WO2024123701A2 - Treatments for pain - Google Patents

Abstract

Engineered strains of yeast and methods for treating pain. Specifically, the present disclosures provide, among other things, engineered strains of Saccharomyces yeast encoding a binding protein that binds to tumor necrosis factor (TNFα) and methods for treating pain in a subject.

Description

TREATMENTS FOR PAIN

CROSS-REFERENCE TO RELATED APPLICATION

[0001 ] This application claims priority to U.S. Provisional Application No. 63/430337, filed December 5, 2022, the entire contents of which are incorporated herein by reference.

TECHNICAL FIELD

[0002] Described herein, are engineered strains of yeast and treatments for pain. Specifically, the present disclosures provide, among other things, engineered strains of Saccharomyces yeast encoding a binding protein that binds to tumor necrosis factor a (TNFa) and methods for treating pain in a subject in need.

BACKGROUND

[0003] The following description of the background of the present technology is provided simply as an aid in understanding the present technology and is not admitted to describe or constitute prior art to the present technology.

[0004] Chronic pain is one of the most common reasons to visit a family physician and is challenging to manage. More than 30% of people in the United States are living with chronic or severe pain. Simultaneously, opioid addiction and overdose rates are on the increase and have become a public health emergency, with pain management being a driving force.

[0005] Visceral pain or visceral sensitivity is a form of chronic pain and is labeled at times as functional pain. Visceral pain is in the internal organs in the midline of the body. It can be difficult to locate and diffuse, and often accompanied by other symptoms such as vomiting, sweating, or a racing heart. The most common causes of visceral pain include inflammation, menstrual cramps, swelling and stretching of the organs, blockages in the bowels or urethra, decreased blood flow, tumors, depression and stress. The visceral pain can be a result of underlying health conditions such as inflammatory bowel disease (IBD), irritable bowel syndrome (IBS) including post-infection irritable bowel syndrome (PI-IBS), postinflammation IBS, stresses, cancer, pancreatitis, indigestion and interstitial cystitis (IC). Generally, the most effective treatment is to target the underlying condition causing the visceral pain. However, that is not always evident and there remains a scarcity of treatments that are potent and efficacious for chronic use.

(0006] Therapeutic polypeptide therapy is a promising option; however, it is currently hampered by issues such as needing to be injected or intravenous infusion requiring patients to travel to healthcare facilities to receive treatment. This can reduce patient compliance and lead to poor patient outcomes. Further, the long-term use of such peptides is associated with severe systemic side effects or loss of effectiveness and currently there are limited alternatives for other routes of administration. For example, oral administration has proven difficult, as the high acidity of stomach fluid can affect the polypeptide potency.

SUMMARY

[0007] In one aspect, the present disclosure provides a method for treating pain in a subject in need thereof, comprising administering (e.g., orally or via suppository) to a subject in need thereof an engineered strain of Saccharomyces yeast encoding a binding protein that binds to tumor necrosis factor a (TNFa).

[0008] In some embodiments, the binding protein comprises a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NO: 21-28, a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NO: 29-34, and a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NO: 35-41.

10009 [ In some embodiments, the binding protein is an antibody. In some embodiments, the antibody is a single domain antibody (sdAb). In some embodiments, the sdAb is a VHH.

[0010] In some embodiments, the binding protein comprises a VHH. In some embodiments, the VHH comprises an amino acid sequence selected from any one of SEQ ID NO: 1-19.

[0011 ] In some embodiments, the binding protein comprises two VHHS. In some embodiments, the two VHHS are homodimers. In some embodiments, the binding protein comprises an amino acid sequence comprising SEQ ID NO: 20. In some embodiments, each of the two VHHS comprise an amino acid sequence selected from any one of SEQ ID NOs: 1- 19. In some embodiments, the two VHHS are heterodimers. In some embodiments, each of the two VHHS independently comprise an amino acid sequence selected from any one of SEQ ID NOs: 1-19. In some embodiments, the binding protein comprises an amino acid sequence comprising SEQ ID NO: 43. In some embodiments, the two VHHS each bind TNFa. In some embodiments, one VHH binds TNFa and one VHH binds a binding target that is not TNFa, for example, IL-17A. In some embodiments, one VHH comprises a second CDR1 comprising an amino acid sequence of SEQ ID NO: 44, a second CDR2 comprising an amino acid sequence of SEQ ID NO: 45, and a second CDR3 comprising an amino acid sequence of SEQ ID NO: 46.

[0012] In some embodiments, the two VHHS are connected via a linker. In some embodiments, the linker comprises an amino acid sequence of GGGGSGGGGSGGGGS (SEQ ID NO: 47).

[0013] In some embodiments, the binding protein comprises a VHH-VHH-FC structure. In some embodiments, the VHH-VHH-FC structure further comprises a linker connecting the two VHH domains. In some embodiments, the VHH-VHH-FC structure further comprises a linker connecting the Fc domain to the two VHH domains.

[0014] In some embodiments, the binding protein comprises an amino acid sequence selected from any one of SEQ ID NO: 1-20 or 43.

[0015] In some embodiments, the pain is chronic. In some embodiments, the pain is somatic pain, visceral pain, referred bone pain, or neuropathic pain. In some embodiments, the visceral pain is abdominal pain. In some embodiments, the pain is stress-induced. In some embodiments, the pain is inflammation-induced. In some embodiments, the pain is induced by an infection. In some embodiments, the pain is induced by a physical injury. In some embodiments, the pain is induced by a disease. In some embodiments, the subject has a functional pain syndrome, fibromyalgia, or temporomandibular dysfunction (TMD). In some embodiments, the subject has irritable bowel syndrome (IBS) or an inflammatory bowel disease (IBD), such as Crohn’s disease, ulcerative colitis, or indeterminate colitis (IC). In some embodiments, the IBS or IBD is quiescent, in remission or relapsed. In some embodiments, the IBS is a post-infection IBS (PI-IBS) or post-inflammation IBS. [0016] In some embodiments, the subject does not have irritable bowel syndrome (IBS) or an inflammatory bowel disease (IBD).

[0017] In some embodiments, the subject has experienced the pain for a month or more.

[0018] In some embodiments, the Saccharomyces yeast further comprises a nucleic acid sequence encoding a dihydrofolate reductase (DHFR) incorporated into the genome. In some embodiments, the DHFR is a mammal DHFR, such as a murine DHFR.

[0019] In some embodiments, the Saccharomyces yeast further comprises one or more exogenous nucleic acids encoding a yeast DFR1.

[0020] In some embodiments, the Saccharomyces yeast comprises a complete or partial deletion of URA3.

[0021] In some embodiments, the Saccharomyces yeast comprises a complete or partial deletion of GAP1.

[0022] In some embodiments, the Saccharomyces yeast is ura3(-/-) and gap 1 (-/-).

{0023] In some embodiments, the binding protein is encoded by a nucleic acid incorporated into the genome of the Saccharomyces yeast. In some embodiments, the nucleic acid encoding the binding protein is incorporated into at least two different chromosomes. In some embodiments, the at least two different chromosomes comprise chromosomes VII and XVI.

[0024] In some embodiments, the binding protein is encoded by a nucleic acid that is not incorporated into the genome of the Saccharomyces yeast.

[0025] In some embodiments, the engineered strain of Saccharomyces yeast is administered daily, every other day, every three days, every four days, every five days, every six days, weekly, every other week, every three weeks, monthly, every other month, every three months, every four months, every five months, or every six months. In some embodiments, the administered daily is at least once daily, at least twice daily, at least three times daily, at least four times daily, at least five times daily, at least six times daily, at least seven times daily, or at least eight times daily. [0026] The foregoing general description and following detailed description are exemplary and explanatory and are intended to provide further explanation of the disclosure as claimed. Other objects, advantages, and novel features will be readily apparent to those skilled in the art from the following brief description of the drawings and detailed description of the disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG. 1 depicts the stress-induced referred pain in rats and mice with trigeminal nerve injury. Panel A shows the study design. Panel B shows referred lower back pain is increased in male CCI-ION rats (left), female CCI-ION (middle) and Sham rats (right) after a repeated force compared to no repeated force swim.

[0028] FIG. 2 depicts the experimental method for treating stress induced pain in mice subject to CCI-ION. Panel A provides for the study design. Panel B shows visceral hypersensitivity measured by Von Frey filament forces (g) that produces a 50% response frequency, as a measure of mechanical sensitivity (EF50), in CCI-ION mice exposed to forced swim and Sham mice exposed to force swim (left) and mice treated with 0.5% lidocaine or PBS (right).

[0029] FIG. 3 depicts the results of female mice with stress induced pain treated with yeast that expressed a binding protein of SEQ ID NO: 20 for 7 days. Mice were gavaged with Saccharomyces boulardii (Sb) control or Sb yeast that expressed a binding protein of SEQ ID NO: 20, daily for 7 days from day 6 to 12. Mice had Von Frey measurements at baseline (day -14), and then on day -3, 3, 5, 6, 8, 10, 12, 14, 16, and 19 (Panel A). The struggle time for each mouse during each force swim day was recorded in the first 5 minutes. ** p<0.01 compared with first day of force swim One-way ANOVA test (Panel B). The feces number for each mouse produced during each force swim day was recorded in the first 5 minutes. ** p<0.01 compared with first day of force swim. One-way ANOVA test (Panel C). Visceral hypersensitivity measured by Von Frey was recorded for mice treated with yeast that expressed a binding protein of SEQ ID NO: 20 or Sb control. **p<0.01 compared with PBS control group. Two-way ANOVA si dak’s multiple comparisons test (Panel D). Facial hypersensitivity measured by Von Frey was recorded for mice treated with yeast that expressed a binding protein of SEQ ID NO: 20 or Sb control. (Panel E).

(0030] FIG. 4 depicts the results of mice with stress induced pain treated with yeast that expressed a binding protein of SEQ ID NO: 20 for 14 days. Mice were gavaged with either PBS, Sb control yeast that does not express anti-(m)TNFa (10⁹ CFU/dose), or yeast that expressed a binding protein of SEQ ID NO: 20 (10⁹ CFU/dose), daily from day 6 to 19 (14 doses total). Mice had Von Frey measurements at baseline (day -14) and then the indicated days (Panel A). The struggle time for each mouse during each force swim day was recorded in the first 5 minutes. White is Group 1 (PBS); Grey is Group 2 (Sb Control Yeast); and Black is Group 3 (yeast that expressed a binding protein of SEQ ID NO: 20) *p<0.05, ** p<0.01 compared with first day of force swim (Panel B). The feces number for each mouse produced during each force swim day was recorded in the first 5 minutes. White is Group 1 (PBS); Grey is Group 2 (Sb Control Yeast); and Black is Group 3 (yeast that expressed a binding protein of SEQ ID NO: 20) (Panel C). Visceral hypersensitivity measured by Von Frey was recorded for Group 1 (PBS), Group 2 (Sb Control yeast), and Group 3 (yeast that expressed a binding protein of SEQ ID NO: 20). * p<0.5 **p<0.01 compared with Group 1. (Panel D). Facial hypersensitivity measured by Von Frey was recorded for Group 1 (PBS), Group 2 (Sb Control yeast), and Group 3 (yeast that expressed a binding protein of SEQ ID NO: 20) (Panel E).

[00311 FIG. 5 depicts the results of mice treated with trinitrobenzene sulfonic acid (TNBS) to induce inflammatory pain and then dosed with oral yeast that expressed a binding protein of SEQ ID NO: 20. Panel A shows the study design. Number of pain behaviors (left) and the length of time of the pain behavior indi ctors in seconds (right) during the 30-minute observation. Minutes of observation were recorded at day -10, day 1, day 3, and day 7 for PBS, control yeast (Sb. Control), and treatment with yeast that expressed a binding protein of SEQ ID NO: 20. Panel B shows behavioral numbers that were recorded for mice treated with PBS (bar on the right of each cluster of 3 bars), Sb Control yeast (bar on the left of each cluster of 3 bars), and yeast that expressed a binding protein of SEQ ID NO: 20 (bar in the center of each cluster of 3 bars). Panel C provides representative pictures of video clips recorded during the pain behavior analysis for control yeast (Sb control; left), yeast that expressed a binding protein of SEQ ID NO: 20 (middle), and PBS (right).

[0032] FIG. 6 depicts the results of treatment yeast that expressed a binding protein of SEQ ID NO: 20 on cytokines in feces. Specifically, this figure shows the fecal cytokine/chemokine (TNFa, MCP-1, KC/GRO, IL12/IL23p40, ILip, and NGAL/LCN2) levels in mice treated with PBS (left grouping of each cluster of 3), Sb Control yeast (center grouping of each cluster of 3), and yeast that expressed a binding protein of SEQ ID NO: 20 (right grouping of each cluster of 3).

[0033] FIG. 7 depicts the effects of treatment with yeast that expressed a binding protein of SEQ ID NO: 20 on psychological stress. Mice were exposed to water avoidance stress (WAS) by placing individual mice on a solid surface surrounded with water for 1 hour per day for 9 consecutive days. Panel A provides for the study design. Panel B shows visceral hypersensitivity measured by Von Frey was recorded for Control yeast (Sb. control) and mice treated with yeast that expressed a binding protein of SEQ ID NO: 20.

[0034] FIG. 8 depicts the effects of treatment with yeast that expressed a binding protein of SEQ ID NO: 43 on inflammation-induced visceral pain in transgenic mice. Panel A provides for the study design. Panel B shows visceral hypersensitivity measured by Von Frey was recorded for PBS-treated mice and mice treated with yeast that expressed a binding protein of SEQ ID NO: 43.

[0035] FIG. 9 depicts oral treatment of visceral pain in mice with PI-IBS. Mice were infected with Citrobacter rodentium (10^A9 CFU) on day 0 and the infection is naturally cleared on day 22. The infection induced gut inflammation even after the clearance of the infection. Fecal lipocalin (A) and colon crypt length (B) were measured on the indicated day post inoculation. (C) Referred visceral hyperalgesia was measured by von Frey test before infection (DO), after FS (D24-26) and 5 days after 7-day (D28-35) treatment with Sb yeast that expressed a binding protein of SEQ ID NO: 20 or PBS treatment (D40) (n=5 per group). (*) P<0.05; (**) P<0.01. DETAILED DESCRIPTION

[0036] The present disclosure provides engineered yeast strains that can be used in treatment of various types of pain, including but not limited to, chronic pain, somatic pain, visceral pain, referred bone pain, or neuropathic pain. The engineered yeast of the disclosure can express one or more binding protein(s) that bind to tumor necrosis factor a (TNFa) or, in some instances, TNFa and another binding target, such as IL- 17 A.

[0037] The most prevalent forms of chronic visceral pain are classified as resultant from functional gastrointestinal disorders such as irritable bowel syndrome (IBS), which affects an estimated 10-15% of the U.S. population (disproportionately affecting more women than men). Visceral pain is a heterogeneous disorder and may involve multiple factors such as infections, inflammations, surgery, and psychological distress. Due to this heterogeneity the current standard of care is unsatisfactory with 7% of patients reporting their pain issue is not solved at all and 37% reporting their pain management was inadequate. A lack of suitable treatments for these disorders is a major contributing factor to the debilitating nature of pain and the large socioeconomic cost accrued by patients, their families, and society.

[0038] Conventional analgesics, such as nonsteroidal anti-inflammatory drugs and opioids, are unsuitable for chronic therapy because they are associated with a high rate of addiction, a lack of efficacy, and, for some inflammatory disorders, the potential to exacerbate the disease. Currently opioids are the most widely used painkillers available, yet a mere 23% of patients with chronic pain find opioids effective. Long-term use of opioids can cause addiction and overdosing often leads to lethal consequences. In fact, the use of potent opioids for visceral pain is associated with higher morbidity and mortality than other pain conditions, significantly contributing to the opioid epidemic.

[0039] Recent findings have linked the microbiota to gastrointestinal disorders characterized by abdominal pain, which suggests that microbes may modulate visceral hypersensitivity and nociception (the neural processes of encoding and processing noxious stimuli) to pain. Although the pathophysiology of IBS is not yet fully elucidated, stress may be a major risk factor for development of IBS and acts as a trigger of pain sensation. Stress modulates both immune responses as well as the gut microbiota and vice versa. Additionally, microbes either directly or through involvement of the immune system activate or sensitize afferent nociceptors. Nociceptor sensory neurons protect organisms from dangers by eliciting pain and driving avoidance. Pain also clearly accompanies gut inflammation and modulation of gut microbiota. Thus, active crosstalk occurs between nociceptor neurons and the immune system to regulate pain, host defense, gut microbiota, and inflammatory diseases. Immune cells at gut nerve terminals and within the spinal cord release mediators that modulate mechanical and thermal sensitivity. In turn, nociceptor neurons release neuropeptides and neurotransmitters from the gut that regulate intestinal vascular, innate, and adaptive immune cell responses. Therefore, the communication between intestinal nociceptor neurons within the mucosal immune system and gut microbiota may be a considerable factor in visceral pain and hypersensitivity.

[0040] TNFa is a current therapeutic target for inflammatory disorders. The US Food and Drug Administration (FDA)-approved anti-TNFa biologies including infliximab, adalimumab, golimumab, and certolizumab pegol, have revolutionized therapy for a variety of chronic inflammatory disorders. However, all of these antibody therapeutics must be administered parenterally. Although systemically delivered anti-TNFa biologies are highly efficacious, their long-term use is often associated with loss of effectiveness due to anti-drug antibody responses and immunosuppressive side effects and systemic toxicities.

[00 1] Accordingly, the present disclosure provides oral biotherapeutics to treat pain (e.g., chronic pain, visceral pain, etc.). In particular, the disclosure provides genetically engineered probiotic yeast (e.g., Saccharomyces boulardii) that secrete anti-inflammatory molecules, such as neutralizing binding proteins against TNFa, alone or a combination with other molecules involved in inflammation and pain, such as IL-17A. The disclosed engineered yeast are believed to regulate pain (e.g., chronic pain, visceral pain, etc.) and hypersensitivity through at least two important mechanisms: direct modulation of gut microbiota via its probiotic effects and reducing gut inflammation via neutralizing proinflammatory cytokines. Thus, the disclosed yeasts provide treatments for pain that provide an excellent safety profile; possesses probiotic activities, potentially helping to reduce intestinal inflammation and microbiota dysbiosis; and provide convenience to the patient and potentially reduce immunogenicity and systemic immunosuppression commonly associated with intravenous (IV) administration of anti-TNFa/IL-17A therapies. Indeed, the disclosed treatments are expected to provide a safety profile that will enable chronic pain relief, unlike current biologies delivered by parenteral injection which have a high rate of anti-drug-antibody responses, systemic toxicities, and often require immuno-suppressants that limit their effectiveness for chronic use.

Definitions

[0042] Embodiments according to the present disclosure will be described more fully hereinafter. Aspects of the disclosure may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[00431 Unless the context indicates otherwise, it is specifically intended that the various features of the disclosure described herein can be used in any combination. Moreover, the disclosure also contemplates that in some embodiments, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0044] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied ( + ) or ( - ) by increments of 1.0 or 0.1, as appropriate, or alternatively by a variation of +/- 15 %, or alternatively 10%, or alternatively 5%, or alternatively 2%. It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the term “about”. It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art. [00451 As used in the description of the invention and the appended claims, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0046] As used herein, the terms “acceptable,” “effective,” or “sufficient” refer to the selection of any components, ranges, dose forms, etc. disclosed herein intend that said component, range, dose form, etc., is suitable for the disclosed purpose.

100471 Also as used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0048] 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 composition or method. “Consisting of’ shall mean excluding more than trace elements of other ingredients for claimed compositions and substantial method steps. Examples and implementations defined by each of these transition terms are within the scope of this disclosure. Accordingly, it is intended that the methods and compositions can include additional steps and components (comprising) or alternatively including steps and compositions of no significance (consisting essentially of) or alternatively, intending only the stated method steps or compositions (consisting of).

[0049] As used herein, “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0050] As used herein, the term “antibody” collectively refers to immunoglobulins or immunoglobulin-like molecules including IgA, IgD, IgE, IgG and IgM, combinations thereof or fragments thereof. Fragments of antibodies may include, for example, Fab fragments and single chain variable fragments (scFv). An antibody generally comprises heavy (H) chains and light (L) chains interconnected by disulfide bonds. There are two types of light chain, lambda (X) and kappa (K). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each heavy and light chain contains a constant region and a variable region (also known as “domains”). In combination, the heavy and the light chain variable regions, also called the “Fab region,” specifically bind to a given antigen. Light and heavy chain variable regions contain a “framework” region interrupted by three hypervariable regions, also called “complementarity-determining regions” or “CDRs.” The extent of the framework region and CDRs has been defined (see Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991). The Kabat database is now maintained online. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species, and framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions.

[0051 [ The CDRs are primarily responsible for binding to an epitope on an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located. Thus, a HCDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, whereas a LCDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found. An antibody will have a specific VH region and the VL region sequence, and thus specific CDR sequences. Antibodies with different specificities generally have different CDRs. Although it is the CDRs that vary from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are called specificity determining residues (SDRs).

[0052] The Fc fragment region (Fc) of an antibody plays a role in modulating immune cell activity. The Fc region functions to guarantee that each antibody generates an appropriate immune response for a given antigen, by binding to a specific class of proteins found on certain cells, such as B lymphocytes, follicular dendritic cells, natural killer cells, macrophages, neutrophils, etc. and are called “Fc receptors.” Because the constant domains of the heavy chains make up the Fc region of an antibody, the classes of heavy chain in antibodies determine their class effects. The heavy chains in antibodies include alpha, gamma, delta, epsilon, and mu, and correlate to the antibody’s isotypes IgA, IgG, IgD, IgE, and IgM, respectively. Thus, different isotypes of antibodies have different class effects due to their different Fc regions binding and activating different types of receptors.

[0053] There are four subclasses of IgG, which is the most abundant antibody isotype found in human serum. The four subclasses, IgGl, IgG2, IgG3, and IgG4, are highly conserved. The amino acid sequence of the constant regions of these peptides are known in the art, e.g., see Rutishauser, U. et al. (1968) “Amino acid sequence of the Fc region of a human gamma G-immunoglobulin” PNAS 61(4): 1414-1421 ; Shinoda et al. (1981) “Complete amino acid sequence of the Fc region of a human delta chain” PNAS 78(2):785-789; and Robinson et al. (1980) “Complete amino acid sequence of a mouse immunoglobulin alpha chain (MOPC 511)” PNAS 77(8)4909-4913.

]0054[ As used herein, the terms “single-domain antibody,” “sdAb,” or “nanobody” is an antibody fragment that has a single monomeric variable antibody domain, which can still bind selectively to a specific antigen.

[0055] Members of the Camelidae family, including South American camelids (e.g. alpaca and llama), produce two isotypes of immunoglobulins, IgG2 and IgG3 that lack light chains and are heavy chain antibodies or HCAbs. The VH domain of HCAbs, called VHH fragments, are a single-domain antibody.

10056] As used herein, the term “pain” refers to an unpleasant sensory and emotional experience associated with, or resembling, actual or potential tissue damage.

[0057] As used herein, the terms “chronic pain” and “persistent pain” refers to pain that continues for generally longer than 12 weeks.

[0058] As used herein, the term “somatic pain” refers to well localized, intermittent or constant pain, when pain receptors in tissue, including but not limited to, skin, muscles, skeletonjoints, and connective tissue, are activated.

[0059] As used herein, the term “visceral pain” refers to pain that is not well localized and occurs when pain receptors in the pelvis, abdomen, chest or intestine are activated. [0060] As used herein, the term “referred bone pain” refers to pain that is perceived in other areas of the body as a result of a pain stimulus in the bones.

(0061 ] As used herein, the term “neuropathic pain” is a result of damage or injury to the nerves.

[0062] As used herein, the term “abdominal pain” refers to pain that occurs anywhere between the chest and groin area.

[0063] As used herein, the term “stress” refers to a change that causes physical, emotional or psychological strain. In general, stress-induced pain occurs from physical, emotional or psychological strain.

[0064] As used herein, the term “inflammation” refers to a process where white blood cells migrate to a stimuli. Inflammation can be short in time (acute) or long-lasting (chronic). In general, pain can be induced by inflammation.

[0065] As used herein, the term “infection” refers to when agents, to include but not limited to, bacteria, virus, yeast and fungi, enter the body and increase in number causing a reaction. In general, pain can be induced by an infection.

[0066] As used herein, the phrase “therapeutically effective amount” with reference to a disclosed engineered strain of yeast means a dose that provides the specific pharmacological effect for which the drug is administered to a subject in need of such treatment. A therapeutically effective amount may be effective to reduce, ameliorate, or eliminate pain and/or improve quality of life in a subject with pain. It is emphasized that a therapeutically effective amount of an engineered strain of Saccharomyces yeast will not always be effective in treating pain in every individual subject, even though such dose is deemed to be a therapeutically effective amount by those of skill in the art. Those skilled in the art can adjust what is deemed to be a therapeutically effective amount in accordance with standard practices as needed to treat a specific subject. A therapeutically effective amount may vary based on, for example, the age and weight of the subject, and/or the subject’s overall health, and/or the severity of the subject’s pain. [0067] As used herein, the terms “treat,” “treatment” or “treating” as used herein with reference to pain refer to reducing, ameliorating, or eliminating the pain and/or improving quality of life in a subject with pain

[0068] The terms “prevent,” “preventing” or “prevention” as used herein with reference to a pain refer to precluding or reducing the risk of developing pain. Prevention may also refer to the prevention of a pain flare or recurrence once an initial pain has been treated or cured.

[0069] The terms “individual,” “subject,” and “patient” are used interchangeably herein, and refer to any individual mammalian subject, e.g., bovine, canine, feline, equine, or human. In specific embodiments, the subject, individual, or patient is a human.

[0070] Tumor Necrosis Factor alpha (TNF alpha; TNFa; TNF; cachectin; cachexin) is an inflammatory cytokine produced by macrophages and monocytes during inflammation.

Engineered Strains of Saccharomyces yeast

[0071] Proinflammatory cytokines (e.g., TNFa) are key immune mediators that contribute to the pathogenesis of many inflammatory diseases. The present disclosure provides engineered strains of Saccharomyces yeast that encode a binding protein that binds to TNFa. The binding protein expressed by the engineered yeast may comprise or consist of a variable heavy (VH) domain of an antibody (e.g., an IgG), a VHH, or a single-domain antibody (sdAB). Such binding proteins are readily expressed using microorganisms, including yeast, and, in the case of VHH and sdAB, may be more stable than conventional antibody fragments. In addition to TNFa, the disclosed binding proteins may bind to one or more additional binding targets. In other words, the binding proteins expressed by the engineered yeast may be bispecific or multi-specific. In general, the one or more additional binding targets will also be inflammatory cytokines or other molecules involved in inflammatory and pain signaling, such as IL-17A. The present disclosure provides for the unexpected discovery that the oral delivery of neutralizing antibodies that binding to cytokines (e.g., TNFa, IL-17A, etc.) via engineered yeast can block the interaction of the cytokines with receptors and provide a treatment for pain (e.g., chronic pain, somatic pain, visceral pain, referred bone pain, or neuropathic pain). [0072] The disclosed engineered strain of Saccharomyces yeast encodes a binding protein that binds to TNFa. In some embodiments, the binding protein is an antibody. In some embodiments the antibody is a single domain antibody (sdAb) or a VHH. Optionally, the binding protein may be bi-specific or multi-specific. In embodiments in which the binding protein is bispecific or multi-specific, the binding protein binds to TNFa and one or more additional binding targets, including but not limited to, inflammatory cytokines or other molecules involved in inflammatory and pain signaling such as IL- 17 A.

[0073] In some embodiments, the single domain antibody is a VHH antibody. Further, the binding protein may comprise more than one sdAb or VHH. For example, in some embodiments, the binding protein comprises a VHH-VHH-FC structure. In some embodiments, the VHH-VHH-FC structure further comprises a linker connecting the two VHH domains. In some embodiments, the VHH-VHH-FC structure further comprises a linker connecting the Fc domain to the two VHH domains. In some embodiments, the binding protein comprises a structure selected from VHH, FC-VHH, VHH-FC, VHH- VHH, VHH- VHH- VHH- VHH, FC-VHH- VHH, VHH-FC-VHH, and VHH-VHH-FC. In some embodiments, the VHH or Fc domains are attached to another VHH or FC domain via a linker sequence. In some embodiments, the binding protein comprises an anti-mouse TNFa binding domain. In some embodiments, the protein comprises an anti-human TNFa binding domain.

[0074] The binding domains encoded by the disclosed engineered yeast will generally possess at least three complementarity determining regions (CDRs): a CDR1, a CDR2, and a CDR3. In some embodiments, the binding protein comprises a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 21-28, a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 29-34, and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 35-41. Table 1 below provides examples of pairings of CDR sequences that can be combined in binding proteins disclosed herein.

[0075] In some embodiments, the CDR1 comprises an amino acid sequence of SEQ ID NO: 21. In some embodiments, the CDR1 comprises an amino acid sequence having at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 21. [0076] In some embodiments, the CDR1 comprises an amino acid sequence of SEQ ID NO:

22. In some embodiments, the CDR1 comprises an amino acid sequence having at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 22.

[0077] In some embodiments, the CDR1 comprises an amino acid sequence of SEQ ID NO:

23. In some embodiments, the CDR1 comprises an amino acid sequence having at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 23.

[0078] In some embodiments, the CDR1 amino acid sequence is SEQ ID NO: 24. In some embodiments, the CDR1 comprises an amino acid sequence having at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 24.

[0079] In some embodiments, the CDR1 comprises an amino acid sequence of SEQ ID NO:

25. In some embodiments, the CDR1 comprises an amino acid sequence having at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 25.

[0080] In some embodiments, the CDR1 comprises an amino acid sequence of SEQ ID NO:

26. In some embodiments, the CDR1 comprises an amino acid sequence having at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 26.

[0081 ] In some embodiments, the CDR1 comprises an amino acid sequence of SEQ ID NO:

27. In some embodiments, the CDR1 comprises an amino acid sequence having at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 27.

[0082] In some embodiments, the CDR1 comprises an amino acid sequence of SEQ ID NO:

28. In some embodiments, the CDR1 comprises an amino acid sequence having at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 28. [0083] In some embodiments, the CDR2 comprises an amino acid sequence of SEQ ID NO:

29. In some embodiments, the CDR2 comprises an amino acid sequence having at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 29.

100841 In some embodiments, the CDR2 comprises an amino acid sequence of SEQ ID NO:

30. In some embodiments, the CDR2 comprises an amino acid sequence having at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 30.

[0085] In some embodiments, the CDR2 comprises an amino acid sequence of SEQ ID NO:

31. In some embodiments, the CDR2 comprises an amino acid sequence having at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 31.

[0086] In some embodiments, the CDR2 comprises an amino acid sequence of SEQ ID NO:

32. In some embodiments, the CDR2 comprises an amino acid sequence having at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 32.

[0087] In some embodiments, the CDR2 comprises an amino acid sequence of SEQ ID NO:

33. In some embodiments, the CDR2 comprises an amino acid sequence having at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 33.

[0088] In some embodiments, the CDR2 comprises an amino acid sequence of SEQ ID NO:

34. In some embodiments, the CDR2 comprises an amino acid sequence having at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34.

[0089] In some embodiments, the CDR3 comprises an amino acid sequence of SEQ ID NO:

35. In some embodiments, the CDR3 comprises an amino acid sequence having at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 35. [0090] In some embodiments, the CDR3 comprises an amino acid sequence of SEQ ID NO:

36. In some embodiments, the CDR3 comprises an amino acid sequence having at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 36.

[0091] In some embodiments, the CDR3 comprises an amino acid sequence of SEQ ID NO:

37. In some embodiments, the CDR3 comprises an amino acid sequence having at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 37.

[0092] In some embodiments, the CDR3 comprises an amino acid sequence of SEQ ID NO:

38. In some embodiments, the CDR3 comprises an amino acid sequence having at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 38.

[0093] In some embodiments, the CDR3 comprises an amino acid sequence of SEQ ID NO:

39. In some embodiments, the CDR3 comprises an amino acid sequence having at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 39.

[0094] In some embodiments, the CDR3 comprises an amino acid sequence of SEQ ID NO:

40. In some embodiments, the CDR3 comprises an amino acid sequence having at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 40.

[0095] In some embodiments, the CDR3 comprises an amino acid sequence of SEQ ID NO:

41. In some embodiments, the CDR3 comprises an amino acid sequence having at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 41.

[0096] In some embodiments, the binding protein comprises a CDR1 with the amino acid sequence of SEQ ID NO: 21; a CDR2 with the amino acid sequence of SEQ ID NO: 29; a CDR3 with the amino acid sequence of SEQ ID NO: 35. [0097] In some embodiments, the binding protein comprises a CDR1 with the amino acid sequence of SEQ ID NO: 22; a CDR2 with the amino acid sequence of SEQ ID NO: 30; a CDR3 with the amino acid sequence of SEQ ID NO: 36.

[0098] In some embodiments, the binding protein comprises a CDR1 with the amino acid sequence of SEQ ID NO: 23; a CDR2 with the amino acid sequence of SEQ ID NO: 30; a CDR3 with the amino acid sequence of SEQ ID NO: 36.

100991 In some embodiments, the binding protein comprises a CDR1 with the amino acid sequence of SEQ ID NO: 24; a CDR2 with the amino acid sequence of SEQ ID NO: 30; a CDR3 with the amino acid sequence of SEQ ID NO: 36.

[01 0] In some embodiments, the binding protein comprises a CDR1 with the amino acid sequence of SEQ ID NO: 25; a CDR2 with the amino acid sequence of SEQ ID NO: 31; a CDR3 with the amino acid sequence of SEQ ID NO: 36.

[0.101] In some embodiments, the binding protein comprises a CDR1 with the amino acid sequence of SEQ ID NO: 24; a CDR2 with the amino acid sequence of SEQ ID NO: 30; a CDR3 with the amino acid sequence of SEQ ID NO: 37.

[0102] In some embodiments, the binding protein comprises a CDR1 with the amino acid sequence of SEQ ID NO: 26; a CDR2 with the amino acid sequence of SEQ ID NO: 29; a CDR3 with the amino acid sequence of SEQ ID NO: 38.

[0103] In some embodiments, the binding protein comprises a CDR1 with the amino acid sequence of SEQ ID NO: 21; a CDR2 with the amino acid sequence of SEQ ID NO: 32; a CDR3 with the amino acid sequence of SEQ ID NO: 39.

[0104] In some embodiments, the binding protein comprises a CDR1 with the amino acid sequence of SEQ ID NO: 21; a CDR2 with the amino acid sequence of SEQ ID NO: 29; a CDR3 with the amino acid sequence of SEQ ID NO: 38.

[0105] In some embodiments, the binding protein comprises a CDR1 with the amino acid sequence of SEQ ID NO: 27; a CDR2 with the amino acid sequence of SEQ ID NO: 33; a CDR3 with the amino acid sequence of SEQ ID NO: 36. [0106| In some embodiments, the binding protein comprises a CDR1 with the amino acid sequence of SEQ ID NO: 27; a CDR2 with the amino acid sequence of SEQ ID NO: 33; a CDR3 with the amino acid sequence of SEQ ID NO: 40.

[0107] In some embodiments, the binding protein comprises a CDR1 with the amino acid sequence of SEQ ID NO: 28; a CDR2 with the amino acid sequence of SEQ ID NO: 34; a CDR3 with the amino acid sequence of SEQ ID NO: 41.

101081 In some embodiments, the binding protein comprises a linker (e.g., a peptide linker). In some embodiments, the linker has the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 47). Other linkers that may be used include, but are not limited to: repeats of glycine, repeats of glycine and serine, and repeats of alanine. In general, linkers that can connect one or more domains of a binding protein for the purposes of expression in an engineered yeast will comprise about 3 to about 20 amino acids. For example, a peptide linker may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.

[0109] In some embodiments, the binding protein comprises an amino acid sequence selected from any one of SEQ ID NOs: 1-20. In some embodiments, the binding protein comprises the amino acid sequence SEQ ID NO: 1. In some embodiments, the binding protein comprises an amino acid sequence that has at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1. In some embodiments, the binding protein comprises the amino acid sequence SEQ ID NO: 20. In some embodiments, the binding protein comprises an amino acid sequence that has at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 20.

[0110] For purposes of this disclosure, “variant antibodies” or “variant” of a binding protein that binds to TNFa, may include, but not limited to (i) antibodies with at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to any one of SEQ ID NOs: 1-20. [0111 As discussed above, the binding proteins may comprise more than one binding domain, for example, more than one VHH. For example, a binding protein suitable for expression by the engineered yeast may comprise two VHHS, wherein each VHH is independently selected from an amino acid sequence selected from any one of SEQ ID NOs: 1-19. The two VHHS may optionally be connected via a linker (e.g., a peptide linker such as SEQ ID NO: 47). SEQ ID NO: 20 is an example of this type of construct for the binding protein.

[0112] Further, the binding proteins may be bispecific or multi-specific. For example, a binding protein suitable for expression by the engineered yeast may comprise a VHH that binds to TNFa (e.g., any one of SEQ ID NOs: 1-19, or a VHH comprising a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 21- 28, a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 29-34, and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 35-41) and a second VHH that binds to a different binding target. For example, the different target may be another molecule involved in inflammation and pain, such as IL- 17 A.

[0113] VHH that bind to IL-17A are also disclosed herein (see, e.g., SEQ ID NOs: 42 and 43 in Table 1 below) and these may be expressed by an engineered yeast (i) separately from/in addition to an anti-TNFa binding protein, or (ii) as part of a bispecific binding protein, such as the one shown as SEQ ID NO: 43 below. For the purposes of the present disclosure a binding protein of the present disclosure may comprise a second set of CDRs, and the second set of CDRs may comprise a CDR1 comprising an amino acid sequence of SEQ ID NO: 44, a CDR2 comprising an amino acid sequence of SEQ ID NO: 45, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 46.

[0114] Indeed, binding proteins that comprise a domain that binds to IL-17A (in addition to a binding protein or domain that binds to TNFa) may comprise a CDR1 that comprises an amino acid sequence having at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 44; a CDR2 that comprises an amino acid sequence having at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 45; and a CDR3 that comprises an amino acid sequence having at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 46. In some embodiments, a binding protein that comprises a domain that binds to IL-17A (in addition to a binding protein or domain that binds to TNFa) may comprise an amino acid sequence having at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 42.

[0115] In some embodiments, a bispecific binding protein that binds to TNFa and IL-17A comprises an amino acid sequence of SEQ ID NO: 43. In some embodiments, a bispecific binding protein that binds to TNFa and IL-17A comprises an amino acid sequence having at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 43.

10116] In some embodiments, the engineered strains of Saccharomyces yeast comprise at least one modification (e.g., mutation, deletion, insertion, etc.) that allows for selection of a particular engineered strain of Saccharomyces yeast (e.g., a selectable marker). In some embodiments, the modification allows for selection comprises a partial deletion of a gene utilized as a selectable marker. In some embodiments, the modification that allows for selection comprises a complete deletion of a gene utilized as a selectable marker.

[0117] For example, the disclosed engineered strains of Saccharomyces yeast may comprise a nucleic acid sequence encoding a dihydrofolate reductase (DHFR) incorporated into the yeast’s genome. In some embodiments, the DHFR is a mammalian DFHR, such as a murine DHFR (MVRPLNCIVAVSQNMGIGKNGDLPWPPLRNEFKYFQRMTTTSSVEGKQNLVIMGR KTWFSIPEKNRPLKDRINIVLSRELKEPPRGAHFLAKSLDDALRLIEQPELASKVDMV WIVGGSSVYQEAMNQPGHLRLFVTRIMQEFESDTFFPEIDLGKYKLLPEYPGVLSEV QEEKGIKYKFEVYEKKD; SEQ ID NO: 48). In some embodiments, the engineered strain of Saccharomyces yeast comprises one or more exogenous nucleic acids encoding a yeast DFR1. In some embodiments, the Saccharomyces yeast comprises a nucleic acid sequence encoding DHFR and one or more exogenous nucleic acids encoding yeast DFR1. In some embodiments, the DFR1 may be a DFR1 from S. boulardii (MAGGKIPIVGIVACLQPEMGIGFRGGLPWRLPSEMKYFRQVTSLTKDPNKKNALIM GRKTWESIPPKFRPLPNRMNVIISRSFKGDFVHDKERSIVQSNSLANAITNLESNFKEH LERIYVIGGGEVYSQIF SITDHWLITKINPLDKNATPAMDTFLDAKKLEEVF SEQDP AQ LKEFLPPKVELPETDCDQRYSLEEKGYCFEFTLYNRK; SEQ ID NO: 49).

[0118] Additionally or alternatively, the disclosed engineered Saccharomyces yeast may comprise a complete or partial deletion of URA3 or GAPE In some embodiments, the disclosed engineered Saccharomyces yeast comprises a complete or partial deletion of URA3. In some embodiments, the disclosed engineered Saccharomyces yeast comprises a complete or partial deletion of GAPE In some embodiments, the disclosed engineered Saccharomyces yeast comprises a complete or partial deletion of URA3 and GAPE In some embodiments, the Saccharomyces yeast is ura3(-/-). In some embodiments, the Saccharomyces yeast is gapl (-/-).

[0119] In some embodiments, the binding protein is encoded by a nucleic acid incorporated into the genome of the Saccharomyces yeast. In some embodiments, the nucleic acid is incorporated into at least one chromosome. In some embodiments, the nucleic acid encoding the binding protein is incorporated into at least 2 different chromosomes. In some embodiments, the nucleic acid encoding the binding protein is incorporated into at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 different chromosomes. In some embodiments, the nucleic acid encoding the binding protein is incorporated into chromosome VIE In some embodiments, the nucleic acid encoding the binding protein is incorporated into chromosome XVI. In some embodiments, the binding protein is encoded by a nucleic acid that is not incorporated into the genome of the Saccharomyces yeast.

[0120] In some embodiments, the engineered strain of Saccharomyces yeast is auxotrophic. In some embodiments, auxotrophic engineered strains of Saccharomyces yeast are less likely to survive in the environment relative to an appropriate reference standard (e.g., a parental strain of Saccharomyces yeast). [01211 In some embodiments, the engineered strain of Saccharomyces yeast is Saccharomyces cerevisiae. In some embodiments, the engineered strain of Saccharomyces yeast is Saccharomyces boulardii.

Treatments for Pain

[0122] Another aspect of the disclosure is directed to treatments for pain (e.g., methods of treating pain, uses of the disclosed engineered yeast, and/or the disclosed engineered yeast for use in treating pain) that generally comprise administering an engineered strain of Saccharomyces yeast as disclosed herein. In some embodiments, the treatment comprises orally administering to a subject in need thereof an engineered strain of Saccharomyces yeast encoding a binding protein that binds to TNFa. In some embodiments, the treatment comprises administering as a suppository an engineered strain of Saccharomyces yeast encoding a binding protein that binds to TNFa. In some embodiments, the binding protein may be monospecific (i.e., bind only to TNFa), bispecific (i.e., bind to TNFa and another binding target), or multi-specific (i.e., bind to TNFa and at least one additional binding target).

[0123] The pain being treated can be chronic or acute. In some embodiments, the subject in need thereof has experienced pain for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 7 days. In some embodiments, the subject in need thereof has experienced pain for between about 1 day to about 2 days, between about 1 day to about 3 days, between about 1 day to about 4 days, between about 2 days to about 3 days, between about 2 days to about 4 days, between about 2 days to about 5 days, between about 3 days to about 4 days, between about 3 days to about 5 days, between about 3 days to about 6 days, between about 4 days to about 5 days, between about 4 days to about 6 days, between about 4 days to about 7 days, between about 5 days to about 6 days, between about 5 days to about 7 days, or between about 6 days to about 7 days. In some embodiments, the subject in need thereof has experienced pain for at least 1 week to about 2 months. In some embodiments, the subject in need thereof has experienced pain for between about 1 week to about 2 weeks, between about 1 week to about 2 weeks, between about 1 week to about 3 weeks, between about 2 weeks to about 3 weeks, between about 2 weeks to about 4 weeks, or between about 3 weeks to about 4 weeks. In some embodiments, the subject has experienced the pain for 1 month or more. In some embodiments, the subject in need thereof has experienced pain between 1 month to about 1.5 months, between about 1 month to about 2 months, or between about 1.5 months to about 2 months. In some embodiments, the subject in need thereof has experienced pain between about 2 months to about 2 years. In some embodiments, the subject in need thereof has experienced pain between about 2 months to about 4 months, between about 2 months to about 6 months, between about 2 months to about 8 months, between about 4 month to about 6 months, between about 4 months to about 8 months, 4 months to about 10 months, between about 6 months to about 8 months, between about 6 months to about 10 months, between about 6 months to about 12 months, between about 8 months to about 10 months, between about 8 months to about 12 months, between about 8 months to about 14 months, between about 10 months to about 12 months, between about 10 months to about 14 months, between about 10 months to about 16 months, between about 12 months to about 14 months, between about 12 months to about 16 months, between about 12 months to about 18 months, between about 14 months to about 16 months, between about 14 months to about 18 months, between about 14 months to about 20 months, between about 16 months to about 18 months, between about 16 months to about 20 months, between about 16 months to about 22 months, between about 18 months to about 20 months, between about 18 months to about 22 months, between about 18 months to about 24 months, between about 20 months to about 22 months, between about 20 months to about 24 months, or between about 22 months to about 24 months. In embodiment, the subject in need thereof has experienced pain between about 2 years to about 20 years or more. In some embodiments, the subject in need thereof has experienced pain for 2 or more years, 3 or more years, 4 or more years, 5 or more years, 6 or more years, 7 or more years, 8 or more years, 9 or more years, 10 or more years, 11 or more years, 12 or more years, 13 or more years, 14 or more years, 15 or more years, 16 or more years, 17 or more years, 18 or more years, 19 or more years, or 20 or more years.

[0124] In some embodiments, the pain is somatic pain. In some embodiments, the pain is visceral pain. In some embodiments, the visceral pain is abdominal pain. In some embodiments, the pain is stress induced. In some embodiments, the pain is inflammation- induced. In some embodiments, the pain is induced by infection. In some embodiments, the pain is induced by stress (e.g., emotional, psychological, mental, etc.). In some embodiments, the pain is induced by a physical injury. In some embodiments, the pain is induced by a disease (e.g., diabetes, rheumatoid arthritis, osteoarthritis, fibromyalgia, cancer, multiple sclerosis, stomach ulcers, AIDS, gallbladder disease, pancreatitis, indigestion and interstitial cystitis).

101251 In some embodiments, the subject has functional pain syndrome, fibromyalgia, or temporomandibular dysfunction (TMD).

[0126] In some embodiments, the subject has irritable bowel syndrome (IBS) or an inflammatory bowel disease (IBD). In some embodiments, the IBD is Crohn’s disease, ulcerative colitis, or indeterminate colitis (IC). In some embodiments, the IBS or IBD is quiescent. In some embodiments, the IBS or IBD is in remission. In some embodiments, the IBS may be a post-infection IBS or post-inflammation IBS. In some embodiments, the IBS or IBD is relapsed. In some embodiments, the subject does not have IBS or IBD.

[0127] In general, an effective amount of the engineered strain of Saccharomyces yeast, is an amount sufficient to effect beneficial or desired results. As disclosed herein, an effective amount would also include an amount sufficient to decrease, prevent the development, or alleviate pain. In some embodiments, the engineered strain of Saccharomyces yeast as disclosed here in is administered to a subject in need thereof. Suitable routes of administration can include oral or suppository administration, as either route would allow the yeast to be provided to the gastrointestinal tract.

[0128] An effective amount can be administered in one or more administrations, applications or dosages. Such delivery is dependent on a number of variables including the time period for which the individual dosage unit is to be used, the bioavailability of the therapeutic agent, the route of administration, etc. It is understood, however, that specific dose levels of the therapeutic agents of the present disclosure for any particular subject depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, and diet of the subject, the time of administration, the rate of excretion, the drug combination, and the severity of the particular disorder being treated and form of administration. Treatment and prevention dosages generally may be titrated to optimize safety and efficacy. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. Typically, dosage-effect relationships from in vitro and/or in vivo tests initially can provide useful guidance on the proper doses for patient administration. In general, one will desire to administer an amount of the compound that is effective to achieve a serum level commensurate with the concentrations found to be effective in vitro. Determination of these parameters is well within the skill of the art. These considerations, as well as effective formulations and administration procedures are well known in the art and are described in standard textbooks.

[0129] Dose regimes may comprise flat dosing (i.e., administering the same dose repeatedly at pre-determined intervals) or comprise a loading dose (i.e. administrating an initial dose that is higher or different than subsequent, serial doses). In some embodiments, administration comprises oral administration.

[0130] In some embodiments, the engineered strain of Saccharomyces yeast is administered at least daily. In some embodiments, the engineered strain of Saccharomyces yeast is administered at least every other day, at least every three days, at least every four days, at least every five days, at least every six days, at least weekly, at least every other week, at least every three weeks, at least monthly, at least every other month, at least every three months, at least every four months, at least every five months, or at least every six months. In some embodiments, the engineered strain of Saccharomyces yeast is administered on day 1, day 2, day 3, day 4, day 5, day 6, and day 7. In some embodiments, the engineered strain of Saccharomyces yeast is administered on day 1, day 3, day 5, day 7, and day 14. In some embodiments, the engineered strain of Saccharomyces yeast is administered at least once daily. In some embodiments, the engineered strain of Saccharomyces yeast is administered at least twice daily. In some embodiments, the engineered strain of Saccharomyces yeast is administered at least three times daily, at least four times daily, at least five times daily, at least six times daily, at least seven times daily, or at least eight times daily.

10.1.311 In some embodiments, the amount of engineered strain of Saccharomyces yeast administered at each dose is 10⁹ CFU/day. In some embodiments, the dose is about 10⁶ CFU/day, about 10⁷ CFU/day, about 10⁸ CFU/day, about 10⁹ CFU/day, about IO¹⁰ CFU/day, or about 10¹¹ CFU/day. [0132] For the purposes of the foregoing treatments (i.e., the treatments disclosed herein), the treatments may comprise administration of at least one of the disclosed engineered yeast strains. For example, methods of treating pain, as described herein may comprise orally administering to a subject in need thereof an engineered strain of Saccharomyces yeast expressing a binding protein that binds to tumor necrosis factor a (TNFa). The binding protein may comprise least three complementarity determining regions (CDRs), which may bind to TNFa, including: a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 21-28, a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 29-34, and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 35-41. In some embodiments, the binding protein may further comprise a domain that binds to a second binding target, such as IL-17A. In such embodiments, the domain that binding to IL-17A may comprise a second set of CDRs including a CDR1 comprising an amino acid sequence of SEQ ID NO: 44, a CDR2 comprising an amino acid sequence of SEQ ID NO: 45, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 46. In some embodiments, the binding protein may be a VHH or a combination of two or more VHHS that are connected via one or more linkers. In such embodiments, one VHH may bind to TNFa (e.g., a VHH comprising an amino acid sequence selected from any one of SEQ ID NOs: 1-19), two or more VHHS may bind to TNFa (e.g., a pair of linked VHHS comprising an amino acid sequence of SEQ ID NO: 20), or one VHH that binds to TNFa and at least one additional VHH (e.g., a VHH comprising an amino acid sequence of SEQ ID NO: 42) that binds to a different binding target, such as IL- 17A (e.g., a pair of linked VHHS comprising an amino acid sequence of SEQ ID NO: 43). In some embodiments, the engineered strain of yeast is Saccharomyces boulardii.

Pharmaceutical Compositions and Kits

[0133] In some aspects, the disclosed engineered strain of Saccharomyces yeast is provided in a pharmaceutical composition, such as a composition comprising the engineered strain of Saccharomyces yeast and a pharmaceutically acceptable carrier, excipient, and/or diluent. Examples of suitable carriers, excipients, and diluents include trehalose, alginate, sucrose, inulin, sodium ascorbate, magnesium sulfate, magnesium stearate, maltodextrin, microcrystalline cellulose, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, minerals, and the like. 101341 The pharmaceutical composition may be prepared for any route of administration, though in general, the pharmaceutical composition is suitable for oral administration. Other suitable routes of administration may include suppository administration.

[0135] In some embodiments, the engineered strain of Saccharomyces yeast is dissolved in water or another pharmaceutically acceptable aqueous carrier in which the conjugate exhibits good solubility, optionally with or without other pharmaceutical acceptable excipients, or preservatives.

10136] Also provided herein are kits. A kit may comprise one or more of the engineered strains of Saccharomyces yeast as disclosed herein, contained in a suitable container, optionally together with instructions for use in a method as disclosed herein. Sequences

Table 1: List of binding protein amino acid sequences

EXAMPLES

[0137] The present technology is further illustrated by the following Examples, which should not be construed as limiting in any way. The examples herein are provided to illustrate advantages of the present technology and to further assist a person of ordinary skill in the art with preparing or using the compositions and systems of the present technology. The examples should in no way be construed as limiting the scope of the present technology, as defined by the appended claims. The examples can include or incorporate any of the variations, aspects, or embodiments of the present technology described above. The variations, aspects, or embodiments described above may also further each include or incorporate the variations of any or all other variations, aspects or embodiments of the present technology.

EXAMPLE 1: Development of yeast expressing a TNFa binding protein

[0138] The probiotic yeast Saccharomyces boulardii (S. boulardii) were engineered to secrete the VHH antibodies, including an anti-(m)TNFa VHH (SEQ ID NO: 20) or an anti- (h)TNFa VHH (SEQ ID NO: 1). For additional methods, reference PCT/US 2022/033037. For the purpose of disclosing technology, Table 2 below gives descriptions of certain embodiments of yeast strains used in the Examples that follow.

[0139] VHHs tarsetins mouse pro-inflammatory and/or pain-sisnalins molecules

[0140] The S. boulardii yeast was engineered by transforming the yeast with a transgene cassette consisting of DNA sequence encoding the amino acid (SEQ ID NO: 20) for a homodimer of an anti-(m)TNF-a VHH fused with Fc and inserting into the PTEF or PTDH genomic loci of the auxotrophic S. boulardii strain. In some embodiments, PCR was used to amplify sequences (e.g., DNA sequences encoding ORFs for gene of interest (GOI) and selectable marker) which were purified and then electroporated into yeast to achieve high- copy insertion. In some embodiments, development and/or generation of engineered strains of Saccharomyces yeast comprises yeast codon optimization of a GOI. Site-specific integration of the transgene cassette was achieved via conventional homologous recombination aided by PTEF or PTDH homology arms flanking the cassette. In some embodiments, successful site-specific integration of the transgene cassette was confirmed by diagnostic PCR of genomic DNA extracted from transformed yeast. In some embodiments, supernatants from positive transformants are assessed, for example by ELISA, for acute expression of the therapeutic polypeptide(s) encoded by a GOI in the supernatant. In some embodiments, one or more (e.g., two, three, four) rounds of clone screening is completed to validate the desired expression levels and purity of clones. In some embodiments, cell banks (CB) from desired clones are generated and further assessed for CB characterization.

[0141 ] VHHs tar getins human pro-inflammatory and/or pain-sisnalins molecules

[0142] The S. boulardii yeast was engineered by transforming the yeast with a transgene cassette containing DNA sequences for an anti-(h)TNF-a VHH-FC fusion and inserting into the PTEF or PTDH genomic loci of the auxotrophic S. boulardii strain. In some embodiments, the transgene cassette may comprise DNA sequences for a VHH that binds to (h)TNFa (e.g., any one of SEQ ID NOs: 1-19, or a VHH comprising a CDR1 with an amino acid sequence selected from the group consisting of SEQ ID NO: 21-28, a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 29-34, and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 35-41). In some embodiments, the transgene cassette includes DNA sequences for a second VHH that binds to a different target molecule involved in inflammation and pain, such as (h)IL-17A (SEQ ID NO: 42). In some embodiments, PCR was used to amplify sequences (e.g., DNA sequences encoding ORFs for GOI and selectable marker) which were purified and then electroporated into yeast to achieve high-copy insertion. In some embodiments, development and/or generation of engineered strains of Saccharomyces yeast comprises yeast codon optimization of a gene of interest (GOI). Site-specific integration of the transgene cassette was achieved via conventional homologous recombination aided by PTEF or PTDH homology arms flanking the cassette. In some embodiments, successful site-specific integration of the transgene cassette was confirmed by diagnostic PCR of genomic DNA extracted from transformed yeast. In some embodiments, supernatants from positive transformants are assessed, for example by ELISA, for acute expression of the therapeutic polypeptide(s) encoded by a GOI in the supernatant. In some embodiments, one or more (e.g., two, three, four) rounds of clone screening is completed to validate the desired expression levels and purity of clones. In some embodiments, cell banks (CB) from desired clones are generated and further assessed for CB characterization. Table 2 - Examples of Yeast Strains

EXAMPLE 2: Stress-induced visceral pain mouse model

[0143] A mouse model of orofacial neuropathic pain was tested following a previously described method (PMID: 32404326, PMID: 18923025). Briefly, mice were subjected to chronic constriction injury to the infraorbital nerve (CCI-ION) followed by subchronic stress induced by 3 days of forced swimming (FS), for 10 minutes at day 1, 20 minutes at day 2, and 20 minutes at day 3 (Fig 2A-2B). Mechanical hyperalgesia of lower back area was measured by the von Frey test for mechanosensation. Stimuli were applied using calibrated fibers with a specific bending force. When the force of the von Frey was applied to the lower back skin, mice displayed avoidance behavior like bowing and walking to withdraw from the pressure. The Ipsilateral side of the orofacial skin (V2 and V3 area) and the paw (fore paw and hind paw) was also measured by the series of calibrated von Frey filaments. The EF50 value was measured, defined as the von Frey filament forces (g) that produces a 50% response frequency, as a measure of mechanical sensitivity. The visceral hypersensitivity as measured by von Frey at the lower back was evident in female mice lasting over one month (Fig. 2C left) and was relieved by the treatment of lidocaine (right).

EXAMPLE 3: Treatment of mice with yeast that expressed a binding protein of SEQ ID NO: 20 for seven days

[0144] The mouse model as described in Example 2 was utilized. The mice had their baseline pain level measured by Von Frey measurements on day 14 and then CCI-ION was performed which is a chronic constriction injury induced by ligation of the infraorbital nerve. 11 days post CCI-ION another Von Frey measurement was performed. The mice were then exposed to force swim conditions for 3 days. The first day of forced swim was for 10 minutes, and days 2 and 3 of the force swim was for 20 minutes to induce stress conditions. Three days later, the mice were orally gavaged with either PBS (n=4) or 10⁹ CFU/day of yeast that expressed a binding protein of SEQ ID NO: 20 (n=7) daily for 7 days (from day 6 to 12). Additional Von Frey measurements were performed on days 6, 8, 10 and 12 during the yeast treatment and day 14, 16, and 19 after withdrawal of the yeast treatment (Fig. 3A). During the forced swim days, the mice struggled significantly less in the subsequent swimming days, which is an indicator of increased depression levels (Fig. 3B). The mice produced similar amounts of feces during all three swim days (Fig. 3C). The visceral hypersensitivity, which is the main cause of pain or discomfort in visceral organs, was measured by the Von Frey, and treatment with yeast that expressed a binding protein of SEQ ID NO: 20 significantly reduced hypersensitivity (i.e. pain) on days 10, 12, and 14 (Fig. 3D). Facial hypersensitivity was also measured by Von Frey and remained unchanged among the two groups (Fig. 3E). See also Fig. 2. EXAMPLE 4: Treatment of mice with yeast that expressed a binding protein of SEQ ID NO: 20 for fourteen days

[0145] The mouse model described in Example 2 was utilized and the baseline Von Frey levels were measured on day 14. Post baseline measurement, CCI-ION was performed. The mice were then exposed to forced swim conditions for 3 days. The first forced swim was for 10 minutes, and the subsequent force swims were for 20 minutes to induce stress conditions. Three days later, the mice were orally gavaged with either Saline (Group PBS; n=5), Control yeast (Parental Sb yeast strain without anti-TNF- a gene insertion; Group Sb. Control; n=5) or 10⁹ CFU/day of yeast that expressed a binding protein of SEQ ID NO: 20 (Group 3; n=5) daily (from day 6 to 19) for 14 days. Von Frey measurements were performed on the indicated days in Fig. 4A. During the forced swim days, the mice struggled significantly less in the subsequent swimming days, which is an indicator of increased depression levels (Fig. 4B). The mice produced similar amounts of feces in different groups during all three swim days (Fig. 4C). The visceral hypersensitivity was measured by the Von Frey, and treatment with yeast that expressed a binding protein of SEQ ID NO: 20 (Group 3) significantly reduced hypersensitivity (i.e. pain) from days 10 to 33 and yeast that expressed a binding protein of SEQ ID NO: 20 treated mice returned to normal baseline 7 days earlier compared to Saline (Group 1) or control yeast (Group 2) (Fig. 4D). Facial hypersensitivity was also measured by Von Frey and remained unchanged among the three groups (Fig. 4E).

EXAMPLE 5: Treatment of mice with yeast that expressed a binding protein of SEQ ID NO: 20 reduces TNBS-induced inflammatory pain

[0146] Mice were pre-sensitized with 1% Trinitrobenzene sulfonic acid (TNBS) on day -7 and injected intrarectally with 2.5% (lOOuL per mouse) of TNBS on day 0. On day -2, the mice were gavaged with PBS, control yeast at 10⁹ CFU/day or yeast that expressed a binding protein of SEQ ID NO: 20 at 10⁹ CFU/day once a day for 9 days until day 6. Pain behaviors such as licking or scratching of abdomen, tail, or lower back, were monitored for 30 minutes by analyzing recorded videos on day -10, day 1, day 3, and day 7. (Fig. 5A). Intrarectal injection of TNBS significantly increased pain behaviors and duration of pain in mice that received control yeast and PBS. Mice that received yeast that expressed a binding protein of SEQ ID NO: 20 demonstrated reduced pain behavior as well as time of pain (Fig. 5). EXAMPLE 6: Treatment of mice with yeast that expressed a binding protein of SEQ ID NO: 20 reduces fecal shedding of pro-inflammatory cytokines and chemokines

[0147] The mouse model as described in Example 2 were gavaged with PBS, 10⁹ CFU/day of control yeast, or 10⁹ CFU/day of yeast that expressed a binding protein of SEQ ID NO: 20 daily as described in Example 4. Feces samples were collected from mice on day -14, day - 10, day 1, day 12, day 26, day 42, and day 61, then evaluated for inflammatory cytokine production by an ultrasensitive Meso Scale Discovery Electrochemiluminescence (MSD) assay. The expressions of proinflammatory cytokines/chemokines, TNFa, MCP-1, KC, IL- 12, IL-lbeta, and NGAL/LCN2 were all upregulated in PBS and control yeast groups.

Higher expression of the proinflammatory cytokines correlated with the symptom of hypersensitivity in control mice, expect IL-12 which remained high at Day 61. Treatment with yeast that expressed a binding protein of SEQ ID NO: 20 significantly reduced gut TNFa expression as early as day 12, which is 6 days after oral yeast treatment. The expressions of other proinflammatory cytokines/chemokines were also reduced in treated groups in general but only reached significance on day 42 for MCP-1, KC, and IL-lbeta (Fig. 6).

EXAMPLE 7: Treatment of mice with yeast that expressed a binding protein of SEQ ID NO: 20 reduces psychological stress-induced hypersensitivity

[01481 Mice were exposed to water avoidance stress (WAS) by placing individual mice on a solid surface surrounded with water for 1 hour per day for 9 consecutive days from Day -8 to Day 0. Mice were orally gavaged with control yeast (5. boulardii) or yeast that expressed a binding protein of SEQ ID NO: 20 for 7 days from day 3 to day 9. Von Frey measurements were performed on day -9, day 1, day 3, day 5, day 7, day 10, day 13, and day 15. (Fig. 7A). WAS induced visceral hyperalgesia in mice lasted for approximately 2 weeks in control yeast treated mice. Treatment with yeast that expressed a binding protein of SEQ ID NO: 20 shortened the duration of hyperalgesia (Fig. 7B). EXAMPLE 8: Treatment of mice with yeast expressing heterodimer VHH that binds to human TNFa and IL-17A blocked inflammation-induced visceral pain in transgenic mice

[0149] Transgenic mice that express human TNFa and IL-17A were given 2% dextran sulfate sodium (DSS) in their drinking water for 7 days. Two days post the initial DSS treatment, mice were gavaged with either PBS or yeast that expressed a binding protein of SEQ ID NO: 43 (i.e., yeast that expressed a heterodimer VHH that bind to TNFa and IL-17A) for 10 days from day 3 to day 12. Von Frey measurements were taken on day 12 and day 13, one day after treatment. (Fig. 8A). Hypersensitivity was reduced in mice treated with yeast that expressed a binding protein of SEQ ID NO: 43 compared to mice treated with PBS (Fig. 8B).

EXAMPLE 9: Oral Treatment of mice with yeast expressing SEQ ID NO: 20 significantly reduced visceral pain in PI-IBS mice

[0150] Post-infectious irritable bowel syndrome (PI-IBS) is a common disorder wherein symptoms of IBS begin after an episode of acute gastroenteritis. To induce PI-IBS in mice, Citrobacter rodentium infection was used. Citrobacter has been widely used to induce PI- IBS in rodents and to study underlying mechanisms of visceral pain. For the PI-IBS model with Citrobacter infection, mice were inoculated with a single dose of 10⁹ CFU of C. rodentium, and both male and female mice developed mild clinical symptoms including intestinal inflammation (Fig. 9A). Although hosts cleared the infection at Day 22 post challenge, animals continued enduring gut inflammation as showed by the upregulation of lipocalin-2 on Day 23 (Fig. 9A) and the elongation of colon crypts on Day 29 (Fig. 9B). After experiencing FS stress, animals in the PBS-treated group exhibited referred hyperalgesia but oral treatment with Sb yeast that expressed a binding protein of SEQ ID NO: 20 attenuated the pain response (Fig. 9C). These results demonstrate that orally administered Sb yeast that expressed a binding protein of SEQ ID NO: 20 are highly effective against persistent referred visceral pain in mice with PI-IBS. [01511 All patents and publications mentioned in the specification are indicative of the levels of those of ordinary skill in the art to which the disclosure pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. 10152 [ The present technology is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the present technology. It is to be understood that this present technology is not limited to particular methods, reagents, compounds compositions or biological systems which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

Claims

WHAT IS CLAIMED IS:

1. A method for treating pain in a subject in need thereof, comprising orally administering to a subject in need thereof an engineered strain of Saccharomyces yeast encoding a binding protein that binds to tumor necrosis factor a (TNFa).

2. The method of claim 1, wherein the binding protein comprises a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NO: 21-28, a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NO: 29-34, and a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NO: 35-41.

3. The method of claim 1 or 2, wherein the binding protein is an antibody.

4. The method of claim 3, wherein the antibody is a single domain antibody (sdAb).

5. The method of claim 4, wherein the sdAb is a VHH.

6. The method of any one of claims 1-5, wherein the binding protein comprises a VHH.

7. The method of claim 6, wherein the VHH comprises an amino acid sequence selected from any one of SEQ ID NO: 1-19.

8. The method of any one of claims 1-5, wherein the binding protein comprises two VHHS.

9. The method of claim 8, wherein the two VHHS are homodimers.

10. The method of claim 9, wherein the binding protein comprises an amino acid sequence comprising SEQ ID NO: 20.

11. The method of claim 9, wherein each of the two VHHS comprise an amino acid sequence selected from any one of SEQ ID NOs: 1-19.

12. The method of claim 8, wherein the two VHHS are heterodimers.

13. The method of claim 12, wherein each of the two VHHS independently comprise an amino acid sequence selected from any one of SEQ ID NOs: 1-19.

14. The method of claim 12, wherein the binding protein comprises an amino acid sequence comprising SEQ ID NO: 43.

15. The method of claim 8, 9, or 12, wherein the two VHHS each bind TNFa.

16. The method of claim 8, 9, or 12, wherein one VHH binds TNFa and one VHH binds a binding target that is not TNFa.

17. The method of claim 16, wherein the binding target that is not TNFa is IL-17A.

18. The method of claim 8, 9, 12, or 15-17, wherein one VHH comprises a second CDR1 comprising an amino acid sequence of SEQ ID NO: 44, a second CDR2 comprising an amino acid sequence of SEQ ID NO: 45, and a second CDR3 comprising an amino acid sequence of SEQ ID NO: 46.

19. The method of any one of claims 8-18, wherein the two VHHs are connected via a linker.

20. The method of claim 19, wherein the linker comprises an amino acid sequence of GGGGSGGGGSGGGGS (SEQ ID NO: 47).

21. The method of any one of claims 1-20, wherein the binding protein comprises a VHH- VHH-FC structure.

22. The method of claim 21, wherein the VHH-VHH-FC structure further comprises a linker connecting the two VHH domains.

23. The method of claim 21 or 22, wherein the VHH-VHH-FC structure further comprises a linker connecting the Fc domain to the two VHH domains.

24. The method of any one of claims 1-6, wherein the binding protein comprises an amino acid sequence selected from any one of SEQ ID NO: 1-20 or 43.

25. The method of any one of claims 1-24, wherein the pain is chronic.

26. The method of any one of claims 1-25, wherein the pain is somatic pain, visceral pain, referred bone pain, or neuropathic pain.

27. The method of any one of claims 1-26, wherein the visceral pain is abdominal pain.

28. The method of any one of claims 1-27, wherein the pain is stress-induced.

29. The method of any one of claims 1-28, wherein the pain is inflammation-induced.

30. The method of any one of claims 1-29, wherein the pain is induced by an infection.

31. The method of any one of claims 1-30, wherein the pain is induced by a physical injury.

32. The method of any one of claims 1-31, wherein the pain is induced by a disease.

33. The method of any one of claims 1-32, wherein the subject has a functional pain syndrome, fibromyalgia, or temporomandibular dysfunction (TMD).

34. The method of any one of claims 1-33, wherein the subject has irritable bowel syndrome (IBS) or an inflammatory bowel disease (IBD).

35. The method of claim 34, wherein the IBD is Crohn’s disease, ulcerative colitis, or indeterminate colitis.

36. The method of claim 34, wherein the IBS or IBD is quiescent, in remission or relapsed.

37. The method of any one of claims 1-36, wherein the subject does not have irritable bowel syndrome (IBS) or an inflammatory bowel disease (IBD).

38. The method of any one of claims 1-37, wherein the subject has experienced the pain for a month or more.

39. The method of any one of claims 1-38, wherein the Saccharomyces yeast further comprises a nucleic acid sequence encoding a dihydrofolate reductase (DHFR) incorporated into the genome.

40. The method of claim 39, wherein the DHFR is a mammal DHFR.

41. The method of any one of claims 1-40, wherein the Saccharomyces yeast further comprises one or more exogenous nucleic acids encoding a yeast DFR1.

42. The method of any one of claims 1- 1, wherein the Saccharomyces yeast comprises a complete or partial deletion of URA3.

43. The method of any one of claims 1-42, wherein the Saccharomyces yeast comprises a complete or partial deletion of GAP1.

44. The method of any one of claims 1-43, wherein the Saccharomyces yeast is ura3(-/-) and gap 1 (-/-).

45. The method of any one of claims 1-44, wherein the binding protein is encoded by a nucleic acid incorporated into the genome of the Saccharomyces yeast.

46. The method of claim 45, wherein the nucleic acid encoding the binding protein is incorporated into at least two different chromosomes.

47. The method of claim 46, wherein the at least two different chromosomes comprise chromosomes VII and XVI.

48. The method of any one of claims 1-47, wherein the binding protein is encoded by a nucleic acid that is not incorporated into the genome of the Saccharomyces yeast.

49. The method of any one of claims 1-48, wherein the engineered strain of Saccharomyces yeast is administered daily, every other day, every three days, every four days, every five days, every six days, weekly, every other week, every three weeks, monthly, every other month, every three months, every four months, every five months, or every six months.

50. The method of claim 49, wherein the administered daily is at least once daily, at least twice daily, at least three times daily, at least four times daily, at least five times daily, at least six times daily, at least seven times daily, or at least eight times daily.