Interleukin-1 alpha chimeric protein

Abstract

The present invention relates, in part, to chimeric proteins, chimeric protein complexes, vaccine compositions, and adjuvants comprising IL-1α or pro-IL-1α, and their use as therapeutics or vaccines. The present invention further relates to methods of treating and vaccinating against various diseases, such as infectious diseases and cancer.

Description

[Technical Field]

[0001] The present invention relates, in part, to chimeric proteins, chimeric protein complexes, adjuvants, or vaccine compositions comprising IL-1α or pro-IL-1α, and their use in the treatment of various infectious diseases, cancer, and other therapeutic indications.

[0002] Sequence Listing The contents of the computer-readable Sequence Listing in XML format ("XML Document") submitted electronically herewith are incorporated herein by reference in their entirety. A computer-readable copy of the Sequence Listing (Filename: ORN-082PC.xml, Created: July 6, 2022, Size: 708,215 bytes) is submitted pursuant to 37 CFR §§ 1.831-1.835. [Background technology]

[0003] Most interleukins are secreted proteins that function as cytokines, mediating intercellular communication during immune responses. IL-1α is a proinflammatory molecule that plays a central role in mediating immune responses due to its broad spectrum of biological functions and range of target cells. IL-1α is a decision-making molecule used by cells to gauge the degree of stress or injury or the severity of an infection, so that either the tissue or the whole body can respond by initiating inflammation or reparative fibrosis. Abnormalities in these sequelae can lead to devastating disruptions of tissue homeostasis, which underlie the pathology of many human diseases.

[0004] Under homeostatic conditions, IL-1α is expressed by multiple hematopoietic and non-hematopoietic cells. IL-1α can be upregulated by a wide variety of inflammatory stimuli, and its biological activity does not depend on proteolytic processing. Thus, cell death due to sterile or infectious injury results in the release of bioactive IL-1α, which signals through the IL-1R and induces an inflammatory response. IL-1R is constitutively expressed by a wide range of cell types, and downstream activation of NF-κB and MAPKs induces the production of proinflammatory mediators, such as cyclooxygenase type 2 (COX-2), IL-6, and tumor necrosis factor (TNF). These proinflammatory mediators further promote the production of IL-1α and IL-1β, amplifying the inflammatory stimulus provided by the initial IL-1α release. Physiological manifestations of IL-1 signaling include fever, hypotension, vasodilation, and increased pain sensitivity.

[0005] Dysregulated IL-1α production is associated with numerous autoinflammatory diseases in promoting host defense against multiple infectious pathogens. Malignant cells, tumor-infiltrating immune cells, and stromal cells can express IL-1α, IL-1β, and IL-1R. IL-1 signaling in tumor tissue and its microenvironment can affect tumor progression in various ways. Even after decades of research, many important questions related to IL-1α remain unanswered. Deregulation of the activity of IL-1α or members of the interleukin family is a major cause of inflammatory and autoimmune diseases. While both IL-1α and IL-1β have the potential to exhibit differentiated biological activities, they can also be potent activators of T cells and NK cells, cell types that play important roles in defense against cancer and infectious diseases. Such responses can include the induction of T cells, memory T cells, and T cell-mediated immunity. Many other cell types are targets of IL-1, relevant for the therapeutic application of IL-1 to cancer, infectious diseases, and other diseases. Therefore, these IL-1 family members represent attractive targets for therapeutic manipulation. The selective activation of desired target cells, such as T cells and NK cells, by IL-1α remains an unsolved challenge in the quest to harness the therapeutic potential of IL-1α. Summary of the Invention [Problem to be solved by the invention]

[0006] Thus, there remains a need for target-selective, safe, and effective IL-1α-based therapeutics with minimal toxicity and improved pharmacokinetic and therapeutic properties. [Means for solving the problem]

[0007] Thus, in some embodiments, the present invention relates to chimeric protein complexes, including chimeric proteins and Fc-based chimeric protein complexes, comprising interleukin-1α (IL-1α), pro-IL-1α, or variants thereof as a signaling factor. As used herein, the term variant includes IL-1α variants or pro-IL-1α variants.

[0008] In some aspects, the present invention relates to a chimeric protein comprising: (a) interleukin-1α (IL-1α), pro-IL-1α, or a variant thereof; and (b) one or more targeting moieties, the targeting moieties comprising a recognition domain that specifically binds to an antigen or receptor of interest, wherein the IL-1α, pro-IL-1α, or variant thereof and the one or more targeting moieties are optionally linked by one or more linkers.

[0009] In some embodiments, the present invention relates to a chimeric protein complex (e.g., an Fc-based chimeric protein complex) comprising: a) interleukin-1α (IL-1α), pro-IL-1α, or a variant thereof; b) one or more targeting moieties, the targeting moieties comprising a recognition domain that specifically binds to an antigen or receptor of interest; and c) an Fc domain, optionally with one or more mutations that reduce or eliminate one or more effector functions of the Fc domain, promote Fc chain pairing in the Fc domain, and / or stabilize the hinge region in the Fc domain.

[0010] In one aspect, the present invention provides a method for producing a polypeptide comprising: (a) a mutant interleukin-1α (IL-1α) or pro-IL-1α, wherein the mutation is a deletion of amino acids 1-6 (Δ1-6) relative to any one of SEQ ID NOs: 1-4; (b) one or more targeting moieties, wherein the targeting moiety comprises a recognition domain that specifically binds to an antigen or receptor of interest; and (c) a connector between (a) and (b), the connector being (i) a flexible linker connecting (a) and (b), and / or (ii) an Fc domain connecting (a) and (b), the Fc domain optionally having one or more mutations that reduce or eliminate one or more effector functions of the Fc domain, promote Fc chain pairing in the Fc domain, and / or stabilize the hinge region in the Fc domain. The present invention provides a chimeric protein or chimeric protein complex (e.g., an Fc-based chimeric protein complex) comprising:

[0011] The present disclosure relates, in part, to the discovery that chimeric proteins or chimeric protein complexes comprising IL-1α, pro-IL-1α, or variants thereof, exhibit substantially reduced or increased IL-1R (IL-1 receptor)-activated signaling activity compared to wild-type IL-1α or wild-type pro-IL-1α or Fc IL-1α or Fc pro-IL-1α or Fc IL-1α(Δ1-6) or Fc pro-IL-1α(Δ113-118) or targeted Fc IL-1α(Δ1-6) or targeted Fc pro-IL-1α(Δ113-118), respectively, although reduced IL-1R-activated signaling activity can be induced and / or restored in target cells when the activity is directed to the target cells via a targeting moiety. Surprisingly, the IL-1α activity induced and / or restored in the target cells achieved through targeting of a chimeric protein or chimeric protein complex comprising IL-1α or a variant thereof can be comparable to or exceed the activity of wild-type IL-1α or wild-type pro-IL-1α or Fc IL-1α or Fc pro-IL-1α or Fc IL-1α(Δ1-6) or Fc pro-IL-1α(Δ113-118) or targeted Fc IL-1α(Δ1-6) or targeted Fc pro-IL-1α(Δ113-118) in the target cells. Furthermore, and equally surprisingly, the activity of the targeted IL-1α of the chimeric protein or chimeric protein complex comprising IL-1α or a variant thereof may be comparable to or exceed the activity of wild-type IL-1α or wild-type pro-IL-1α or Fc IL-1α or Fc pro-IL-1α or Fc IL-1α(Δ1-6) or Fc pro-IL-1α(Δ113-118) or targeted Fc IL-1α(Δ1-6) or targeted Fc pro-IL-1α(Δ113-118).Importantly, the IL-1α-containing IL-1α chimeric proteins and chimeric protein complexes described herein exhibit substantial and surprising selectivity for target cells over non-target cells compared to wild-type IL-1α, which may be substantially more than is merely achieved by chimeric proteins containing targeted IL-1α having, for example, a deletion of amino acids 1-6 (Δ1-6) relative to SEQ ID NO: 1 or 3. Taken together, the IL-1α compositions and variants thereof described herein can be used to achieve a unique combination of highly potent and highly cell target selective IL-1R signaling activation.

[0012] The present disclosure also relates, in part, to the discovery that chimeric proteins or chimeric protein complexes comprising IL-1α, pro-IL-1α, or variants thereof, exhibit increased (but not decreased) IL-1R-activating signaling activity compared to wild-type IL-1α or wild-type pro-IL-1α and / or an equivalent IL-1α having a deletion of amino acids 1 to 6 (Δ1-6) relative to SEQ ID NO: 1 or 3, or an equivalent pro-IL-1α having a deletion of amino acids 113 to 118 (Δ113-118) relative to SEQ ID NO: 2 or 4, but without any additional mutations other than Δ1-6 in IL-1α or Δ113-118 in pro-IL-1α. Importantly, and equally surprising, such chimeric proteins and chimeric protein complexes comprising IL-1α, pro-IL-1α, or the above variants described herein can exhibit substantial and surprising selectivity for target cells over non-target cells compared to wild-type IL-1α or wild-type pro-IL-1α and / or an equivalent IL-1α having a deletion of amino acids 1 to 6 (Δ1-6) relative to SEQ ID NO: 1 or 3, or an equivalent pro-IL-1α having a deletion of amino acids 113 to 118 (Δ113-118) relative to SEQ ID NO: 2 or 4, but without any additional mutations other than Δ1-6 in IL-1α or Δ113-118 in pro-IL-1α.

[0013] In some embodiments, the IL-1α or pro-IL-1α is modified, i.e., mutant, and comprises one or more mutations in IL-1α (or pro-IL-1α). In some embodiments, the one or more mutations reduce or increase the biological activity of the IL-1α or pro-IL-1α. For example, the one or more mutations may reduce or increase the affinity and / or activity of the IL-1α or pro-IL-1α for a therapeutic receptor. In one embodiment, the modified IL-1α or modified pro-IL-1α comprises one or more mutations that reduce or increase its affinity and / or activity for IL-1R. In another embodiment, the modified IL-1α or modified pro-IL-1α comprises one or more mutations that reduce or increase its affinity and / or activity for IL-1R or IL-1RAcP. In some embodiments, decreased affinity and / or activity of the modified IL-1α or modified pro-IL-1α for a therapeutic receptor, e.g., IL-1R or IL-1RAcP, can be induced and / or restored by targeting or directing the chimeric protein or chimeric protein complex comprising the modified IL-1α or modified pro-IL-1α to a target cell via a targeting moiety. In some embodiments, the modified IL-1α or pro-IL-1α variant comprising one or more mutations that decrease or increase its affinity and / or activity for IL-1R or IL-1RAcP further comprises one or more mutations that reduce undesired disulfide pairing, improving product uniformity and the pharmaceutical properties of the chimeric protein or chimeric protein complex, while maintaining or avoiding a substantial decrease in induction and / or restoration of IL-1R-activating activity by the modified IL-1α or pro-IL-1α when directed / targeted to a target cell via a targeting moiety.

[0014] In some embodiments, incorporation of wild-type IL-1α into a chimeric protein or chimeric protein complex, e.g., via gene fusion or gene conjugation (e.g., complex formation), reduces the biological activity of IL-1α. For example, wild-type IL-1α incorporated into a chimeric protein or chimeric protein complex may have reduced affinity and / or activity for a therapeutic receptor compared to wild-type IL-1α. In some embodiments, reduced affinity and / or activity of wild-type IL-1α for a therapeutic receptor, e.g., IL-1R, can be induced and / or restored by directing or targeting the IL-1α-containing chimeric protein or chimeric protein complex to a target cell via a targeting moiety. In some embodiments, induction and / or restoration of IL-1α-mediated IL-1R activation in target cells may reach levels comparable to or greater than those achieved with wild-type (non-chimeric) IL-1α. In some embodiments, the IL-1α is a variant that includes one or more mutations that reduce undesired disulfide pairing, improving the uniformity and pharmaceutical properties of the chimeric protein or chimeric protein complex product, while maintaining or avoiding a substantial reduction in IL-1R activation by the modified IL-1α compared to wild-type IL-1α in the context of the chimeric protein or chimeric protein complex, including maintaining or avoiding a substantial reduction in restoration and / or induction of IL-1R activation by the modified IL-1α when directed to or targeted to a target cell via a targeting moiety.

[0015] In some embodiments, the chimeric protein complexes, including the chimeric proteins and Fc-based chimeric protein complexes, further comprise one or more additional signaling factors, such as, but not limited to, interferons, interleukins, and tumor necrosis factors, which may be modified. In various embodiments, the chimeric protein complexes, including the chimeric proteins and Fc-based chimeric protein complexes of the present invention, have improved safety and / or improved therapeutic activity and / or improved pharmacokinetic profile (e.g., increased serum half-life) compared to untargeted and / or unmodified IL-1α or unmodified wild-type IL-1α.

[0016] In various embodiments, the chimeric protein complexes, including the chimeric protein complexes and Fc-based chimeric protein complexes, comprise one or more targeting moieties having a recognition domain (e.g., an antigen recognition domain, including, but not limited to, various antibody formats, including single-domain antibodies) that specifically binds to a target of interest (e.g., an antigen, a receptor). In various embodiments, the targeting moieties have a recognition domain that specifically binds to a target of interest (e.g., an antigen, a receptor), including a recognition domain present on one or more types of immune cells, which may include, but are not limited to, T cells, cytotoxic T lymphocytes, T helper cells, regulatory T cells (Treg), natural killer (NK) cells, natural killer T (NKT) cells, anti-tumor and tumor-associated macrophages (e.g., M1 and M2 macrophages), B cells, regulatory B (Breg) cells, neutrophils, monocytes, bone marrow-derived cells, and dendritic cells. In various embodiments, the targeting moiety has a recognition domain that specifically binds to a target of interest (e.g., an antigen, receptor), including targets present on one or more tumor cells, endothelial cells, epithelial cells, mesenchymal cells, stromal cells, or other cell types characteristic and / or unique to a particular organ and / or tissue, including cell types specifically associated with a disease. In some embodiments, the recognition domain specifically binds to a target of interest (e.g., an antigen, receptor) and effectively recruits one or more immune cells. In some embodiments, the target of interest (e.g., an antigen, receptor) may be present on one or more tumor cells. In some embodiments, chimeric proteins, including Fc-based chimeric protein complexes, of the present invention may recruit immune cells to a site of action (such as, by way of non-limiting example, the tumor microenvironment), e.g., immune cells that can kill and / or suppress tumor cells or modulate other immune cells.In some embodiments, the chimeric proteins, including Fc-based chimeric protein complexes, may regulate immune cells at a site of action or recruit immune cells to a site of action (such as, by way of non-limiting example, the tumor microenvironment) associated with autoimmune, inflammatory, infectious, metabolic, and / or cardiovascular disease. In some embodiments, the recognition domain specifically binds to a target of interest (e.g., an antigen, receptor) that is part of a non-cellular structure.

[0017] In some embodiments, the chimeric proteins or chimeric protein complexes described herein comprise IL-1α or pro-IL-1α mutants (which are examples of cytokines / signaling factors that may be used in the present invention) conjugated to one or more targeting moieties with reduced or increased biological activity. In some embodiments, the chimeric proteins or chimeric protein complexes of the present invention comprise AcTakines (Activity-on-Target cytokines) with one or more mutant cytokines that remain inactive during transit through the body and only manifest their full agonist activity upon binding to target cells. In some embodiments, the chimeric proteins or chimeric protein complexes target mutant IL-1α to CD8 + In vivo, such chimeric proteins or chimeric protein complexes have a significantly reduced toxicity profile compared to wild-type (WT) IL-1α and target CD8 T cells to antigens. + It may act as an adjuvant to potently promote T cell responses. In some embodiments, the chimeric protein or chimeric protein complex targets mutant IL-1α to NK cells or other immune cell types or surrogate cell types.

[0018] In various embodiments, the chimeric proteins and chimeric protein complexes, including Fc-based chimeric protein complexes, are used to treat a variety of diseases or disorders, such as cancer, infectious diseases, immune disorders, autoimmune diseases, cardiovascular diseases, wounds, ischemia-related diseases, neurodegenerative diseases, metabolic diseases, and many other diseases and disorders, and the present invention encompasses a variety of treatment methods. In various embodiments, the adjuvants, the chimeric proteins, and the chimeric protein complexes are used to vaccinate or treat a variety of diseases or disorders, such as infectious diseases. The present invention encompasses a variety of treatment methods or vaccination methods for such diseases or disorders.

[0019] Another aspect of the present invention relates to a method of treating a subject suffering from an infectious disease, comprising administering a chimeric protein or chimeric protein complex described herein. In some embodiments, the chimeric protein or chimeric protein complex comprises: (i) IL-1α, pro-IL-1α, or a variant thereof; (ii) one or more targeting moieties, the targeting moieties comprising a recognition domain that specifically binds to an antigen or receptor of interest; and (iii) a connector between (i) and (ii), wherein the connector is: (1) an Fc domain, optionally having one or more mutations that reduce or eliminate one or more effector functions of the Fc domain, promote Fc chain pairing in the Fc domain, and / or stabilize a hinge region in the Fc domain connecting (i) and (ii), and / or (2) a flexible linker connecting (i) and (ii), wherein the variant IL-1α or pro-IL-1α is characterized by a reduced or increased affinity or activity at an IL-1 receptor.

[0020] Yet another aspect of the present invention relates to a method of treating a subject suffering from cancer, comprising administering a chimeric protein or chimeric protein complex described herein. In some embodiments, the chimeric protein or chimeric protein complex comprises: (i) IL-1α, pro-IL-1α, or a variant thereof; (ii) one or more targeting moieties, the targeting moieties comprising a recognition domain that specifically binds to an antigen or receptor of interest; and (iii) a connector between (i) and (ii), wherein the connector is: (1) an Fc domain, optionally having one or more mutations that reduce or eliminate one or more effector functions of the Fc domain, promote Fc chain pairing in the Fc domain, and / or stabilize the hinge region in the Fc domain connecting (i) and (ii), and / or (2) a flexible linker connecting (i) and (ii), wherein the variant IL-1α or pro-IL-1α is characterized by a reduced or increased affinity or activity at an IL-1 receptor.

[0021] One aspect of the present application relates to a vaccine composition comprising a chimeric protein or chimeric protein complex described herein. In some embodiments, the vaccine composition comprises (a) an adjuvant and (b) an antigen suitable for inducing an immune response. The adjuvant comprises (i) IL-1α, pro-IL-1α, or a variant thereof (which is an example of a signal transduction factor described herein), (ii) one or more targeting moieties comprising a recognition domain that specifically binds to an antigen or receptor of interest, and (iii) a linker between (i) and (ii). The binder comprises (1) an Fc domain, optionally having one or more mutations that reduce or eliminate one or more effector functions of the Fc domain, promote Fc chain pairing in the Fc domain, and / or stabilize the hinge region in the Fc domain connecting (i) and (ii), and / or (2) a flexible linker connecting (i) and (ii), and the mutant IL-1α or pro-IL-1α is characterized by low or high affinity or activity at the IL-1 receptor.

[0022] In some embodiments, the vaccine adjuvants described herein are CD8 + The present disclosure also provides, in part, an adjuvant, chimeric protein, or chimeric protein complex comprising IL-1α, pro-IL-1α, or a variant thereof, comprising wild-type IL-1α or wild-type pro-IL-1α or Fc IL-1α or Fc pro-IL-1α or Fc IL-1α(Δ1-6) or Fc pro-IL-1α(Δ113-118) or targeted Fc IL-1α(Δ1-6) or targeted Fc The present invention also relates to the discovery that IL-1α, pro-IL-1α, or variants thereof exhibit a substantial decrease or increase in IL-1α activity compared to wild-type IL-1α or wild-type pro-IL-1α or Fc IL-1α or Fc pro-IL-1α or Fc IL-1α(Δ1-6) or Fc pro-IL-1α(Δ1-6) or targeted Fc IL-1α(Δ1-6) or targeted ... This may be equivalent to or greater than the induction and / or restoration of pro-IL-1α(Δ113-118).

[0023] Importantly, the adjuvants, chimeric proteins, and chimeric protein complexes comprising the mutant IL-1α or pro-IL-1α described herein exhibit substantial and surprising selectivity for target cells over non-target cells, which may substantially exceed that achieved solely by, for example, wild-type IL-1α or wild-type pro-IL-1α or Fc IL-1α or Fc pro-IL-1α or Fc IL-1α(Δ1-6) or Fc pro-IL-1α(Δ113-118) or targeted Fc IL-1α(Δ1-6) or targeted Fc pro-IL-1α(Δ113-118). In summary, a unique combination of highly potent and highly cell target-selective signaling activation can be achieved using the adjuvants, chimeric proteins, or chimeric protein complexes described herein.

[0024] In some embodiments, decreased affinity and / or activity of IL-1α or pro-IL-1α for its receptor, e.g., IL-1 receptor, can be induced and / or restored by directing or targeting the adjuvant, chimeric protein, or chimeric protein complex comprising IL-1α or pro-IL-1α to target cells via a targeting moiety. In some embodiments, induction and / or restoration of IL-1α-mediated activation in target cells may reach levels equivalent to or greater than that achieved by wild-type (non-chimeric) IL-1α.

[0025] In one embodiment, the modified IL-1α or pro-IL-1α comprises one or more mutations that reduce or increase its affinity and / or activity for the IL-1 receptor. In another embodiment, the modified IL-1α or pro-IL-1α comprises one or more mutations that reduce or increase its affinity and / or activity for IL-1R1 or IL-1RAcP (co-receptor). In one embodiment, the modified IL-1α or pro-IL-1α comprises one or more mutations that reduce or increase its affinity and / or activity for IL-1R1 and one or more mutations that reduce or increase its affinity and / or activity for IL-1RAcP. In some embodiments, the reduced affinity and / or activity of the modified IL-1α or pro-IL-1α for a receptor, e.g., IL-1R1, IL-1RAcP, can be induced and / or restored by directing or targeting the adjuvant, chimeric protein, or chimeric protein complex comprising the modified IL-1α or pro-IL-1α to a target cell via a targeting moiety.

[0026] In some embodiments, the chimeric protein complexes, including the adjuvants, the chimeric proteins, and the Fc-based chimeric protein complexes, comprise one or more additional signaling factors or cytokines, such as, but not limited to, optionally modified interferons, interleukins, and tumor necrosis factors. In various embodiments, the adjuvants, chimeric proteins, or chimeric protein complexes, including the Fc-based chimeric protein complexes of the present invention, have improved safety and / or improved therapeutic activity and / or improved pharmacokinetic profile (e.g., increased serum half-life) compared to untargeted and / or unmodified IL-1α or pro-IL-1α, or unmodified wild-type IL-1α or pro-IL-1α.

[0027] In various embodiments, the adjuvants, the chimeric proteins, and the chimeric protein complexes, including Fc-based chimeric protein complexes, comprise one or more targeting moieties having a recognition domain (e.g., an antigen recognition domain, including, but not limited to, various antibody formats, including single-domain antibodies) that specifically binds to a target of interest (e.g., an antigen, a receptor). In various embodiments, the targeting moieties have a recognition domain that specifically binds to a target of interest (e.g., an antigen, a receptor), including targets present on immune cells, which may include, but are not limited to, one or more of cancer cells, T cells, cytotoxic T lymphocytes, helper T cells, regulatory T cells (Treg), natural killer (NK) cells, natural killer T (NKT) cells, macrophages (e.g., M1 and M2 macrophages), B cells, regulatory B (Breg) cells, neutrophils, monocytes, bone marrow-derived cells, and dendritic cells. In various embodiments, the targeting moiety has a recognition domain that specifically binds to a target of interest (e.g., antigen, receptor), including targets present on one or more cancer cells, endothelial cells, epithelial cells, mesenchymal cells, stromal cells, or other cell types characteristic and / or unique to a particular organ and / or tissue, including cell types specifically associated with a disease. In some embodiments, the recognition domain specifically binds to a target of interest (e.g., antigen, receptor) and effectively recruits one or more immune cells.

[0028] In some embodiments, the adjuvant, the chimeric protein, or the chimeric protein complex comprising an Fc-based chimeric protein complex may recruit immune cells to a site of action, e.g., immune cells capable of producing an anti-infectious effect or modulating other immune cells. In some embodiments, the adjuvant, the chimeric protein, or the chimeric protein complex comprising an Fc-based chimeric protein complex may modulate or recruit immune cells to a site of action.

[0029] In some embodiments, the present invention provides a method for treating a leukemia comprising administering to a subject an effective amount of an IL-1α signal transduction factor (IL-1α), pro-IL-1α, or a variant thereof (which is an example of a signal transduction factor described herein); (ii) one or more targeting moieties, the targeting moiety comprising a recognition domain that specifically binds to an antigen or receptor of interest; and (iii) a linker between (i) and (ii), the linker comprising: (1) an Fc domain, optionally reducing or eliminating one or more effector functions of the Fc domain and promoting Fc chain pairing in the Fc domain; and / or (ii) a linker between (i) and (ii), the linker comprising: (1) an Fc domain, the Fc domain optionally reducing or eliminating one or more effector functions of the Fc domain and promoting Fc chain pairing in the Fc domain; and / or (ii) a linker between (i) and (ii), the Fc domain and / or (ii) and (b) an adjuvant comprising a chimeric protein or chimeric protein complex comprising (i) an Fc domain having one or more mutations that stabilize the hinge region in the Fc domain that binds (i) and (ii), and / or (2) the linker being a flexible linker that connects (i) and (ii), wherein the mutant IL-1α or the pro-IL-1α is characterized by low or high affinity or activity at the IL-1 receptor, and (b) an antigen suitable for inducing an immune response.

[0030] 1A-F, 2A-H, 3A-H, 4A-D, 5A-F, 6A-J, 7A-D, 8A-F, 9A-J, 10A-F, 11A-L, 12A-L, 13A-F, 14A-L, 15A-L, 16A-J, 17A-J, 18A-F, and 19A-F show various non-limiting and exemplary schematic diagrams of chimeric protein complexes (e.g., Fc-based chimeric protein complexes) of the invention. In some embodiments, each schematic diagram is a composition of the invention. Where applicable in the figures, "TM" refers to a "targeting moiety" as described herein, "SA" refers to a "signaling agent" as described herein,

[0031] [ka]

[0032] is an optional "linker" as described herein, the two parallel long rectangles are, for example, human Fc domains derived from IgG1, IgG2, or IgG4 as described herein, and optionally with effector knockout and / or stabilizing mutations, also as described herein, and the two parallel long rectangles, one with a protrusion and the other with a recess, are, for example, human Fc domains derived from IgG1, IgG2, or IgG4 as described herein, with knob-in-hole and / or ion-pair (a / k / a charge pair, ion bond, or charged residue pair) mutations as described herein, and optionally with effector knockout and / or stabilizing mutations, also as described herein. [Brief explanation of the drawings]

[0033] [Figure 1A] 1 shows an example of a homodimeric two-chain complex. This figure shows an example of the structure of the homodimeric two-chain complex. [Figure 1B] 1 shows an example of a homodimeric two-chain complex. This figure shows an example of the structure of the homodimeric two-chain complex. [Figure 1C] 1 shows an example of a homodimeric two-chain complex. This figure shows an example of the structure of the homodimeric two-chain complex. [Figure 1D] 1 shows an example of a homodimeric two-chain complex. This figure shows an example of the structure of the homodimeric two-chain complex. [Figure 1E] 1 shows an example of a homodimeric two-chain complex. This figure shows an example of the structure of the homodimeric two-chain complex. [Figure 1F] 1 shows an example of a homodimeric two-chain complex. This figure shows an example of the structure of the homodimeric two-chain complex. [Figure 2A] 1 shows an example of a homodimeric two-chain complex with two targeting moieties (TM) (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. [Figure 2B]1 shows an example of a homodimeric two-chain complex with two targeting moieties (TM) (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. [Figure 2C] 1 shows an example of a homodimeric two-chain complex with two targeting moieties (TM) (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. [Figure 2D] 1 shows an example of a homodimeric two-chain complex with two targeting moieties (TM) (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. [Figure 2E] 1 shows an example of a homodimeric two-chain complex with two targeting moieties (TM) (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. [Figure 2F] 1 shows an example of a homodimeric two-chain complex with two targeting moieties (TM) (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. [Figure 2G] Shown are examples of homodimeric two-chain complexes with two targeting moieties (TM) (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, the boxed constructs have a signaling agent (SA) between TM1 and TM2 or between TM1 and Fc. [Figure 2H]Shown are examples of homodimeric two-chain complexes with two targeting moieties (TM) (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, the boxed constructs have a signaling agent (SA) between TM1 and TM2 or between TM1 and Fc. [Figure 3A] An example of a homodimeric two-chain complex with two signaling factors (in some embodiments, there may be more signaling factors, as described herein) is shown. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 3B] An example of a homodimeric two-chain complex with two signaling factors (in some embodiments, there may be more signaling factors, as described herein) is shown. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 3C] An example of a homodimeric two-chain complex with two signaling factors (in some embodiments, there may be more signaling factors, as described herein) is shown. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 3D] An example of a homodimeric two-chain complex with two signaling factors (in some embodiments, there may be more signaling factors, as described herein) is shown. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 3E] An example of a homodimeric two-chain complex with two signaling factors (in some embodiments, there may be more signaling factors, as described herein) is shown. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 3F]An example of a homodimeric two-chain complex with two signaling factors (in some embodiments, there may be more signaling factors, as described herein) is shown. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 3G] An example of a homodimeric two-chain complex with two signaling factors (in some embodiments, there may be more signaling factors, as described herein) is shown. In some embodiments, the positions of SA1 and SA2 are interchangeable. In some embodiments, the construct shown in the box has a TM between SA1 and SA2 or a TM at the N- or C-terminus. [Figure 3H] An example of a homodimeric two-chain complex with two signaling factors (in some embodiments, there may be more signaling factors, as described herein) is shown. In some embodiments, the positions of SA1 and SA2 are interchangeable. In some embodiments, the construct shown in the box has a TM between SA1 and SA2 or a TM at the N- or C-terminus. [Figure 4A] An example of a heterodimeric two-chain complex with split TM and SA chains, i.e., TM on the knob chain of Fc and SA on the hole chain of Fc, is shown. [Figure 4B] An example of a heterodimeric two-chain complex with split TM and SA chains, i.e., TM on the knob chain of Fc and SA on the hole chain of Fc, is shown. [Figure 4C] An example of a heterodimeric two-chain complex with split TM and SA chains, i.e., TM on the knob chain of Fc and SA on the hole chain of Fc, is shown. [Figure 4D] An example of a heterodimeric two-chain complex with split TM and SA chains, i.e., TM on the knob chain of Fc and SA on the hole chain of Fc, is shown. [Figure 5A]1 shows an example of a heterodimeric two-chain complex with split TM and SA chains, i.e., with both TM on the knob chain of Fc and SA on the hole chain of Fc, and with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 5B] 1 shows an example of a heterodimeric two-chain complex with split TM and SA chains, i.e., both TM on the knob chain of Fc and SA on the hole chain of Fc, and with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 5C] 1 shows an example of a heterodimeric two-chain complex with split TM and SA chains, i.e., both TM on the knob chain of Fc and SA on the hole chain of Fc, and with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 5D] 1 shows an example of a heterodimeric two-chain complex with split TM and SA chains, i.e., both TM on the knob chain of Fc and SA on the hole chain of Fc, and with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 5E]1 shows an example of a heterodimeric two-chain complex with split TM and SA chains, i.e., both TM on the knob chain of Fc and SA on the hole chain of Fc, and with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 5F] 1 shows an example of a heterodimeric two-chain complex with split TM and SA chains, i.e., both TM on the knob chain of Fc and SA on the hole chain of Fc, and with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 6A] Illustrated are examples of heterodimeric two-chain complexes with split TM and SA chains, i.e., TM on the knob chain of Fc and SA on the hole chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In these orientations / configurations, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 6B] Illustrated are examples of heterodimeric two-chain complexes with split TM and SA chains, i.e., TM on the knob chain of Fc and SA on the hole chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In these orientations / configurations, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 6C]Illustrated are examples of heterodimeric two-chain complexes with split TM and SA chains, i.e., TM on the knob chain of Fc and SA on the hole chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In these orientations / configurations, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 6D] Illustrated are examples of heterodimeric two-chain complexes with split TM and SA chains, i.e., TM on the knob chain of Fc and SA on the hole chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In these orientations / configurations, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 6E] Illustrated are examples of heterodimeric two-chain complexes with split TM and SA chains, i.e., TM on the knob chain of Fc and SA on the hole chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In these orientations / configurations, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 6F]Illustrated are examples of heterodimeric two-chain complexes with split TM and SA chains, i.e., TM on the knob chain of Fc and SA on the hole chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In these orientations / configurations, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 6G] Illustrated are examples of heterodimeric two-chain complexes with split TM and SA chains, i.e., TM on the knob chain of Fc and SA on the hole chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In these orientations / configurations, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 6H] Illustrated are examples of heterodimeric two-chain complexes with split TM and SA chains, i.e., TM on the knob chain of Fc and SA on the hole chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In these orientations / configurations, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 6I]Illustrated are examples of heterodimeric two-chain complexes with split TM and SA chains, i.e., TM on the knob chain of Fc and SA on the hole chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In these orientations / configurations, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 6J] Illustrated are examples of heterodimeric two-chain complexes with split TM and SA chains, i.e., TM on the knob chain of Fc and SA on the hole chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In these orientations / configurations, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 7A] An example of a heterodimeric two-chain complex with split TM and SA chains, i.e., SA on the knob chain of Fc and TM on the hole chain of Fc, is shown. [Figure 7B] An example of a heterodimeric two-chain complex with split TM and SA chains, i.e., SA on the knob chain of Fc and TM on the hole chain of Fc, is shown. [Figure 7C] An example of a heterodimeric two-chain complex with split TM and SA chains, i.e., SA on the knob chain of Fc and TM on the hole chain of Fc, is shown. [Figure 7D] An example of a heterodimeric two-chain complex with split TM and SA chains, i.e., SA on the knob chain of Fc and TM on the hole chain of Fc, is shown. [Figure 8A]1 shows an example of a heterodimeric two-chain complex with split TM and SA chains, i.e., SA on the knob chain of Fc and TM both on the hole chain of Fc, and with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 8B] 1 shows an example of a heterodimeric two-chain complex with split TM and SA chains, i.e., SA on the knob chain of Fc and TM both on the hole chain of Fc, and with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 8C] 1 shows an example of a heterodimeric two-chain complex with split TM and SA chains, i.e., SA on the knob chain of Fc and TM both on the hole chain of Fc, and with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 8D] 1 shows an example of a heterodimeric two-chain complex with split TM and SA chains, i.e., SA on the knob chain of Fc and TM both on the hole chain of Fc, and with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 8E]1 shows an example of a heterodimeric two-chain complex with split TM and SA chains, i.e., SA on the knob chain of Fc and TM both on the hole chain of Fc, and with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 8F] 1 shows an example of a heterodimeric two-chain complex with split TM and SA chains, i.e., SA on the knob chain of Fc and TM both on the hole chain of Fc, and with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 9A] Illustrated are examples of heterodimeric two-chain complexes with split TM and SA chains, i.e., SA on the knob chain of Fc and TM on the hole chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In these orientations / configurations, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 9B] Illustrated are examples of heterodimeric two-chain complexes with split TM and SA chains, i.e., SA on the knob chain of Fc and TM on the hole chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In these orientations / configurations, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 9C]Illustrated are examples of heterodimeric two-chain complexes with split TM and SA chains, i.e., SA on the knob chain of Fc and TM on the hole chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In these orientations / configurations, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 9D] Illustrated are examples of heterodimeric two-chain complexes with split TM and SA chains, i.e., SA on the knob chain of Fc and TM on the hole chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In these orientations / configurations, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 9E] Illustrated are examples of heterodimeric two-chain complexes with split TM and SA chains, i.e., SA on the knob chain of Fc and TM on the hole chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In these orientations / configurations, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 9F]Illustrated are examples of heterodimeric two-chain complexes with split TM and SA chains, i.e., SA on the knob chain of Fc and TM on the hole chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In these orientations / configurations, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 9G] Illustrated are examples of heterodimeric two-chain complexes with split TM and SA chains, i.e., SA on the knob chain of Fc and TM on the hole chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In these orientations / configurations, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 9H] Illustrated are examples of heterodimeric two-chain complexes with split TM and SA chains, i.e., SA on the knob chain of Fc and TM on the hole chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In these orientations / configurations, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 9I]Illustrated are examples of heterodimeric two-chain complexes with split TM and SA chains, i.e., SA on the knob chain of Fc and TM on the hole chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In these orientations / configurations, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 9J] Illustrated are examples of heterodimeric two-chain complexes with split TM and SA chains, i.e., SA on the knob chain of Fc and TM on the hole chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In these orientations / configurations, one SA is on the knob chain and one SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 10A] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc. [Figure 10B] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc. [Figure 10C] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc. [Figure 10D] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc. [Figure 10E] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc. [Figure 10F] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc. [Figure 11A] 1 shows an example of a heterodimeric two-chain complex with TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 11B] 1 shows an example of a heterodimeric two-chain complex with TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 11C] 1 shows an example of a heterodimeric two-chain complex with TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 11D] 1 shows an example of a heterodimeric two-chain complex with TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 11E]1 shows an example of a heterodimeric two-chain complex with TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 11F] 1 shows an example of a heterodimeric two-chain complex with TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 11G] 1 shows an example of a heterodimeric two-chain complex with TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 11H] 1 shows an example of a heterodimeric two-chain complex with TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 11I]1 shows an example of a heterodimeric two-chain complex with TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 11J] 1 shows an example of a heterodimeric two-chain complex with TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 11K] 1 shows an example of a heterodimeric two-chain complex with TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 11L] 1 shows an example of a heterodimeric two-chain complex with TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 12A] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the knob chain of the Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 12B] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 12C] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 12D] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 12E] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 12F] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 12G]An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 12H] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 12I] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 12J] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 12K] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 12L]An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the knob chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 13A] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the hole chain of Fc. [Figure 13B] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the hole chain of Fc. [Figure 13C] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the hole chain of Fc. [Figure 13D] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the hole chain of Fc. [Figure 13E] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the hole chain of Fc. [Figure 13F] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the hole chain of Fc. [Figure 14A] 1 shows an example of a heterodimeric two-chain complex with TM and SA on the same chain, i.e., both SA and TM on the whole chain of Fc, and with two targeting moieties (in some embodiments, there are more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 14B]1 shows an example of a heterodimeric two-chain complex with TM and SA on the same chain, i.e., both SA and TM on the whole chain of Fc, and with two targeting moieties (in some embodiments, there are more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 14C] 1 shows an example of a heterodimeric two-chain complex with TM and SA on the same chain, i.e., both SA and TM on the whole chain of Fc, and with two targeting moieties (in some embodiments, there are more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 14D] 1 shows an example of a heterodimeric two-chain complex with TM and SA on the same chain, i.e., both SA and TM on the whole chain of Fc, and with two targeting moieties (in some embodiments, there are more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 14E] 1 shows an example of a heterodimeric two-chain complex with TM and SA on the same chain, i.e., both SA and TM on the whole chain of Fc, and with two targeting moieties (in some embodiments, there are more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 14F]1 shows an example of a heterodimeric two-chain complex with TM and SA on the same chain, i.e., both SA and TM on the whole chain of Fc, and with two targeting moieties (in some embodiments, there are more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 14G] 1 shows an example of a heterodimeric two-chain complex with TM and SA on the same chain, i.e., both SA and TM on the whole chain of Fc, and with two targeting moieties (in some embodiments, there are more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 14H] 1 shows an example of a heterodimeric two-chain complex with TM and SA on the same chain, i.e., both SA and TM on the whole chain of Fc, and with two targeting moieties (in some embodiments, there are more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 14I] 1 shows an example of a heterodimeric two-chain complex with TM and SA on the same chain, i.e., both SA and TM on the whole chain of Fc, and with two targeting moieties (in some embodiments, there are more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 14J]1 shows an example of a heterodimeric two-chain complex with TM and SA on the same chain, i.e., both SA and TM on the whole chain of Fc, and with two targeting moieties (in some embodiments, there are more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 14K] 1 shows an example of a heterodimeric two-chain complex with TM and SA on the same chain, i.e., both SA and TM on the whole chain of Fc, and with two targeting moieties (in some embodiments, there are more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 14L] 1 shows an example of a heterodimeric two-chain complex with TM and SA on the same chain, i.e., both SA and TM on the whole chain of Fc, and with two targeting moieties (in some embodiments, there are more targeting moieties, as described herein). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 15A] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the whole chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 15B] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the whole chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 15C]An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the whole chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 15D] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the whole chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 15E] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the whole chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 15F] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the whole chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 15G] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the whole chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 15H]An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the whole chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 15I] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the whole chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 15J] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the whole chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 15K] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the whole chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 15L] An example of a heterodimeric two-chain complex is shown, with the TM and SA on the same chain, i.e., both SA and TM on the whole chain of Fc, and with two signaling factors (in some embodiments, there may be more signaling factors, as described herein). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 16A]1 shows an example of a heterodimeric two-chain complex with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein), and with an SA on the knob Fc and a TM on each chain. In some embodiments, TM1 and TM2 may be the same. [Figure 16B] 1 shows an example of a heterodimeric two-chain complex with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein), and with an SA on the knob Fc and a TM on each chain. In some embodiments, TM1 and TM2 may be the same. [Figure 16C] 1 shows an example of a heterodimeric two-chain complex with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein), and with an SA on the knob Fc and a TM on each chain. In some embodiments, TM1 and TM2 may be the same. [Figure 16D] 1 shows an example of a heterodimeric two-chain complex with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein), and with an SA on the knob Fc and a TM on each chain. In some embodiments, TM1 and TM2 may be the same. [Figure 16E] 1 shows an example of a heterodimeric two-chain complex with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein), and with an SA on the knob Fc and a TM on each chain. In some embodiments, TM1 and TM2 may be the same. [Figure 16F] 1 shows an example of a heterodimeric two-chain complex with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein), and with an SA on the knob Fc and a TM on each chain. In some embodiments, TM1 and TM2 may be the same. [Figure 16G]1 shows an example of a heterodimeric two-chain complex with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein), and with an SA on the knob Fc and a TM on each chain. In some embodiments, TM1 and TM2 may be the same. [Figure 16H] 1 shows an example of a heterodimeric two-chain complex with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein), and with an SA on the knob Fc and a TM on each chain. In some embodiments, TM1 and TM2 may be the same. [Figure 16I] 1 shows an example of a heterodimeric two-chain complex with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein), and with an SA on the knob Fc and a TM on each chain. In some embodiments, TM1 and TM2 may be the same. [Figure 16J] 1 shows an example of a heterodimeric two-chain complex with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein), and with an SA on the knob Fc and a TM on each chain. In some embodiments, TM1 and TM2 may be the same. [Figure 17A] 1 shows an example of a heterodimeric two-chain complex with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein), and with an SA on the whole Fc and a TM on each chain. In some embodiments, TM1 and TM2 may be the same. [Figure 17B] 1 shows an example of a heterodimeric two-chain complex with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein), and with an SA on the whole Fc and a TM on each chain. In some embodiments, TM1 and TM2 may be the same. [Figure 17C]1 shows an example of a heterodimeric two-chain complex with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein), and with an SA on the whole Fc and a TM on each chain. In some embodiments, TM1 and TM2 may be the same. [Figure 17D] 1 shows an example of a heterodimeric two-chain complex with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein), and with an SA on the whole Fc and a TM on each chain. In some embodiments, TM1 and TM2 may be the same. [Figure 17E] 1 shows an example of a heterodimeric two-chain complex with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein), and with an SA on the whole Fc and a TM on each chain. In some embodiments, TM1 and TM2 may be the same. [Figure 17F] 1 shows an example of a heterodimeric two-chain complex with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein), and with an SA on the whole Fc and a TM on each chain. In some embodiments, TM1 and TM2 may be the same. [Figure 17G] 1 shows an example of a heterodimeric two-chain complex with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein), and with an SA on the whole Fc and a TM on each chain. In some embodiments, TM1 and TM2 may be the same. [Figure 17H] 1 shows an example of a heterodimeric two-chain complex with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein), and with an SA on the whole Fc and a TM on each chain. In some embodiments, TM1 and TM2 may be the same. [Figure 17I]1 shows an example of a heterodimeric two-chain complex with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein), and with an SA on the whole Fc and a TM on each chain. In some embodiments, TM1 and TM2 may be the same. [Figure 17J] 1 shows an example of a heterodimeric two-chain complex with two targeting moieties (in some embodiments, there may be more targeting moieties, as described herein), and with an SA on the whole Fc and a TM on each chain. In some embodiments, TM1 and TM2 may be the same. [Figure 18A] An example of a heterodimeric two-chain complex is shown, having two signaling factors (in some embodiments, there may be more signaling factors as described herein) and having split SA and TM chains, i.e., SA on the knob Fc and TM on the hole Fc. [Figure 18B] An example of a heterodimeric two-chain complex is shown, having two signaling factors (in some embodiments, there may be more signaling factors as described herein) and having split SA and TM chains, i.e., SA on the knob Fc and TM on the hole Fc. [Figure 18C] An example of a heterodimeric two-chain complex is shown, having two signaling factors (in some embodiments, there may be more signaling factors as described herein) and having split SA and TM chains, i.e., SA on the knob Fc and TM on the hole Fc. [Figure 18D] An example of a heterodimeric two-chain complex is shown, having two signaling factors (in some embodiments, there may be more signaling factors as described herein) and having split SA and TM chains, i.e., SA on the knob Fc and TM on the hole Fc. [Figure 18E]An example of a heterodimeric two-chain complex is shown, having two signaling factors (in some embodiments, there may be more signaling factors as described herein) and having split SA and TM chains, i.e., SA on the knob Fc and TM on the hole Fc. [Figure 18F] An example of a heterodimeric two-chain complex is shown, having two signaling factors (in some embodiments, there may be more signaling factors as described herein) and having split SA and TM chains, i.e., SA on the knob Fc and TM on the hole Fc. [Figure 19A] An example of a heterodimeric two-chain complex is shown, having two signaling factors (in some embodiments, there may be more signaling factors as described herein) and having split SA and TM chains, i.e., TM on the knob Fc and SA on the hole Fc. [Figure 19B] An example of a heterodimeric two-chain complex is shown, having two signaling factors (in some embodiments, there may be more signaling factors as described herein) and having split SA and TM chains, i.e., TM on the knob Fc and SA on the hole Fc. [Figure 19C] An example of a heterodimeric two-chain complex is shown, having two signaling factors (in some embodiments, there may be more signaling factors as described herein) and having split SA and TM chains, i.e., TM on the knob Fc and SA on the hole Fc. [Figure 19D] An example of a heterodimeric two-chain complex is shown, having two signaling factors (in some embodiments, there may be more signaling factors as described herein) and having split SA and TM chains, i.e., TM on the knob Fc and SA on the hole Fc. [Figure 19E]An example of a heterodimeric two-chain complex is shown, having two signaling factors (in some embodiments, there may be more signaling factors as described herein) and having split SA and TM chains, i.e., TM on the knob Fc and SA on the hole Fc. [Figure 19F] An example of a heterodimeric two-chain complex is shown, having two signaling factors (in some embodiments, there may be more signaling factors as described herein) and having split SA and TM chains, i.e., TM on the knob Fc and SA on the hole Fc. [Figure 20] NF-κB reporter activity in HEK293T cells treated with recombinant IL1α or recombinant Fc-IL1α is shown. [Figure 21] Figure 1 shows NF-κB reporter activity in HEK293T cells with and without CD8 expression treated with recombinant IL1α or recombinant Fc-IL1α. Note that anti-CD8 VHH-Fc-IL1α is more potent than approximately 0.01 nM recombinant IL1α (boxed area). [Figure 22A] Figure 1 shows NF-κB reporter activity in CD8-expressing and non-expressing HEK293T cells treated with recombinant anti-CD8 VHH-Fc-IL-1α carrying the C141 mutation. [Figure 22B] Figure 1 shows NF-κB reporter activity in CD8-expressing and non-expressing HEK293T cells treated with recombinant anti-CD8 VHH-Fc-IL-1α carrying the C141 mutation. [Figure 22C] Figure 1 shows NF-κB reporter activity in CD8-expressing and non-expressing HEK293T cells treated with recombinant anti-CD8 VHH-Fc-IL-1α carrying the C141 mutation. [Figure 23A] Figure 1 shows NF-κB reporter activity in HEK293T cells expressing and not expressing CD8 treated with recombinant anti-CD8 VHH-Fc-IL1α carrying the A44T, N29A, or N29G mutations. [Figure 23B]Figure 1 shows NF-κB reporter activity in HEK293T cells expressing and not expressing CD8 treated with recombinant anti-CD8 VHH-Fc-IL-1α carrying the A44T, N29A, or N29G mutations. [Figure 23C] Figure 1 shows NF-κB reporter activity in HEK293T cells expressing and not expressing CD8 treated with recombinant anti-CD8 VHH-Fc-IL-1α carrying the A44T, N29A, or N29G mutations. [Figure 23D] Figure 1 shows NF-κB reporter activity in HEK293T cells expressing and not expressing CD8 treated with recombinant anti-CD8 VHH-Fc-IL-1α carrying the A44T, N29A, or N29G mutations. [Figure 24A] Figure 1 shows NF-κB reporter activity in CD8-expressing and non-expressing HEK293T cells treated with recombinant anti-CD8 VHH-Fc-IL1α carrying the D151 mutation. [Figure 24B] Figure 1 shows NF-κB reporter activity in CD8-expressing and non-expressing HEK293T cells treated with recombinant anti-CD8 VHH-Fc-IL-1α carrying the D151 mutation. [Figure 24C] Figure 1 shows NF-κB reporter activity in CD8-expressing and non-expressing HEK293T cells treated with recombinant anti-CD8 VHH-Fc-IL-1α carrying the D151 mutation. [Figure 25A] Figure 1 shows NF-κB reporter activity in CD8-expressing and non-expressing HEK293T cells treated with recombinant anti-CD8 VHH-Fc-IL1α carrying the M15G mutation. [Figure 25B] Figure 1 shows NF-κB reporter activity in CD8-expressing and non-expressing HEK293T cells treated with recombinant anti-CD8 VHH-Fc-IL1α carrying the R16G mutation. [Figure 25C] Figure 1 shows NF-κB reporter activity in CD8-expressing and non-expressing HEK293T cells treated with recombinant anti-CD8 VHH-Fc-IL-1α carrying the I18G mutation. [Figure 26A]Figure 1 shows NF-κB reporter activity in CD8-expressing and non-expressing HEK293T cells treated with recombinant anti-CD8 VHH-Fc-IL1α carrying the R16A mutation. [Figure 26B] Figure 1 shows NF-κB reporter activity in CD8-expressing and non-expressing HEK293T cells treated with recombinant anti-CD8 VHH-Fc-IL-1α carrying the I18A mutation. [Figure 26C] Figure 1 shows NF-κB reporter activity in CD8-expressing and non-expressing HEK293T cells treated with recombinant anti-CD8 VHH-Fc-IL-1α carrying the H46A mutation. [Figure 26D] Figure 1 shows NF-κB reporter activity in CD8-expressing and non-expressing HEK293T cells treated with recombinant anti-CD8 VHH-Fc-IL-1α carrying the H46G mutation. [Figure 26E] Figure 1 shows NF-κB reporter activity in CD8-expressing and non-expressing HEK293T cells treated with recombinant anti-CD8 VHH-Fc-IL-1α carrying the A58H mutation. [Figure 26F] Figure 1 shows NF-κB reporter activity in CD8-expressing and non-expressing HEK293T cells treated with recombinant anti-CD8 VHH-Fc-IL-1α carrying the A58N mutation. [Figure 26G] Figure 1 shows NF-κB reporter activity in CD8-expressing and non-expressing HEK293T cells treated with recombinant anti-CD8 VHH-Fc-IL-1α carrying the I68G mutation. [Figure 26H] Figure 1 shows NF-κB reporter activity in CD8-expressing and non-expressing HEK293T cells treated with recombinant anti-CD8 VHH-Fc-IL-1α carrying the D64G / D65A mutations. [Figure 27A] Figure 1 shows the biological activity of CD8-targeted human IL-1α Fc-constructs with the del1-6 mutation, with or without the C141 mutation. [Figure 27B]Figure 1 shows the biological activity of CD8-targeted human IL-1α Fc-constructs with the del1-6 mutation, with or without the C141 mutation. [Figure 27C] Figure 1 shows the biological activity of CD8-targeted human IL-1α Fc-constructs with the del1-6 mutation, with or without the C141 mutation. [Figure 27D] Figure 1 shows the biological activity of CD8-targeted human IL-1α Fc-constructs with the del1-6 mutation, with or without the C141 mutation. [Figure 27E] Figure 1 shows the biological activity of CD8-targeted human IL-1α Fc-constructs with the del1-6 mutation, with or without the C141 mutation. [Figure 27F] Figure 1 shows the biological activity of CD8-targeted human IL-1α Fc-constructs with the del1-6 mutation, with or without the C141 mutation. [Figure 28A] Figure 1 shows the biological activity of CD8-targeted human IL-1α Fc-constructs carrying the del1-6 mutation with an additional mutation at S31 (PC = parental HekBlue-IL-1β cell line; hCD8 = HekBlue-IL-1β cell line stably transfected with CD8). [Figure 28B] Figure 1 shows the biological activity of CD8-targeted human IL-1α Fc-constructs carrying the del1-6 mutation with an additional mutation at C141 (PC = parental HekBlue-IL-1β cell line; hCD8 = HekBlue-IL-1β cell line stably transfected with CD8). [Figure 28C] Figure 1 shows the biological activity of CD8-targeted human IL-1α Fc-constructs carrying the del1-6 mutation with an additional mutation at C141 (PC = parental HekBlue-IL-1β cell line; hCD8 = HekBlue-IL-1β cell line stably transfected with CD8). [Figure 28D]Figure 1 shows the biological activity of CD8-targeted human IL-1α Fc-constructs carrying the del1-6 mutation with an additional mutation at N29 (PC = parental HekBlue-IL-1β cell line; hCD8 = HekBlue-IL-1β cell line stably transfected with CD8). [Figure 28E] Figure 1 shows the biological activity of CD8-targeted human IL-1α Fc-constructs carrying the del1-6 mutation with an additional mutation at N29 (PC = parental HekBlue-IL-1β cell line; hCD8 = HekBlue-IL-1β cell line stably transfected with CD8). [Figure 28F] Figure 1 shows the biological activity of CD8-targeted human IL-1α Fc-constructs carrying the del1-6 mutation with an additional mutation at N29 (PC = parental HekBlue-IL-1β cell line; hCD8 = HekBlue-IL-1β cell line stably transfected with CD8). [Figure 28G] Figure 1 shows the biological activity of CD8-targeted human IL-1α Fc-constructs carrying the del1-6 mutation with an additional mutation at S31 (PC = parental HekBlue-IL-1β cell line; hCD8 = HekBlue-IL-1β cell line stably transfected with CD8). [Figure 28H] Figure 1 shows the biological activity of CD8-targeted human IL-1α Fc-constructs carrying the del1-6 mutation with an additional mutation at S31 (PC = parental HekBlue-IL-1β cell line; hCD8 = HekBlue-IL-1β cell line stably transfected with CD8). [Figure 29A] Figure 1 shows the biological activity of CD8-targeted human IL-1α Fc-constructs with C141 mutation, del1-6 mutation and N29 mutation. [Figure 29B] Figure 1 shows the biological activity of CD8-targeted human IL-1α Fc-constructs with C141 mutation, del1-6 mutation and N29 mutation. [Figure 29C] Figure 1 shows the biological activity of CD8-targeted human IL-1α Fc-constructs with C141 mutation, del1-6 mutation and N29 mutation. [Figure 29D] Figure 1 shows the biological activity of CD8-targeted human IL-1α Fc-constructs with C141 mutation, del1-6 mutation and N29 mutation. [Figure 29E] Figure 1 shows the biological activity of CD8-targeted human IL-1α Fc-constructs with C141 mutation, del1-6 mutation and N29 mutation. [Figure 29F] Figure 1 shows the biological activity of CD8-targeted human IL-1α Fc-constructs with C141 mutation, del1-6 mutation and N29 mutation. [Figure 29G] Figure 1 shows the biological activity of CD8-targeted human IL-1α Fc-constructs with C141 mutation, del1-6 mutation and S31 mutation. [Figure 29H] Figure 1 shows the biological activity of CD8-targeted human IL-1α Fc-constructs with C141 mutation, del1-6 mutation and S31 mutation. [Figure 29I] Figure 1 shows the biological activity of CD8-targeted human IL-1α Fc-constructs with C141 mutation, del1-6 mutation and S31 mutation. [Figure 29J] Figure 1 shows the biological activity of CD8-targeted human IL-1α Fc-constructs with C141 mutation, del1-6 mutation and S31 mutation. [Figure 30] 1 shows the biological activity of CD8-targeted Fc-IL-1α variants with IL-1α fused to the C-terminus or N-terminus on parental HekBlue-IL-1β cells (PC) and CD8+ HekBlue-IL-1β cells. [Figure 31] 1 shows the biological activity of CD8-targeted single peptide IL-1α variants on parental HekBlue-IL-1β cells (PC) and CD8+ HekBlue-IL-1β cells. [Figure 32] Figure 1 shows the biological activity of CD8-targeted Fc-IL-1α(Δ1-6) C141H or M15G using different CD8-targeting domains (VHH or OKT8 scFv) on mock- or CD8-transiently transfected HEK293T cells. [Figure 33]1 shows the biological activity of monovalent and bivalent CD8-targeted Fc-IL-1α variants on parental HekBlue-IL-1β cells (PC) and CD8+ HekBlue-IL-1β cells. [Figure 34] 1 shows the biological activity of NKp46-targeted Fc-IL-1α variants on isolated NK cells. DETAILED DESCRIPTION OF THE INVENTION

[0034] In one aspect, the present invention provides a chimeric protein, chimeric protein complex, vaccine composition, or adjuvant comprising interleukin-1α (IL-1α), pro-IL-1α, or a variant thereof. IL-1α is a proinflammatory cytokine and an important immune system regulator. IL-1α is constitutively expressed as a 31 kDa precursor by epithelial cells, endothelial cells, and keratinocytes. Unlike IL-1β, IL-1α exhibits basal activity in its immature, unprocessed form (i.e., pro-IL-1α exhibits basal activity). IL-1α is released from damaged cells and binds to the IL-1R1 receptor (see Afonina et al., Immunity 42, 991-1004, 2015). IL-1α expression is rapidly upregulated by a variety of molecular patterns associated with danger and pathogens. IL-1α is highly promiscuous in its intracellular localization, and upon secretion from these cells, it functions as an IL-1R agonist or as a membrane-bound molecule. While the other IL-1R ligand, IL-1β, requires inflammasome activation for its maturation and pyroptosis for its secretion, IL-1α is bioavailable in a broader range of cellular scenarios. Because IL-1α is bioactive in both its pro- and mature forms, both pyroptotic and necrotic cell death produce bioactive IL-1α.

[0035] IL-1α is translated as pro-IL-1α, and this precursor form undergoes several post-translational modifications. Specifically, pro-IL-1α is phosphorylated at Ser90, myristoylated at Lys82, and acetylated at Lys82 (Di Paolo et al., Nat Immunol. 2016 July 19;17(8):906-913). Cleavage of human pro-IL-1α at Phe118 is mediated by calpain, a calcium-dependent neutral protease. Calpain can cleave pro-IL-1α in cells or under cell-free conditions.

[0036] In various embodiments, the IL-1α of the present invention comprises a truncated pro-IL-1α that exhibits activity toward or binds to an IL-1α receptor. For example, in one embodiment, the IL-1α of the present invention comprises amino acids 128 to 267 of pro-IL-1α having the amino acid sequence of SEQ ID NO: 2 or 4 (e.g., as reported by Mosley, Bruce, et al., "Determination of the minimum polypeptide lengths of the functionally active sites of human interleukins 1 alpha and 1 beta," PNAS 84.13 (1987): 4572-4576, which is incorporated herein by reference in its entirety). In some embodiments, the truncated pro-IL-1α is truncated to obtain, for example, a truncated IL-1α having an N-terminus at N102, S104, S117, or L119 of the amino acid sequence of SEQ ID NO: 2 or 4 (e.g., as reported by Afonina, et al., "Granzyme B-dependent Proteolysis Acts as a Switch to Enhance the Proinflammatory Activity of IL-1α," Mol Cell. 44:265-278 (2011) and Afonina et al., "Proteolytic Processing of Interleukin-1 Family Cytokines: Variations on a Common Theme," Immunity 42.6 (2015): 991-1004 (2015) (which are incorporated herein by reference in their entireties)).

[0037] In various embodiments, the present invention provides chimeric protein complexes, such as chimeric proteins or Fc-based chimeric protein complexes, comprising IL-1α or variants thereof fused to one or more targeting moieties. In some embodiments, the present invention provides chimeric protein complexes, such as chimeric proteins or Fc-based chimeric protein complexes, comprising pro-IL-1α or variants thereof fused to one or more targeting moieties.

[0038] In some embodiments, the mutant IL-1α or mutant pro-IL-1α is human IL-1α or human pro-IL-1α. In some embodiments, the mutant IL-1α or mutant pro-IL-1α has reduced or increased affinity and / or activity for the IL-1 receptor. In some embodiments, the affinity and / or activity of the mutant IL-1α or mutant pro-IL-1α for the IL-1 receptor is substantially reduced, eliminated, or increased. In some embodiments, the reduced affinity or activity of the mutant IL-1α or mutant pro-IL-1α at the IL-1 receptor is reversible and / or inducible by binding to one or more targeting moieties or by inclusion in a chimeric protein complex.

[0039] In one embodiment, the wild-type IL-1α is IL-1α (wild type) (SEQ ID NO: 1) SAPFSFLSNVKYNFMRIIKYEFILNDALNQSIIRANDQYLTAAALHNLDEAVKFDMGAYKSSKDDAKITVILRISKTQLYVTAQDEDQPVLLKEMPEIPKTITGSETNLIFFWETHGTKNYFTSVAHPNLFIATKQDYWVCLAGGPPSITDFQILENQA It has the amino acid sequence:

[0040] In one embodiment, the wild-type pro-IL-1α is pro-IL-1α (wild type) (SEQ ID NO: 2) MAKVPDMFEDLKNCYSENEEDSSSSIDHLSLNQKSFYHVSYGPLHEGCMDQSVSLSISETSKTSKLTFKESMVVVATNGKVLKKRRLSLSQSITDDDLEAIANDSEEEIIKPRSAPFSFLSNVKYNFMRIIKYEFI LNDALNQSIIRANDQYLTAAALHNLDEAVKFDMGAYKSSKDDAKITVILRISKTQLYVTAQDEDQPVLLKEMPEIPKTITGSETNLIFFWETHGTKNYFTSVAHPNLFIATKQDYWVCLAGGPPSITDFQILENQA It has the amino acid sequence:

[0041] In one embodiment, the wild-type IL-1α is IL-1α (wild type) (SEQ ID NO: 3) SAPFSFLSNVKYNFMRIIKYEFILNDALNQSIIRANDQYLTAAALHNLDEAVKFDMGAYKSSKDDAKITVILRISKTQLYVTAQDEDQPVLLKEMPEIPKTITGSETNLLFFWETHGTKNYFTSVAHPNLFIATKQDYWVCLAGGPPSITDFQILENQA It has the amino acid sequence:

[0042] In one embodiment, the wild-type pro-IL-1α is pro-IL-1α (wild type) (SEQ ID NO: 4) MAKVPDMFEDLKNCYSENEEDSSSSIDHLSLNQKSFYHVSYGPLHEGCMDQSVSLSISETSKTSKLTFKESMVVVATNGKVLKKRRLSLSQSITDDDLEAIANDSEEEIIKPRSAPFSFLSNVKYNFMRIIKYEFI LNDALNQSIIRANDQYLTAAALHNLDEAVKFDMGAYKSSKDDAKITVILRISKTQLYVTAQDEDQPVLLKEMPEIPKTITGSETNLLFFWETHGTKNYFTSVAHPNLFIATKQDYWVCLAGGPPSITDFQILENQA It has the amino acid sequence:

[0043] In some embodiments, the mutant human IL-1α has an amino acid sequence at least 95%, or 96%, or 97%, or 98%, or 99% identical to SEQ ID NO: 1 or 3. In some embodiments, the mutant human pro-IL-1α has an amino acid sequence at least 95%, or 96%, or 97%, or 98%, or 99% identical to SEQ ID NO: 2 or 4.

[0044] In some embodiments, the human IL-1α has the amino acid sequence of wild-type human IL-1α.

[0045] In some embodiments, the mutant IL-1α has a del1-6 (i.e., "Δ1-6") mutation with respect to SEQ ID NO: 1 or 3, wherein the del1-6 mutation is a deletion of amino acid residues numbered 1 to 6 of SEQ ID NO: 1 or 3.

[0046] In some embodiments, the mutant pro-IL-1α has a del113-118 (i.e., "Δ113-118") mutation with respect to SEQ ID NO: 2 or 4, where the del113-118 mutation is a deletion of amino acid residues numbered 113 to 118 of SEQ ID NO: 2 or 4.

[0047] In some embodiments, the mutant IL-1α has one or more substitution mutations selected from C141, N29, S31, P3, M15, R16, I17, I18, L24, N25, D26, L28, I33, L40, A44, H46, V52, F54, M56, A58, Y59, K60, D64, D65, K67, I68, V70, L72, L79, Y80, P89, L91, E94, P99, K100, E106, F111, W113, K119, S124, P128, I132, Q136, T134, V140, L142, D151, F152, Q153 (wherein the numbering of the residues is based on SEQ ID NO: 1 or 3). In some embodiments, one or more of these mutations result in a modified human IL-1α that has reduced or increased binding affinity to type I IL-1 and reduced or increased biological activity. In some embodiments, the mutant IL-1α includes any of the following: C141A, C141S, N29A, N29D, N29G, S31A, S31G, M15A, M15G, M15S, R16A, R16K, R16G, I18A, I18G, I18L, L24K, L24S, N25A, N25G, D26V, L28A, L28G, I33A, I33G, A44G, A44S, A44T, A44N, A44H, H46A, H46G, A58G, A58S, A58T, and having one or more substitution mutations selected from A58N, A58H, A58F, Y59A, K60A, K60G, D64A, D64G, D65A, K67A, I68A, I68G, V70A, Y80A, K100A, K100D, W113F, Q136A, Q136C, C141H, D151A, D151K, D151Y, F152Q, F152N, F152S, Q153A, and Q153G (wherein the numbering of the above residues is based on SEQ ID NO: 1 or 3).

[0048] In some embodiments, the mutant pro-IL-1α comprises the following amino acids: N141, S143, P115, M127, R128, I129, I130, L136, N137, D138, L140, I145, L152, A156, H158, V164, F166, M168, A170, A171, K172, D176, D177, K179, I180, V182, L183, L184, L185, L186, L187, L188, L189, L190, L191, L192, L193, L194, L195, L196, L197, L198, L199, L200, L201, L202, L203, L204, L205, L206, L207, L210, L211, L212, L213, L214, L215, L216, L217, L220, L225, L226, L227, L230, L231, L232, L233, L234, L235, L240, L241, L242, L243, L244, L245, L252, L256, L258, L260, L262, L264, L266, L268, L270, L271, L272, L273, L274, L275, L276, L277, L280, L282, L283, L284, L285, L290, L291, L29 184, L191, Y192, P201, L203, E206, P211, K212, E218, F223, W225, K231, S236, P240, I244, Q248, T246, V252, C253, L254, D263, F264, Q265 (wherein the numbering of these residues is based on SEQ ID NO: 2 or 4). In some embodiments, one or more of these mutations results in a modified human pro-IL-1α that has reduced or increased binding affinity to type I IL-1 and reduced or increased biological activity. In some embodiments, the mutant pro-IL-1α is selected from the group consisting of N141A, N141D, N141G, S143A, S143G, M127S, R128A, R128K, I130A, I130L, L136K, L136S, N137A, N137G, D138V, L140A, L140G, A156G, A156S, A156T, A156N, A156H, H158A, H158G, A170G, A170S, A170T, A170N, A170H, A170S, A170T ... 0T, A170N, A170H, A170F, Y171A, K172A, K172G, D176A, D176G, D177A, I180A, I80G, V182A, Y192A, K212A, K212D, W225F, Q248C, D263K, F264Q, F264N, F264S, Q265A, and Q265G (wherein the numbering of the above residues is based on SEQ ID NO: 2 or 4).

[0049] In embodiments, the one or more additional mutations result in decreased or increased activity compared to IL-1α having the amino acid sequence of SEQ ID NO: 1 or 3 with a deletion of amino acids 1-6 (Δ1-6), or compared to pro-IL-1α having the amino acid sequence of SEQ ID NO: 2 or 4 with a deletion of amino acids 113-118 (Δ113-118). In such embodiments, the one or more mutations in IL-1α or pro-IL-1α result in decreased or increased activity that is reversible and / or inducible by binding to one or more targeting moieties or by inclusion in the chimeric protein or chimeric protein complex.

[0050] In other embodiments, the one or more additional mutations increase activity compared to IL-1α having the amino acid sequence of SEQ ID NO: 1 or 3 with a deletion of amino acids 1 to 6 (Δ1-6), and optionally the one or more additional mutations are selected from amino acid substitutions at positions selected from N29 and S31 with respect to SEQ ID NO: 1 or 3, and optionally the substitutions are selected from N29A, N29D, N29G, S31A, and S31G.

[0051] In other embodiments, the one or more additional mutations result in increased activity compared to pro-IL-1α having the amino acid sequence of SEQ ID NO: 2 or 4, which has a deletion of amino acids 113 to 118 (Δ113-118), and optionally the one or more additional mutations are selected from amino acid substitutions at positions selected from N141 and S143 with respect to SEQ ID NO: 2 or 4, and optionally the substitutions are selected from N141A, N141D, N141G, S143A, and S143G.

[0052] In some embodiments, the one or more additional mutations increase the activity of the chimeric proteins and chimeric protein complexes of the present application. In such embodiments, the present application provides chimeric proteins and chimeric protein complexes that have at least about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 100-fold, at least about 150-fold, or about 10-50-fold, about 50-100-fold, about 100-150-fold, about 150-200-fold, or more than 200-fold greater biological activity when comparing biological activity on a plurality of cells that express or overexpress the antigen targeted by the targeting moiety to a plurality of cells that do not express or barely express the antigen targeted by the targeting moiety.

[0053] In various embodiments, the mutations allow for one or more of the following to be attenuated or reduced: one or more of reduced binding affinity, reduced intrinsic activity, and reduced specific biological activity of IL-1α or pro-IL-1α compared to the unaltered or unmutated, i.e., wild-type form of IL-1α or pro-IL-1α (e.g., comparing an altered (e.g., mutant) form of IL-1α to the wild-type form of IL-1α), or compared to wild-type IL-1α or wild-type pro-IL-1α or Fc IL-1α or Fc pro-IL-1α or Fc IL-1α(Δ1-6) or Fc pro-IL-1α(Δ113-118) or targeted Fc IL-1α(Δ1-6) or targeted Fc pro-IL-1α(Δ113-118). In some embodiments, mutations that attenuate or reduce binding or affinity include mutations that substantially reduce or eliminate binding or activity. In some embodiments, the mutations that attenuate or reduce binding or affinity are different from mutations that substantially reduce or eliminate binding or activity. As a result, in various embodiments, the mutations allow for improved safety of IL-1α, e.g., reduced systemic toxicity, reduced side effects, and reduced off-target effects, compared to unmutated, i.e., wild-type, IL-1α (e.g., comparing an altered (e.g., mutant) form of IL-1α to a wild-type form of IL-1α), or compared to wild-type IL-1α or wild-type pro-IL-1α or Fc IL-1α or Fc pro-IL-1α or Fc IL-1α(Δ1-6) or Fc pro-IL-1α(Δ113-118) or targeted Fc IL-1α(Δ1-6) or targeted Fc pro-IL-1α(Δ113-118).

[0054] In various embodiments, the mutations allow for an increase in the activity of IL-1α or pro-IL-1α compared to the unaltered or unmutated, i.e., wild-type, form of IL-1α or pro-IL-1α (e.g., comparing the modified (e.g., mutant) form of IL-1α to the wild-type form of IL-1α), or compared to wild-type IL-1α or wild-type pro-IL-1α or Fc IL-1α or Fc pro-IL-1α or Fc IL-1α(Δ1-6) or Fc pro-IL-1α(Δ113-118) or targeted Fc IL-1α(Δ1-6) or targeted Fc pro-IL-1α(Δ113-118), e.g., one or more of increased binding affinity, increased intrinsic activity, and increased specific biological activity. As a result, in various embodiments, the mutations allow for improved safety of IL-1α, e.g., reduced systemic toxicity, reduced side effects, and reduced off-target effects, compared to unmutated, i.e., wild-type, IL-1α (e.g., comparing an altered (e.g., mutant) form of IL-1α to a wild-type form of IL-1α), or compared to wild-type IL-1α or wild-type pro-IL-1α or Fc IL-1α or Fc pro-IL-1α or Fc IL-1α(Δ1-6) or Fc pro-IL-1α(Δ113-118) or targeted Fc IL-1α(Δ1-6) or targeted Fc pro-IL-1α(Δ113-118).

[0055] In various embodiments, IL-1α or pro-IL-1α is modified to have one or more mutations that reduce or increase its binding affinity or activity for one or more of its receptors. In some embodiments, IL-1α or pro-IL-1α is modified to have one or more mutations that substantially reduce, eliminate, or increase binding affinity or activity for the receptor. In some embodiments, the activity provided by wild-type IL-1α or pro-IL-1α is an activator at the receptor (e.g., activation of a cellular effect at a therapeutic site). For example, wild-type IL-1α or pro-IL-1α may activate its receptor. In such embodiments, the mutation reduces, eliminates, or increases the activator activity of the modified IL-1α or pro-IL-1α at the receptor. For example, the mutation may reduce, increase, or eliminate the activating signal that the modified IL-1α or pro-IL-1α delivers to a target cell.

[0056] In some embodiments, the activity provided by the wild-type IL-1α (or pro-IL-1α) is antagonism at the receptor (e.g., blocking or suppressing a cellular effect at the treatment site). For example, the wild-type IL-1α or pro-IL-1α may antagonize or inhibit the receptor. In these embodiments, the mutation reduces, eliminates, or increases the antagonistic activity of the modified IL-1α or pro-IL-1α at the receptor. For example, the mutation may reduce, increase, or eliminate the inhibitory signal that the modified IL-1α or pro-IL-1α delivers to target cells.

[0057] In some embodiments, the decreased affinity or activity of the IL-1α or pro-IL-1α at the receptor is reversible and / or inducible by binding to one or more of the targeting moieties, while in other embodiments, the decreased affinity or activity of the IL-1α or pro-IL-1α at the receptor is not substantially reversible and / or inducible by the activity of one or more of the targeting moieties.

[0058] In various embodiments, the chimeric protein or chimeric protein complex of the invention reduces off-target effects because the IL-1α or pro-IL-1α of the invention has mutations that weaken or eliminate binding affinity or activity at the receptor. In various embodiments, this reduction in side effects is observed, for example, in comparison to wild-type IL-1α or wild-type pro-IL-1α, or Fc IL-1α or Fc pro-IL-1α, or Fc IL-1α(Δ1-6) or Fc pro-IL-1α(Δ113-118), or targeted Fc IL-1α(Δ1-6) or targeted Fc pro-IL-1α(Δ113-118). In various embodiments, the IL-1α or pro-IL-1α is active on target cells because the targeting moiety(s) compensate for the lack / deficiency of binding (e.g., and / or avidity) required for substantial activation. In various embodiments, the modified IL-1α or pro-IL-1α is substantially inactive on its way to the site of therapeutic activity and has substantially its effect on the specifically targeted cell type, thereby significantly reducing undesirable side effects.

[0059] In various embodiments, substantially reducing or eliminating binding or activity at these receptors reduces or eliminates sequestration of the therapeutic chimeric protein from its site of therapeutic action, thereby improving the therapeutic efficacy of IL-1α or pro-IL-1α. For example, in some embodiments, this eliminates the need for high doses of the vaccine compositions to compensate for attrition at other receptors. The ability to reduce such doses further reduces the potential for side effects.

[0060] In various embodiments, the modified IL-1α or pro-IL-1α is modified to improve the affinity, e.g., binding (e.g., K DIn various embodiments, the affinity of the modified IL-1α or pro-IL-1α for the receptor can be reduced to attenuate activity (including receptor agonism or antagonism). In such embodiments, the affinity of the modified IL-1α or pro-IL-1α for the receptor can be reduced by one or more mutations that reduce, substantially reduce, or eliminate the affinity of wild-type IL-1α or wild-type pro-IL-1α or Fc IL-1α or Fc pro-IL-1α or Fc IL-1α(Δ1-6) or Fc pro-IL-1α(Δ113-118) or targeted Fc IL-1α(Δ1-6) or targeted Fc Compared to pro-IL-1α(Δ113-118), it is about 1%, or about 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 10% to 20%, about 20% to 40%, about 50%, about 40% to 60%, about 60% to 80%, or about 80% to 100%. In some embodiments, the binding affinity is greater than or equal to wild-type IL-1α or wild-type pro-IL-1α or Fc IL-1α or Fc pro-IL-1α or Fc IL-1α(Δ1-6) or Fc pro-IL-1α(Δ113-118) or targeted Fc IL-1α(Δ1-6) or targeted Fc Compared to pro-IL-1α(Δ113-118), it is about 1 / 2 or less, about 1 / 3 or less, about 1 / 4 or less, about 1 / 5 or less, about 1 / 6 or less, about 1 / 7 or less, about 1 / 8 or less, about 1 / 9 or less, about 1 / 10 or less, about 1 / 15 or less, about 1 / 20 or less, about 1 / 25 or less, about 1 / 30 or less, about 1 / 35 or less, about 1 / 40 or less, about 1 / 45 or less, about 1 / 50 or less, about 1 / 100 or less, about 1 / 150 or less, or about 1 / 10 to 1 / 50, about 1 / 50 to 1 / 100, about 1 / 100 to 1 / 150, about 1 / 150 to 1 / 200, or less than 1 / 200.

[0061] In various embodiments, the modified IL-1α or pro-IL-1α is modified to improve the affinity, e.g., binding (e.g., K DIn various embodiments, the affinity of the modified IL-1α or pro-IL-1α for the receptor can be increased to enhance activity (including receptor agonism or antagonism). In such embodiments, the affinity of the modified IL-1α or pro-IL-1α for the receptor can be increased by one or more mutations that increase or substantially increase the affinity of wild-type IL-1α or wild-type pro-IL-1α or Fc IL-1α or Fc pro-IL-1α or Fc IL-1α(Δ1-6) or Fc pro-IL-1α(Δ113-118) or targeted Fc IL-1α(Δ1-6) or targeted Fc Compared to pro-IL-1α(Δ113-118), it is about 1%, or about 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 10% to 20%, about 20% to 40%, about 50%, about 40% to 60%, about 60% to 80%, or about 80% to 100% higher. In some embodiments, the activity and / or binding affinity is greater than or equal to wild-type IL-1α or wild-type pro-IL-1α or Fc IL-1α or Fc pro-IL-1α or Fc IL-1α(Δ1-6) or Fc pro-IL-1α(Δ113-118) or targeted Fc IL-1α(Δ1-6) or targeted Fc Compared to pro-IL-1α(Δ113-118), it is at least about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 100-fold, at least about 150-fold, or about 10-50-fold, about 50-100-fold, about 100-150-fold, about 150-200-fold, or more than 200-fold.

[0062] In various embodiments, the modified IL-1α or pro-IL-1α comprises one or more mutations that reduce or increase the intrinsic activity of IL-1α or pro-IL-1α by, for example, about 75%, or about 70%, or about 60%, or about 50%, or about 40%, or about 30%, or about 25%, or about 20%, or about 10%, or about 5%, or about 3%, or about 1%, compared to wild-type IL-1α or wild-type pro-IL-1α or Fc IL-1α or Fc pro-IL-1α or Fc IL-1α(Δ1-6) or Fc pro-IL-1α(Δ113-118) or targeted Fc IL-1α(Δ1-6) or targeted Fc pro-IL-1α(Δ113-118). In some embodiments, the modified IL-1α or pro-IL-1α contains one or more mutations that decrease or increase the affinity of IL-1α (or pro-IL-1α) for its receptor(s) such that it is lower or higher than the binding affinity of the targeting moiety(s) for its receptor(s). In some embodiments, this difference in binding affinity is between the IL-1α / receptor and the targeting moiety / receptor on the same cell. In some embodiments, this difference in binding affinity allows the mutant IL-1α or pro-IL-1α to have a localized, on-target effect, minimizing off-target effects that underlie the side effects observed with wild-type IL-1α or wild-type pro-IL-1α. In some embodiments, the binding affinity is about 2-fold or less, or about 5-fold or less, or about 10-fold or less, or about 15-fold or less, or about 25-fold or less, or about 50-fold or less, or about 100-fold or less, or about 150-fold or less, or is at least about 2-fold, or at least about 5-fold, or at least about 10-fold, or at least about 15-fold, or at least about 25-fold, or at least about 50-fold, or at least about 100-fold, or at least about 150-fold less.

[0063] Receptor binding activity can be measured using methods known in the art. For example, affinity and / or avidity can be assessed by Scatchard plot analysis and computer approximation of binding data (e.g., Scatchard, 1949), or by reflectance interferometry under flow conditions (reported by Brecht et al. (1993)), the entire contents of which are incorporated herein by reference.

[0064] In various embodiments, the modified IL-1α or pro-IL-1α is at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72% identical to known wild-type IL-1α or wild-type pro-IL-1α. , or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90% %, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% sequence identity).

[0065] In various embodiments, the modified IL-1α is at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72 ...75%, or at least about 75%, or at least about 75%, or at least about 76%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90% , or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% sequence identity).

[0066] In various embodiments, the modified IL-1α or pro-IL-1α comprises an amino acid sequence having one or more amino acid mutations. In some embodiments, the one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and truncations. In some embodiments, the amino acid mutations are amino acid substitutions, which may include conservative and / or non-conservative substitutions.

[0067] "Conservative substitutions" may be made, for example, on the basis of similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the amino acid residues involved. The 20 naturally occurring amino acids can be divided into six standard amino acid groups: (1) hydrophobic: Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0068] As used herein, a "conservative substitution" is defined as the replacement of an amino acid with another amino acid within the same group of the six standard amino acid groups. For example, replacing Asp with Glu retains a single negative charge in the modified polypeptide. Furthermore, glycine and proline can be substituted for each other based on their ability to disrupt alpha helices.

[0069] As used herein, a "non-conservative substitution" is defined as the replacement of an amino acid with another amino acid from a different group of the six standard amino acid groups (1) to (6) above.

[0070] In various embodiments, substitutions also include non-classical amino acids (e.g., selenocysteine, pyrrolysine, N-formylmethionine, β-alanine, GABA and δ-aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoroamino acids, designer amino acids such as β-methyl amino acids, C α-methyl amino acids, N α-methyl amino acids, and amino acid analogs in general).

[0071] In some embodiments, the reduced affinity or activity of the modified IL-1α or pro-IL-1α at a therapeutic receptor is inducible and / or reversible by binding to a targeting moiety or by inclusion of a targeting moiety in a chimeric protein or chimeric protein complex disclosed herein, including, for example, an Fc-based chimeric protein complex. In some embodiments, the activity of IL-1α or pro-IL-1α is reduced or attenuated by its fusion with another protein, optionally including fusion with a targeting moiety described herein. In other embodiments, the activity of IL-1α or pro-IL-1α is reduced or attenuated by modifying the IL-1α or pro-IL-1α, e.g., by introducing a mutation described herein. In some embodiments, the reduced activity can be restored and / or induced by binding the IL-1α or pro-IL-1α to a targeting moiety or by the action of the bound targeting moiety. In embodiments, the targeting moiety induces the activity of IL-1α through its binding or its activity. In some embodiments, the loss of affinity or activity at the receptor is inducible and / or reversible by binding to one or more targeting moieties described herein or by inclusion in a chimeric protein complex, such as an Fc-based chimeric protein complex, as described herein.

[0072] targeting part In various embodiments, the chimeric protein, chimeric protein conjugate, vaccine composition, or adjuvant of the present invention further comprises one or more targeting moieties having a recognition domain that specifically binds to a target of interest (e.g., an antigen, a receptor). In some embodiments, the chimeric protein, chimeric protein conjugate, vaccine composition, or adjuvant may comprise two, three, four, five, six, seven, eight, nine, ten, or more targeting moieties. In exemplary embodiments, the chimeric protein, chimeric protein conjugate, vaccine composition, or adjuvant of the present invention comprises two or more targeting moieties. In such embodiments, the chimeric protein, chimeric protein conjugate, vaccine composition, or adjuvant can target two different types of cells (e.g., to form synapses) or the same type of cells (e.g., to achieve the effect of a more concentrated signaling factor). In some embodiments, the chimeric protein, chimeric protein conjugate, vaccine composition, or adjuvant of the present invention comprises a targeting moiety that recognizes IL-1α, pro-IL-1α, or a variant thereof, Flt3L, and two targeting moieties that recognize PD-1 or PD-L1.

[0073] In some embodiments, a chimeric protein, chimeric protein conjugate, vaccine composition, or adjuvant of the invention comprises a targeting moiety that is IL-1α, pro-IL-1α, or a variant thereof, Flt3L, and one targeting moiety that recognizes PD-1 or PD-L1. In some embodiments, a chimeric protein, chimeric protein conjugate, vaccine composition, or adjuvant of the invention comprises a targeting moiety that is IL-1α, pro-IL-1α, or a variant thereof, Flt3L, and two targeting moieties that recognize PD-1 or PD-L1.

[0074] In various embodiments, the desired target (e.g., antigen, receptor) can be present on one or more immune cells, which may include, but are not limited to, T cells, cytotoxic T lymphocytes, helper T cells, natural killer (NK) cells, natural killer T (NKT) cells, anti-tumor or tumor-associated macrophages (e.g., M1 or M2 macrophages), B cells, Breg cells, dendritic cells, or a subset thereof. In some embodiments, the recognition domain specifically binds to the desired target (e.g., antigen, receptor) and effectively recruits, directly or indirectly, one or more immune cells. In some embodiments, the desired target (e.g., antigen, receptor) can be present on one or more tumor cells. In some embodiments, the chimeric protein, chimeric protein complex, vaccine composition, or adjuvant can, for example, directly or indirectly recruit immune cells to a treatment site (e.g., a site containing one or more diseased cells or cells to be modulated to achieve a therapeutic effect). In some embodiments, the chimeric protein, chimeric protein complex, vaccine composition, or adjuvant can directly or indirectly recruit immune cells, e.g., immune cells capable of killing and / or suppressing tumor cells, to a site of action (such as, but not limited to, the tumor microenvironment).

[0075] In various embodiments, the chimeric protein, chimeric protein complex, vaccine composition, or adjuvant has a targeting moiety having a recognition domain that specifically binds to a target (e.g., an antigen, receptor) that is part of a non-cellular structure. In some embodiments, the antigen or receptor is not an integral component of an intact cell or cellular structure. In some embodiments, the antigen or receptor is an extracellular antigen or receptor. In some embodiments, the target is a non-proteinaceous non-cellular marker, which includes, but is not limited to, extracellular deposits such as nucleic acids or cholesterol, including DNA or RNA, such as, for example, DNA released from necrotic tumor cells.

[0076] In some embodiments, the target of interest (e.g., antigen, receptor) is part of the noncellular components of the stroma or extracellular matrix (ECM) or a marker associated therewith. As used herein, stroma refers to the connective and supportive framework of a tissue or organ. The stroma can include a collection of cells, such as fibroblasts / myofibroblasts / glial cells, epithelial, adipose, immune, vascular, smooth muscle, and immune cells, along with the extracellular matrix (ECM) and extracellular molecules. In various embodiments, the target of interest (e.g., antigen, receptor) is part of the noncellular components of the stroma, such as the extracellular matrix and extracellular molecules. As used herein, ECM refers to the noncellular components present in all tissues and organs. The ECM consists of a large collection of biochemically distinct components, including, but not limited to, proteins, glycoproteins, proteoglycans, and polysaccharides. These ECM components are typically produced by neighboring cells and secreted into the ECM by exocytosis. Upon secretion, ECM components often assemble to form a complex network of macromolecules. In various embodiments, a chimeric protein, chimeric protein complex, vaccine composition, or adjuvant of the invention comprises a targeting moiety that recognizes a target (e.g., an antigen or receptor or non-protein molecule) located on any component of the ECM. Examples of components of the ECM include, but are not limited to, proteoglycans, non-proteoglycan polysaccharides, fibers, and other ECM proteins or ECM non-proteins, e.g., polysaccharides and / or lipids, or ECM-associated molecules (e.g., proteins or non-proteins, e.g., polysaccharides, nucleic acids, and / or lipids).

[0077] In some embodiments, the targeting moiety recognizes a target (e.g., antigen, receptor) on an ECM proteoglycan. Proteoglycans are glycosylated proteins. A basic proteoglycan unit comprises a core protein with one or more covalently attached glycosaminoglycan (GAG) chains. Proteoglycans have a net negative charge that attracts positively charged sodium ions (Na+), which attract water via osmosis and keep the ECM and resident cells hydrated. Proteoglycans can also capture and store growth factors within the ECM. Examples of proteoglycans that may be targeted by the chimeric proteins, chimeric protein complexes, vaccine compositions, or adjuvants of the present invention include, but are not limited to, heparan sulfate, chondroitin sulfate, and keratan sulfate. In certain embodiments, the targeting moiety recognizes a target (e.g., antigen, receptor) on a non-proteoglycan polysaccharide, such as hyaluronic acid.

[0078] In some embodiments, the targeting moiety recognizes a target (e.g., antigen, receptor) on ECM fibers. ECM fibers include collagen fibers and elastin fibers. In some embodiments, the targeting moiety recognizes one or more epitopes on collagen or collagen fibers. Collagen is the most abundant protein in the ECM. Collagen exists in the ECM as a fibrous protein and provides structural support for resident cells. In one or more embodiments, the targeting moiety recognizes and binds to various types of collagen present in the ECM, including, but not limited to, fibrillar collagens (types I, II, III, V, XI), facit collagens (types IX, XII, XIV), short-chain collagens (types VIII, X), basement membrane collagens (type IV), and / or type VI, VII, or XIII collagen. Elastin fibers provide elasticity to tissues, allowing them to stretch as needed and then return to their original state. In some embodiments, the targeting moiety recognizes one or more epitopes on elastin or elastin fibers.

[0079] In some embodiments, the targeting moiety recognizes one or more ECM proteins, including, but not limited to, tenascin, fibronectin, fibrin, laminin, or nidogen / entactin.

[0080] In certain embodiments, the targeting moiety recognizes and binds to tenascin. The tenascin (TN) family of glycoproteins includes at least four members: tenascin-C, tenascin-R, tenascin-X, and tenascin-W. The primary structure of tenascin proteins contains several common motifs ordered in the same contiguous sequence: amino-terminal heptad repeats, epidermal growth factor (EGF)-like repeats, fibronectin type III domain repeats, and a carboxyl-terminal fibrinogen-like globular domain. Each protein member is associated with typical variations in the number and nature of the EGF-like and fibronectin type III repeats. Isoform variants also exist, particularly for tenascin-C. More than 27 splice variants and / or isoforms of tenascin-C are known. In certain embodiments, the targeting moiety recognizes and binds to tenascin-CA1. Similarly, tenascin-R also has various splice variants and isoforms. Tenascin-R typically exists as a dimer or trimer. Tenascin-X is the largest member of the tenascin family and is known to exist as a trimer. Tenascin-W exists as a trimer. In some embodiments, the targeting moiety recognizes one or more epitopes on the tenascin protein. In some embodiments, the targeting moiety recognizes monomeric, dimeric, trimeric, and / or hexameric forms of the tenascin protein.

[0081] In certain embodiments, the targeting moiety recognizes and binds to fibronectin. Fibronectin is a glycoprotein that connects cells to collagen fibers in the ECM, allowing cells to migrate through the ECM. Upon binding to integrins, fibronectin unfolds to form functional dimers. In some embodiments, the targeting moiety recognizes monomeric and / or dimeric forms of fibronectin. In some embodiments, the targeting moiety recognizes one or more epitopes on fibronectin. In exemplary embodiments, the targeting moiety recognizes fibronectin extracellular domain A (EDA) or fibronectin extracellular domain B (EDB). Elevated EDA levels are associated with various diseases and disorders, including psoriasis, rheumatoid arthritis, diabetes, and cancer. In some embodiments, the targeting moiety recognizes fibronectin containing the EDA isoform and can be used to target chimeric proteins, chimeric protein complexes, vaccine compositions, or adjuvants to diseased cells, including cancer cells. In some embodiments, the targeting moiety recognizes fibronectin containing the EDB isoform. In various embodiments, such targeting moieties can be used to target the chimeric protein, chimeric protein conjugate, vaccine composition, or adjuvant to tumor cells, including tumor neovasculature.

[0082] In certain embodiments, the targeting moiety recognizes and binds to fibrin, another protein substance often present in the matrix network of ECM. Fibrin is formed by the action of the protease thrombin on fibrinogen, thereby polymerizing it. In some embodiments, the targeting moiety recognizes one or more epitopes on fibrin. In some embodiments, the targeting moiety recognizes both monomeric and polymerized forms of fibrin.

[0083] In certain embodiments, the targeting moiety recognizes and binds to laminin. Laminin is a major component of basement membranes and the underlying protein network for cells and organs. Laminin is a heterotrimeric protein comprising an α chain, a β chain, and a γ chain. In some embodiments, the targeting moiety recognizes one or more epitopes on laminin. In some embodiments, the targeting moiety recognizes monomeric, dimeric, and trimeric forms of laminin.

[0084] In certain embodiments, the targeting moiety recognizes and binds to nidogen or entactin. Nidogen / entactin are a family of highly conserved sulfated glycoproteins. They are major structural components of basement membranes and function to connect laminin with the collagen IV network in basement membranes. Members of this family include nidogen-1 and nidogen-2. In various embodiments, the targeting moiety recognizes an epitope on nidogen-1 and / or nidogen-2.

[0085] In various embodiments, the targeting moiety comprises an antigen recognition domain that recognizes an epitope present on any of the targets described herein. In one embodiment, the antigen recognition domain recognizes one or more linear epitopes present on a protein. As used herein, a linear epitope refers to any continuous sequence of amino acids present on a protein. In another embodiment, the antigen recognition domain recognizes one or more conformational epitopes present on a protein. As used herein, a conformational epitope refers to a portion of one or more amino acids (which may be discontinuous) that forms a three-dimensional surface with characteristics and / or shape and / or tertiary structure that can be recognized by the antigen recognition domain.

[0086] In various embodiments, the targeting moiety may bind to full-length and / or mature and / or isoforms and / or splice variants and / or fragments and / or any other natural or synthetic analogs, variants, or mutants of any of the targets described herein. In various embodiments, the targeting moiety may bind to any form of protein described herein, including monomers, dimers, trimers, tetramers, heterodimers, multimers, and associated forms. In various embodiments, the targeting moiety may bind to any post-translationally modified form of protein described herein, such as glycosylated and / or phosphorylated forms.

[0087] In various embodiments, the targeting moiety comprises an antigen recognition domain that recognizes an extracellular molecule such as DNA. In some embodiments, the targeting moiety comprises an antigen recognition domain that recognizes DNA. In certain embodiments, DNA is shed into the extracellular space from necrotic or apoptotic tumor cells or other diseased cells.

[0088] In various embodiments, the targeting moiety comprises an antigen recognition domain that recognizes one or more non-cellular structures associated with atherosclerotic plaques. Two types of atherosclerotic plaques are known: Fibro-lipid (fibro-fatty) plaques are characterized by the accumulation of lipid-laden cells beneath the intima of an artery. Beneath the endothelium lies a fibrous cap that covers the atherosclerotic plaque core. The core contains lipid-laden cells (macrophages and smooth muscle cells) with increased tissue cholesterol and cholesterol ester content, fibrin, proteoglycans, collagen, elastin, and necrotic cellular debris. In advanced plaques, the central core of the plaque typically contains extracellular cholesterol deposits (released from dead cells), which form areas of cholesterol crystals with empty needle-like spaces. The periphery of the plaque contains younger foam cells and capillaries. Fibrous plaques are also localized subintima within the arterial wall, resulting in wall thickening and proliferation, and sometimes, scattered, localized narrowing of the lumen, accompanied by slight atrophy of the muscularis. Fibrous plaques contain collagen fibers (eosinophilic), calcium deposits (hematoxylinophilic), and lipid-laden cells. In some embodiments, the targeting moiety recognizes and binds to one or more noncellular components of these plaques, such as fibrin, proteoglycans, collagen, elastin, necrotic cell debris, and calcium or other mineral deposits or precipitates. In some embodiments, the necrotic cell debris is nucleic acid, e.g., DNA or RNA released from dying cells.

[0089] In various embodiments, the targeting moiety comprises an antigen recognition domain that recognizes one or more non-cellular structures present in brain plaques associated with neurodegenerative diseases. In some embodiments, the targeting moiety recognizes and binds to one or more non-cellular structures localized in amyloid plaques present in the brains of patients with Alzheimer's disease. For example, the targeting moiety recognizes and binds to the peptide amyloid beta. The peptide amyloid beta is a major component of amyloid plaques. In some embodiments, the targeting moiety recognizes and binds to one or more non-cellular structures present in brain plaques present in patients with Huntington's disease. In various embodiments, the targeting moiety recognizes and binds to one or more non-cellular structures present in plaques associated with other neurodegenerative or musculoskeletal diseases, such as Lewy body dementia and inclusion body myositis.

[0090] In some embodiments, the chimeric proteins, chimeric protein conjugates, vaccine compositions, or adjuvants of the invention can have two or more targeting moieties that bind to non-cellular structures. In some embodiments, there are two targeting moieties, one that targets cells and one that targets non-cellular structures. In various embodiments, the targeting moieties can directly or indirectly recruit cells, such as disease cells and / or effector cells. In some embodiments, the chimeric proteins, chimeric protein conjugates, vaccine compositions, or adjuvants can alter the balance of immune cells in favor of immune attack of tumors, or are used in methods involving altering said balance. For example, the chimeric protein, chimeric protein conjugate, vaccine composition, or adjuvant can shift the ratio of immune cells at a clinically important site in favor of cells capable of killing and / or suppressing tumors (e.g., T cells, cytotoxic T lymphocytes, helper T cells, natural killer (NK) cells, natural killer T (NKT) cells, anti-tumor macrophages (e.g., M1 macrophages), B cells, dendritic cells, or a subset thereof) and against cells that protect tumors (e.g., myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs); tumor-associated neutrophils (TANs), M2 macrophages, tumor-associated macrophages (TAMs), or a subset thereof). In some embodiments, the chimeric protein, chimeric protein conjugate, vaccine composition, or adjuvant can increase the ratio of effector T cells to regulatory T cells.

[0091] For example, in some embodiments, the recognition domain specifically binds to a target (e.g., an antigen, receptor) associated with a T cell. In some embodiments, the recognition domain directly or indirectly recruits a T cell. In certain embodiments, the recognition domain specifically binds to an effector T cell. In some embodiments, the recognition domain directly or indirectly recruits an effector T cell, for example, in some embodiments, to a therapeutic site (e.g., a site having one or more diseased cells or cells to be modulated to achieve a therapeutic effect). Examples of effector T cells include cytotoxic T cells (e.g., αβ TCR, CD3 + , CD8 + , CD45RO + );CD4 + Effector T cells (e.g., αβTCR, CD3 + , CD4 + , CCR7 + , CD62Lhi, IL-7R / CD127 + );CD8 + Effector T cells (e.g., αβTCR, CD3 + , CD8 + , CCR7 + , CD62Lhi, IL-7R / CD127 + effector memory T cells (e.g., CD62Llow, CD44 + , TCR, CD3 + , IL-7R / CD127 + , IL-15R + , CCR7low); central memory T cells (e.g., CCR7 + , CD62L + , CD27 + or CCR7hi, CD44 + , CD62Lhi, TCR, CD3 + , IL-7R / CD127 + , IL-15R + );CD62L + Effector T cells; early effector memory T cells (CD27 + CD62L - ) and late effector memory T cells (CD27 -CD62L - ) (TemE and TemL, respectively) containing CD8 + Effector memory T cells (TEM); CD127 ( + )CD25(low / -) effector T cells; CD127( - )CD25( - ) Effector T cells; CD8 + Stem cell memory effector cells (TSCM) (e.g., CD44(low)CD62L(high)CD122(high)sca( + )); TH1 effector T cells (e.g., CXCR3 + , CXCR6 + and CCR5 + ; or αβTCR, CD3 + , CD4 + , IL-12R + , IFNγR + , CXCR3 + ), TH2 effector T cells (e.g., CCR3 + , CCR4 + and CCR8 + ; or αβTCR, CD3 + , CD4 + , IL-4R + , IL-33R + , CCR4 + , IL-17RB + , CRTH2 + ); TH9 effector T cells (e.g., αβTCR, CD3 + , CD4 + ); TH17 effector T cells (e.g., αβTCR, CD3 + , CD4 + , IL-23R + , CCR6 + , IL-1R + );CD4 + CD45RO + CCR7 + Effector T cells, ICOS + Effector T cells; CD4 + CD45RO + CCR7( -) effector T cells; and effector T cells that secrete IL-2, IL-4, and / or IFN-γ.

[0092] Examples of T cell antigens of interest include, for example, the following (including extracellular domains, if applicable): CD8, CD3, SLAMF4, IL-2Rα, 4-1BB / TNFRSF9, IL-2Rβ, ALCAM, B7-1, IL-4R, B7-H3, BLAME / SLAMFS, CEACAM1, IL-6R, CCR3, IL-7Rα, CCR4, CXCR1 / ILSRA, CCR5, CCR6, IL-10Rα, CCR7, IL-10Rβ, CCRS, IL-12Rβ1, CCR9, IL-12Rβ2, CD2, IL-13Rα1, IL-13, CD3, CD4, ILT2 / CDS5j, ILT3 / CDS5k, ILT4 / CDS5d, ILT5 / CDS5a, and lutegrin.α4 / CD49d, CDS, integrin αE / CD103, CD6, integrin αM / CD11b, CDS, integrin αX / CD11c, integrin β2 / CDlS, KIR / CD15S, CD27 / TNFRSF7, KIR2DL1, CD2S, KIR2DL3, CD30 / TNFRSFS, KIR2DL4 / CD15Sd, CD31 / PECAM-1, KIR2DS4, CD40 ligand / TNFSF 5, LAG-3, CD43, LAIR1, CD45, LAIR2, CDS3, leukotriene B4-R1, CDS4 / SLAMF5, NCAM-L1, CD94, NKG2A, CD97, NKG2C, CD229 / SLAMF3, NKG2D, CD2F-10 / SLAMF9, NT-4, CD69, NTB-A / SLAMF6, common gamma chain / IL-2Rγ, osteopontin, CRACC / SLAMF7, PD-1, C RTAM, PSGL-1, CTLA-4, RANK / TNFRSF11A, CX3CR1, CX3CL1, L-selectin, CXCR3, SIRPβ1, CXCR4, SLAM, CXCR6, TCCR / WS X-1, DNAM-1, thymopoietin, EMMPRIN / CD147, TIM-1, EphB6, TIM-2, Fas / TNFRSF6, TIM-3, Fas ligand / TNFSF6, TIM-4, FcγR III / CD16, TIM-6, TNFR1 / TNFRSF1A, granulysin, TNFRIII / TNFRSF1B, TRAILR1 / TNFRSF10A, ICAM-1 / CD54, TRAILR2 / TNFRSF10B, ICAM-2 / CD102, TRAILR3 / TNFRSF10C, IFN-γR1, TRAILR4 / TNFRSF10D, IFN-γR2, TSLP, IL-1R1, and TSLPR. In various embodiments, the targeting moiety of the chimeric protein, chimeric protein complex, vaccine composition, or adjuvant binds to one or more of these exemplary T cell antigens.

[0093] By way of non-limiting example, in various embodiments, a chimeric protein, chimeric protein conjugate, vaccine composition, or adjuvant comprises a targeting moiety directed against one or more of the checkpoint markers expressed on T cells, e.g., PD-1, CD28, CTLA4, ICOS, BTLA, KIR, LAG3, CD137, OX40, CD27, CD40L, TIM3, and A2aR.

[0094] For example, in some embodiments, the recognition domain specifically binds to a target (e.g., an antigen, receptor) associated with a B cell. In some embodiments, the recognition domain directly or indirectly recruits the B cell to, for example, a therapeutic site (e.g., a site containing one or more diseased cells or cells to be modulated to achieve a therapeutic effect). Examples of B cell antigens of interest include, for example, CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD38, CD39, CD40, CD70, CD72, CD73, CD74, CDw75, CDw76, CD77, CD78, CD79a / b, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD89, CD98, CD126, CD127, CDw130, CD138, CDw150, CS1, and B-cell maturation antigen (BCMA). In various embodiments, the targeting moiety of the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex binds to one or more of these exemplary B cell antigens.

[0095] Further, for example, in some embodiments, the recognition domain specifically binds to a target (e.g., an antigen, receptor) associated with natural killer cells. In some embodiments, the recognition domain directly or indirectly recruits natural killer cells to, for example, in some embodiments, a therapeutic site (e.g., a site having one or more diseased cells or cells to be modulated to achieve a therapeutic effect). Examples of natural killer cell antigens of interest include, for example, TIGIT, 2B4 / SLAMF4, KIR2DS4, CD155 / PVR, KIR3DL1, CD94, LMIR1 / CD300A, CD69, LMIR2 / CD300c, CRACC / SLAMF7, LMIR3 / CD300LF, DNAM-1, LMIR5 / CD300LB, Fc-epsilon RII, LMIR6 / CD300LE, Fc-γRl / CD64, MICA, Fc-γRIIB / CD32b, MICB, Fc-γRIIC / CD32c, MULT-1, Fc-γRIIA / CD32a, Nectin-2 / CD112, Fc-γRIII / CD16, NKG2A, FcRH1 / IRTA5, NK G2C, FcRH2 / IRTA4, NKG2D, FcRH4 / IRTA1, NKp30, FcRH5 / IRTA2, NKp44, Fc-receptor-like 3 / CD16-2, NKp46 / NCR1, NKp80 / KLRF1, NTB-A / SLAMF6, Rae-1, Rae-1α, Rae-1β, Rae-1δ, H60, Rae-1ε, ILT2 / CD85j, Rae-1γ, ILT3 / CD85k, TREM-1, ILT4 / CD85d, TREM-2, ILT5 / CD85a, TREM-3, KIR / CD158, TREML1 / TLT-1, KIR2DL1, ULBP-1, KIR2DL3, ULBP-2, KIR2DL4 / CD158d, and ULBP-3. In various embodiments, the targeting moiety of the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex binds to one or more of these exemplary NK cell antigens.

[0096] Also, in some embodiments, the recognition domain specifically binds to a target (e.g., an antigen, receptor) associated with macrophages / monocytes. In some embodiments, the recognition domain directly or indirectly recruits macrophages / monocytes, for example, in some embodiments, to a therapeutic site (e.g., a site having one or more diseased cells or cells to be modulated to achieve a therapeutic effect). Examples of macrophage / monocyte antigens of interest include, for example, SIRP1a, B7-1 / CD80, ILT4 / CD85d, B7-H1, ILT5 / CD85a, common beta chain, integrin α4 / CD49d, BLAME / SLAMF8, integrin αX / CDllc, CCL6 / C10, integrin β2 / CD18, CD155 / PVR, integrin β3 / CD61, CD31 / PECAM-1, latexin, CD36 / SR-B3, leukotriene B4R1, CD40 / TNFRSF5, LIMPIIISR-B2, CD43, LMIR1 / CD300A, CD45, LMIR2 / CD300c, CD68, LMIR3 / CD300LF, CD84 / SLAMF5, LMIR5 / CD300LB, CD97, LMIR6 / CD300LE, CD163, LRP-1, CD2F-10 / SLAMF9, MARCO, CRACC / SLAMF7, MD-1, ECF-L, MD-2, EMMPRIN / CD147, MGL2, endoglin / CD105, osteoactivin / GPNMB, Fc-γRI / CD64, osteopontin, Fc-γRIIB / CD32b, PD-L2, Fc-γRIIC / CD32c, Siglec-3 / CD33, Fc-γRIIA / CD32a, SIGNR1 / CD209, Fc-γRIII / CD16, SLAM, GM-CSFRα, TCCR / WSX-1, ICAM-2 / CD102, TLR3, IFN-γRl, TLR4, IFN-γR2, TREM-l, IL-lRII, TREM-2, ILT2 / CD85j, TREM-3, ILT3 / CD85k, TREML1 / TLT-1, 2B4 / SLAMF4, IL-10Rα, ALCAM, IL-10Rβ, aminopeptidase N / ANPEP, ILT2 / CD85j, common β chain, ILT3 / CD85k, ClqR1 / CD93, ILT4 / CD85d, CCR1, ILT5 / CD85a, CCR2, integrin α4 / CD49d, CCR5, integrin αM / CDllb, CCR8, integrin αX / CDllc, CD155 / PVR, integrin β2 / CD18, CD14, integrin β3 / CD61, CD36 / SR-B3, LAIR1, CD43, LAIR2, CD45, leukotriene B4-R1, CD68, LIMPIIISR-B2, CD84 / SLAMF5, LMIR1 / CD300A, CD97, LMIR2 / CD300c, LMIR3 / CD300LF, coagulation factor III / tissue factor, LMIR5 / CD300LB, CX3CR1, CX3CL1, LMIR6 / CD300LE, CXCR4, LRP-1, CXCR6, M-CSFR, DEP-1 / CD148, MD-1, DNAM-1, MD-2, EMMPRIN / CD147, MMR, endoglin / CD105, NCAM-L1, Fc-γRI / C These include D64, PSGL-1, Fc-γRIIIICD16, RP105, G-CSFR, L-selectin, GM-CSFRα, Siglec-3 / CD33, HVEM / TNFRSF14, SLAM, ICAM-1 / CD54, TCCR / WSX-1, ICAM-2 / CD102, TREM-1, IL-6R, TREM-2, CXCR1 / IL-8RA, TREM-3, and TREML1 / TLT-1. In various embodiments, the targeting moiety of the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex binds to one or more of these exemplary macrophage / monocyte antigens.

[0097] Also, in some embodiments, the recognition domain specifically binds to a target (e.g., an antigen, receptor) associated with a dendritic cell. In some embodiments, the recognition domain directly or indirectly recruits dendritic cells to, for example, a therapeutic site (e.g., a site having one or more diseased cells or cells to be modulated to achieve a therapeutic effect). Examples of dendritic cell antigens of interest include, for example, CLEC9A, XCR1, RANK, CD36 / SRB3, LOX-1 / SR-E1, CD68, MARCO, CD163, SR-A1 / MSR, CD5L, SREC-1, CL-P1 / COLEC12, SREC-II, LIMPIIISRB2, RP105, TLR4, TLR1, TLR5, TLR2, TLR6, TLR3, TLR9, 4-IBB ligand / TNFSF9, IL-12 / IL-23p40, 4-Amino-1, 8-naphthalimide, ILT2 / CD85j, CCL21 / 6Ckine, ILT3 / CD85k, 8-oxo-dG, ILT4 / CD85d, 8D6A, ILT5 / CD85a, A2B5, lutegrin α4 / CD49d, Aag, integrin β2 / CD18, AMICA, Langerin, B7-2 / CD86, leukotriene B4Rl, B7-H3, LMIR1 / CD300A, BLAME / SLAMF8, LMIR2 / CD300c, ClqR1 / CD93, LMIR3 / CD300LF, CCR6, LMIR5 / CD300LBCCR7, LMIR6 / CD300LE, CD40 / TNFRSF5, MAG / Siglec-4-a, CD43, M CAM, CD45, MD-1, CD68, MD-2, CD83, MDL-1 / CLEC5A, CD84 / SLAMF5, MMR, CD97, NCAMLl, CD2F-10 / SLAMF9, osteoactivin GPNMB, Che rn23, PD-L2, CLEC-1, RP105, CLEC-2, CLEC-8, Siglec-2 / CD22, CRACC / SLAMF7, Siglec-3 / CD33, DC-SIGN, Siglec-5, DC-SIGNR / CD299, Siglec-6, DCAR, Siglec-7, DCIR / CLEC4A, Siglec-9, DEC-205, Siglec-10, Dectin-1 / CLEC7A, Siglec-F, Dectin-2 / CLEC6A,These include SIGNR1 / CD209, DEP-1 / CD148, SIGNR4, DLEC / CLEC4C, SLAM, EMMPRIN / CD147, TCCR / WSX-1, Fc-γR1 / CD64, TLR3, Fc-γRIIB / CD32b, TREM-1, Fc-γRIIC / CD32c, TREM-2, Fc-γRIIA / CD32a, TREM-3, Fc-γRIII / CD16, TREML1 / TLT-1, ICAM-2 / CD102, and vanilloid R1. In various embodiments, the targeting moiety of the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex binds to one or more of these exemplary DC antigens.

[0098] In some embodiments, the recognition domain specifically binds to a target (e.g., antigen, receptor) on an immune cell selected from, but not limited to, megakaryocytes, platelets, erythrocytes, mast cells, basophils, neutrophils, myeloid cells, monocytes, eosinophils, or a subset thereof. In some embodiments, the recognition domain localizes the megakaryocytes, platelets, erythrocytes, mast cells, basophils, neutrophils, myeloid cells, monocytes, eosinophils, or a subset thereof, for example, in some embodiments, directly or indirectly, to a treatment site (e.g., a site having one or more diseased cells or cells to be modulated to achieve a therapeutic effect). In some embodiments, the immune cell is selected from T cells, B cells, dendritic cells, macrophages, neutrophils, mast cells, monocytes, erythrocytes, myeloid cells, myeloid-derived suppressor cells, NKT cells, and NK cells, or derivatives thereof.

[0099] In some embodiments, the recognition domain specifically binds to a target (e.g., antigen, receptor) associated with megakaryocytes and / or platelets. Examples of megakaryocyte and / or platelet antigens of interest include, for example, GPIIb / IIIa, GPIb, vWF, PF4, and TSP. In various embodiments, the targeting moiety of the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex binds to one or more of these exemplary megakaryocyte and / or platelet antigens.

[0100] In some embodiments, the recognition domain specifically binds to a target (e.g., antigen, receptor) associated with an erythrocyte. Examples of erythrocyte antigens of interest include, for example, CD34, CD36, CD38, CD41a (platelet glycoprotein IIb / IIIa), CD41b (GPIIb), CD71 (transferrin receptor), CD105, glycophorin A, glycophorin C, c-kit, HLA-DR, H2 (MHC-II), and Rh antigen. In various embodiments, the targeting moiety of the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex binds to one or more of these exemplary erythrocyte antigens.

[0101] In some embodiments, the recognition domain specifically binds to a target (e.g., antigen, receptor) associated with a mast cell. Examples of mast cell antigens of interest include, for example, SCFR / CD117, Fc ε RI, CD2, CD25, CD35, CD88, CD203c, C5R1, CMA1, FCERL1A, FCER2, TPSABl. In various embodiments, the targeting moiety of the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex binds to one or more of these mast cell antigens.

[0102] In some embodiments, the recognition domain specifically binds to a target (e.g., antigen, receptor) associated with a basophil. Examples of basophil antigens of interest include, for example, Fc ε RI, CD203c, CD123, CD13, CD107a, CD107b, and CD 164. In various embodiments, the targeting moiety of the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex binds to one or more of these basophil antigens.

[0103] In some embodiments, the recognition domain specifically binds to a target (e.g., antigen, receptor) associated with a neutrophil. Examples of neutrophil antigens of interest include, for example, 7D5, CD10 / CALLA, CD13, CD16 (FcRIII), CD18 proteins (LFA-1, CR3, and p150, 95), CD45, CD67, and CD177. In various embodiments, the targeting moiety of the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex binds to one or more of these neutrophil antigens.

[0104] In some embodiments, the recognition domain specifically binds to a target (e.g., antigen, receptor) associated with eosinophils. Examples of eosinophil antigens of interest include, for example, CD35, CD44, and CD69. In various embodiments, the targeting moiety of the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex binds to one or more of these eosinophil antigens.

[0105] In various embodiments, the recognition domain may specifically bind to any suitable target, antigen, receptor, or cell surface marker known to those of skill in the art. In some embodiments, the antigen or cell surface marker is a tissue-specific marker. Examples of tissue-specific markers include endothelial cell surface markers such as ACE, CD14, CD34, CDH5, ENG, ICAM2, MCAM, NOS3, PECAM1, PROCR, SELE, SELP, TEK, THBD, VCAM1, and VWF; smooth muscle cell surface markers such as ACTA2, MYH1O, MYH11, MYH9, and MYOCD; fibroblast (stromal) cell surface markers such as ALCAM, CD34, COL1A1, COL1A2, COL3A1, FAP, and PH-4; epithelial cell surface markers such as CD1D, K6IRS2, KRT1O, KRT13, KRT17, KRT18, KRT19, KRT4, KRT5, KRT8, MUCl, and TACSTD1; and CD13, TFNA, and alpha beta 3 (α). Vβ3), E-selectin, and other angiogenic markers; and adipocyte surface markers, such as ADIPOQ, FABP4, and RETN. In various embodiments, the targeting moiety of the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex binds to one or more of these antigens. In various embodiments, the targeting moiety of the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex binds to cells bearing one or more of these antigens.

[0106] In some embodiments, the recognition domain specifically binds to a target (e.g., an antigen, a receptor) associated with a tumor cell. In some embodiments, the recognition domain directly or indirectly recruits tumor cells. For example, in some embodiments, the direct or indirect recruitment of tumor cells is to one or more effector cells (e.g., immune cells described herein) that can kill and / or suppress the tumor cells.

[0107] Tumor cells, or cancer cells, refer to uncontrolled proliferation of cells or tissues and / or abnormally increased cell survival and / or abnormally increased inhibition of apoptosis that interferes with the normal functioning of bodily organs and systems. For example, tumor cells include benign and malignant cancers, polyps, hyperplasias, and dormant tumors or micrometastases. Examples of tumor cells include basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colon and rectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, stomach cancer (including gastrointestinal cancer), glioblastoma, liver cancer, hepatoma, intraepithelial neoplasia, kidney or renal cancer, and tumors of the liver and kidney. cancer), laryngeal cancer, leukemia, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), melanoma, myeloma, neuroblastoma, oral cancer (lip, tongue, tonsil, and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, salivary gland carcinoma, sarcoma, skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, cancer of the urinary system, vulvar cancer, lymphomas including Hodgkin's lymphoma and non-Hodgkin's lymphoma, and B-cell lymphomas (including low-grade / follicular non-Hodgkin's lymphoma (NHL)), small lymphocytic (SL) NHL, intermediate-grade / follicular NH These include, but are not limited to, L, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleaved cell NHL, bulky mass disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and other carcinomas and sarcomas, and post-transplant lymphoproliferative disorder (PTLD), as well as cells of abnormal blood vessel proliferation associated with phacomatosis, edema (e.g., associated with brain tumors), and Meig syndrome.

[0108] Tumor cells or cancer cells also include, but are not limited to, carcinomas, such as various subtypes (including, for example, adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, and transitional cell carcinoma), sarcomas (including, for example, bone and soft tissue), leukemias (including, for example, acute myeloid, acute lymphoblastic, chronic myeloid, chronic lymphocytic, and hairy cell), lymphomas and myelomas (including, for example, Hodgkin's and non-Hodgkin's lymphoma, light chain, non-secretory MGUS, and plasmacytoma), and central nervous system cancers (e.g., brain tumors (e.g., gliomas (e.g., astrocytoma, oligodendroglioma, and ependymoma), meningioma, pituitary adenoma, and neuroma), and spinal cord tumors (e.g., meningioma and neurofibroma)).

[0109] Examples of tumor antigens include MART-1 / Melan-A, gp100, dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein (ADAbp), cyclophilin b, colorectal-associated antigen (CRC)-0017-1A / GA733, carcinoembryonic antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1, prostate-specific antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2, and PSA-3, prostate-specific membrane antigen (PSMA), T-cell receptor / CD3-zeta chain, and tumor antigens of the MAGE family (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-A13, MAGE-A14, MAGE-A15, MAGE-A16, MAGE-A17, MAGE-A18, MAGE-A19, MAGE-A20, MAGE-A21, MAGE-A22, MAGE-A23, MAGE-A24, MAGE-A25, MAGE-A26, MAGE-A27, MAGE-A28, MAGE-A29, MAGE-A30, MAGE-A31, MAGE-A29, MAGE-A21, MAGE-A22, MAGE-A23, MAGE-A24, MAGE-A25, MAGE-A26, MAGE-A27, MAGE-A28, MAGE-A29, MAGE-A20, MAGE-A21, MAGE-A22, MAGE-A23, MAGE-A24, MAGE-A25, MAGE E-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), tumor antigens of the GAGE ​​family (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4, GA GE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2 / neu, p21ras, RCAS1, α-fetoprotein, E-cadherin, α-catenin, β-catenin and γ-catenin, p120ctn, gp100 Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, connexin 37, Ig-idiotypes, p15, gp75, GM2 and GD2 gangliosides, viral products such as human papillomavirus proteins, tumor antigens of the Smad family, lmp-1, NA, EBV-encoded nuclear antigen (EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1These tumor antigens include, but are not limited to, CT-7, c-erbB-2, CD19, CD20, CD22, CD30, CD33, CD37, CD47, CS1, CD38, ASGPR, CD56, CD70, CD74, CD138, AGS16, MUC1, GPNMB, Ep-CAM, PD-L1, PD-L2, PMSA, and BCMA (TNFRSF17). In various embodiments, the targeting moiety of the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex binds to one or more of these tumor antigens. In one embodiment, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex binds to HER2. In another embodiment, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex binds to PD-L2.

[0110] In some embodiments, the chimeric protein complex, such as the chimeric protein or Fc-based chimeric protein complex, of the present invention comprises, in combination with any of the signal transduction factors described herein (e.g., IL-1α or a variant thereof), (i) one or more targeting moieties directed against immune cells selected from T cells, B cells, dendritic cells, macrophages, NK cells, or a subset thereof, and (ii) one or more targeting moieties directed against tumor cells. In one embodiment, the chimeric protein complex, such as the chimeric protein or Fc-based chimeric protein complex, of the present invention comprises, in combination with any of the signal transduction factors described herein, (i) a targeting moiety directed against T cells (including, but not limited to, effector T cells), and (ii) a targeting moiety directed against tumor cells. In one embodiment, the chimeric protein complex, such as the chimeric protein or Fc-based chimeric protein complex, of the present invention comprises, in combination with any of the signal transduction factors described herein, (i) a targeting moiety directed against B cells, and (ii) a targeting moiety directed against tumor cells. In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention comprises (i) a targeting moiety directed to dendritic cells and (ii) a targeting moiety directed to tumor cells, in combination with any of the signaling factors described herein. In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention comprises (i) a targeting moiety directed to macrophages and (ii) a targeting moiety directed to tumor cells, in combination with any of the signaling factors described herein. In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention comprises (i) a targeting moiety directed to NK cells and (ii) a targeting moiety directed to tumor cells, in combination with any of the signaling factors described herein.

[0111] By way of non-limiting example, in various embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention may be used in combination with any of the signal transduction factors described herein (e.g., IL-1α or a variant thereof) in combination with (i) any of the signal transduction factors, such as CD8, SLAMF4, IL-2Rα, 4-1BB / TNFRSF9, IL-2Rβ, ALCAM, B7-1, IL-4R, B7-H3, BLAME / S LAMFS, CEACAM1, IL-6R, CCR3, IL-7Rα, CCR4, CXCRl / ILSRA, CCR5, CCR6, IL-10Rα, CCR7, IL-10Rβ, CCRS, IL-12Rβ1 , CCR9, IL-12Rβ2, CD2, IL-13Rα1, IL-13, CD3, CD4, ILT2 / CDS5j, ILT3 / CDS5k, ILT4 / CDS5d, ILT5 / CDS5a, lutegrinα4 / CD49d, CDS, integrin αE / CD103, CD6, integrin αM / CD11b, CDS, integrin αX / CD11c, integrin β2 / CDlS, KIR / CD15S, CD27 / TNFRSF7, KIR2DL1, CD2S, KIR2DL3, CD30 / TNFRSFS, KIR2DL4 / CD15Sd, CD31 / PECAM-1, KIR2DS4, CD40 ligand / TNFSF5, LAG-3, CD43, LAI R1, CD45, LAIR2, CDS3, leukotriene B4-R1, CDS4 / SLAMF5, NCAM-L1, CD94, NKG2A, CD97, NKG2C, CD229 / SLAMF3, NKG2D, CD2F-10 / SLAMF9, NT-4, CD69, NTB-A / SLAMF6, common gamma chain / IL-2Rγ, osteopontin, CRACC / SLAMF7, PD-1, CRTAM, PSGL-1, CTLA-4, RANK / TNFRSF11A , CX3CR1, CX3CL1, L-selectin, CXCR3, SIRPβ1, CXCR4, SLAM, CXCR6, TCCR / WSX-1, DNAM-1, thymopoietin, EMMPRIN / CD147, TIM-1, Eph B6, TIM-2, Fas / TNFRSF6, TIM-3, Fas ligand / TNFSF6, TIM-4, FcγRIII / CD16, TIM-6, TNFR1 / TNFRSF1A, granulysin, TNFRIII / TNF and (ii) a targeting moiety directed against T cells, mediated by targeting to RSF1B, TRAILR1 / TNFRSF10A, ICAM-1 / CD54, TRAILR2 / TNFRSF10B, ICAM-2 / CD102, TRAILR3 / TNFRSF10C, IFN-γR1, TRAILR4 / TNFRSF10D, IFN-γR2, TSLP, IL-1R1, or TSLPR, and

[0112] As a non-limiting example, in various embodiments, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the invention comprises, in addition to any signal transduction factor described herein, (i) a checkpoint marker expressed on T cells, e.g., one or more of PD-1, CD28, CTLA4, ICOS, BTLA, KIR, LAG3, CD137, OX40, CD27, CD40L, TIM3, and A2aR, and (ii) a targeting moiety directed against a targeting moiety directed against a tumor cell.

[0113] In various embodiments, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the present invention comprise one or more targeting moieties to PD-1. In some embodiments, the vaccine compositions, adjuvants, chimeric proteins, or chimeric protein complexes comprise one or more targeting moieties that selectively bind to a PD-1 polypeptide. In some embodiments, the vaccine compositions, adjuvants, chimeric proteins, or chimeric protein complexes comprise one or more of an antibody, antibody derivative or antibody format, peptide or polypeptide, or fusion protein that selectively binds to a PD-1 polypeptide.

[0114] In one embodiment, the targeting moiety comprises the anti-PD-1 antibody pembrolizumab (also known as MK-3475, Keytruda), or a fragment thereof. Pembrolizumab and other humanized anti-PD-1 antibodies are disclosed in Hamid, et al. (2013) New England Journal of Medicine 369(2):134-44, US 8,354,509, and WO2009 / 114335, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, pembrolizumab, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:7 and / or a light chain comprising the amino acid sequence of SEQ ID NO:8.

[0115] In one embodiment, the targeting moiety comprises the anti-PD-1 antibody, nivolvam (also known as BMS-936558, MDX-1106, ONO-4538, Opdivo), or a fragment thereof. Nivolvam (clone 5C4) and other human monoclonal antibodies that specifically bind to PD-1 are disclosed in U.S. Pat. No. 8,008,449 and WO 2006 / 121168, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, nivolvam or an antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:9 and / or a light chain comprising the amino acid sequence of SEQ ID NO:10.

[0116] In one embodiment, the targeting moiety comprises the anti-PD-1 antibody pidilizumab (also known as CT-011, hBAT, or hBAT-1), or a fragment thereof. Pidilizumab and other humanized anti-PD-1 monoclonal antibodies are disclosed in US2008 / 0025980 and WO2009 / 101611, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, the anti-PD-1 antibody or antigen-binding fragment thereof for use in the methods provided herein comprises an amino acid sequence selected from SEQ ID NOS:15-18 of US2008 / 0025980: SEQ ID NO:15 of US2008 / 0025980 (SEQ ID NO:11); SEQ ID NO:16 of US2008 / 0025980 (SEQ ID NO:12); SEQ ID NO:17 of US2008 / 0025980 (SEQ ID NO:13); or SEQ ID NO:18 of US2008 / 0025980 (SEQ ID NO:14). and / or a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 20-24 of US2008 / 0025980: SEQ ID NO: 20 of US2008 / 0025980 (SEQ ID NO: 15); SEQ ID NO: 21 of US2008 / 0025980 (SEQ ID NO: 16); SEQ ID NO: 22 of US2008 / 0025980 (SEQ ID NO: 17); SEQ ID NO: 23 of US2008 / 0025980 (SEQ ID NO: 18) and SEQ ID NO: 24 of US2008 / 0025980 (SEQ ID NO: 19).

[0117] In one embodiment, the targeting moiety comprises a light chain comprising SEQ ID NO: 18 (SEQ ID NO: 14) of US2008 / 0025980 and a heavy chain comprising SEQ ID NO: 22 (SEQ ID NO: 17) of US2008 / 0025980.

[0118] In one embodiment, the targeting moiety comprises AMP-514 (also known as MEDI-0680).

[0119] In one embodiment, the targeting moiety comprises the PD-L2-Fc fusion protein AMP-224, which is disclosed in WO2010 / 027827 and WO2011 / 066342, the entire disclosures of which are incorporated herein by reference. In such an embodiment, the targeting moiety may comprise a targeting domain comprising SEQ ID NO:4 (SEQ ID NO:20) of WO2010 / 027827 and / or a B7-DC fusion protein comprising SEQ ID NO:83 (SEQ ID NO:21) of WO2010 / 027827.

[0120] In some embodiments, the targeting moiety comprises the peptide AUNP12 or any other peptide disclosed in US2011 / 0318373 or US8,907,053. For example, the targeting moiety may comprise AUNP12 (i.e., compound 8 or SEQ ID NO: 49 of US2011 / 0318373), which has the following sequence: [ka]

[0121] It has.

[0122] In one embodiment, the targeting moiety comprises the anti-PD-1 antibody 1E3 or a fragment thereof, as disclosed in US 2014 / 0044738, the entire disclosure of which is incorporated herein by reference. In an exemplary embodiment, 1E3 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:23; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:24.

[0123] In one embodiment, the targeting moiety comprises the anti-PD-1 antibody 1E8, or a fragment thereof, as disclosed in US 2014 / 0044738, the entire disclosure of which is incorporated herein by reference. In an exemplary embodiment, 1E8, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:25; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:26.

[0124] In one embodiment, the targeting moiety comprises the anti-PD-1 antibody 1H3 or a fragment thereof, as disclosed in US 2014 / 0044738, the entire disclosure of which is incorporated herein by reference. In an exemplary embodiment, 1H3 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:27; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:28.

[0125] In one embodiment, the targeting moiety comprises a VHH directed against PD-1, e.g., as disclosed in US 8,907,065 and WO 2008 / 071447, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, the VHH directed against PD-1 comprises SEQ ID NOs: 347-351 of US 8,907,065 (SEQ ID NO: 347 of US 8,907,065 (SEQ ID NO: 29); SEQ ID NO: 348 of US 8,907,065 (SEQ ID NO: 30); SEQ ID NO: 349 of US 8,907,065 (SEQ ID NO: 31); SEQ ID NO: 350 of US 8,907,065 (SEQ ID NO: 32); or SEQ ID NO: 351 of US 8,907,065 (SEQ ID NO: 33)).

[0126] In some embodiments, the targeting moiety comprises any one of the anti-PD-1 antibodies or fragments thereof as disclosed in US2011 / 0271358 and WO2010 / 036959, the entire contents of which are incorporated herein by reference. In exemplary embodiments, the antibody or antigen-binding fragment thereof for use in the methods provided herein is selected from the group consisting of SEQ ID NOS:25-29 of US2011 / 0271358 (SEQ ID NO:25 of US2011 / 0271358 (SEQ ID NO:34); SEQ ID NO:26 of US2011 / 0271358 (SEQ ID NO:35); SEQ ID NO:27 of US2011 / 0271358 (SEQ ID NO:36); SEQ ID NO:28 of US2011 / 0271358 (SEQ ID NO:37); US2011 / 0271358 (SEQ ID NO:38); and / or a light chain comprising an amino acid sequence selected from SEQ ID NOs: 30 to 33 of US2011 / 0271358 (SEQ ID NO: 30 (SEQ ID NO: 39) of US2011 / 0271358; SEQ ID NO: 31 (SEQ ID NO: 40) of US2011 / 0271358; SEQ ID NO: 32 (SEQ ID NO: 41) of US2011 / 0271358; SEQ ID NO: 33 (SEQ ID NO: 42) of US2011 / 0271358).

[0127] In various embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the present invention comprises one or more antibodies, or antibody fragments thereof, directed against PD-1 selected from TSR-042 (Tesaro, Inc.), REGN2810 (Regeneron Pharmaceuticals, Inc.), PDR001 (Novartis Pharmaceuticals), and BGB-A317 (BeiGene Ltd.).

[0128] In various embodiments, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the invention comprise one or more targeting moieties to PD-L1. In some embodiments, the vaccine compositions, adjuvants, chimeric proteins, or chimeric protein complexes comprise one or more targeting moieties that selectively bind to a PD-L1 polypeptide. In some embodiments, the vaccine compositions, adjuvants, chimeric proteins, or chimeric protein complexes comprise one or more of an antibody, antibody derivative or antibody format, peptide or polypeptide, or fusion protein that selectively binds to a PD-L1 polypeptide.

[0129] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody MEDI4736 (also known as durvalumab), or a fragment thereof. MEDI4736 is selective for PD-L1 and blocks the binding of PD-L1 to the PD-1 and CD80 receptors. MEDI4736 and antigen-binding fragments thereof for use in the methods provided herein comprise a heavy chain and a light chain or a heavy chain variable region and a light chain variable region. The sequence of MEDI4736 is disclosed in WO2016 / 06272, the entire contents of which are incorporated herein by reference. In an exemplary embodiment, MEDI4736 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:43; and / or a light chain comprising the amino acid sequence of SEQ ID NO:44.

[0130] In an exemplary embodiment, MEDI4736, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4 (SEQ ID NO:45) of WO2016 / 06272; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:3 (SEQ ID NO:46) of WO2016 / 06272.

[0131] In some embodiments, the targeting moiety comprises the anti-PD-L1 antibody atezolizumab (also known as MPDL3280A, RG7446), or a fragment thereof. In an exemplary embodiment, atezolizumab or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:47; and / or a light chain comprising the amino acid sequence of SEQ ID NO:48.

[0132] In some embodiments, the targeting moiety comprises the anti-PD-L1 antibody avelumab (also known as MSB0010718C), or a fragment thereof. In an exemplary embodiment, atezolizumab, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:49; and / or a light chain comprising the amino acid sequence of SEQ ID NO:50.

[0133] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody BMS-936559 (also known as 12A4, MDX-1105), or a fragment thereof, as disclosed in US 2013 / 0309250 and WO 2007 / 005874, the entire disclosures of which are incorporated by reference herein. In an exemplary embodiment, BMS-936559, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO:51); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:52).

[0134] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 3G10, or a fragment thereof, as disclosed in US 2013 / 0309250 and WO 2007 / 005874, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, 3G10, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO:53); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:54).

[0135] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 10A5, or a fragment thereof, as disclosed in US 2013 / 0309250 and WO 2007 / 005874, the entire disclosures of which are incorporated by reference herein. In an exemplary embodiment, 10A5, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO:55); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:56).

[0136] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 5F8, or a fragment thereof, as disclosed in US 2013 / 0309250 and WO 2007 / 005874, the entire disclosures of which are incorporated by reference herein. In an exemplary embodiment, 5F8, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO:57); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:58).

[0137] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 10H10, or a fragment thereof, as disclosed in US 2013 / 0309250 and WO 2007 / 005874, the entire disclosures of which are incorporated by reference herein. In an exemplary embodiment, 10H10, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO:59); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:60).

[0138] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 1B12, or a fragment thereof, as disclosed in US 2013 / 0309250 and WO 2007 / 005874, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, 1B12, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO:61); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:62).

[0139] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 7H1, or a fragment thereof, as disclosed in US 2013 / 0309250 and WO 2007 / 005874, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, 7H1, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO:63); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:64).

[0140] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 11E6, or a fragment thereof, as disclosed in US 2013 / 0309250 and WO 2007 / 005874, the entire disclosures of which are incorporated by reference herein. In an exemplary embodiment, 11E6, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO:65); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:66).

[0141] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 12B7, or a fragment thereof, as disclosed in US 2013 / 0309250 and WO 2007 / 005874, the entire disclosures of which are incorporated by reference herein. In an exemplary embodiment, 12B7, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO:67); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:68).

[0142] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 13G4, or a fragment thereof, as disclosed in US 2013 / 0309250 and WO 2007 / 005874, the entire disclosures of which are incorporated by reference herein. In an exemplary embodiment, 13G4, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO:69); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:70).

[0143] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 1E12, or a fragment thereof, as disclosed in US 2014 / 0044738, the entire disclosure of which is incorporated herein by reference. In an exemplary embodiment, 1E12, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO:71); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:72).

[0144] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 1F4, or a fragment thereof, as disclosed in US 2014 / 0044738, the entire disclosure of which is incorporated herein by reference. 1F4, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO:73); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:74).

[0145] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2G11, or a fragment thereof, as disclosed in US 2014 / 0044738, the entire disclosure of which is incorporated herein by reference. In an exemplary embodiment, 2G11, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO:75); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:76).

[0146] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 3B6, or a fragment thereof, as disclosed in US 2014 / 0044738, the entire disclosure of which is incorporated herein by reference. In an exemplary embodiment, 3B6, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO:77); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:78).

[0147] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 3D10, or a fragment thereof, as disclosed in US 2014 / 0044738 and WO 2012 / 145493, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, 3D10, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO:79); and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO:80).

[0148] In some embodiments, the targeting moiety comprises any one of the anti-PD-L1 antibodies disclosed in US2011 / 0271358 and WO2010 / 036959, the entire contents of which are incorporated by reference herein. In exemplary embodiments, the antibody or antigen-binding fragment thereof for use in the methods provided herein is selected from the group consisting of SEQ ID NOS: 34-38 of US2011 / 0271358 (SEQ ID NO: 34 of US2011 / 0271358 (SEQ ID NO: 81); SEQ ID NO: 35 of US2011 / 0271358 (SEQ ID NO: 82); SEQ ID NO: 36 of US2011 / 0271358 (SEQ ID NO: 83); SEQ ID NO: 37 of US2011 / 0271358 (SEQ ID NO: 84); and / or a light chain comprising an amino acid sequence selected from SEQ ID NOs: 39 to 42 of US2011 / 0271358 (SEQ ID NO: 39 of US2011 / 0271358 (SEQ ID NO: 86); SEQ ID NO: 40 of US2011 / 0271358 (SEQ ID NO: 87); SEQ ID NO: 41 of US2011 / 0271358 (SEQ ID NO: 88); SEQ ID NO: 42 of US2011 / 0271358 (SEQ ID NO: 89)).

[0149] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2.7A4, or a fragment thereof, as disclosed in WO2011 / 066389, US8,779,108, and US2014 / 0356353. The entire disclosures of these patents are incorporated by reference herein. In an exemplary embodiment, 2.7A4, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:2 (SEQ ID NO:90) of WO2011 / 066389; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:7 (SEQ ID NO:91) of WO2011 / 066389.

[0150] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2.9D10, or a fragment thereof, as disclosed in WO2011 / 066389, US8,779,108, and US2014 / 0356353. The entire disclosures of these patents are incorporated by reference herein. In an exemplary embodiment, 2.9D10, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:12 (SEQ ID NO:92) of WO2011 / 066389; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:17 (SEQ ID NO:93) of WO2011 / 066389.

[0151] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2.14H9, or a fragment thereof, as disclosed in WO2011 / 066389, US8,779,108, and US2014 / 0356353. The entire disclosures of these patents are incorporated by reference herein. In an exemplary embodiment, 2.14H9, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:22 (SEQ ID NO:94) of WO2011 / 066389; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:27 (SEQ ID NO:95) of WO2011 / 066389.

[0152] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2.20A8, or a fragment thereof, as disclosed in WO2011 / 066389, US8,779,108, and US2014 / 0356353. The entire disclosures of these patents are incorporated by reference herein. In an exemplary embodiment, 2.20A8, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:32 (SEQ ID NO:96) of WO2011 / 066389; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:37 (SEQ ID NO:97) of WO2011 / 066389.

[0153] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 3.15G8, or a fragment thereof, as disclosed in WO2011 / 066389, US8,779,108, and US2014 / 0356353. The entire disclosures of these patents are incorporated by reference herein. In an exemplary embodiment, 3.15G8, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:42 (SEQ ID NO:98) of WO2011 / 066389; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:47 (SEQ ID NO:99) of WO2011 / 066389.

[0154] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 3.18G1, or a fragment thereof, as disclosed in WO2011 / 066389, US8,779,108, and US2014 / 0356353. The entire disclosures of these patents are incorporated by reference herein. In an exemplary embodiment, 3.18G1, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:52 (SEQ ID NO:100) of WO2011 / 066389; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:57 (SEQ ID NO:101) of WO2011 / 066389.

[0155] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2.7A4OPT, or a fragment thereof, as disclosed in WO2011 / 066389, US8,779,108, and US2014 / 0356353. The entire disclosures of these patents are incorporated by reference herein. In an exemplary embodiment, 2.7A4OPT, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:62 (SEQ ID NO:102) of WO2011 / 066389; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:67 (SEQ ID NO:103) of WO2011 / 066389.

[0156] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2.14H9OPT, or a fragment thereof, as disclosed in WO2011 / 066389, US8,779,108, and US2014 / 0356353. The entire disclosures of these patents are incorporated by reference herein. In an exemplary embodiment, 2.14H9OPT, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:72 (SEQ ID NO:104) of WO2011 / 066389; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:77 (SEQ ID NO:105) of WO2011 / 066389.

[0157] In some embodiments, the targeting moiety comprises any one of the anti-PD-L1 antibodies disclosed in WO2016 / 061142, the entire contents of which are incorporated herein by reference. In exemplary embodiments, the antibodies or antigen-binding fragments thereof for use in the methods provided herein are selected from the group consisting of SEQ ID NOs: 18, 30, 38, 46, 50, 54, 62, 70, and 78 of WO2016 / 061142 (SEQ ID NO: 18 of WO2016 / 061142 (SEQ ID NO: 106); SEQ ID NO: 30 of WO2016 / 061142 (SEQ ID NO: 107); SEQ ID NO: 38 of WO2016 / 061142 (SEQ ID NO: 109); SEQ ID NO: 40 of WO2016 / 061142 (SEQ ID NO: 110); SEQ ID NO: 41 of WO2016 / 061142 (SEQ ID NO: 111); SEQ ID NO: 42 of WO2016 / 061142 (SEQ ID NO: 112); SEQ ID NO: 43 of WO2016 / 061142 (SEQ ID NO: 113); SEQ ID NO: 44 of WO2016 / 061142 (SEQ ID NO: 114); SEQ ID NO: 45 of WO2016 / 061142 (SEQ ID NO: 115); SEQ ID NO: 46 of WO2016 / 061142 (SEQ ID NO: 116); SEQ ID NO: 47 of WO2016 / 061142 (SEQ ID NO: 117); SEQ ID NO: 49 of WO2016 SEQ ID NO: 108); SEQ ID NO: 46 of WO2016 / 061142 (SEQ ID NO: 109); SEQ ID NO: 50 of WO2016 / 061142 (SEQ ID NO: 110); SEQ ID NO: 54 of WO2016 / 061142 (SEQ ID NO: 111); SEQ ID NO: 62 of WO2016 / 061142 (SEQ ID NO: 112); SEQ ID NO: 70 of WO2016 / 061142 (SEQ ID NO: 113) and SEQ ID NO: 78 of WO2016 / 061142 (SEQ ID NO: 114). 4)); and / or a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 22, 26, 34, 42, 58, 66, 74, 82, and 86 of WO2016 / 061142 (SEQ ID NO: 22 of WO2016 / 061142 (SEQ ID NO: 115); SEQ ID NO: 26 of WO2016 / 061142 (SEQ ID NO: 116); SEQ ID NO: 34 of WO2016 / 061142 (SEQ ID NO: 117); SEQ ID NO: 35 of WO2016 / 061142 (SEQ ID NO: 118); SEQ ID NO: 36 of WO2016 / 061142 (SEQ ID NO: 119); SEQ ID NO: 40 of WO2016 / 061142 (SEQ ID NO: 120); 42 (SEQ ID NO: 118) of WO2016 / 061142; SEQ ID NO: 58 of WO2016 / 061142 (SEQ ID NO: 119) of WO2016 / 061142; SEQ ID NO: 66 of WO2016 / 061142 (SEQ ID NO: 120) of WO2016 / 061142; SEQ ID NO: 74 of WO2016 / 061142 (SEQ ID NO: 121) of WO2016 / 061142; SEQ ID NO: 82 of WO2016 / 061142 (SEQ ID NO: 122) and SEQ ID NO: 86 of WO2016 / 061142 (SEQ ID NO: 123) of WO2016 / 061142.

[0158] In one embodiment, the targeting moiety comprises any one of the anti-PD-L1 antibodies disclosed in WO2016 / 022630, the entire contents of which are hereby incorporated by reference.In exemplary embodiments, the antibodies or antigen-binding fragments thereof for use in the methods provided herein are selected from the group consisting of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, and 46 of WO2016 / 022630 (SEQ ID NO: 2 of WO2016 / 022630 (SEQ ID NO: 124); SEQ ID NO: 6 of WO2016 / 022630 (SEQ ID NO: 125); SEQ ID NO: 10 of WO2016 / 022630 (SEQ ID NO: 126); SEQ ID NO: 14 of WO2016 / 022630 (SEQ ID NO: 127); SEQ ID NO: 15 of WO2016 / 022630 (SEQ ID NO: 128); SEQ ID NO: 16 of WO2016 / 022630 (SEQ ID NO: 129); SEQ ID NO: 2 of WO2016 / 022630 (SEQ ID NO: 130); SEQ ID NO: 2 of WO2016 / 022630 (SEQ ID NO: 131); SEQ ID NO: 2 of WO2016 / 022630 (SEQ ID NO: 132); SEQ ID NO: 2 of WO2016 / 022630 (SEQ ID NO: 133); SEQ ID NO: 2 of WO2016 / 022630 (SEQ ID NO: 134); SEQ ID NO: 2 of WO2016 / 022630 (SEQ ID NO: 135); SEQ ID NO: 2 of WO2016 / 022630 (SEQ ID NO: 1 SEQ ID NO: 18 of WO2016 / 022630 (SEQ ID NO: 128); SEQ ID NO: 22 of WO2016 / 022630 (SEQ ID NO: 129); SEQ ID NO: 26 of WO2016 / 022630 (SEQ ID NO: 130); SEQ ID NO: 30 of WO2016 / 022630 (SEQ ID NO: 131); SEQ ID NO: 34 of WO2016 / 022630 (SEQ ID NO: 132); SEQ ID NO: 38 of WO2016 / 022630 (SEQ ID NO: 133); SEQ ID NO: 42 of WO2016 / 022630 (SEQ ID NO: 134); and SEQ ID NO: 46 of WO2016 / 022630 (SEQ ID NO: 13 5)); and / or a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, and 48 of WO2016 / 022630 (SEQ ID NO: 4 of WO2016 / 022630 (SEQ ID NO: 136); SEQ ID NO: 8 of WO2016 / 022630 (SEQ ID NO: 137); SEQ ID NO: 12 of WO2016 / 022630 (SEQ ID NO: 138); SEQ ID NO: 16 of WO2016 / 022630 (SEQ ID NO: 139); SEQ ID NO: 20 of WO2016 / 022630 (SEQ ID NO: 140); W The present invention relates to a light chain comprising an amino acid sequence selected from SEQ ID NO: 24 of WO2016 / 022630 (SEQ ID NO: 141); SEQ ID NO: 28 of WO2016 / 022630 (SEQ ID NO: 142); SEQ ID NO: 32 of WO2016 / 022630 (SEQ ID NO: 143); SEQ ID NO: 36 of WO2016 / 022630 (SEQ ID NO: 144); SEQ ID NO: 40 of WO2016 / 022630 (SEQ ID NO: 145); SEQ ID NO: 44 of WO2016 / 022630 (SEQ ID NO: 146); and SEQ ID NO: 48 of WO2016 / 022630 (SEQ ID NO: 147)).

[0159] In one embodiment, the targeting moiety comprises any one of the anti-PD-L1 antibodies disclosed in WO2015 / 112900, the entire contents of which are hereby incorporated by reference. In exemplary embodiments, the antibody or antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 38, 50, 82, and 86 of WO2015 / 112900 (SEQ ID NO: 38 of WO2015 / 112900 (SEQ ID NO: 148); SEQ ID NO: 50 of WO2015 / 112900 (SEQ ID NO: 149); SEQ ID NO: 82 of WO2015 / 112900 (SEQ ID NO: 150); and SEQ ID NO: 86 of WO2015 / 112900 (SEQ ID NO: 151)); and / or a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 42, 46, 54, 58, 62, 66, 70, 74, and 78 of WO2015 / 112900 (SEQ ID NO: 152). SEQ ID NO: 42 of WO2015 / 112900 (SEQ ID NO: 152); SEQ ID NO: 46 of WO2015 / 112900 (SEQ ID NO: 153); SEQ ID NO: 54 of WO2015 / 112900 (SEQ ID NO: 154); SEQ ID NO: 58 of WO2015 / 112900 (SEQ ID NO: 155); SEQ ID NO: 62 of WO2015 / 112900 (SEQ ID NO: 156); SEQ ID NO: 66 of WO2015 / 112900 (SEQ ID NO: 157); SEQ ID NO: 70 of WO2015 / 112900 (SEQ ID NO: 158); SEQ ID NO: 74 of WO2015 / 112900 (SEQ ID NO: 159); and SEQ ID NO: 78 of WO2015 / 112900 (SEQ ID NO: 160).

[0160] In some embodiments, the targeting moiety comprises any one of the anti-PD-L1 antibodies disclosed in WO2010 / 077634 and US 8,217,149, the entire disclosures of which are incorporated by reference herein. In an exemplary embodiment, an anti-PD-L1 or antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:20 (SEQ ID NO:161) of WO2010 / 077634; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:21 (SEQ ID NO:162) of WO2010 / 077634.

[0161] In one embodiment, the targeting moiety comprises any one of the anti-PD-L1 antibodies obtainable from the hybridomas available under CNCM Accession Nos. CNCM I-4122, CNCM I-4080, and CNCM I-4081, as disclosed in US20120039906, the entire disclosures of which are hereby incorporated by reference.

[0162] In one embodiment, the targeting moiety comprises a VHH directed against a PD-L1 antibody, e.g., as disclosed in US 8,907,065 and WO 2008 / 071447, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, the VHH directed against PD-L1 comprises SEQ ID NOS: 394-399 of US 8,907,065 (SEQ ID NO: 394 (SEQ ID NO: 163) of US 8,907,065; SEQ ID NO: 395 (SEQ ID NO: 164) of US 8,907,065; SEQ ID NO: 396 (SEQ ID NO: 165) of US 8,907,065; SEQ ID NO: 397 (SEQ ID NO: 166) of US 8,907,065; SEQ ID NO: 398 (SEQ ID NO: 167) of US 8,907,065; SEQ ID NO: 399 (SEQ ID NO: 168) of US 8,907,065).

[0163] In various embodiments, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the invention comprise one or more targeting moieties to PD-L2. In some embodiments, the vaccine compositions, adjuvants, chimeric proteins, or chimeric protein complexes comprise one or more targeting moieties that selectively bind to a PD-L2 polypeptide. In some embodiments, the vaccine compositions, adjuvants, chimeric proteins, or chimeric protein complexes comprise one or more of an antibody, antibody derivative or antibody format, peptide or polypeptide, or fusion protein that selectively binds to a PD-L2 polypeptide.

[0164] In one embodiment, the targeting moiety comprises a VHH directed against PD-L2, e.g., as disclosed in US8,907,065 and WO2008 / 071447, the entire disclosures of which are hereby incorporated by reference. In an exemplary embodiment, the VHH against PD-L2 comprises SEQ ID NOs: 449 to 455 of US 8,907,065 (SEQ ID NO: 449 of US 8,907,065 (SEQ ID NO: 169); SEQ ID NO: 450 of US 8,907,065 (SEQ ID NO: 170); SEQ ID NO: 451 of US 8,907,065 (SEQ ID NO: 171); SEQ ID NO: 452 of US 8,907,065 (SEQ ID NO: 172); SEQ ID NO: 453 of US 8,907,065 (SEQ ID NO: 173); SEQ ID NO: 454 of US 8,907,065 (SEQ ID NO: 174); and SEQ ID NO: 455 of US 8,907,065 (SEQ ID NO: 175)).

[0165] In some embodiments, the targeting moiety comprises any one of the anti-PD-L2 antibodies disclosed in US2011 / 0271358 and WO2010 / 036959, the entire contents of which are incorporated by reference herein. In exemplary embodiments, the antibody or antigen-binding fragment thereof for use in the methods provided herein is selected from the group consisting of SEQ ID NOS: 43-47 of US2011 / 0271358 (SEQ ID NO: 43 of US2011 / 0271358 (SEQ ID NO: 176); SEQ ID NO: 44 of US2011 / 0271358 (SEQ ID NO: 177); SEQ ID NO: 45 of US2011 / 0271358 (SEQ ID NO: 178); SEQ ID NO: 46 of US2011 / 0271358 (SEQ ID NO: 179); and SEQ ID NO: 47 of US2011 / 0271358 (SEQ ID NO: 179). and / or a light chain comprising an amino acid sequence selected from SEQ ID NOs: 48 to 51 of US2011 / 0271358 (SEQ ID NO: 48 of US2011 / 0271358 (SEQ ID NO: 181); SEQ ID NO: 49 of US2011 / 0271358 (SEQ ID NO: 182); SEQ ID NO: 50 of US2011 / 0271358 (SEQ ID NO: 183); and SEQ ID NO: 51 of US2011 / 0271358 (SEQ ID NO: 184)).

[0166] In various embodiments, the targeting moieties of the invention align with any of the sequences disclosed herein by at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97% , at least about 98%, at least about 99%, or 100% identical (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or to any of the sequences disclosed herein). or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, about 99% or about 100% sequence identity).

[0167] In various embodiments, targeting moieties of the invention can include any combination of heavy chain, light chain, heavy chain variable region, light chain variable region, complementarity determining region (CDR), and framework region sequences that target PD-1, PD-L1, and / or PD-L2 as disclosed herein.

[0168] Additional antibodies, antibody derivatives or antibody formats, peptides or polypeptides, or fusion proteins that selectively bind to or target PD-1, PD-L1, and / or PD-L2 are disclosed in WO2011 / 066389, US2008 / 0025980, US2013 / 0034559, US8,779,108, US2014 / 0356353, US8,609,089, US2010 / 028330, US2012 / 0114649, WO2010 / 027827, WO2011 / 066342, US8,907,065, WO2016 / 062722, WO2009 / 101611, WO 2010 / 027827, WO2011 / 066342, WO2007 / 005874, WO2001 / 014556, US2011 / 0271358 , WO2010 / 036959, WO2010 / 077634, US8,217,149, US2012 / 0039906, WO2012 / 14549 3, US2011 / 0318373, US8,779,108, US2014 / 0044738, WO2009 / 089149, WO2007 / 00587, WO2016 / 061142, WO2016 / 02263, WO2010 / 077634, and WO2015 / 112900, the entire disclosures of which are hereby incorporated by reference.

[0169] In one embodiment, a chimeric protein, vaccine, adjuvant, or chimeric protein complex of the invention comprises (i) a targeting moiety directed against T cells, e.g., mediated by targeting to CD8, and (ii) a targeting moiety directed against tumor cells, in conjunction with any of the signal transduction factors described herein (e.g., IL-1α, pro-IL-1α, or variants thereof). In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the invention comprises a targeting moiety directed against CD8 on T cells and a second targeting moiety directed against PD-L1 or PD-L2 on tumor cells.

[0170] In one embodiment, a chimeric protein, vaccine, adjuvant, or chimeric protein complex of the invention comprises (i) a targeting moiety directed against T cells, e.g., mediated by targeting to CD4, and ii) a targeting moiety directed against tumor cells, in conjunction with any signal transduction factor described herein (e.g., IL-1α or a variant thereof). In one embodiment, a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the invention comprises a targeting moiety directed against CD4 on T cells and a second targeting moiety directed against PD-L1 or PD-L2 on tumor cells.

[0171] In one embodiment, the chimeric protein, vaccine, adjuvant, or chimeric protein complex of the invention comprises (i) a targeting moiety directed against T cells, e.g., mediated by targeting to CD3, CXCR3, CCR4, CCR9, CD70, CD103, or one or more immune checkpoint markers, and ii) a targeting moiety directed against tumor cells, in conjunction with any signal transduction factor described herein (e.g., IL-1α or a variant thereof). In one embodiment, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, of the invention comprises a targeting moiety directed against CD3 on T cells and a second targeting moiety directed against PD-L1 or PD-L2 on tumor cells.

[0172] In some embodiments, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the present invention have one or more targeting moieties directed against CD3 expressed on T cells. In some embodiments, the vaccine compositions, adjuvants, chimeric proteins, or mer protein complexes have one or more targeting moieties that selectively bind to a CD3 polypeptide. In some embodiments, the vaccine compositions, adjuvants, chimeric proteins, or mer protein complexes comprise one or more of an antibody, antibody derivative or antibody format, peptide or polypeptide, or fusion protein that selectively binds to a CD3 polypeptide.

[0173] In one embodiment, the targeting moiety comprises the anti-CD3 antibody muromonab-CD3 (also known as Orthoclone OKT3), or a fragment thereof. Muromonab-CD3 is disclosed in U.S. Patent No. 4,361,549 and Wilde et al. (1996) 51:865-894, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, muromonab-CD3, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence of (SEQ ID NO: 185); and / or a light chain comprising the amino acid sequence of (SEQ ID NO: 186).

[0174] In one embodiment, the targeting moiety comprises the anti-CD3 antibody otelixizumab, or a fragment thereof. Otelixizumab is disclosed in U.S. Patent Publication No. 20160000916 and Chatenoud et al. (2012) 9:372-381, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, otelixizumab, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 187; and / or a light chain comprising the amino acid sequence of SEQ ID NO: 188.

[0175] In one embodiment, the targeting moiety comprises the anti-CD3 antibody teplizumab (also known as MGA031 and hOKT3γ1(Ala-Ala)), or a fragment thereof. Teplizumab is disclosed in Chatenoud et al. (2012) 9:372-381, the entire disclosure of which is incorporated herein by reference. In an exemplary embodiment, teplizumab, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 189; and / or a light chain comprising the amino acid sequence of SEQ ID NO: 190.

[0176] In one embodiment, the targeting moiety comprises the anti-CD3 antibody visilizumab (also known as Nuvion®; HuM291), or a fragment thereof. Visilizumab is disclosed in US Pat. No. 5,834,597 and WO2004052397 and Cole et al., Transplantation (1999) 68:563-571, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, visilizumab, or an antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 191; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 192.

[0177] In one embodiment, the targeting moiety comprises the anti-CD3 antibody foralarumab (also known as NI-0401), or a fragment thereof. In various embodiments, the targeting moiety comprises any one of the anti-CD3 antibodies disclosed in US20140193399, US7,728,114, US20100183554, and US8,551,478, the entire disclosures of which are hereby incorporated by reference.

[0178] In exemplary embodiments, an anti-CD3 antibody or antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:2 and SEQ ID NO:6 of US 7,728,114 (SEQ ID NO:2 of US 7,728,114 (SEQ ID NO:193) and SEQ ID NO:6 of US 7,728,114 (SEQ ID NO:194)); and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:4 and SEQ ID NO:8 of US 7,728,114 (SEQ ID NO:4 of US 7,728,114 (SEQ ID NO:195) and SEQ ID NO:8 of US 7,728,114 (SEQ ID NO:196)).

[0179] In some embodiments, the targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 of US 7,728,114; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4 of US 7,728,114. In some embodiments, the targeting moiety comprises any one of the anti-CD3 antibodies disclosed in US 2016 / 0168247, the entire contents of which are incorporated herein by reference. In exemplary embodiments, an antibody or antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 6-9 of US2016 / 0168247 (SEQ ID NO: 6 of US2016 / 0168247 (SEQ ID NO: 197); SEQ ID NO: 7 of US2016 / 0168247 (SEQ ID NO: 198); SEQ ID NO: 8 of US2016 / 0168247 (SEQ ID NO: 199); SEQ ID NO: 9 of US2016 / 0168247 (SEQ ID NO: 200)); and / or a light chain comprising an amino acid sequence selected from SEQ ID NOs: 10-12 of US2016 / 0168247 (SEQ ID NO: 10 of US2016 / 0168247 (SEQ ID NO: 201); SEQ ID NO: 11 of US2016 / 0168247 (SEQ ID NO: 202); SEQ ID NO: 12 of US2016 / 0168247 (SEQ ID NO: 203)).

[0180] In some embodiments, the targeting moiety comprises any one of the anti-CD3 antibodies disclosed in US2015 / 0175699, the entire contents of which are incorporated herein by reference. In an exemplary embodiment, an antibody or antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising an amino acid sequence selected from SEQ ID NO:9 (SEQ ID NO:204) of US2015 / 0175699; and / or a light chain comprising an amino acid sequence selected from SEQ ID NO:10 (SEQ ID NO:205) of US2015 / 0175699.

[0181] In one embodiment, the targeting moiety comprises any one of the anti-CD3 antibodies disclosed in US 8,784,821, the entire contents of which are hereby incorporated by reference. In exemplary embodiments, the antibody or antigen-binding fragment thereof for use in the methods provided herein is selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98 and 114 of US8,784,821 (SEQ ID NO: 2 of US8,784,821 (SEQ ID NO: 206); SEQ ID NO: 18 of US8,784,821 (SEQ ID NO: 207); SEQ ID NO: 34 of US8,784,821 (SEQ ID NO: 208); SEQ ID NO: 50 of US8,784,821 (SEQ ID NO: 209); SEQ ID NO: 66 of US8,784,821 (SEQ ID NO: 210); SEQ ID NO: 82 of US8,784,821 (SEQ ID NO: 211); SEQ ID NO: 98 of US8,784,821 (SEQ ID NO: 212); and SEQ ID NO: 114 of US8,784,821 (SEQ ID NO: 213 )); and / or a light chain comprising an amino acid sequence selected from SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106 and 122 of US 8,784,821 (SEQ ID NO: 10 of US 8,784,821 (SEQ ID NO: 214); SEQ ID NO: 26 of US 8,784,821 (SEQ ID NO: 215); SEQ ID NO: 42 of US 8,784,821 (SEQ ID NO: 216); SEQ ID NO: 58 of US 8,784,821 (SEQ ID NO: 217); SEQ ID NO: 74 of US 8,784,821 (SEQ ID NO: 218); SEQ ID NO: 90 of US 8,784,821 (SEQ ID NO: 219); SEQ ID NO: 106 of US 8,784,821 (SEQ ID NO: 220); and SEQ ID NO: 122 of US 8,784,821 (SEQ ID NO: 221)).

[0182] In some embodiments, the targeting moiety comprises any one of the anti-CD3 binding constructs disclosed in US2015 / 0118252, the entire contents of which are incorporated herein by reference. In exemplary embodiments, an antibody or antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 6 and 86 of US20150118252 (SEQ ID NO: 6 of US20150118252 (SEQ ID NO: 222) and SEQ ID NO: 86 of US20150118252 (SEQ ID NO: 223)); and / or a light chain comprising an amino acid sequence selected from SEQ ID NO: 3 of US20150175699 (SEQ ID NO: 3 of US20150118252 (SEQ ID NO: 224)).

[0183] In certain embodiments, the targeting moiety comprises any one of the anti-CD3 binding proteins disclosed in US2016 / 0039934, the entire contents of which are hereby incorporated by reference. In exemplary embodiments, the antibody or antigen-binding fragment thereof for use in the methods provided herein is selected from SEQ ID NOS: 6-9 of US2016 / 0039934 (SEQ ID NO: 6 of US2016 / 0039934 (SEQ ID NO: 225); SEQ ID NO: 7 of US2016 / 0039934 (SEQ ID NO: 226); SEQ ID NO: 8 of US2016 / 0039934 (SEQ ID NO: 227); and SEQ ID NO: 9 of US2016 / 0039934 (SEQ ID NO: 228). 28)); and / or a light chain comprising an amino acid sequence selected from SEQ ID NOs: 1 to 4 of US2016 / 0039934 (SEQ ID NO: 1 of US2016 / 0039934 (SEQ ID NO: 229); SEQ ID NO: 2 of US2016 / 0039934 (SEQ ID NO: 230); SEQ ID NO: 3 of US2016 / 0039934 (SEQ ID NO: 231); and SEQ ID NO: 4 of US2016 / 0039934 (SEQ ID NO: 232)).

[0184] In various embodiments, the targeting moieties of the invention align with any of the sequences disclosed herein by at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, or about 100%, or about 101%, or about 102%, or about 103%, or about 104%, or about 105%, or about 106%, or about 107%, or about 108%, or about 109%, or about 110%, or about 111%, or about 112%, or about 113%, or about 114%, or about 115%, or about 116%, or about 117%, or about 118%, or about 119%, or about 120%, or about 121%, or about 122%, or about 123%, or about 124%, or about 125%, or about 126%, or about 127%, or about 128%, or about 129%, or about 130%, or about 131%, or about 132%, or about 133%, The CD3 targeting sequence may comprise a sequence that is about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, about 99%, or about 100% sequence identity).

[0185] In various embodiments, the targeting moieties of the present invention may comprise any combination of CD3-targeting heavy chain, light chain, heavy chain variable region, light chain variable region, complementarity determining region (CDR), and framework region sequences disclosed herein. In various embodiments, a targeting moiety of the invention may comprise any of the heavy chain, light chain, heavy chain variable region, light chain variable region, complementarity determining region (CDR), and framework region sequences of a CD3-specific antibody, including, but not limited to, X35-3, VIT3, BMA030 (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12.5, Fl 11-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3 / RW2-8C8, T3 / RW2-4B6, OKT3D, M-T301, SMC2, WT31, and F101.01. These CD3-specific antibodies are well known in the art and are described, inter alia, in Tunnacliffe (1989), Int. Immunol. 1, 546-550, the entire disclosure of which is hereby incorporated by reference.

[0186] Additional antibodies, antibody derivatives or formats, peptides or polypeptides, or fusion proteins that selectively bind or target CD3 are disclosed in U.S. Patent Publication No. 2016 / 0000916, U.S. Patent Nos. 4,361,549, 5,834,597, 6,491,916, 6,406,696, 6,143,297, 6,750,325, and WO2004 / 052397, the entire disclosures of which are incorporated herein by reference.

[0187] In one embodiment, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises, for example, (i) a targeting moiety directed against T cells, mediated by targeting to PD-1, and (ii) a targeting moiety directed against tumor cells, in combination with any signal transduction factor described herein (e.g., 1L-1α or a variant thereof).

[0188] As a non-limiting example, in various embodiments, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises any signal transduction factor described herein (e.g., 1L-1α or a variant thereof) in combination with (i) a targeting moiety directed against B cells, e.g., mediated by targeting to CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD38, CD39, CD40, CD70, CD72, CD73, CD74, CDw75, CDw76, CD77, CD78, CD79a / b, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD89, CD98, CD126, CD127, CDw130, CD138, or CDw150, and (ii) a targeting moiety directed against tumor cells. In certain embodiments, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises a targeting moiety directed against CD20.

[0189] In one embodiment, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises (i) a targeting moiety directed against B cells, e.g., mediated by targeting to CD19, CD20, or CD70, and (ii) a targeting moiety directed against tumor cells, in combination with any of the signal transduction factors described herein (e.g., 1L-1α or a variant thereof).

[0190] In one embodiment, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises (i) a targeting moiety directed against B cells, e.g., mediated by targeting to CD20, and (ii) a targeting moiety directed against tumor cells, in combination with any signal transduction factor described herein (e.g., 1L-1α or a variant thereof). In some embodiments, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises a targeting moiety directed against CD20 on B cells and a second targeting moiety directed against PD-L1 or PD-L2 on tumor cells. By way of example, in some embodiments, the CD20 targeting moiety has the sequence: QVQLQESGGGLAQAGGSLRLSCAASGRTFSMGWFRQAPGKEREFVAAITYSGGSPYYASSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAANPTYGSDWNAENWGQGTQVTVSS (SEQ ID NO: 288) It is a recombinant heavy chain antibody (VHH) having the following structure:

[0191] By way of non-limiting example, in various embodiments, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex may comprise any of the signal transduction factors described herein (e.g., 1L-1α or a variant thereof) in combination with (i) any of the signal transduction factors, e.g., 2B4 / SLAMF4, KIR2DS4, CD155 / PVR, KIR3DL1, CD94, LMIR1 / CD300A, CD69, LMIR2 / CD300A, CD69, LMIR2 / CD300B, LMIR3 / CD300C, LMIR4 / CD300D, LMIR5 / CD300E, LMIR6 / CD300F, LMIR7 / CD300G, LMIR8 / CD300H, LMIR9 / CD300I, LMIR10 / CD300I, LMIR11 / CD300I, LMIR12 / CD300I, LMIR13 / CD300I, LMIR14 / CD300I, LMIR15 / CD300I, LMIR16 / CD300I, LMIR17 / CD300I, LMIR18 / CD300I, LMIR19 / CD300I, LMIR20 / CD300I, LMIR21 / CD300I, LMIR22 / CD300I, LMIR23 / CD300I, LMIR24 / CD300I, LMIR25 / CD300I, LMIR26 / CD300I, LMIR27 / CD300I, LMIR28 / CD300I, LMIR29 / CD300I, LMIR29 / CD300I, LMIR29 / CD300I, LMIR29 / CD300I, LMIR c, CRACC / SLAMF7, LMIR3 / CD300LF, DNAM-1, LMIR5 / CD300LB, Fc-EpsilonRII, LMIR6 / CD300LE, Fc-γRl / CD64, MICA, Fc-γRIIB / CD32b, MICB, Fc-γRIIC / CD32c, MULT-1, Fc-γRIIA / CD32a, Nectin-2 / CD112, Fc-γRIII / CD16, NKG2A, Fc RH1 / IRTA5, NKG2C, FcRH2 / IRTA4, NKG2D, FcRH4 / IRTA1, NKp30, FcRH5 / IRTA2, NKp44, Fc-receptor-like 3 / CD16-2, NKp46 / NCR1, NKp80 / KLRF1, NTB-A / SLAMF6, Rae-1, Rae-1α, Rae-1β, Rae-1δ, H60, Rae-1ε, ILT2 / CD85j, Rae-1γ, ILT3 / CD8 and (ii) a targeting moiety directed against NK cells, mediated by targeting to 5k, TREM-1, ILT4 / CD85d, TREM-2, ILT5 / CD85a, TREM-3, KIR / CD158, TREML1 / TLT-1, KIR2DL1, ULBP-1, KIR2DL3, ULBP-2, KIR2DL4 / CD158d, or ULBP-3.

[0192] In one embodiment, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises (i) a targeting moiety directed against NK cells, e.g., mediated by targeting to Kir1α, DNAM-1, or CD64, and (ii) a targeting moiety directed against tumor cells, in combination with any of the signal transduction factors described herein (e.g., IL-1α or a variant thereof).

[0193] In one embodiment, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises (i) a targeting moiety directed against NK cells, e.g., mediated by targeting to KIR1, and (ii) a targeting moiety directed against tumor cells, in conjunction with any of the signal transduction factors described herein (e.g., IL-1α or variants thereof). In one embodiment, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises a targeting moiety directed against KIR1 on NK cells and a second targeting moiety directed against PD-L1 or PD-L2 on tumor cells.

[0194] In one embodiment, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises (i) a targeting moiety directed against NK cells, e.g., mediated by targeting to TIGIT or KIR1, and (ii) a targeting moiety directed against tumor cells, in conjunction with any of the signal transduction factors described herein (e.g., IL-1α or variants thereof). In some embodiments, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises a targeting moiety directed against TIGIT on NK cells and a second targeting moiety directed against PD-L1 or PD-L2 on tumor cells.

[0195] By way of non-limiting example, in various embodiments, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex may comprise any of the signal transduction factors described herein (e.g., IL-1α or a variant thereof) in combination with (i) any of the signal transduction factors, e.g., CLEC9A, XCR1, RANK, CD36 / SRB3, LOX-1 / SR-E1, CD68, MARCO, CD163, SR-A1 / MSR, CD5L, SREC-1, CL-P1 / COL1, CLEC9A, XCR1, RANK, CD36 / SRB3, LOX-1 / SR-E1, CD68, MARCO, CD163, SR-A1 / MSR, CD5L, SREC- ... EC12, SREC-II, LIMPIIISRB2, RP105, TLR4, TLR1, TLR5, TLR2, TLR6, TLR3, TLR9, 4-IBB ligand / TNFSF9, IL-12 / IL-23p40, 4-A mino-1, 8-naphthalimide, ILT2 / CD85j, CCL21 / 6Ckine, ILT3 / CD85k, 8-oxo-dG, ILT4 / CD85d, 8D6A, ILT5 / CD85a, A2B5, lutegrin α4 / CD49d, Aag, integrin β2 / CD18, AMICA, Langerin, B7-2 / CD86, leukotriene B4 Rl, B7-H3, LMIR1 / CD300A, BLAME / SLAMF8, LMIR2 / CD300c, ClqR1 / CD93, LMIR3 / CD300LF, CCR6, LMIR5 / CD300LBCCR7, LMIR6 / CD300LE, CD40 / TNFRS F5, MAG / Siglec-4-a, CD43, MCAM, CD45, MD-1, CD68, MD-2, CD83, MDL-1 / CLEC5A, CD84 / SLAMF5, MMR, CD97, NCAMLl, CD2F-10 / SLAMF9, OsteoactivinGPNMB, Chern23, PD-L2, CLEC-1, RP105, CLEC-2, Siglec-2 / CD22, CRACC / SLAMF7, Siglec-3 / CD33, DC-SIGN, Siglec-5, DC-SIGNR / CD299, Siglec-6, DCAR, Siglec-7, DCIR / CLEC4A, Siglec-9, DEC-205, Siglec-10, Dectin-1 / CLEC7A, Siglec-F, Dectin-2 / CLEC6A, SIGNR1 / CD209, DEP-1 / CD148, SIG and (ii) a targeting moiety directed against dendritic cells mediated by targeting to NR4, DLEC, SLAM, EMMPRIN / CD147, TCCR / WSX-1, Fc-γR1 / CD64, TLR3, Fc-γRIIB / CD32b, TREM-1, Fc-γRIIC / CD32c, TREM-2, Fc-γRIIA / CD32a, TREM-3, Fc-γRIII / CD16, TREML1 / TLT-1, ICAM-2 / CD102, or vanilloid R1, and

[0196] In one embodiment, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises (i) a targeting moiety directed against dendritic cells, e.g., mediated by targeting to CLEC-9A, DC-SIGN, CD64, CLEC-4A, or DEC205, and (ii) a targeting moiety directed against tumor cells, in conjunction with any of the signal transduction factors described herein (e.g., IL-1α or variants thereof). In certain embodiments, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises a targeting moiety directed against CLEC9A on dendritic cells and a second targeting moiety directed against PD-L1 or PD-L2 on tumor cells.

[0197] In one embodiment, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises (i) a targeting moiety directed against dendritic cells, e.g., mediated by targeting to CLEC9A, and (ii) a targeting moiety directed against tumor cells, in conjunction with any of the signal transduction factors described herein (e.g., IL-1α or variants thereof). In some embodiments, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises a targeting moiety directed against CLEC9A on dendritic cells and a second targeting moiety directed against PD-L1 or PD-L2 on tumor cells.

[0198] In one embodiment, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises (i) a targeting moiety directed against dendritic cells, e.g., mediated by targeting to XCR1, and (ii) a targeting moiety directed against tumor cells, in conjunction with any of the signal transduction factors described herein (e.g., IL-1α or variants thereof). In some embodiments, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises a targeting moiety directed against XCR1 on dendritic cells and a second targeting moiety directed against PD-L1 or PD-L2 on tumor cells.

[0199] In one embodiment, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises (i) a targeting moiety directed against dendritic cells, e.g., mediated by targeting to RANK, and (ii) a targeting moiety directed against tumor cells, in conjunction with any of the signal transduction factors described herein (e.g., IL-1α or variants thereof). In some embodiments, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises a targeting moiety directed against RANK on dendritic cells and a second targeting moiety directed against PD-L1 or PD-L2 on tumor cells.

[0200] By way of non-limiting example, in various embodiments, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex may comprise any of the signal transduction factors described herein (e.g., IL-1α or a variant thereof) in combination with (i) any of the signal transduction factors, e.g., SIRP1a, B7-1 / CD80, ILT4 / CD85d, B7-H1, ILT5 / CD85a, common beta chain, integrin α4 / CD49d, BLAME / SLAMF8, integrin αX / CDllc, CCL6 / C10, integrin β2 / CD18, CD155 / PVR, integrin β3 / CD61, CD31 / PECAM-1, Latexin, CD36 / SR-B3, Leukotriene B4R1, CD40 / TNFRSF5, LIMPIIISR-B2, CD43, LMIR1 / CD300A, CD45, LMIR2 / CD300c, CD68, LMIR3 / CD3 00LF, CD84 / SLAMF5, LMIR5 / CD300LB, CD97, LMIR6 / CD300LE, CD163, LRP-1, CD2F-10 / SLAMF9, MARCO, CRACC / SLAMF7, MD-1, ECF-L, MD-2, EMMPRIN / CD147 , MGL2, endoglin / CD105, osteoactivin / GPNMB, Fc-γRI / CD64, osteopontin, Fc-γRIIB / CD32b, PD-L2, Fc-γRIIC / CD32c, Siglec-3 / CD33, Fc-γRIIA / CD32a, SIGN R1 / CD209, Fc-γRIII / CD16, SLAM, GM-CSFRα, TCCR / WSX-1, ICAM-2 / CD102, TLR3, IFN-γRl, TLR4, IFN-γR2, TREM-l, IL-lRII, TREM-2, ILT2 / CD85j, TREM- 3, ILT3 / CD85k, TREML1 / TLT-1, 2B4 / SLAMF4, IL-10Rα, ALCAM, IL-10Rβ, aminopeptidase N / ANPEP, ILT2 / CD85j, common β chain, ILT3 / CD85k, ClqR1 / CD93, ILT4 / CD85d, CCR1, ILT5 / CD85a, CCR2, CD206, integrin α4 / CD49d, CCR5, integrin αM / CDllb, CCR8, integrin αX / CDllc, CD155 / PVR, integrin β2 / CD18, CD14, integrin β3 / CD61,CD36 / SR-B3, LAIR1, CD43, LAIR2, CD45, leukotriene B4-R1, CD68, LIMPIIISR-B2, CD84 / SLAMF5, LMIR1 / CD300A, CD97, LMIR2 / CD300c, CD163, LMIR3 / CD300LF, coagulation factor III / tissue factor, LMIR5 / CD300LB, CX3CR1, CX3CL1, LMIR6 / CD300LE, CXCR4, LRP-1, CXCR6, M-CSFR, DEP-1 / CD148, MD-1, DNAM-1, MD-2, EMMPRIN / CD147, MMR, endoglin / CD1 and (ii) a targeting moiety with directivity for monocytes / macrophages mediated by targeting to: (i) NCAM-L1, Fc-γRI / CD64, PSGL-1, Fc-γRIIIICD16, RP105, G-CSFR, L-selectin, GM-CSFRα, Siglec-3 / CD33, HVEM / TNFRSF14, SLAM, ICAM-1 / CD54, TCCR / WSX-1, ICAM-2 / CD102, TREM-1, -6R, TREM-2, CXCR1 / IL-8RA, TREM-3, or TREML1 / TLT-1; and

[0201] In one embodiment, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises (i) a targeting moiety directed against monocytes / macrophages, e.g., mediated by targeting to B7-H1, CD31 / PECAM-1, CD163, CCR2, or the macrophage mannose receptor CD206, and (ii) a targeting moiety directed against tumor cells, in combination with any of the signal transduction factors described herein (e.g., IL-1α or variants thereof).

[0202] In one embodiment, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises (i) a targeting moiety directed against monocytes / macrophages, e.g., mediated by targeting to SIRP1a, and (ii) a targeting moiety directed against tumor cells, in conjunction with any of the signal transduction factors described herein (e.g., IL-1α or variants thereof). In some embodiments, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises a targeting moiety directed against SIRP1a on macrophages and a second targeting moiety directed against PD-L1 or PD-L2 on tumor cells.

[0203] In various embodiments, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises one or more targeting moieties directed against one or more of the following checkpoint markers: PD-1 / PD-L1 or PD-L2, CD28 / CD80 or CD86, CTLA4 / CD80 or CD86, ICOS / ICOSL or B7RP1, BTLA / HVEM, KIR, LAG3, CD137 / CD137L, OX40 / OX40L, CD27, CD40L, TIM3 / Gal9, CD47, CD70, and A2aR. In one embodiment, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises (i) a targeting moiety directed against a checkpoint marker on T cells, e.g., PD-1, and (ii) a targeting moiety directed against a tumor cell, e.g., PD-L1 or PD-L2, in combination with any of the signal transduction factors described herein (e.g., IL-1α or variants thereof). In one embodiment, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises a targeting moiety directed against PD-1 on T cells and a second targeting moiety directed against PD-L1 on tumor cells. In another embodiment, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises a targeting moiety directed against PD-1 on T cells and a second targeting moiety directed against PD-L2 on tumor cells.

[0204] In some embodiments, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises two or more targeting moieties directed against the same or different immune cells. In some embodiments, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises (i) one or more targeting moieties directed against an immune cell selected from T cells, B cells, dendritic cells, macrophages, NK cells, or a subset thereof, and (ii) one or more targeting moieties directed against either the same or different immune cells selected from T cells, B cells, dendritic cells, macrophages, NK cells, or a subset thereof, in combination with any of the signal transduction factors described herein (e.g., IL-1α or a variant thereof).

[0205] In one embodiment, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises one or more targeting moieties directed against T cells and one or more targeting moieties directed against the same or different T cells. In one embodiment, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises one or more targeting moieties directed against T cells and one or more targeting moieties directed against B cells. In one embodiment, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises one or more targeting moieties directed against T cells and one or more targeting moieties directed against dendritic cells. In one embodiment, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises one or more targeting moieties directed against T cells and one or more targeting moieties directed against macrophages. In one embodiment, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises one or more targeting moieties directed against T cells and one or more targeting moieties directed against NK cells. For example, in an exemplary embodiment, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex may comprise a targeting moiety for CD8 and a targeting moiety for Clec9A. In another exemplary embodiment, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex may comprise a targeting moiety for CD8 and a targeting moiety for CD3. In another exemplary embodiment, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex may comprise a targeting moiety for CD8 and a targeting moiety for PD-1.

[0206] In various embodiments, the chimeric proteins or chimeric protein complexes disclosed herein comprise a CD8 binding agent that is a protein-based agent capable of specifically binding to CD8. In various embodiments, the CD8 binding agents of the invention are protein-based agents capable of specifically binding to CD8 without functionally modulating (e.g., partially or fully neutralizing) CD8.

[0207] In various embodiments, the CD8 binding agents of the present invention comprise a targeting moiety capable of specific binding. In various embodiments, the CD8 binding agents comprise a targeting moiety having an antigen recognition domain, such as an antibody or a derivative thereof. In one embodiment, the CD8 binding agent comprises a targeting moiety that is an antibody. In various embodiments, the antibody is a full-length multimeric protein comprising two heavy chains and two light chains. Each heavy chain comprises one variable region (e.g., V H ) and at least three constant regions (e.g., CH1, CH2, and CH3), and each light chain contains one variable region (V L ) and one constant region (C L ) The variable regions determine the specificity of the antibody. Each variable region contains three hypervariable regions, also known as complementarity-determining regions (CDRs), flanked by four relatively conserved framework regions (FRs). The three CDRs, termed CDR1, CDR2, and CDR3, contribute to the binding specificity of the antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody.

[0208] In some embodiments, the CD8 binding agent comprises a targeting moiety that is an antibody derivative or antibody format. In some embodiments, the CD8 binding agent is a targeting moiety described in U.S. Patents or U.S. Patent Publication Nos. 7,417,130, 2004 / 132094, 5,831,012, 2004 / 023334, 7,250,297, 6,818,418, 2004 / 209243, 7,838,629, 7,186,524, or 6,004,746. , 5,475,096, 2004 / 146938, 2004 / 157209, 6,994,982, 6,794,144, 2010 / 239633, 7,803,907, 2010 / 119446, and / or 7,166,697, the contents of which are hereby incorporated by reference in their entireties. The targeting moiety may be a antibody, recombinant heavy chain antibody (VHH), single chain antibody (scFv), shark heavy chain antibody (VNAR), microprotein (cysteine ​​knot protein, knottin), darpin; tetranectin; affibody; transbody; anticalin; adnectin; affilin; affimer, microbody; aptamer; alterase; plastic antibody; phylomer; stradobody; maxibody; shrimpbody; finomer, armadillo repeat protein, Kunitz domain, avimer, atrimer, probody, immunobody, triomab, troibody; pepbody; vatibody, unibody; duobody, Fv, Fab, Fab', F(ab')2, peptidomimetic molecule, or synthetic molecule. See also Storz MAbs. 2011 May-Jun;3(3):310-317.

[0209] In some embodiments, the CD8 binding agent comprises a targeting moiety that is a single domain antibody, such as a VHH. The VHH may be derived from an organism that produces VHH antibodies, e.g., camelids, sharks, etc., or the VHH may be an engineered VHH. VHHs are antibody-derived therapeutic proteins that contain the unique structural and functional properties of naturally occurring heavy chain antibodies. VHH technology is based on fully functional camelid antibodies that lack light chains. These heavy chain antibodies contain a single variable domain (VHH). H H) and two constant domains (CH2 and CH3).

[0210] In one embodiment, the CD8 binding agent comprises a VHH. In some embodiments, the VHH is a humanized or camelized VHH.

[0211] In some embodiments, the VHH is a fully human VHH. H In some embodiments, the fully human V domain is a human VH domain, e.g., a human body (Crescendo Biologics, Cambridge, UK). H The domains, e.g., human bodies, are monovalent, bivalent, or trivalent. In some embodiments, the fully human V H The domains, e.g., humabodies, may be monospecific or multispecific, such as monospecific, bispecific, or trispecific. H Domains such as the human body are described, for example, in WO2016 / 113555 and WO2016 / 113557, the disclosures of which are incorporated by reference in their entireties.

[0212] In some embodiments, the CD8-binding agent comprises a targeting moiety that is a VHH comprising a single amino acid chain having four "framework regions" or FRs, and three "complementarity-determining regions" or CDRs. As used herein, "framework region" or "FR" refers to a region in a variable domain that is located between the CDRs. As used herein, "complementarity-determining region" or "CDR" refers to a variable region in a VHH that comprises an amino acid sequence capable of specifically binding to an antigenic target. In various embodiments, the CD8-binding agent comprises a VHH having a variable domain comprising at least one CDR1, CDR2, and / or CDR3 sequence.

[0213] In some embodiments, the targeting moiety comprises an anti-CD8 antibody described in WO2019033043, the entire disclosure of which is incorporated herein by reference. In some embodiments, the anti-CD8 antibody comprises at least one heavy chain variable region comprising the amino acid sequence of CDR H1: GFNIKDTYIH (SEQ ID NO: 293); CDR H2: RIDPANDNTLYASKFQG (SEQ ID NO: 294); CDR H2: RIDPANDNTLYARKFQG (SEQ ID NO: 295); CDR H3: GRGYGYYVFDH (SEQ ID NO: 296); or CDR H3: TRGYGYYVFDT (SEQ ID NO: 297).

[0214] In some embodiments, the anti-CD8 antibody comprises at least one light chain variable region comprising the amino acid sequence of CDR L1: SISQY (SEQ ID NO: 298); CDR L1: SISKY (SEQ ID NO: 299); CDR L2: SGSTLQ (SEQ ID NO: 300); CDR L3: HNENPL (SEQ ID NO: 301); CDR L3: HNEFPV (SEQ ID NO: 302); CDR L3: HNEFPP (SEQ ID NO: 303); CDR L3: VNEFPP (SEQ ID NO: 304); CDR L3: VNEFPV (SEQ ID NO: 305).

[0215] In some embodiments, the targeting moiety comprises an anti-CD8 antibody described in WO2019023148 ​​(the entire disclosure of which is hereby incorporated by reference). In some embodiments, the anti-CD8 antibody comprises at least one heavy chain variable region comprising the amino acid sequence of: CDR H1: GFIFSNYG (SEQ ID NO: 306); CDR H2: IWYDGSNK (SEQ ID NO: 307); CDR H3: ARSYDMLTGSGDYYGL (SEQ ID NO: 308). In some embodiments, the anti-CD8 antibody comprises at least one light chain variable region comprising the amino acid sequence of: CDR L1: QDITNY (SEQ ID NO: 309); CDR L2: GAS; CDR L3: QQYNNYPLT (SEQ ID NO: 310).

[0216] In some embodiments, the targeting moiety comprises an anti-CD8 antibody described in WO2015184203, the entire disclosure of which is incorporated herein by reference. In some embodiments, the anti-CD8 antibody comprises at least one heavy chain variable region comprising the amino acid sequence of: CDR H1: SGYTGTDYNMH (SEQ ID NO: 311); CDR H2: YIYPYTGGTGYNQKFKN (SEQ ID NO: 312); CDR H1: DFGMN (SEQ ID NO: 313); CDR H2: LIYYDGSNKFY (SEQ ID NO: 314); CDR H3: PHYDGYYHFFDS (SEQ ID NO: 315). In some embodiments, the anti-CD8 antibody comprises at least one light chain variable region comprising the amino acid sequence of CDR L1: RASESVDSYDNSLMH (SEQ ID NO: 316); CDR L2: LASNLES (SEQ ID NO: 317); CDR L3: QQNNEDPYT (SEQ ID NO: 318); CDR L1: KGSQDINNYLA (SEQ ID NO: 319); CDR L2: NTDILHT (SEQ ID NO: 320); CDR L3: YQYNNGYT (SEQ ID NO: 321).

[0217] In some embodiments, the targeting moiety comprises an anti-CD8 antibody described in WO2018170096 (the entire disclosure of which is incorporated herein by reference). In some embodiments, the anti-CD8 antibody comprises at least one heavy chain variable region comprising the amino acid sequence of: CDR H1: GYTFTSY (SEQ ID NO: 322); CDR H2: DPSDNY (SEQ ID NO: 333); CDR H3: PKSAYAFDVGGYAMDY (SEQ ID NO: 334). In some embodiments, the anti-CD8 antibody comprises at least one light chain variable region comprising the amino acid sequence of: CDR L1: RTSENIDSYLT (SEQ ID NO: 335); CDR L2: AATLLAD (SEQ ID NO: 336); CDR L3: QHYYSTPWT (SEQ ID NO: 337).

[0218] In some embodiments, the targeting moiety comprises an anti-CD8 antibody described in WO2014164553 (the entire disclosure of which is incorporated herein by reference). In some embodiments, the anti-CD8 antibody comprises at least one heavy chain variable region comprising the amino acid sequence of CDR H1: GFNIKD (SEQ ID NO: 338); CDR H2: RIDPANDNT (SEQ ID NO: 339); CDR H3: GYGYYVFDH (SEQ ID NO: 340). In some embodiments, the anti-CD8 antibody comprises at least one light chain variable region comprising the amino acid sequence of CDR L1: RTSRSISQYLA (SEQ ID NO: 341); CDR L2: SGSTLQS (SEQ ID NO: 342); CDR L3: QQHNENPLT (SEQ ID NO: 343).

[0219] In various embodiments, the chimeric proteins or chimeric protein complexes disclosed herein comprise a CD4 binding agent that is a protein-based substance capable of specifically binding to CD4. In various embodiments, the CD4 binding agents of the present invention are protein-based substances capable of specifically binding to CD4 without functionally modulating (e.g., partially or completely neutralizing) CD4.

[0220] In various embodiments, the CD4 binding agents of the present invention comprise a targeting moiety capable of specific binding. In various embodiments, the CD4 binding agents comprise a targeting moiety having an antigen recognition domain, such as an antibody or derivative thereof. In one embodiment, the CD4 binding agent comprises a targeting moiety that is an antibody. In various embodiments, the antibody is a full-length multimeric protein comprising two heavy chains and two light chains. Each heavy chain comprises one variable region (e.g., V H ) and at least three constant regions (e.g., CH1, CH2, and CH3), and each light chain comprises one variable region (V L ) and one constant region (C L ) The variable regions determine the specificity of the antibody. Each variable region contains three hypervariable regions, also known as complementarity-determining regions (CDRs), flanked by four relatively conserved framework regions (FRs). The three CDRs, termed CDR1, CDR2, and CDR3, contribute to the binding specificity of the antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody.

[0221] In some embodiments, the CD4 binding agent comprises a targeting moiety that is an antibody derivative or antibody format. In some embodiments, the CD4 binding agent is a targeting moiety that ... , 5,475,096, 2004 / 146938, 2004 / 157209, 6,994,982, 6,794,144, 2010 / 239633, 7,803,907, 2010 / 119446, and / or 7,166,697, the contents of which are hereby incorporated by reference in their entireties. The targeting moiety may be a antibody, recombinant heavy chain antibody (VHH), single chain antibody (scFv), shark heavy chain antibody (VNAR), microprotein (cysteine ​​knot protein, knottin), darpin; tetranectin; affibody; transbody; anticalin; adnectin; affilin; affimer, microbody; aptamer; alterase; plastic antibody; phylomer; stradobody; maxibody; shrimpbody; finomer, armadillo repeat protein, Kunitz domain, avimer, atrimer, probody, immunobody, triomab, troibody; pepbody; vatibody, unibody; duobody, Fv, Fab, Fab', F(ab')2, peptidomimetic molecule, or synthetic molecule. See also Storz MAbs. 2011 May-Jun;3(3):310-317.

[0222] In some embodiments, the CD4 binding agent comprises a targeting moiety that is a single domain antibody, such as a VHH. The VHH may be derived from an organism that produces VHH antibodies, e.g., camelids, sharks, etc., or the VHH may be an engineered VHH. The VHH is an antibody-derived therapeutic protein that contains the unique structural and functional properties of naturally occurring heavy chain antibodies. VHH technology is based on the fully functional antibodies of camelids, which do not possess a single variable domain (VHH). These heavy chain antibodies contain a single variable domain (VHH). H It contains a nucleotide sequence (H) and two constant domains (CH2 and CH3).

[0223] In one embodiment, the CD4 binding agent comprises a VHH. In some embodiments, the VHH is a humanized VHH or a camelized VHH. In some embodiments, the VHH is a fully human VHH. H In some embodiments, the fully human V domain is a human VH domain, e.g., a human body (Crescendo Biologics, Cambridge, UK). H The domains, e.g., humanbodies, are monovalent, bivalent, or trivalent. In some embodiments, fully human V H Domains, e.g., humabodies, can be monospecific or multispecific, such as monospecific, bispecific, or trispecific. Exemplary fully human V H Domains such as the human body are described, for example, in WO2016 / 113555 and WO2016 / 113557, the entire disclosures of which are incorporated by reference.

[0224] In some embodiments, the CD4 binding agent comprises a targeting moiety that is a VHH comprising a single amino acid chain having four "framework regions" or FRs and three "complementarity-determining regions" or CDRs. As used herein, "framework region" or "FR" refers to a region in a variable domain located between the CDRs. As used herein, "complementarity-determining region" or "CDR" refers to a variable region in a VHH comprising an amino acid sequence capable of specifically binding to an antigenic target. In various embodiments, the CD4 binding agent comprises a VHH having a variable domain comprising at least one CDR1, CDR2, and / or CDR3 sequence. In some embodiments, the targeting moiety comprises an anti-CD4 antibody described in WO2020082045 (the entire disclosure of which is incorporated herein by reference). In some embodiments, the anti-CD4 antibody comprises at least one heavy chain variable region comprising the amino acid sequence of CDRH1: GYTFTAHI (SEQ ID NO: 344); CDRH2: IKPQYGAV (SEQ ID NO: 345); or CDRH3: AR. In some embodiments, the anti-CD4 antibody comprises at least one light chain variable region comprising the amino acid sequence of CDRL1: QGVGSD (SEQ ID NO: 346); CDRL2: HTS; or CDRL3: QVLQF (SEQ ID NO: 347).

[0225] In some embodiments, the targeting moiety comprises an anti-CD4 antibody described in WO2018170096 (the entire disclosure of which is incorporated herein by reference). In some embodiments, the anti-CD4 antibody comprises at least one heavy chain variable region comprising the amino acid sequence of CDRH1: GYTFTSN (SEQ ID NO: 348); CDRH2: YPRSGN (SEQ ID NO: 349); or CDRH3: RVPYFDH (SEQ ID NO: 350). In some embodiments, the anti-CD4 antibody comprises at least one light chain variable region comprising the amino acid sequence of CDRL1: KASQSVGNNVA (SEQ ID NO: 351); CDRL2: YASNRYT (SEQ ID NO: 352); or CDRL3: QQHYSSPFT (SEQ ID NO: 353).

[0226] In some embodiments, the targeting moiety comprises an anti-CD4 antibody described in WO2016156570 (the entire disclosure of which is hereby incorporated by reference). In some embodiments, the anti-CD4 antibody comprises at least one CDR1 comprising the amino acid sequence of CDR1: GYWMY (SEQ ID NO: 354); CDR1: SYSMG (SEQ ID NO: 355); CDR1: FNAMG (SEQ ID NO: 356); or CDR1: VMG. In some embodiments, the anti-CD4 antibody comprises at least one CDR2 comprising the amino acid sequence of CDR2: AISPGGGSTYYPDSVK (SEQ ID NO: 357); CDR2: AISWSGDETSYADSVK (SEQ ID NO: 358); CDR2: TIARAGATKYADSVKG (SEQ ID NO: 359); or CDR2: AVRWSSTGIYYTQYAD (SEQ ID NO: 360). In some embodiments, the anti-CD4 antibody comprises at least one CDR3 comprising the amino acid sequence of CDR3:SLTATHTYEYDY (SEQ ID NO: 361); CDR3:DRWWRPAGLQWDY (SEQ ID NO: 362); CDR3:RVFDLPNDY (SEQ ID NO: 363); or CDR3:DTYNSNPARWDGYDF (SEQ ID NO: 364).

[0227] In some embodiments, the targeting moiety comprises an anti-CD4 antibody described in WO2012145238 (the entire disclosure of which is hereby incorporated by reference). In some embodiments, the anti-CD4 antibody comprises at least one heavy chain variable region comprising the amino acid sequence of CDRH1: AYVIS (SEQ ID NO: 365); CDRH2: EIYPGSGSSYYNEKFKG (SEQ ID NO: 366); or CDRH3: SGDGSKFVY (SEQ ID NO: 367). In some embodiments, the anti-CD4 antibody comprises at least one light chain variable region comprising the amino acid sequence of CDRL1: KASQSVDYCGDSYMN (SEQ ID NO: 368); CDRL2: VASNLES (SEQ ID NO: 369); or CDRL3: QQSLQDPPT (SEQ ID NO: 370).

[0228] In some embodiments, the targeting moiety comprises an anti-CD4 antibody described in WO2008134046 (the entire disclosure of which is hereby incorporated by reference). In some embodiments, the anti-CD4 antibody comprises at least one heavy chain variable region comprising the amino acid sequence of CDRH1: GYTFTSYVIH (SEQ ID NO: 371); CDRH2: YINPYNDGTDYDEKFK (SEQ ID NO: 372); or CDRH3: EKDNYATGAWFAY (SEQ ID NO: 373). In some embodiments, the anti-CD4 antibody comprises at least one light chain variable region comprising the amino acid sequence of CDRL1: KSSQSLLYSTNQKNY (SEQ ID NO: 374); CDRL2: WASTRES (SEQ ID NO: 375); or CDRL3: QQYYSYRT (SEQ ID NO: 376).

[0229] In some embodiments, the targeting moiety comprises an anti-CD4 antibody described in WO2009012944, the entire disclosure of which is incorporated herein by reference. In some embodiments, the anti-CD4 antibody comprises at least one heavy chain variable region comprising the amino acid sequence of CDRH1:SYVIH (SEQ ID NO:377); CDRH1:GFTFSNYAMS (SEQ ID NO:378); or CDRH2:AISDHSTNTYYP (SEQ ID NO:379); CDRH3:EKDNYATGAWFAY (SEQ ID NO:380); or CDRH3:ARKYGGDYDPF (SEQ ID NO:381). In some embodiments, the anti-CD4 antibody comprises at least one light chain variable region comprising the amino acid sequence of CDRL1: KSSQSLLYSTNQKNYL (SEQ ID NO: 382); CDRL1: KSSGSLLYSTNQKNYL (SEQ ID NO: 383); CDRL1: KASQDINNY (SEQ ID NO: 384); CDRL2: WASTRES (SEQ ID NO: 385); CDRL2: YTSTLQPGVPS (SEQ ID NO: 386); CDRL3: QQYYSYRT (SEQ ID NO: 387); or CDRL3: YDNLLF (SEQ ID NO: 388).

[0230] In some embodiments, the targeting moiety comprises an anti-CD4 antibody described in WO2004005350, the entire disclosure of which is hereby incorporated by reference. In some embodiments, the anti-CD4 antibody comprises at least one heavy chain variable region comprising the amino acid sequence of CDRH1:TFGVH (SEQ ID NO:389) CDRH1:TAGVH (SEQ ID NO:390); or CDRH1:TFGVA (SEQ ID NO:391); CDRH2:VIWRSGITDYNVPFMS (SEQ ID NO:392); CDRH2:VIARSGITDYNVPFMS (SEQ ID NO:393); CDRH2:VIWASGITDYNVPFMS (SEQ ID NO:394); CDRH3:NDPGTGFAY (SEQ ID NO:395); CDRH3:NDPGTGAAY (SEQ ID NO:396); or CDRH3:NDPGTGFAA (SEQ ID NO:397). In some embodiments, the anti-CD4 antibody comprises at least one light chain variable region comprising the amino acid sequence of CDRL1: RASENIYSYLA (SEQ ID NO: 398); CDRL1: RASENIYSALA (SEQ ID NO: 399); CDRL2: DAKTLAE (SEQ ID NO: 400); CDRL3: QHHYGNPPT (SEQ ID NO: 401); CDRL3: QHAYGNPPT (SEQ ID NO: 402); or CDRL3: QHHAGNPPT (SEQ ID NO: 403).

[0231] In some embodiments, the targeting moiety comprises an anti-CD4 antibody described in WO2004083247 (the entire disclosure of which is hereby incorporated by reference). In some embodiments, the anti-CD4 antibody comprises at least one heavy chain variable region comprising the amino acid sequence of CDRH1: DYVIN (SEQ ID NO: 404); CDRH2: EIYPGSGSDYYNENLKD (SEQ ID NO: 405); or CDRH3: KGENGNSLAFAY (SEQ ID NO: 406). In some embodiments, the anti-CD4 antibody comprises at least one light chain variable region comprising the amino acid sequence of CDRL1: QSVDYDGDSYMN (SEQ ID NO: 407); CDRL2: AASNLES (SEQ ID NO: 408); or CDRL3: QQSIQDPCT (SEQ ID NO: 409).

[0232] In some embodiments, the targeting moiety comprises an anti-CD4 antibody described in WO2014100139, the entire disclosure of which is hereby incorporated by reference. In some embodiments, the anti-CD4 antibody has the following amino acid sequence: Anti-CD4 antibody MV1, heavy chain MEWSGVFMFLLSVTAGVHSQVQLQQSGPEVVKPGASVKMSCKASGYTFTSYVIHWVRQKPGQGLDWIGYINPYNDGTDYDEKFKGKATLTSDTSTSTAYMELSSLRSEDTAVYYCAREKD NYATGAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE PKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 410) and at least one heavy chain comprising:

[0233] In some embodiments, the anti-CD4 antibody has the following amino acid sequence: Anti-CD4 antibody MV1, light chain MEWSGVFIFLLSVTAGVHSDIVMTQSPDSLAVSLGERVTMNCKSSQSLLYSTNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYYRTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 411) and at least one light chain comprising:

[0234] In some embodiments, the targeting moiety comprises an anti-CD4 antibody described in WO2004083247, the entire disclosure of which is hereby incorporated by reference. In some embodiments, the anti-CD4 antibody has the following amino acid sequence: EEQLVESGGGLVKPGGSLRLSCAASGFSFSDCRMYWLRQAPGKGLEWIGVISVKSENYGANYAESVRGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCSASYYRYDVGAFAYGQGTLVTVSS (SEQ ID NO: 412) and at least one heavy chain comprising:

[0235] In some embodiments, the anti-CD4 antibody comprises at least one of the following amino acid sequences: DIVMTQSPDSLAVSLGERATINCRASKSVSTSGYSYIYWYQQKPGQPPKLLIYLASILESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSRELPTFGQGTKVEIK (SEQ ID NO: 413) and at least one light chain comprising:

[0236] In one embodiment, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises one or more targeting moieties directed against B cells and one or more targeting moieties directed against the same or different B cells. In one embodiment, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises one or more targeting moieties directed against B cells and one or more targeting moieties directed against T cells. In one embodiment, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises one or more targeting moieties directed against B cells and one or more targeting moieties directed against dendritic cells. In one embodiment, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises one or more targeting moieties directed against B cells and one or more targeting moieties directed against macrophages. In one embodiment, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises one or more targeting moieties directed against B cells and one or more targeting moieties directed against NK cells.

[0237] In one embodiment, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises one or more targeting moieties directed against dendritic cells and one or more targeting moieties directed against the same or different dendritic cells. In one embodiment, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises one or more targeting moieties directed against dendritic cells and one or more targeting moieties directed against T cells. In one embodiment, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises one or more targeting moieties directed against dendritic cells and one or more targeting moieties directed against B cells. In one embodiment, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises one or more targeting moieties directed against dendritic cells and one or more targeting moieties directed against macrophages. In one embodiment, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises one or more targeting moieties directed against dendritic cells and one or more targeting moieties directed against NK cells.

[0238] In one embodiment, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises one or more targeting moieties directed against macrophages and one or more targeting moieties directed against the same or different macrophages. In one embodiment, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises one or more targeting moieties directed against macrophages and one or more targeting moieties directed against T cells. In one embodiment, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises one or more targeting moieties directed against macrophages and one or more targeting moieties directed against B cells. In one embodiment, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises one or more targeting moieties directed against macrophages and one or more targeting moieties directed against dendritic cells. In one embodiment, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises one or more targeting moieties directed against macrophages and one or more targeting moieties directed against NK cells.

[0239] In one embodiment, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises one or more targeting moieties directed against NK cells and one or more targeting moieties directed against the same or different NK cells. In one embodiment, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises one or more targeting moieties directed against NK cells and one or more targeting moieties directed against T cells. In one embodiment, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises one or more targeting moieties directed against NK cells and one or more targeting moieties directed against B cells. In one embodiment, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises one or more targeting moieties directed against NK cells and one or more targeting moieties directed against macrophages. In one embodiment, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises one or more targeting moieties directed against NK cells and one or more targeting moieties directed against dendritic cells.

[0240] In one embodiment, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises a targeting moiety directed against a tumor cell and a second targeting moiety directed against the same or a different tumor cell, wherein the targeting moiety is capable of binding to any of the tumor antigens described herein.

[0241] In some embodiments, the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex is administered to or from a mammalian cell selected from the group consisting of adipocytes (e.g., white adipocytes, brown adipocytes), liver lipid cells, liver cells, kidney cells (e.g., kidney parietal cells, renal salivary gland, mammary gland, etc.), duct cells (e.g., seminal vesicles, prostate, etc.), intestinal brush border cells (with microvilli), exocrine gland striate duct cells, gallbladder epithelial cells, small efferent tract non-ciliated cells, epididymal principal cells, epididymal basal cells, endothelial cells, ameloblast epithelial cells (tooth enamel secretion), ear cells, and the like. Crescentic epithelial cells of the vestibular system (secreting proteoglycans), interdental epithelial cells of the organ of Corti (secreting the tectorial membrane that covers hair cells), loose connective tissue fibroblasts, corneal fibroblasts (keratocytes), tendon fibroblasts, bone marrow reticular fibroblasts, non-epithelial fibroblasts, pericytes, nucleus pulposus cells of the intervertebral disc, cementoblasts / cementocytes (secreting root bone-like Ivan cells), odontoblasts / odontocytes (secreting tooth dentin), hyaline chondrocytes, fibrochondrocytes , elastic chondrocytes, osteoblasts / osteocytes, bone precursor cells (osteoblast stem cells), vitreous cells of the vitreous body of the eye, stellate cells of the perilymphatic space of the ear, hepatic stellate cells (Ito cells), pancreatic stellate cells, skeletal muscle cells, satellite cells, cardiac muscle cells, smooth muscle cells, myoepithelial cells of the iris, myoepithelial cells of exocrine glands, exocrine secretory epithelial cells (e.g., salivary gland cells, mammary gland cells, lacrimal gland cells, sweat gland cells, sebaceous gland cells, prostate cells, gastric gland cells, pancreatic acinar cells, lung cells), hormone-secreting cells (e.g., pituitary cells, neurosecretory cells, intestinal and airway cells, thyroid gland cells, The targeting moieties comprise one or more targeting moieties having a recognition domain that binds to a target of interest (e.g., antigen, receptor), including those found on one or more cells selected from: epithelial cells, parathyroid cells, adrenal cells, Leydig cells of the testes, pancreatic islet cells, keratinizing epithelial cells, moist stratified barrier epithelial cells, neural cells (e.g., sensory transducer cells, autonomic neuron cells, sensory organ and peripheral neural support cells, and central nervous system neurons, and glial cells such as interneurons, principal cells, astrocytes, oligodendrocytes, and ependymal cells).

[0242] Targeted Moiety Format In some embodiments, the targeting moiety of the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex is a protein-based substance with specific binding capability, such as an antibody or a derivative thereof. In certain embodiments, the targeting moiety comprises an antibody. In various embodiments, the antibody is a full-length multimeric protein comprising two heavy chains and two light chains. Each heavy chain comprises one variable region (e.g., V H ) and at least three constant regions (e.g., CH1, CH2, and CH3), and each light chain contains one variable region (V L ) and one constant region (C L ) The variable regions determine the specificity of the antibody. Each variable region contains three hypervariable regions, also known as complementarity-determining regions (CDRs), flanked by four relatively conserved framework regions (FRs). The three CDRs are termed CDR1, CDR2, and CDR3 and contribute to the binding specificity of the antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody.

[0243] In some embodiments, the targeting moiety comprises an antibody derivative or antibody format. In some embodiments, the targeting moiety of the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex is an antibody described in U.S. Patent or U.S. Patent Publication Nos. 7,417,130, 2004 / 132094, 5,831,012, 2004 / 023334, 7,250,297, 6,818 , No. 418, No. 2004 / 209243, No. 7,838,629, No. 7,186,524, No. 6,004,746, No. 5,475,096, No. 2004 / 146938, 2004 / 157209, 6,994,982, 6,794,144, 2010 / 239633, 7,803,907, Single domain antibodies, recombinant heavy chain antibodies (VHH), single chain antibodies (scFv), shark heavy chain antibodies (VNAR), microproteins (cysteine ​​knot proteins, knottins), darpins; tetranectins; affibodies; transbodies; anticalins; adnectins; affilins; microbodies; peptide aptamers; alterases; plastic antibodies; phylomers; stradobodies; maxibodies; shrimp bodies; phylomers; armadillo repeat proteins (armadillo repeat proteins), as described in Patent Publication Nos. 2010 / 119446 and / or 7,166,697 (the entire contents of which are incorporated herein by reference). repeat protein); Kunitz domain, avimer, atrimer, probody, immunobody, triomab, troibody, pepbody, vaccibody, unibody; affimer, duobody, Fv, Fab, Fab', F(ab')2, peptidomimetic molecule, or synthetic molecule. See also Storz MAbs. 2011 May-Jun;3(3):310-317.

[0244] In one embodiment, the targeting moiety comprises a VHH derived from an organism that produces VHH antibodies, such as a camelid or shark, or a single-domain antibody, such as an engineered VHH. VHHs are antibody-derived therapeutic proteins that contain the unique structural and functional properties of naturally occurring heavy-chain antibodies. VHH technology is based on fully functional antibodies derived from camelids that lack light chains. These heavy-chain antibodies contain a single variable domain (VHH) and two constant domains (CH2 and CH3). VHHs are commercially available under the registered trademarks NANOBODY or NANOBODIES.

[0245] In certain embodiments, the targeting moiety comprises a VHH. In some embodiments, the VHH is a humanized or camelized VHH.

[0246] In some embodiments, the VHH is a fully human VHH. H The fully human VH domains include domains, e.g., humanbodies (Crescendo Biologics, Cambridge, UK). In some embodiments, the fully human VH domains, e.g., humanbodies, are monovalent, bivalent, or trivalent. In some embodiments, the fully human VH domains, e.g., humanbodies, are monospecific or multispecific, such as monospecific, bispecific, or trispecific. Exemplary fully human VH domains, e.g., humanbodies, are described, for example, in WO2016 / 113555 and WO2016 / 113557, the entire disclosures of which are incorporated herein by reference.

[0247] In various embodiments, the targeting moiety of the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex is a protein-based substance capable of specifically binding to a cellular receptor, such as a natural ligand for the cellular receptor. In various embodiments, the cellular receptor is present on one or more immune cells, including, but not limited to, T cells, cytotoxic T lymphocytes, helper T cells, natural killer (NK) cells, natural killer T (NKT) cells, anti-tumor macrophages (e.g., M1 macrophages), B cells, dendritic cells, or a subset thereof. In some embodiments, the cellular receptor is present on megakaryocytes, platelets, erythrocytes, mast cells, basophils, neutrophils, eosinophils, or a subset thereof.

[0248] In some embodiments, the targeting moiety is a natural ligand, such as a chemokine. Examples of chemokines that can be included in the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, of the present invention include, but are not limited to, CCL1, CCL2, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL29, CCL30, CCL31, CCL32, CCL33, CCL34, CCL35, CCL36, CCL37, CCL38, CCL39, CCL40, CCL41, CCL42, CCL43, CCL44, CCL45, CCL46, CCL47, CCL48, CCL49, CCL50, CCL51, CCL52, CCL53, CCL54, CCL55, CCL56, CCL57, CCL58, CCL59, CCL60, CCL61, CCL62, CCL63, CCL64, CCL65, CCL66, CCL67, CCL68, CCL69, CCL70, CCL71, CCL72, CCL73, CCL74, CCL75, CCL76, CCL77, CCL78, CCL79, CCL80, CCL81, CCL82, CCL83, CCL84, CCL85, CCL86, CCL87, CCL88, CCL89, CCL90, CCL91, Examples of targeting moieties include 22, CCL23, CCL24, CLL25, CCL26, CCL27, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, XCL1, XCL2, CX3CL1, HCC-4, and LDGF-PBP. In an exemplary embodiment, the targeting moiety can be XCL1, a chemokine that recognizes and binds to the dendritic cell receptor XCR1. In another exemplary embodiment, the targeting moiety is CCL1, a chemokine that recognizes and binds to CCR8. In another exemplary embodiment, the targeting moiety is CCL2, a chemokine that recognizes and binds to CCR2 or CCR9. In another exemplary embodiment, the targeting moiety is CCL3, a chemokine that recognizes and binds to CCR1, CCR5, or CCR9. In another exemplary embodiment, the targeting moiety is CCL4, a chemokine that recognizes and binds to CCR1 or CCR5 or CCR9. In another exemplary embodiment, the targeting moiety is CCL5, a chemokine that recognizes and binds to CCR1 or CCR3 or CCR4 or CCR5. In another exemplary embodiment, the targeting moiety is CCL6, a chemokine that recognizes and binds to CCR1. In another exemplary embodiment, the targeting moiety is CCL7, a chemokine that recognizes and binds to CCR2 or CCR9. In another exemplary embodiment, the targeting moiety is CCL8, a chemokine that recognizes and binds to CCR1 or CCR2 or CCR2B or CCR5 or CCR9.In another exemplary embodiment, the targeting moiety is CCL9, a chemokine that recognizes and binds to CCR1. In another exemplary embodiment, the targeting moiety is CCL10, a chemokine that recognizes and binds to CCR1. In another exemplary embodiment, the targeting moiety is CCL11, a chemokine that recognizes and binds to CCR2 or CCR3 or CCR5 or CCR9. In another exemplary embodiment, the targeting moiety is CCL13, a chemokine that recognizes and binds to CCR2 or CCR3 or CCR5 or CCR9. In another exemplary embodiment, the targeting moiety is CCL14, a chemokine that recognizes and binds to CCR1 or CCR9. In another exemplary embodiment, the targeting moiety is CCL15, a chemokine that recognizes and binds to CCR1 or CCR3. In another exemplary embodiment, the targeting moiety is CCL16, a chemokine that recognizes and binds to CCR1, CCR2, CCR5, or CCR8. In another exemplary embodiment, the targeting moiety is CCL17, a chemokine that recognizes and binds CCR4. In another exemplary embodiment, the targeting moiety is CCL19, a chemokine that recognizes and binds CCR7. In another exemplary embodiment, the targeting moiety is CCL20, a chemokine that recognizes and binds CCR6. In another exemplary embodiment, the targeting moiety is CCL21, a chemokine that recognizes and binds CCR7. In another exemplary embodiment, the targeting moiety is CCL22, a chemokine that recognizes and binds CCR4. In another exemplary embodiment, the targeting moiety is CCL23, a chemokine that recognizes and binds CCR1. In another exemplary embodiment, the targeting moiety is CCL24, a chemokine that recognizes and binds CCR3. In another exemplary embodiment, the targeting moiety is CCL25, a chemokine that recognizes and binds CCR9. In another exemplary embodiment, the targeting moiety is CCL26, a chemokine that recognizes and binds CCR3. In another exemplary embodiment, the targeting moiety is CCL27, a chemokine that recognizes and binds to CCR10.In another exemplary embodiment, the targeting moiety is CCL28, a chemokine that recognizes and binds to CCR3 or CCR10. In another exemplary embodiment, the targeting moiety is CXCL1, a chemokine that recognizes and binds to CXCR1 or CXCR2. In another exemplary embodiment, the targeting moiety is CXCL2, a chemokine that recognizes and binds to CXCR2. In another exemplary embodiment, the targeting moiety is CXCL3, a chemokine that recognizes and binds to CXCR2. In another exemplary embodiment, the targeting moiety is CXCL4, a chemokine that recognizes and binds CXCR3B. In another exemplary embodiment, the targeting moiety is CXCL5, a chemokine that recognizes and binds CXCR2. In another exemplary embodiment, the targeting moiety is CXCL6, a chemokine that recognizes and binds to CXCR1 or CXCR2. In another exemplary embodiment, the targeting moiety is CXCL8, a chemokine that recognizes and binds to CXCR1 or CXCR2. In another exemplary embodiment, the targeting moiety is CXCL9, a chemokine that recognizes and binds to CXCR3. In another exemplary embodiment, the targeting moiety is CXCL10, a chemokine that recognizes and binds to CXCR3. In another exemplary embodiment, the targeting moiety is CXCL11, a chemokine that recognizes and binds to CXCR3 or CXCR7. In another exemplary embodiment, the targeting moiety is CXCL12, a chemokine that recognizes and binds to CXCR4 or CXCR7. In another exemplary embodiment, the targeting moiety is CXCL13, a chemokine that recognizes and binds CXCR5. In another exemplary embodiment, the targeting moiety is CXCL16, a chemokine that recognizes and binds CXCR6. In another exemplary embodiment, the targeting moiety is LDGF-PBP, a chemokine that recognizes and binds CXCR2. In another exemplary embodiment, the targeting moiety is XCL2, a chemokine that recognizes and binds XCR1. In another exemplary embodiment, the targeting moiety is CX3CL1, a chemokine that recognizes and binds to CX3CR1.

[0249] In some embodiments, the targeting moiety is a natural ligand, such as Flt3-like tyrosine kinase 3 ligand (Flt3L) or a truncated region thereof (e.g., a region capable of binding Flt3). In some embodiments, the targeting moiety is the extracellular domain of Flt3L. In some embodiments, the targeting moiety comprises a Flt3L domain, wherein the Flt3L domain is a single-chain dimer, optionally wherein one Flt3L domain is linked to another Flt3L domain via one or more linkers, wherein the linkers are flexible linkers. In some embodiments, the targeting moiety of the present invention comprises a Flt3L domain, wherein the Flt3L domain is a single-chain dimer and an Fc domain, optionally comprising one or more mutations that reduce or eliminate one or more effector functions of the Fc domain, promote Fc chain pairing in the Fc domain, and / or stabilize the hinge region in the Fc domain. In some embodiments, the targeting moiety recognizes CD20. In some embodiments, the targeting moiety recognizes PD-L1. In some embodiments, the targeting moiety recognizes Clec9A.

[0250] In various embodiments, the targeting moiety of the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex comprises various combinations of targeting moieties. In an exemplary embodiment, the targeting moiety of the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex may comprise two targeting moieties, both of which are antibodies or derivatives thereof. In another exemplary embodiment, the targeting moiety of the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex may comprise two targeting moieties, both of which are natural ligands for a cellular receptor. In a further exemplary embodiment, the targeting moiety of the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex may comprise two targeting moieties, one of which is an antibody or derivative thereof and the other of which is a natural ligand for a cellular receptor.

[0251] In various embodiments, the targeting moiety of the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex functionally modulates (including, but not limited to, partially or completely neutralizes) a target of interest (e.g., an antigen, receptor), e.g., substantially inhibits, reduces, or neutralizes the biological effect of the antigen. For example, various recognition domains may be directed against one or more tumor antigens that actively suppress or have the ability to suppress the immune system of tumor-bearing patients. For example, in some embodiments, the targeting moiety of the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex functionally modulates one or more immunosuppressive signals (e.g., checkpoint inhibitors), such as TIM-3, BTLA, PD-1, CTLA-4, B7-H4, GITR, galectin-9, HVEM, PD-L1, PD-L2, B7-H3, CD244, CD160, TIGIT, SIRPα, ICOS, CD172a, and TMIGD2. For example, in some embodiments, the targeting moiety of the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex is genetically modified to disrupt, block, reduce, and / or inhibit the transmission of immunosuppressive signals, including, but not limited to, the binding of PD-1 to PD-L1 or PD-L2, and / or the binding of CTLA-4 to one or more of AP2M1, CD80, CD86, SHP-2, and PPP2R5A.

[0252] In various embodiments, the targeting moiety of the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex binds to but does not functionally modulate a target of interest (e.g., an antigen, receptor), e.g., the recognition domain is or resembles a binding antibody. For example, in various embodiments, the recognition domain only targets an antigen or receptor without substantially inhibiting, reducing, or functionally modulating the biological action of the antigen or receptor. For example, some of the above-described antibody formats (e.g., compared to whole antibodies) have the ability to target difficult-to-access epitopes, providing a broader range of specific binding surfaces. In various embodiments, the recognition domain binds to an epitope that is physically distant from the antigen or receptor site (e.g., the active site of the antigen) important for its biological activity.

[0253] Such non-neutralizing binding is used in various embodiments of the present invention, including methods in which the vaccine compositions, adjuvants, chimeric proteins, or chimeric protein complexes are used to directly or indirectly recruit active immune cells to a required site via an effector antigen. For example, in various embodiments, the vaccine compositions, adjuvants, chimeric proteins, or chimeric protein complexes may be used to directly or indirectly recruit cytotoxic T cells to tumor cells via CD8 in methods of reducing or eliminating tumors (e.g., chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, may contain an anti-CD8 recognition domain and a recognition domain for a tumor antigen). In such embodiments, it is desirable to directly or indirectly recruit CD8-expressing cytotoxic T cells but not functionally modulate CD8 activity. In contrast, in these embodiments, CD8 signaling is an important part of the tumor reduction or elimination process. By way of further example, in various embodiments, the vaccine compositions, adjuvants, chimeric proteins, or chimeric protein complexes are used to directly or indirectly recruit dendritic cells (DCs) via CLEC9A (e.g., chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, may comprise an anti-CLEC9A recognition domain and a recognition domain for a tumor antigen). In such embodiments, it is desirable to directly or indirectly recruit DCs that express CLEC9A, but not functionally modulate CLEC9A activity. In contrast, in these embodiments, CLEC9A signaling is an important part of tumor reduction or elimination.

[0254] In various embodiments, the recognition domain of the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex binds to XCR1 on dendritic cells, for example, and in some embodiments, the recognition domain comprises all or a portion of XCL1 or a non-neutralizing anti-XCR1 agent.

[0255] In various embodiments, the recognition domain of the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex binds to an immunomodulatory antigen (e.g., an immunostimulatory antigen or an immunosuppressive antigen). In various embodiments, the immunomodulatory antigen is one or more of 4-1BB, OX-40, HVEM, GITR, CD27, CD28, CD30, CD40, ICOS ligand; OX-40 ligand, LIGHT (CD258), GITR ligand, CD70, B7-1, B7-2, CD30 ligand, CD40 ligand, ICOS, ICOS ligand, CD137 ligand, and TL1A. In various embodiments, the immunostimulatory antigen is expressed on tumor cells. In various embodiments, the recognition domain of the vaccine composition, adjuvant, chimeric protein, or chimeric protein complex binds to but does not functionally modulate such immunostimulatory antigens, thus allowing the recruitment of cells expressing these antigens without reducing or eliminating their potential tumor reduction or elimination capabilities.

[0256] In various embodiments, the recognition domains of the vaccine compositions, adjuvants, chimeric proteins, or chimeric protein complexes may be associated with chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, that contain two recognition domains with neutralizing activity, or two recognition domains with non-neutralizing (e.g., binding) activity, or one recognition domain with neutralizing activity and one recognition domain with non-neutralizing (e.g., binding) activity.

[0257] Fc domain The fragment crystallizable domain (Fc domain) is the tail region of an antibody that interacts with Fc receptors located on the cell surface of cells involved in the immune system, e.g., B lymphocytes, dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils, and mast cells. In IgG, IgA, and IgD antibody isotypes, the Fc domain is composed of two identical protein fragments, derived from the second and third constant domains of the antibody's two heavy chains. In IgM and IgE antibody isotypes, the Fc domain is composed of the three heavy chain constant domains (C) in each polypeptide chain. H domains 2-4).

[0258] In some embodiments, the Fc-based chimeric protein complex of the present technology comprises an Fc domain. In some embodiments, the Fc domain is selected from IgG, IgA, IgD, IgM, or IgE. In some embodiments, the Fc domain is selected from IgG1, IgG2, IgG3, or IgG4.

[0259] In some embodiments, the Fc domain is selected from human IgG, IgA, IgD, IgM, or IgE. In some embodiments, the Fc domain is selected from human IgG1, IgG2, IgG3, or IgG4.

[0260] In some embodiments, the Fc domain of the Fc-based chimeric protein complex comprises the CH2 and CH3 regions of IgG. In some embodiments, the IgG is human IgG. In some embodiments, the human IgG is selected from IgG1, IgG2, IgG3, or IgG4.

[0261] In some embodiments, the Fc domain comprises one or more mutations. In some embodiments, the mutations to the Fc domain reduce or eliminate an effector function of the Fc domain. In some embodiments, the mutated Fc domain has reduced affinity or binding to a target receptor. For example, in some embodiments, the mutations to the Fc domain reduce or eliminate binding of the Fc domain to an FcγR. In some embodiments, the FcγR is selected from FcγRI; FcγRIIa, 131R / R; FcγRIIa, 131H / H, FcγRIIb; and FcγRIII. In some embodiments, the mutations to the Fc domain reduce or eliminate binding to a complement protein, such as C1q. In some embodiments, the mutations to the Fc domain reduce or eliminate binding to both an FcγR and a complement protein, such as C1q.

[0262] In some embodiments, the Fc domain comprises a LALA mutation to reduce or eliminate effector function of the Fc domain. For example, in some embodiments, the LALA mutation comprises L234A and L235A substitutions in human IgG (e.g., IgG1) (numbering based on the commonly used CH2 residue numbering for human IgG1 according to EU regulations (PNAS, Edelman et al., 1969;63(1)78-85)).

[0263] In some embodiments, the Fc domain of a human IgG comprises a mutation that reduces or eliminates an effector function of the Fc domain, for example, in some embodiments, the mutation is selected from L234A, L234F, L235A, L235E, L235Q, K322A, K322Q, D265A, P329G, P329A, P331G, and P331S.

[0264] In some embodiments, the Fc domain comprises a FALA mutation that reduces or eliminates the effector function of the Fc domain, for example, in some embodiments, the FALA mutation comprises F234A and L235A substitutions in human IgG4.

[0265] In some embodiments, the Fc domain of human IgG4 comprises mutations at one or more of F234, L235, K322, D265, and P329 to reduce or eliminate effector function of the Fc domain. For example, in some embodiments, the mutations are selected from F234A, L235A, L235E, L235Q, K322A, K322Q, D265A, P329G, and P329A.

[0266] In some embodiments, the mutation in the Fc domain stabilizes the hinge region of the Fc domain. For example, in some embodiments, the Fc domain contains a mutation at position S228 of human IgG to stabilize the hinge region. In some embodiments, the mutation is S228P.

[0267] In some embodiments, mutations to the Fc domain promote chain pairing of the Fc domain, hi some embodiments, chain pairing is promoted by ionic pairing (a / k / a charge pairs, ionic bonds, or charged residue pairs).

[0268] In some embodiments, the Fc domain contains mutations at the following amino acid residue positions of another IgG to promote ion pairing: D356, E357, L368, K370, K392, D399, and K409.

[0269] For example, in some embodiments, a human IgG Fc domain contains one of the combinations of mutations in Table 1 to promote ion pairing.

[0270] [Table 1]

[0271] In some embodiments, chain pairing is promoted by knobs-in-hole mutations. In some embodiments, the Fc domain contains one or more mutations that enable knobs-in-hole interactions in the Fc domain. In some embodiments, a first Fc chain is engineered to express a "knob" and a second Fc chain is engineered to express a complementary "hole." For example, in some embodiments, a human IgG Fc domain contains the mutations in Table 2 that enable knobs-in-hole interactions.

[0272] [Table 2]

[0273] In some embodiments, the Fc domain in the chimeric protein complex (e.g., Fc-based chimeric protein complex) of the present technology comprises any combination of the mutations disclosed above. For example, in some embodiments, the Fc domain comprises mutations that promote ion-pairing and / or knobs-in-hole interactions. For example, in some embodiments, the Fc domain comprises mutations that have one or more of the following properties: promote ion-pairing, induce knobs-in-hole interactions, reduce or eliminate effector function of the Fc domain, and stabilize the Fc (e.g., in the hinge).

[0274] For example, in some embodiments, a human IgG Fc domain contains mutations disclosed in Table 3 that promote ion pairing in the Fc domain and / or promote knob-in-hole interactions.

[0275] [Table 3-1]

[0276] [Table 3-2]

[0277] For example, in some embodiments, a human IgG Fc domain contains mutations disclosed in Table 4 that promote ion pairing of the Fc domain and / or promote knob-in-hole interactions, or a combination thereof. In some embodiments, "Chain 1" and "Chain 2" in Table 4 are interchangeable (e.g., Chain 1 can have Y407T and Chain 2 can have T366Y).

[0278] [Table 4-1]

[0279] [Table 4-2]

[0280] [Table 4-3]

[0281] [Table 4-4]

[0282] [Table 4-5]

[0283] For example, in some embodiments, a human IgG Fc domain comprises mutations disclosed in Table 5 that reduce or eliminate FcγR and / or complement binding in the Fc domain. In some embodiments, the mutations in Table 5 are present in both chains.

[0284] [Table 5-1]

[0285] [Table 5-2]

[0286] [Table 5-3]

[0287] In some embodiments, the Fc domain in the chimeric protein complexes (e.g., Fc-based chimeric protein complexes) of the present technology is a homodimer, i.e., the Fc region in the chimeric protein complex comprises two identical protein fragments.

[0288] In some embodiments, the Fc domain in the chimeric protein complexes (e.g., Fc-based chimeric protein complexes) of the present technology is a heterodimer, i.e., the Fc domain comprises two non-identical protein fragments.

[0289] In some embodiments, the heterodimeric Fc domain is engineered using ion-pairing and / or knobs-in-holes mutations described herein. In some embodiments, the heterodimeric chimeric protein complex (e.g., Fc-based chimeric protein complex) has a trans orientation / configuration. In a trans orientation / configuration, the targeting moiety and the signaling factor, in some embodiments, are not present on the same polypeptide chain in the chimeric protein complex (e.g., Fc-based chimeric protein complex) of the invention.

[0290] In some embodiments, the Fc domain includes or begins with the core hinge region of wild-type human IgG1, which region includes the sequence Cys-Pro-Pro-Cys. In some embodiments, the Fc domain also includes the upper hinge, or a portion thereof (e.g., DKTHTCPPC, see WO2009053368), EPKSCDKTHTCPPC, or EPKSSDKTHTCPPC (see Lo et al., Protein Engineering vol. 11 no. 6 pp. 495-500, 1998).

[0291] Chimeric protein complexes The chimeric protein complexes (e.g., Fc-based chimeric protein complexes) of the present technology comprise at least one Fc domain disclosed herein, at least one signal transduction agent (SA) disclosed herein, such as IL-1α or pro-IL-1α, and at least one targeting moiety (TM) disclosed herein.

[0292] It is understood that the chimeric protein complexes (e.g., Fc-based chimeric protein complexes) of the present invention may comprise two fusion proteins, each comprising an Fc domain. In some embodiments, the chimeric protein complexes (e.g., Fc-based chimeric protein complexes) are heterodimers. In some embodiments, the heterodimeric chimeric protein complexes (e.g., Fc-based chimeric protein complexes) have a trans configuration / structure. In some embodiments, the heterodimeric Fc-based chimeric protein complexes have a cis configuration / structure.

[0293] In some embodiments, the heterodimeric Fc domain is modified using ion-pairing and / or knobs-in-holes mutations described herein. In some embodiments, the heterodimeric chimeric protein complex (e.g., Fc-based chimeric protein complex) has a trans configuration. In a trans configuration, the targeting moiety and the signaling factor are, in some embodiments, not present on the same polypeptide chain in the chimeric protein complex (e.g., Fc-based chimeric protein complex) of the present invention. In a trans configuration, the targeting moiety and the signaling factor are, in some embodiments, present on separate polypeptide chains in the chimeric protein complex (e.g., Fc-based chimeric protein complex). In a cis configuration, the targeting moiety and the signaling factor are, in some embodiments, present on the same polypeptide chain in the chimeric protein complex (e.g., Fc-based chimeric protein complex).

[0294] In some embodiments where two or more targeting moieties are present in the heterodimeric protein complexes described herein, one targeting moiety may be present in a trans configuration (relative to the signaling factor), while another targeting moiety may be present in a cis configuration (relative to the signaling factor). In some embodiments, the signaling factor and targeting moiety are present on the same end / side (N- or C-terminus) of the Fc domain. In some embodiments, the signaling factor and targeting moiety are present on different ends / sides (N- or C-terminus) of the Fc domain.

[0295] In some embodiments in which two or more targeting moieties are present in a heterodimeric protein complex described herein, the targeting moieties can be found on the same Fc chain or on two different Fc chains in the heterodimeric protein complex (in the latter case, the targeting moieties must be trans relative to each other since they are on different Fc chains). In some embodiments in which two or more targeting moieties are present on the same Fc chain, the targeting moieties can be on the same or different sides / ends of the Fc chain (N- or C-terminus).

[0296] In some embodiments in which two or more targeting moieties are present in a heterodimeric protein complex described herein, the targeting moieties are found on the same Fc chain or on two different Fc chains in the heterodimeric protein complex (in the latter case, the targeting moieties must be in trans relative to each other since they are on different Fc chains). In some embodiments in which two or more signaling factors are present on the same Fc chain, the signaling factors may be present on the same or different sides / ends of the Fc chain (N- or C-terminus).

[0297] In some embodiments where more than one signaling factor is present in a heterodimeric chimeric protein complex described herein (e.g., an Fc-based chimeric protein complex), one signaling factor may be present in a trans configuration (relative to the targeting moiety) while another signaling factor may be present in a cis configuration (relative to the targeting moiety).

[0298] In some embodiments, the heterodimeric chimeric protein complex (eg, an Fc-based chimeric protein complex) does not comprise a signaling factor, eg, IL-1α, and a targeting moiety on a single polypeptide.

[0299] In some embodiments, the chimeric protein complex (e.g., an Fc-based chimeric protein complex) has improved in vivo half-life compared to a chimeric protein that does not have an Fc or is not a heterodimeric complex. In some embodiments, the chimeric protein complex (e.g., an Fc-based chimeric protein complex) has improved solubility, stability, and other pharmacological properties compared to a chimeric protein that does not have an Fc or is not a heterodimeric complex.

[0300] Heterodimeric chimeric protein complexes (e.g., Fc-based chimeric protein complexes) are composed of two different polypeptides. In some embodiments described herein, the targeting domain is present on a different polypeptide from the signal transduction factor, e.g., IL-1α, thus allowing for the creation of proteins containing only one targeting domain copy and, similarly, only one type of signal transduction factor, e.g., IL-1α (which can control for potential interference with desired properties). Furthermore, in some embodiments, the use of only one targeting domain (e.g., VHH) can avoid cross-linking of antigens on the cell surface, which can induce undesirable effects. Furthermore, in some embodiments, the use of only one signal transduction factor, e.g., IL-1α, can mitigate molecular "crowding" and potential interference with avidity-mediated induction or recovery of effector function, depending on the targeting domain. Furthermore, in some embodiments, heterodimeric chimeric protein complexes (e.g., Fc-based chimeric protein complexes) may have two targeting moieties, which may be located on two different polypeptides. For example, in some embodiments, the C-termini of both targeting moieties (e.g., VHHs) can be masked to avoid potential or pre-existing autoantibodies (e.g., VHH autoantibodies or pre-existing antibodies). Furthermore, in some embodiments, a heterodimeric chimeric protein complex (e.g., an Fc-based chimeric protein complex) having a targeting domain on a different polypeptide than, for example, a signaling factor, e.g., IL-1α (e.g., a wild-type signaling factor, e.g., wild-type IL-1α) can preferentially "cross-link" two cell types (e.g., tumor cells and immune cells). Furthermore, in some embodiments, a heterodimeric chimeric protein complex (e.g., an Fc-based chimeric protein complex) has two signaling factors, each on a different polypeptide, allowing for more complex effector responses.

[0301] Furthermore, in some embodiments, heterodimeric chimeric protein complexes (e.g., Fc-based chimeric protein complexes) having a variety of combinations of targeting moieties and signaling factors, e.g., IL-1α, are provided in a practical manner, e.g., having a targeting domain on a polypeptide different from a signal transduction product, e.g., IL-1α. For example, in some embodiments, a polypeptide having any of the targeting moieties described herein can be combined "off the shelf" with a polypeptide having any of the signaling factors described herein, allowing for the rapid generation of various combinations of targeting moieties and signaling factors in a single chimeric protein complex (e.g., Fc-based chimeric protein complex).

[0302] In some embodiments, the chimeric protein complex (e.g., Fc-based chimeric protein complex) comprises one or more linkers. In some embodiments, the chimeric protein complex (e.g., Fc-based chimeric protein complex) comprises a linker connecting an Fc domain, a signaling factor, e.g., IL-1α, and a targeting moiety. In some embodiments, the chimeric protein complex (e.g., Fc-based chimeric protein complex) comprises a linker linking each signaling factor, e.g., IL-1α, and a targeting moiety (or, in the case of two or more targeting moieties, linking a signaling factor, e.g., IL-1α, to one of the targeting moieties). In some embodiments, the chimeric protein complex (e.g., Fc-based chimeric protein complex) comprises a linker linking each signaling factor, e.g., IL-1α, to the Fc domain. In some embodiments, the Fc-based chimeric protein complex comprises a linker linking each targeting moiety to the Fc domain. In some embodiments, the chimeric protein complex (e.g., Fc-based chimeric protein complex) comprises a linker linking a targeting moiety to another targeting moiety. In some embodiments, the chimeric protein complex (eg, an Fc-based chimeric protein complex) comprises a linker that connects a signaling factor, eg, IL-1α, to another signaling factor.

[0303] In some embodiments, a chimeric protein complex (e.g., an Fc-based chimeric protein complex) comprises two or more targeting moieties, which may be the same or different targeting moieties.

[0304] In some embodiments, a chimeric protein complex (e.g., an Fc-based chimeric protein complex) comprises two or more signaling factors, which may be the same or different targeting moieties.

[0305] For example, in some embodiments, the chimeric protein complex (e.g., an Fc-based chimeric protein complex) comprises an Fc domain, at least two signaling agents (SAs), and at least two targeting moieties (TMs), wherein the Fc domain, signaling agents, and targeting moieties are selected from any of the Fc domains, signaling agents, and targeting moieties disclosed herein. In some embodiments, the Fc domain is a homodimer.

[0306] In various embodiments, the chimeric protein complex (eg, an Fc-based chimeric protein complex) takes the form of any of the schematic diagrams in Figures 1A-F.

[0307] In various embodiments, the chimeric protein complex (eg, an Fc-based chimeric protein complex) takes the form of any of the schematic diagrams in Figures 2A-H.

[0308] In various embodiments, the chimeric protein complex (eg, an Fc-based chimeric protein complex) takes the form of any of the schematic diagrams in Figures 3A-H.

[0309] In various embodiments, the chimeric protein complex (eg, an Fc-based chimeric protein complex) takes the form of any of the schematic diagrams in Figures 4A-D.

[0310] In various embodiments, the chimeric protein complex (eg, an Fc-based chimeric protein complex) takes the form of any of the schematic diagrams in Figures 5A-F.

[0311] In various embodiments, the chimeric protein complex (eg, an Fc-based chimeric protein complex) takes the form of any of the schematic diagrams in Figures 6A-J.

[0312] In various embodiments, the chimeric protein complex (eg, an Fc-based chimeric protein complex) takes the form of any of the schematic diagrams in Figures 7A-D.

[0313] In various embodiments, the chimeric protein complex (eg, an Fc-based chimeric protein complex) takes the form of any of the schematic diagrams in Figures 8A-F.

[0314] In various embodiments, the chimeric protein complex (eg, an Fc-based chimeric protein complex) takes the form of any of the schematic diagrams in Figures 9A-J.

[0315] In various embodiments, the chimeric protein complex (eg, an Fc-based chimeric protein complex) takes the form of any of the schematic diagrams in Figures 10A-F.

[0316] In various embodiments, the chimeric protein complex (eg, an Fc-based chimeric protein complex) takes the form of any of the schematic diagrams in Figures 11A-L.

[0317] In various embodiments, the chimeric protein complex (eg, an Fc-based chimeric protein complex) takes the form of any of the schematic diagrams in Figures 12A-L.

[0318] In various embodiments, the chimeric protein complex (eg, an Fc-based chimeric protein complex) takes the form of any of the schematic diagrams in Figures 13A-F.

[0319] In various embodiments, the chimeric protein complex (eg, an Fc-based chimeric protein complex) takes the form of any of the schematic diagrams in Figures 14A-L.

[0320] In various embodiments, the chimeric protein complex (eg, an Fc-based chimeric protein complex) takes the form of any of the schematic diagrams in Figures 15A-L.

[0321] In various embodiments, the chimeric protein complex (eg, an Fc-based chimeric protein complex) takes the form of any of the schematic diagrams in Figures 16A-J.

[0322] In various embodiments, the chimeric protein complex (eg, an Fc-based chimeric protein complex) takes the form of any of the schematic diagrams in Figures 17A-J.

[0323] In various embodiments, the chimeric protein complex (eg, an Fc-based chimeric protein complex) takes the form of any of the schematic diagrams in Figures 18A-F.

[0324] In various embodiments, the chimeric protein complex (eg, an Fc-based chimeric protein complex) takes the form of any of the schematic diagrams in Figures 19A-F.

[0325] In some embodiments, the signaling agent is linked to the targeting moiety, and the targeting moiety is linked to the Fc domain on the same end (see Figures 1A-F). In some embodiments, the Fc domain is a homodimer.

[0326] In some embodiments, the signaling agent and targeting moiety are linked to the Fc domain, and the targeting moiety and signaling agent are linked on the same end (see Figures 1A-F). In some embodiments, the Fc domain is a homodimer.

[0327] In some embodiments, the targeting moiety is linked to a signaling factor and the signaling factor is linked on the same end to the Fc domain (see Figures 1A-F). In some embodiments, the Fc domain is a homodimer.

[0328] In some embodiments, the homodimeric chimeric protein complex (e.g., an Fc-based chimeric protein complex) comprises two or more targeting moieties. In some embodiments, there are four targeting moieties and two signaling factors, and the targeting moieties are linked to the Fc domain and the signaling factors are linked to the targeting moieties on the same end (see Figures 2A-H). In some embodiments, the Fc domain is a homodimer. In some embodiments, there are four targeting moieties and two signaling factors, and two targeting moieties are linked to the Fc domain and two targeting moieties are linked to the signaling factors, which are linked to the Fc domain on the same end (see Figures 2A-H). In some embodiments, the Fc domain is a homodimer. In some embodiments, there are four targeting moieties and two signaling factors, and two targeting moieties are linked to each other, and one targeting moiety from each pair is linked to the Fc domain on the same end, and the signaling factors are linked to the Fc domain on the same end (see Figures 2A-H). In some embodiments, the Fc domain is a homodimer. In some embodiments where there are four targeting moieties and two signaling agents, two targeting moieties are linked to each other, one targeting m...

Claims

**Claim 1** (a) A mutant interleukin-1α (IL-1α) wherein the mutation is a deletion of amino acids 1-6 (Δ1-6) with respect to SEQ ID NO: 1 or 3, or a mutant pro-IL-1α wherein the mutation is a deletion of amino acids 113-118 (Δ113-118) with respect to SEQ ID NO: 2 or 4, and (b) One or more targeting moieties, said targeting moieties comprising a recognition domain that specifically binds to a target antigen or receptor, and (c) A linker between (a) and (b), wherein (i) A flexible linker that links (a) and (b), and / or (ii) An Fc domain that links (a) and (b), optionally having one or more mutations that reduce or eliminate one or more effector functions of the Fc domain, promote Fc chain pairing in the Fc domain, and / or stabilize the hinge region in the Fc domain is said linker, and comprising a chimeric protein or chimeric protein complex. **Claim 2** The chimeric protein or chimeric protein complex according to claim 1, wherein the mutant interleukin-1α (IL-1α) further comprises an amino acid substitution at position C141 with respect to SEQ ID NO: 1 or 3, optionally C141S or C141A or C141H, or the mutant pro-IL-1α further comprises an amino acid substitution at position C253 with respect to SEQ ID NO: 2 or 4, optionally C253S or C253A or C253H. **Claim 3** The mutant IL-1α is one or more additional mutations selected from amino acid substitutions at positions selected from N29, S31, P3, M15, R16, I17, I18, L24, N25, D26, L28, I33, L40, A44, H46, V52, F54, M56, A58, Y59, K60, D64, D65, K67, I68, V70, L72, L79, Y80, P89, L91, E94, P99, K100, E106, F111, W113, K119, S124, P128, I132, Q136, T134, V140, L142, D151, F152, Q153, and combinations thereof (wherein said positions are with respect to SEQ ID NO: 1 or 3), optionally including the mutations selected from N29A, N29D, N29G, S31A, S31G, M15A, M15G, M15S, R16A, R16K, R16G, I18A, I18G, I18L, L24K, L24S, N25A, N25G, D26V, L28A, L28G, I33A, I33G, A44G, A44S, A44T, A44N, A44H, H46A, H46G, A58G, A58S, A58T, A58N, A58H, A58F, Y59A, K60A, K60G, D64A, D64G, D65A, K67A, I68A, I68G, V70A, Y80A, K100A, K100D, W113F, Q136A, Q136C, D151A, D151K, D151Y, F152Q, F152N, F152S, Q153A, and Q153G, and combinations thereof, or The variant pro-IL-1α is one or more additional mutations selected from amino acid substitutions at positions selected from N141, S143, P115, M127, R128, I129, I130, L136, N137, D138, L140, I145, L152, A156, H158, V164, F166, M168, A170, A171, K172, D176, D177, K179, I180, V182, L184, L191, Y192, P201, L203, E206, P211, K212, E218, F223, W225, K231, S236, P240, I244, Q248, T246, V252, C253, L254, D263, F264, Q265, and combinations thereof (wherein said positions are with respect to SEQ ID NO: 2 or 4), optionally N141A, N141D, N141G, S143A, S143G, M127S, R128A, R128K, I130A, I130L, L136K, L136S, N137A, N137G, D138V, L140A, L140G, A156G, A156S, A156T, A156N, A156H, H158A, H158G, A170G, A170S, A170T, A170N, A170H, A170F, Y171A, K172A, K172G, D176A, D176G, D177A, I180A, I80G, V182A, Y192A, K212A, K212D, W225F, Q248C, D263K, F264Q, F264N, F264S, Q265A, and Q265G, and combinations thereof, including said mutations selected from The chimeric protein or chimeric protein complex according to claim 1.

4. By said one or more additional mutations, compared to IL-1α having the amino acid sequence of SEQ ID NO: 1 or 3 having a deletion of amino acids 1-6 (Δ1-6), or compared to pro-IL-1α having the amino acid sequence of SEQ ID NO: 2 or 4 having a deletion of amino acids 113-118 (Δ113-118), the activity is reduced, and optionally, by said one or more mutations, the activity is inducibly and / or recoverably reduced by binding to one or more target moieties or by inclusion in said chimeric protein or chimeric protein complex, or Due to said one or more further mutations, the activity is increased as compared to IL-1α having the amino acid sequence of SEQ ID NO: 1 or 3 with a deletion of amino acids 1-6 (Δ1-6), and optionally, said one or more further mutations are selected from amino acid substitutions at positions selected from N29 and S31 with respect to SEQ ID NO: 1 or 3, and optionally, said substitutions are selected from N29A, N29D, N29G, S31A, and S31G, or, Due to said one or more further mutations, the activity is increased as compared to pro-IL-1α having the amino acid sequence of SEQ ID NO: 2 or 4 with a deletion of amino acids 113-118 (Δ113-118), and optionally, said one or more further mutations are selected from amino acid substitutions at positions selected from N141 and S143 with respect to SEQ ID NO: 2 or 4, and optionally, said substitutions are selected from N141A, N141D, N141G, S143A, and S143G, and / or (a) said IL-1α comprises an amino acid sequence having at least 97%, or at least 98%, or at least 99% identity with SEQ ID NO: 1 or 3, or, (b) said Pro-IL-1α comprises an amino acid sequence having at least 97%, or at least 98%, or at least 99% identity with SEQ ID NO: 2 or 4, The chimeric protein or chimeric protein complex according to claim 2.

5. Due to said one or more mutations of said mutant IL-1α or mutant Pro-IL-1α, the activity is decreased or increased, and / or the affinity for IL-1R or IL-1RAcP is decreased or increased, and optionally, said IL-1α or Pro-IL-1α exhibits a decrease or increase in activity and / or a decrease or increase in affinity for IL-1R1, or, said IL-1α or Pro-IL-1α exhibits a decrease or increase in activity and / or a decrease or increase in affinity for IL-1RAcP, The chimeric protein or protein complex according to claim 1.

6. The targeting moiety comprises a recognition domain that recognizes and / or binds to an antigen or receptor on tumor cells, endothelial cells, epithelial cells, mesenchymal cells, tumor stroma or stromal cells, ECM and / or immune cells, organ cells, and / or tissue cells, and optionally, the immune cells are selected from T cells, B cells, dendritic cells, macrophages, neutrophils, mast cells, monocytes, erythrocytes, myeloid cells, myeloid-derived suppressor cells, NKT cells, and NK cells, or derivatives thereof. The chimeric protein or chimeric protein complex according to claim 1.

7. wherein the targeting moiety comprises a single domain antibody, a full-length antibody or fragment thereof, a recombinant heavy chain antibody (VHH), a single-chain antibody (scFv), a human antibody, a shark heavy chain antibody (VNAR), a microprotein (e.g., a cysteine knot protein, a knottin), a darpin, an anticalin, an adnectin, an aptamer, an Fv, a Fab, a Fab', or an F(ab') ２ and / or comprises a recognition domain which is The recognition domain functionally regulates the antigen or receptor of interest, or the recognition domain binds to the antigen or receptor of interest but does not functionally regulate it. The chimeric protein or chimeric protein complex according to claim 1.

8. Comprising two or more targeting moieties and / or further comprising one or more additional modified signaling factors, and optionally, the chimeric protein complex comprises two signaling factors or two targeting moieties, or two of both, or the chimeric protein complex comprises three signaling factors or three targeting moieties, or three of both. The chimeric protein or chimeric protein complex according to claim 1.

9. The linker between (a) and (b) is a flexible linker and / or The flexible linker is substantially composed of glycine residues and serine residues, and optionally, (i) contains (Gly 4 Ser)n (where n is 1 to 8), (ii) contains (Gly 2 Ser)n (where n is 1 to 20), or (iii) contains one or more of SEQ ID NOs: 435 to 442, or the flexible linker is substantially composed of GGGSGGGSGGGGGSGGGGGS (SEQ ID NO: 257). The flexible linker consists essentially of (GGGGGS)n (n = 1 to 4) (SEQ ID NOs: 249 to 252), LE, (Gly)8 (SEQ ID NO: 258), (Gly)6 (SEQ ID NO: 259), (EAAAK)n (n = 1 to 3) (SEQ ID NOs: 260 to 262), A(EAAAK)nA (n = 2 to 5) (SEQ ID NOs: 263 to 266), AEAAAKKEAAAKA (SEQ ID NO: 263), A(EAAAK)4ALE A(EAAAK)4A (SEQ ID NO: 267), PAPAP (SEQ ID NO: 268), KESGSVSSSQLAQFRSLD (SEQ ID NO: 269), EGKSSSGSGSKSKST (SEQ ID NO: 270), GSAGSAAGSGEF (SEQ ID NO: 271), and (XP)n (wherein X represents any amino acid, for example, Ala, Lys, or Glu). The chimeric protein according to claim 1.

10. Comprising an Fc domain, wherein the Fc domain optionally has one or more mutations that reduce or eliminate one or more effector functions of the Fc domain, promote Fc chain pairing in the Fc domain, and / or stabilize the hinge region of the Fc domain, and optionally, the Fc domain is selected from IgG, IgA, IgD, IgM, or IgE, or human IgG, IgA, IgD, IgM, or IgE, and / or, the IgG is selected from IgG1, IgG2, IgG3, or IgG4, and optionally selected from human IgG1, IgG2, IgG3, or IgG4, and / or the Fc chain pairing is promoted by ion pair formation and / or knob-in-hole pairing, and optionally, due to the one or more mutations to the Fc domain, ion pair formation occurs between Fc chains in the Fc domain, or due to the one or more mutations to the Fc domain, knob-in-hole pairing occurs in the Fc domain, and / or, due to the one or more mutations to the Fc domain, the effector function of the Fc domain is reduced or eliminated. The chimeric protein complex according to claim 1.

11. A heterodimer having a trans or cis orientation / configuration with respect to each other with respect to any targeting moiety and IL-1α or pro-IL-1α, or with respect to each other with respect to any targeting moiety, or with respect to each other with respect to any IL-1α or pro-IL-1α, and / or wherein said Fc contains L234A, L235A, and K322Q substitutions (by EU numbering) in human IgG1, and / or wherein said Fc is human IgG1 and optionally contains one or more mutations of L234, L235, K322, D265, P329, and P331 (by EU numbering), and / or wherein said chimeric protein complex has the orientation and / or configuration described in any one of FIGS. 7B, 1A-F, 2A-H, 3A-H, 4A-D, 5A-F, 6A-J, 7A, 7C-D, 8A-F, 9A-J, 10A-F, 11A-L, 12A-L, 13A-F, 14A-L, 15A-L, 16A-J, 17A-J, 18A-F, and 19A-F, The chimeric protein complex according to claim 10.

12. A recombinant nucleic acid encoding one or more of the chimeric proteins or chimeric protein complexes according to any one of claims 1 to 11, or a constituent polypeptide thereof.

13. A host cell comprising one or more of the chimeric proteins or chimeric protein complexes according to any one of claims 1 to 11, or a recombinant nucleic acid encoding a constituent polypeptide thereof.

14. In the manufacture of a medicament for the treatment of cancer, i) the chimeric protein or chimeric protein complex according to any one of claims 1 to 11, ii) one or more of the chimeric proteins or chimeric protein complexes according to any one of claims 1 to 11, or a recombinant nucleic acid encoding a constituent polypeptide thereof, or iii) one or more host cells comprising the chimeric proteins or chimeric protein complexes according to any one of claims 1 to 11, or a recombinant nucleic acid encoding a constituent polypeptide thereof for use, wherein the cancer is selected from one or more of melanoma, basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer, cancer of the brain and central nervous system, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, glioblastoma, liver cancer, hepatocellular carcinoma, intraepithelial neoplasia, kidney cancer or renal carcinoma, laryngeal cancer, leukemia, liver cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell lung cancer, multiple myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, salivary gland cancer, sarcoma, Kaposi's sarcoma, skin cancer, squamous cell carcinoma, gastric cancer, testicular cancer, thyroid cancer, uterine cancer or endometrial cancer, urinary system cancer, vulvar cancer, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocyte (SL) NHL, intermediate follicular NHL, intermediate diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleaved cell NHL, bulky lesion NHL, mantle cell lymphoma, AIDS-related lymphoma, Waldenström macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myelogenous leukemia, carcinoma, post-transplant lymphoproliferative disorder (PTLD), phakomatosis, edema, and abnormal vascular proliferation associated with Meigs syndrome, said use.

15. In the manufacture of a medicament for the treatment of an autoimmune disease or disorder, i) the chimeric protein or chimeric protein complex according to any one of claims 1 to 11, ii) one or more of the chimeric proteins or chimeric protein complexes according to any one of claims 1 to 11, or recombinant nucleic acids encoding the constituent polypeptides thereof, or iii) host cells containing one or more of the chimeric proteins or chimeric protein complexes according to any one of claims 1 to 11, or recombinant nucleic acids encoding the constituent polypeptides thereof for use, wherein the autoimmune disease or disorder may be selected from Crohn's disease, diabetes, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis or juvenile rheumatoid arthritis, and ulcerative colitis, said use.

16. a) an adjuvant comprising the chimeric protein or chimeric protein complex according to any one of claims 1 to 11, and (b) an antigen suitable for inducing an immune response and a vaccine composition comprising the same. **Claim 17** wherein the targeting moiety comprises a recognition domain that recognizes and / or binds to an antigen or receptor on an immune cell, the immune cell is a T cell, and / or the targeting moiety comprises a recognition domain that recognizes and / or binds to CD8, CD3, CD4, Clec9A, XCR1 or SIRP1α, and / or further comprising an aluminum gel or salt, optionally wherein the aluminum gel or salt is selected from aluminum hydroxide, aluminum phosphate, and aluminum sulfate, and / or further comprising a further adjuvant selected from an oil-in-water emulsion formulation, a saponin adjuvant, a Freund's adjuvant, a toll-like receptor ligand, a cytokine, and chitosan, The vaccine composition according to claim 16. **Claim 18** Suitable for preventing or alleviating a disease or disorder that is an infectious disease, an autoimmune disease, or cancer, optionally wherein the disease or disorder is an infectious disease, and / or the infectious disease is optionally an infectious disease caused by a pathogen selected from bacteria, viruses, fungi, or parasites, optionally wherein the pathogen is a virus, optionally wherein the virus is (a) an influenza virus, optionally selected from influenza A, B, C, and D viruses, or (b) a member of the Coronaviridae family, optionally (i) a betacoronavirus, optionally selected from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), HCoV-HKU1, and HCoV-OC43, or (ii) an alphacoronavirus, optionally selected from HCoV-NL63 and HCoV-229E, and optionally wherein the virus is SARS-CoV-2, and / or The virus is an influenza virus, and optionally the antigen is an influenza virus antigen, an antigenic fragment thereof, or a nucleic acid encoding the same, and the viral antigen is optionally a hemagglutinin (HA) protein, a matrix 2 (M2) protein, and neuraminidase, or an antigenic fragment thereof, or a nucleic acid encoding the same, and / or the disease or disorder is selected from diphtheria, tetanus, pertussis, influenza, pneumonia, hepatitis A, hepatitis B, polio, yellow fever, human papillomavirus (HPV) infection, anthrax, rabies, Japanese encephalitis, meningitis, measles, mumps, rubella, gastroenteritis, smallpox, typhoid fever, varicella (chickenpox), rotavirus, and herpes zoster, and / or the antigen is one or more antigens of the following vaccines, namely, DTP (diphtheria-tetanus-pertussis vaccine), DTaP (diphtheria-tetanus-acellular pertussis vaccine), Hib (Haemophilus influenzae type b) conjugate vaccine, pneumococcal conjugate vaccine, hepatitis A vaccine, polio vaccine, yellow fever vaccine, hepatitis B vaccine, DTaP / Tdap / Hib combination vaccine, human papillomavirus (HPV) vaccine, anthrax vaccine, and rabies vaccine, and / or the cancer is selected from one or more of basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer, cancers of the brain and central nervous system, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, cancers of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, glioblastoma, liver cancer, hepatocellular carcinoma, intraepithelial neoplasia, kidney cancer or renal carcinoma, laryngeal cancer, leukemia, liver cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell lung carcinoma, melanoma, myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, salivary gland cancer, sarcoma, Kaposi's sarcoma, skin cancer, squamous cell carcinoma, gastric cancer, testicular cancer, thyroid cancer, uterine cancer or endometrial cancer, urinary system cancer, vulvar cancer, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B cell lymphoma, low grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocyte (SL) NHL, intermediate follicular NHL, intermediate diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-cleaved cell NHL, bulky lesion NHL, mantle cell lymphoma, AIDS-related lymphoma, Waldenström macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myelogenous leukemia, carcinoma, post-transplant lymphoproliferative disorder (PTLD), phakomatosis, edema, and abnormal vascular proliferation associated with Meigs syndrome, and / or the autoimmune disease or disorder is selected from Crohn's disease, diabetes, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis or juvenile rheumatoid arthritis, and ulcerative colitis, The vaccine composition according to claim 16.

19. the adjuvant is the chimeric protein or chimeric protein complex, or a nucleic acid encoding a component thereof, and / or the antigen is a protein or an antigenic fragment of a protein, or the antigen is a protein or an antigenic fragment of a protein, or a nucleic acid encoding a component thereof, and / or The nucleic acid is mRNA, optionally the mRNA comprising one or more non-standard nucleotides, wherein the non-standard nucleotide is optionally selected from pseudouridine and 5-methoxyuridine, or the nucleic acid is DNA, optionally the DNA selected from linear DNA, DNA fragment, or DNA plasmid, and / or the antigen is a 2019-nCoV protein, an antigenic fragment thereof, or a nucleic acid encoding the same, wherein the 2019-nCoV protein is optionally selected from the spike surface glycoprotein, membrane glycoprotein M, envelope protein E, and nucleocapsid phosphoprotein N, optionally the spike surface glycoprotein is the S1 subunit or the S2 subunit, or an antigenic fragment thereof, optionally the spike surface glycoprotein comprises the amino acid sequence of SEQ ID NO: 500, the membrane glycoprotein precursor M comprises the amino acid sequence of SEQ ID NO: 501, the envelope protein E comprises the amino acid sequence of SEQ ID NO: 502, and the nucleocapsid phosphoprotein N comprises the amino acid sequence of SEQ ID NO: 503, or comprises an amino acid sequence having at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99% identity with any of the amino acid sequences, or comprises an antigenic fragment of any of the amino acid sequences, The vaccine composition according to claim 16.

20. Formulated for intravenous administration, and / or the vaccine composition is formulated for administration to the lung, optionally formulated for administration by inhalation, optionally formulated for administration by aerosol or nebulizer, optionally formulated for administration by liquid spray, dry powder dispersion, and metered dose, and / or When the adjuvant or vaccine composition is administered to a patient, it has (a) low toxicity, (b) the ability to stimulate an immune response against the antigen that persists for a long period, (c) substantial stability, (d) the ability to induce a humoral immune response and / or cell-mediated immunity against the antigen, (e) the ability to selectively interact with a population of antigen-presenting cells, (f) the ability to specifically induce a TH1 and / or TH2 cell-specific immune response against the antigen, and / or (g) the ability to selectively increase the level of an antibody isotype that is appropriate for the antigen and is optionally IgA, and / or When the adjuvant or vaccine composition is administered to a patient, it stimulates a CD8+ T cell response against the antigen, and / or when the adjuvant or vaccine composition is administered to a patient, it stimulates the activation of IL-1R, and / or when the adjuvant or vaccine composition is administered to a patient, it does not substantially cause one or more of fever, neutrophilia, and the release of acute-phase proteins The vaccine composition according to claim 16

21. Use of the vaccine composition according to claim 16 in the manufacture of a medicament for vaccination against an infectious disease, wherein the adjuvant and antigen of the vaccine composition may be administered simultaneously, and / or the adjuvant and antigen of the vaccine composition may be co-formulated, and / or the adjuvant and antigen of the vaccine composition may be administered sequentially, and / or the adjuvant and antigen of the vaccine composition may be administered multiple times, and / or the adjuvant of the vaccine composition may be administered by multiple booster doses and the antigen of the vaccine composition may be administered once The use

22. Use of the chimeric protein or chimeric protein complex according to any one of claims 1 to 11 in the manufacture of a medicament for the treatment of an infectious disease, wherein the infectious disease may optionally be an infectious disease caused by a pathogen selected from bacteria, viruses, fungi, or parasites, and / or The pathogen is (a) an influenza virus, optionally the influenza virus selected from influenza A, B, C, and D viruses, or (b) a member of the Coronaviridae family, optionally (i) a betacoronavirus, optionally the betacoronavirus selected from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), HCoV-HKU1, and HCoV-OC43, or (ii) an alphacoronavirus, optionally the alphacoronavirus selected from HCoV-NL63 and HCoV-229E, which may be a virus that is a member of the Coronaviridae family selected from the above. The use.

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