 96 well plate. Absorbance was read at 450 nm (A450) for measuring. Clones selected for Sanger sequencing are bolded (intermediate binding: A450 sample between 3 times A450 of positive control 2 and 2.5).

[0049] Figures 18A-18B depict absorbance data from screening ELISA for hTNF-a binding of triple mutants. Figure 18A: LNXGGQIK (SEQ ID NO: 120). Figure 18B: QNXGGQIK (SEQ ID NO: 121). There were two positive controls. Positive control 1, related to the single mutant that was used for generating the new mutations (well A4 on each 96 well plate); LDFGGQIK (SEQ ID NO: 103) in plate LNXGGQIK and QDFGGQIK (SEQ ID NO: 102) in plate QNXGGOIK). KDFGGQIK (CDR3+1, wt; SEQ ID NO:8; MG1363[pAGX2701]) was used as positive control 2 (well Al on each 96 well plate). MG1363[pTlNX] was used as negative control (well A2 on each 96 well plate). 0.1% casein was added to A3 on each 96 well plate. Absorbance was read at 450 nm (A450) for measuring. Clones selected for Sanger sequencing are stippled for high binders (high binding: A450 sample > average A450 of positive control 1 x 1.2 = 2.5) and bolded for intermediate binders (intermediate binding: A450 sample between 3 times A450 of positive control 2 and 2.5).

[0050] Figure 19 depicts an overview of all herein- identified amino acids that increase hTNF-oc binding with their corresponding codons. The original amino acid with corresponding codon for both positions is indicated in the column “original.” As described in Figures 17A- 17B and Figures 18A-18B, high binders (“High”) and intermediate binders (“Intermediate”) are indicated in the table header. indicates binders selected for expression. Sequences in the top row of table, from left to right, SEQ ID NO: 118; SEQ ID NO: 119; SEQ ID NO: 120; SEQ ID NO:121.

[0051] Figure 20 depicts binding to hTNF-a data of double mutants derived from single mutant LDFGGQIK (SEQ ID NO: 103). A450: absorbance at 450 nm. For each mutant, the amino acid changes in CDR3+1 are underlined. MG1363[pAGX2934] serves as a positive control (LDFGGQIK; SEQ ID NO: 103), MG1363[pAGX2701] serves as the reference (KDFGGQIK, original CDR3+1, wt; SEQ ID NO:8), and MG1363[pTlNX] serves as the negative control. Sequences in legend, from top to bottom, SEQ ID NO:8; SEQ ID NO: 103; SEQ ID NO: 115; SEQ ID NO: 115; SEQ ID NO: 122; SEQ ID NO: 122; SEQ ID NO: 122; SEQ ID NO:-123.

[0052] Figure 21 depicts a protein gel stained with Imperial Protein Stain of expression samples of VHH domains in crude culture supernatants of mutants with multiple codons. MG1363[pTlNX] serves as negative control and to set the reference for the signal intensity of Usp45. Codons for the mutated amino acid are indicated in the column after the plasmid name. For each mutant, the amino acid changes in CDR3+1 are underlined. Expected sizes in kDa are indicated in the left column. Markl2 unstained standard was used as the Molecular Weight Marker (MWM), with the 14.4 kDa and 55.4 kDa reference markers are indicated by arrowheads. All protein bands corresponding to VHH domains or Usp45 are enclosed by a rectangle and marked accordingly. The signal intensity of each band was visualized by Odyssey CLx and is indicated in the column VHH or Usp45, respectively. To correct for small differences in expression and loading, the VHH signal is normalized versus Usp45 and by setting Usp45 of MG1363[pTlNX] as reference. Sequences from top to bottom, SEQ ID NO: 115; SEQ ID NO: 122; SEQ ID NO:94.

[0053] Figure 22 depicts overview of hTNF-a neutralization data of double mutants derived from single mutant LDFGGQIK (SEQ ID NO: 103). A 1/3 dilution series of culture supernatant (BRPMIE), mixed with an equal volume of 10 lU/ml hTNF-a and 2 pg/ml Actinomycin D, was added to a suspension of L929 cells. Untreated cells and cells treated with hTNF-a and Actinomycin D served as controls. After 20 to 24 h incubation, CellTiter 96® Aqueous One Solution Reagent was added, and the absorbance was read at 490 nm for measuring and 700 nm as a reference to determine the amount of cell death. The result is expressed as % neutralization versus hTNF-a controls. For each mutant, the amino acid change in CDR3+1 is underlined. MG1363[pAGX2701] serves as positive control (original CDR3+1, wt) and MG1363[pTlNX] serves as negative control. Sequences in legend, from top to bottom, SEQ ID NO:8; SEQ ID NO: 103; SEQ ID NO: 115; SEQ ID NO: 115; SEQ ID NO: 122; SEQ ID NO: 122; SEQ ID NO: 122; SEQ ID NO: 123.

[0054] Figure 23 depicts protein gel stained with Imperial Protein Stain of expression samples of VHH domains in crude culture supernatants of a double mutant with 2 different codons. MG1363[pTlNX] serves as the negative control and to set the reference for the signal intensity of Usp45. Codons for the mutated amino acid are indicated in the column after the plasmid name. The amino acid changes in CDR3+1 are underlined. Expected sizes in kDa are indicated in the left column. Markl2 unstained standard was used as Molecular Weight Marker (MWM; 14.4 kDa and 55.4 kDa reference markers are indicated by arrowheads). All protein bands corresponding to VHH domains or Usp45 are enclosed by a rectangle and marked accordingly. The signal intensity of each band was visualized by Odyssey CLx and is indicated in the column VHH or Usp45, respectively. To correct for the minor differences in expression and loading, the VHH signal is normalized versus Usp45 and by setting Usp45 of MG1363[pTlNX] as reference. Sequences from top to bottom, SEQ ID NO:8; SEQ ID NO: 102.

[0055] Figure 24 depicts hTNF-a neutralization data (left y-axis) and binding to hTNF-a (right y-axis) of single mutants QDFGGQIK (SEQ ID NO: 102). For each mutant, the amino acid changes in CDR3+1 are underlined. MG1363[pAGX2933] serves as the positive control (QDFGGQIK; SEQ ID NO: 102), MG1363[pAGX2701] serves as the reference (KDFGGQIK, original CDR3+1, wt; SEQ ID NO:8), and MG1363[pTlNX] as the negative control. Sequences in legend, from top to bottom, SEQ ID NO:8; SEQ ID NO: 102; SEQ ID NO: 102; SEQ ID NO:8; SEQ ID NO: 102; SEQ ID NO: 102.

[0056] Figure 25 depicts binding to hTNF-a data of triple mutants of LNXGGQIK (SEQ ID NO: 120). A450: absorbance read at 450 nm. For each mutant, the amino acid changes in CDR3+1 are underlined. Double mutant LNFGGQIK (SEQ ID NO: 115) serves as the positive control (MG1363[pAGX2939]), MG1363[pAGX2701] serves as the reference (KDFGGQIK, original CDR3+1, wt; SEQ ID NO:8), and MG1363[pTlNX] as the negative control. Sequences in legend, from top to bottom, SEQ ID NO:8; SEQ ID NO: 115; SEQ ID NO: 115; SEQ ID NO:87; SEQ ID NO:87; SEQ ID NO:92; SEQ ID NO:91; SEQ ID NO:95; SEQ ID NO:94; SEQ ID NO: 156; SEQ ID NO: 157; SEQ ID NO: 158.

[0057] Figure 26 depicts hTNF-a neutralization data of triple mutants derived from LNXGGQIK (SEQ ID NO: 120). The result is expressed as % neutralization versus hTNF-a controls. For each mutant, the amino acid change in CDR3+1 is underlined. MG1363[pAGX2701] serves as the positive control (original CDR3+1, wt), and MG1363[pTlNX] serves as the negative control. Sequences in legend, from top to bottom, SEQ ID NO:8; SEQ ID NO:115; SEQ ID NO:115; SEQ ID NO:87; SEQ ID NO:87; SEQ ID NO:91; SEQ ID NO:92; SEQ ID NO:94; SEQ ID NO:95; SEQ ID NO: 156; SEQ ID NO: 157; SEQ ID NO:158.

[0058] Figure 27 depicts binding to hTNF-a data of single, double, and triple mutants related to QDFGGQIK (SEQ ID NO: 102). A 1/2 dilution series of culture supernatant (BRPMIE) was analyzed for binding to hTNF-a. Bound single domain antibodies were detected with MonoRab™ rabbit anti-camelid VHH IgG, followed by goat anti-rabbit IgG-HRP and TMB solution for detection. The reaction was stopped by adding 0.5 M HC1, and the absorbance was read at 450 nm for measuring and 595 nm as reference. For each mutant, the amino acid changes in CDR3+1 are underlined. MG1363[pAGX2933] serves as the positive control (QDFGGQIK; SEQ ID NO: 102), MG1363[pAGX2701] serves as the reference (KDFGGQIK, original CDR3+1, wt; SEQ ID NO:8), and MG1363[pTlNX] as the negative control. Sequences in legend, from top to bottom, SEQ ID NO:8; SEQ ID NO: 102; SEQ ID NO: 106; SEQ ID NO:96; SEQ ID NO: 116; SEQ ID NO:93; SEQ ID NO:89; SEQ ID NO:88; SEQ ID NO:88.

[0059] Figure 28 depicts hTNF-a neutralization data of single, double, and triple mutants related to QDFGGQIK (SEQ ID NO: 102). A 1/3 (one-third) dilution series of culture supernatant (BRPMIE), mixed with an equal volume of 10 lU/ml hTNF-a and 2 pg/ml Actinomycin D, was added to a suspension of L929 cells. Untreated cells and cells treated with hTNF-a and Actinomycin D served as controls. After 20 to 24 h incubation, CellTiter 96® Aqueous One Solution Reagent was added, and the absorbance was read at 490 nm for measuring and 700 nm as a reference to determine the amount of cell death. The result is expressed as % neutralization versus hTNF-a controls. For each mutant, the amino acid change in CDR3+1 is underlined. MG1363[pAGX2701] serves as the positive control (original CDR3+1, wt), and MG1363[pTlNX] serves as the negative control. Sequences in legend, from top to bottom, SEQ ID NO:8; SEQ ID NO: 102; SEQ ID NO: 106; SEQ ID NO:96; SEQ ID NO:116; SEQ ID NO:93; SEQ ID NO:89; SEQ ID NO:88; SEQ ID NO:88.

[0060] Figure 29 depicts the expression of VHH domains by L. lactis strains comprising the indicated plasmid (pTINX or pAGX plasmid name) in crude culture supernatants of a selection of mutants. Expression was analyzed by equivalents of 1 ml of crude culture supernatant of the L. lactis strains. Right panel: protein gel stained with Imperial Protein Stain. Markl2 unstained standard was used as the Molecular Weight Marker (MWM). 14.4 kDa and 55.4 kDa reference markers are indicated by arrowheads. Left panel: western blot. Reference molecular weight markers of 15 kDa and 25 kDa are indicated by arrowheads. MG1363[pTlNX] serves as the negative control, and MG1363[pAGX2701] (original CDR3+1, wild type = wt) was used as the positive control. The amino acid sequence by single letter code is indicated in the column to the right of the column listing the plasmid name. For each mutant, the amino acid changes in CDR3+1 are underlined. Expected sizes in kDa are indicated in the left column. Sequences in legend, from top to bottom, SEQ ID NO:8; SEQ ID NO: 102; SEQ ID NO: 103; SEQ ID NO: 106; SEQ ID NO:96; SEQ ID NO: 115; SEQ ID NO: 122; SEQ ID NO: 123; SEQ ID NO:90; SEQ ID NO: 116; SEQ ID NO:93; SEQ ID NO:89; SEQ ID NO:87; SEQ ID NO: 157; SEQ ID NO:94; SEQ ID NO:92; SEQ ID NO:91; SEQ ID NO:156; SEQ ID NO:87; SEQ ID NO:95; SEQ ID NO:158; SEQ ID NO:88; SEQ ID NO:88.

[0061] Figure 30 depicts binding to hTNF-a data of selected mutants and wild-type parent VHHTNAOOlh (pAGX2701). MG1363[pTlNX] serves as the negative control. A ₄₅₀ : absorbance at 450 nm. Sequences in legend, from top to bottom, SEQ ID NO:87; SEQ ID NO:88; SEQ ID NO:89; SEQ ID NO:90; SEQ ID NO:91; SEQ ID NO:92; SEQ ID NO:93; SEQ ID NO:94; SEQ ID NO:95; SEQ ID NO:96; SEQ ID NO:8.

[0062] Figure 31 depicts an overview of hTNF-a neutralization of selected mutants and wild-type parent VHHTNAOOlh (pAGX2701). pTINX serves as the negative control. The neutralization is expressed as % neutralization versus hTNF-a controls. MG1363[pTlNX] serves as the negative control. Sequences in legend, from top to bottom, SEQ ID NO:87; SEQ ID NO:88; SEQ ID NO:89; SEQ ID NO:90; SEQ ID NO:91; SEQ ID NO:92; SEQ ID NO:93; SEQ ID NO:94; SEQ ID NO:95; SEQ ID NO:96; SEQ ID NO:8.

DETAILED DESCRIPTION

Definitions

[0063] As used in the specification, embodiments, and claims, the singular forms “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof. Similarly, use of “a compound” for treatment or preparation of medicaments as described herein contemplates using one or more compounds of this invention for such treatment or preparation unless the context clearly dictates otherwise.

[0064] As used herein, the term “comprising” is intended to mean that the compositions and methods include the recited elements, but not excluding others. “Consisting essentially of’ when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline (PBS), preservatives, and the like. “Consisting of’ shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this invention. Embodiments defined by each of these transition terms are within the scope of this invention.

[0065] Polypeptides and nucleic acids have a form of directionality when discussed in certain orientations. A nucleic acid is discussed in a 5’ to 3’ direction, or sense direction, which relates for example, to when a nucleic acid is translated into a polypeptide. A polypeptide is described in an amino terminal (N-terminal or N-terminus) to carboxy terminal (C-terminal or C-terminus) orientation. Polypeptide sequences that reflect the use of, for example, Xi, X2, and X3 describe what options can occur at that sequence. For example, Leu34Gly, indicates a substitution at position 34 of a polypeptide sequence of a leucine for a glycine.

[0066] As used herein, the term “expressing” a gene or polypeptide or “producing” a polypeptide (e.g., a TNF-alpha antibody and one or more bioactive polypeptides), or “secreting” a polypeptide is meant to include “capable of expressing” and “capable of producing,” or “capable of secreting,” respectively. For example, a microorganism that contains an exogenous nucleic acid can, under sufficient conditions (e.g., sufficient hydration and/or in the presence of nutrients), express and secrete a polypeptide encoded by an exogenous nucleic acid. However, the microorganism may not always actively express the encoded polypeptide. The microorganism e.g., bacterium) may be dried (e.g., freeze-dried) and in that state can be considered dormant (i.e., is not actively producing polypeptide). However, once the microorganism is subjected to sufficient conditions, e.g., is administered to a subject and is released (e.g., in the gastro-intestinal tract of the subject), it may begin expressing and secreting polypeptide. Thus, a microorganism “expressing” a gene or polypeptide, “producing” a polypeptide, or “secreting” a polypeptide of the current disclosure includes the microorganism in its “dormant” state in which biochemical processes within the microorganism are substantially slowed down or halted. As used herein, “secrete” means that the protein is exported outside the cell and into the culture medium/supernatant or other extracellular milieu.

[0067] As used herein, the term “constitutive” in the context of a promoter (or by extension relating to gene expression or secretion of a polypeptide) refers to a promoter that allows for continual transcription of its associated gene. A constitutive promoter compares to an “inducible” promoter.

[0068] As used herein, the terms “inducible” and “inducibly” in the context of a promoter (or by extension relating to gene expression or secretion of a polypeptide) refer to a promoter that allows for increased transcription of the gene it is operably linked to when in the presence of an inducer of said promoter.

[0069] The term “about” in relation to a reference numerical value, and its grammatical equivalents as used herein, can include the reference numerical value itself and a range of values plus or minus 10% from that reference numerical value. For example, the term “about 10” includes 10 and any amount from and including 9 to 11. In some cases, the term “about” in relation to a reference numerical value can also include a range of values plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that reference numerical value. In some embodiments, “about” in connection with a number or range measured by a particular method indicates that the given numerical value includes values determined by the variability of that method. [0070] The term “chromosomally integrated” or “integrated into a chromosome” or any variation thereof means that a nucleic acid sequence (e.g., gene; open reading frame; exogenous nucleic acid encoding a polypeptide; promoter; expression cassette; and the like) is located on (integrated into) a microbial e.g., bacterial) chromosome, i.e., is not located on an episomal vector, such as a plasmid. In some embodiments, in which the nucleic acid sequence is chromosomally integrated, the polypeptide encoded by such chromosomally integrated nucleic acid is constitutively expressed. For example, an exemplary nucleic acid sequence, which is chromosomally integrated, may inducibly express the polypeptide the integrated nucleic acid encodes.

[0071] The “percentage identity” between polypeptide sequences can be calculated using commercially available algorithms, which compare a reference sequence with a query sequence. In some embodiments, polypeptide sequence identity can be 70%, at least 70%, 75%, at least 75%, 80%, at least 80%, 85%, at least 85%, 90%, at least 90%, 92%, at least 92%, 95%, at least 95%, 97%, at least 97%, 98%, at least 98%, 99%, or at least 99% or 100% identical to a reference polypeptide, or a fragment thereof (e.g., as measured by BLASTP or CLUSTAL, or other alignment software) using default parameters. Similarly, nucleic acids can also be described with reference to a starting nucleic acid, e.g., they can be 50%, at least 50%, 60%, at least 60%, 70%, at least 70%, 75%, at least 75%, 80%, at least 80%, 85%, at least 85%, 90%, at least 90%, 95%, at least 95%, 97%, at least 97%, 98%, at least 98%, 99%, at least 99%, or 100% identical to a reference nucleic acid or a fragment thereof (e.g., as measured by BLASTN or CLUSTAL, or other alignment software using default parameters). When one molecule is said to have a certain percentage of sequence identity with a larger molecule, it means that when the two molecules are optimally aligned, the percentage of residues in the smaller molecule finds a match residue in the larger molecule in accordance with the order by which the two molecules are optimally aligned, and the “%” (percent) identity is calculated in accord with the length of the smaller molecule.

Single domain antibody (sdAb)

[0072] As used herein, “VHH” and “VHH antibody” refer to a single domain antibody (sdAb) obtained from, recombinantly designed based on, or derived from a camelid domain antibody (camelid dAb). A “VHH antibody” is also referred to in the art as a nanobody (Nb). Camelid dAbs are antibody single variable domain polypeptides, which are derived from species including camel, llama, alpaca, dromedary, and guanaco, and comprise heavy chain antibodies naturally devoid of a light chain: VHH. VHH molecules are about 10X smaller than IgG molecules, and as single polypeptides, they are very stable, resisting extreme pH and temperature conditions.

[0073] Camelid antibodies are described in, for example, U.S. Pat. Nos. 5,759,808; 5,800,988; 5,840,526; 5,874,541; 6,005,079; 6,015,695, and U.S. Patent Publication Nos. 2006/0034845A1 and 2015/0353635, the contents of each of which are incorporated herein in their entirety. Further exemplary information on camelid antibodies can be found, for instance, in Muyldermans, S., Annu. Rev. Biochem. 2013, 82: 775-97; Muyldermans, S., Annu. Rev. Anim. Biosci. 2021, 9: 401-421.

[0074] The amino acid sequence and structure of a VHH antibody is comprised of four framework regions (FR), or framework sequences or "FR's", which are referred to in the art and herein as "Framework region 1" or "FR1"; as "Framework region 2" or"FR2"; as "Framework region 3" or "FR3"; and as "Framework region 4" or "FR4", respectively. Framework regions are separated by three complementary determining regions or "CDR's," which are referred to in the art as "Complementarity Determining Region 1" or "CDR1"; as "Complementarity Determining Region 2" or "CDR2", and as "Complementarity Determining Region 3" or "CDR3", respectively.

[0075] In particular, a camelid VHH against TNF-a, according to the present disclosure, may have the structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0076] In some aspects, the camelid VHH against TNF-a may have the structure: FR1- CDR1-FR2-CDR2-FR3-CDR3-FR4-Hinge.

[0077] Exemplary sequences for CDR1, CDR2, and CDR3 are shown in Table 1. Exemplary sequences, including framework sequences (FR1, FR2, FR3, and FR4) and hinge, are shown in Table 2. See also Table 3. In some embodiments, the CDR3 includes the C-terminal amino acid (referred to herein as “CDR3+1”). The C-terminal amino acid is the N-terminal amino acid of FR4 and is not an insertion of another amino acid. FR4 thus would be understood to have one less amino acid at its N-terminus.

[0078] As used herein, “VHHTNA###” is the general designation herein for a single variable domain antibody against TNF. The designation “VHHTNA###h” indicates the presence of the hinge region in the single variable domain antibody. Table 1

[0079] A consensus CDR3 sequence is X1X2X3GGQI (SEQ ID NO:97) where Xi is Lys,

Leu, or Gin; X2 is Asp or Asn; and X3 is Phe, Pro, Thr, Arg, Gin, or Glu. A consensus CDR3+1 sequence is X1X2X3GGQIK (SEQ ID NO:98) where Xi is Lys, Leu, or Gin; X2 is Asp or Asn; and X3 is Phe, Pro, Thr, Arg, Gin, or Glu.

Table 2

[0080] Exemplary sequences for the framework region are shown in Table 3.

Table 3

[0081] In some embodiments, the framework sequences of a VHH antibody of the disclosure are:

FR1: QVQLVESGGGLVQPGGSLTLSCAASGFDFG (SEQ ID NO:33);

FR2: WVRQTPGKGLEWVS (SEQ ID NO:34);

FR3: RFTVSRDNAKNMMYLQMNSLASEDTAVYYCA (SEQ ID NO: 35); and

FR4: KGQGTQVTVSS (SEQ ID NO:36).

[0082] In some embodiments, the framework sequences of a VHH antibody of the disclosure are:

FR1: QVQLQESGGGLVQPGGSLRLSCAASGRTFS (SEQ ID NO:37);

FR2: WFRQAPGKEREFVA (SEQ ID NO:38);

FR3: RFAISRDIAKNTVDLTMNNLEPEDTAVYYCA (SEQ ID NO: 39); and

FR4: WGQGTQVTVSS (SEQ ID NO:40).

[0083] For a VHH antibody having CDR3+1, FR4 is GQGTQVTVSS (SEQ ID NO:99), i.e., the N-terminal K of SEQ ID NO:36 or the N-terminal W of SEQ ID NO:40 is not present. [0084] A VHH antibody may be humanized. As used herein, “humanized” refers to an optimized VHH such that immunogenicity upon administration in human patients is minor or nonexistent. Humanizing a polypeptide comprises a step of replacing one or more of the Camelidae amino acids with their human counterpart as found in the human consensus sequence, without that polypeptide losing its typical character, the humanization does not significantly affect the antigen binding capacity of the resulting polypeptide. In an embodiment of a humanized form of a VHH antibody, some, most, or all of the amino acids outside the CDR domains have been replaced with amino acids from human immunoglobulins, whereas some, most or all amino acids within one or more CDR regions are unchanged. Small additions, deletions, insertions, substitutions, or modifications of amino acids are permissible as long as they do not abrogate the ability of the VHH antibody to bind to a particular antigen. A “humanized” VHH antibody retains an antigenic specificity similar to that of the original VHH antibody.

[0085] Humanized camelid VHH polypeptides are taught, for example, in U.S. Patent Publication Nos. 2006/0034845A1 and 2010/0022452, the teachings of which are incorporated herein in their entirety. See also Vincke et al., 2009, “General strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold,” J. Biol. Chem. 284(5): 3273-84. PMID: 19010777. These references provide general as well as specific guidance to specific residues in the framework regions of a VHH sdAb to mutate in order to generate humanized camelid VHH polypeptides. See, e.g., Table 2 of U.S. Patent Publication No. 2010/0022452.

[0086] A “chimeric antibody” refers to a VHH antibody in which the variable region is derived from one species, and the constant regions are derived from another species, such as a VHH antibody in which the variable region is derived from a camelid VHH and the constant regions are derived from a human antibody.

[0087] Camelid VHH sdAbs can further comprise a hinge sequence at the C-terminus of the sdAb. Exemplary hinge sequences include EPKTPKPQ (SEQ ID NO:6), EPKTPKPQPQPQ (SEQ ID NO:75) or AHHSEDPS (SEQ ID NO:83). In some embodiments, the hinge can be EPKTPKPQ (SEQ ID NO:6). The hinge sequence enhances the production and secretion of the VHH sdAb from certain host cells.

[0088] Camelid VHH sdAbs are particularly well suited to be produced by Lactococcus lactis (see, e.g., Vandenbroucke, K., et al., Mucosal Immunol. 2010, 3(1): 49-56. Signal peptide sequences (also referred to as a “secretion leader sequence” and “secretion signal sequence”) can be selected from endogenous L. lactis signal peptides. Examples of signal peptide sequences (also referred to as secretion leader sequences and secretion signal sequence) include the signal peptide sequence of L. lactis MG1363 ps356 endolysin (UniProtKB A2RJJ4; also referred to herein as SS21), N-acetylglucosaminidase/peptidoglycan hydrolase AcmD (UniProtKB A2RIL8), gamma-glutamyl-diamino acid-endopeptidase llmg_1594 (UniProt A2RLK0; also referred to herein as SS09), secreted 45 kDa protein precursor Usp45 (UniProtKB P22865) and a K4N mutation thereof, N-acetylmuramoyl-L- alanine amidase/peptidoglycan hydrolase AcmB (UniProtKB Q8KKF9), hypothetical protein/Immunogenic secreted protein homolog llmg_0904 (UniProtKB A2RJP5), hypothetical protein/putative secreted protein llmg_0918 (UniProtKB A2RJQ9), cell wall surface anchor family protein llmg_1127 (UniProtKB A2RKB1), hypothetical protein/putative secreted protein llmg_1800 (UniProtKB A2RM44), hypothetical protein/ORFlO llmg_1399 (UniProtKB A2RU19), putative transglyco sylase llmg_0760 (UniProtKB A2RJB2), cell surface antigen I/II precursor CluA (UniProtKB A2RU18), hypothetical protein/Glucosyltransferase-I llmg_0458 (UniProtKB A2RIG7), hypothetical protein/putative secreted protein llmg_0877 (UniProtKB A2RJU9) as shown in Table 4 below:

Table 4

TNF and TNF Activities

[0089] Single domain antibody polypeptides are provided that bind human Tumor Necrosis Factor-alpha (TNF-a). TNF-a is also known as cachectin, DIF, Tnfa, TNFalpha, TNF alpha, TNF-alpha, Tnfsfla, and tumor necrosis factor-alpha. Relevant structural information for human TNF-a can be found, for example, at UniProt Accession Number UniProtKB - P01375 (TNFA_HUMAN) “Human TNF-a” refers to the TNF-a comprising the following amino acid sequence:

UniProtKB - P01375 (TNFA_HUMAN) 77-233

VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPS EGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPE GAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL(SEQ ID NO: 1). The sequence of the antigen used to inject llama to generate camelid VHHs was UniProtKB - P01375 (TNFA_HUMAN) 77-233. The human TNF-alpha protein was E. coli derived and was a mixture of TNF-alpha protein with and without an N-terminal methionine. The TNF-alpha protein sequence, including the N-terminal methionine, is shown here:

MVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVV PSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETP EGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL (SEQ ID NO: 2).

[0090] Binding of an antibody polypeptide disclosed herein to TNF-a neutralizes TNF-a activity. TNF is a multifunctional pro inflammatory cytokine mainly secreted by macrophages. TNF-a can bind to, and thus functions through its receptors TNFRSF1A/TNFR1 and TNFRSF1B/TNFBR. A “TNF-a activity” thus includes, but is not limited to, binding to TNFR1 and binding to TNFR2. TNF-a is a cytokine involved in the regulation of a wide spectrum of biological processes including cell proliferation, differentiation, apoptosis, lipid metabolism, and coagulation. TNF plays a vital role in the typical immune response through the regulation of a number of pathways encompassing an immediate inflammatory reaction with significant innate immune involvement as well as cellular activation with subsequent proliferation and programmed cell death or necrosis. TNF is a potent pyrogen causing fever by direct action or by stimulation of interleukin- 1 secretion and is implicated in the induction of cachexia. Under certain conditions, it can stimulate cell proliferation and induce cell differentiation. TNF is central to the development of autoimmune disease, cancer, and protection against infectious pathogens. TNF has been implicated in a number of diseases, such as rheumatoid arthritis, ankylosing spondylitis, Crohn’s disease, autoimmune diseases, insulin resistance, psoriasis, tuberculosis, autosomal dominant polycystic kidney disease, and cancer. Mutations in TNF gene affect susceptibility to cerebral malaria, septic shock, and Alzheimer's disease. As well as myriad other activities, TNF can be a product of T cells and can act on T cells. TNF can promote the activation and proliferation of naive and effector T cells, but also can induce apoptosis of highly activated effector T cells, further determining the size of the pathogenic or protective conventional T cell pool. Moreover, TNF can have divergent effects on regulatory T cells. It can both downregulate their suppressive capacity but also contribute in other instances to their development or accumulation. Biologies that block TNF or stimulate TNFR2, therefore, have the potential to strongly modulate the balance between effector T cells and Treg cells, which [0091] could impact disease in both positive and negative manners. TNF impairs regulatory T-cells (Treg) function in individuals with rheumatoid arthritis. An antibody or a functional fragment thereof, as disclosed herein having a TNF-a neutralizing activity, would inhibit one or more of these listed activities.

[0092] The VHH antibodies of the disclosure advantageously neutralize TNF-a activity. While not limited to a particular mechanism, the described VHH antibodies are believed to bind to TNF-a and prevent or substantially inhibit TNF-a from binding to its receptors, thereby neutralizing TNF-a activity. As used herein, “neutralize TNF-a activity” refers to inhibition of TNF-a binding to TNFR1, inhibition of binding to TNF-a TNFR2, inhibition of binding to both TNFR1 and TNFR2, inhibition of at least activity resulting from TNF binding to TNFR1 and/or binding to TNFR2.

[0093] TNF-a neutralization can be assessed by an assay known in the art. An exemplary assay for identifying a VHH antibody that neutralizes TNF-a activity is as follows. A suspension of L929 cells at 2 x 10 ⁵ cells/ml is added to a flat bottom 96 well assay plate and incubated overnight at 37°C, 5% CO2 in a humidified incubator. In a separate plate an equal volume of a dilution series of the samples (dilution series of the anti-hTNF-a standard (Cimzia), negative controls, and dilution series of crude culture supernatants of the control L. lactis strain and L. lactis expressing anti-hTNF-a mutants) is mixed with an equal volume of 10 lU/ml hTNF-a and 2 pg/ml Actinomycin D. 100 pl of this mixture is added to the cells. Untreated cells and cells treated with hTNF a and Actinomycin D serve as controls. The plates are incubated for 20 to 24 h at 37°C, 5% CO2 in a humidified incubator. Afterward, 20 pl CellTiter 96® AQueous One Solution Reagent is added to the wells, and the plates are incubated for another 4 h at 37°C, 5% CO2 in a humidified incubator. The absorbance is read at 490 nm for measuring and 700 nm as a reference to determine the amount of cell death. The result is expressed as % neutralization versus hTNF a controls. Clones that show increased hTNF-a neutralization in comparison to the reference (z.e., the initial sequence used at the start of the optimization) are retained.

[0094] In some embodiments, neutralization can be assessed in vitro using an assay described in the examples; the in vitro assay can be performed with a cell supernatant from cells expressing an sdAb fused to a signal peptide, or with a purified sdAb. In an embodiment, the VHH antibody neutralizes TNF-a with at least about 90% neutralization versus hTNF-a control as assessed by the assay described in the Examples. In an embodiment, the VHH antibody neutralizes TNF-a at least about 90. 5%, at least about 91%, at least about 92%, or at least about 93% neutralization versus hTNF-a control.

[0095] In an embodiment, the VHH antibody neutralizes TNF-a in a standard assay with an IC50 of 50nM or less. Neutralization may also be assessed using the in vivo reporting assay described in Buurman, D.J., et al., PLOS ONE 2018, 13(12): e0208922.

[0096] As used herein, “specific binding” refers to the binding of an antigen by an antibody polypeptide with a dissociation constant (Ka) of about 1 pM or lower as measured, for example, by surface plasmon resonance (SPR). Suitable assay systems include the BIAcore™ surface plasmon resonance system and BIAcore™ kinetic evaluation software (e.g., version 2.1). In an embodiment, binding and Kd are assessed using a BIAcore device. Biotinylated protein is captured by the biotin CAPture reagent (hTNF-a), and then periplasmic extract samples flow over the captured protein. Binding and Kd are then determined by measuring surface plasmon resonance. The affinity or Kd for a specific binding interaction may be about 1 pM or lower, about 500 nM or lower, or about 300 nM or lower.

Construct and Expression Cassette

[0097] The described TNF-oc single domain antibody (sdAb) can be encoded by a polynucleic acid, arranged in the form of an expression cassette.

[0098] The term “expression cassette” or “expression unit” is used in accordance with its generally accepted meaning in the art, and refers to a nucleic acid containing one or more genes and sequences controlling the expression of the one or more genes. Exemplary expression cassettes contain at least one promoter sequence and at least one open reading frame.

[0099] The terms “polycistronic expression cassette” “polycistronic expression unit” or “polycistronic expression system” are used herein interchangeably and in accordance with their generally accepted meaning in the art. They refer to a nucleic acid sequence wherein the expression of two or more genes is regulated by common regulatory mechanisms, such as promoters, operators, and the like. The term polycistronic expression unit, as used herein, is synonymous with multicistronic expression unit. Examples of polycistronic expression units are without limitation bicistronic, tricistronic, and tetracistronic expression units. Any mRNA comprising two or more, such as 3, 4, 5, 6, 7, 8, 9, 10, or more, open reading frames or coding regions encoding individual expression products such as proteins, polypeptides and/or peptides is encompassed within the term polycistronic. A polycistronic expression cassette includes at least one promoter, and at least two open reading frames controlled by the promoter, wherein an intergenic region is optionally placed between the two open reading frames.

[00100] In some examples, the “polycistronic expression cassette” includes one or more endogenous genes and one or more exogenous genes that are transcriptionally controlled by a promoter that is endogenous to the microorganism (e.g., LAB). The endogenous gene may be eno, gapB, usp45, rplS, pyk, rpmB, pdhD, sodA, or lufA. The polycistronic expression unit or system as described herein can be transcriptionally controlled by a promoter that is exogenous to the microorganism e.g., LAB). By “exogenous promoter” to the microorganism is meant that the promoter contains a mutation to the endogenous promoter or is not a promoter naturally found in that microorganism. By “endogenous promoter” is meant a promoter that is naturally found in the microorganism or tied to a specific gene. In a further embodiment, the translationally or transcriptionally coupled one or more endogenous genes and one or more exogenous genes as described herein are transcriptionally controlled by the native promoter of (one of) said one or more endogenous genes. In another embodiment, the polycistronic expression unit is transcriptionally controlled by the native promoter of (one of) said one or more endogenous genes comprised in said polycistronic expression unit (i.e., an endogenous promoter of the gene). In another embodiment, the polycistronic expression unit is operably linked to a Gram-positive endogenous promoter. In an exemplary embodiment, the promoter may be positioned upstream of, i.e., 5’ of the open reading frame(s) to which it is operably linked. In a further embodiment, the promoter is the native promoter of the 5’ most, i.e., most upstream, endogenous gene in the polycistronic expression unit. Accordingly, in some examples, the polycistronic expression unit contains an endogenous gene and one or more exogenous genes transcriptionally coupled to the 3’ end of said one or more endogenous gene, for example, wherein said one or more exogenous gene(s) is (are) the most 3’ gene(s) of the polycistronic expression unit.

[00101] As used herein, the term “translationally coupled” is synonymous with “translationally linked” or “translationally connected.” These terms, in essence, relate to polycistronic expression cassettes or units. Two or more genes, open reading frames, or coding sequences are said to be translationally coupled when a common regulatory element(s) such as in particular a common promoter effects the transcription of said two or more genes as one mRNA encoding said two or more genes, open reading frames or coding sequences, which can be subsequently translated into two or more individual polypeptide sequences. The skilled person will appreciate that bacterial operons are naturally occurring polycistronic expression systems or units in which two or more genes are translationally or transcriptionally coupled.

[00102] By “promoter” is meant generally a region on a nucleic acid molecule, for example DNA molecule, to which an RNA polymerase binds and initiates transcription. A promoter is, for example, positioned upstream, i.e., 5’, of the sequence the transcription of which it controls. The skilled person will appreciate that the promoter may be associated with additional native regulatory sequences or regions, e.g. operators. The precise nature of the regulatory regions needed for expression may vary from organism to organism, but shall in general include a promoter region which, in prokaryotes, contains both the promoter (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal the initiation of protein synthesis. Such regions will normally include those 5’-non-coding sequences involved with the initiation of transcription and translation, such as the Pribnow-box (cf. TATA-box), Shine-Dalgarno sequence, and the like.

[00103] A promoter employed in the present disclosure is in some cases expressed constitutively in the bacterium. The use of a constitutive promoter avoids the need to supply an inducer or other regulatory signal for expression to take place. In some cases, the promoter directs expression at a level at which the bacterial host cell remains viable, i.e., retains some metabolic activity, even if growth is not maintained. Advantageously then, such expression may be at a low level. For example, where the expression product accumulates intracellularly, the level of expression may lead to accumulation of the expression product at less than about 10% of cellular protein, about or less than about 5%, for example, about 1-3%. The promoter may be homologous to the bacterium employed, z. e. , one found in that bacterium in nature. For example, a Lactococcal promoter may be used in a Lactococcus. An exemplary promoter for use in Lactococcus lactis (or other Lactococci) is “Pl” derived from the chromosome of Lactococcus lactis (Waterfield, N. R. et al., Gene 1995, 165( 1) :9- 15) . Other examples of a promoter include, the usp45 promoter, the gapB promoter, and the hllA promoter. Other useful promoters are described in U.S. Patent Nos. 8,759,088 and 9,920,324, the disclosures of which are incorporated herein by reference in their entirety. In some examples, a nucleic acid encoding the TNF-oc antibody described herein is placed under an hllA promoter.

[00104] The terms “signal peptide,” “signal peptide sequence,” “secretion leader sequence,” “secretion leader,” “secretion signal,” and “secretion signal sequence” are used interchangeably herein. The terms are used in accordance with their art recognized meaning and generally refer to a nucleic acid sequence, which encodes a “signal peptide” or “secretion signal peptide” or “secretion leader peptide” causes a polypeptide being expressed by a microorganism and comprising the signal peptide to be secreted by the microorganism, i.e., causes the polypeptide to leave the intracellular space, e.g., be secreted into the surrounding medium, or be anchored in the cell wall with at least a portion of the polypeptide be exposed to the surrounding medium, e.g. on the surface of the microorganism. The signal peptide may be the signal peptide from a gene selected from L. lactis MG1363 ps356 endolysin (UniProtKB A2RJJ4; SEQ ID NO:41), N-acetylglucosaminidase/peptidoglycan hydrolase AcmD (UniProtKB A2RIL8; SEQ ID NO:43), gamma-glutamyl-diamino acid-endopeptidase llmg_1594 (UniProt A2RLK0; SEQ ID NO:45), secreted 45 kDa protein precursor Usp45 (UniProtKB P22865; SEQ ID NO:47) and a K4N mutation thereof (P22865**; SEQ ID NO:49), N-acetylmuramoyl-L- alanine amidase/peptidoglycan hydrolase AcmB (UniProtKB Q8KKF9; SEQ ID NO:51), hypothetical protein/Immunogenic secreted protein homolog llmg_0904 (UniProtKB A2RJP5; SEQ ID NO:53), hypothetical protein/putative secreted protein llmg_0918 (UniProtKB A2RJQ9; SEQ ID NO:55), cell wall surface anchor family protein llmg_1127 (UniProtKB A2RKB1; SEQ ID NO:57), hypothetical protein/putative secreted protein llmg_1800 (UniProtKB A2RM44; SEQ ID NO:59), hypothetical protein/ORFlO Umg_1399 (UniProtKB A2RL19; SEQ ID NO:61), putative transglyco sylase llmg_0760 (UniProtKB A2RJB2; SEQ ID NO:63), cell surface antigen I/II precursor CluA (UniProtKB A2RL18; SEQ ID NO:65), hypothetical protein/Glucosyltransferase-I llmg_0458 (UniProtKB A2RIG7; SEQ ID NO:67), hypothetical protein/putative secreted protein llmg_0877 (UniProtKB A2RJL9; SEQ ID NO:69), and variants thereof having 1, 2, or 3 variant amino acid positions. Other signal peptides are known in the art. See, e.g., WO 2021/059240.

[00105] The term “operably linked” refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control sequence “operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. For example, a promoter is said to be operably linked to a gene, open reading frame, or coding sequence if the linkage or connection allows or effects transcription of said gene. In a further example, a 5’ and a 3’ gene, cistron, open reading frame, or coding sequence are said to be operably linked in a polycistronic expression unit if the linkage or connection allows or effects translation of at least the 3’ gene. For example, DNA sequences, such as, e.g., a promoter and an open reading frame (ORF), are said to be operably linked if the nature of the linkage between the sequences does not (1) result in the introduction of a frame- shift mutation, (2) interfere with the ability of the promoter to direct the transcription of the open reading frame, or (3) interfere with the ability of the open reading frame to be transcribed by the promoter region sequence.

[00106] As used herein, the term “intergenic region” is synonymous with “intergenic linker” or “intergenic spacer.” An intergenic region is defined as a polynucleic acid sequence between adjacent (i.e., located on the same polynucleic acid sequence) genes, open reading frames, cistrons or coding sequences. By extension, the intergenic region can include the stop codon of the 5’ gene and/or the start codon of the 3’ gene that are linked by said intergenic region. As defined herein, the term intergenic region specifically relates to intergenic regions between adjacent genes in a polycistronic expression unit. For example, an intergenic region as defined herein, can be found between adjacent genes in an operon. Accordingly, in an embodiment, the intergenic region as defined herein is an operon intergenic region.

[00107] In some examples, the intergenic region, linker or spacer is selected from intergenic regions preceding, i.e., 5’ to, more particularly immediately 5’ to, rplW, rpfP, rpmD, rplB, rpsG, rpsE or rplN of a Gram-positive bacterium. In an embodiment, said Gram-positive bacterium is a lactic acid bacterium (LAB), for example, a Lactococcus species, e.g., Lactococcus lactis, and any subspecies or strain thereof. In an embodiment, said intergenic region encompasses the start codon of rplW, rplP, rpmD, rplB, rpsG, rpsE, or rplN and/or the stop codon of the preceding, i.e. 5’, gene. In a preferred embodiment, the invention relates to a Gram-positive bacterium or a recombinant nucleic acid as described herein, wherein the endogenous gene and the one or more exogenous genes are transcriptionally coupled by intergenic region or regions active in the Gram-positive bacterium, for example, wherein the intergenic region or regions is endogenous to said Gram-positive bacterium, for example, wherein the endogenous intergenic region is selected from intergenic regions preceding rplW, rpf P, rpmD, rplB, rpsG, rpsE, rplN, rplM, rplE, and rplF.

[00108] The skilled person will appreciate that if the intergenic region encompasses a 5’ stop codon and/or a 3’ start codon, these respective codons, in some cases, are not present in the genes that are linked by said intergenic regions, in order to avoid double start and/or stop codons, which may affect correct translation initiation and/or termination. Methods for identifying intergenic regions are known in the art. By means of further guidance, intergenic regions can for instance, be identified based on the prediction of operons, and associated promoters and open reading frames, for which software is known and available in the art. Exemplary intergenic regions (IRs) are described in, for example, international patent publication WO2012/164083, the disclosure of which is incorporated herein by reference in its entirety.

Microorganisms

[00109] The described single domain antibody (sdAb) may be produced by a microorganism comprising the polynucleic acid described above.

[00110] The microorganism can be a Gram-positive bacterium, such as an LAB, a Bifidobacterium, or a Staphylococcus. The LAB can be a Lactococcus species bacterium. An exemplary LAB species includes a Lactobacillus species, a Streptococcus species, or an Enterococcus species.

[00111] Exemplary Lactobacillus species include Lactobacillus acetotolerans, Lactobacillus acidophilus, Lactobacillus agilis, Lactobacillus algidus, Lactobacillus alimentarius, Lactobacillus amylolyticus, Lactobacillus amylophilus, Lactobacillus amylovorus, Lactobacillus animalis, Lactobacillus aviarius, Lactobacillus aviarius subsp. araffinosus, Lactobacillus aviarius subsp. aviarius, Lactobacillus bavaricus, Lactobacillus bifermentans, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus bulgaricus, Lactobacillus carnis, Lactobacillus casei, Lactobacillus casei subsp. alactosus, Lactobacillus casei subsp. casei, Lactobacillus casei subsp. pseudoplantarum, Lactobacillus casei subsp. rhamnosus, Lactobacillus casei subsp. tolerans, Lactobacillus catenaformis, Lactobacillus cellobiosus, Lactobacillus collinoides, Lactobacillus confusus, Lactobacillus coryniformis, Lactobacillus coryniformis subsp. coryniformis, Lactobacillus coryniformis subsp. torquens, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus curvatus subsp. curvatus, Lactobacillus curvatus subsp. melibiosus, Lactobacillus delbrueckii, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. delbrueckii, Lactobacillus delbrueckii subsp. lactis, Lactobacillus divergens, Lactobacillus farciminis, Lactobacillus fermentum, Lactobacillus fornicalis, Lactobacillus fructivorans, Lactobacillus fructosus, Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus graminis, Lactobacillus halotolerans, Lactobacillus hamsteri, Lactobacillus helveticus, Lactobacillus heterohiochii, Lactobacillus hilgardii, Lactobacillus homohiochii, Lactobacillus iners, Lactobacillus intestinalis, Lactobacillus jensenii, Lactobacillus johnsonii, Lactobacillus kandleri, Lactobacillus kefiri, Lactobacillus kefiranofaciens, Lactobacillus kefirgranum, Lactobacillus kunkeei, Lactobacillus lactis, Lactobacillus leichmannii, Lactobacillus lindneri, Lactobacillus malefermentans, Lactobacillus mali, Lactobacillus maltaromicus, Lactobacillus manihotivorans, Lactobacillus minor, Lactobacillus minutus, Lactobacillus mucosae, Lactobacillus murinus, Lactobacillus nagelii, Lactobacillus oris, Lactobacillus panis, Lactobacillus parabuchneri, Lactobacillus paracasei, Lactobacillus paracasei subsp. paracasei, Lactobacillus paracasei subsp. tolerans, Lactobacillus parakefiri, Lactobacillus paralimentarius, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus perolens, Lactobacillus piscicola, Lactobacillus plantarum, Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus rimae, Lactobacillus rogosae, Lactobacillus ruminis, Lactobacillus sakei, Lactobacillus sakei subsp. camosus, Lactobacillus sakei subsp. sakei, Lactobacillus salivarius, Lactobacillus salivarius subsp. salicinius, Lactobacillus salivarius subsp. salivarius, Lactobacillus sanfranciscensis, Lactobacillus sharpeae, Lactobacillus suebicus, Lactobacillus trichodes, Lactobacillus uli, Lactobacillus vaccinostercus, Lactobacillus vaginalis, Lactobacillus viridescens, Lactobacillus vitulinus, Lactobacillus xylosus, Lactobacillus yamanashiensis, Lactobacillus yamanashiensis subsp. mali, Lactobacillus yamanashiensis subsp. Yamanashiensis, Lactobacillus zeae, Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium longum, and Bifidobacterium inf antis. In some examples, the LAB is Lactococcus lactis (LL).

[00112] In further examples, the bacterium can be selected from the group consisting of Enterococcus alcedinis, Enterococcus aquimarinus, Enterococcus asini, Enterococcus avium, Enterococcus caccae, Enterococcus camelliae, Enterococcus canintestini, Enterococcus canis, Enterococcus casseliflavus, Enterococcus cecorum, Enterococcus columbae, Enterococcus devriesei, Enterococcus diestrammenae, Enterococcus dispar, Enterococcus durans, Enterococcus eurekensis, Enterococcus faecalis, Enterococcus faecium, Enterococcus gallinarum, Enterococcus gilvus, Enterococcus haemoperoxidus, Enterococcus hermanniensis, Enterococcus hirae, Enterococcus italicus, Enterococcus lactis, Enterococcus lemanii, Enterococcus malodoratus, Enterococcus moraviensis, Enterococcus mundtii, Enterococcus olivae, Enterococcus pollens, Enterococcus phoeniculicola, Enterococcus plantarum, Enterococcus pseudoavium, Enterococcus quebecensis, Enterococcus raffinosus, Enterococcus ratti, Enterococcus rivorum, Enterococcus rotai, Enterococcus saccharolyticus, Enterococcus silesiacus, Enterococcus solitarius, Enterococcus sulfureus, Enterococcus termitis, Enterococcus thailandicus, Enterococcus ureasiticus, Enterococcus ureilyticus, Enterococcus viikkiensis, Enterococcus villorum, and Enterococcus xiangfangensis,

[00113] In further examples, the bacterium can be selected from the group consisting of Streptococcus agalactiae, Streptococcus anginosus, Streptococcus bovis, Streptococcus canis, Streptococcus constellatus, Streptococcus dysgalactiae, Streptococcus equinus, Streptococcus iniae, Streptococcus intermedins, Streptococcus milleri, Streptococcus mitis, Streptococcus mutans, Streptococcus oralis, Streptococcus parasanguinis, Streptococcus peroris, Streptococcus pneumoniae, Streptococcus pseudopneumoniae, Streptococcus pyogenes, Streptococcus ratti, Streptococcus salivarius, Streptococcus tigurinus, Streptococcus thermophilus, Streptococcus sanguinis, Streptococcus sobrinus, Streptococcus suis, Streptococcus uberis, Streptococcus vestibularis, Streptococcus viridans, and Streptococcus zooepidemicus.

[00114] In a particular aspect of the present disclosure, the Gram-positive food-grade bacterial strain is Lactococcus lactis or any of its subspecies, including Lactococcus lactis subsp. Cremoris, Lactococcus lactis subsp. Hordniae, and Lactococcus lactis subsp. Lactis. Exemplary recombinant Gram-positive bacterial strains can be a biologically contained system, such as the plasmid-free Lactococcus lactis strain MG1363, that lost the ability of normal growth and acid production in milk (Gasson, M.J., J. Bacteriol. 1983, 154: 1-9); or the threonine- and pyrimidine- auxotroph derivative L. lactis strains (Sorensen etal.,Appl. Environ. Microbiol. 2000, 66: 1253- 1258; Glenting et al., Appl. Environ. Microbiol. 2002, 68: 5051-5056).

[00115] Particularly useful is an L. lactis strain having the following features:

• Thymidylate synthase gene (thyA', Gene ID: 4798358) is absent to warrant environmental containment (Steidler, L., et al., Nat. Biotechnol. 2003, 21(7): 785-789).

• Trehalose-6-phosphate phosphorylase gene (trePP-, Gene ID: 4797140) is absent, to allow accumulation of exogenously added trehalose.

• Trehalose-6-phosphate phosphatase gene (ptsB', Gene ID: 1036914) is positioned downstream of usp45 (Gene ID: 4797218) to facilitate conversion of trehalose- 6-phosphate to trehalose. The otsB expression unit was transcriptionally and translationally coupled to usp45 by use of the intergenic region (IR) preceding the highly expressed L. lactis MG1363 50S ribosomal protein L30 gene IrpmD', Gene ID: 4797873).

• The constitutive promoter of the HU-like DNA-binding protein gene (P/z/ZA; Gene ID: 4797353) is preceding the putative phosphotransferase genes in the trehalose operon (ire PTS', Umg_0453 and llmg_0454; Gene ID: 4797778 and Gene ID: 4797093 respectively) to potentiate trehalose uptake. • The gene encoding a cellobiose- specific PTS system IIC component (Gene ID: 4796893), ptcC, is deleted (AptcC). This mutation ascertains trehalose retention after accumulation.

[00116] The microorganism described herein may comprise another polynucleic acid encoding one or more additional therapeutic polypeptides. The one or more additional therapeutic polypeptides may include: Interleukin (IL)-10, trefoil factor (TFF)l, TFF2, TFF3, elastase inhibitors (e.g., elafin), protease inhibitors, matrix metalloproteinase (MMP) inhibitors, transforming growth factor-^ (TGF-|3), keratinocyte growth factor (KGF), neutralizing antibodies/decoy molecules to IL-1, IL-2, IL-4, IL-5, IL-6, CXCL8 (formerly IL-8), IL-9, IL- 12, IL-13, IL-17, IL-18, IL-21, IL-22 and IL-23, interferon gamma (IFNy), granulocytemacrophage colony stimulating factor (GM-CSF), G-CSF, and/or KGF. The microorganism may constitutively produce and secrete one or more of these therapeutic polypeptides.

Pharmaceutical Compositions and Treatment

[00117] The present disclosure further provides pharmaceutical compositions containing a microorganism (e.g., LAB) as described herein, e.g., a microorganism (e.g., LAB) in accordance with any of the above described modifications, and further containing a pharmaceutically acceptable carrier.

[00118] The term “pharmaceutically acceptable” is used herein in accordance with its art- recognized meaning and refers to carriers that are compatible with the other ingredients of a pharmaceutical composition, and are not deleterious to the recipient thereof. Non-limiting examples of suitable excipients, diluents, and carriers include preservatives, inorganic salts, acids, bases, buffers, nutrients, vitamins, fillers, and extenders such as starch, sugars, mannitol, and silicic derivatives; binding agents such as carboxymethyl cellulose and other cellulose derivatives, alginates, gelatin, and polyvinyl pyrolidone; moisturizing agents such as glycerol/ disintegrating agents such as calcium carbonate and sodium bicarbonate; agents for retarding dissolution such as paraffin; resorption accelerators such as quaternary ammonium compounds; surface active agents such as acetyl alcohol, glycerol monostearate; adsorptive carriers such as kaolin and bentonite; carriers such as propylene glycol and ethyl alcohol, and lubricants such as talc, calcium and magnesium stearate, and solid polyethyl glycols.

[00119] The compositions of the present disclosure can be prepared in any known or otherwise effective dosage or product form suitable for delivery of the microorganism (e.g., bacteria) to the mucosa, which would include pharmaceutical compositions and dosage forms as well as nutritional product forms. [00120] In some embodiments, the pharmaceutical composition (z.e., formulation) is an oral pharmaceutical composition. In some examples according to this embodiment, the formulation or pharmaceutical composition comprises the non-pathogenic microorganism in a dried form (e.g., dry-powder form; e.g., freeze-dried form) or in compacted form thereof, optionally in combination with other dry carriers. Oral formulations will generally include an inert diluent carrier or an edible carrier.

[00121] In some examples, oral formulations may include compounds providing controlled release, sustained release, or prolonged release of the microorganism and thereby provide controlled release of the desired protein encoded therein. These dosage forms (e.g., tablets or capsules) typically contain conventional and well known excipients, such as lipophilic, polymeric, cellulosic, insoluble, and/or swellable excipients. Controlled release formulations may also be used for any other delivery sites including intestinal, colon, bioadhesion, or sublingual delivery (z.e., dental mucosal delivery) and bronchial delivery. When the compositions of the invention are to be administered rectally or vaginally, pharmaceutical formulations may include suppositories and creams. In this instance, the host cells are suspended in a mixture of common excipients also, including lipids. Each of the aforementioned formulations are well known in the art and are described, for example, in the following references: H. Ansel et al. (1990, Pharmaceutical dosage forms and drug delivery systems, 5th edition, William and Wilkins); Chien 1992, Novel drug delivery system, 2nd edition, M. Dekker); Prescott et al. (1989, Novel drug delivery and its therapeutic application, J. Wiley & Sons); and Gazzaniga et al., Int. J. Pharm. 1994, 108:77-83.

[00122] The present disclosure further provides a microbial suspension (e.g., bacterial suspension) containing a microorganism (e.g., LAB) in accordance with any of the modifications, and further containing a solvent and a stabilizing agent. In some examples, the solvent can be selected from water, oil, and any combination thereof. For example, the present disclosure provides a bacterial suspension containing an LAB of the present disclosure, an aqueous mixture (e.g., a drink), and a stabilizing agent. Exemplary stabilizing agents are selected from a protein or polypeptide (e.g., glycoprotein), a peptide, a mono-, di- or polysaccharide, an amino acid, a gel, a fatty acid, a polyol (e.g., sorbitol, mannitol, or inositol), a salt (e.g., an amino acid salt), or any combination thereof.

[00123] The present disclosure further provides a microorganism as described herein (e.g., an LAB in accordance with any of the above embodiments), a composition as described herein, or a pharmaceutical composition as described herein, for use in the treatment of any of the following diseases / conditions related to inflammation (e.g., inflammatory conditions), such as a mucosal or skin inflammation, inflammatory bowel disease (IBD), Crohn’s disease (CD), ulcerative colitis (UC), psoriasis, irritable bowel syndrome (IBS), oral mucositis (OM), recurrent aphthous stomatitis (RAS), mTOR inhibitor associated stomatitis (mlAS), graft-versus-host disease (GVHD), oral pemphigus vulgaris (OPV), oral lichen planus (OLP), mucous membrane pemphigoid (MMP), vulvar lichen planus (VLP), vulvar lichen sclerosus (VLS), vulvar lichen simplex (VLSi), vulvar pemphigus vulgaris (VPV), Lipschiitz ulcer, vulvodynia, granulomatosis with polyangiitis (GPA), alopecia, lung inflammation, ocular inflammation, and inflammatory pain. The treatable inflammation may also include rheumatoid arthritis (RA), ankylosing spondylitis (AS), juvenile chronic arthritis (JCA), neurological inflammation, Behcet’s disease, rheumatoid vasculitis, Churg Strauss syndrome, Kawasaki’s arteritis, Takayasu’s arteritis, giant cell arteritis, polyarteritis nodosa, and cryoglobulinemic vasculitis. The provided microorganism, provided composition, or provided pharmaceutical composition may also be used in the treatment of other pathological conditions with brain/gut related pathologies, such as myalgic encephalomyelitis/chronic fatigue syndrome (CFS), depression, and/or Parkinson’s disease (PD).

[00124] The terms “treatment”, “treating”, and the like, as used herein, means ameliorating or alleviating characteristic symptoms or manifestations of a disease or condition. As used herein, these terms also encompass preventing or delaying the onset of a disease or condition or of symptoms associated with a disease or condition, including reducing the severity of a disease or condition or symptoms associated therewith prior to affliction with said disease or condition. Such prevention or reduction prior to affliction refers to the administration of the compound or composition of the invention to a patient that is not at the time of administration afflicted with the disease or condition. “Preventing” also encompasses preventing the recurrence or relapseprevention of a disease or condition or of symptoms associated therewith, for instance, after a period of improvement. Treatment of a subject “in need thereof’ conveys that the subject has a disease or condition, and the therapeutic method of the invention is performed with the intentional purpose of treating the specific disease or condition.

[00125] As used herein, the term “therapeutically effective amount” refers to an amount of a non-pathogenic microorganism or a composition of the present disclosure that will elicit a desired therapeutic effect or response when administered according to the desired treatment regimen. In some cases, the compounds or compositions are provided in a unit dosage form, for example a tablet or capsule, which contains an amount of the active component equivalent with the therapeutically effective amount when administered once, or multiple times per day.

[00126] The amount of secreted single domain antibody (sdAb) can be determined based on cfu, for example, following the methods described in Steidler et al., Science 2000 289(5483): 1352-1355, or by using ELISA. For example, a particular microorganism may secrete at least about 1 ng (nanogram) to about 1 pg of active polypeptide per 10 ⁹ cfu (colony forming units). Based thereon, the skilled person can calculate the range of antigen polypeptide secreted at other cfu doses.

[00127] Therapeutically effective amounts may be administered in connection with any dosing regimen as described herein. The daily dose of active polypeptide may be administered in 1, 2, 3, 4, 5, or 6 portions throughout the day. Further, the daily doses may be administered for any number of days, with any number of rest periods between administration periods.

[00128] The term “mucosa” or “mucous membrane” is used herein in accordance with its art recognized meaning. The “mucosa” can be any mucosa found in the body, such as oral mucosa, rectal mucosa, gastric mucosa, intestinal mucosa, urethral mucosa, vaginal mucosa, ocular mucosa, buccal mucosa, bronchial or pulmonary mucosa, and nasal or olfactory mucosa. Mucosa may also refer to surface mucosa, e.g., those found in fish and amphibians.

[00129] The term “mucosal delivery” as used herein is used in accordance with its art recognized meaning, i.e., delivery to the mucosa, e.g., via contacting a composition of the present disclosure with a mucosa. Oral mucosal delivery includes buccal, sublingual, and gingival routes of delivery. Accordingly, in some embodiments, “mucosal delivery” includes gastric delivery, intestinal delivery, rectal delivery, buccal delivery, pulmonary delivery, ocular delivery, nasal delivery, vaginal delivery and oral delivery. The person of ordinary skill will understand that oral delivery can affect delivery to distal portions of the gastrointestinal tract.

[00130] In some embodiments, the microorganism (e.g., LAB), optionally contained in a composition (e.g., a pharmaceutical composition) of the present disclosure or a unit dosage form of the present disclosure, will be administered, once, twice, three, four, five, or six times daily, e.g., using an oral formulation. In some embodiments, the microorganism is administered every day, every other day, once per week, twice per week, three times per week, or four times per [00131] week. In other embodiments, treatment occurs once every two weeks. In other embodiments, treatment occurs once every three weeks. In other embodiments, treatment occurs once per month.

[00132] The duration of a treatment cycle is, for example, 7 days to the subject’s lifetime, as needed to treat or reverse a disease condition, or prevent relapse. In some embodiments, a treatment cycle lasts for 21 days to about 2 years. In some embodiments, a treatment cycle lasts for 21 days, 30 days, or 42 days to 1.5 years. In other embodiments, the subject will have a treatment cycle that lasts from 21 days, 30 days, or 42 days, to 1 year. In other embodiments, the subject will have a treatment cycle that lasts from 21 days, 30 days, or 42 days to 11 months. In other embodiments, the subject will have a treatment cycle that lasts from 21 days, 30 days, or 42 days to 10 months. In other embodiments, the subject will have a treatment cycle that lasts from 21 days, 30 days or 42 days to 9 months. In other embodiments, the subject will have a treatment cycle that lasts from 21 days, 30 days, or 42 days to 8 months. In other embodiments, the subject will have a treatment cycle that lasts from 21 days, 30 days, or 42 days to 7 months. In other embodiments, the subject will have a treatment cycle that lasts from 21 days, 30 days, or 42 days to 6 months. In other embodiments, the subject will have a treatment cycle that lasts from 21 days, 30 days, or 42 days to 5 months. In other embodiments, the subject will have a treatment cycle that lasts from 21 days, 30 days, or 42 days to 4 months. In other embodiments, the subject will have a treatment cycle that lasts from 21 days, 30 days, or 42 days to 3 months. In other embodiments, the subject will have a treatment cycle that lasts from 21 days, 30 days, or 42 days to 2 months.

[00133] The pharmaceutical compositions of the present disclosure can be prepared by any known or otherwise effective method for formulating or manufacturing the selected dosage form. For example, the microorganisms can be formulated along with common, e.g., pharmaceutically acceptable carriers, such as excipients and diluents, formed into oral tablets, capsules, sprays, lozenges, treated substrates (e.g., oral or topical swabs, pads, or disposable, non-digestible substrate treated with the compositions of the present invention); oral liquids (e.g., suspensions, solutions, emulsions), powders, suppositories, or any other suitable dosage form. In some embodiments, the present disclosure provides a method for the manufacture of a pharmaceutical composition. Exemplary methods include: contacting the microorganism (e.g., the non- pathogenic bacterium) with a pharmaceutically acceptable carrier, thereby forming the pharmaceutical composition. In some examples, the method further includes steps for growing the microorganism in a medium. The method may further include freeze-drying a liquid containing the microorganism, wherein the liquid optionally includes the pharmaceutically acceptable carrier.

[00134] The current disclosure further provides unit dosage forms comprising a certain amount of a non-pathogenic microorganism optionally in combination with a food-grade or pharmaceutically acceptable carrier, wherein said non-pathogenic microorganism (e.g., the non- pathogenic Gram-positive bacterium) comprises: a polynucleic acid encoding a single domain antibody to TNF-oc. Exemplary unit dosage forms contain from about 1 x 10 ³ to about 1 x 10 ¹⁴ colony-forming units (cfu) of the non-pathogenic microorganism (e.g., a non-pathogenic grampositive bacterium). Other exemplary unit dosage forms contain from about 1 x 10 ⁴ to about 1 x 10 ¹³ colony-forming units (cfu) of a non-pathogenic microorganism (e.g., a non-pathogenic Gram-positive bacterium), or from about 1 x 10 ⁴ to about 1 x 10 ¹² colony-forming units (cfu) of a non-pathogenic microorganism (e.g., a non-pathogenic Gram-positive bacterium). In other embodiments, the unit dosage form comprises from about 1 x 10 ⁵ to about 1 x 10 ¹² colonyforming units (cfu), or from about 1 x 10 ⁶ to about 1 x 10 ¹² colony-forming units (cfu) of the non-pathogenic microorganism (e.g., the non-pathogenic Gram-positive bacterium). In other embodiments, the unit dosage form comprises from about 1 x 10 ⁸ to about 1 x 10 ¹² colonyforming units (cfu), or from about 1 x 10 ⁹ to about 1 x 10 ¹² colony-forming units (cfu) of the non-pathogenic microorganism (e.g., the non-pathogenic Gram-positive bacterium). In yet other embodiments, the unit dosage form comprises from about 1 x 10 ⁹ to about 1 x 10 ¹¹ colonyforming units (cfu), or from about 1 x 10 ⁹ to about 1 x IO ¹⁰ colony-forming units (cfu) of the non-pathogenic microorganism (e.g., the non-pathogenic Gram-positive bacterium). In yet other embodiments, the unit dosage form comprises from about 1 x 10 ⁷ to about 1 x 10 ¹¹ colonyforming units (cfu), or from about 1 x 10 ⁸ to about 1 x IO ¹⁰ colony-forming units (cfu) of the non-pathogenic microorganism (e.g., the non-pathogenic Gram-positive bacterium).

[00135] In yet other embodiments, the unit dosage form comprises from about 1 x 10 ⁹ to about 1 x IO ¹⁰ colony-forming units (cfu), or from about 1 x 10 ⁹ to about 100 x 10 ⁹ colonyforming units (cfu) of the non-pathogenic microorganism (e.g., the non-pathogenic Grampositive bacterium).

[00136] The unit dosage form can have any physical form or shape. In some embodiments, the unit dosage form is adapted for oral administration. In some examples, according to these embodiments, the unit dosage form is in the form of a capsule, a tablet, or a granule. Exemplary capsules include capsules filled with micro-granules. In some embodiments, the non-pathogenic microorganism (e.g., the non-pathogenic Gram-positive bacterium) contained in the dosage form is in a dry-powder form. For example, the microorganism is in a freeze-dried powder form, which is optionally compacted and coated.

[00137] The current disclosure further provides kits containing (1) a microorganism e.g., LAB) according to any of the embodiments disclosed herein, a composition containing a microorganism (e.g., LAB) according to any of the embodiments described herein, a pharmaceutical composition containing a microorganism (e.g., LAB) according to any of the embodiments described herein, or a unit dosage form containing a microorganism (e.g., LAB) according to any of the embodiments described herein; and (2) instructions for administering the microorganism (e.g., LAB), the composition, the pharmaceutical composition, or the unit dosage form to a mammal, e.g., a human (e.g., a human patient).

[00138] The present disclosure further provides pharmaceutical compositions comprising an isolated antibody polypeptide comprising a single variable domain that specifically binds a human Tumor Necrosis Factor-alpha (TNF-a) as described herein, and optionally a pharmaceutically acceptable carrier. Exemplary pharmaceutically acceptable carriers are described elsewhere herein. These isolated antibody polypeptide compositions of the present disclosure can be prepared in any known or otherwise effective dosage or product form suitable for administration of the antibody polypeptide to a patient in need thereof. The compositions can be formulated to provide quick, sustained, or delayed release of the active ingredient(s) after administration. Suitable pharmaceutical compositions and processes for preparing them are known in the art. See, e.g. , Remington, THE SCIENCE AND PRACTICE OF PHARMACY, A. Gennaro, et al., eds., 21st ed., Mack Publishing Co. (2005).

[00139] In certain embodiments, a method of treating an inflammatory condition or a pathological condition in a patient in need of such treatment may comprise administering to the patient a therapeutically effective amount of the isolated antibody, or antigen binding portion thereof, as described herein. Exemplary inflammatory conditions and pathological conditions are disclosed elsewhere herein. Also provided is the use of an isolated antibody, or antigen binding portion thereof, of the disclosure, or a pharmaceutically acceptable salt thereof, for treating an inflammatory condition or a pathological condition in a patient in need of such treatment. Also provided is the use of an antibody, or antigen binding portion thereof, of the disclosure, or a pharmaceutically acceptable salt thereof, for the preparation of a medicament for treating an inflammatory condition or a pathological condition in a patient in need of such treatment.

[00140] In certain embodiments, the pharmaceutical composition may be administered alone or in combination therapy, (/'.<?., simultaneously or sequentially) with one or more therapeutic agents for treating an inflammatory or pathological condition.

[00141] Any suitable method or route can be used to administer the antibody, or antigen binding portion thereof, or the pharmaceutical composition. Routes of administration include, for example, intravenous, intraperitoneal, subcutaneous, or intramuscular administration. A therapeutically effective dose of administered antibody depends on numerous factors, including, for example, the type and severity of the disease being treated, the use of combination therapy, the route of administration of the antibody, or antigen binding portion thereof, or pharmaceutical composition, and the weight of the patient. A non-limiting range for a therapeutically effective amount of an antibody is 0.1-20 milligram/kilogram (mg/kg), and in an aspect, 1-10 mg/kg, relative to the body weight of the patient.

[00142] A kit useful for treating an inflammatory or pathological condition in a human patient is provided. A kit useful for treating or preventing an inflammatory or pathological condition in a human patient is also provided. The kit can comprise (a) a dose of an isolated antibody, or antigen binding portion thereof, of the present disclosure and (b) instructional material for using the isolated antibody, or antigen binding portion thereof , in the method of treating an inflammatory condition or a pathological condition, or for using the isolated antibody, or antigen binding portion thereof, in the method of preventing an inflammatory condition or a pathological condition, in a patient.

[00143] These products, compositions, and methods can be better understood by reference to the Examples that follow, but those skilled in the art will appreciate that these are only illustrative of the invention as described more fully in the numbered embodiments and claims that follow. Additionally, throughout this application, various publications are cited. The disclosures of these publications are hereby incorporated by reference in their entirety. EXAMPLES

Example 1: Preparing camelid VHH library and selecting hTNF-a neutralizing antihuman TNF-alpha clones

[00144] Two llamas (Lama glama)wcvc immunized with tumor necrosis factor-alpha (TNF- a). Both llamas were immunized with a combination of human TNF-a (hTNF-a) and murine TNF-a (mTNF-a). Total RNA was isolated from each lama for the construction of VHH libraries. Two rounds of VHH phage display were done to improve binding to hTNF-a (and mTNF-a libraries). Periplasmic extracts were prepared from the resulting clones. Immunizations up to periplasmic extracts were performed by Fairjourney Biologies, Porto, Portugal. The resulting clones were first selected for increased hTNF-a binding, and subsequently, the selected clones were tested for hTNF-a neutralization. Binding and neutralization were assayed using periplasmic extracts

[00145] The resulting clones were first selected for increased hTNF-a binding, and subsequently, the selected clones were tested for hTNF-a neutralization. Binding and neutralization were assayed using periplasmic extracts, which were 1/10 dilutions. Periplasmic extracts were prepared by osmotic shock procedure. A suspension of L929 cells at 2 x 10 ⁵ cells/ml was added to a flat bottom 96 well assay plate and incubated overnight at 37°C, 5% CO2 in a humidified incubator. In a separate plate, an equal volume of a dilution series of the samples was mixed with an equal volume of 10 lU/ml hTNF-a and 2 pg/ml Actinomycin D. 100 pl of this mixture was added to the cells. Untreated cells and cells treated with hTNF a and Actinomycin D serve as controls. The plates were incubated for 20 to 24 h at 37°C, 5% CO2 in a humidified incubator. Afterward, 20 pl CellTiter 96® AQueous One Solution Reagent was added to the wells, and the plates were incubated for another 4 h at 37°C, 5% CO2 in a humidified incubator. The absorbance was read at 490 nm for measuring and 700 nm as a reference to determine the amount of cell death. The result is expressed as % neutralization versus hTNF a controls. Clones that show increased hTNF-a neutralization in comparison to the reference (/'.<?., the initial sequence used at the start of the optimization) were retained. Binding affinity and Kd were also assayed using a Biacore device. In brief, the biotinylated protein was captured by the biotin CAPture reagent (hTNF-a), and then periplasmic extract samples were flowed over the captured protein. Binding and Kd were determined by measuring surface plasmon resonance. From these data, three clones were identified as having adequate hTNF-a neutralizing capacity. The neutralization and binding affinity (dissociation constant) data for the three selected clones is shown in Table 5.

Table 5

[00146] The three selected clones were sequenced. The amino acid sequences of the three clones are shown in Table 6. The CDRs are indicated. The CDR regions were defined based on Kabat numeration using appropriate software (Dondelinger et al., 2018, “Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface/Residue Definition”. Front Immunol. 9:2278. PMID 30386328).

Table 6

[00147] The three selected clones were sequenced and subcloned into an expression module. A schematic view of the VHHTNAOOlh expression module pAGX2701 is depicted in Figure 1. The sequence of the expression module for VHHTNAOOlh expression module pAGX2701 is provided in Figures 2A and 2B. The sequence of the expression module for VHHTNA002h expression module pAGX2702 is provided in Figures 2C and 2D. The sequence of the expression module for VHHTNA003h expression module pAGX2703 is provided in Figures 2E and 2F.

Example 2: Evaluation of hTNF-a neutralizing single-domain antibodies

[00148] Of VHHTNAOOlh, VHHTNA002h, and VHHTNA003h, VHHTNAOOlh has the best hTNF-a neutralizing capacity. However, as shown by the data in Table 4, VHHTNAOOlh’ s affinity is rather low (4 x 10 ³ /s) as compared to VHHTNA002h and VHHTNA003h, respectively 1.5 x 10 ⁴ /s and 9 x 10 ⁵ /s. During the screening of perip lasmic extracts of all original clones, one clone (FJ1808MP05E02) was identified that has a CDR3 that is highly similar to the CDR3 of VHHTNAOOlh; residue at the N-terminus of the CDR3 differed, as shown in Table 7. Clone FJ1808MP05E02 did not neutralize hTNF-a, likely due to mutations in CDR1 and CDR2 (see Table 7). Table 7: Alignment of the 3 selected hTNF-a neutralizing clones VHHTNAOOlh, VHHTNA002h, and VHHTNA003h and of FJ1808MP05E02

¹ result of 1/10 diluted periplasmic extract.

[00149] Given these data and observations, it was decided not to study mutations to CDR1 and CDR2 at this stage but rather, to focus on mutating CDR3, and including the 1 amino acid position at the C-terminal end, which is designated herein as CDR3+1 and is composed of 8 amino acids (the 7 amino acids of the CDR3 and the amino acid flanking the C-terminal amino acid of the CDR3). Two approaches were undertaken: 1) exchange CDR3+1 from VHHTNAOOlh with the same region from antibodies with higher affinity (such as VHHTNA002h and VHHTNA003h); and 2) to improve hTNF-a neutralization by mutation of CDR3+1 of VHHTNAOOlh.

Example 3: CDR3 Exchange

[00150] As described above, VHHTNAOOlh has a higher neutralizing capacity and lower affinity as compared to VHHTNA002h and VHHTNA003h. The amino acid sequences of VHHTNAOOlh, VHHTNA002h, and VHHTNA003h have about 60% homology. The region around CDR3 is shown in Figure 3. The first approach was to change the CDR3 of VHHTNAOOlh to be the CDR3 of VHHTNA002h or of VHHTNA003h. [00151] VHHTNAOOlh, in which the CDR3 was replaced by the CDR3 from VHHTNA002h was named VHHTNA015h (carried by pAGX2906). VHHTNAOOlh in which the CDR3 was replaced by the CDR3 from VHHTNA003h was named VHHTNA016h (carried by pAGX2907).

[00152] The size and extent of expression was examined by Western blot. It was determined that both new configurations VHHTNA015h and VHHTNA016h were expressed less as compared to VHHTNAOOlh (see Figure 4). In addition, the hTNF-a neutralizing capacity was assessed. The hTNF-a neutralizing capacity of the new configurations, VHHTNA015h, and VHHTNA016h, was lost (not detectable).

Example 4: Mutagenesis of CDR3+1 from VHHTNAOOlh

[00153] Single amino acid changes were introduced in the CDR3+1 of VHHTNAOOlh. The CDR3+1 from VHHTNAOOlh is composed of 8 amino acids - KDFGGQIK -(SEQ ID NO:8; CDR3 sequences are shown in Table 7); it was decided to include the position flanking the C-terminal, i.e.. the N-terminal lysine of framework region 4 in this mutation strategy. Thus, KDFGGQIK (SEQ ID NO: 8) is the amino acid sequence subjected to mutagenesis. The two glycine residues in the middle of CDR3+1 were left unmutated. In total, six (6) single mutations were introduced. In the following Table 8, “X” indicates any amino acid.

Table 8

¹ The plasmid name ending in “d” reflects that this refers to a plasmid used in mutagenesis and effectively represents an array of plasmids wherein the “X” in the mutated sequence indicates the position is mutagenized but does indicate a specific amino acid. Plasmid names without the “d” were assigned to specific clones having a specific amino acid. For instance, the name “pAGX2908” was assigned to this clone CDFGGQIK (ttg) (SEQ ID NO: 101).

[00154] At each codon encoding the X residue, random triplets were introduced by using primers with random nucleotides for that position (see Table 9), carried on plasmids pAGX2908d to pAGX2913d.

Table 9

[00155] Following transformation, the resulting primary colonies were grown to saturation in GM17E, and crude cultures were screened in ELISA for binding to hTNF-a. A fixed dilution was used, and only one well of the 96-well plate per colony. In brief, from each colony, a single 1/250 dilution of culture medium, grown overnight at 32°C in GM17E, was analyzed for binding to hTNF-a. Bound single domain antibodies were detected with rabbit anti- llama-biotin, followed by streptavidin-HRP and TMB solution for detection. The reaction was stopped by adding 0.5 M HC1, and the absorbance was read at 450 nm (A450) for measuring and 595 nm as reference. MG1363[pAGX2701] was used as a positive control (Al on each 96 well plate).

[00156] MG1363[pTlNX] was used as negative control (A2 on each 96 well plate). 0.1% casein was added to A3 on each 96 well plate.

[00157] The data are shown in Figures 5A-5F. High binding clones, defined as having at least 20% above the average absorbance of the positive control pAGX2701, were selected for Sanger sequencing and further characterization. These clones are stippled in Figures 5A-5F. Intermediate binding clones, defined as in between high binding clones and the positive control, were also selected for Sanger sequencing and further characterization. These clones are bolded in Figures 5A-5F.

[00158] The selection resulted in a total of 63 clones that included - 21 high binders and 42 intermediate binders. The 63 clones were sequenced. Some clones showed clean sequences high-quality sequence reads with unambiguously allocated bases, whereas others showed mixed reads. Therefore, clones with unique sequences were streaked to a single colony and, depending on the quality of the first sequence, 3, 5, or 10 colonies for each clone were analyzed. In this way, 26 unique sequences were obtained. An overview of all these identified amino acids with their corresponding codons is summarized in Figure 6. Two different proline codons for pAGX2910 appeared in the high binders as well as in the intermediate binders, in which case the highest binders were selected for further use.

[00159] Twenty-nine (29) clones were assayed for hTNF-a binding. The 29 clones consist of seven clones for each of pAGX2908d (XDFGGQIK; SEQ ID NO: 100), pAGX2910d (KDXGGQIK; SEQ ID NO: 107), and pAGX2912d (KDFGGQXK; SEQ ID NO: 110), 3 clones from pAGX2911d (KDFGGXIK; SEQ ID NO: 108), and 1 clone from each of pAGX2909d (KXFGGQIK; SEQ ID NO: 105) and pAGX2913d (KDFGGQIX; SEQ ID NO: 113). Each clone was grown to saturation overnight at 32°C in GM17E, and a 1/2 dilution series of the culture supernatant were tested in ELISA for binding to hTNF-a. Bound single domain antibodies were detected with MonoRab™ rabbit anti-camelid VHH IgG, followed by goat anti-rabbit IgG-HRP and TMB solution for detection. The reaction was stopped by adding 0.5 M HC1, and the absorbance was read at 450 nm (A450) for measuring and 595 nm as reference.

[00160] The results of the hTNF-a binding assays are shown in Figures 7A-7E. [00161] Mutation of KI (pAGX2908d) improved binding to hTNF-a when the lysine is changed to alanine, cysteine (2 codons), glutamine, or leucine (2 codons). However, when the lysine is changed to histidine, binding to hTNF-a is less efficient. See Figure 7A. Four of the seven pAGX2908d clones were retained: (i) CDFGGQIK (ttg; SEQ ID NO: 101) (named pAGX2908) (ii) QDFGGQIK (gaa; SEQ ID NO: 102) (named pAGX2933); (iii) LDFGGQIK (eta; SEQ ID NO: 103) (named pAGX2934); and (iv) ADFGGQIK (gca; SEQ ID NO: 104) (named pAGX2935).

[00162] Mutation of D2 (pAGX2909d) improved binding to hTNF-a when the aspartic acid at position 2 is changed to asparagine. See Figure 7E. One pAGX2909d clone was retained: KNFGGQIK (aat; SEQ ID NO: 106) (named pAGX2909).

[00163] Mutation of F3 (pAGX2910d) improved binding to hTNF-a, when the phenylalanine at position 3 is changed to proline (3 codons). Most other mutants - phenylalanine to aspartic acid, glycine, arginine, or leucine - show less or equal binding to hTNF-a. See Figure 7B. One pAGX2910d clone was retained: KDPGGQIK proline (cca; SEQ ID NO:96) (named pAGX2910).

[00164] Mutation of Q6 (pAGX291 Id) improved binding to hTNF-a when the glutamine at position 6 is changed to cysteine. When the glutamine is changed to glutamic acid or histidine, however, binding to hTNF a is less efficient. See Figure 7C. One pAGX2911d clone was retained: KDFGGCIK (tgc; SEQ ID NO: 109) (named pAGX2911).

[00165] Mutation of 17 (pAGX2912d) had substantially no impact on binding to hTNF-a when the isoleucine is changed to glutamine or leucine (2 codons). However, when the isoleucine is changed to glycine, proline, tyrosine, or aspartic acid, binding to hTNF-a is reduced. See Figure 7D. Two ofthe clones were retained: KDFGGQQK (cag; SEQ ID NO: 111) (named pAGX2912) and KDFGGQLK (etc; SEQ ID NO: 112) (named pAGX2955).

[00166] Mutation of K8 (pAGX2913d) improved binding to hTNF-a when the lysine is changed to arginine. See Figure 7E. This clone was retained: KDFGGQQIR (aga; SEQ ID NO: 114) (named pAGX2913).

[00167] The data confirms that many of the mutants showed improved binding to hTNF-a, compared to binding of the original clone - VHHTNAOOlh (KDFGGQIK (SEQ ID NO:8) (pAGX2701)). The ten mutants with improved binding to hTNF-a were retained for further characterization. Example 5: Validation of single mutant VHH

[00168] The ten mutants identified in Example 4, a negative control (pTINX) and positive control (pAGX2701), were prepared for protein expression in a buffered minimal medium, BRPMIE. The culture supernatants were analyzed in a western blot, in an hTNF-a binding ELISA, and in an hTNF-a neutralization assay.

[00169] In the western blot, equivalents of 1 ml of culture supernatant of L. lactis strains were analyzed. The recombinant single domain antibodies (sdAbs) were detected with goat antillama IgG, followed by IRDye 800CW donkey anti-goat IgG. Fluorescent signals were visualized by Odyssey CLx. The data are shown in Figure 8. All mutant VHH-domains are equally well recognized. Differences in expected molecular weights of these single domain antibodies (13.40 to 13.48 kDa) are very small and do not fully explain the differences in mobility observed. The explanation of the mobility differences remains unknown.

[00170] The hTNF-a binding ELISA was performed substantially as described in Example 4. A 1/2 dilution series of culture supernatant (BRPMIE) was analyzed for binding to hTNF-a. Bound sdAbs were detected with MonoRab™ rabbit anti-camelid VHH IgG, followed by goat anti-rabbit IgG-HRP and TMB solution. The reaction was stopped by adding 0.5 M HC1 and the absorbance was read at 450 nm (A450) for measuring and 595 nm as reference.

[00171] The assay of hTNF-a neutralization was performed as follows. A 1/3 dilution series of culture supernatant (BRPMIE), mixed with an equal volume of 10 lU/ml hTNF-a and 2 pg/ml Actinomycin D, was added to a suspension of L929 cells. Untreated cells and cells treated with hTNF-a and Actinomycin D served as controls. CellTiter 96® AQueous One Solution Reagent (Promega Corporation, Madison, WI) was added after 20 to 24 h incubation, and the absorbance was read at 490 nm for measuring and 700 nm as a reference to determine the amount of cell death. The result is expressed as % neutralization versus hTNF-a controls. MG1363[pAGX2701] serves as the positive control (VHHTNAOOlh (KDFGGQIK (SEQ ID NO:8); original wild type CDR3+1). MG1363[pTlNX] serves as the negative control. For each mutant, the amino acid change in CDR3+1 is underlined.

[00172] Table 10 is a summary of the EC50 dilution of hTNF-a binding data and the IC50 dilution of hTNF-a neutralization data of single mutants. “Ratio vs wt” is the ratio of the mutant EC50 or IC50 versus (vs) the wild type (wt) sequence and is included to the immediate right of the respective dilutions. Table 10 [00173] The hTNF-a binding confirmed the improved binding to hTNF-a of mutants having an amino acid change at position 1 to glutamine (QDFGGQIK; SEQ ID NO: 102) or leucine (LDFGGQIK; SEQ ID NO: 103), at position 2 to asparagine (KNFGGQIK; SEQ ID NO: 106), and at position 3 to proline (KDPGGQIK; SEQ ID NO:96), that was observed during screening. See Figure 9 A and Figures 7A-7C. This improved binding is also evidenced by the EC50 dilution of these mutants versus the wild type sequence (Table 9). These 4 mutants with mutations in the first half of the CDR3+1 also showed improved hTNF-a neutralization. The hTNF-a neutralization is substantially better - by about a factor 2.5 - than the hTNF-a neutralization of the wild-type CDR3+1 (KDFGGQIK; SEQ ID NO:8) (see IC50 dilution in Table 9).

[00174] The improved binding to hTNF-a by mutation of position 1 to alanine (ADFGGQIK; SEQ ID NO: 104) or cysteine (CDFGGQIK; SEQ ID NO: 101) observed in Figure 9A, was also evidenced by the EC50 dilution of these mutants versus the wild type sequence. However, neither of these mutants shows improved hTNF-a neutralization. See Table 9. These data suggest that improving hTNF-a binding does not necessarily lead to improved hTNF-a neutralization.

[00175] Two mutations in the second half of CDR3+1, the mutation of position 7 to leucine (KDFGGQLK; SEQ ID NO: 112) and the mutation of position s to arginine (KDFGGQIR; SEQ ID NO: 114), showed improved binding to hTNF-a. See Figure 9B and Figure 7E. However, only the variant having position 8 mutated to arginine (KDFGGQIR; SEQ ID NO: 114) also showed improved hTNF-a neutralization. Neutralization of hTNF-a by KDFGGQIR (; SEQ ID NO: 114) is only slightly better than that of the wild type CDR3+1 ( (KDFGGQIK; SEQ ID NO:8) and was not as pronounced as mutations in the first half of CDR3+1. These data suggest that improving hTNF-a neutralization does not necessarily correlate with improved hTNF-a binding.

[00176] From these data, the following observations were made. hTNF-a binding and neutralization are improved when:

■ lysine at position 1 is changed to leucine or glutamine;

■ aspartic acid at position 2 is changed to asparagine;

■ phenylalanine is changed to proline; or

■ position 8 is changed to arginine, but the effect is less as compared to the mutations listed above. [00177] As the next steps to try to further improve hTNF-a binding and neutralization, two approaches were undertaken: (i) combining mutations described in Examples 4 and 5; and (ii) performing a second round of mutations in available single mutants.

Example 6: Combining mutations

[00178] Table 11 lists the mutants that were identified as having both improved hTNF-a binding and neutralization (see Table 10 ):

Table 11

[00179] Eight (8) combinations of the single mutations were prepared. See Table 12.

Table 12

[00180] For the construction of pAGX2939 to pAGX2946, oligonucleotides oAGX9654 to oAGX9662 (Table 13) were used to introduce the second and third amino acid change in the respective single mutants.

Table 13

[00181] Expression of the resulting double and triple mutant sdAbs was performed in crude culture supernatants of L. lactis strain MG1363 containing pAGXNNNN (mutant sdAbs), pAGX2701 VHHTNAOOlh (KDFGGQIK (SEQ ID NO:8); original wild type CDR3+1 ; positive control), or pTINX (negative control). Expression of the clones was performed in buffered minimal medium, and the culture supernatant was analyzed by Coomassie Brilliant Blue (CBB) stained SDS-PAGE and in a western blot (respectively the right and left panel of Figure 10). Equivalents of 1 ml of culture supernatant of the L. lactis strains were analyzed. For the western blot, recombinant sdAbs were detected with goat anti-llama IgG, followed by IRDye 800CW donkey anti-goat IgG. Fluorescent signals were visualized by Odyssey CLx.

[00182] The mutant sdAbs were analyzed for binding to hTNF-a (see Figure 11, Figure 13, and Figure 15) and for their capacity to neutralize hTNF-a (see Figure 12, Figure 14, and Figure 16).

[00183] Binding to hTNF-a for mutants derived from QDFGGQIK (SEQ ID NO: 102) (pAGX2933) is shown in Figure 11 and neutralization of hTNF-a for the same mutants is shown Figure 12. The best binding to hTNF-a is observed when all three amino acid changes in the first half of CDR3+1 are combined: QNPGGQIK (SEQ ID NO:88). This triple mutant QNPGGQIK (SEQ ID NO:88) also neutralizes hTNF-a very efficiently. When either glutamine at position 1 or asparagine at position 2 is combined with proline at position 3 (QDPGGQIK (SEQ ID NO:93) or KNPGGQIK (SEQ ID NO:89)), binding to hTNF-a is improved as compared to single mutant_QDFGGQIK (SEQ ID NO: 102). Binding to hTNF-a of these mutants

[00184] is equally efficient. Double mutant KNPGGQIK (SEQ ID NO:89) neutralizes hTNF-a equally efficiently as the triple mutant QNPGGQIK (SEQ ID NO:88) and better than QDPGGQIK (SEQ ID NO:93). Double mutant QNFGGQIK (SEQ ID NO: 116) showed no improvement in hTNF-a binding, but did improve hTNF-a neutralization observed with the single mutant QDFGGQIK (SEQ ID NO: 102). [00185] Binding to hTNF-a and neutralization of hTNF-a for mutants derived from LDFGGQIK (SEQ ID NO: 103) (pAGX2934) are shown in respectively Figure 13 and Figure 14. Also, here the triple mutant LNPGGQIK (SEQ ID NO: 87) shows the best binding to hTNF-a and the best hTNF-a neutralization capacity. The combination of leucine at position 1 and proline at position 3 (LDPGGQIK; (SEQ ID NO:90) was not yet available at the time of this expression study. For comparison and completeness, the results of a later expression study for LDPGGQIK (SEQ ID NO:90) are also shown in these data. The double mutant LDPGGQIK (SEQ ID NO:90) showed greatly improved binding to hTNF-a and hTNF-a neutralization. The second-best double mutant for binding to hTNF-a and neutralization of hTNF-a capacity is KNPGGQIK (SEQ ID NO:89). If asparagine at position 2 is combined with leucine at position 1 (LNFGGQIK: (SEQ ID NO: 115), undetectable or minimal improvement in hTNF-a binding is observed, and there is no improvement in hTNF-a neutralization capacity. This modest increase in hTNF-a binding of double mutant LNFGGQIK (SEQ ID NO: 115), might be related to growth retardation and, accordingly, lesser production of the single-domain antibody.

[00186] Figure 15 shows binding to hTNF-a, and Figure 16 demonstrates neutralization of hTNF-a for mutations in the second half of CDR3+1 compared to relevant controls. The combination of leucine at position 7 and arginine at position 8 (KDFGGQLR; (SEQ ID NO: 117) showed improved binding to hTNF-a versus both single mutants but were far less efficient as compared to double mutant KNPGGQIK (SEQ ID NO:-89). KDFGGQLR (SEQ ID NO: 117) binds hTNF-a equally well as LNFGGQIK (SEQ ID NO: 115), which showed a slower growth rate. Mutations in the second half of CDR3+1, leucine at position 7, arginine at position 8, or a combination of both KDFGGQLR (SEQ ID NO: 117) did not improve hTNF-a neutralization.

Example 7: Second round of random mutagenesis

[00187] A second round of mutagenesis was performed as another approach to further investigate the improvement of hTNF-a binding and neutralization. The second round of mutagenesis was performed with two single mutants in which lysine at position 1 of CDR3+1 was changed: QDFGGQIK (SEQ ID NO: 102) (pAGX2933) and LDFGGQIK (SEQ ID NO: 103) (pAGX2934). In these two single mutants, random amino acids were introduced at position 2 by using oligonucleotides randomized at the appropriate codon position (oAGX9663 and oAGX9664; see Table 12). The resulting constructs were named pAGX2947d (LXFGGQIK: (SEQ ID NO: 118) and pAGX2948d (QXFGGQIK: (SEQ ID NO: 119), respectively. [00188] In addition, because double mutants were not yet available at the time of this second round of mutagenesis, position 2 was fixed in both single mutants to asparagine (N), and random mutagenesis at position 3 (LNXGGOIK (SEQ ID NO: 120) and QNXGGQIK (SEQ ID NO: 121), respectively) was performed. Random amino acids for position 3 were introduced by using oligonucleotides randomized at the codon positions (oAGX9665 and oAGX9666; see Table 14). The resulting constructs were named pAGX2949d (LNXGGOIK: (SEQ ID NO: 120) and pAGX2950d (ONXGGOIK: (SEQ ID NO: 121), respectively.

Table 14

[00189] In summary:

1) LXFGGQIK (SEQ ID NO: 118) (pAGX2947d): random mutagenesis of position 2 in pAGX2934.

2) QXFGGQIK (SEQ ID NO: 119) (pAGX2948d): random mutagenesis of position 2 in pAGX2933.

3) LNXGGOIK (SEQ ID NO: 120) (pAGX2949d): position 2 fixed to asparagine and random mutagenesis of position 3 in pAGX2934.

4) ONXGGOIK (SEQ ID NO: 121) (pAGX2950d): position 2 fixed to asparagine and random mutagenesis of position 3 in pAGX2933.

[00190] Primary colonies of these 4 mutagenesis experiments were screened in ELISA for binding to hTNF-a. In brief, from each colony grown overnight at 32°C in GM17E, a single 1/250 dilution of culture medium was analyzed for binding to hTNF-a. Bound single-domain antibodies were detected with rabbit anti-llama, followed by goat anti-rabbit HRP and TMB solution for detection. The reaction was stopped by adding 0.5 M HC1, and the absorbance was read at 450 nm for measuring and 595 nm as reference.

[00191] Two (2) positive controls were used in each screening. Positive control 1 was the plasmid that was used as template for the mutagenesis; MG1363[pAGX2934] (LDFGGQIK (SEQ ID NO: 103); top plate) and MG1363[pAGX2947] (QDFGGQIK (SEQ ID NO: 102); bottom plate). Because double mutants LNFGGQIK (SEQ ID NO: 115) and QNFGGQIK (SEQ ID NO: 116) were not yet available at the time of the construction and screening of the triple mutants, the same single mutants were used: MG1363[pAGX2934] (LDFGGQIK (SEQ ID NO: 103); top plate) and MG1363[pAGX2947] (QDFGGQIK (SEQ ID NO: 102); bottom plate). With the average absorbance of positive control 1, the lower limit for the high binding clones was calculated. The second positive control depends on the construct. KDFGGQIK ((SEQ ID NO:8) wt CDR3+1; MG1363[pAGX2701]) is used as the positive control 2 in the screening of the double mutants, and KNFGGQIK (SEQ ID NO: 106) (MG1363[pAGX2909]) as the positive control 2 in the screening of the triple mutants. The average absorbance of the positive control 2 is used to calculate the lower limit for the intermediate binding clones. MG1363[pTlNX] was used as the negative control.

[00192] Results of the double mutants are shown in Figures 17A-17B and those of the triple mutants in Figure 18A-18B. Both high binding clones (absorbance (A450) at least 20% above the average absorbance of positive control 1; stippled) and intermediate binding clones (A450 in between the good binders and 3 times the average absorbance of positive control 2; bolded) were selected for Sanger sequencing. Almost all high binders were identified in the 96- well plate LNXGGQIK (SEQ ID NO: 120) (pAGX2949) (see Figure 18A). In total, the nucleotide sequences of 45 high binders and 60 intermediate binders were determined. Twenty- six (26) unique sequences were identified. An overview of all identified amino acids with the corresponding codons is summarized in Figure 19. Details about the clones selected for each construction are described in the following.

[00193] In the following, clones were analyzed for expression, binding to hTNF-a, and/or neutralization of hTNF-a. Equivalents of 1 ml of culture supernatant of L. lactis strains grown in buffered minimal medium, BRPMIE, were analyzed for expression, as described, for instance, in Example 5. A 1/2 dilution series of culture supernatant (BRPMIE) was analyzed for binding to hTNF-a, as described previously (see, for instance, in Example 5). A 1/3 dilution series of culture supernatant (BRPMIE) was analyzed for binding to hTNF-a, as described previously (see, for instance, in Example 5).

A. LXFGGQIK. pAGX2947d

[00194] The nucleotide sequences of 14 intermediate binders in mutagenesis LXFGGQIK (SEQ ID NO: 118) (pAGX2947d, see Figure 17A, LXFGGQIK) were determined. Some clones showed clean sequences, whereas others showed mixed reads. Five (5) clones with unique sequences were streaked to a single colony; 3 or 9 colonies for each clone were analyzed, depending on the quality of the first sequence. Three (3) amino acid substitutions at position X (see Figure 19) were found: asparagine (aac and aat (which is identical to pAGX2939)), serine (age, tcc, and teg), and threonine (acc). All these mutants, as well as double mutant LNFGGQIK (SEQ ID NO: 115) (pAGX2939; described in Example 6), single mutant LDFGGQIK (SEQ ID NO: 103) (pAGX2934), the original single domain antibody (KDFGGQIK, (SEQ ID NO:8); wt CDR3+1), plus a negative control (pTINX) were prepared for expression of the different VHH in buffered minimal medium, BRPMIE.

[00195] The hTNF-a binding data are shown in Figure 20, and the expression data are shown in Figure 21.

[00196] As seen in Figure 20, mutation of position 2 to asparagine with codon aac (LNFGGQIK; SEQ ID NO; 115) showed the best hTNF-a binding. Double mutant LNFGGQIK (SEQ ID NO: 115) (pAGX2939) with the codon aat for asparagine is less efficient. The decreased efficiency is believed to be due to impaired growth of the bacteria and, consequently, less expression of the sdAb (Figure 21). The clone with the alternative codon for asparagine (aac) was stored as pAGX2947.

[00197] Mutation of position 2 to serine (LSFGGQIK: SEQ ID NO: 122) shows slightly improved hTNF-a binding, depending on the codon usage. This is related to better expression of the different VHH domains, as can be seen in Figure 21. The clone with the age codon for serine shows the better expression and hTNF-a binding of the three clones.

[00198] Mutation of position 2 to threonine (LTFGGQIK: SEQ ID NO: 123) does not show improved hTNF-a binding and is comparable to the original aspartic acid in that position.

[00199] The culture supernatant was also tested for the capacity to neutralize hTNF-a (Figure 22). Double mutant LNFGGQIK (SEQ ID NO: 115) with codon aac for asparagine showed the best hTNF-a neutralization. Double mutant LNFGGQIK (SEQ ID NO: 115) (pAGX2939) with the codon aat for asparagine is less efficient due to impaired growth of the bacteria.

[00200] Double mutant LSFGGQIK (SEQ ID NO: 122) shows the same trend in hTNF-a neutralization capacity as for hTNF-a binding, with codon age for serine being better than the teg codon and better than the tec codon.

[00201] Mutation of position 2 to threonine (LTFGGQIK; SEQ ID NO: 123) shows improved hTNF-a neutralization comparable to the best LSFGGQIK (SEQ ID NO: 122). Double mutants ESFGGQIK (SEQ ID NO: 122) and LTFGGQIK (SEQ ID NO: 123) do not show improved hTNF-a neutralization as compared to the single mutant they are derived from; LDFGGQIK (SEQ ID NO: 103).

[00202] From this round of mutagenesis, it can be concluded that double mutant LNFGGQIK (SEQ ID NO: 115) with aac coding for asparagine showed much better binding to hTNF-a and only minor improved hTNF-a neutralization than single mutant LDFGGQIK (SEQ ID NO: 103).

B. QXFGGOIK. pAGX2948d

[00203] In this mutagenesis, only 3 intermediate binding clones were identified (see Figure 17B, QXFGGOIK: SEQ ID NO: 119). None of them had a mutant amino acid. Only one clone had an alternative coding for aspartic acid: gac codon instead of gat codon. This single mutant was stored as pAGX2948.

[00204] The two clones with different codons for aspartic acid, plus a negative control (pTINX) were prepared for expression of the different VHH in buffered minimal medium, BRPMIE, as well as for binding to hTNF-a, and hTNF-a neutralization. A comparison of both configurations showed a minor difference between the codons in expression (see Figure 23). Binding to hTNF-a and hTNF-a neutralization was comparable for both single mutants (see Figure 24).

C. LNXGGOIK. pAGX2949d

[00205] For LNXGGQIK (SEQ ID NO: 120), 44 high binding clones and 36 intermediate

[00206] binding clones were identified (see Figure 18 A, LNXGGQIK). Some clones showed clean sequences, while others showed mixed reads, which is a result of mixed clones. Sequence analysis showed position X of LNXGGQIK substituted with codons for almost all amino acids except for tryptophan (see Figure 19). The high binding clones showing the highest absorbance, with unique amino acids and the cleanest sequences were focused on. These clones were streaked to a single colony and analyzed (3 colonies for each clone). The (10) clones were selected for expression analysis (indicated with in Figure 19). The original single domain antibody was included as reference (wt; CDR3+1, KDFGGQIK (SEQ ID NO:8), MG1363[pAGX2701]), MG1363[pTlNX] as the negative control, and double mutant LNFGGQIK (SEQ ID NO: 115) (MG1363[pAGX2939]) as the positive control. Given that the latter strain has a growth defect, there is a reduction in the amount of sdAb that can be recovered compared to other sdAb expressing strains. Therefore, MG1363[pAGX2947] was also included; MG1363[pAGX2947] has the same CDR3+1 sequence, LNFGGQIK (SEQ ID NO: 115), and shows better growth (see Figure 21). The triple mutant LNPGGQIK (SEQ ID NO:87) made in Example 6 (pAGX2944), has a different codon for proline (cca) as compared to the clone isolated during this mutagenesis (cct; pAGX2963), which was also included.

[00207] Three codons were identified for triple mutant LNRGGQIK (SEQ ID NO:94): agg, egg, and cga. The expression was compared in a Coomassie-stained protein gel (Figure 21); only the clone with agg (pAGX3002) was included for this mutant for data presented in Figure 25 and Figure 26, because this mutant was slightly better expressed than the other mutants with codon egg or cga.

[00208] Binding to hTNF-a and hTNF-a neutralization was also assayed. The data are shown in Figure 26.

[00209] Changing position 3 to proline leads to the most efficient hTNF-a binding and hTNF-a neutralization properties. When the triple mutant LNPGGQIK (SEQ ID NO:87; pAGX2963) obtained in this experiment is compared with the pAGX2944 clone having the same triple mutant described in Example 6, it is evident that the codon usage for proline has no detectable influence on the binding efficiency (Figure 25) or expression. LNPGGQIK (SEQ ID NO:87) mutant with proline codon cca (pAGX2944) shows better hTNF-a neutralization than the LNPGGQIK (SEQ ID NO:87) mutant with proline codon cct (pAGX2963) (Figure 26). This difference in neutralization may reflect a difference in expression since VHH levels were not normalized in these assays

[00210] Mutation of position 3 to threonine (LNTGGQIK: SEQ ID NO:95), glutamine (LNQGGQIK: SEQ ID NO:92) or glutamic acid (LNEGGQIK: SEQ ID NO:91) showed equal binding efficiency to hTNF-a and slightly less than LNPGGQIK (SEQ ID NO:87). (Figure 25) Neutralization of hTNF-a shows more pronounced differences between these three mutants, with LNTGGQIK (SEQ ID NO:95) being less efficient than LNEGGQIK(SEQ ID NO:91) or LNQGGQIK (SEQ ID NO:92). (Figure 26).

[00211] The triple mutant with histidine at position 3 (LNHGGQIK: SEQ ID NO: 156) neutralizes hTNF-a equally efficiently as LNTGGQIK (SEQ ID NO:95), while binding to hTNF-a was less efficient. Triple mutants with arginine (LNRGGQIK: SEQ ID NO:94) or histidine (LNHGGQIK: SEQ ID NO: 156) at position 3 bound more efficiently to hTNF-a than the better-growing double mutant LNFGGQIK (SEQ ID NO: 115) (pAGX2947). Mutation of position 3 to tyrosine (SEQ ID NO: 158) or alanine (SEQ ID NO: 157) showed less efficient binding to hTNF-a than the better-growing double mutant (pAGX2947) and better than double mutant pAGX2939.

[00212] The following configurations were retained:

■ LNEGGQIK (SEQ ID NO:91) (gag; clone pAGX2949d-C9.3) named pAGX2949.

■ LNPGGQIK (SEQ ID NO:87) (cct; clone pAGX2949d-E4.3) stored as pAGX2963. This is the same amino acid as in pAGX2944 but coded by a different codon.

■ LNQGGQIK (SEQ ID NO:92) (caa; clone pAGX2949d-H9.3) stored as pAGX2964.

■ LNTGGQIK (SEQ ID NO:95) (act; clone pAGX2949d-D5.1) stored as pAGX3001.

■ LNRGGQIK (SEQ ID NO:94) (agg; clone pAGX2949d-B8.2) stored as pAGX3002.

D. QNXGGOIK. pAGX2950d

[00213] For QNXGGQIK (SEQ ID NO: 121), 1 high binding and 7 intermediate binding clones were identified (see Figure 18B, QNXGGQIK (SEQ ID NO: 121)). Most of these clones encoded proline at position X; codon ccc in the high binding clone (retained as pAGX2950) and in the intermediate binding clones: codon cca (as in pAGX2945) or codon ccg. Binding to

[00214] hTNF-a by pAGX2950 was compared to a selection of double and triple mutants made in Example 6. The data are shown in Figure 27. The best binding is observed when the first three amino acids of CDR3+1 are changed to QNPGGQIK (SEQ ID NO:88). The codon usage for proline, cca versus ccc, has no discernable influence on the expression or the binding efficiency. Single mutants KNFGGQIK (SEQ ID NO: 106) and KDPGGQIK (SEQ ID NO:96) bind equally efficiently to hTNF-a. QDFGGQIK (SEQ ID NO: 102), the single mutant with glutamine at position 1 (pAGX2933), binds better to hTNF-a than the other two single mutants. Introducing asparagine at position 2, which results in QNFGGQIK (SEQ ID NO: 116) did not improve binding to hTNF-a. The binding efficiency of double mutants KNPGGQIK (SEQ ID NO:89) and QDPGGQIK (SEQ ID NO:93) is situated between the triple mutant QNPGGQIK (SEQ ID NO:88) and the single mutant QDFGGQIK (SEQ ID NO: 102).

Example 8: Validation of double and triple mutant VHH

[00215] The culture supernatant of a selection of mutants was analyzed by Coomassie Brilliant Blue (CBB) stained SDS-PAGE (right panel of Figure 29), in a western blot (left panel of Figure 29) and by hTNF-a binding (Figure 20, Figure 25, and Figure 27) and hTNF-a neutralization assay (Figure 22, Figure 26, and Figure 28). Because the double mutant ENFGGQIK (SEQ ID NO: 115) (pAGX2939) showed severe growth retardation, it was replaced by pAGX2947, which expresses the same mutant amino acid, but uses a different codon as was isolated during the second round of mutagenesis (see Example 7). As shown in Figure 15 and Figure 16, double mutant KDFGGQER (SEQ ID NO: 117) (pAGX2946) shows only marginal improvement of hTNF-a binding and neutralization.

[00216] CBB stained SDS-PAGE in Figure 29 shows that most sdAbs are highly expressed. In western blots, all the mutant VHH-domains are recognized. While overall, the correlation between western blot and CBB stained SDS-PAGE is good, in some instances, the intensities do not always correlate with protein quantity. For example, pAGX2909 and pAGX2943 show a more intense band on the Coomassie-stained gel than on the western blot. For pAGX2934, the opposite is observed: detection on the western blot is relatively more intense than via CBB staining. As already mentioned above, a larger variation in mobility is observed than what is expected based on differences in molecular weight of these single domain antibodies; 13.39 to 13.48 kDa.

[00217] From the above-described examples, the following conclusions can be made:

■ Random mutagenesis of single amino acids in CDR3+1 of VHHTNAOOlh, a hTNF-a neutralizing single domain antibody, can improve binding to hTNF-a and neutralization of hTNF-a. ■ Enhanced hTNF-a binding is correlated with improved neutralization of hTNF- a, but is not necessarily predictive of improved neutralization of hTNF-a.

■ It is possible to combine single mutants to further increase binding to hTNF-a and neutralization of hTNF-a.

■ The best hTNF-a binding and neutralization are observed in triple CDR3+1 mutants.

■ Binding and neutralization of hTNF-a by VHHTNAOOlh can be greatly improved by changing CDR 3 from KDFGGQIK (SEQ ID NO:8) to LNPGGQIK (SEQ ID NO:87).

■ Exchanging the CDR3 from different single domain antibodies with different affinities for hTNF-a was not successful in improving binding and neutralization.

Example 9: Selection of VHH camelid single-domain antibodies (VHH sdAb) that are able to neutralize human TNF-a

[00218] A review of the hTNF-a binding and hTNF-a neutralization data led to the selection of the following mutant VHH sdAbs. Table 15 lists the name, the plasmid, and the sequences of the three complementarity-determining regions (CDRs) for each VHH.

Table 15

[00219] The hTNF-a binding data of the ten selected VHH and wild-type parent VHHTNAOOlh are depicted in Figure 30. The hTNF-a neutralization data of the selected VHH are depicted in Figure 31.

[00220] Of the ten mutant VHH, VHHTNA032h has the best neutralizing property, and VHHTNAO33h has the second-best neutralizing property, compared to the unmutated wild-type parent VHHTNAOOlh. These two sdAbs are triple mutants of VHHTNAOOlh. The other eight VHH’s all possessed good neutralization properties. [00221] Accordingly, these single-domain antibodies (VHH) are contemplated as particularly useful for therapeutic applications. In a particular application, delivery by oral administration of, such as non-pathogenic, Gram-positive bacteria, e.g., lactic acid bacteria such as Lactococcus lactis, genetically engineered to express an anti-hTNF-a sdAb for accumulation and/or secretion is of medical interest. Anti-TNF-a VHH may be humanized according to well- described procedures such as disclosed in Vincke et al., 2009 (“General strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold,” J. Biol. Chem. 284(5): 3273-84, PMID: 19010777) or as in WO 2006122825A9 (Single domain vhh antibodies against von Willebrand factor, vwf). The recombinant bacteria may also be equipped/engineered to produce other adventitious molecules (such as metabolites, proteins, enzymes, adherence factors, RNAi). Such molecules may not relate to the therapeutic or beneficial effect but help to formulate the product, deliver, or retain the polypeptide to a specific substrate or body location. Such polypeptides may be expressed as a separate polypeptide or may be fused to the active polypeptide in the form of a fusion protein, optionally via a linker. A “fusion protein” is one that generally consists of one or more different proteins or polypeptide fragments of a protein that are joined together with or without a linker or spacer. A linker in a fusion protein can consist of one or more amino acids but are generally short. Linkers can be flexible or rigid. A polypeptide linker can be designed to be cleavable.

Materials and Methods

[00222] The following materials and methods were employed in carrying out the work described in the examples.

I. Materials

Strains

[00223] Lactococcus lactis (L. lactis) subsp. cremoris, strain MG 1363.

Plasmids

[00224] An overview of all plasmids used and generated in this study is listed in TABLE 16. In the table, CDR3+1, which, as used herein, indicates a CDR3 and further includes the C- terminal amino acid, is presented using a single-letter amino acid code. X represents any random amino acid. Underlined amino acids correspond to mutations as compared to the original CDR3+1 of VHHTNAOOlh; KDFGGQIK (SEQ ID NO: 8).

[00225] All single domain antibody (sdAb) genes are under the control of the constitutive promoter of the HU-like DNA-binding protein gene (P/zZZA; Gene ID: 4797353) and are preceded by sequences encoding the signal sequence from ps356 endolysin (SS21; ps356; Gene ID: 4798697; UniProtKB A2RJJ4) or the signal sequence of gamma-glutamyl-diamino acid- endopeptidase (SS09; Umg_1594; Gene ID: 4798983; UniProtKB A2RLK0).

Table 16

² "CDR3+1” refer to CDRs from sdAb’s based on VHHTNA001 include the flanking C-terminal residue.

Table 17 Reagents

Stock Solutions

• 20% w/v glucose stock solution (Merck #1.08337).

• 25 mg/ml erythromycin solution

• 100 mM CaCh stock solution.

• 0.5 M NaHCCh stock solution.

• 0.5 M Na2CO3 stock solution.

• 2.5% casein.

• 10X PBS.

• 100% Tween 20 (Sigma P1379).

• Wash buffer: lx PBS - 0.05% Tween 20.

• TMB stabilized chromagen (Biosource SB02).

• 0.5 M HC1 (hydrochloric acid).

• 5 mg/ml sodium deoxycholate.

• 100% trichloroacetic acid solution.

• 10 ml 4X NuPAGE® LDS Sample Buffer (Invitrogen NP0007).

• 250 pl 10X NuPAGE™ Sample Reducing Agent (Invitrogen NP0004).

• 500 ml 20X NuPAGE™ MES SDS Running Buffer (Invitrogen NP0002).

• 15 ml NuPAGE® Antioxidant (Invitrogen NP0005).

• Imperial™ Protein Stain (Invitrogen 24617).

• 500 ml Intercept™ (PBS) Blocking Buffer (LI-COR #927-70001).

• 0.05% trypsin with EDTA (Life Technologies #25200-056)

• Trypan Blue Stain (Life Technologies #15250-061)

• 1 mg/ml Actinomycin D (Calbiochem #114666) dissolved in dimethyl sulfoxide (DMSO).

• CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega #G3582) Culture media

• M17 broth (Oxoid #CM0817).

• GM17 is M17 supplemented with 0.5% glucose.

• GM17E is GM17 supplemented with 5 pg/ml erythromycin.

• RPMI 1640 Medium, HEPES (Gibco™ #52400-025)

• BRPMI: RPMI 1640 with an increased glucose concentration to 0.5% and additional buffering with NaHCO3/Na2CO3 to a pH between 8.1 and 8.5.

• Agar plates.

• lOOOx Penicillin-Streptomycin (Lonza #DE17-602E)

• DMEM medium with glutamine and L-glutamine, without sodium pyruvate (Lonza #BE12-741-F) • Fetal Bovine Serum (Life Technologies, #10100-147)

Oligonucleotides

[00226] Table 18 is a list of oligonucleotides used in these examples. “N” indicates a random nucleotide.

Table 18

Enzymes

• Herculase II Fusion DNA polymerase was purchased from Agilent Technologies (#600677) and was used according to the instructions of the manufacturer.

• T4 Polynucleotide Kinase was purchased from New England Bio labs (#M020 I S) and was used according to the manufacturer’s instructions.

• T4 DNA Ligase was purchased from Roche (#10 716 359 001) and was used according to the manufacturer’s instructions.

• PurelT ExoZAP PCR Clean-Up was purchased from Ampliqon (#A620603) and was used according to the manufacturer’s instructions.

DNA purification

• Qiagen Plasmid Mini kit (100) (Qiagen cat#12125) was purchased from QIAGEN and was used to the specifications of the manufacturer.

• Qiagen MinElute PCR Purification kit (250) (Qiagen cat#28006) was purchased from QIAGEN and was used to the specifications of the manufacturer.

DNA molecular weight marker

• Tracklt™ 1 Kb plus DNA Ladder (Thermo Fisher Scientific, Invitrogen, Waltham, MA; • #10488-085) was used for size estimation. The DNA Ladder contains the following relevant molecular weight markers (MWM) in base pairs (bp): 100, 200, 300, 400, 500, 650, 850, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 10000 and 15000.

Protein molecular weight markers

• In near-infrared detection, performed with an Odyssey® CLx Infrared Imaging System, Chameleon® Duo Pre-stained Protein Ladder (LLCOR Biosciences, Lincoln, NE; #928- 60000) from LLwas used for size estimation. The standard includes 11 protein bands: 8, 15, 25, 30, 38, 50, 70, 90, 125, 160, and 260 kDa.

• Markl2™ Unstained Standard (Thermo Fisher Scientific, Invitrogen, Waltham, MA; #LC5677) was used for size estimation in protein gels for Coomassie staining. The standard includes 12 protein bands: 2.5, 3.5, 6, 14.4, 21.5, 31, 36.5, 55.4, 66.3, 97.4, 125, 116.3, and 200 kDa.

Protein gels and blots

• NuPAGE™ Novex™ 10% Bis-Tris Protein Gels, 1.0 mm, 12-well (Thermo Fisher Scientific, Invitrogen, Waltham, MA; #NP0302BOX) and was used according to the specifications of the manufacturer.

• NuPAGE™ Novex™ 10% Bis-Tris Midi Protein Gels, 26-well (Thermo Fisher Scientific, Invitrogen, Waltham, MA; #WG1203BOX) and was used according to the specifications of the manufacturer.

• iBlot™ 2 NC Mini Stacks (Thermo Fisher Scientific, Invitrogen, Waltham, MA; #IB23002) were used according to the specifications of the manufacturer.

• iBlot™ 2 NC Regular Stacks (Thermo Fisher Scientific, Invitrogen, Waltham, MA; #IB23001) were used according to the specifications of the manufacturer.

Proteins

• hTNF-a: recombinant (E. coli) - stock solution: 1 mg/ml; lot number MH091026A; 6.8 x 10 ⁷ lU/mg MH091026A (Q-Biologicals NV, Ghent, Belgium).

Antibodies

• Goat polyclonal anti- llama IgG (Bethyl Laboratories A160-100A).

• Rabbit anti-llama-biotin (Mybiosource #MBS687396).

• MonoRab™ rabbit anti-camelid VHH IgG (Genscript Biotech, Piscataway, NJ; #A01860).

• IRDye 800 CW Donkey Anti-Goat (LLCOR #926-32214).

• Goat anti-rabbit IgG-HRP (Southern Biotech 4050-05).

• Streptavidin-HRP (BD Biosciences #554066)

Storage of bacterial strains

Bacterial strains were stored in ActoBio Therapeutics’ strain collection in Microbank™ bacterial and fungal preservation system (Pro-lab Diagnostics, Cat # PL. 170), used according to the specifications of the manufacturer.

II. Methods

[00227] Sanger sequencing was performed by Eurofins Genomics (Constance, Germany).

Expression

[00228] Two types of expressions were employed. For screening colonies in a hTNF-a binding assay, we used cultures grown in microtiter plates or in tubes for 16h at 32°C in GM17E. For expression, samples for loading on a protein gel or for analysis in a hTNF-a binding or neutralization assay; we used a standard operating procedure. Briefly, L. lactis strains were grown at 32°C for 3 h in GM17E followed by 3 h expression in BRPMIE. The bacteria were removed by centrifugation, and the culture supernatant was used in a hTNF-a binding or neutralization assay. The culture supernatant was precipitated with trichloroacetic acid for loading on a protein gel.

Protein gel analysis

[00229] Equivalents of 1 ml of culture supernatant were loaded on a 10% Bis-Tris Midi protein gel and run at 200 V in MES -buffer for about 35 min. When the protein gel had to be stained with Coomassie Brilliant Blue (Imperial™ Protein Stain, we used Markl2 as a molecular weight marker. When the protein gel was blotted, we used the Chameleon Duo pre-stained protein ladder. Coomassie-stained protein gels can be visualized with the 700 nm channel of the Odyssey CEx or scanned with a flatbed scanner.

Western blot

[00230] After blotting, the membrane is blocked in Intercept blocking buffer for at least 2 h at room temperature. Recombinant VHH-domains were detected with goat anti- llama IgG. This step is done overnight at 5°C. The next day the membrane is washed 3 times for 5 min with Intercept blocking buffer and then incubated at room temperature with IRDye 800CW donkey anti-goat IgG. Unbound antibody is removed by washing the membrane 3 times for 5 min with Intercept blocking buffer, followed by a washing step with lx PBS. Fluorescent signals were visualized with the Odyssey CEx. The intensity of the signals at 700 and 800 nm are chosen to see clear signals without introducing too much background signal. hTNF-a binding ELISA

[00231] Maxisorp F96 plates (Nunc #442404) were coated with 0.5 pg/ml hTNF-a (Q- Biologicals #MH091026A) overnight at 5°C. After blocking with 0.1% casein, samples were added. Bound single-domain antibodies were detected with rabbit anti-llama-biotin (Mybiosource #MBS687396), followed by streptavidin- HRP (BD #554066) and TMB solution (Life Technologies #SB02) for detection. The reaction was stopped by adding 0.5 M HC1, and the absorbance was read at 450 nm for measuring and 595 nm as reference. hTNF-a neutralization assay

[00232] A suspension of L929 cells at 2 x 10 ⁵ cells/ml was added to a flat bottom 96 well assay plate and incubated overnight at 37°C, 5% CO2 in a humidified incubator. In a separate plate, an equal volume of a dilution series of the samples was mixed with an equal volume of 10 lU/ml hTNF-a and 2 pg/ml Actinomycin D. 100 pl of this mixture was added to the cells. Untreated cells and cells treated with hTNF-a and Actinomycin D served as controls. The plates were incubated for 20 to 24 h at 37°C, 5% CO2 in a humidified incubator. Afterward, 20 pl CellTiter 96® AQueous One Solution Reagent was added to the wells, and the plates were incubated for another 4 h at 37°C, 5% CO2 in a humidified incubator. The absorbance was read at 490 nm for measuring and 700 nm as a reference to determine the amount of cell death. The result is expressed as % neutralization versus hTNF-a controls.

EXEMPLARY EMBODIMENTS

Embodiment 1. An antibody polypeptide comprising a single variable domain that specifically binds a human Tumor Necrosis Factor-alpha (TNF-a) comprising the amino acid sequence of SEQ ID NO:1, wherein the single variable domain comprises:

(a) a complementary determining region 1 (CDR1) sequence having up to one amino acid substitution in the amino acid sequence of Ile-Tyr-Trp-Met-Thr (SEQ ID NO:3),

(b) a complementary determining region 2 (CDR2) sequence having up to one amino acid substitution in the amino acid sequence of Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg-Arg-Tyr- Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4), and

(cl) a complementary determining region 3 (CDR3) sequence having from one to three amino acid substitutions in the amino acid sequence of Lys-Asp-Phe-Gly-Gly-Gln-Ile (SEQ ID NO: 5), or

(c2) a complementary determining region 3 (CDR3) sequence that is a CDR3+1 sequence having from one to three amino acid substitutions in the amino acid sequence of Lys- Asp-Phe-Gly-Gly-Gln-Ile-Lys (SEQ ID NO: 8).

Embodiment 2. The antibody polypeptide of embodiment 1, wherein:

(a) the CDR1 sequence is Ile-Tyr-Trp-Met-Thr (SEQ ID NOG), and

(b) the CDR2 sequence is Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg-Arg-Tyr-Ala-Asp- Ser-Val-Glu-Gly (SEQ ID NO:4).

Embodiment 3. The antibody polypeptide of embodiment 2, wherein

(cl) the CDR3 sequence is Xi- X2--X3-Gly-Gly-Gln-Ile (SEQ ID NO:97), wherein Xi is Lys, Leu, or Gin; X2 is Asp or Asn; and X3 is Phe, Pro, Thr, Arg, Gin, or Glu, or

(c2) the CDR3+1 sequence is Xi- X2--X ₃ -Gly-Gly-Gln-Ile-Lys (SEQ ID NO:98), wherein Xi is Lys, Leu, or Gin; X2 is Asp or Asn; and X3 is Phe, Pro, Thr, Arg, Gin, or Glu.

Embodiment 4. The antibody polypeptide of any one of embodiments 1 to 3, wherein the third amino acid residue in the CDR3 or CDR3+1 sequence is Pro.

Embodiment 5. The antibody polypeptide of any one of embodiments 1 to 3, wherein the second amino acid residue in the CDR3 or CDR3+1 sequence is asparagine (Asn). Embodiment 6. The antibody polypeptide of embodiment 5, where the CDR3 sequence is: LNTGGQI (SEQ ID NO:25; TNA037), LNRGGQI (SEQ ID NO:23; TNAO38), LNQGGQI (SEQ ID NO: 19; TNA036), LNEGGQI (SEQ ID NO: 17; TNA035), LNPGGQI (SEQ ID NO:9; TNA032), QNPGGQI (SEQ ID NO: 11; TNAO33), or KNPGGQI (SEQ ID NO: 13; TNA031).

Embodiment 7. The antibody polypeptide of embodiment 5, where the CDR3+1 sequence is: LNTGGQIK (SEQ ID NO:95; TNA037), LNRGGQIK (SEQ ID NO:94; TNAO38), LNQGGQIK (SEQ ID NO:92; TNA036), LNEGGQIK (SEQ ID NO:91; TNA035), LNPGGQIK (SEQ ID NO:87; TNA032), QNPGGQIK (SEQ ID NO:88; TNAO33), or KNPGGQIK (SEQ ID NO:89; TNA031).

Embodiment 8. The antibody polypeptide of any one of embodiments 1 to 3, wherein the second amino acid residue of the CDR3 or CDR3+1 sequence is Asp.

Embodiment 9. The antibody polypeptide of any one of embodiments 1 to 3, wherein the first amino acid residue of the CDR3 or CDR3+1 sequence is leucine (Leu).

Embodiment 10. The antibody polypeptide of any one of embodiments 1 to 3, wherein the first amino acid residue of the CDR3 sequence is glutamine (Gin).

Embodiment 11. The antibody polypeptide of embodiment 1, wherein the CDR1, CDR2, and CDR3 sequences are:

(a) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg-Arg- Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and LNPGGQI (SEQ ID NO:9; TNA032);

(b) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg-Arg- Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and QNPGGQI (SEQ ID NO: 11; TNAO33);

(c) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg-Arg- Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and KNPGGQI (SEQ ID NO: 13; TNA031);

(d) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg-Arg- Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and LDPGGQI (SEQ ID NO: 15; TNA028);

(e) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg-Arg- Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and LNEGGQI (SEQ ID NO: 17; TNA035); (f) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg-Arg- Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and LNQGGQI (SEQ ID NO: 19; TNA036);

(g) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg-Arg- Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and QDPGGQI (SEQ ID NO:21; TNA030);

(h) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg-Arg- Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and LNRGGQI (SEQ ID NO:23; TNAO38);

(i) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg-Arg- Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and LNTGGQI (SEQ ID NO:25; TNA037); or

(j) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg-Arg- Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and KDPGGQI (SEQ ID NO:27; TNA019).

Embodiment 12. The antibody polypeptide of embodiment 1, wherein the CDR1, CDR2, and CDR3+1 sequences are:

(a) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg-Arg- Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and LNPGGQIK (SEQ ID NO:87; TNA032);

(b) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg-Arg- Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and QNPGGQIK (SEQ ID NO:88; TNAO33);

(c) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg-Arg- Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and KNPGGQIK (SEQ ID NO:89; TNA031);

(d) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg-Arg- Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and LDPGGQIK (SEQ ID NO:90; TNA028);

(e) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg-Arg- Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and LNEGGQIK (SEQ ID NO:91; TNA035);

(f) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg-Arg- Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and LNQGGQIK (SEQ ID NO:92; TNA036);

(g) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg-Arg- Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and QDPGGQIK (SEQ ID NO:93; TNA030);

(h) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg-Arg- Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and LNRGGQIK (SEQ ID NO:94; TNAO38);

(i) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg-Arg- Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and LNTGGQIK (SEQ ID NO:95; TNA037); or (j) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg-Arg- Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and KDPGGQIK (SEQ ID NO:96; TNA019).

Embodiment 13. A pharmaceutical composition comprising an antibody polypeptide of any one of embodiments 1 to 12 and optionally a pharmaceutically acceptable carrier.

Embodiment 14. Use of an antibody polypeptide of any one of embodiments 1 to 12 or the pharmaceutical composition of Embodiment 13in the treatment of an inflammatory condition or a pathological condition.

Embodiment 15. Use of an antibody polypeptide of any one of embodiments 1 to 12 or the pharmaceutical composition of Embodiment 13for the preparation of a medicament for the treatment of an inflammatory condition or a pathological condition.

Embodiment 16: A method of treating an inflammatory condition or a pathological condition in a patient in need thereof comprising administering to the patient a therapeutically effective amount of the an antibody polypeptide of any one of embodiments 1 to 12 or the pharmaceutical composition of Embodiment 13.

Embodiment 17. A Gram-positive bacterium comprising:

(a) a polynucleic acid encoding the antibody polypeptide of any one of embodiments 1 to 12;

(b) a vector comprising a polynucleic acid encoding the antibody polypeptide of any one of embodiment s 1 to 12;

(c) a polycistronic expression unit comprising, in 5’ to 3’ order:

(i) a promoter endogenous to a Gram-positive bacterium;

(ii) a gene endogenous to the Gram-positive bacterium;

(iii) an intergenic region active in the Gram-positive bacterium; and

(iv) either (1) a polynucleic acid encoding the antibody polypeptide of any one of claims 1 to 12, or (2) a vector comprising a polynucleic acid encoding the antibody polypeptide of any one of embodiment s 1 to 12. Embodiment 18. Use of the Gram-positive bacterium of embodiment 17 in the treatment of an inflammatory condition or a pathological condition.

Embodiment 19. Use of the Gram-positive bacterium of embodiment 17 for the preparation of a medicament for the treatment of an inflammatory condition or a pathological condition.

Embodiment 20: A method of treating an inflammatory condition or a pathological condition in a patient in need thereof comprising administering to the patient a therapeutically effective amount of the Gram-positive bacterium of embodiment 17.

FURTHER EMBODIMENTS

Embodiment 101. An antibody polypeptide comprising a single variable domain that specifically binds a human Tumor Necrosis Factor-alpha (TNF-a) comprising the amino acid sequence of SEQ ID NO:1, wherein the single variable domain comprises:

(a) a complementary determining region 1 (CDR1) sequence having up to one amino acid substitution in the amino acid sequence of Ile-Tyr-Trp-Met-Thr (SEQ ID NOG),

(b) a complementary determining region 2 (CDR2) sequence having up to one amino acid substitution in the amino acid sequence of Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg-Arg-Tyr-Ala- Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4), and

(c) a complementary determining region 3 (CDR3) sequence having from one to three amino acid substitutions in the amino acid sequence of Lys-Asp-Phe-Gly-Gly-Gln-Ile (SEQ ID NO: 5).

Embodiment 102. The antibody polypeptide of embodiment 101, wherein the CDR3 sequence is a CDR3+1 sequence having from one to three amino acid substitutions in the amino acid sequence of Lys-Asp-Phe-Gly-Gly-Gln-Ile-Lys (SEQ ID NO: 8).

Embodiment 3. The antibody polypeptide of embodiment 101 or 102, wherein:

(a) the CDR1 sequence is Ile-Tyr-Trp-Met-Thr (SEQ ID NOG), and

(b) the CDR2 sequence is Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg-Arg-Tyr-Ala-Asp-Ser-Val- Glu-Gly (SEQ ID NOG). Embodiment 104. The antibody polypeptide of embodiment 101 or embodiment 103, wherein

(c) the CDR3 sequence is Xi- X2--X3-Gly-Gly-Gln-Ile (SEQ ID NO:97), wherein Xi is Lys, Leu, or Gin; X2 is Asp or Asn; and X3 is Phe, Pro, Thr, Arg, Gin, or Glu.

Embodiment 105. The antibody polypeptide of embodiment 102 or embodiment 103, wherein

(c) the CDR3+1 sequence is Xi- X2--X3-Gly-Gly-Gln-Ile-Lys (SEQ ID NO:98), wherein Xi is Lys, Leu, or Gin; X2 is Asp or Asn; and X3 is Phe, Pro, Thr, Arg, Gin, or Glu.

Embodiment 106. The antibody polypeptide of any one of embodiments 101 to 105, wherein the third amino acid residue in the CDR3 or CDR3+1 sequence is Pro.

Embodiment 107. The antibody polypeptide of embodiment 106, wherein the CDR3 sequence is: KDPGGQI (SEQ ID NO: 27; TNA019), QDPGGQI (SEQ ID NO: 21; TNA030), LDPGGQI (SEQ ID NO: 15; TNA028), QNPGGQI (SEQ ID NO:.11; TNAO33), or KNPGGQI (SEQ ID NO: 13; TNA031).

Embodiment 108. The antibody polypeptide of embodiment 106, wherein the CDR3+1 sequence is: KDPGGQIK (SEQ ID NO: 96; TNA019), QDPGGQIK (SEQ ID NO: 93; TNA030), LDPGGQIK (SEQ ID NO: 90; TNA028), QNPGGQIK (SEQ ID NO:88; TNAO33), or KNPGGQIK (SEQ ID NO:89; TNA031).

Embodiment 109. The antibody polypeptide of any one of embodiments 101 to 105, wherein the second amino acid residue in the CDR3 or CDR3+1 sequence is asparagine (Asn).

Embodiment 110. The antibody polypeptide of embodiment 109, where the CDR3 sequence is: LNTGGQI (SEQ ID NO:25; TNA037), LNRGGQI (SEQ ID NO:23; TNAO38), LNQGGQI (SEQ ID NO: 19; TNA036), LNEGGQI (SEQ ID NO: 17; TNA035), LNPGGQI (SEQ ID NO:9; TNA032), QNPGGQI (SEQ ID NO: 11; TNAO33), or KNPGGQI (SEQ ID NO: 13; TNA031). Embodiment 111. The antibody polypeptide of embodiment 109, where the CDR3+1 sequence is: LNTGGQIK (SEQ ID NO:95; TNA037), LNRGGQIK (SEQ ID NO:94; TNAO38), LNQGGQIK (SEQ ID NO:92; TNA036), LNEGGQIK (SEQ ID NO:91; TNA035), LNPGGQIK (SEQ ID NO:87; TNA032), QNPGGQIK (SEQ ID NO:88; TNAO33), or KNPGGQIK (SEQ ID NO:89; TNA031).

Embodiment 112. The antibody polypeptide of any one of embodiments 101 to 105, wherein the second amino acid residue of the CDR3 or CDR3+1 sequence is aspartic acid (Asp).

Embodiment 113. The antibody polypeptide of embodiment 112, where the CDR3 sequence is: KDPGGQI (SEQ ID NO: 27; TNA019), QDPGGQI (SEQ ID NO: 21; TNA030), or LDPGGQI (SEQ ID NO: 15; TNA028).

Embodiment 114. The antibody polypeptide of embodiment 112, where the CDR3+1 sequence is: KDPGGQIK (SEQ ID NO: 96; TNA019), QDPGGQIK (SEQ ID NO: 93; TNA030), or LDPGGQIK (SEQ ID NO: 90; TNA028).

Embodiment 115. The antibody polypeptide of any one of embodiments 101 to 105, wherein the first amino acid residue of the CDR3 or CDR3+1 sequence is leucine (Leu).

Embodiment 116. The antibody polypeptide of embodiment 115, where the CDR3 region sequence is: LNTGGQI (SEQ ID NO:25; TNA037), LNRGGQI (SEQ ID NO:23; TNAO38), LNQGGQI (SEQ ID NO: 19; TNA036), LNEGGQI (SEQ ID NO: 17; TNA035), LDPGGQI (SEQ ID NO: 15; TNA028), or LNPGGQI (SEQ ID NO:9; TNA032).

Embodiment 117. The antibody polypeptide of embodiment 115, where the CDR3+1 region sequence is: LNTGGQIK (SEQ ID NO:95; TNA037), LNRGGQIK (SEQ ID NO:94; TNAO38), LNQGGQIK (SEQ ID NO:92; TNA036), LNEGGQIK (SEQ ID NO:91; TNA035), LDPGGQIK (SEQ ID NO: 90; TNA028), or LNPGGQIK (SEQ ID NO:87; TNA032).

Embodiment 118. The antibody polypeptide of any one of embodiments 101 to 105, wherein the first amino acid residue of the CDR3 sequence is glutamine (Gin). Embodiment 119. The antibody polypeptide of embodiment 118, where the CDR3 sequence is: QDPGGQI (SEQ ID NO:21; TNA030) or QNPGGQI (SEQ ID NO:11; TNAO33).

Embodiment 120. The antibody polypeptide of embodiment 118, where the CDR3+1 sequence is: QDPGGQIK (SEQ ID NO:93; TNA030) or QNPGGQIK (SEQ ID NO:88; TNAO33).

Embodiment 121. The antibody polypeptide of embodiment 101, wherein the CDR 1 ,

CDR2, and CDR3 sequences are:

(a) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg-Arg- Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and LNPGGQI (SEQ ID NO:9; TNA032);

(b) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg-Arg- Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and QNPGGQI (SEQ ID NO: 11; TNAO33);

(c) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg-Arg- Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and KNPGGQI (SEQ ID NO: 13; TNA031);

(d) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg-Arg- Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and LDPGGQI (SEQ ID NO: 15; TNA028);

(e) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg-Arg- Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and LNEGGQI (SEQ ID NO: 17; TNA035);

(f) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg-Arg- Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and LNQGGQI (SEQ ID NO: 19; TNA036);

(g) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg-Arg- Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and QDPGGQI (SEQ ID NO:21; TNA030);

(h) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg-Arg- Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and LNRGGQI (SEQ ID NO:23; TNAO38);

(i) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg-Arg- Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and LNTGGQI (SEQ ID NO:25; TNA037); or

(j) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg-Arg- Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and KDPGGQI (SEQ ID NO:27; TNA019).

Embodiment 122. The antibody polypeptide of embodiment 102, wherein the CDR1,

CDR2, and CDR3+1 sequences are: (a) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg-Arg- Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and LNPGGQIK (SEQ ID NO:87; TNA032);

(b) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg-Arg- Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and QNPGGQIK (SEQ ID NO:88; TNAO33);

(c) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg-Arg- Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and KNPGGQIK (SEQ ID NO:89; TNA031);

(d) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg-Arg- Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and LDPGGQIK (SEQ ID NO:90; TNA028);

(e) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg-Arg- Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and LNEGGQIK (SEQ ID NO:91; TNA035);

(f) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg-Arg- Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and LNQGGQIK (SEQ ID NO:92; TNA036);

(g) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg-Arg- Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and QDPGGQIK (SEQ ID NO:93; TNA030);

(h) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg-Arg- Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and LNRGGQIK (SEQ ID NO:94; TNAO38);

(i) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg-Arg- Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and LNTGGQIK (SEQ ID NO:95; TNA037); or

(j) Ile-Tyr-Trp-Met-Thr (SEQ ID NO: 3); Glu-Ile-Asn-Thr-Asn-Gly-Leu-Ile-Arg-Arg- Tyr-Ala-Asp-Ser-Val-Glu-Gly (SEQ ID NO: 4); and KDPGGQIK (SEQ ID NO:96; TNA019).

Embodiment 123. The antibody polypeptide of any one of embodiments 101 to 122, wherein the single variable domain comprises:

(a) a framework 1 (FR1) sequence having at least 80% sequence identity to QVQLVESGGGLVQPGGSLTLSCAASGFDFG (SEQ ID NO:33),

(b) a framework 2 (FR2) sequence having at least 80% sequence identity to

WVRQTPGKGEEWVS (SEQ ID NO:34)

(c) a framework 3 (FR3) sequence having at least 80% sequence identity to

RFTVSRDNAKNMMYLQMNSLASEDTAVYYCA (SEQ ID NO:35), and

(d) a framework 4 (FR4) sequence having at least 80% sequence identity to

KGQGTQVTVSS (SEQ ID NO:36) for CDR3 or a framework 4 (FR4) sequence having at least 80% sequence identity to GQGTQVTVSS (SEQ ID NO:99) for CDR3+1. Embodiment 124. The antibody polypeptide of any one of embodiments 101 to 122, wherein:

(a) the FR1 sequence has at least 90% sequence identity to QVQLVESGGGLVQPGGSLTLSCAASGFDFG (SEQ ID NO:33);

(b) the FR2 sequence has at least 90% sequence identity to WVRQTPGKGEEWVS (SEQ ID NO:34);

(c) the FR3 sequence has at least 90% sequence identity to RFTVSRDNAKNMMYLQMNSLASEDTAVYYCA (SEQ ID NO:35); and

(d) the FR4 sequence has at least 90% sequence identity to KGQGTQVTVSS (SEQ ID NO: 36) for CDR3 or a framework 4 (FR4) sequence having at least 80% sequence identity to GQGTQVTVSS (SEQ ID NO:99) for CDR3+1.

Embodiment 125. The antibody polypeptide of any one of embodiments 101 to 124, wherein the single variable domain is humanized.

Embodiment 126. The antibody polypeptide of any one of embodiments 101 to 125, wherein the single variable domain further comprises a hinge sequence at the carboxy terminus (C-terminus).

Embodiment 127. The antibody polypeptide of embodiment 126, wherein the hinge sequence comprises EPKTPKPQ (SEQ ID NO: 6), EPKTPKPQPQPQ (SEQ ID NO:75) or AHHSEDPS (SEQ ID NO:83).

Embodiment 128. The antibody polypeptide of any one of embodiments 101 to 127 having a TNF-a neutralizing activity.

Embodiment 129. A polynucleic acid encoding the antibody polypeptide of any one of embodiments 101 to 128.

Embodiment 130. The polynucleic acid of embodiment 129, further encoding a secretion leader sequence fused to the antibody polypeptide, wherein the secretion leader is an endogenous L. lactis secretion leader sequence of a gene selected from L. lactis MG1363 ps356 endolysin (UniProtKB A2RJJ4; SEQ ID NO:42), N-acetylglucosaminidase/peptidoglycan hydrolase AcmD (UniProtKB A2RIL8; SEQ ID NO:44), gamma-glutamyl-diamino acid- endopeptidase llmg_1594 (UniProt A2RLK0; SEQ ID NO:46), secreted 45 kDa protein precursor Usp45 (UniProtKB P22865; SEQ ID NO:48) and a K4N mutation thereof (P22865**; SEQ ID NO:50), N-acetylmuramoyl-L-alanine amidase/peptidoglycan hydrolase AcmB (UniProtKB Q8KKF9; SEQ ID NO:52), hypothetical protein/Immunogenic secreted protein homolog llmg_0904 (UniProtKB A2RJP5; SEQ ID NO:54), hypothetical protein/putative secreted protein llmg_0918 (UniProtKB A2RJQ9; SEQ ID NO:56), cell wall surface anchor family protein llmg_l 127 (UniProtKB A2RKB 1 ; SEQ ID NO:58), hypothetical protein/putative secreted protein llmg_1800 (UniProtKB A2RM44; SEQ ID NO:60), hypothetical protein/ORFlO llmg_1399 (UniProtKB A2RL19; SEQ ID NO:62), putative transglyco sylase llmg_0760 (UniProtKB A2RJB2; SEQ ID NO:64), cell surface antigen I/II precursor CluA (UniProtKB A2RL18; SEQ ID NO:66), hypothetical protein/Glucosyltransferase-I llmg_0458 (UniProtKB A2RIG7; SEQ ID NO:68), hypothetical protein/putative secreted protein llmg_0877 (UniProtKB A2RJL9; SEQ ID NO:70), and variants thereof having 1, 2, or 3 variant amino acid positions and variants thereof having 1, 2, or 3 variant amino acid positions.

Embodiment 131. The polynucleic acid of either embodiment 129 or embodiment 130, further comprising an hllA promoter (P/z//A), wherein the expression of the antibody polypeptide or the antibody polypeptide fused with the secretion leader is driven by Phil A.

Embodiment 132. A polycistronic expression unit comprising, in 5’ to 3’ order:

(a) a promoter endogenous to a Gram-positive bacterium;

(b) a gene endogenous to the Gram-positive bacterium;

(c) an intergenic region active in the Gram-positive bacterium; and

(d) the polynucleic acid of either embodiment 129 or embodiment 130.

Embodiment 133. The polycistronic expression unit of embodiment 132, wherein the endogenous gene is: eno, gapB, usp45, rplS, pyk, rpmB, pdhD, sodA, or tufA.

Embodiment 134. The polycistronic expression unit of either embodiment 132 or embodiment 133, wherein the endogenous promoter is the native promoter of the endogenous gene. Embodiment 135. The polycistronic expression unit of any one of embodiments 132 to 134, wherein the intergenic region is the intergenic region preceding rplW, rplP, rpmD, rplB, rpsG, rpsE, rplN, rplM, rplE, or rplF.

Embodiment 136. A vector comprising the polynucleic acid of any one of embodiments 129 to 131, or the polycistronic expression unit of any one of embodiments 132 to 135.

Embodiment 137. A Gram-positive bacterium comprising the polynucleic acid of any one of embodiments 129 to 131, the polycistronic expression unit of any one of embodiments 131 to 135, or the vector of embodimentl 36.

Embodiment 138. The Gram-positive bacterium of embodiment 137, being a lactic acid bacterium (LAB), a Bifidobacterium, or a Staphylococcus.

Embodiment 139. The Gram-positive bacterium of either embodiment 137 or embodiment 138, wherein the LAB is: a Lactococcus species, a Lactobacillus species, a Streptococcus species, or an Enterococcus species.

Embodiment 140. The Gram-positive bacterium of any one of embodiments 137 to

139, wherein the LAB is a L. lactis.

Embodiment 141. The Gram-positive bacterium of any one of embodiments 137 to

140, wherein the polynucleic acid is chromo somally integrated in the Gram-positive bacterium.

Embodiment 142. The Gram-positive bacterium of any one of embodiments 137 to

141, wherein the antibody polypeptide is constitutively expressed.

Embodiment 143. The Gram-positive bacterium of any one of embodiments 137 to

142, wherein the antibody polypeptide is secreted or is displayed at the surface of the Grampositive bacterium. Embodiment 144. The Gram-positive bacterium of embodiment 140, wherein the polynucleic acid encoding the antibody polypeptide is integrated into the chromosome of L. lactis, forming a polycistronic expression unit at the eno locus, wherein the polycistronic expression unit at the eno locus comprises, in 5’ to 3’ order, an eno promoter (Peno), eno, an intergenic region preceding rmpD, and a fusion protein having a Usp45 secretion leader (SSusp45) fused to the N-terminus of the antibody polypeptide.

Embodiment 145. The Gram-positive bacterium of embodiment 140, wherein the polynucleic acid encoding the antibody polypeptide is integrated into the chromosome of L. lactis, forming a polycistronic expression unit at the gapB locus, wherein the polycistronic expression unit at the gapB locus comprises, in 5’ to 3’ order, a gapB promoter (PgapB), gapB, an intergenic region preceding rpmD, and a fusion protein having a Usp45 secretion leader (SSusp45) fused to the N-terminus of the antibody polypeptide.

Embodiment 146. The Gram-positive bacterium of embodiment 140, wherein the polynucleic acid encoding the antibody polypeptide is integrated into the chromosome of L. lactis, forming a polycistronic expression unit at the rplS locus, wherein the polycistronic expression unit at the rplS locus comprises, in 5’ to 3’ order, a rplS promoter (P rplS), rplS, an intergenic region preceding rmpD, and a fusion protein having a Usp45 secretion leader (SSusp45) fused to the N-terminus of the antibody polypeptide.

Embodiment 147. The Gram-positive bacterium of embodiment 140, wherein the polynucleic acid encoding the antibody polypeptide is integrated into the chromosome of L. lactis, forming a polycistronic expression unit at the pyk locus, wherein the polycistronic expression unit at the pyk locus comprises, in 5’ to 3’ order, a pyk promoter (Ppyk), pyk, an intergenic region preceding rmpD, and a fusion protein having a Usp45 secretion leader (SSusp45) fused to the N-terminus of the antibody polypeptide.

Embodiment 148. The Gram-positive bacterium of embodiment 140, wherein the polynucleic acid encoding the antibody polypeptide is integrated into the chromosome of L. lactis, forming a polycistronic expression unit at the rpmB locus, wherein the polycistronic expression unit at the rpmB locus comprises, in 5’ to 3’ order, an rpmB promoter (PrpmB), rpmB, an intergenic region preceding rmpD, and a fusion protein having a Usp45 secretion leader (SSusp45) fused to the N-terminus of the antibody polypeptide.

Embodiment 149. The Gram-positive bacterium of embodiment 140, wherein the polynucleic acid encoding the antibody polypeptide is integrated into the chromosome of L. lactis, forming a polycistronic expression unit at the pdhD locus, wherein the polycistronic expression unit at the pdhB locus comprises, in 5’ to 3’ order, an pdhD promoter PpdhD), pdhD, an intergenic region preceding rmpD, and a fusion protein having a Usp45 secretion leader (SSusp45) fused to the N-terminus of the antibody polypeptide.

Embodiment 150. The Gram-positive bacterium of embodiment 140, wherein the polynucleic acid encoding the antibody polypeptide is integrated into the chromosome of L. lactis, forming a polycistronic expression unit at the sodA locus, wherein the polycistronic expression unit at the sodA locus comprises, in 5’ to 3’ order, an sodA promoter (PsodA), sodA, an intergenic region preceding rmpD, and a fusion protein having a Usp45 secretion leader (SSusp45) fused to the N-terminus of the antibody polypeptide.

Embodiment 151. The Gram-positive bacterium of embodiment 140, wherein the polynucleic acid encoding the antibody polypeptide is integrated into the chromosome of L. lactis, forming a polycistronic expression unit at the tufA locus, wherein the polycistronic expression unit at the tufA locus comprises, in 5’ to 3’ order, an tufA promoter (PlufA), tufA, an intergenic region preceding rmpD, and a fusion protein having a Usp45 secretion leader (SSusp45) fused to the N-terminus of the antibody polypeptide.

Embodiment 152. The Gram-positive bacterium of any one of embodiments 144 to 151, wherein the L. lactis further comprises one or more of the following genetic modifications: a) deletion of a thymidylate synthase gene (thyAf, b) inactivation or deletion of a trehalose-6-phosphate phosphorylase gene (trePPf, c) inactivation or deletion of a gene encoding a cellobiose- specific PTS system IIC component ( /cC); d) integration of an exogenous otsB, forming a polycistronic expression unit at the usp45 locus, wherein the polycistronic expression unit at the usp45 locus comprises, in 5’ to 3’ order, a usp45 promoter Pusp45 usp45, an intergenic region preceding rpmD, and the exogenous otsB and e) expression of ptsl and ptsll under the control of a hllA promoter (P/z/ZA).

Embodiment 153. A pharmaceutical composition comprising the Gram-positive bacterium of any one of embodiments 137 to 152 and a pharmaceutically acceptable carrier.

Embodiment 154: A method of treating an inflammatory condition in a patient in need thereof comprising administering to the patient a therapeutically effective amount of the Gram-positive bacterium of any one of embodiments 137 to 152, or the pharmaceutical composition of embodiment 153.

Embodiment 155. The method of embodiment 154, wherein the inflammatory condition is: mucosal inflammation, skin inflammation, inflammatory bowel disease (IBD), Crohn’s disease (CD), ulcerative colitis (UC), psoriasis, irritable bowel syndrome (IBS), oral mucositis (OM), recurrent aphthous stomatitis (RAS), mTOR inhibitor associated stomatitis (mlAS), graft-versus-host disease (GVHD), oral pemphigus vulgaris (OPV), oral lichen planus (OLP), mucous membrane pemphigoid (MMP), vulvar lichen planus (VLP), vulvar lichen sclerosus (VLS), vulvar lichen simplex (VLSi), vulvar pemphigus vulgaris (VPV), Lipschiitz ulcer, vulvodynia, granulomatosis with polyangiitis (GPA), alopecia, lung inflammation, ocular inflammation, or inflammatory pain.

Embodiment 156: A method of treating a pathological condition in a patient in need thereof comprising: administering to the patient a therapeutically effective amount of the Grampositive bacterium of any one of embodiments 137 to 152, or the pharmaceutical composition of embodiment 153, wherein the pathological condition is: myalgic encephalomyelitis, chronic fatigue syndrome (CFS), depression, or Parkinson’s disease (PD).

Embodiment 157: Use of the LAB of any one of embodiments 137 to 152 or the composition of embodiment 153 in the treatment of an inflammatory condition. Embodiment 158: Use of the LAB of any one of embodiments 137 to 152 or the composition of embodiment 153 for the preparation of a medicament for the treatment of an inflammatory condition.

Embodiment 159: Use of the LAB of any one of embodiments 137 to 152 or the composition of embodiment 153 in the treatment of a pathological condition.

Embodiment 160: Use of the LAB of any one of embodiments 137 to 152 or the composition of embodiment 153 for the preparation of a medicament for the treatment of a pathological condition.

Embodiment 161. A pharmaceutical composition comprising an antibody polypeptide of any one of embodiments 101 to 128 and optionally a pharmaceutically acceptable carrier.

Embodiment 162. Use of an antibody polypeptide of any one of embodiments 101 to 128 or the pharmaceutical composition of Embodiment 161 in the treatment of an inflammatory condition or a pathological condition.

Embodiment 163. Use of an antibody polypeptide of any one of embodiments 101 to 128 or the pharmaceutical composition of embodiment 161 for the preparation of a medicament for the treatment of an inflammatory condition or a pathological condition.

Embodiment 164: A method of treating an inflammatory condition or a pathological condition in a patient in need thereof comprising administering to the patient a therapeutically effective amount of the antibody polypeptide of any one of embodiments 101 to 128 or the pharmaceutical composition of Embodiment 161.

Embodiment 165. The method of embodiment 164, wherein the inflammatory condition is: mucosal inflammation, skin inflammation, inflammatory bowel disease (IBD), Crohn’s disease (CD), ulcerative colitis (UC), psoriasis, irritable bowel syndrome (IBS), oral mucositis (OM), recurrent aphthous stomatitis (RAS), mTOR inhibitor associated stomatitis (mlAS), graft-versus-host disease (GVHD), oral pemphigus vulgaris (OPV), oral lichen planus (OLP), mucous membrane pemphigoid (MMP), vulvar lichen planus (VLP), vulvar lichen sclerosus (VLS), vulvar lichen simplex (VLSi), vulvar pemphigus vulgaris (VPV), Lipschiitz ulcer, vulvodynia, granulomatosis with polyangiitis (GPA), alopecia, lung inflammation, ocular inflammation, or inflammatory pain.