Single domain antibodies against gasdermin D and uses thereof

Abstract

The present invention relates to single domain antibodies against Gasdermin D (GSDMD). The single domain antibodies can be used in medical applications, preferably for preventing and / or treating inflammatory diseases or conditions in a subject and / or for determining the presence or absence of GSDMD oligomers in a sample obtained from a subject.

Description

[Technical Field]

[0001] This invention was made with government support under DP1-GM106409 awarded by the NIH. The government has certain rights in this invention.

[0002] Technical Field The present invention relates to the field of biomedical sciences and provides single domain antibodies against Gasdermin D (GSDMD). The single domain antibodies can be used in medical applications, preferably for preventing and / or treating inflammatory diseases or conditions in a subject and / or for determining the presence or absence of GSDMD oligomers in a sample obtained from a subject. [Background technology]

[0003] Background of the Invention Pyroptosis is an inflammatory cell death pathway typically triggered by microbial infection. It involves inflammasome activation and the maturation of the proinflammatory cytokines interleukin-1β (IL-1β) and interleukin-18 (IL-18). Pyroptosis exerts tumor-suppressive functions and elicits antitumor immune responses. Treatment regimens, including chemotherapy, radiation therapy, targeted therapy, and immunotherapy, induce pyroptosis in cancer, which enhances local and systemic antitumor immunity. On the other hand, pyroptosis in normal cells is a side effect of anticancer treatment. Furthermore, dysregulation of the inflammatory response is a key driver in many debilitating diseases linked to acute and chronic inflammation, such as sepsis, atherosclerosis, inflammatory bowel disease, nonalcoholic steatohepatitis, lung cancer, familial Mediterranean fever, and autoinflammatory diseases, such as cryopyrin-associated periodic fever syndrome. Therefore, inhibitors that specifically target pyroptotic cell death may be therapeutically useful in the clinic for the treatment of these diseases.

[0004] Gasdermin family proteins are pyroptosis enforcers and mediate the final common step in all inflammasome pathways. Gasdermin D (GSDMD) is a pore-forming protein that executes pyroptosis by creating pores in the plasma membrane. GSDMD pore formation is critical for the release of the proinflammatory cytokines interleukin-1β (IL-1β) and interleukin-18 (IL-18) downstream of canonical inflammasome activation. GSDMD pore formation triggers cell death by pyroptosis after canonical and non-canonical inflammasome activation. Aberrant activation of IL-1β and IL-18 signaling and pyroptosis are the molecular basis for most pathologies involving systemic inflammation, including monogenic and polygenic autoinflammatory diseases, autoinflammatory diseases caused by abiotic factors and infections (e.g., SARS-CoV-2), and certain types of inflammation-driven cancer. Therefore, GSDMD is considered a key effector protein of the inflammasome pathway.

[0005] Inflammasomes are cytoplasmic multiprotein complexes containing sensor proteins, inflammatory caspases, and in some, but not all, adaptor proteins that connect the two. They can be activated by a repertoire of endogenous and exogenous stimuli, leading to the enzymatic activation of canonical caspase-1, noncanonical caspase-11 (or the human equivalents caspase-4 and caspase-5), or caspase-8, resulting in the secretion of IL-1β and IL-18 and pyroptotic (caspase-1, -11, -4, -5) or apoptotic (caspase-8) cell death.

[0006] The subsequent activation of caspase-1 is not only responsible for the maturation of the proinflammatory cytokines IL-1β and IL-18, but also for the cleavage of GSDMD at its interdomain linker, resulting in the N-terminal (GSDMD NT ) is released from the autoinhibitory C-terminal control and NTThis allows the construction of pores in the plasma membrane. As a result, the mature cytokines IL-1β and IL-18, as well as potentially other proinflammatory danger-associated molecular patterns (DAMPs), are released, and the plasma membrane becomes permeable to DNA-intercalating dyes such as propidium iodide or DRAQ7. Finally, the entire cell ruptures, releasing larger cytosolic components, including tetrameric lactate dehydrogenase (LDH).

[0007] In vitro reconstituted GSDMD pores consist of 31–34 monomers, forming pores with an estimated inner diameter of 22 nm. NT GSDMD undergoes a dramatic conformational change involving a transition of the short β-sheet and helices in the extended domain into two β-hairpins with an extended β-sheet that constitutes a membrane-spanning pore. CT It is unclear whether the loss of GSDMD is sufficient to induce the conformational change, or whether this occurs only in conjunction with oligomerization. Apart from assays reporting plasma membrane permeability to various dyes or cell death, endogenous GSDMD pore formation has not been studied in detail molecularly in live cells, mainly due to the lack of suitable tools. Pyroptotic cells are very delicate and not amenable to staining methods that involve fixation and multiple washing steps. Furthermore, upon inflammasome activation, the fluorescent derivative GSDMD is released. NT was rarely observed at the plasma membrane and mostly in intracellular compartments or structures.

[0008] Because GSDMD oligomerization has been linked to pyroptosis, diagnostic tools are needed to identify whether GSDMD oligomerization is occurring in cell systems, and therapeutic tools are also needed to prevent or halt processes linked to or caused by GSDMD oligomerization.

[0009] Both of these goals have been achieved by the present invention.

[0010] To provide more insight into the oligomerization of GSDMD, which leads to pore formation and ultimately pyroptosis in live cell models, we generated single-domain antibodies, also called nanobodies, against the human GSDMD protein. Nanobodies are single-domain antibodies derived from the variable domains of heavy-chain-only antibodies (VHH) present in camelids. Their small size, specificity, and functionality in the cytosol make them useful tools for studying target proteins in cellular systems. GSDMD NT We generated several antagonistic GSDMD nanobodies that inhibit pyroptosis and IL-1β release by blocking oligomerization of GSDMD. NT still partitions to the plasma membrane, thus monomeric GSDMD NT It was concluded that GSDMD exhibits a conformation suitable for insertion into the plasma membrane and oligomerizes only after insertion. Surprisingly, the existence of an unexpected layer of negative caspase-1 regulation by functional GSDMD pores was established in the present invention, and inhibitory nanobodies were found to exhibit great potential for preventing inflammatory cell death in primary human macrophages when administered to the extracellular environment. This is particularly interesting because GSDMD has been linked to an ever-growing list of (auto)inflammatory, metabolic, and neurodegenerative diseases, as well as cancer, and is therefore an excellent drug target. Summary of the Invention

[0011] The present invention will be described in detail below. The features of the present invention are described in individual paragraphs. However, this does not mean that a feature described in a paragraph is separate from one or more features described in other paragraphs. Rather, a feature described in a paragraph can be combined with one or more features described in other paragraphs.

[0012] The present invention relates to single domain antibodies against GSDMD.

[0013] The single domain antibody of the present invention is characterized by comprising an amino acid sequence including framework region 1 (FR1), complementarity determining region 1 (CDR1), FR2, CDR2, FR3, CDR3, and FR4. FR1 is located at the N-terminus of the amino acid chain, and FR4 is located at the C-terminus of the amino acid chain.

[0014] GSDMD1 and variants In one aspect, the single domain antibody of the invention comprises: TIFF2026508175000001.tif11134. Additionally, the single domain antibody is characterized by comprising: May contain TIFF2026508175000002.tif24158.

[0015] The single domain antibody of the present invention can also be characterized by comprising the amino acid sequence defined in SEQ ID NO:8, which consists of SEQ ID NOs:1 to 7. The single domain antibody of the present invention consisting of the amino acid sequence defined by SEQ ID NO:8 can alternatively be VHH AK-A04, VHH GSDMD-1 , GSDMD1, or VHH-1.

[0016] The single domain antibodies of the invention can also be characterized as variants of the single domain antibodies of the invention, characterized in that they comprise the amino acid sequence defined in SEQ ID NO: 8. The variants are characterized in that they comprise an amino acid sequence that is at least 80%, 90%, 95%, or 99% identical to the amino acid sequence defined in SEQ ID NO: 8. In preferred embodiments, the variants comprise the CDR regions defined in SEQ ID NOs: 2, 4, and 6, with variations occurring in the framework regions.

[0017] GSDMD2 and variants In one aspect, the single domain antibody of the invention comprises: TIFF2026508175000003.tif11129. Additionally, the single domain antibody is characterized by comprising: May contain TIFF2026508175000004.tif24158.

[0018] The single domain antibody of the present invention can also be characterized by comprising the amino acid sequence defined in SEQ ID NO:16, which consists of SEQ ID NOs:9 to 15. The single domain antibody of the present invention consisting of the amino acid sequence defined by SEQ ID NO:16 includes VHH AK-C03, VHH GSDMD-2 , GSDMD2, or VHH-2.

[0019] The single domain antibodies of the invention can also be characterized as variants of the single domain antibodies of the invention, characterized in that they comprise the amino acid sequence defined in SEQ ID NO: 16. Variants are characterized in that they comprise an amino acid sequence that is at least 80%, 90%, 95%, or 99% identical to the amino acid sequence defined in SEQ ID NO: 16. In preferred embodiments, the variants comprise the CDR regions defined in SEQ ID NOs: 10, 12, and 14, with variations occurring in the framework regions.

[0020] GSDMD3 and variants In one embodiment, the single domain antibody of the present invention is characterized in that it comprises a CDR1 defined by SEQ ID NO: 18, a CDR2 defined by SEQ ID NO: 20, and a CDR3 defined by SEQ ID NO: 22. Additionally, the single domain antibody may comprise an FR1 defined by SEQ ID NO: 17, an FR2 defined by SEQ ID NO: 19, an FR3 defined by SEQ ID NO: 21, and an FR4 defined by SEQ ID NO: 23.

[0021] The single domain antibody of the present invention can also be characterized by comprising the amino acid sequence defined in SEQ ID NO:24, which consists of SEQ ID NOs:17 to 23. The single domain antibody of the present invention consisting of the amino acid sequence defined by SEQ ID NO:24 is referred to as VHH AK-H12, GSDMD3, or VHH-3.

[0022] The single domain antibodies of the invention can also be characterized as variants of the single domain antibodies of the invention, characterized in that they comprise the amino acid sequence defined in SEQ ID NO: 24. The variants are characterized in that they comprise an amino acid sequence that is at least 80%, 90%, 95%, or 99% identical to the amino acid sequence defined in SEQ ID NO: 24. In preferred embodiments, the variants comprise the CDR regions defined in SEQ ID NOs: 18, 20, and 22, with variations occurring in the framework regions.

[0023] GSDMD4 and variants In one embodiment, the single domain antibody of the present invention is characterized in that it comprises a CDR1 defined by SEQ ID NO: 26, a CDR2 defined by SEQ ID NO: 28, and a CDR3 defined by SEQ ID NO: 30. Additionally, the single domain antibody may comprise an FR1 defined by SEQ ID NO: 25, an FR2 defined by SEQ ID NO: 27, an FR3 defined by SEQ ID NO: 29, and an FR4 defined by SEQ ID NO: 31.

[0024] The single domain antibody of the present invention can also be characterized by comprising the amino acid sequence defined in SEQ ID NO:32, which consists of SEQ ID NOs:25 to 31. The single domain antibody of the present invention consisting of the amino acid sequence defined by SEQ ID NO:32 is referred to as VHH AK-C10, GSDMD4, or VHH-4.

[0025] The single domain antibodies of the invention can also be characterized as variants of the single domain antibodies of the invention, characterized in that they comprise an amino acid sequence as defined in SEQ ID NO: 32. Variants are characterized in that they comprise an amino acid sequence that is at least 80%, 90%, 95%, or 99% identical to the amino acid sequence as defined in SEQ ID NO: 32. In preferred embodiments, the variants comprise the CDR regions as defined in SEQ ID NOs: 26, 28, and 30, with variations occurring in the framework regions.

[0026] GSDMD5 and variants In one embodiment, the single domain antibody of the present invention is characterized in that it comprises a CDR1 defined by SEQ ID NO: 34, a CDR2 defined by SEQ ID NO: 36, and a CDR3 defined by SEQ ID NO: 38. Additionally, the single domain antibody may comprise an FR1 defined by SEQ ID NO: 33, an FR2 defined by SEQ ID NO: 35, an FR3 defined by SEQ ID NO: 37, and an FR4 defined by SEQ ID NO: 39.

[0027] The single domain antibody of the present invention can also be characterized by comprising the amino acid sequence defined in SEQ ID NO:40, which consists of SEQ ID NOs:33 to 39. The single domain antibody of the present invention consisting of the amino acid sequence defined by SEQ ID NO:40 is referred to as VHH AK-G04, GSDMD5, or VHH-5.

[0028] The single domain antibodies of the invention can also be characterized as variants of the single domain antibodies of the invention, characterized in that they comprise an amino acid sequence as defined in SEQ ID NO: 40. The variants are characterized in that they comprise an amino acid sequence that is at least 80%, 90%, 95%, or 99% identical to the amino acid sequence as defined in SEQ ID NO: 40. In preferred embodiments, the variants comprise the CDR regions as defined in SEQ ID NOs: 34, 36, and 38, with variations occurring in the framework regions.

[0029] GSDMD6 and variants In one embodiment, the single domain antibody of the present invention is characterized in that it comprises a CDR1 defined by SEQ ID NO: 42, a CDR2 defined by SEQ ID NO: 44, and a CDR3 defined by SEQ ID NO: 46. Additionally, the single domain antibody may comprise an FR1 defined by SEQ ID NO: 41, a FR2 defined by SEQ ID NO: 43, a FR3 defined by SEQ ID NO: 45, and a FR4 defined by SEQ ID NO: 47.

[0030] The single domain antibody of the present invention can also be characterized by comprising the amino acid sequence defined in SEQ ID NO:48, which consists of SEQ ID NOs:41 to 47. The single domain antibody of the present invention consisting of the amino acid sequence defined by SEQ ID NO:48 is referred to as VHH AK-G08, GSDMD6, or VHH-6.

[0031] The single domain antibodies of the invention can also be characterized as variants of the single domain antibodies of the invention, characterized in that they comprise an amino acid sequence as defined in SEQ ID NO: 48. The variants are characterized in that they comprise an amino acid sequence that is at least 80%, 90%, 95%, or 99% identical to the amino acid sequence as defined in SEQ ID NO: 48. In preferred embodiments, the variants comprise the CDR regions as defined in SEQ ID NOs: 42, 44, and 46, with variations occurring in the framework regions.

[0032] GSDMD7 and variants In one embodiment, the single domain antibody of the present invention is characterized in that it comprises a CDR1 defined by SEQ ID NO: 50, a CDR2 defined by SEQ ID NO: 52, and a CDR3 defined by SEQ ID NO: 54. Additionally, the single domain antibody may comprise a FR1 defined by SEQ ID NO: 49, a FR2 defined by SEQ ID NO: 51, a FR3 defined by SEQ ID NO: 53, and a FR4 defined by SEQ ID NO: 55.

[0033] The single domain antibody of the present invention can also be characterized by comprising the amino acid sequence defined in SEQ ID NO: 56, which consists of SEQ ID NOs: 49 to 55. The single domain antibody of the present invention consisting of the amino acid sequence defined by SEQ ID NO: 56 is called GSDMD7 or LS-01-A02.

[0034] The single domain antibodies of the invention can also be characterized as variants of the single domain antibodies of the invention, characterized in that they comprise the amino acid sequence defined in SEQ ID NO: 56. The variants are characterized in that they comprise an amino acid sequence that is at least 80%, 90%, 95%, or 99% identical to the amino acid sequence defined in SEQ ID NO: 56. In preferred embodiments, the variants comprise the CDR regions defined in SEQ ID NOs: 50, 52, and 54, with variations occurring in the framework regions.

[0035] GSDMD8 and variants In one embodiment, the single domain antibody of the present invention is characterized in that it comprises a CDR1 defined by SEQ ID NO: 58, a CDR2 defined by SEQ ID NO: 60, and a CDR3 defined by SEQ ID NO: 62. Additionally, the single domain antibody may comprise a FR1 defined by SEQ ID NO: 57, a FR2 defined by SEQ ID NO: 59, a FR3 defined by SEQ ID NO: 61, and a FR4 defined by SEQ ID NO: 63.

[0036] The single domain antibody of the present invention can also be characterized by comprising the amino acid sequence defined in SEQ ID NO:64, which consists of SEQ ID NOs:57 to 63. The single domain antibody of the present invention consisting of the amino acid sequence defined by SEQ ID NO:64 is called GSDMD8 or LS-01-D09.

[0037] The single domain antibodies of the present invention can also be characterized as variants of the single domain antibodies of the present invention, characterized in that they comprise an amino acid sequence as defined in SEQ ID NO: 64. The variants are characterized in that they comprise an amino acid sequence that is at least 80%, 90%, 95%, or 99% identical to the amino acid sequence as defined in SEQ ID NO: 64. In preferred embodiments, the variants comprise the CDR regions as defined in SEQ ID NOs: 58, 60, and 62, with variations occurring in the framework regions.

[0038] GSDMD9 and variants In one embodiment, the single domain antibody of the present invention is characterized in that it comprises a CDR1 defined by SEQ ID NO: 66, a CDR2 defined by SEQ ID NO: 68, and a CDR3 defined by SEQ ID NO: 70. Additionally, the single domain antibody may comprise an FR1 defined by SEQ ID NO: 65, an FR2 defined by SEQ ID NO: 67, an FR3 defined by SEQ ID NO: 69, and an FR4 defined by SEQ ID NO: 71.

[0039] The single domain antibody of the present invention can also be characterized by comprising the amino acid sequence defined in SEQ ID NO:72, which consists of SEQ ID NOs:65 to 71. The single domain antibody of the present invention consisting of the amino acid sequence defined by SEQ ID NO:72 is called GSDMD9 or LS-01-F06.

[0040] The single domain antibodies of the invention can also be characterized as variants of the single domain antibodies of the invention, characterized in that they comprise the amino acid sequence defined in SEQ ID NO: 72. Variants are characterized in that they comprise an amino acid sequence that is at least 80%, 90%, 95%, or 99% identical to the amino acid sequence defined in SEQ ID NO: 72. In preferred embodiments, the variants comprise the CDR regions defined in SEQ ID NOs: 66, 68, and 70, with variations occurring in the framework regions.

[0041] GSDMD10 and variants In one embodiment, the single domain antibody of the present invention is characterized in that it comprises a CDR1 defined by SEQ ID NO: 74, a CDR2 defined by SEQ ID NO: 76, and a CDR3 defined by SEQ ID NO: 78. Additionally, the single domain antibody may comprise an FR1 defined by SEQ ID NO: 73, a FR2 defined by SEQ ID NO: 75, a FR3 defined by SEQ ID NO: 77, and a FR4 defined by SEQ ID NO: 79.

[0042] The single domain antibody of the present invention can also be characterized by comprising the amino acid sequence defined in SEQ ID NO:80, which consists of SEQ ID NOs:73 to 79. The single domain antibody of the present invention consisting of the amino acid sequence defined by SEQ ID NO:80 is called GSDMD10 or LS-02-A07.

[0043] The single domain antibodies of the invention can also be characterized as variants of the single domain antibodies of the invention, characterized in that they comprise an amino acid sequence as defined in SEQ ID NO: 80. Variants are characterized in that they comprise an amino acid sequence that is at least 80%, 90%, 95%, or 99% identical to the amino acid sequence as defined in SEQ ID NO: 80. In preferred embodiments, the variants comprise the CDR regions as defined in SEQ ID NOs: 74, 76, and 78, with variations occurring in the framework regions.

[0044] GSDMD11 and variants In one embodiment, the single domain antibody of the present invention is characterized in that it comprises a CDR1 defined by SEQ ID NO: 82, a CDR2 defined by SEQ ID NO: 84, and a CDR3 defined by SEQ ID NO: 86. Additionally, the single domain antibody may comprise an FR1 defined by SEQ ID NO: 81, a FR2 defined by SEQ ID NO: 83, a FR3 defined by SEQ ID NO: 85, and a FR4 defined by SEQ ID NO: 87.

[0045] The single domain antibody of the present invention can also be characterized by comprising the amino acid sequence defined in SEQ ID NO: 88, which consists of SEQ ID NOs: 81 to 87. The single domain antibody of the present invention consisting of the amino acid sequence defined by SEQ ID NO: 88 is called GSDMD11 or LS-02-A12.

[0046] The single domain antibodies of the invention can also be characterized as variants of the single domain antibodies of the invention, characterized in that they comprise the amino acid sequence defined in SEQ ID NO: 88. Variants are characterized in that they comprise an amino acid sequence that is at least 80%, 90%, 95%, or 99% identical to the amino acid sequence defined in SEQ ID NO: 88. In preferred embodiments, the variants comprise the CDR regions defined in SEQ ID NOs: 82, 84, and 86, with variations occurring in the framework regions.

[0047] GSDMD12 and variants In one embodiment, the single domain antibody of the present invention is characterized in that it comprises a CDR1 defined by SEQ ID NO: 90, a CDR2 defined by SEQ ID NO: 92, and a CDR3 defined by SEQ ID NO: 94. Additionally, the single domain antibody may comprise an FR1 defined by SEQ ID NO: 89, an FR2 defined by SEQ ID NO: 91, an FR3 defined by SEQ ID NO: 93, and an FR4 defined by SEQ ID NO: 95.

[0048] The single domain antibody of the present invention can also be characterized by comprising the amino acid sequence defined in SEQ ID NO:96, which consists of SEQ ID NOs:89 to 95. The single domain antibody of the present invention consisting of the amino acid sequence defined by SEQ ID NO:96 is called GSDMD12 or LS-02-B10.

[0049] The single domain antibodies of the invention can also be characterized as variants of the single domain antibodies of the invention, characterized in that they comprise the amino acid sequence defined in SEQ ID NO: 96. Variants are characterized in that they comprise an amino acid sequence that is at least 80%, 90%, 95%, or 99% identical to the amino acid sequence defined in SEQ ID NO: 96. In preferred embodiments, the variants comprise the CDR regions defined in SEQ ID NOs: 90, 92, and 94, with variations occurring in the framework regions.

[0050] GSDMD13 and variants In one embodiment, the single domain antibody of the present invention is characterized in that it comprises a CDR1 defined by SEQ ID NO: 98, a CDR2 defined by SEQ ID NO: 100, and a CDR3 defined by SEQ ID NO: 102. Additionally, the single domain antibody may comprise an FR1 defined by SEQ ID NO: 97, an FR2 defined by SEQ ID NO: 99, an FR3 defined by SEQ ID NO: 101, and an FR4 defined by SEQ ID NO: 103.

[0051] The single domain antibody of the present invention can also be characterized by comprising the amino acid sequence defined in SEQ ID NO: 104, which consists of SEQ ID NOs: 97 to 103. The single domain antibody of the present invention consisting of the amino acid sequence defined by SEQ ID NO: 104 is called GSDMD13 or LS-02-C09.

[0052] The single domain antibodies of the invention can also be characterized as variants of the single domain antibodies of the invention, characterized in that they comprise an amino acid sequence as defined in SEQ ID NO: 104. Variants are characterized in that they comprise an amino acid sequence that is at least 80%, 90%, 95%, or 99% identical to the amino acid sequence as defined in SEQ ID NO: 104. In preferred embodiments, the variants comprise the CDR regions as defined in SEQ ID NOs: 98, 100, and 102, with variations occurring in the framework regions.

[0053] GSDMD14 and variants In one embodiment, the single domain antibody of the present invention is characterized in that it comprises a CDR1 defined by SEQ ID NO: 106, a CDR2 defined by SEQ ID NO: 108, and a CDR3 defined by SEQ ID NO: 110. Additionally, the single domain antibody may comprise a FR1 defined by SEQ ID NO: 105, a FR2 defined by SEQ ID NO: 107, a FR3 defined by SEQ ID NO: 109, and a FR4 defined by SEQ ID NO: 111.

[0054] The single domain antibody of the present invention can also be characterized by comprising the amino acid sequence defined in SEQ ID NO: 112, which consists of SEQ ID NOs: 105 to 111. The single domain antibody of the present invention consisting of the amino acid sequence defined by SEQ ID NO: 112 is called GSDMD14 or LS-02-C10.

[0055] The single domain antibodies of the invention can also be characterized as variants of the single domain antibodies of the invention, characterized in that they comprise the amino acid sequence defined in SEQ ID NO: 112. Variants are characterized in that they comprise an amino acid sequence that is at least 80%, 90%, 95%, or 99% identical to the amino acid sequence defined in SEQ ID NO: 112. In preferred embodiments, the variants comprise the CDR regions defined in SEQ ID NOs: 106, 108, and 110, with variations occurring in the framework regions.

[0056] GSDMD15 and variants In one embodiment, the single domain antibody of the present invention is characterized in that it comprises a CDR1 defined by SEQ ID NO: 114, a CDR2 defined by SEQ ID NO: 116, and a CDR3 defined by SEQ ID NO: 118. Additionally, the single domain antibody may comprise an FR1 defined by SEQ ID NO: 113, a FR2 defined by SEQ ID NO: 115, a FR3 defined by SEQ ID NO: 117, and a FR4 defined by SEQ ID NO: 119.

[0057] The single domain antibody of the present invention can also be characterized by comprising the amino acid sequence defined in SEQ ID NO: 120, which consists of SEQ ID NOs: 113 to 119. The single domain antibody of the present invention consisting of the amino acid sequence defined by SEQ ID NO: 120 is called GSDMD15 or LS-02-D10.

[0058] The single domain antibodies of the invention can also be characterized as variants of the single domain antibodies of the invention, characterized in that they comprise an amino acid sequence as defined in SEQ ID NO: 120. Variants are characterized in that they comprise an amino acid sequence that is at least 80%, 90%, 95%, or 99% identical to the amino acid sequence as defined in SEQ ID NO: 120. In preferred embodiments, the variants comprise the CDR regions as defined in SEQ ID NOs: 114, 116, and 118, with variations occurring in the framework regions.

[0059] GSDMD16 and variants In one embodiment, the single domain antibody of the present invention is characterized in that it comprises a CDR1 defined by SEQ ID NO: 122, a CDR2 defined by SEQ ID NO: 124, and a CDR3 defined by SEQ ID NO: 126. Additionally, the single domain antibody may comprise an FR1 defined by SEQ ID NO: 121, a FR2 defined by SEQ ID NO: 123, a FR3 defined by SEQ ID NO: 125, and a FR4 defined by SEQ ID NO: 127.

[0060] The single domain antibody of the present invention can also be characterized by comprising the amino acid sequence defined in SEQ ID NO: 128, which consists of SEQ ID NOs: 121 to 127. The single domain antibody of the present invention consisting of the amino acid sequence defined by SEQ ID NO: 128 is called GSDMD16 or SN-62-E01.

[0061] The single domain antibodies of the invention can also be characterized as variants of the single domain antibodies of the invention, characterized in that they comprise an amino acid sequence as defined in SEQ ID NO: 128. Variants are characterized in that they comprise an amino acid sequence that is at least 80%, 90%, 95%, or 99% identical to the amino acid sequence as defined in SEQ ID NO: 128. In preferred embodiments, the variants comprise the CDR regions as defined in SEQ ID NOs: 122, 124, and 126, with variations occurring in the framework regions.

[0062] GSDMD17 and variants In one embodiment, the single domain antibody of the present invention is characterized in that it comprises a CDR1 defined by SEQ ID NO: 130, a CDR2 defined by SEQ ID NO: 132, and a CDR3 defined by SEQ ID NO: 134. Additionally, the single domain antibody may comprise an FR1 defined by SEQ ID NO: 129, a FR2 defined by SEQ ID NO: 131, a FR3 defined by SEQ ID NO: 133, and a FR4 defined by SEQ ID NO: 135.

[0063] The single domain antibody of the present invention can also be characterized by comprising the amino acid sequence defined in SEQ ID NO: 136, which consists of SEQ ID NOs: 129 to 135. The single domain antibody of the present invention consisting of the amino acid sequence defined by SEQ ID NO: 136 is called GSDMD17 or SN-62-E02.

[0064] The single domain antibodies of the invention can also be characterized as variants of the single domain antibodies of the invention, characterized in that they comprise the amino acid sequence defined in SEQ ID NO: 136. Variants are characterized in that they comprise an amino acid sequence that is at least 80%, 90%, 95%, or 99% identical to the amino acid sequence defined in SEQ ID NO: 136. In preferred embodiments, the variants comprise the CDR regions defined in SEQ ID NOs: 130, 132, and 134, with variations occurring in the framework regions.

[0065] GSDMD18 and variants In one embodiment, the single domain antibody of the present invention is characterized in that it comprises a CDR1 defined by SEQ ID NO: 138, a CDR2 defined by SEQ ID NO: 140, and a CDR3 defined by SEQ ID NO: 142. Additionally, the single domain antibody may comprise a FR1 defined by SEQ ID NO: 137, a FR2 defined by SEQ ID NO: 139, a FR3 defined by SEQ ID NO: 141, and a FR4 defined by SEQ ID NO: 143.

[0066] The single domain antibody of the present invention can also be characterized by comprising the amino acid sequence defined in SEQ ID NO: 144, which consists of SEQ ID NOs: 137 to 143. The single domain antibody of the present invention consisting of the amino acid sequence defined by SEQ ID NO: 144 is called GSDMD18 or SN-62-F05.

[0067] The single domain antibodies of the invention can also be characterized as variants of the single domain antibodies of the invention, characterized in that they comprise an amino acid sequence as defined in SEQ ID NO: 144. Variants are characterized in that they comprise an amino acid sequence that is at least 80%, 90%, 95%, or 99% identical to the amino acid sequence as defined in SEQ ID NO: 144. In preferred embodiments, the variants comprise the CDR regions as defined in SEQ ID NOs: 138, 140, and 142, with variations occurring in the framework regions.

[0068] GSDMD19 and variants In one embodiment, the single domain antibody of the present invention is characterized in that it comprises a CDR1 defined by SEQ ID NO: 146, a CDR2 defined by SEQ ID NO: 148, and a CDR3 defined by SEQ ID NO: 150. Additionally, the single domain antibody may comprise a FR1 defined by SEQ ID NO: 145, a FR2 defined by SEQ ID NO: 147, a FR3 defined by SEQ ID NO: 149, and a FR4 defined by SEQ ID NO: 151.

[0069] The single domain antibody of the present invention can also be characterized by comprising the amino acid sequence defined in SEQ ID NO: 152, which consists of SEQ ID NOs: 145 to 151. The single domain antibody of the present invention consisting of the amino acid sequence defined by SEQ ID NO: 152 is called GSDMD19 or SN-62-F09.

[0070] The single domain antibodies of the invention can also be characterized as variants of the single domain antibodies of the invention, characterized in that they comprise an amino acid sequence as defined in SEQ ID NO: 152. Variants are characterized in that they comprise an amino acid sequence that is at least 80%, 90%, 95%, or 99% identical to the amino acid sequence as defined in SEQ ID NO: 152. In preferred embodiments, the variants comprise the CDR regions as defined in SEQ ID NOs: 146, 148, and 150, with variations occurring in the framework regions.

[0071] GSDMD20 and variants In one embodiment, the single domain antibody of the present invention is characterized in that it comprises a CDR1 defined by SEQ ID NO: 154, a CDR2 defined by SEQ ID NO: 156, and a CDR3 defined by SEQ ID NO: 158. Additionally, the single domain antibody may comprise a FR1 defined by SEQ ID NO: 153, a FR2 defined by SEQ ID NO: 155, a FR3 defined by SEQ ID NO: 157, and a FR4 defined by SEQ ID NO: 159.

[0072] The single domain antibody of the present invention can also be characterized by comprising the amino acid sequence defined in SEQ ID NO: 160, which consists of SEQ ID NOs: 153 to 159. The single domain antibody of the present invention consisting of the amino acid sequence defined by SEQ ID NO: 160 is called GSDMD20 or SN-62-G04.

[0073] The single domain antibodies of the invention can also be characterized as variants of the single domain antibodies of the invention, characterized in that they comprise an amino acid sequence as defined in SEQ ID NO: 160. Variants are characterized in that they comprise an amino acid sequence that is at least 80%, 90%, 95%, or 99% identical to the amino acid sequence as defined in SEQ ID NO: 160. In preferred embodiments, the variants comprise the CDR regions as defined in SEQ ID NOs: 154, 156, and 158, with variations occurring in the framework regions.

[0074] GSDMD21 and variants In one embodiment, the single domain antibody of the present invention is characterized in that it comprises a CDR1 defined by SEQ ID NO: 162, a CDR2 defined by SEQ ID NO: 164, and a CDR3 defined by SEQ ID NO: 166. Additionally, the single domain antibody may comprise an FR1 defined by SEQ ID NO: 161, a FR2 defined by SEQ ID NO: 163, a FR3 defined by SEQ ID NO: 165, and a FR4 defined by SEQ ID NO: 167.

[0075] The single domain antibody of the present invention can also be characterized by comprising the amino acid sequence defined in SEQ ID NO: 168, which consists of SEQ ID NOs: 161 to 167. The single domain antibody of the present invention consisting of the amino acid sequence defined by SEQ ID NO: 168 is called GSDMD21 or SN-62-G11.

[0076] The single domain antibodies of the invention can also be characterized as variants of the single domain antibodies of the invention, characterized in that they comprise an amino acid sequence as defined in SEQ ID NO: 168. Variants are characterized in that they comprise an amino acid sequence that is at least 80%, 90%, 95%, or 99% identical to the amino acid sequence as defined in SEQ ID NO: 168. In preferred embodiments, the variants comprise the CDR regions as defined in SEQ ID NOs: 162, 164, and 166, with variations occurring in the framework regions.

[0077] GSDMD22 and variants In one embodiment, the single domain antibody of the present invention is characterized in that it comprises a CDR1 defined by SEQ ID NO: 170, a CDR2 defined by SEQ ID NO: 172, and a CDR3 defined by SEQ ID NO: 174. Additionally, the single domain antibody may comprise an FR1 defined by SEQ ID NO: 169, a FR2 defined by SEQ ID NO: 171, a FR3 defined by SEQ ID NO: 173, and a FR4 defined by SEQ ID NO: 175.

[0078] The single domain antibody of the present invention can also be characterized by comprising the amino acid sequence defined in SEQ ID NO: 176, which consists of SEQ ID NOs: 169 to 175. The single domain antibody of the present invention consisting of the amino acid sequence defined by SEQ ID NO: 176 is referred to as GSDMD22 or SN-62-G12.

[0079] The single domain antibodies of the invention can also be characterized as variants of the single domain antibodies of the invention, characterized in that they comprise an amino acid sequence as defined in SEQ ID NO: 176. Variants are characterized in that they comprise an amino acid sequence that is at least 80%, 90%, 95%, or 99% identical to the amino acid sequence as defined in SEQ ID NO: 176. In preferred embodiments, the variants comprise the CDR regions as defined in SEQ ID NOs: 170, 172, and 174, with variations occurring in the framework regions.

[0080] GSDMD23 and variants In one embodiment, the single domain antibody of the present invention is characterized in that it comprises a CDR1 defined by SEQ ID NO: 178, a CDR2 defined by SEQ ID NO: 180, and a CDR3 defined by SEQ ID NO: 182. Additionally, the single domain antibody may comprise an FR1 defined by SEQ ID NO: 177, a FR2 defined by SEQ ID NO: 179, a FR3 defined by SEQ ID NO: 181, and a FR4 defined by SEQ ID NO: 183.

[0081] The single domain antibody of the present invention can also be characterized by comprising the amino acid sequence defined in SEQ ID NO: 184, which consists of SEQ ID NOs: 177 to 183. The single domain antibody of the present invention consisting of the amino acid sequence defined by SEQ ID NO: 184 is called GSDMD23 or SN-62-H06.

[0082] The single domain antibodies of the invention can also be characterized as variants of the single domain antibodies of the invention, characterized in that they comprise the amino acid sequence defined in SEQ ID NO: 184. Variants are characterized in that they comprise an amino acid sequence that is at least 80%, 90%, 95%, or 99% identical to the amino acid sequence defined in SEQ ID NO: 184. In preferred embodiments, the variants comprise the CDR regions defined in SEQ ID NOs: 178, 180, and 182, with variations occurring in the framework regions.

[0083] Epitopes of the Single Domain Antibodies of the Invention Preferably, the single domain antibody of the present invention is directed against an epitope located in the N-terminal domain of GSDMD. The N-terminal domain of GSDMD is involved in oligomerization. Therefore, the single domain antibody of the present invention that binds to an epitope located in the N-terminal domain of GSDMD can inhibit the ability of GSDMD to oligomerize and thereby form pores.

[0084] The single domain antibody against GSDMD of the present invention can specifically bind to an epitope within the N-terminal domain of GSDMD, which epitope has the sequence of GSDMD (SEQ ID NO:185): It is a discontinuous epitope defined by amino acids L16, H18, G19, E21, F22, Q75, G77, R78, F80, S122, S124, D126, P127, N128, Q131, E162, R217, L231, and F232 of TIFF2026508175000005.tif63128.

[0085] The present invention also relates to polynucleotides encoding the single domain antibodies of the present invention.

[0086] The present invention also relates to polynucleotides comprising one or more nucleic acid sequences encoding the single domain antibodies of the present invention. The polynucleotides of the present invention can be selected from RNA, such as mRNA, DNA, such as genomic DNA, cDNA, or synthetic DNA, analogs thereof, or combinations thereof. Preferably, the polynucleotide is mRNA.

[0087] The polynucleotides of the present invention can be used to transfect cells by methods known in the art. For example, AAV-mediated gene therapy can be used. Adeno-associated virus (AAV) is a small, non-enveloped virus that was adapted for use as a gene transfer vehicle 30 years ago. AAV can transduce a wide range of species and tissues without any evidence of toxicity in vivo, generating relatively mild innate and adaptive immune responses. Upon transfection, cells will produce the single-domain antibodies of the present invention. The single-domain antibodies thus produced can bind to cytosolic GSDMD in cells and thereby inhibit cellular pyroptosis. Therefore, the present invention also relates to single-domain antibodies that can bind to cytosolic GSDMD in cells and thereby inhibit cellular pyroptosis, and the single-domain antibodies against GSDMD are produced by cells when they are transfected with nucleic acids encoding the single-domain antibodies of the present invention.

[0088] The present invention also relates to host cells comprising a polynucleotide encoding a single domain antibody of the present invention; optionally, the host cell is selected from eukaryotic cells, including, but not limited to, hamster cell lines (CHO and their derivatives), mouse cell lines (such as C127, NS0, SP2 / 0, YB2 / 0, XB2 / 09, and all their derivatives), or human cell lines (HEK and their derivatives, such as EXPI293, HT-1080, PER.C6, or HuH-7). Also included are monkey-derived cell lines, such as Vero cells and their derivatives, and insect cells, such as SF-9 cells and their derivatives. Polynucleotides encoding the single domain antibodies of the present invention can also be recombinantly produced in bacterial cells or yeast cells, such as Pichia pastoris.

[0089] The present invention also relates to a pharmaceutical composition comprising a single domain antibody of the present invention or a polynucleotide encoding a single domain antibody of the present invention, and a pharmaceutically acceptable carrier.

[0090] The single domain antibodies of the present invention can prevent the oligomerization of GSDMD. Because GSDMD oligomerization leads to pore formation and ultimately to cellular pyroptosis, preventing GSDMD oligomerization can be a therapeutic approach for stopping or preventing pyroptosis and conditions and symptoms associated with pyroptosis. Therefore, the present invention also relates to the single domain antibodies against GSDMD of the present invention, or the polynucleotides encoding the single domain antibodies of the present invention, or the pharmaceutical compositions of the present invention, for use in therapy.

[0091] In particular, the present invention provides a method for the treatment of acute inflammation, chronic inflammation, sepsis, particularly sepsis-induced blood-brain barrier loss, septic shock, non-alcoholic steatohepatitis, lung cancer, familial Mediterranean fever (FMF), autoinflammatory diseases, cryopyrin-associated periodic fever syndromes (CAPS), non-alcoholic fatty liver disease, Alzheimer's disease, Parkinson's disease, age-related macular degeneration, atherosclerosis, asthma and allergic airway inflammation, gout, Crohn's disease, ulcerative colitis, inflammatory bowel disease, hypertension, nephropathy, myocardial infarction, multiple sclerosis, experimental autoimmune encephalitis, influenza, and the like in a subject. The present invention relates to a single domain antibody against GSDMD of the present invention, or a polynucleotide encoding the single domain antibody of the present invention, or a pharmaceutical composition of the present invention, for use in a method for treating or preventing an inflammatory disease or condition selected from the group consisting of hyperinflammation after fluenza infection, graft-versus-host disease, stroke, silicosis, asbestosis, mesothelioma, type 1 diabetes, type 2 diabetes, obesity-induced inflammation, insulin resistance, rheumatoid arthritis, myelodysplastic syndrome, contact hypersensitivity, chikungunya virus-induced joint inflammation, and traumatic brain injury.

[0092] In a preferred embodiment, mRNA encoding the antibody of the present invention is used in a therapeutic method. The mRNA can be taken up by cells, and the cells will produce the single domain antibody of the present invention. The single domain antibody can then, for example, prevent oligomerization or further oligomerization of GSDMD monomers in the cytosol of the cell. The existing pore is repaired by membrane shedding and / or internalization. This can stop and / or prevent the process leading to pyroptosis. In another embodiment, the single domain antibody itself can be used in a therapeutic method. In both cases, the uptake of nucleic acids or single domain antibodies is enhanced in cells that already present pores formed by GSDMD oligomerization. This is because the pores provide easier entry for extracellular molecules. Therefore, cells that already present pores will take up externally provided single domain antibodies or nucleic acids encoding externally provided single domain antibodies more quickly and in increased amounts compared to cells that do not present pores. This allows for targeting of diseased cells, which provides better therapeutic efficacy and fewer adverse effects.

[0093] The single domain antibodies of the present invention may be suitable for determining whether oligomerization of GSDMD occurs in a cell line.

[0094] Thus, the present invention also relates to a method for determining the presence or absence of GSDMD oligomers in a sample obtained from a subject, the method comprising contacting the sample with a reporter system comprising two single domain antibodies against GSDMD, wherein both single domain antibodies compete for the same epitope in GSDMD, wherein binding of the single domain antibodies to the epitope does not affect the ability of GSDMD to oligomerize, and wherein, upon binding of both single domain antibodies to GSDMD, the reporter system provides a detectable reporter signal indicative of the presence of GSDMD oligomers.

[0095] In the present invention, the sample may be selected from the group including serum, plasma, and whole blood.

[0096] The method of the present invention for determining the presence or absence of GSDMS oligomers in a sample obtained from a subject can be a sandwich ELISA, in which a first single-domain antibody against GSDMD can be used as a primary antibody to capture GSDMD, and a second single-domain antibody against GSDMD can be used as a secondary antibody, wherein the secondary antibody is labeled.

[0097] The method of the present invention for determining the presence or absence of GSDMS oligomers in a sample obtained from a subject can also be an HTRF / FRET-based assay, in which a first fluorescently labeled single domain antibody against GSDMD can be used as a fluorescent donor and a second fluorescently labeled single domain antibody against GSDMD can be used as a fluorescent acceptor.

[0098] An example of the diagnostic method of the present invention is schematically disclosed in FIG.

[0099] In the diagnostic method of the present invention, the first single domain antibody against GSDMD and the second single domain antibody against GSDMD can be single domain antibodies of the present invention, and the first single domain antibody and the second single domain antibody can be the same single domain antibody or different single domain antibodies.

[0100] When GSDMD oligomers are determined to be present in a sample obtained from a subject, it can be concluded that the pyroptosis process is ongoing. In these subjects, a therapeutic approach would be most beneficial, as the therapeutic single domain antibodies of the present invention can halt or prevent pyroptosis. Therefore, the present invention also provides a method for treating acute inflammation, chronic inflammation, sepsis, septic shock, non-alcoholic steatohepatitis, lung cancer, familial Mediterranean fever (FMF), autoinflammatory diseases, cryopyrin-associated periodic fever syndrome (CAPS), non-alcoholic fatty liver disease, Alzheimer's disease, Parkinson's disease, age-related macular degeneration, atherosclerosis, asthma and allergic airway inflammation, gout, Crohn's disease, ulcerative colitis, inflammatory bowel disease, hypertension, nephropathy, myocardial infarction, multiple sclerosis, experimental autoimmune encephalitis, hyperinflammation after influenza infection, graft-versus-host disease, stroke, silicosis, asbestos, and other conditions in subjects. The present invention relates to a single domain antibody against Gasdermin D (GSDMS), or a polynucleotide encoding the single domain antibody, or a pharmaceutical composition of the present invention for use in a method for treating or preventing an inflammatory disease or condition selected from the group consisting of inflammatory bowel disease, mesothelioma, type 1 diabetes, type 2 diabetes, obesity-induced inflammation, insulin resistance, rheumatoid arthritis, myelodysplastic syndrome, contact hypersensitivity, chikungunya virus-induced joint inflammation, and traumatic brain injury, wherein the presence of GSDMD oligomers has been determined in a sample obtained from the subject. DETAILED DESCRIPTION OF THE INVENTION

[0101] definition As used herein, the term "comprise / s / ing" means including or encompassing the disclosed features as well as additional features not specifically mentioned. The term "comprise / es / ing" can also be used in the sense of "consist / s / ing of" the indicated features and therefore not including additional features other than those indicated. Thus, the subject matter of the present invention may be characterized by additional features in addition to those as indicated.

[0102] The term "single-domain antibody" as used herein is interchangeable with the term "nanobody" and defines a recombinant antigen-specific antibody that consists of only one single monomeric variable antibody domain (usually corresponding to the variable region of a heavy chain antibody (VHH)). Nanobodies can be derived from naturally occurring heavy chain antibodies. They offer several advantages over conventional antibodies due to their small size.

[0103] Sequence identity can be determined by skilled artisans.For example, sequence identity can be calculated using BLASTP as disclosed in the prior art (see, for example, Altschul et al. (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402; Altschul et al. (2005) "Protein database searches using compositionally adjusted substitution matrices", FEBS J. 272:5101-5109), preferably using version BLASTP 2.2.29+ (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi), preferably using the following settings: · Field "Enter Query Sequence": Query subrange: None · Field "Choose Search Set": Database: Non-redundant protein sequences (nr); Optional parameters: None Field "Program Selection": Algorithm: blastp (protein-protein BLAST) Algorithm parameters: Field "General parameters": Maximum target sequence: 100; Short query: Automatically adjust parameters for short input sequences; Expectation threshold: 10; Word size: 3; Maximum matches in query range: 0 Algorithm parameters: Field "Scoring parameters": Matrix: BLOSUM62; Gap costs: Presence: 11, Extension: 1; Composition adjustment: Conditional composition score matrix adjustment · Algorithm parameters: Field "Filters and Masking": Filter: none; Mask: none

[0104] Results are filtered for sequences with query coverage greater than 35%.

[0105] Preferably, the variant may contain one or more conservative substitutions relative to the amino acids contained in the exemplary sequences SEQ ID NOs:8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 104, 112, 120, 128, 136, 144, 152, 160, 168, 176, or 184.

[0106] "Conservative substitution" refers to the substitution of one amino acid with another amino acid, where the substitution results in a silent change. This means that one or more amino acid residues in the amino acid sequence of the present invention can be replaced with another amino acid of similar polarity, which acts as a functional equivalent. Substitutes for amino acids in the sequence can be selected from other members of the class to which the amino acid belongs (i.e., conservative substitutions). For example, one polar amino acid can be substituted with another polar amino acid, one amino acid that is positively or negatively charged, respectively, can be substituted with another amino acid that is positively or negatively charged, respectively, and so on. Classes of amino acids are, for example, nonpolar (hydrophobic) amino acids, including alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids, including glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids, including arginine, lysine, and histidine; and negatively charged (acidic) amino acids, including aspartic acid and glutamic acid.

[0107] As used herein, "derivative" and "derivatives" should be understood as all progeny cell lines derived therefrom or emerging therefrom upon modification or further development. Polypeptide expression using cell lines can be achieved by using a variety of transfection systems. Non-limiting examples are, for example, lipid-based transfection or viral transduction techniques, which are very well known to those skilled in the art.

[0108] The patient or subject of the present invention can be a mammal, preferably a human.

[0109] These therapeutic and prophylactic aspects of the present invention are preferably achieved by administering an effective amount of a single domain antibody of the present invention, or a polynucleotide encoding a single domain antibody of the present invention, or a pharmaceutical composition of the present invention, for a time and under conditions sufficient to achieve the appropriate therapeutic or prophylactic effect.

[0110] A "therapeutically effective amount" refers to an amount that is prophylactically and / or therapeutically effective, or an amount sufficient to provide a prophylactic and / or therapeutic effect. A therapeutically effective amount is an amount that produces biological activity and will depend, inter alia, on the individual. The amount will vary depending on the health and physical condition of the individual being treated, the taxonomic group of the individual being treated, the degree of protection desired, the formulation of the composition, the evaluation of the medical condition, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine testing.

[0111] References to "treatment" and "prevention" herein should be considered in their broadest context. The term "treatment" does not necessarily imply that a subject is treated until complete recovery. Similarly, "prevention" does not necessarily mean that a subject will not eventually suffer from a disease state. Thus, treatment and prevention include ameliorating the symptoms of a particular condition, or preventing or otherwise reducing the risk of developing a particular condition. The term "prevention" can be considered as reducing the severity or onset of a particular condition. "Treatment" can also reduce or delay the severity or progression of an existing condition.

[0112] The administration of the single domain antibody of the present invention, or the polynucleotide encoding the single domain antibody of the present invention, or the pharmaceutical composition of the present invention can be carried out by various administration methods. Non-limiting examples include, but are not limited to, intravenous administration, intraarterial administration, intraperitoneal administration, intramuscular administration, pulmonary administration, and inhalation administration. The dosage regimen will be determined by the attending physician and other clinical factors. As is well known to those skilled in the art, the dosage for any one patient will vary and may depend on many factors, including, for example, size, age, sex, time and route of administration, and stage of disease.

[0113] The present invention is further explained by the accompanying figures and examples which are intended to illustrate but not to limit the invention. [Brief explanation of the drawings]

[0114] [Figure 1] Identification of GSDMD-specific nanobodies. (A) Scheme of GSDMD nanobody generation and selection by phage display. (B) Sequence alignment of six GSDMD-specific nanobodies with complementarity-determining regions (CDRs) indicated. (C) SUMO-GSDMD or the control protein SUMO was immobilized on an ELISA plate, and binding of the indicated concentrations of HA-His-tagged VHHs was quantified by ELISA with anti-HA HRP. (D) LUMIER assay: HEK293T cells were cotransfected with the indicated HA-tagged VHHs and expression vectors for the indicated protein-Renilla luciferase fusions. 24 h after transfection, cell lysates were generated, and VHH-HAs were immunoprecipitated with immobilized anti-HA. Coelerentazine was added, and the luminescence of the copurified Renilla luciferase was measured and normalized to the luminescence of the lysate. Data represent the mean (with individual data points) ± SEM from three independent experiments. [Figure 2-1]VHHGSDMD-1 and VHHGSDMD-2 suppress pyroptotic cell death. (A) HEK293T cells were co-transfected with the indicated HA-tagged nanobody and GSDMDNT expression vector or empty vector. LDH release was measured 24 hours after transfection and normalized to cells lysed in Triton X-100 (n=4). (B-E) PMA-differentiated THP-1 macrophages constitutively expressing the indicated HA-tagged nanobody or wt control were stimulated with 0.1 μg / mL LFn-MxiH and 1.0 μg / mL PA (MxiH) for 1 hour to activate NLRC4 (B, D) or with 200 ng / mL ultrapure LPS for 3 hours and 10 μM nigericin (Nig) for 1 hour to activate NLRP3 (C, E). LDH release was then measured as in A (B, C) or IL-1β secretion was measured by homogeneous time-resolved fluorescence (HTRF) (D, E). (F, G) PMA-differentiated THP-1 macrophages were stimulated with NLRC4 and NLRP3 activators as described above, but in the presence of 100 nM DRAQ7. DRAQ7 uptake was monitored over 5 hours in an Incucyte Live-Cell Imaging system. A representative image (F) and a graph of normalized DRAQ7 uptake (n = 3) after 1 hour are shown. Scale bar: 100 μm. (H) Overview of transduction of primary human macrophages with lentiviral vectors encoding various nanobodies and C1C-EGFP under the control of a bidirectional doxycycline (dox)-inducible promoter. Stimulation with the NLRC4 activator MxiH induces cell death by pyroptosis unless the expressed nanobody inhibits GSDMD pore formation, resulting in enrichment of the respective transduced (C1C-EGFP-positive) cells. (I–K) Primary M-CSF-differentiated monocyte-derived human macrophages were transduced with lentiviral vectors packaging Vpx-Vpr and encoding C1C-EGFP and the indicated nanobodies.Twenty-four hours after transduction, gene expression was induced with dox, and 24 hours later, cells were treated with the NLRC4 activator MxiH as in B and D. One hour after treatment, cells were harvested, fixed, and analyzed by flow cytometry to determine cell number over 30 seconds (I), the percentage of C1C-EGFP+ cells, and therefore VHH-expressing cells (J), and the percentage of C1C-EGFP+ cells forming ASC specks (K). Data represent the mean (with individual data points) ± SEM from three independent experiments or donors, unless otherwise noted. [Figure 2-2] See description of Figure 2-1. [Figure 2-3] See description of Figure 2-1. [Figure 3-1]This shows that VHHGSDMD-1 and VHHGSDMD-2 prevent GSDMDNT oligomerization while still allowing membrane localization. (A) THP-1 cell lines expressing C1C-mCherry (dox-inducible) and the indicated VHH-EGFP fusions (constitutive) were differentiated with PMA, treated with dox for 24 hours, and stimulated with the NLRC4 agonist MxiH for 1 hour as described in 2A, but in the presence of 40 μM VX. Cells were harvested, and ASC specks were quantified by flow cytometry. Data represent the mean (with individual data points) ± SEM from three independent experiments. (B, C) PMA-differentiated THP-1 macrophages expressing the indicated HA-tagged nanobodies were stimulated with MxiH for 1 hour as described in 2A. Cells were lysed in SDS-PAGE buffer containing 100 mM DTT (B) or SDS-PAGE buffer without reducing agents (C) and subjected to SDS-PAGE and immunoblotting with GSDMD and GAPDH antibodies. Representative immunoblots from at least three independent experiments are shown. (D) HEK 293T cells stably expressing the indicated VHH-EGFP fusions were transfected with expression vectors for GSDMDNT-mCherry I104N (left) or GSDMD-mCherry I104N (right) and analyzed by live confocal imaging. Representative data from three independent experiments containing 6–22 images per condition are shown. Scale bar = 10 μm. (E) PMA-differentiated THP-1 cells constitutively expressing GSDMD-mNeonGreen_ins and the indicated HA-tagged nanobodies were stimulated with MxiH for 1 h as described in 2A. Representative images are shown. Scale bar = 10 μm. [Figure 3-2] See description of Figure 3-1. [Figure 3-3] See description of Figure 3-1. [Figure 4-1]Inhibition of pore formation by VHHGSDMD induces caspase-1-dependent apoptosis. (A-D) THP-1 cell lines expressing C1C-mCherry (dox-inducible) and the indicated VHH-EGFP fusions (constitutive) were differentiated with PMA, treated with dox for 24 hours, and stimulated with the NLRC4 agonist MxiH for 1 hour as described in 2B, with 5 μM staurosporine (stau) to induce apoptosis. Stimulation was performed in the absence or presence of VX as indicated. (A) A representative cell from three independent experiments was recorded by live confocal microscopy. Scale bar = 10 μm. (B) Cells were harvested and analyzed by flow cytometry to quantify C1C-mCherry specks. (C / D) Cells were harvested and stained for cleaved caspase-3, and the percentage of cells positive for cleaved caspase-3 was quantified by flow cytometry (C). Representative histograms of two exemplary cell lines with the indicated treatments are presented in (D). (E-L) PMA-differentiated THP-1 wt (E-I, K, L) or THP-1 ΔASC (E, J, K) cells constitutively expressing the indicated HA-tagged nanobodies were stimulated with MxiH for 1 h as described in 2A. Experiments were performed in the presence of 40 μM VX, 50 μM Z-VAD, 30 μM, 20 μM, and 4 μM caspase-3 / 7 inhibitors, or 5 / 10 / 20 / 30 ng / mL perfringolysin O (PFO), as indicated. (E, K) Cells and supernatants were harvested to measure caspase-3 / 7 (E) or caspase-1 (K, L) activity using the Caspase-Glo assay. Activity was normalized to cell number using CTB values. Data represent the mean (with individual data points) ± SEM from three independent experiments. (F-J) Cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. Of note, the two caspase-3 blots were developed separately, with a longer exposure for the cleaved form of caspase-3 (F). Data represent the mean (with individual data points) ± SEM from three independent experiments for all flow cytometry and caspase activity assays.Representative immunoblot or microscopic images of at least three independent experiments are shown. [Figure 4-2] See description of Figure 4-1. [Figure 4-3] See description of Figure 4-1. [Figure 5-1] Recombinant nanobodies inhibit pyroptosis when administered extracellularly. (A-B) PMA-differentiated THP-1 cells were treated with MxiH for 1 hour as described in 2A in the presence of increasing concentrations (2, 20, 50, 100, and 200 μg / mL) of the indicated recombinant nanobodies. (A) LDH release was measured and normalized to cells lysed in Triton X-100. (B) IL-1β secretion into the supernatant was quantified by HTRF. (C-D) M-CSF-differentiated primary human macrophages from three independent donors were treated as in 5A / B, and LDH release (C) and IL-1β secretion (D) were quantified as before. (E) THP-1 cells treated as in 5A / B in the presence of DRAQ7 were recorded with an Incucyte Live-Cell Imaging system. One representative image is shown. Red arrows indicate apoptotic cells. Data for LDH and IL-1β release represent means (with individual data points) ± SEM from three independent experiments or donors. (F) shows that fluorescent nanobodies cannot enter intact cells but can indeed enter pyroptotic cells (presumably through GSDMD pores). [Figure 5-2] See description of Figure 5-1. [Figure 6]Figure 6A shows an average distance tree depicting the percentage identity between selected GSDMD-specific nanobody sequences. Figure 6B shows that inhibition of pore formation by antagonistic GSDMD nanobodies enhances caspase-1 activity. PMA-differentiated THP-1 WT cells constitutively expressing the indicated HA-tagged nanobodies were stimulated with MxiH for 1 hour as described in Figure 2A. Experiments were performed in the presence of increasing PFO concentrations (0, 5, 10, 20, 30, 60, 120, 240, and 480 ng / mL). Cells and supernatants were harvested and caspase-1 activity was measured using the Caspase-Glo assay. Activity was corrected for cell number per sample using CTB values ​​and normalized to MxiH-treated cells expressing VHHNP-1 (shown as a dashed line). Data represent the mean (with individual data points) ± SEM from three independent experiments. ***P<0.001, and ****P<0.0001 (unpaired two-tailed Student's t-test). [Figure 7] VHHGSDMD-1 and VHHGSDMD-2 inhibit pyroptosis. (A) THP-1 cell lines constitutively expressing the indicated HA-tagged nanobodies were fixed and stained for HA, and histograms of HA signals from a representative experiment are shown. (B-D) Primary GM-CSF-differentiated monocyte-derived human macrophages were transduced and stimulated as described in 2I-K. One hour after treatment, cells were harvested, fixed, and analyzed by flow cytometry to determine the cell number over 30 seconds (B), the percentage of C1C-EGFP+ cells, and therefore VHH-expressing cells (C), and the percentage of C1C-EGFP+ cells forming ASC specks (D). Data represent the mean (with individual data points) ± SEM from three independent donors. [Figure 8]This shows that VHHGSDMD-1 and VHHGSDMD-2 do not interfere with inflammasome assembly. (A) THP-1 cell lines expressing C1C-mCherry (dox-inducible) and the indicated VHH-EGFP fusions (constitutive) were differentiated with PMA, treated with dox for 24 hours, and stimulated with 200 ng / mL ultrapure LPS for 3 hours and 10 μM nigericin (Nig) in the presence of VX for 1 hour to activate NLRP3. Cells were harvested, and ASC specks were quantified by flow cytometry. Data represent the mean (with individual data points) ± SEM from three independent experiments. [Figure 9-1]Inhibition of pore formation by antagonistic GSDMD nanobodies enhances caspase-1 activity and induces caspase-1-dependent apoptosis. (A) Primary GM-CSF-differentiated monocyte-derived human macrophages were transduced and stimulated as described in 2I-K. Images of three independent donors were recorded by live confocal microscopy. Scale bar = 10 μm. (B-C) PMA-differentiated THP-1 cell lines expressing C1C-mCherry (dox-inducible) and the indicated VHH-EGFP fusions (constitutive) were differentiated with PMA, treated with dox for 24 hours, and stimulated with 200 ng / mL ultrapure LPS for 3 hours and 10 μM nigericin (Nig) for 1 hour to activate NLRP3 (B), or with 0.1 μg / mL LFn-MxiH and 1.0 μg / mL PA (MxiH) for 1 hour to activate NLRC4 (C). Cells were harvested and analyzed by flow cytometry to quantify C1C-mCherry specks (B), or stained for cleaved caspase-3. Representative histograms of two control cell lines with the indicated treatments are presented in (C). Experiments shown in panels B and C were performed in parallel with those shown in Figure 4B–D, but represent triggers (B) or individual cell lines (C) that were not shown in the main figure. (D–E) PMA-differentiated THP-1 ΔASC cells constitutively expressing the indicated HA-tagged nanobodies were stimulated with MxiH for 1 h as described in Figure 2B. 5 μM staurosporine (Stau) was used as a positive control to induce apoptosis. Stimulation was performed in the absence or presence of VX as indicated. Cells were harvested and stained for cleaved caspase-3, and the percentage of cells positive for cleaved caspase-3 was quantified by flow cytometry (D). Representative histograms of cleaved caspase-3 staining for the indicated treatments are presented in (E). (F-H) PMA-differentiated THP-1 WT or THP-1 ΔASC cells constitutively expressing the indicated HA-tagged nanobodies were stimulated with MxiH for 1 h as described in Figure 2B. LDH release was measured and normalized to cells lysed in Triton X-100 (F).Cells and supernatants were harvested and caspase-8 activity was measured using the Caspase-Glo assay; activity was corrected for cell number per sample using CTB values ​​(G). Cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies (H). Representative immunoblots from three independent experiments are shown. Data represent the mean (with individual data points) ± SEM from three independent experiments unless otherwise indicated. [Figure 9-2] See description of Figure 9-1. [Figure 10] Identification of six GSDMD-targeting nanobodies is shown. a) Amino acid sequence alignment of GSDMD-targeting nanobodies showing the three complementarity-determining regions (CDRs 1-3). b) Average distance tree based on the amino acid sequences of the nanobodies. The tree displays the average distance with percent identity and was calculated using the software Jalview. c) Determination of binding affinity using surface plasmon resonance (SPR). Chemically biotinylated GSDMD was immobilized on a sensor chip, and nanobodies were injected as analytes at the indicated concentrations for 120 seconds, followed by dissociation for 300 seconds. The dissociation constants (KD) were determined from the binding and dissociation fits by applying a 1:1 binding model. d) Epitope binning assay. Chemically biotinylated GSDMD was immobilized on an SPR sensor chip, and competitive binding of nanobodies was tested in a pairwise fashion. Binding of a second nanobody to a distinct epitope can be observed as a second binding event in the SPR sensorgram. e, Interaction matrix of VHH-1 to 6. f, Binning of nanobodies according to their properties in competitive binding assays. [Figure 11]VHH-1, VHH-2, and VHH-3 inhibit the formation of functional GSDMD pores in vitro. a) Liposomes composed of POPC, PE, and CL in a 32:55:13 ratio were loaded with the self-quenching dye calcein. GSDMD (or GSDMD-3C) and nanobody were added at an equimolar ratio (0.5 μM). After the addition of 0.2 μM caspase-4 (or 3C protease), calcein release was monitored by detecting fluorescence emitted at 525 nm after excitation at 485 nm. b) GSDMD, nanobody, and caspase-4 were incubated at 37°C for 180 minutes, and calcein release was monitored every minute. VX-765 was used at a concentration of 0.125 μM. One representative experiment out of three independent experiments is shown. c, GSDMD-3C, nanobody, and 3C protease were incubated at 37°C for 45 minutes, and calcein release was detected every minute. One representative experiment out of three is shown. d, Unfolding temperatures of GSDMD, nanobody, and GSDMD-nanobody complex were measured using nanoDSF. Unfolding temperatures of GSDMD and GSDMD-nanobody complex after addition of 5 μM GSDMD and 5 μM nanobody are shown. N=x, data are expressed with SD. e, Unfolding temperatures of GSDMD and GSDMD-nanobody complex. [Figure 12] Crystal structures of GSDMD in complex with VHH-2 and VHH-6 are shown. a) Cartoon representation of the GSDMD-VHH-2-VHH-6 structure, consisting of two heterotrimeric complexes. b) Cartoon representation of one of the two heterotrimeric GSDMD-VHH-2-VHH-6 complexes found in the structure. c) SEC-MALS analysis of the GSDMD-VHH-2-VHH-6 complex using an S200 10 / 300 column. d) Interaction of the N- and C-terminal domains of two GSDMD molecules. e) Superposition of the complex of the GSDMD NTD and GSDMD' CTD with the previous GSDMD crystal structure (PDB 6N9O). [Figure 13]The interfaces between GSDMD and VHH-2 and VHH-6 are shown. a, Cartoon representation of the GSDMD-VHH-2-VHH-6 structure showing the GSDMD NTD, GSDMD' CTD, VHH-2, and VHH-6. CDR-1, CDR-2, and CDR-3 are highlighted in yellow, orange, and cyan, respectively. b, Electrostatic surface representation of the GSDMD-nanobody complex. The epitopes of VHH-2 and VHH-6 are highlighted with dotted lines. c, GSDMD-VHH-2 interface. Residues directly involved in the interaction are labeled. Salt bridges and hydrogen bonds are indicated by dotted lines. d, GSDMD-VHH-6 interface. As in c, residues directly involved in the interaction are labeled, and salt bridges and hydrogen bonds are indicated by dotted lines. [Figure 14] Mechanism of pyroptosis inhibition. a) Recombinant GSDMD was incubated with equimolar amounts of VHH-1 or VHH-2 and caspase-4 at 37°C for 4 hours. GSDMD cleavage by caspase-4 was analyzed by SDS-PAGE. b) Superposition of nanobody-bound GSDMD NTD with the cryo-EM structure (6VFE) of activated GSDMD NTD. [Figure 15] Nanobodies and GSDMD variants are shown. a) SEC elution chromatogram and b) SDS-PAGE of recombinantly expressed and purified nanobodies VHH-1 to VHH-6. c) SEC elution chromatogram and SDS-PAGE analysis of wild-type human GSDMD. d) SEC elution chromatogram and SDS-PAGE analysis of human GSDMD variant (1-484; residues 184-194 and 247-272 deleted). [Figure 16]SPR-based epitope binning is shown. a) Epitope binning assay using VHH-1 in the first titration step, followed by a second titration step with one nanobody from a pool of five (VHH-1, VHH-2, VHH-3, VHH-5, and VHH-6). Chemically biotinylated human full-length GSDMD was immobilized on an SPR sensor chip, and competitive binding of nanobodies was tested in a pairwise manner. b) Epitope binning assay using VHH-2 in the first titration step. Binding of the second nanobody to a distinct epitope can be confirmed for VHH-6 as the second binding event in the SPR sensorgram. c-e) Same as in a, starting with VHH-3, VHH-5, and VHH-6, respectively. [Figure 17] Figure 1 shows that VHH-1, VHH-2, and VHH-3 inhibit the formation of functional GSDMD pores in vitro. Replicate liposome leakage assays. Data from three independent experiments are represented on individual graphs. Each experiment was performed in technical replicates and data are presented with SEM. a, Leakage assay using GSDMD and caspase-4 for cleavage. b, Leakage assay using GSDMD-3C and 3C protease for cleavage. [Figure 18] Figure 1 shows the effect of nanobodies on the thermal stability of GSDMD. a-f, Melting temperatures were measured using nanoDSF. The melting temperatures of GSDMD and nanobodies alone were measured at concentrations of 5 μM and 50 μM, respectively. In titration experiments, 5 μM of GSDMD was mixed with increasing concentrations of each nanobody (1-50 μM). g, Summary of melting temperatures. [Figure 19] 1 shows a schematic diagram illustrating a diagnostic method using a single domain antibody of the invention to determine the presence or absence of GSDMD oligomers in a sample: A: GSDMD in membrane before and after extraction from the membrane; B: sandwich ELISA scheme; C: HTRF / FRET-based assay scheme. [Figure 20]A diagram showing the results of the LUMIER assay is shown: HEK293T cells were co-transfected with expression vectors for the indicated HA-tagged nanobodies and the indicated protein-Renilla luciferase fusions. 24 hours after transfection, cells were lysed and VHH-HA was immunoprecipitated with immobilized anti-HA. Coelenterazine-h was added and the luminescence of the co-purified Renilla luciferase was measured and normalized to the luminescence of the lysate. Data represent the mean (with individual data points) ± SEM from three independent experiments. [Figure 21] Figure 1 shows data on the specificity of the nanobodies of the invention. A: VHHGSDMD-1 and VHHGSDMD-2 inhibit pyroptosis induced by overexpressed GSDMDNT, but not by overexpressed its related counterpart GSDMENT. HEK293T cells were co-transfected with the indicated HA-tagged nanobody expression vector and empty vector, GSDMENT. LDH release was measured 24 hours after transfection and normalized to cells lysed in 1% Triton X-100 (n=3). B: Partial inhibition of mouse GsdmDNT pore formation and uptake of extracellularly administered recombinant antagonistic GSDMD nanobody. HEK293T cells were co-transfected with the indicated HA-tagged nanobody expression vector and empty vector, GSDMDNT, mouse GsdmDNT (mGsdmDNT), or GSDMENT. LDH release was measured 24 hours after transfection and normalized to cells lysed in 1% Triton X-100. C: Mouse iMACs were treated with 200 ng / mL ultrapure LPS for 3 hours and 10 μM nigericin (Nig) for 1 hour in the presence of increasing concentrations (2, 20, 50, 100, and 200 μg / mL) of the indicated recombinant nanobodies to activate NLRP3. LDH release was measured and normalized as in B. Data represent the mean (with individual data points) ± SEM from three independent experiments. [Figure 22]This shows that VHHGSDMD-1 and VHHGSDMD-2 prevent GSDMDNT oligomerization but still allow membrane localization of overexpressed GSDMDNT. (A) HEK293T cells stably expressing VHHGSDMD-1-EGFP were transfected with expression vectors for the plasma membrane marker emiRFP670-CAAX and the indicated GSDMD variants fused to mCherry. Representative images are shown at the top. The distribution of the indicated GSDMD variants in emiRFP670- and mCherry-positive cells was counted, and the mean ± SEM from three independent experiments with at least n = 30 cells per condition is shown at the bottom. PM, plasma membrane. Scale bar, 10 μm. [Figure 23] This shows that nanobodies that prevent oligomerization still allow membrane localization of processed GSDMDNT. (A, B) PMA-differentiated THP-1 cells constitutively expressing GSDMD-mNeonGreen_ins (GSDMD-mNG_ins) and the indicated HA-tagged nanobodies were stimulated with 1.0 μg / mL PA and 0.1 μg / mL LFn-MxiH (MxiH) for 1 hour to activate NLRC4. The plasma membranes of cells in (A) and (B) were stained with CellMask™ Orange (CMO) before stimulation. Cells were recorded by live-cell confocal microscopy, and representative images are shown (A). Fluorescence intensity along the orange diagonal line is displayed to indicate plasma membrane localization of GSDMDNT-mNG (A). The percentage of cells with obvious plasma membrane localization of GSDMDNT-mNG was counted and the mean ± SEM from three independent experiments with at least n = 330 cells per condition is displayed (B). [Figure 24]This shows that nanobodies that prevent oligomerization still allow membrane localization of processed GSDMDNT. PMA-differentiated THP-1 cells constitutively expressing GSDMD-mNG_ins and the indicated HA-tagged nanobodies were stained with CMO and left untreated (left) or stimulated with MxiH (right) and recorded as described in Figure 23A. Additional representative images and intensity sections of the experiment in Figure 23A are shown. Representative images of at least three independent experiments are shown. Scale bar, 10 μm. [Figure 25] This shows that nanobodies that prevent oligomerization still allow membrane localization of processed GSDMDNT. PMA-differentiated THP-1 cells constitutively expressing VHHGSDMD-1-HA were stimulated with MxiH for 15, 30, and 60 minutes. Cells were fixed, stained with fluorescent wheat germ agglutinin (WGA), fixed again, permeabilized, and stained for cleaved GSDMDNT (anti-GSDMDNT) and DNA (Hoechst 33342). Cells were recorded by confocal microscopy, and representative images are shown (A). Fluorescence intensity along the orange diagonal line is displayed to indicate plasma membrane localization of GSDMDNT-mNG. The percentage of cells with clear plasma membrane localization of GSDMDNT was counted, and the mean ± SEM from three independent experiments with at least n = 100 cells per condition is shown (B). [Figure 26-1]This shows that GSDMDNT-mNG localizes to internal structures after pore formation. (A, B) PMA-differentiated THP-1 cells constitutively expressing GSDMD-mNG_ins and VHHNP-1-HA were stained with CMO and stimulated with MxiH as in Figure 24. (A) Stimulated cells were tracked over time by live-cell confocal microscopy (3-minute intervals; time after treatment is indicated). Merged images with enhanced brightness are shown on the right for later time points. Intracellular vesicular structures positive for GSDMD-mNG and CMO are highlighted with yellow arrows. Representative images from at least three independent experiments are shown. Scale bar, 10 μm. (B) A Z-stack containing a representative cell was recorded 60 minutes after stimulation. XY sections and matching XZ and YZ sections are shown centered relative to the cell. [Figure 26-2] See description of Figure 26-1. [Figure 27]We show that inhibition of pore formation by antagonistic GSDMD nanobodies induces caspase-1-dependent apoptosis, and that extracellular nanobodies inhibit NLRP3-dependent pyroptosis. (A) THP-1 cell lines expressing the indicated HA-tagged VHHs were differentiated with PMA and stimulated with 1.0 μg / mL PA and 0.1 μg / mL LFn-MxiH (MxiH) for 1 hour in the presence of 100 nM SYTOX Green nucleic acid stain to activate NLRC4. Cells were recorded by live-cell confocal microscopy, including bright-field recording. The absolute numbers of SYTOX Green-positive cells and cells with pyroptotic and apoptotic morphologies were counted per tile scan (675 μm × 675 μm). Mean values ​​± SEM from three independent experiments are shown. (B, C) Recombinant antagonistic GSDMD nanobodies inhibit pyroptosis when administered extracellularly. PMA-differentiated THP-1 cells were treated with 200 ng / mL ultrapure LPS for 3 hours and 10 μM nigericin (Nig) for 1 hour in the presence of increasing concentrations (2, 20, 50, 100, and 200 μg / mL) of the indicated recombinant nanobodies to activate NLRP3. (B) LDH release was measured and normalized to cells lysed in Triton X-100. (C) IL-1β in the supernatant was quantified by HTRF. Mean values ​​± SEM from three independent experiments are shown. (Data for LDH and IL-1β release represent mean values ​​(with individual data points) ± SEM from three independent experiments or donors. *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001 (unpaired, two-tailed Student's t-test)). [Figure 28]Inhibition of pore formation by antagonistic GSDMD nanobodies induces caspase-1-dependent apoptosis. (A, B) PMA-differentiated THP-1 cells were treated with MxiH, 100 ng / mL PFO, or 5 μM Stau for 1, 2, 3, 4, and 20 hours in the presence of 200 μg / mL of the indicated nanobody. The reducing capacity, as a readout of viability, was measured by CellTiter-Blue® (CTB) assay and normalized to untreated cells in the presence of VHHNP-1. As a positive control, cells were incubated with 1% Triton X-100. Where indicated, cells were treated with 40 μM caspase-3 / 7 inhibitor (casp-3 / 7i) for 4 hours (B). (Data for CTB assays represent the mean (with individual data points) ± SEM from three independent experiments or donors. *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001 (unpaired, two-tailed Student's t-test)). [Figure 29]We show that extracellular antagonistic GSDMD nanobodies prevent pyroptosis and enter cells in a caspase-1-dependent manner. (A) M-CSF-differentiated primary human macrophages from independent donors were treated as previously described, and LDH release was quantified as before. (B) THP-1 cells were differentiated with PMA, labeled with CMO, and stimulated with MxiH in the presence of a total of 200 μg / mL VHHNP-1 (60 μg / mL nanobody labeled with AF647). Where indicated, stimulation was performed in the presence of 40 μM VX (B). Cells were recorded by live-cell confocal microscopy, including bright-field recording. Cell regions (mostly containing single cells) were identified using CMO staining with Imaris. Cell areas were scored as VHH-positive (VHH+) when the intensity of VHHNP-1-AF647 was at least 80 (corresponding to approximately 75% of the average intensity outside the cells in the first dataset). The percentage of VHH+ cell area was normalized to the total cell area. Mean values ​​± SEM from three independent experiments are shown. (C) M-CSF-differentiated primary human macrophages derived from cells of three independent donors were treated with MxiH for 1 hour as described in 2A in the presence of increasing concentrations (2, 20, 50, 100, and 200 μg / mL) of the indicated recombinant nanobodies. IL-1β secretion into the supernatant was quantified by HTRF. (Data for LDH release and IL-1β secretion represent the mean (with individual data points) ± SEM from three independent experiments or donors. *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001 (unpaired two-tailed Student's t-test)). [Figure 30-1]Figure 29B shows that uptake of extracellularly administered recombinant nanobodies relies on caspase-1 and GSDMD pore formation. (A, B) THP-1 cells (A) or THP-1 cell lines expressing the indicated HA-tagged VHHs (B) were differentiated with PMA, labeled with CMO, and stimulated with MxiH in the presence of fluorescent VHHNP-1 as described in Figure 29B. Cells were recorded by live-cell confocal microscopy, including bright-field recordings, and representative images of three independent experiments are shown. VHHNP-1 AF647 in endosomes is indicated by yellow arrows, while cytosolic VHHNP-1 AF647 is highlighted by white arrows. Quantitation of VHH uptake is shown in Figure 29B. Scale bar, 50 μm. NS, not significant (unpaired two-tailed Student's t-test). [Figure 30-2] See description of Figure 30-1. [Figure 31] Pyroptotic cells and cells with transient GSDMD pores show different amounts of DNA dye uptake. (A-C) PMA-differentiated THP-1 cells were treated with MxiH as previously described, but in the presence of SYTOX Green and 200 μg / mL of the indicated nanobody, in the absence (A, C) or presence (B) of VX. Cells were recorded using an Incucyte Live-Cell Imaging system, and the integrated SYTOX Green intensity per cell area was displayed over time (A, B). SYTOX Green-positive nuclei were identified, and the mean fluorescence intensity was extracted. Nuclei were sorted into the indicated bins of SYTOX Green intensity, and the resulting histogram is displayed (C). Dye influx in the presence of inhibitory VHHGSDMD-1 (left) and VHHGSDMD-1 (right) was compared to the control nanobody VHHNP-1 (pyroptotic cells). Representative data from at least three independent experiments are shown. [Figure 32-1]Nanobodies are taken up by pyroptotic cells and cells with transient GSDMD pores in a caspase-1-dependent manner. (A-E) PMA-differentiated THP-1 cells constitutively expressing C1C-EGFP (THP-1C1C-EGFP) were treated with MxiH as previously described in the presence of 30 μg / mL VHHASC AF647 and 200 μg / mL VHHGSDMD-1 (A) or VHHNP-1 (B). Cells were stimulated in the absence (top) or presence (bottom) of VX. Cells were recorded by live-cell confocal microscopy, and representative images are shown. Scale bar, 50 μm. Three exemplary cells with assembled inflammasomes (C1C-EGFP specks) are shown on the right in color-coded insets. Nuclei and C1C-EGFP specks were detected, and the percentage of cells with C1C-EGFP specks (C), the percentage of cells with complete VHH ASC AF647 uptake throughout the cell (represented by AF647-positive nuclei) (D), and the percentage of C1C-EGFP specks positive for AF647 (indicating minimal VHH uptake) (E) were quantified. Mean values ​​± SEM from three independent experiments with at least n = 250 cells per condition (typically > 500 cells) are shown. [Figure 32-2] See description of Figure 32-1. [Figure 32-3] See description of Figure 32-1. [Example]

[0115] Example 1 – Materials and Methods cell line Human embryonic kidney (HEK) 293T cells (ATCC Cat# CRL-3216, RRID:CVCL_0063) were cultured in DMEM GlutaMax™ medium (Gibco) containing 10% FBS; THP-1 cells (ATCC TIB-202) were cultured in RPMI 1640 GlutaMax™ medium (Gibco) containing 10% FBS and 50 μM 2-mercaptoethanol. All genetically modified cell lines were generated by lentiviral transduction using lentiviruses generated with the packaging vectors psPax2 and pMD2.G (gift from Didier Trono, Ecole polytechnique federale de Lausanne, Switzerland). VHHs were expressed under the control of the human elongation factor-1α promoter (pEF1α). GSDMD-1 , VHH GSDMD-2 , VHH GSDMD-3 , VHH NP-1 , or VHH ASC Constitutively expressing THP-1 or HEK293T cell lines were generated using lentiviral vectors constructed by Gateway cloning (Thermo Fisher Scientific) using vectors modified from pRLL (a gift from Susan Lindquist, Whitehead Institute of Biomedical Research), followed by selection in 0.75 μg / mL puromycin (Life Technologies). Cell lines inducibly expressing the C1C-EGFP or C1C-mCherry inflammasome reporter were generated using lentivirus engineered with a derivative of pInducer20, followed by selection in 500 μg / mL geneticin (Gibco). These cell lines formed the basis for further lentiviral transduction to incorporate constitutively expressed nanobodies as described above. EGFP-tagged VHHs GSDMD-1 , VHH GSDMD-2 , VHH NP-1 , or VHH ASCTHP-1 ΔASC cells expressing VHHs were generated without subsequent antibiotic selection but instead sorted on a BD FacsAria Fusion cell sorter for EGFP positivity as an indicator of VHH expression. All expression levels were verified by flow cytometry; for EGFP-tagged VHHs, C1C-EGFP, or C1C-mCherry, by examining a right shift; or for HA-tagged VHHs, by staining with a 1:1000 dilution of anti-HA antibody (B6 HA.11 epitope tag) combined with a secondary anti-mouse AF-488 antibody (1:500). Cells were fixed in 4% formaldehyde and analyzed using a MACSQuant on a BD FACSCanto flow cytometer. Cell lines are routinely tested for mycoplasma contamination. All experiments involving lentiviruses were performed in a biosafety level 2 laboratory.

[0116] primary cells Human CD14 + Monocytes were isolated from human whole blood buffy coats obtained from the blood bank of the University Hospital of Bonn, with the consent of healthy donors, according to a protocol approved by the University of Bonn's Institutional Review Board (local ethics votes Lfd. Nt. 075 / 14). PBMCs were isolated using Ficoll-Paque™ PLUS (VWR), which creates a gradient where PBMCs accumulate between the Ficoll and plasma layers. CD14 (human) monocytes were isolated by positive selection using CD14 MicroBeads (Miltenyi Biotec). + Monocytes were isolated from other PBMCs and cultured for 3 days in RPMI 1640 GlutaMax™ medium supplemented with 10% FBS, PenStrep (500 U / mL), and sodium pyruvate (1 mM) with the cytokines 100 ng / mL recombinant human M-CSF (Immunotools) or 500 U / mL recombinant human GM-CSF (Immunotools). +Monocytes were differentiated into M-CSF- or GM-CSF-induced macrophages. GSDMD-1 , VHH GSDMD-2 , or VHH NP-1 To transiently transduce VHH and C1C-EGFP inflammasome reporter into primary macrophages, they were transduced for 6 hours with a lentivirus expressing both an HA-tagged VHH and a C1C-EGFP inflammasome reporter under a bidirectional doxycycline-inducible promoter derived from the lentiviral vector pInducer20bi-NA, a derivative of pInducer20-NA. Additionally, to promote expression in primary human macrophages, lentivirus was constructed using the packaging vectors psPax2 and pMD2.G, as described above, and the pCAGGs VPx-VPr vector, which inhibits SAMHD1. The following day, expression of both VHH and C1C-EGFP was induced with 1 μg / mL doxycycline for 24 hours.

[0117] protein Expression and purification of His-SUMO-GSDMD and His-SUMO Lfn-MxiH PAs Expression vectors for human His-SUMO-GSMD and His-SUMO were generated by Gibson cloning of GSDMD into the vector pET28. Proteins were expressed in Escherichia coli (E. coli) LOBSTR cells in Terrific Broth, induced with 0.2 or 1 mM IPTG at an OD of 0.6. Cells were grown at 18°C ​​for 24 hours and lysed by sonication in a French press or a Bandelin Sonopuls HD2070 with a TT13 tip. Proteins were then purified by Ni-NTA affinity chromatography using Ni-NTA agarose beads (Qiagen) and gel filtration on a HiLoad 16 / 600 Superdex 75 pg column in a buffer containing 20 mM HEPES pH 7.4, 150 mM NaCl, and 10% glycerol.

[0118] Nanobody expression and purification The nanobody coding sequences of various anti-GSDMD VHHs and the control VHHNP-1 were cloned into pHEN6-based bacterial periplasmic expression vectors with C-terminal LPETG-His6 (large-scale) or HA-His6 (small-scale) tags using conventional restriction enzyme cloning with NcoI and BstEII. E. coli WK6 was transformed with the nanobody expression vectors and grown in Terrific Broth. Expression was induced with 1 mM IPTG at an OD of 0.6, followed by incubation at 30°C for 16 hours. The bacterial pellet was resuspended in TES buffer (200 mM Tris-HCl pH 8.0, 0.65 mM EDTA, 0.5 M sucrose), followed by overnight osmotic shock in 0.25x TES at 4°C. Finally, the nanobodies were Ni-NTA purified using Ni-NTA agarose beads (Qiagen), followed by gel filtration on a HiLoad 16 / 600 Superdex 75 pg column in a buffer containing 20 mM HEPES pH 7.4, 150 mM NaCl, and 10% glycerol. The nanobodies used in ELISA experiments were purified on a small scale and only desalted on a PD MiniTrap G-25 column (GE Healthcare Life Sciences) after Ni-NTA purification. For the use of recombinant nanobodies in the culture medium of primary cells, an endotoxin removal procedure was performed using Pierce™ High Capacity Endotoxin Removal Spin Columns (Thermo Fischer Scientific).

[0119] antibody The following antibodies were used: rabbit polyclonal anti-GSDMD (Atlas Antibodies Cat# HPA044487, RRID:AB_2678957), mouse anti-GAPDH clone 0411 (Santa Cruz Biotechnology Cat# sc-47724, RRID:AB_627678), mouse anti-vinculin clone hVIN-1 (Sigma-Aldrich Cat# V9131, RRID:AB_477629), rabbit anti-caspase-7 clone D2Q3L (Cell Signaling Technology Cat# 12827T, RRID:AB_2687912), rabbit anti-PARP clone 46D11 (Cell Signaling Technology Cat# 9532S, RRID:AB_659884), rabbit anti-DFNA5 / GSDME clone EPR19859 (Abcam Cat# ab215191, RRID:AB_627678). RRID:AB_2737000), mouse anti-caspase-8 clone 1C12 (Cell Signaling Technology Cat# 9746S, RRID:AB_2275120), rabbit anti-caspase-3 clone D3R6Y (Cell Signaling Technology Cat# 14220, RRID:AB_2798429), mouse anti-caspase-9 clone C9 (Cell Signaling Technology Cat# 9508S, RRID:AB_2068620), rabbit polyclonal anti-BID (Cell Signaling Technology Cat# 2002S, RRID:AB_10692485), goat polyclonal anti-rabbit IgG(H+L)-HRP (Invitrogen Cat#31460, RRID:AB_228341), goat polyclonal anti-mouse IgG(H+L)-HRP (Invitrogen Cat# 31430, RRID:AB_228307), mouse anti-HA-HRP clone 6E2 (Cell Signaling Technology Cat# 2999S, RRID:AB_1264166), mouse anti-HA.11 epitope tag clone 16B12 (BioLegend Cat# 901503, RRID:AB_2565005), goat polyclonal anti-mouse IgG(H+L)-Alexa Fluor™ 488 (Invitrogen Cat# A-11029, RRID:AB_2534088), rabbit anti-cleaved caspase-3 (Asp175) clone 5A1E (Cell Signaling Technology Cat# 9664S, RRID:AB_2070042), goat polyclonal anti-rabbit IgG(H+L)-Alexa Fluor™ Plus 647 (Invitrogen Cat# A32733, RRID:AB_2633282), rabbit polyclonal anti-E-tag-HRP (Bethyl Cat# A190-133P, RRID:AB_345222). .

[0120] Small Compound Inhibitors and Reagents The following small compound inhibitors and reagents were used: caspase-3 / 7 inhibitor I (Sigma), CRID3 (MCC-950) (Tocris), doxycycline (Biomol), LPS-EK Ultrapure (Invivogen), MG-132 (Selleckchem), N-ethylmaleimide (NEM) (Sigma), nigericin sodium salt (Biomol), PMA (phorbol 12-myristate 13-acetate) (Sigma), Roche cOmplete™ Mini Protease Inhibitor Cocktail (Sigma Aldrich), staurosporine (Enzo), Vx-765 / vernacasane (Selleckchem), Z-VAD(Ome)-FMK (MedChemExpress).

[0121] Generation of nanobody libraries To raise heavy chain-only antibodies (VHHs) against human GSDMD, alpacas were immunized four times with 200 μg of GSDMD using Imject™ Alum Adjuvant (Thermo Fisher Scientific) according to locally approved protocols. A VHH plasmid library in the M13 phagemid vector pD (pJSC) was generated as previously described (Schmidt et al., 2016b; Koenig et al., 2021). Briefly, RNA was extracted from peripheral blood lymphocytes and used as a template to generate cDNA using three sets of primers: random hexamers, oligo(dT), and primers specific for the constant region of the alpaca heavy chain gene. The VHH coding sequence was amplified by PCR using VHH-specific primers, cut with AscI and NotI, and ligated into the M13 phagemid vector (pJSC) linearized with the same restriction enzymes. The ligation reaction was electroporated into E. coli TG1 cells (Agilent), and the resulting ampicillin-resistant colonies were picked, pooled, and stored as glycerol stocks.

[0122] Identification of nanobodies using VHH phage display and panning GSDMD-specific VHHs were obtained by phage display and panning. E. coli TG1 cells containing the VHH library were infected with helper phage VCSM13 to produce phages displaying the encoded VHHs as pIII fusion proteins. The phages in the supernatant were purified and concentrated by precipitation. Phages displaying GSDMD-specific VHHs were enriched using biotinylated GSDMD immobilized on Dynabeads™ MyOne™ Streptavidin T1 (Life Technologies). The retained phages were used to infect E. coli ER2738 and subjected to a second round of panning. 96 E. coli ER2837 colonies obtained in the second panning were grown in 96-well plates, and VHH expression was induced with IPTG. The VHHs leaked into the supernatant were tested for specificity using ELISA plates coated with the control protein SUMO or SUMO-GSDMD. Bound VHHs were detected with HRP-conjugated rabbit anti-E-Tag antibody (1:10,000) and the chromogenic substrate tetramethylbenzidine (TMB) (Life Technologies). The reaction was stopped with 1 M HCl, and the absorbance at 450 nm was recorded using a SpectraMax i3 instrument and SoftMax Pro 6.3 Software (Molecular Devices). Positive candidates were sequenced, and representative nanobodies were cloned into bacterial expression vectors for further analysis.

[0123] Nanobody ELISA To test nanobody candidates, SUMO-GSDMD or SUMO in PBS was immobilized on an ELISA plate overnight at a concentration of 1 μg / mL. The immobilized antigen was then incubated with HA-tagged nanobodies in 10% FBS / PBS at 10-fold dilutions ranging from 100 nM to 1 pM. Nanobodies were detected using a mouse anti-HA HRP antibody (1:5000) and developed with the chromogenic substrate TMB. The reaction was stopped with 0.5 M HCl, and absorbance was then measured at 450 nm using a SpectraMax i3 instrument and SoftMax Pro 6.3 Software (Molecular Devices).

[0124] LUMIER assay To test the functionality of our VHHs in the reducing environment of the cell cytosol, we performed a LUMIER assay. HEK293T cells (2.5 10 per well in a 24-well plate) were cultured using LUMIER assay. 5 Cells were cultured (seeded the day before) in 0.25 μg of pCAGGS VHH-HA expression vector and 0.25 μg of Renilla fusion bait protein GSDMD, GSDMD 4A , GSDMD NT-4A , GSDMD CT , or control NLRP1 CARDThe cells were co-transfected using PEI Max (Polysciences). Lumitrac 600 white high-binding 96-well plates (Labomedic) were coated with 20 μg / mL of mouse anti-HA.11 epitope tag clone 16B12 antibody in PBS. The next day, transfected HEK293T cells were lysed in LUMIER lysis buffer (50 mM Hepes-KOH pH 7.9, 150 mM NaCl, 2 mM EDTA, 0.5% Triton X-100, 5% glycerol, and Roche cOmplete™ Mini protease inhibitor cocktail) and applied to both HA-coated Lumitrac plates and uncoated Lumitrac plates as lysate controls. Upon addition of the Renilla luciferase substrate, coelenterazine-h, luminescence was measured for both the immunoprecipitated (IP) lysates and lysate controls using a SpectraMax i3 instrument and SoftMax Pro 6.3 Software (Molecular Devices). Values ​​plotted are IP luminescence values ​​normalized by lysate values.

[0125] Quantification of cell death by LDH release To quantify pyroptotic cell death, THP-1 cells differentiated with PMA (50 μg / mL for 18 hours, followed by a 24-hour rest period) or primary human macrophages differentiated with M-CSF were plated (3·10 per well in 24-well plates) in the presence of intracellularly expressed or exogenously added recombinant VHHs. 5In cells, we activated the NLRP3 and / or NLRC4 inflammasomes. NLRP3 was activated with 10 μM nigericin (Nig), a potassium ionophore derived from Streptomyces hygroscopicus, in OptiMEM for 1 hour. This was preceded by a 3-hour priming with 200 ng / mL ultra-pure LPS, in the presence or absence of 40 μM VX or 2.5 μM CRID3, added 30 minutes before and during stimulation. NLRC4 inflammasomes were activated with recombinant MxiH (0.1 μg / mL) fused to the N-terminal domain of anthrax lethal factor (LFn-MxiH), which can be delivered to the cytosol by utilizing a channel formed by protective antigen (PA) (1.0 μg / mL), in OptiMEM, in the presence or absence of 40 μM VX. Extracellular administration of recombinant VHHs at increasing concentrations (1, 2, 20, 50, 100, and 200 μg / mL) was performed simultaneously with inflammasome stimulation. To measure pyroptotic cell death in HEK293T cells, cells (5 10 per well in a 24-well plate) were cultured. 5 Cells) were transfected with 0.50 μg of HA-tagged VHH and 0.25 μg of GSDMD using Lipofectamine™ 2000 (L2000) transfection reagent (Invitrogen). NT Cells were co-transfected with either 0.1% TNF-α or 0.1% EMP or empty vector for 4 hours. Supernatants were collected 24 hours after transient transfection and subjected to lactate dehydrogenase (LDH) release assays using an LDH cytotoxicity detection kit (TaKaRa or Roche) according to the manufacturer's instructions. Absorbance at 492 nm was measured using a SpectraMax i3 instrument and SoftMax Pro 6.3 Software (Molecular Devices). After subtracting the medium background signal, LDH release was normalized using a control sample in which cells were lysed in 1% Triton X-100.

[0126] Cytokine quantification by HTRF To quantify IL-1β secretion, the supernatant obtained through the procedure described above for the LDH release assay was subjected to a human IL-1β homogeneous time-resolved fluorescence (HTRF) assay (Cisbio) according to the manufacturer's instructions. Emissions at 616 nm and 665 nm were measured using a SpectraMax i3 instrument after excitation at 340 nm, and IL-1β levels were calculated using SoftMax Pro 6.3 Software (Molecular Devices) based on a standard curve.

[0127] Quantification of cell death by DRAQ7 uptake To quantify cell death over time, 4 10 cells per well were added. 4 PMA-differentiated THP-1 cells were seeded in 96-well plates and treated as described above for the LDH assay. The stimulation medium was supplemented with the non-cell-permeable DNA dye DRAQ7 (1:3000) (Biolegend), and uptake was analyzed using the Incucyte Live-Cell Imaging System (Sartorius). Cells were imaged every 5 minutes for a total of 5 hours using an Incucyte SX5 instrument, with four images acquired per well. The number of DRAQ7-positive nuclei (cell death count) and cell confluency were analyzed using Incucyte 2021C software. For every single image, the cell death count was corrected by subtracting the value at the beginning of the experiment. The corrected cell death count was further normalized to cell confluency due to division, and then the average of all four images was calculated and plotted over time.

[0128] Flow cytometry-based quantification To quantify the amount of successfully transduced nonpyroptotic primary human (G)M-CSF macrophages upon lentiviral transduction of an EGFP-tagged C1C inflammasome reporter and HA-tagged VHHs, EGFP was used. +The percentage of cells was quantified by flow cytometry. At the same time, to compare the amount of nonpyroptotic cells per sample, measurements were performed over a fixed time period of 30 seconds. Because only pyroptotic cells do not survive the flow cytometry procedure and therefore cannot be measured, the reduction in measured cell mass is an indirect indicator of pyroptotic cell death. Furthermore, recruitment of C1C-EGFP to ASC specks was confirmed by a lower width and higher height signal, as described elsewhere, indicating the presence of EGFP. + For these procedures, 1 10 5 Primary macrophages were treated in 24-well plates and transduced with lentivirus as described above. NLRC4 inflammasomes were activated with 0.1 μg / mL LFn-MxiH in combination with 1.0 μg / mL PA for 1 hour. Cells were harvested by trypsinization, fixed in 4% formaldehyde, and analyzed using a BD FACSCanto flow cytometer.

[0129] To assess the success of inflammasome formation in the presence of EGFP-tagged VHHs, PMA-differentiated THP-1 macrophages expressing both VHH-EGFP and C1C-mCherry were stimulated for 1 hour with either 0.1 μg / mL LFn-MxiH in combination with 1.0 μg / mL PA or 200 ng / mL LPS (pre-treatment for 3 hours) and 10 μM nigericin. To prevent loss of responder cells due to caspase-1-dependent pyroptosis, cells were stimulated in the presence of 40 μM VX. The number of speckled C1C-mCherry+ single cells was measured. To demonstrate that inhibitory anti-GSDMD VHHs induce the speckled response without pyroptosis, experiments were also performed in the absence of VX, allowing pyroptotic cell death and cell loss. Staining for cleaved caspase-3 in PMA-differentiated THP-1 macrophages was performed as a measure of apoptotic cell death. 3·10 5THP-1 macrophages were treated in 24-well plates with 0.1 μg / mL LFn-MxiH in combination with 1.0 μg / mL PA for 1 hour in the presence or absence of 40 μM VX. Staurosporine is a nonselective inhibitor of several kinases and served here as a positive control for intrinsic apoptosis and caspase-3 activation. After fixation, cells were permeabilized and stained with rabbit anti-cleaved caspase-3 primary antibody (1:2000) followed by Intracellular Staining Permeabilization Wash Buffer (Biolegend) combined with goat anti-rabbit Alexa Fluor™ Plus 647-conjugated highly cross-absorbing secondary antibody (1:500). Single cells expressing EGFP-tagged VHHs were analyzed for their cleaved caspase-3 activity using a BD LSRFortessa SORP flow cytometer. + Cell populations were analyzed. All flow cytometry data were analyzed using FlowJo 10.7.1 software.

[0130] Live cell confocal microscopy For live cell confocal microscopy experiments, 4 PMA-differentiated THP-1 cells, or 2 10 4 GM-CSF-differentiated primary human macrophages were cultured in 15µ slide 8-well Ibidi chambers or black clear-bottom TC-treated PhenoPlate™ 96-well microscopy plates (Perkin Elmer). NLRC4 inflammasomes were activated with 0.1µg / mL LFn-MxiH and 1.0µg / mL PA for 1 hour in imaging medium (RPMI containing 10% FBS, 50µM 2-mercaptoethanol, 30mM Hepes, and phenol red-free). HEK293T cells constitutively expressing EGFP-tagged VHHs were seeded (9-10 per well) in Ibidi chambers coated with poly-L-lysine (Sigma, MW 70,000-150,000). 4Then, they were transfected with 0.25 μg of the expression vector GSDMD I104N-mcherry or GSDMD NT I104N-mCherry was transiently transfected for 5 hours. Images were recorded at least every 10 minutes using a HC PL APO CS2 63x1.20 water objective on a Leica SP8 Lightning confocal microscope (37°C, 5% CO2). Images were processed using ImageJ 2.3.0 software.

[0131] Immunoblotting To detect the presence and / or cleavage of our protein of interest, we treated 3–4 10 μL of lysed ... 5 (per well in a 24-well plate) or 1.25 10 6PMA-differentiated THP-1 cells (per well in a 6-well plate) were lysed in 100 μL or 300 μL of RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 2 mM EDTA, 0.1% SDS, Roche cOmplete™ Mini protease inhibitor cocktail) to generate immunoblot samples. Immediately before running, the samples were supplemented with a final concentration of 1× SDS-PAGE sample buffer (50 mM Tris pH 6.8, 0.01% bromophenol blue, 10% glycerol, 2% SDS) with or without 100 mM DTT, sheared, and heated at 95°C for 5 minutes. Proteins were separated by SDS-PAGE using 10% or 12% homemade SDS-PAGE gels. Separated proteins were transferred to PVDF membranes (0.45 μm, Merck) by semi-dry transfer. All immunoblots were blocked for at least 2 h in 5% nonfat dry milk (NFDM) in TBS-T (0.05% Tween-20) and probed with the following primary antibody dilutions: anti-GSDMD 1:500, anti-GAPDH 1:1000, anti-vinculin 1:1000, anti-caspase-3 1:500, anti-caspase-7 1:500, anti-caspase-8 1:500, anti-caspase-9 1:500, anti-PARP 1:500, anti-GSDME 1:500, and anti-BID 1:500. All primary antibodies were added in NFDM solution. After overnight incubation at 4°C, immunoblots were probed with HRP-conjugated secondary antibodies (1:3000) in NFDM solution for 2 h. Chemiluminescent signals were induced by Western Lightning® Plus-ECL (Perkin Elmer) except for immunoblots of GSDMD, caspase-3, caspase-7, caspase-8, caspase-9, and BID, which required Western Lightning Ultra (Perkin Elmer).Signals were detected using a Fusion Advancer imaging system (Vilber) and images were acquired using EvolutionCapt SL6 software (Vilber).

[0132] Caspase-Glo activity and cell titer blue assay To quantify the activity of caspase-1, caspase-3 / 7, and caspase-8, 5-10 μg / ml of cells cultured in a 96-well plate were used. 4 PMA-differentiated THP-1 cells were activated with recombinant LFn-MxiH (0.1 μg / mL) in OptiMEM with 1.0 μg / mL PA for 1 hour. The cells and supernatant were then mixed with an equal volume of caspase-Glo reagent for 1 hour at room temperature according to the manufacturer's instructions (Promega). The mixture was then added to a Lumitrac plate, and luminescence was measured using a SpectraMax i3 instrument and SoftMax Pro 6.3 Software (Molecular Devices). A blank control value of OptiMEM alone mixed with caspase-Glo reagent was subtracted from the measured values. In parallel, a cell titer blue (CTB) assay was performed using CellTiter-Blue® reagent (Promega) according to the manufacturer's instructions to determine the viability of cultured cells. The metabolic activity of live cells can be measured by the conversion of resazurin to highly fluorescent resofurin. Fluorescence was measured at 585 nm. Because of the linear relationship between fluorescence and cell number, we were able to estimate the relative cell number per cell line. Values ​​from control wells without cells were subtracted, and the control VHHNP-1-expressing cell line was set to 1. The final CTB value was used to correct for discrepancies in cell number between different cell lines in the caspase-Glo assay. For the caspase-1 Glo assay, values ​​were further normalized to MxiH-treated VHH52 samples to highlight the increased caspase-1 activity in apoptotic cells compared with pyroptotic cells.

[0133] Protein expression and purification The coding sequence of human GSDMD 1-484 was cloned into a pET28-derived bacterial expression vector providing an N-terminal His6-SUMO-tag. For crystallization experiments, residues 247-272 were deleted (GSDMD 1-484) to prevent precipitation during crystallization. Δ247-272 ), or deletion of residues 184–194 and 247–272 (GSDMD Δ184-194 / Δ247-272 The expression construct was transformed into E. coli Rosetta (DE3) cells, and the cells were cultured at OD in 2×LB medium containing 0.5% glucose and 50 μg / ml kanamycin at 37°C. 600 The cells were grown until a RI of 0.8 was reached. Expression was induced with 0.2 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and 0.6% (w / v) lactose overnight at 20°C. Cells were harvested and lysed by sonication in lysis buffer containing 25 mM Tris (pH 8.0), 200 mM NaCl, 10% glycerol, 10 mM imidazole, and 5 mM DTT. Cell lysates were cleared by centrifugation at 20,000 rpm for 45 minutes, and the His-SUMO fusion protein was enriched on Ni-NTA beads (Thermo Fisher Scientific). Proteins were eluted with a buffer containing 25 mM Tris (pH 8.0), 200 mM NaCl, 300 mM imidazole, and 5 mM DTT. Buffer exchange to 25 mM Tris (pH 8.0), 200 mM NaCl, 5% glycerol, and 5 mM DTT was performed using a PD10 column (Cytiva). The sample was incubated overnight at 4°C with SUMO protease ULP1 (prepared in-house), followed by a second round of Ni-NTA chromatography to remove uncleaved protein, ULP1 protease, and the His-SUMO tag. The sample was then subjected to size-exclusion chromatography using an S200 column (GE Healthcare) and a buffer containing 20 mM HEPES (pH 8.0), 200 mM NaCl, and 5 mM DTT.

[0134] E. coli WK6 cells were transformed with the pHEN6 vector for bacterial periplasmic expression of GSDMD-targeting nanobodies. VHH-1, VHH-2, and VHH-3 were expressed with a C-terminal LPTEG-His tag, and VHH-4, VHH-5, and VHH-6 were expressed with a C-terminal HA-His tag. Cells were grown at OD in TB medium containing 100 μg / ml ampicillin at 37°C. 600 The cells were grown until a pH of 0.6 was reached. Expression was induced with 1 mM IPTG overnight at 20°C. Cells were harvested, and periplasmic extracts were generated using osmotic shock. For this purpose, cell pellets were resuspended in TES buffer (20 mM Tris (pH 8.0), 0.65 mM EDTA, 0.5 M sucrose) and incubated for 1 h, followed by incubation in 0.25x TES for at least 1 h at 4°C. The lysate was clarified by centrifugation at 20,000 rpm for 45 min, and His-tagged nanobodies were enriched using Ni-NTA beads. The beads were washed with a buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, and 10 mM imidazole, and the protein was eluted in the same buffer supplemented with 0.5 M imidazole. Elution fractions containing the protein were pooled and subjected to gel filtration using an S75 16 / 600 column (GE Healthcare) in a buffer containing 20 mM HEPES pH 7.5, and 150 mM NaCl.

[0135] The coding sequence of human caspase-4 was cloned into the pACEBac1-His-SUMO expression vector. The plasmid was amplified in Escherichia coli DH10 cells. Baculovirus was produced by transfection of bacmid DNA into Sf9 insect cells using the transfection reagent cellfectin (Mirus Bio, Madison, WI). Transfections were performed using 0.7 x 10 6 Cells were incubated at 27°C in a 6-well format with 0.6 x 10 cells / well. After 3 days, the initial virus stock V0 was harvested and 0.6 x 106 V1 virus was harvested 3 days later and used to infect a 20 ml culture of Sf9 cells grown to a density of 1.0 × 10 cells / ml. 6 Sf9 cultures grown to a density of 1.5 x 10 cells / ml were infected with 2% V1. After an additional 3 days, V2 virus was harvested. Expression cultures were infected with 1.5 x 10 6 Cells were infected with 1% virus at a cell density of 1000 cells / ml, and protein expression was allowed to continue for 48 hours at 27°C. Cells were harvested and lysed by sonication in a buffer containing 50 mM Tris (pH 8.0), 150 mM NaCl, 5 mM imidazole, and 2 mM β-ME. Cell lysates were clarified by centrifugation at 20,000 rpm for 45 minutes. His-SUMO-caspase-4 was purified using Ni-NTA affinity chromatography. Elution fractions containing the His-SUMO fusion protein were pooled and concentrated to a concentration of 10 mg / ml using a 30K Amicon column. Samples were incubated overnight at 4°C to enhance protease autoactivation. For liposome leakage assays, a GSDMD variant in which the caspase cleavage site was mutated to a 3C protease cleavage site (positions 273-280 were LEVLFQGP instead of LTDGVPAE) was used.

[0136] Protein crystallization and data collection Wild-type GSDMD and GSDMD in complex with nanobodies VHH-1 to VHH-6 and VHH-2 + VHH-6 combinations Δ184-194 / Δ247-272 Screening of crystallization conditions for VHH-2 and VHH-6 was performed by sitting drop vapor diffusion using commercial kits from Molecular Dimensions (Maumee, OH, USA) and Jena Bioscience (Jena, Germany). Δ184-194 / Δ247-272Initial crystals of a sample containing α-glucan were obtained at a protein concentration of 20 mg / ml using a reservoir solution containing 0.07 M NaCl, 22% (v / v) PEG 400, and 0.05 M Na3Cit, pH 4.5, at 20°C. Optimization of crystallization conditions resulted in well-diffracting crystals grown at 20 mg / ml in a reservoir solution consisting of 0.04 M NaCl, 25.8% (v / v) PEG 400, and 0.05 M Na3Cit, pH 4.4, at 20°C.

[0137] Crystals were frozen in liquid nitrogen in reservoir solution plus PEG 400 at a final concentration of 35%. X-ray diffraction data were collected at beamline P13 of the PETRA III synchrotron at the Deutsches Elektronen-Synchrotron (DESY) in Hamburg, Germany, at a wavelength of λ = 0.976255 Å. Diffraction data were processed with the program XDS. Phases were determined by molecular replacement. For GSDMD, the previous crystal structure of human GSDMD (PDB: 6N9O) was used as a search model. To account for possible movements between the N- and C-terminal domains of GSDMD, the structure was split into GSDMD-NTD and GSDMD-CTD, resulting in two separate search models. In addition, the structure of the BC2 nanobody (PDB: 5IVO) was used as a search model for VHH-2 and VHH-6. Manual model building and refinement were performed in Coot and Phenix, respectively. The crystal structure was validated by the MolProbity server. The structural diagram was prepared using PyMOL (The PyMOL Molecular Graphics System, Version 2.0 Schrodinger, LLC). Interacting residues at the protein interface were determined with PDBePISA (https: / / www.ebi.ac.uk / pdbe / pisa / ). Intermolecular salt bridges and hydrogen bonds are indicated by dashed lines, and all residues whose buried surface area contributes to the interface >10 Å are labeled. The structural diagram was prepared using PyMOL (The PyMOL Molecular Graphics System, Version 2.0 Schrodinger, LLC).

[0138] Surface plasmon resonance Surface plasmon resonance experiments were performed using a Biacore 8K instrument (GE Healthcare). The flow system was cleaned using the "Desorb" function of the maintenance kit (Desorb Kit, GE Healthcare). The system was rinsed with running buffer (20 mM HEPES (pH 8.0), 200 mM NaCl, 5 mM DTT, 0.05% Tween 20), and all steps were performed at a chip temperature of 25 °C. Chemically biotinylated GSDMD at a concentration of 100 nM was immobilized on flow cell 2 of a Series S Sensor Chip CAP for 180 s using a biotin capture kit and a flow rate of 30 μl / min. The system was washed for 600 s with running buffer at a flow rate of 30 μl / min. Binding affinity was determined using multicycle kinetics. To account for different binding affinities, nanobodies were injected at various concentrations (VHH-1, VHH-2, VHH-3, VHH-5: 0.5–32 nM, VHH-4 and VHH-6: 64–4096 nM) at a flow rate of 30 μl / min. The association step lasted 120 s and the dissociation step lasted 300 s.

[0139] For epitope binning experiments, nanobodies were tested in pairs for competitive binding. The first analyte was injected at a concentration of 128 nM for 120 seconds at a flow rate of 10 μl / min. This step was followed by a 60-second dissociation step. A mixture of the first and second analytes (both at 128 nM) was then injected at a flow rate of 10 μl / min for 120 seconds, followed by a 30-second dissociation step. After each cycle, the surface was regenerated for 120 seconds using the regeneration solution from the capture kit at a flow rate of 10 μl / min. Data were referenced by subtracting a blank cycle (no analyte injected) and the reference flow cell (flow cell 1). Data were analyzed using Biacore Insight Evaluation Software. Dissociation constants were determined based on fits using a 1:1 binding model.

[0140] Liposome leakage assay Lipids for LUV production were obtained from Avanti Polar Lipids and dissolved in chloroform to a final concentration of 25 mg / ml. Liposomes were generated by mixing 80 μl of phosphatidylcholine (POPC), 128 μl of phosphatidylethanolamine (POPE), and 64 μl of cardiolipin in a glass tube. The chloroform was evaporated under a steady stream of nitrogen, and the lipids were rehydrated in 400 μl of an 80 mM calcein solution in HO (pH 7.0). The liposome suspension was thoroughly vortexed and subjected to five freeze-thaw cycles, followed by 31 extrusions through a 100 nm pore size polycarbonate membrane using an Avanti mini-extruder (Avanti Polar Lipids, Inc., Alabaster, AL). The extruded liposomes were passed through a PD-10 column equilibrated with 20 mM HEPES (pH 7.4), 150 mM NaCl, and 1 mM EDTA to remove excess calcein. For this purpose, 100 μl of liposomes were loaded onto the column, and 200 μl elution fractions were collected. The homogeneity and quality of the resulting liposomes were controlled using DLS and evaluation of packaging by measuring fluorescence at 525 nm after dissolution with 1% Triton-X 100, respectively. Fractions containing liposomes of good quality were pooled and diluted 1:10 in buffer.

[0141] For the liposome leakage assay, 120 μl of liposome solution, 0.5 μM GSDMD, 0.2 μM His-SUMO-caspase-4, and 0.5 μM VHH were mixed in a final volume of 200 μl in a dark-well glass-bottom plate and incubated for 180 min at 37° C. In a second experiment, 120 μl of liposome solution, 0.5 μM GSDMD-3C, 0.2 μM 3C-protease (homemade), and 0.5 μM VHH were mixed in a final volume of 200 μl in a dark-well glass-bottom plate and incubated for 45 min at 37° C. Fluorescence emitted at 525 nm upon excitation at 485 nm was measured every minute using a plate reader.

[0142] Thermal shift assay NanoDSF was used to determine the effect of GSDMD-targeting nanobodies on the thermal stability of GSDMD. Samples containing various concentrations of protein were loaded into glass capillaries and applied to a nanoDSF instrument, Prometheus NT.48 (Nanotemper). The samples were heated from 20°C to 90°C at a gradient of 1.5°C / min, and protein unfolding was monitored by detecting shifts in fluorescence at 330 nm and 350 nm. Data were analyzed using Nanotemper PR.ThermControl software.

[0143] Multi-angle light scattering (MALS) GSDMD-VHH GSDMD-2 -VHH GSDMD-6 For SEC-MALS analysis, GSDMD Δ184-194 / Δ247-272 ,VHH GSDMD-2 , VHH GSDMD-6 , or mixed with both nanobodies at equimolar concentrations (188.8 μM) and injected onto a Superose 6 10 / 300 GL column equilibrated with GSDMD-SEC buffer. The chromatographic system was equipped with a triangular light scattering detector 380 (miniDAWN, Wyatt) and a refractive index detector (Optilab T-rEX, Wyatt). Data were collected every 0.5 seconds at a flow rate of 0.5 ml / min and analyzed using ASTRA V software (Wyatt).

[0144] Caspase cleavage assay Recombinant GSDMD (15 μM) was incubated with an equimolar amount of VHH GSDMD-1 or VHH GSDMD-2 and caspase-4 (6 μM) for 4 h at 37° C. GSDMD cleavage by caspase-4 was analyzed by SDS-PAGE at the indicated time points.

[0145] Inflammasome activation To induce the human NLRC4 inflammasome, recombinantly purified Shigella flexneri needle protein MxiH fused to Bacillus anthracis LFn (LFn-MxiH, 0.1 μg / mL) was delivered into the cytosol for 1 hour using 1.0 μg / mL Bacillus anthracis protective antigen (PA), as described in the art. MxiH binds to human NAIP, which then initiates NLRC4 oligomerization. NLRP3 is an indirect sensor of potassium efflux and perturbations in intracellular homeostasis. To stimulate NLRP3, cells were primed with 200 ng / mL ultrapure LPS for 3 hours, and NLRP3 was activated by the addition of 10 μM nigericin (Nig), a potassium ionophore derived from Streptomyces hygroscopicus, for 1 hour. Where indicated, caspase-1 activity was inhibited with 40 μM VX-765 or NLRP3 was inhibited with 2.5 μM CRID3 for 30 min before and during stimulation.

[0146] Quantification of cell death by CellTiter-Blue assay (reducing activity) The CellTiter-Blue® (CTB) assay was performed using CellTiter-Blue® reagent (Promega) according to the manufacturer's instructions to determine the reducing capacity, and therefore the viability, of untreated or stimulated cells. Cells in 96-well plates were treated as for the LDH assay. The supernatant was aspirated and replaced with 100 μL of CTB reagent, followed by incubation at 37° C. for 1 hour. Samples were excited with light at a wavelength of 560 nm, and fluorescence was measured at 585 nm using a SpectraMax i3 instrument.

[0147] Quantification of expression levels, inflammasome assembly, and caspase-3 cleavage by flow cytometry Transduction of primary human M-CSF macrophages with lentivirus encoding the C1C-EGFP inflammasome reporter and VHH-HA was assessed by quantifying the percentage of C1C-EGFP-positive cells by flow cytometry. To estimate the total number of intact cells per sample, cells were treated identically, resuspended in the same volume, and measured by flow cytometry for a fixed time period of 30 seconds. The reduction in cells per volume served as an indirect indicator of pyroptotic cell death. Caspase-1 CARD -EGFP (C1C-EGFP) is caspase-1 CARD and ASC CARD This study recapitulates the recruitment of unprocessed caspase-1 to nascent ASC specks through homotypic interactions between C1C-EGFP and C1C-EGFP, and can therefore be used as a fluorescent reporter of ASC specks and, therefore, inflammasome assembly. To quantify C1C-EGFP specks as a surrogate for inflammasome assembly, we exploited the unique redistribution of EGFP fluorescence from the cytosol to speck-bound forms, resulting in distinct cell populations exhibiting higher fluorescence intensity, EGFP(H), and a narrower width of the fluorescent signal, EGFP(W). We first gated on C1C-EGFP-positive cells [EGFP(A)] and then plotted the height versus width of the C1C-EGFP signal. For these experiments, 1·10 cells in 24 wells were used. 5Transduced primary macrophages or PMA-differentiated THP-1 derivatives were stimulated as described above. Cells were harvested by trypsinization, fixed in 4% formaldehyde, and analyzed using a BD FACSCanto flow cytometer. To quantify NLRP3 and NLRC4 inflammasome assembly in the presence of cytosolic VHH-EGFP fusions, PMA-differentiated THP-1 macrophages expressing both VHH-EGFP and C1C-mCherry were stimulated as described above. To prevent loss of responder cells due to caspase-1-dependent pyroptosis, cells were stimulated in the presence of 40 μM VX. The percentage of speckled C1C-mCherry-positive cells was measured using a BD LSRFortessa SORP flow cytometer. Experiments were also performed in the absence of VX, revealing that pyroptotic cells were lost during sample processing, whereas untreated and apoptotic cells could be analyzed by flow cytometry. To measure cleaved caspase-3 in PMA-differentiated THP-1 macrophages, experiments were performed as described for apoptotic cell death. 3 10 cells were cultured in wells of a 24-well plate. 5 THP-1 macrophages were treated with 1.0 μg / mL recombinant Bacillus anthracis PA and 0.1 μg / mL LFn-MxiH for 1 hour in the presence of 40 μM VX where indicated. Staurosporine, a nonselective inhibitor of several kinases, was added for 20 hours as a positive control for intrinsic apoptosis and caspase-3 activation. After fixation, cells were stained with rabbit anti-cleaved caspase-3 primary antibody (1:2000) and goat anti-rabbit Alexa Fluor Plus 647-conjugated secondary antibody (1:500). The percentage of cells positive for cleaved caspase-3 was measured using a BD LSRFortessa SORP flow cytometer. All flow cytometry data were analyzed using FlowJo 10.7.1 software.

[0148] Confocal microscopy For live-cell confocal microscopy experiments, PMA-differentiated THP-1 cells or GM-CSF-differentiated primary human macrophages were plated in 15 μl slides, 8-well Ibidi chambers (9·10 4 cells), or black clear-bottom TC-treated PhenoPlate™ 96-well microscopy plates (Perkin Elmer) (2–5 10 4 Cells were cultured in a 1000-well plate (Cells). Where indicated, cells were stained with CellMask™ Orange Plasma membrane stain (1:10,000, Thermo Fisher Scientific) for 10 minutes at 37°C, followed by three washes with Opti-MEM. NLRC4 inflammasomes were activated with PA and LFn-MxiH for 1 hour in imaging medium (RPMI containing 10% FBS, 50 μM 2-mercaptoethanol, 30 mM HEPES, and no phenol red) at the concentrations described above. To stain endogenous proteins in microscopy samples, cells were seeded and treated as described above and fixed in 4% formaldehyde in PBS for 20 minutes; where indicated, cells were stained with 5 μg / mL WGA AF647 and fixed again. To stain intracellular proteins, cells were permeabilized with 0.5% Triton X-100 and stained with Hoechst 33342 (Thermo Fisher Scientific) and rabbit anti-cleaved GSDMD in PBS, as indicated. NTThe cells were stained with either TOM20 antibody (1:500) + goat anti-rabbit IgG AF488 (1:1000) or mouse anti-TOM20 antibody (1:500) + goat anti-mouse IgG AF647 (1:1000) + 10% goat serum. Most images were recorded with a HC PL APO CS2 63x / 1.20NA water objective on a Leica SP8 Lightning confocal microscope. Images in Figure 4G and Figures 26A, 32, A–C were recorded with a HC PL APO CS2 63x / 1.20NA water objective on a Leica Stellaris 8 microscope. HEK293T cells constitutively expressing VHH-EGFP fusions were seeded (9-10 per well) into Ibidi chambers coated with poly-L-lysine (molecular weight 70,000–150,000) (Sigma-Aldrich). 4 cells). Among them, GSDMD and GSDMD NT In the first experiment, cells were transiently transfected with expression vectors for the variants fused with mCherry. NT To facilitate the observation of GSDMD, we used the attenuated GSDMD mutant I104N. Because the mutant behaved almost like WT GSDMD in our assays, WT GSDMD will be used in subsequent experiments. Where indicated, cells were cotransfected with an expression vector for emiRFP670 with a C-terminal CAAX motif (emiRFP670-CAAX). emi-RFP670-CAAX is prenylated to tether the fluorescent protein to the plasma membrane, allowing for assessment of membrane localization. Five hours after transfection, images were recorded at least every 10 minutes using a HC PL APO CS2 63x1.20 water objective on a Leica SP8 Lightning confocal microscope (37°C, 5% CO2). Alternatively, cells were fixed 12 hours after transfection, DNA was stained, and images were recorded using the same microscopy settings.

[0149] Image analysis Images were processed using ImageJ 2.3.0 software. To quantify the influx of fluorescent nanobodies, the cell detection tool in Imaris (Bitplane) was used to detect cellular regions containing mostly accurately segmented cells, but occasionally clusters of cells, using the CMO channel (detection of cells without nuclear or vesicular staining; cell type = membrane; cell minimum diameter = 12 μm; membrane detail = 1 μm; cell filter type = local contrast; intensity manual threshold = 4; quality manual threshold = 0.090; filtering objects between 120 and 10,000 voxels). Regions, and VHHs were analyzed. NP-1 The fluorescence intensity in the -AF647 channel was extracted. NP-1 If the mean intensity in the -AF647 channel was greater than 80, the cell area was scored as VHH+. The percentage of VHH+ cell area within the total cell area was calculated and plotted. SYTOX green-positive nuclei were detected with the spot detection tool in Imaris (estimated diameter = 8 μm, quality > 10). Cells with distinct morphological features or fluorescence distribution were counted manually with the help of the counting function in Imaris. Fluorescent GSDMD NT To quantify the plasma membrane (PM) distribution of the fusions, fluorescence intensity cross sections along a line cutting the cell were analyzed for each cell. If the fluorescence colocalized only with plasma membrane markers, the localization was classified as "PM." If the fluorescence was found only in the cytosol and dropped off at the plasma membrane, the localization was counted as "cytosol." If fluorescence above background was found in the cytosol but fluorescence was still elevated at the PM, the localization was scored as "cytosol + PM." When quantifying the distribution of GSDMD-mNG_ins, "clear plasma membrane localization" was defined as follows: upon analysis of the intensity cross sections, the cell exhibited a clear plasma membrane signal (a clear peak of fluorescence at the edge of the cell, i.e., the fluorescence appears as a relatively thin line), which required that the focal plane traverse the body of the cell and not at the bottom or top of the cell; and B) GSDMD NT -mNG showed an equally sharp fluorescence peak that colocalized with the plasma membrane staining (as evident in the intensity plot; GSDMD NTThis distinction was primarily used to exclude false positives, i.e., cells cut off at the top or bottom by the focal plane (no obvious ring-like signal of the plasma membrane marker), or cells with a large nucleus in the focal plane with little adjacent cytoplasm. In the latter case, GSDM NT Although GSDMD-mNG staining also occasionally appeared as a ring, the fluorescence was observed at a broader rim with a less rapid increase and without colocalization with the plasma membrane. Notably, the apparently low percentage of responding cells can be explained by a) membrane staining perpendicular to the focal plane and b) a lower number of cells with sufficient GSDMD-mNG or endogenous GSDMD expression and cleavage. SYTOX green intensity in Incucyte experiments was analyzed with Incucyte software to extract integrated fluorescence per field. To quantify SYTOX green intensity per nucleus, nuclei were detected in CellProfiler using the "IdentifyPrimaryObjects" function (default settings, minimum radius = 10 pixel units, maximum radius = 40 pixel units), and the mean fluorescence intensity was extracted with the "MeasureObjectIntensity" function. From a z-stack (5 slices, 2 μm intervals), VHH ASC To quantify the localization of -AF647 to C1C-EGFP specks or nuclei, we first detected both structures using the Imaris spot detection routine (specks: C1C-EGFP channel, estimated diameter = 2 μm, quality > 10; nuclei: Hoechst channel, estimated diameter = 8 μm, quality > 1) and located the central VHH ASC - C1C-EGFP specks with AF647 intensity > 65 and average VHH ASC Nuclei with AF647 intensity >100 were counted. ASC The percentage of AF647-positive nuclei was calculated using the VHH (to correct for the variable background of cells that were C1C-EGFP negative but PI- / VHH positive before treatment). NP-1 and corrected by the percentage of positive cells observed in cells stimulated in the presence of VX.

[0150] Example 2 – Identification of GSDMD-specific single domain antibodies Specific inhibitors of GSDMD are scarce, and other than complete knockouts and overexpressed point mutants, no tools are available to perturb GSDMD and study pore formation in molecular detail in living cells. To overcome these drawbacks, single-domain antibodies (also referred to herein as nanobodies or VHHs) were raised against the human GSDMD protein. Alpacas (Vicugna pacos) were immunized with bacterially expressed recombinant full-length GSDMD protein. Using phage display, we positively selected GSDMD-specific VHHs, yielding six hits that differ substantially in their complementarity-determining regions (CDRs) (Figure 1A, B). ELISA experiments at decreasing concentrations confirmed their specificity for GSDMD (Figure 1C). To test the functionality of six GSDMD-specific nanobodies in the cytosol of live cells, a LUMIER assay was performed: HEK 293T cells were co-transfected with expression vectors for HA-tagged nanobodies (VHH-HA) and various variants of GSDMD fused to Renilla luciferase (GSDMD-Renilla). The HA-tagged nanobodies were then immunoprecipitated from the lysates. If the nanobodies bind to GSDMD in cells, GSDMD and luciferase activity are co-immunoprecipitated, resulting in a luminescent signal after the addition of the Renilla luciferase substrate coelenterazine. This allows for the expression of VHHs in the cytosol. GSDMD-2 and VHH GSDMD-3 Robust GSDMD binding of VHHs, as well as VHHs GSDMD-1 and VHH GSDMD-5 To determine the domain of GSDMD to which the nanobody binds, we performed a 100-kDa antibody assay using the GSDMD nanobody. NT and GSDMD CT A fusion of GSDMD to Renilla luciferase was also included. NTOverexpression of the full-length and N-terminal domains of GSDMD mutant 4A, which are no longer membrane bound and do not induce pyroptosis, alone kills cells by pyroptosis. GSDMD-1 and VHH GSDMD-2 clearly binds to the N-terminal domain of GSDMD, whereas VHH GSDMD-3 and VHH GSDMD-5 The binding of β-glucan was affected by the 4A mutations, making clear conclusions impossible.

[0151] Example 3 - VHH GSDMD-1 and VHH GSDMD-2 suppresses pyroptosis We next investigated whether the identified nanobodies perturbed GSDMD function when expressed in cells. NT To test the effect of nanobodies on inducible LDH release, HEK 293T cells were transfected with GSDMD NT and the expression vectors for the indicated GSDMD nanobodies. GSDMD-1 , and to some extent VHH GSDMD-2 is GSDMD NT In HEK293T cells overexpressing α-glucan, LDH release was inhibited, but GSDME NT In HEK293T cells overexpressing VHH, the inhibition of cell death was not observed (Figure 21A), whereas the control nanobody and other GSDMD nanobodies had no effect on cell death, which is consistent with the results of the VHH GSDMD-1 and VHH GSDMD-2 These results suggest that VHHs can inhibit pyroptosis (Figure 2A). To confirm this in a more relevant cell type, we generated a human myeloid THP-1 cell line that constitutively expresses HA-tagged nanobodies under the strong EF1α promoter. GSDMD-3 was not expressed sufficiently in these cell lines and was therefore excluded from further analysis (Figure 7A). NP-1 ) were used as a negative control, while VHH ASC is ASCCARD This served as a positive control because it interferes with inflammasome formation and IL-1β release by impairing the interaction. THP-1 cells were differentiated into macrophages with PMA and activated with either the Shigella needle protein MxiH delivered with an anthrax toxin delivery system to induce NLRC4 inflammasome activation, or LPS and nigericin to activate the NLRP3 inflammasome. Robust LDH and IL-1β release was observed in the control cell line, and responses to both triggers were suppressed by the caspase-1 inhibitor VX-765 (VX), confirming their complete dependence on caspase-1. Responses to LPS and nigericin treatment were mediated by NLRP3, as demonstrated by sensitivity to the NLRP3 inhibitor CRID3 (Figure 2B-E). Remarkably, VHH GSDMD-1 and VHH GSDMD-2 Both VHHs completely blocked LDH (Figure 2B, C) and IL-1β (Figure 2D, E) release to background levels following activation of the NLRC4 and NLRP3 inflammasomes. Inflammasomes were then activated and followed by microscopy, and plasma membrane permeability was monitored over time by quantifying uptake of DRAQ7, a membrane-impermeable, far-red fluorescent DNA dye. While DRAQ7 uptake was detected within 1 h of treatment in almost all control cells, VHHs GSDMD-1 and VHH GSDMD-2 In THP-1 macrophages expressing VHH, no uptake of DRAQ7 was evident (Fig. 2F, G). Consistent with these findings, cells expressing antagonistic nanobodies, in contrast to negative controls, do not exhibit any hallmarks of pyroptotic cell death (Fig. 2F). GSDMD-1 and VHH GSDMD-2 It was concluded that cytosolic expression of completely suppresses GSDMD-mediated effector function.

[0152] Next, we also investigated the functionality of the inhibitory GSDMD nanobody in primary human M-CSF- and GM-CSF-differentiated macrophages. To this end, we transfected primary human macrophages with our previously described fluorescent inflammasome reporter, caspase-1, which is recruited to ASC specks via the caspase recruitment domain (CARD) of caspase-1. CARD Macrophages were transduced with lentivirus encoding a nanobody of interest in addition to α-EGFP (C1C-EGFP) (Figure 2H). To overcome SAMHD1 restriction in macrophages, lentivirus was produced in cells expressing a fusion protein of SIVmac251 Vpx and HIV-1 NL4.3 Vpr. When Vpx-Vpr was packaged into lentiviral particles, Vpr bound to the structural protein Gag, thus delivering Vpx into target cells, which mediates Cullin-4a-mediated proteasomal degradation of SAMHD1. This resulted in transduction efficiencies of approximately 10-30% as measured by flow cytometry (Figure 2I, Figure 7C). Upon treatment with MxiH, an NLRC4 activation trigger, a strong reduction in cell number was observed, as pyroptotic cells were too fragile to survive processing for flow cytometry (Figure 2I, Figure 7B). At the same time, antagonistic VHHs were transfected so that EGFP-positive cells accounted for 60–70% of the single cell population at this time point. GSDMD-1 or VHH GSDMD-2 Macrophages expressing C1C-EGFP and C1C-EGFP preferentially survived (Figure 2J, Figure 7C), whereas macrophages expressing the control nanobody VHH NP-1 It was observed that cells expressing antagonistic VHHs provided no survival advantage, suggesting that only cells expressing antagonistic VHHs were able to survive the lethal trigger and therefore VHHs GSDMD-1 and VHH GSDMD-2We show that VHH prevents pyroptosis in primary human macrophages. To verify that MxiH treatment induced inflammasome assembly, we detected the recruitment of C1C-EGFP to ASC specks by measuring the width and height of the C1C-EGFP signal by flow cytometry. Robust inflammasome activation was observed in the majority of MxiH-treated cells that survived NLRC4 activation by expression of the antagonistic GSDMD nanobody, indicating that surviving cells responded to MxiH and that survival was not due to any impairment of inflammasome assembly (Figure 2K, Figure 7D). Altogether, these results demonstrate the role of VHH in both THP-1 and primary human macrophages upon NLRP3 and NLRC4 inflammasome activation. GSDMD-1 and VHH GSDMD-2 The results show a strong inhibitory effect of pyroptosis on the cytosol.

[0153] Example 4 - Antagonistic nanobodies prevent oligomerization but not GSDMD NT still allows membrane localization of Next, we sought to determine the mechanism by which the GSDMD nanobody inhibits pyroptosis. To exclude any effect on inflammasome assembly, we analyzed ASC speck formation in THP-1 macrophages in which various VHH-EGFP fusions, as well as the C1C-mCherry inflammasome reporter, were inducibly expressed. Flow cytometry analysis upon MxiH delivery (in the presence of VX to prevent pyroptosis and thus cell loss) or stimulation with LPS and nigericin demonstrated that the nanobody did not affect ASC speck formation (Figures 3A and 8A).

[0154] Next, VHH GSDMD-1 or VHH GSDMD-2We analyzed whether binding of MxiH altered GSDMD levels or its processing by caspase-1. Lysates from MxiH-treated THP-1 macrophages were separated by SDS-PAGE under reducing conditions and analyzed by immunoblotting. NT No difference was detected in the cleavage to produce (Fig. 3B).

[0155] It has previously been shown that GSDMD oligomers appear as high molecular weight bands under non-reducing conditions. Therefore, THP1 cell lysates were analyzed after SDS-PAGE in the absence of DTT. In wt THP-1 cells and cells expressing a control nanobody, the formation of dimers and higher-order oligomers could be observed in the absence of DTT, whereas VHH GSDMD-1 or VHH GSDMD-2 The nanobody was no longer present in lysates from cells expressing GSDMD (Figure 3C). NT This demonstrates that the oligomerization of

[0156] Antagonistic nanobodies inhibit the formation of monomeric GSDMD by preventing oligomerization NT With a stabilized system now established, it became possible to determine whether monomeric protein is sufficient for membrane localization or whether oligomerization is a prerequisite. Therefore, we transfected HEK 293T cells stably expressing a VHH-EGFP fusion with either full-length GSDMD-mCherry I104N or GSDMD. NT We transfected cells with an expression vector for GSDMD-mCherry I104N and followed its localization by live-cell confocal microscopy. The I104N mutant has been reported to be less active, and therefore favors the detection of GSDMD pores before cells die by pyroptosis. The GSDMD-mCherry signal was uniformly distributed in the cytosol regardless of the nanobody expressed, confirming that full-length GSDMD did not insert into the plasma membrane as expected (Figure 3D). NT -mCherry I104N, a control nanobody VHHNP-1 When GSDMD was expressed in HEK 293T cells expressing GSDMD, most cells appeared to be pyroptotic, but fluorescence was observed to be found almost entirely in internal structures or organelles in pyroptotic cells. NT This suggests that GSDMDs do not accumulate at the plasma membrane and are rapidly removed, for example, by membrane repair processes involving shedding of membrane vesicles. As was speculated earlier in the art, some GSDMDs NT It cannot be excluded that GSDMD is also recruited to other cell membranes. NT In cells, VHH GSDMD-1 or VHH GSDMD-2 When co-expressed with GSDMD NT -mCherry partitioned almost entirely into the plasma membrane, where it colocalized with VHH-EGFP (Figure 3D). Cells no longer showed signs of pyroptosis, consistent with the expected inhibition of GSDMD pore formation. Similar results were observed with wt GSDMD. NT -mCherry. Therefore, monomeric GSDMD NT can insert into the plasma membrane, indicating that the conformational changes required for membrane integration occur in monomeric GSDMD after removal of the autoinhibitory C-terminus. NT However, oligomerization only occurs after insertion into the plasma membrane, and therefore oligomeric GSDMD does not insert into the membrane. NT This strongly suggests that it is most likely not necessary to go through a prepore state composed of GSDMD. From these observations, two additional important conclusions are possible: 1) GSDMD NT is localized almost exclusively to the plasma membrane, making it less likely that insertion into other organelles, such as mitochondria, contributes to pyroptosis. NT -mCherry GSDMD NTInternal structures were only observed when GSDMD was able to form pores, i.e., in cells with a control nanobody, suggesting that the observed structures result from internalization of the GSDMD pore and, therefore, an endocytic repair mechanism that removes the GSDMD pore.

[0157] We attempted to visualize GSDMD pores in pyroptotic cells after inflammasome activation in THP-1 cells inducibly expressing GSDMD with an mNeonGreen insertion after amino acid 270 (GSDMD-mNeonGreen_ins), but were unsuccessful. NT Insertion of a fluorescent protein between GSDMD and the caspase-1 cleavage site inhibits GSDMD after caspase-1 cleavage. NT -mNeonGreen is produced. GSDMD NT GSDMD generated by caspase-1 cleavage, similar to our observations in HEK 293T cells expressing -mCherry NT The majority of -mNeonGreen localized to internal structures but not to the plasma membrane. GSDMD-1 and VHH GSDMD-2 However, GSDMD was expressed at the plasma membrane in HEK 293T cells. NT Given that they stabilized the monomer, antagonistic nanobodies also inhibited cleaved GSDMD after inflammasome-mediated caspase-1 activation in relevant cell types. NT Therefore, we investigated whether GSDMD-mNeonGreen_ins could stabilize the VHH. GSDMD-1 -HA or VHH GSDMD-2 We generated THP-1 cells expressing HA. When inflammasome activation was induced in these cells, GSDMD was detected at the plasma membrane of the cells, which did not exhibit the morphological characteristics of pyroptosis. NT -mNeonGreen was observed (Figure 3E), indicating that GSDMD was released by cleavage of full-length GSDMD. NTIt was also confirmed that the GSDMD nanobody inserted into the plasma membrane as a monomer before oligomerizing to form a pore. NT Interfering with oligomerization of GSDMD, leaving its cleavage and membrane localization intact in living cells, has important implications for the mechanism of pore formation: monomeric GSDMD NT We propose that β-glucanase can directly insert into plasma membranes without the need for prior oligomerization, constructing pores in target membranes monomer by monomer.

[0158] Example 5 – Inhibition of pore formation by antagonistic GSDMD nanobodies enhances caspase-1 activity and induces caspase-1-dependent apoptosis When THP-1 macrophages expressing various VHH-EGFP fusions in combination with the C1C-mCherry inflammasome reporter were analyzed in more detail, VHHs were significantly inhibited, as indicated by ASC speck formation 1 hour after treatment. GSDMD-1 and VHH GSDMD-2 Inflammasome assembly was observed in the presence of VHH (Figure 4A). Interestingly, cells bearing ASC specks exhibited blebs or were fragmented into multiple vesicular fragments, a morphology more typically associated with apoptosis. However, in the presence of VHH NP-1Control-expressing cells bearing ASC specks were rounded with a "balloon-like" morphology, as expected for cells undergoing pyroptosis (Figure 4A). Similar apoptotic morphology could be observed in primary human macrophages transduced with the antagonistic GSDMD nanobody and C1C-EGFP as described above (Figure 9A, Figure 2H). Although ASC specks were observed by microscopy in pyroptotic THP-1 cells expressing the control nanobody, no speckled cells were detected when treated cells were trypsinized and analyzed by flow cytometry (Figure 4B, Figure 9B). This confirms that pyroptotic cells rupture during processing. Notably, caspase-1 is typically inhibited with VX, allowing quantification of inflammasome assembly by flow cytometry. In contrast, when cells expressing antagonistic GSDMD nanobodies were treated with inflammasome activators, ASC specks were readily detected by flow cytometry in approximately 60% of NLRC4-activated cells and 25% of NLRP3-activated cells, demonstrating that ASC speck assembly in apoptotic cells can be analyzed by flow cytometry (Figures 4B and 9B). ASC THP-1 macrophages expressing VHH do not assemble ASC specks, as previously shown in the art. GSDMD-1 or VHH GSDMD-2 This is another indication that cells in the presence of VHH do not die by pyroptosis despite inflammasome activation. To investigate true apoptosis, various PMA-differentiated THP-1 cell lines were next stained for cleaved caspase-3 upon MxiH treatment, and the percentage of cells positive for cleaved caspase-3 was quantified by flow cytometry. Staurosporine treatment was used as a positive control and resulted in over 60% of cells positive for cleaved caspase-3 (Figure 4C). GSDMD-1 Expressing cells and VHHs GSDMD-2Both VX-expressing cells showed a clear population of cells positive for cleaved caspase-3, indicating the presence of this apoptotic effector caspase (Figures 4C and 4D, 9C). Interestingly, caspase-3 activation appeared to be caspase-1 dependent, as it was strongly reduced in the presence of VX. Direct activation of caspase-8 by recruitment and autoproteolytic activation on ASC specks was reported earlier in the art. Because caspase-3 activation was largely blocked by VX, caspase-1-independent activation of caspase-8 does not appear to contribute significantly to caspase-3 activation. However, the remaining proportion of cells positive for cleaved caspase-3 after VX treatment may result from direct activation of caspase-8 on ASC specks. Consistent with this interpretation, VX-expressing cells expressing VHHs that prevent ASC speck formation. ASC When NLRC4-stimulated inflammasomes were expressed, residual caspase-3 activation was no longer observed. Flow cytometry analysis of caspase-3 cleavage in ΔASC THP-1 cells expressing various VHHs also demonstrated a complete dependence on ASC speck formation (Fig. 9D, E). Theoretically, caspase-1 activation in NLRC4-stimulated inflammasomes could be mediated by caspase-1. CARD and NLRC4 CARD Although direct interaction of VHH with caspase-3 may be ASC-independent, the lack of LDH release in ΔASC THP-1 macrophages makes ASC-independent activation of caspase-1 unlikely in our setting (Figure 9F). Flow cytometry does not allow for the measurement of pyroptotic cells, and therefore, flow cytometry-based analysis may not detect cleaved caspase-3 in control cell lines that still undergo pyroptosis. Therefore, to measure caspase-3 activity independently of cell death or rupture, we performed a caspase Glo assay to measure caspase-3 / 7 activity in THP-1 macrophages upon MxiH treatment. Notably, caspase activity is measured in samples derived from cells and supernatants. Strong caspase-3 / 7 activity was observed in VHHs. GSDMD-1 and VHHGSDMD-2 This was observed in MxiH-treated cells expressing VHH but not in cells expressing a control nanobody. Again, this activity was completely dependent on ASC, as no caspase-3 / 7 activity was observed in ASC knockout cells (Figure 4D). Analysis of cell lysates by immunoblotting confirmed the cleavage of caspase-3, caspase-7, and the caspase-3 substrates PARP and GSDME only in samples with caspase-3 activity, i.e., cells in which the antagonistic GSDMD nanobody prevented pore formation (Figure 4E). Notably, cleavage of GSDMDE in cells expressing the antagonistic GSDMD nanobody does not appear to be sufficient to construct functional GSDME pores, as no pyroptosis, IL-1β release, or DRAQ7 uptake was observed (Figure 2C, E, F). Thus, GSDME is involved in the regulation of VHH activity. GSDMD Caspase-3 activation also plays a major role in the death of GSDMD-expressing cells. p20 This explains the presence of the band (Figure 3B), because the addition of a caspase-3 / 7 inhibitor prevents the formation of this cleavage product (Figure 4F). p20 VHH GSDMD-1 VHHs are formed only in cell lines expressing VHHs. GSDMD-2 This is likely the result of differences in VHHs masking different epitopes on GSDMD. GSDMD-1 Expression THP-1 macrophages and VHH GSDMD-2In addition to the presence of cleaved caspase-3 in THP-1 macrophages expressing caspase-3, processed caspase-8, processed caspase-9, and cleaved tBID were also detected in those samples with caspase-3 activity (Figure 4G, Figure 9G,H), indicating that both the intrinsic and extrinsic apoptotic pathways, or a feedback mechanism, may be involved. Theoretically, caspase-1, caspase-8, and caspase-9 can all catalyze the cleavage of caspase-3. However, caspase-3 activity was found to appear to be dependent on caspase-1 and ASC, suggesting that inflammasome-activated caspase-1 appears to be a key regulator of the alternative cell death program. Consistently, caspase-8 and caspase-9 activity was found to be highly dependent on caspase-1 activity and inflammasome formation, as their cleavage was strongly reduced in the presence of VX and in ΔASC THP-1 macrophages (Figure 4H, I). Only for caspase-8, there was some residual processing visible even in the absence of GSDMD VHH (Figure 4H). However, this caspase-8 activation was completely ASC-dependent, as it was absent in THP-1 ΔASC cells (Figure 4I). This suggests that a small fraction of caspase-8 is cleaved at the ASC speck independently of caspase-1, as previously concluded in the art. Next, we quantified caspase-1 activity in THP-1 macrophages upon MxiH stimulation using the caspase-1 Glo assay. Surprisingly, caspase-1 activity was significantly reduced in THP-1 macrophages stimulated with MxiH, even in the absence of VHH. GSDMD-1 or VHH GSDMD-2 In the presence of VHH NP-1We found that the activity of caspase-1 in pyroptotic cells was increased by up to sixfold compared to cells expressing GSDMD pores (Figure 4J). This is noteworthy, as ASC speck assembly was comparable in all samples (Figure 8A). We therefore hypothesized that the ability to form GSDMD pores has a profound effect on caspase-1 activity, suggesting that GSDMD pores downregulate caspase-1 activity through a previously unexplored mechanism. Because the enhanced caspase-1 activity observed in the absence of GSDMD pores was sufficient to activate caspase-3 and apoptosis, caspase-1 ultimately serves as a master regulator of downstream cell death. One possible explanation for the relatively low caspase-1 activity in pyroptotic cells may be that, although cell supernatants were included in the analysis, ion flux through the GSDMD pore and / or dilution of cellular material into the supernatant compromises the stability or activity of caspase-1. Therefore, VHH GSDMD-1 or VHH GSDMD-2 THP-1 macrophages expressing MxiH were treated with perfringolysin O (PFO), a pore-forming toxin derived from Clostridium perfringens, which forms pores of 25-30 nm diameter, i.e., similar in size to, if not slightly larger than, GSDMD pores. To avoid additional activation of NLRP3 by potassium efflux through PFO pores, NLRC4 stimulation experiments were performed in the presence of the NLRP3 inhibitor CRID3. The enhancement of caspase-1 activity in the absence of GSDMD pores decreased with increasing concentrations of PFO (Figure 4L, Figure 6B), suggesting that caspase-1 activity depends on changes in the cell caused by pore formation. This indicates that some specific property of GSDMD pores impairs caspase-1 activity. In summary, we propose that the apoptosis observed in the absence of functional GSDMD pores is entirely inflammasome-dependent, with a central role for enhanced caspase-1 activity required for the processing of initiator and effector caspases.

[0159] Example 6 – Recombinant antagonistic GSDMD nanobodies inhibit pyroptosis when administered extracellularly Although GSDMD is a highly sought-after drug target, the development of specific GSDMD inhibitors has not been successful to date. For any therapeutic application of the potent pyroptosis-inhibiting nanobodies we discovered, delivery into the cytosol of target cells would be a prerequisite. Although nanobodies cannot cross intact cell membranes, we observed that fluorescent nanobodies could enter pyroptotic cells, likely through GSDMD pores. Therefore, we speculate that the early GSDMD pore would allow the passage of exogenously administered antagonistic GSDMD nanobodies. Therefore, we expressed and purified the identified GSDMD nanobodies in bacteria. Next, increasing concentrations of the nanobodies were added to the culture medium of THP-1 macrophages treated with MxiH as an NLRC4 inflammasome activator. A dose-dependent reduction in LDH release was observed, and importantly, higher concentrations of VHH GSDMD-1 and VHH GSDMD-2 In contrast, increasing amounts of VHH reduced LDH release to background levels. NP-1 Addition of 29% IL-1β did not affect LDH release (Figure 5A). IL-1β secretion was also substantially reduced, although the highest concentration did not completely suppress cytokine release (Figure 5B). To confirm these findings in a physiologically relevant in vitro model, we repeated the same experiment in primary human M-CSF macrophages (Figures 5C, D, 29C). LDH release was inhibited in a dose-dependent manner, and IL-1β secretion was reduced to background levels at the highest concentration. We hypothesize that the nanobody enters inflammasome-bearing cells at the time of the formation of the first GSDMD pore, prior to the lytic phase of pyroptosis. Thus, the cytosolic nanobody may prevent any further GSDMD pore assembly, which appears to be sufficient to allow cell survival. Fluorescent GSDMD NTBased on our experiments with fusions, early GSDMD pores are likely rapidly removed by membrane repair processes. Because cytokines can still be released through early, sublytic GSDMD pores, early pore formation may well explain remaining IL-1β secretion. Altogether, these results indicate that nanobodies, when administered extracellularly, are potent inhibitors of inflammasome-induced pyroptosis and therefore have interesting therapeutic potential. Importantly, early GSDMD pore formation may not be a terminal event, since cells can still be rescued by antagonistic GSDMD nanobodies.

[0160] Example 7 - Discussion of Results I GSDMD pore formation is an effector mechanism that mediates cell death by pyroptosis and the non-conventional secretion of IL-1β and IL-18. The function of GSDMD was previously demonstrated in a loss-of-function screen of seminal fluid, in which soluble full-length GSDMD and GSDMD NT The structure of the pore has been elucidated by structural biology. However, key molecular aspects of pore formation are unknown, primarily because pyroptotic cells cannot easily tolerate sample preparation for microscopy and flow cytometry, and the process cannot be easily studied in relevant cells. In this study, two GSDMD nanobodies, VHHs, GSDMD-1 and VHH GSDMD-2 These are the GSDMD NT potently inhibits pyroptosis by preventing oligomerization of GSDMD and thus monomeric GSDMD NT This stabilizes the pore formation step, making it amenable to molecular studies. Importantly, the monomeric GSDMD NTrevealed that GSDMD was still able to insert into the plasma membrane, thus demonstrating that cleavage of GSDMD is sufficient to mediate the conformational changes required for membrane insertion. This is therefore the first time that this step can be observed in (living) human cells, supporting the conclusion that pores can grow monomer-by-monomer in the plasma membrane. These results demonstrate that in artificial membranes, human GSDMD NT This is consistent with previous in vitro findings showing that smaller assemblies of GSDMDs can construct pores by transforming from arc-shaped to slit-shaped and finally ring-shaped assemblies. Similarly, atomistic molecular dynamics simulations have shown that small GSDMDs NT It was predicted that the assembly could already form ion-conducting membrane pores and provide a plausible route for pore drilling in intact biolayers. Earlier studies had shown that GsdmA, a ring-like assembly bound to the membrane in a globular conformation, resembles the conformation of the N-terminal domain in full-length gasdermin. NT or GSDMD NT proposed the formation of a GsdmA3 or GSDMD prepore composed of β-barrel subunits. This model implied a coordinated conformational change in all subunits that results in the final β-barrel structure that inserts into the plasma membrane. Our data suggest that even if oligomerization is prevented by nanobodies, GSDMD NT This suggests that the GsdmA3 monomer undergoes a conformational change that allows membrane insertion, suggesting that prepore assembly is not required for membrane insertion and is therefore unlikely to be important for pore formation. Recent atomic force microscopy data on GsdmA3 pores experimentally confirmed membrane penetration of growing pores with various morphologies in vitro. Mobile prepore-like assemblies were observed to attach to and disappear from the membrane, but none of them were observed to penetrate the membrane. Nevertheless, these findings do not completely exclude the existence of two distinct parallel pathways to pore formation.

[0161] Because inhibitory nanobodies stabilize GSDMD in a monomeric intermediate, they can serve as valuable tools to further elucidate the molecular details of membrane insertion and pore formation in relevant cell types using live-cell microscopy. Importantly, when oligomerization is inhibited, GSDMD NT Fluorescent fusions of GSDMD were found to insert almost exclusively into the plasma membrane, indicating that the plasma membrane is indeed the site of GSDMD. NT This demonstrates that GSDMD is the primary target membrane for the pore. Consequently, similar antagonistic nanobodies may reveal which membranes the N-termini of other GSDMD family members target. NT Internal fluorescent structures containing fluorescent fusions of GSDMD were only observed when pore formation was possible, i.e., in the absence of antagonistic nanobodies. Thus, stabilization of the monomers may allow for the formation of GSDMD before and after pore formation. NT Therefore, it becomes possible to distinguish the localization of GSDMD. NT The intracellular structures containing VHHs are the result of pore formation and very likely constitute GSDMD pores removed from the plasma membrane by an endocytic membrane repair process, suggesting that membrane shedding, as described for MLKL, is not the only mechanism for positioning GSDMD pores. The inhibitory GSDMD nanobodies also provided new insights into the interconnectedness of various cell death pathways in macrophages. GSDMD-1 or VHH GSDMD-2Despite the presence of fully cleaved endogenous GSDMD in cells expressing GSDMD, macrophages undergo apoptosis dependent on inflammasome assembly, ASC speckling, and caspase-1 activity, as previously reported for GSDMD KO cells and cells expressing catalytically inactive caspase-1. Importantly, we report for the first time that the absence of inflammasome activation in the absence of GSDMD pore formation strongly enhanced caspase-1 activity. Only this enhanced activity resulted in the efficient cleavage of caspase-3, caspase-7, and their substrates. Thus, we propose a key regulatory role for caspase-1 activity, which appears to be reduced upon GSDMD pore formation. The formation of similarly sized PFO pores, as observed in the presence of functional GSDMD pores, also appears to reduce caspase-1 activity, suggesting that caspase-1 activity is altered by ion flow or some other direct or indirect consequence of pore formation. It is possible that the most active cleaved form of caspase-1, the (p33 / p10)2 form, can be stabilized by preventing or delaying the secondary cleavage between CARD and p20, which is associated with loss of activity. GSDMD pore formation may also provide caspase-1 or other caspases with some undetected feedback signal that attenuates their activity, such as altered ion concentrations or changes in the physical properties of the cytosol, such as reduction potential. Interestingly, the apoptosis observed in our system followed kinetics comparable to pyroptosis, with MxiH-stimulated cells already exhibiting morphology of pyroptosis or apoptosis, respectively, within 20–30 min of treatment. Remarkably, despite efficient cleavage of GSDME in cells with inflammasomes assembled in the absence of the GSDMD pore, GSDME NT No pyroptosis mediated by GSDME was observed, confirming earlier findings suggesting that GSDME-induced lytic cell death does not play a major role in macrophages. In contrast, overexpression of GSDME in HEK 293T cellsNT , and GSDME cleaved by caspase-3 in keratinocytes is sufficient to initiate pyroptosis. NT However, for example, GSDMD, which has been proposed to require ROS for full activation, NT This suggests that inflammasomes may be subject to additional layers of regulation, as proposed for GSDMD. As the number of diseases in which inflammasomes and GSDMD play a detrimental role continues to grow, there is growing interest in specific GSDMD inhibitors. Therefore, antagonistic nanobodies, VHHs, GSDMD-1 and VHH GSDMD-2 Our proof-of-concept experiments highlight several intriguing therapeutic potentials. At the crossroads of intracellular signaling and DAMPs during pathogenic threats, targeting GSDMD would prevent inflammation not only upon canonical inflammasome stimulation but also upon non-canonical inflammasome stimulation. We were able to show that exogenous addition of nanobodies significantly reduced pyroptosis and the release of the pro-inflammatory cytokine IL-1β in both PMA-differentiated THP-1 macrophages and M-CSF-differentiated primary human macrophages. The nanobodies enter cells upon the formation of the first sublytic GSDMD pores and inhibit further GSDMD activation. NT We propose that this would preclude oligomerization, and thus pore formation and pyroptosis. One additional benefit of the therapeutic application of recombinant nanobodies is that the nanobodies in this scenario would be able to bind to the already assembled GSDMD. NTThe goal is to target only cells with pores, thus gaining access only to cells associated with the inflammatory response. The high specificity for both the target protein and the cellular state makes antagonistic GSDMD nanobodies of therapeutic interest. This is particularly interesting because other reported inhibitors are often cysteine-reactive compounds and therefore lack specificity. The possibility of tailoring nanobodies to bivalent or multivalent molecules may constitute a good basis for further optimization. Finally, nanobody-mediated survival of macrophages with cleaved GSDMD may rely on membrane repair processes, which can be studied in more detail using this system.

[0162] In conclusion, antagonistic GSDMD nanobodies are able to inhibit GSDMD NT We have shown that stabilizing an informative intermediate of the ATPase provides an unprecedented mode of intervention. This functional perturbation not only enabled mechanistic insight into membrane insertion, pore formation, and pore removal from the plasma membrane, but also provides an intriguing proof-of-concept for therapeutic applications of recombinant nanobodies.

[0163] Example 8 – Identification of GSDMD-specific single domain antibodies II GSDMD-targeting nanobodies were generated by immunization of alpacas with full-length recombinant human GSDMD protein. Positive hits from the sera were identified by enzyme-linked immunosorbent assay (ELISA) as described elsewhere. Potential binders varied by at least 7.6% in their amino acid sequences and showed great diversity in the length and composition of their complementarity-determining region 3 (CDR3) (Figure 10A, B). Nanobody binding was confirmed using surface plasmon resonance (SPR) spectroscopy, and binding affinity was determined by applying multi-cycle kinetics (Figure 10C). Nanobodies VHH-1, VHH-2, VHH-3, and VHH-5 bound to GSDMD with high affinity in the nanomolar range, exhibiting rapid binding and slow dissociation rates. The tightest binder was VHH-1, with a dissociation constant of 0.55 nM. VHH-2, VHH-3, and VHH-5 had dissociation constants of 8.24 nM, 2.19 nM, and 4.14 nM, respectively. In contrast, VHH-4 and VHH-6 exhibited significantly lower binding affinities in the micromolar range. Due to its low affinity, VHH-4 was excluded from further SPR experiments.

[0164] The binding epitopes of nanobodies on GSDMD were analyzed using an SPR-based epitope binning assay (Figures 10D-F, 15). Chemically biotinylated GSDMD was immobilized on an SPR sensor chip, and nanobodies were injected as analytes in a pairwise manner to test whether they compete for overlapping or distinct epitopes on GSDMD (Figure 10D). VHH-1 and VHH-5 competed with all other nanobodies for overlapping epitopes (Figure 15). VHH-2 and VHH-3 competed for a single epitope, but additional binding of VHH-6 to both nanobodies was observed (Figure 10E). Based on these observations, VHH-1 and VHH-5, as well as VHH-2 and VHH-3, were grouped into a single epitope bin, whereas VHH-6 was present alone (Figure 10F).

[0165] Example 9 – Single domain antibodies of the invention inhibit the assembly of functional GSDMD pores in vitro We used an in vitro liposome leakage assay to test whether nanobody binding affected the formation of functional GSDMD pores. GSDMD and nanobodies were added to calcein-loaded liposomes at an equimolar ratio, and calcein release through GSDMD pores was followed by measuring fluorescence at 525 nm after the addition of caspase-4 (Figure 11A). As a control, the caspase inhibitor VX-765, which completely inhibited calcein release, was used (Figure 11B). Addition of VHH-1 inhibited calcein release to the same extent as VX-765, indicating that the assembly of functional GSDMD pores was completely suppressed. VHH-2 and VHH-3 also had an inhibitory effect, although to a lesser extent than VHH-1. Addition of VHH-6 slowed calcein release, but still reached the maximum fluorescence observed without the addition of nanobodies. VHH-4 tended to increase calcein leakage, whereas VHH-5 had no significant effect on GSDMD pore formation (Fig. 11B). Comparable results were obtained when GSDMD-3C, a construct in which the caspase recognition site was replaced by the 3C protease recognition site, and 3C protease were used in the experiment. Also in this experiment, VHH-1, VHH-2, and VHH-3 inhibited calcein release through GSDMD pores, whereas VHH-4, VHH-5, and VHH-6 did not inhibit GSDMD pore assembly (Fig. 11C).

[0166] The thermal stability of the nanobodies and their effect on the thermostability of GSDMD were further analyzed using a thermal shift assay by nanodifferential scanning fluorimetry (Figures 11D and 11E, and Figure 18). Titration of the nanobodies against GSDMD at increasing concentrations revealed a fluorescence shift peak distinct from that observed for GSDMD or the nanobody alone, indicating complex formation. At equimolar concentrations, three inhibitory nanobodies (VHH-1, VHH-2, and VHH-3) increased the thermal stability of GSDMD by up to 9.4°C. In contrast, the non-inhibitory nanobodies VHH-4 and VHH-5 had a slight destabilizing effect, decreasing the melting temperature of GSDMD by up to 2.5°C. VHH-6 slowed pore formation in a leakage assay and increased the thermal stability of GSDMD by 4.2°C.

[0167] Example 10 – Crystal structure of GSDMD in complex with two nanobodies To map the nanobody epitope in detail and shed light on the molecular mechanism by which VHH-1, VHH-2, and VHH-3 inhibit GSDMD pore formation, we initiated crystallization studies of the GSDMD-nanobody complex. For this purpose, we used a GSDMD construct in which the linker region (residues 247–272) and residues 184–194 in the NTD were deleted to prevent precipitation during crystallization. Crystallization studies of a ternary complex consisting of GSDMD, VHH-2, and VHH-6 were successful, and well-diffracting crystals were reproducibly grown using this protein combination. The crystal structure of the complex was determined at 1.9 Å resolution by molecular replacement using the structures of human GSDMD (PDB 6N9O) and the BC2 nanobody (PDB 5IVO) as search models. GSDMD and the nanobody were found in a 1:1:1 stoichiometry, and the two nanobodies were clearly distinguished by their characteristic CDR regions. Two heterotrimeric GSDMD-VHH-2-VHH-6 complexes form the asymmetric unit of the crystal lattice, with excellent stereochemistry and an R of 21.2%. ワーク and 24.9% R フリー (Figure 12A, Table 1).

[0168] Table 1. Crystallographic data collection and refinement statistics related to Figures 12 and 13. TIFF2026508175000006.tif247163 a The values ​​in parentheses are for the highest resolution shell. R フリー The value is equivalent to the R value, but is calculated for 5% of reflections that are randomly selected and omitted from the refinement process.

[0169] The two GSDMD molecules found in the structure form a dimeric complex in which the NTD of one GSDMD molecule tightly interacts with the CTD of the other, spanning 4018 Å when counting both molecules. 2 In the single heterotrimeric complex, VHH-2 binds to the NTD of GSDMD, whereas VHH-6 interacts with not only the NTD but also the CTD, stabilizing the folding of the separate NTD and CTD in the GSDMD molecule (Figure 12B).

[0170] Because GSDMD has not previously been reported to form dimers, we hypothesized that the observed complex formation might be a crystallization artifact. Therefore, we performed SEC-MALS analysis and found that the complex of GSDMD, VHH-2, and VHH-6 exhibited a molecular weight of 69.2 kDa, consistent with a 1:1:1 complex with a calculated molecular weight of 79.7 kDa. For this reason, we conclude that dimerization of the two heterotrimeric complexes occurred during crystallization. The interaction between the N- and C-terminal domains of the two GSDMD molecules resembles the interaction between both domains observed in the previous GSDMD structure (PDB 6N9O), and superposition of the complex with the previous structure results in a root-mean-square deviation of 2.311 Å across 2244 Ca atoms (Figure 12D, E).

[0171] Example 11 – Binding interface of GSDMD-VHH interaction The CDR1, CDR2, and CDR3 segments of VHH-2 contain 10, 7, and 11 residues, respectively, and the nanobody backbone is stabilized by a conserved disulfide bond between C22 and C95. The interface between VHH-2 and GSDMD is primarily formed by CDR1 and CDR3 of VHH-2, while CDR2 does not significantly contribute to the interaction. In contrast, for VHH-6, all three CDRs are involved in binding to GSDMD, and the nanobody backbone also contacts GSDMD (Figure 13A). Electrostatic interactions are important for the interaction of both nanobodies with GSDMD. The positively charged CDRs of VHH-2 bind to an acidic cleft on the surface of GSDMD, including residues E21, D126, and E162. The CDRs of VHH-6 contact acidic residues D224, D226, D228, D234, and D275, and the VHH-6 backbone contacts residues E448 and E459 on the GSDMD surface (Figure 13B). Binding of VHH-2 increases the contact area by 1521 Å, counting both molecules. 2 This results in a buried surface area of ​​1000 kJ / s. A clear salt bridge is formed by Glu21 ​​in the GSDMD NTD and Arg99 in CDR3 of VHH-2. Furthermore, Arg78 on the GSDMD surface forms hydrogen bonds with Tyr100 and Thr101 in CDR3, and an additional hydrogen bond can be found between CDR3 residue Trp108 and Asn128 on the GSDMD surface.

[0172] Binding of VHH2 to GSDMD resulted in 1521 Å, counting both molecules. 2This results in a buried surface area of ​​1000. A distinct salt bridge forms between R99 in the CDR3 of GSDMD2 and E21 in the GSDMD N-terminal domain, complemented by a weak interaction to E162 of GSDMD on the other side. Furthermore, the backbone carboxyl groups of neighboring residues Y100 and T101 in CDR3 form intermolecular hydrogen bonds with R78 of GSDMD. Another hydrogen bond forms between CDR3 residue W108 and N128 on the GSDMD surface. Additional hydrophobic contacts are formed between F232 of GSDMD and V105, Y106, R26, and W28 of GSDMD2, as well as Y100 in the CDR3 of GSDMD2, which is sandwiched between H18 and F80 of GSDMD (Figure 13C).

[0173] VHH-6 was essential for the crystallization of the high-resolution GSDMD-nanobody complex by acting as a crystallization chaperone. The CDR3 of VHH-6 is particularly long, containing 15 residues, and is stabilized by an additional disulfide bond between Cys100 and Cys110, which contributes to its indistinguishable specificity (Figure 13D). Various interactions can be found between CDR1 and 2 and the NTD of GSDMD. Residues Asn32 and Gln33 in CDR1 form hydrogen bonds with Gln237 and Gln241, while CDR2 residue Thr53 contacts Asp224 on the GSDMD NTD (Figure 13D). CDR3 of VHH-6 forms several hydrogen bonds and salt bridges with the NTD of GSDMD. A prominent salt bridge is found between residues Arg98 and Asp11 in the CDR3 of VHH-6 and residues Asp234 and Arg238 on the GSDMD, respectively. Several residues in the VHH-6 backbone, including residues 39, 42-45, 47, 95, and 112-115, as well as residues 104-112 in CDR3, contact the CTD of GSDMD, which may contribute to VHH-6's role in facilitating crystallization.

[0174] Example 12 – Pyroptosis is inhibited by blocking oligomerization of the GSDMD NTD We demonstrate that VHH-1, VHH-2, and VHH-3 inhibited the assembly of functional GSDMD pores in vitro, raising the question of the mechanism by which pore formation is inhibited. Both nanobodies were found to bind to overlapping epitopes on the GSDMD NTD in SPR-based epitope binning experiments, and neither nanobody affected GSDMD cleavage by caspase-4, as observed on SDS-PAGE (Figure 14A). We superimposed the structure of the nanobody-bound GSDMD NTD with the cryo-EM structure of the activated GSDMD NTD previously determined by the Wu lab (PDB 6VFE). The superposition indicates that VHH-2 and VHH-6 bind to the globular portion of the activated NTD. VHH-6 binds to the top of the spherical rim of the GSDMD pore and does not interfere with oligomerization, whereas VHH-2 binds at the oligomerization interface of a single N-terminus, thus sterically inhibiting pore assembly (Figure 14B). Whether blocking GSDMD pore assembly also affects membrane binding and insertion remains to be investigated.

[0175] Example 13 - Discussion of Results II GSDMD is the executioner of pyroptosis and mediates the final common step of all inflammasome pathways. Because pyroptosis is implicated in many diseases, a deep understanding of the mechanisms underlying GSDMD pore formation and regulatory processes is essential. Inhibiting GSDMD is an attractive strategy for treating excessive inflammation and requires molecules that specifically interact with GSDMD. To date, three small molecule inhibitors (necrosulfonamide, disulfiram, and dimethyl fumarate) have been known to covalently modify Cys191 in the GSDMD NTD, effectively preventing pyroptosis in cells and suppressing inflammatory responses in mouse models. However, this class of inhibitors has significant drawbacks: due to their cysteine ​​reactivity, the compounds are not specific for GSDMD, and off-target binding may cause undesirable side effects in humans. In this study, we characterized six unique GSDMD-targeting nanobodies that bind to the GSDMD NTD with varying affinities. Three of these nanobodies (VHH-1, VHH-2, and VHH-3) inhibited GSDMD pore formation in an in vitro liposome leakage assay. High-resolution crystal structures of GSDMD in complex with one inhibitory and one non-inhibitory nanobody revealed that the inhibitory nanobody sterically blocks GSDMD pore assembly by binding to an epitope present at the oligomerization interface of the activated GSDMD NTD. Pore formation is inhibited, but caspase cleavage is unaffected.

[0176] Our nanobodies offer a novel mechanism for pyroptosis inhibition by sterically inhibiting oligomerization instead of targeting reactive cysteine ​​residues in GSDMD. Cellular assays and in vivo studies are needed to explore the full potential of nanobodies as GSDMD-specific drugs. GSDMD is an intracellular protein, and delivery of antibodies and nanobodies to the cytoplasm has long been limited by their inability to cross the plasma membrane. Nevertheless, recent studies have demonstrated that nanobodies can be delivered to the cytoplasm of cells using cell-penetrating peptide fusions, and nanobody mRNA can be delivered using gene therapy approaches. Our SPR-based binding experiments were performed in the presence of 5 mM DTT, indicating that nanobodies maintain their binding ability even under reducing conditions and should be unaffected by the reducing environment of the cytoplasm. Instead of being applied as a drug itself, our nanobody could also facilitate the development and characterization of new GSDMD-specific small molecule inhibitors by its function as a crystallization chaperone, which enabled the generation of fast-growing, reproducible, and well-diffracting GSDMD crystals for X-ray crystallography and high-resolution structure determination.

[0177] Apart from that, nanobodies are versatile tools and find wide applications in many research areas. For example, nanobodies can be fluorescently labeled to visualize their target proteins for fluorescence microscopy approaches or engineered to translocate their antigens to specific cellular compartments. Taken together, our non-inhibitory and inhibitory nanobodies are useful tools for studying GSDMD biology by using them as specific inhibitors, fluorescent labels in imaging approaches, or crystallization chaperones for high-resolution GSDMD structures, and can be used as templates for the development of nanobody-based GSDMD inhibitory drugs.

[0178] Example 14 -Further characterization of single domain antibodies with respect to their specificity and mechanism of pore formation Further data provided in Figures 20-32 further showed that human GSDMD nanobodies did not bind to mouse GsdmD in the LUMIER assay (Figure 20A). GSDMD-1 and VHH GSDMD-2 overexpressed GSDMD NT inhibits pyroptosis induced by α-glucan, but overexpression of its related GSDME NT It was further found that VHH expressed in the cytosol did not inhibit pyroptosis induced by VHH (Fig. 21A). GSDMD-1 overexpressed mouse GsdmD NT It was also established that recombinant extracellular VHH inhibits pyroptosis induced by VHH (Figure 21B). GSDMD-1 It was also found that VHHs did not inhibit pyroptosis induced in mouse macrophages (Figure 21C), suggesting that inhibition of mouse GsdmD is not very potent. Further data also provided lessons regarding GSDMD pore formation. GSDMD-1 In the plasma membrane of HEK293T cells, monomeric GSDMD NT -stabilizes mCherry. Quantitation of membrane localization is shown in Figures 22A and 22B. Analysis of GSDMD point mutants supports the interpretation of membrane localization as membrane insertion. VHH GSDMD-1 and VHH GSDMD-2 GSDMD at the plasma membrane after cleavage by caspase-1 in THP-1 cells NT It was also confirmed that VHH stabilizes mNeonGreen. Quantitation of membrane localization is shown in Figures 23A / B and 24. GSDMD-1 endogenous GSDMD at the plasma membrane after cleavage by caspase-1 in THP-1 cells NT It was also confirmed that cleaved GSDMD stabilizes the cleaved GSDMD (staining with antibody and quantification of membrane localization are shown in Figure 25A / B).

[0179] Additional data indicate that GSDMD pores are rapidly removed from the plasma membrane, likely by a mechanism involving endocytosis of the plasma membrane stretches containing GSDMD pores (Figure 26A / B). As quantified in Figure 27A, VHHs expressed in the cytosol GSDMD-1 and VHH GSDMD-2 has also been shown to transition cells from pyroptosis to apoptosis.

[0180] Additional data are also relevant to therapeutic applications. GSDMD-1 and VHH GSDMD-2 It is also shown that VHH inhibits cell death after activation of the NLRP3 inflammasome (Fig. 27B / C). GSDMD-1 and VHH GSDMD-2 The inhibition / delay of cell death by GSDMD nanobodies was also confirmed using different readouts, independent of plasma membrane integrity (Figure 28A / B). The data also confirm the observed transition from pyroptosis to apoptosis. It was also confirmed that nanobody uptake into pyroptotic cells relies on the GSDMD pore (Figures 29A / B, 30A / B). In this regard, pyroptotic cells are rounded but not ruptured cells with GSDMD pores. Cells with sublytic GSDMD pores [i.e., cells stimulated in the presence of extracellular GSDMD nanobodies] were further demonstrated and quantified to uptake DNA dyes (Figure 31) and nanobodies (Figure 32).

Claims

1. A single domain antibody against gasdermin D (GSDMD), comprising an amino acid sequence including framework region 1 (FR1), complementarity determining region 1 (CDR1), FR2, CDR2, FR3, CDR3, and FR4; (a) a CDR1 defined by SEQ ID NO:2, a CDR2 defined by SEQ ID NO:4, and a CDR3 defined by SEQ ID NO:6; or (b) a CDR1 defined by SEQ ID NO:10, a CDR2 defined by SEQ ID NO:12, and a CDR3 defined by SEQ ID NO:14; or (c) a CDR1 defined by SEQ ID NO: 18, a CDR2 defined by SEQ ID NO: 20, and a CDR3 defined by SEQ ID NO: 22; or (d) a CDR1 defined by SEQ ID NO:26, a CDR2 defined by SEQ ID NO:28, and a CDR3 defined by SEQ ID NO:30; or (e) a CDR1 defined by SEQ ID NO:34, a CDR2 defined by SEQ ID NO:36, and a CDR3 defined by SEQ ID NO:38; or (f) a CDR1 defined by SEQ ID NO:42, a CDR2 defined by SEQ ID NO:44, and a CDR3 defined by SEQ ID NO:46; or (g) a CDR1 defined by SEQ ID NO: 50, a CDR2 defined by SEQ ID NO: 52, and a CDR3 defined by SEQ ID NO: 54; or (h) a CDR1 defined by SEQ ID NO:58, a CDR2 defined by SEQ ID NO:60, and a CDR3 defined by SEQ ID NO:62; or (i) a CDR1 defined by SEQ ID NO:66, a CDR2 defined by SEQ ID NO:68, and a CDR3 defined by SEQ ID NO:70; or (j) a CDR1 defined by SEQ ID NO: 74, a CDR2 defined by SEQ ID NO: 76, and a CDR3 defined by SEQ ID NO: 78; or (k) a CDR1 defined by SEQ ID NO: 82, a CDR2 defined by SEQ ID NO: 84, and a CDR3 defined by SEQ ID NO: 86; or (l) a CDR1 defined by SEQ ID NO: 90, a CDR2 defined by SEQ ID NO: 92, and a CDR3 defined by SEQ ID NO: 94; or (m) a CDR1 defined by SEQ ID NO: 98, a CDR2 defined by SEQ ID NO: 100, and a CDR3 defined by SEQ ID NO: 102; or (n) a CDR1 defined by SEQ ID NO: 106, a CDR2 defined by SEQ ID NO: 108, and a CDR3 defined by SEQ ID NO: 110; or (o) a CDR1 defined by SEQ ID NO: 114, a CDR2 defined by SEQ ID NO: 116, and a CDR3 defined by SEQ ID NO: 118; or (p) a CDR1 defined by SEQ ID NO: 122, a CDR2 defined by SEQ ID NO: 124, and a CDR3 defined by SEQ ID NO: 126; or (q) a CDR1 defined by SEQ ID NO: 130, a CDR2 defined by SEQ ID NO: 132, and a CDR3 defined by SEQ ID NO: 134; or (r) a CDR1 defined by SEQ ID NO: 138, a CDR2 defined by SEQ ID NO: 140, and a CDR3 defined by SEQ ID NO: 142; or (s) a CDR1 defined by SEQ ID NO: 146, a CDR2 defined by SEQ ID NO: 148, and a CDR3 defined by SEQ ID NO: 150; or (t) a CDR1 defined by SEQ ID NO: 154, a CDR2 defined by SEQ ID NO: 156, and a CDR3 defined by SEQ ID NO: 158; or (u) a CDR1 defined by SEQ ID NO: 162, a CDR2 defined by SEQ ID NO: 164, and a CDR3 defined by SEQ ID NO: 166; or (v) a CDR1 defined by SEQ ID NO: 170, a CDR2 defined by SEQ ID NO: 172, and a CDR3 defined by SEQ ID NO: 174; or (w) a CDR1 defined by SEQ ID NO: 178, a CDR2 defined by SEQ ID NO: 180, and a CDR3 defined by SEQ ID NO: 182 A single domain antibody comprising:

2. 2. The single domain antibody against GSDMD of claim 1, wherein the single domain antibody comprises an amino acid sequence selected from the group comprising the amino acid sequence defined in SEQ ID NO: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 104, 112, 120, 128, 136, 144, 152, 160, 168, 176, or 184, or a variant thereof, wherein the variant comprises an amino acid sequence that is at least 80%, 90%, 95%, or 99% identical to an amino acid sequence selected from the group comprising the amino acid sequence defined in SEQ ID NO: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 104, 112, 120, 128, 136, 144, 152, 160, 168, 176, or 184.

3. The single domain antibody is capable of specifically binding to an epitope within the N-terminal domain of GSDMD, the epitope having the sequence of GSDMD (SEQ ID NO:185):

3. The single domain antibody against GSDMD of claim 1 or 2, wherein the single domain antibody is a discontinuous epitope defined by amino acids L16, H18, G19, E21, F22, Q75, G77, R78, F80, S122, S124, D126, P127, N128, Q131, E162, R217, L231, and F232.

4. A polynucleotide encoding the single domain antibody of any one of claims 1 to 3.

5. The polynucleotide of claim 4, wherein the polynucleotide is selected from RNA, such as mRNA, DNA, such as genomic DNA, cDNA, or synthetic DNA, analogs thereof, or combinations thereof, preferably the polynucleotide is mRNA.

6. A single domain antibody against GSDMD according to any one of claims 1 to 3, wherein the single domain antibody is capable of binding to cytosolic GSDMD in a cell and thereby inhibiting pyroptosis of the cell, and the single domain antibody against GSDMD is produced by a cell when the cell is transfected with the polynucleotide of claim 4 or 5.

7. A host cell comprising the polynucleotide of claim 4 or 5.

8. A pharmaceutical composition comprising the single domain antibody against GSDMD of any one of claims 1 to 3 or the polynucleotide of claim 4 or 5, and a pharmaceutically acceptable carrier.

9. A single domain antibody against GSDMD according to any one of claims 1 to 3, or a polynucleotide according to claim 4 or 5, or a pharmaceutical composition according to claim 8, for use in therapy.

10. In subjects, the following diseases have been reported: acute inflammation, chronic inflammation, sepsis, especially sepsis-induced loss of the blood-brain barrier, septic shock, non-alcoholic steatohepatitis, lung cancer, familial Mediterranean fever (FMF), autoinflammatory diseases, cryopyrin-associated periodic fever syndromes (CAPS), non-alcoholic fatty liver disease, Alzheimer's disease, Parkinson's disease, age-related macular degeneration, atherosclerosis, asthma and allergic airway inflammation, gout, Crohn's disease, ulcerative colitis, inflammatory bowel disease, hypertension, nephropathy, myocardial infarction, multiple sclerosis, experimental autoimmune encephalitis, influenza virus infection.

9. The single domain antibody against GSDMD of any one of claims 1 to 3, or the polynucleotide of claim 4 or 5, or the pharmaceutical composition of claim 8, for use in a method for treating or preventing an inflammatory disease or condition selected from the group consisting of hyperinflammation after infection, graft-versus-host disease, stroke, silicosis, asbestosis, mesothelioma, type 1 diabetes, type 2 diabetes, obesity-induced inflammation, insulin resistance, rheumatoid arthritis, myelodysplastic syndrome, contact hypersensitivity, chikungunya virus-induced joint inflammation, and traumatic brain injury.

11. A method for determining the presence or absence of GSDMD oligomers in a sample obtained from a subject, the method comprising contacting the sample with a reporter system comprising two single domain antibodies to GSDMD, wherein both single domain antibodies compete for the same epitope in GSDMD, binding of the single domain antibodies to the epitope does not affect the ability of GSDMD to oligomerize, and upon binding of both single domain antibodies to GSDMD, the reporter system provides a detectable reporter signal indicating the presence of GSDMD oligomers.

12. 12. The method of claim 11, wherein the sample is selected from the group comprising serum, plasma, and whole blood.

13. The presence or absence of or by sandwich ELISA, in which a first single domain antibody against GSDMD is used as a primary antibody to capture GSDMD, and a second single domain antibody against GSDMD is used as a secondary antibody, the secondary antibody being labeled; or and determining the activity of GSDMD in a HTRF / FRET-based assay, wherein a first fluorescently labeled single domain antibody against GSDMD is used as a fluorescent donor and a second fluorescently labeled single domain antibody against GSDMD is used as a fluorescent acceptor.

13. The method of claim 11 or 12.

14. 14. The method of any one of claims 11 to 13, wherein the first single domain antibody against GSDMD and the second single domain antibody against GSDMD are the single domain antibodies of any one of claims 1 to 3, and wherein the first single domain antibody and the second single domain antibody can be the same single domain antibody or different single domain antibodies.

15. 1. A method for treating or preventing an inflammatory disease or condition in a subject, comprising administering to a subject a single domain antibody, or a polynucleotide encoding the single domain antibody, or a pharmaceutical composition comprising the single domain antibody or polynucleotide, the method comprising administering to a subject a single domain antibody or polynucleotide encoding ... the inflammatory disease or condition is selected from the group comprising acute inflammation, chronic inflammation, sepsis, in particular loss of the blood-brain barrier caused by sepsis, septic shock, non-alcoholic steatohepatitis, lung cancer, familial Mediterranean fever (FMF), autoinflammatory diseases, cryopyrin-associated periodic fever syndromes (CAPS), non-alcoholic fatty liver disease, Alzheimer's disease, Parkinson's disease, age-related macular degeneration, atherosclerosis, asthma and allergic airway inflammation, gout, Crohn's disease, ulcerative colitis, inflammatory bowel disease, hypertension, nephropathy, myocardial infarction, multiple sclerosis, experimental autoimmune encephalitis, hyperinflammation after influenza infection, graft-versus-host disease, stroke, silicosis, asbestosis, mesothelioma, type 1 diabetes, type 2 diabetes, obesity-induced inflammation, insulin resistance, rheumatoid arthritis, myelodysplastic syndrome, contact hypersensitivity, chikungunya virus-induced joint inflammation, and traumatic brain injury; The presence of GSDMD oligomers is determined in a sample obtained from the subject. A single domain antibody against gasdermin D (GSDMD), or a polynucleotide encoding the single domain antibody, or a pharmaceutical composition comprising the single domain antibody or polynucleotide.

16. A single domain antibody against Gasdermin D (GSDMD), or a polynucleotide encoding the single domain antibody, for use according to claim 15, wherein the single domain antibody against GSDMD is a single domain antibody according to any one of claims 1 to 3.

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