Cellular signaling complexes and their uses

Abstract

Disclosed herein are cell signaling complexes, such as chimeric cytokine complexes, and their uses. In some embodiments, the chimeric cytokine complex comprises a protein cage polypeptide, a plurality of engineered Fc antibody domains bound to the protein cage polypeptide, and one or more cytokines linked to each of the plurality of engineered Fc antibody domains. Also disclosed are Fc-cytokine complexes and their uses. In some embodiments, the Fc-cytokine complex comprises engineered Fc antibody domains and one or more cytokines linked to the engineered Fc antibody domains.

Description

[Technical Field]

[0001] Cross-reference of related applications

[0001] This application claims priority to U.S. Patent Application No. 63 / 511,968, filed on 5 July 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] Reference to electronic sequence listings

[0002] This application includes a sequence listing filed through the Patent Center, which is incorporated herein by reference in its entirety. The aforementioned .xml copy, made on 15 May 2024, is named 04_NNTNZ00100WO_sequence_listing.xml and has a size of 35,143 bytes.

[0003] Technical field

[0003] This disclosure generally relates to the field of cell signaling complexes, and more particularly to cell signaling complexes such as chimeric cytokine complexes and their use. [Background technology]

[0004] background

[0004] Commonly used mechanisms for activating cellular pathways from the cell surface are receptor oligomerization or receptor clustering. For example, receptor oligomerization / clustering has been demonstrated by co-stimulation receptor binding by ligand and agonist antibodies [1], as well as the binding of cell signaling molecules such as cytokines to specific cell receptors [2]. The receptor often includes an extracellular domain on the cell surface, a transmembrane domain that crosses the cell membrane, and an intracellular domain located inside the cell. In these receptor clustering paradigms, the extracellular domain of the receptor can be engaged by cell signaling molecules, which can lead to both a conformational change that is translated across the membrane and contributes to activation / signaling, and / or oligomerization of multiple receptors that bring together an intracellular signaling complex that drives activation / signaling.

[0005]

[0005] As previously mentioned, cytokines are a well-known class of cell signaling proteins. The well-established standard mechanism for cytokines is the dimerization of extracellular cytokine receptors, often via the engagement of two JAK (Janus kinase)-associated receptor subunits in the form of heterodimers and sometimes homodimers [2, 3]. JAK dimerization results in the phosphorylation of STAT (signaling and transcription activator) transcription factors, driving cell activation through gene regulation [4].

[0006]

[0006] Cytokines can be further classified into interleukins, interferons, chemokines, lymphokines, colony-stimulating factors (CSFs), and tumor necrosis factors (TGFs). One important family of cytokines is the common γ chain (γc) family of cytokines, which plays an important role in the proliferation and survival of T cells. Some members of the γc family of cytokines include interleukin-2 (IL-2), IL-7, and IL-15.

[0007]

[0007] The functional IL-2-engaged IL-2 receptor signaling complex consists of IL-2Rα, IL-2Rβ, and γc subunit (K) as high-affinity heterotrimers. d about 10 -11 M) or an intermediate affinity dimer complex (K) consisting of only IL-2Rβ and γc with an affinity of approximately 100 times. d about 10 -9 It can consist of continuous binding / complexation of M) [2, 5]. IL-2 has low affinity for the IL-2Rα subunit (K d about 10 -8M) However, this binding does not induce signaling. Naive T cells are thought to start with low levels of IL-2Rα, but IL-2Rα expression is upregulated immediately after T cell receptor (TCR) activation. IL-2Rβ and γc are constitutively expressed on lymphopiohematopoietic cells, including low-density expression on naive T cells [5, 6]. In the activated T cell scenario, IL-2 engages with the upregulated IL-2Rα subunit and the low-expression intermediate-affinity IL-2Rβ, as well as the γc heterodimer, which results in low activation after or inefficient recruitment of all receptor subunits to assemble the high-affinity signaling complex. Therefore, approaches to enhance IL-2 receptor signaling can take the form of (i) increasing the affinity of IL-2 to the signal-ready IL-2Rβ / γc heterodimer to effectively enhance signaling stability, (ii) increasing the recruitment and assembly of all three IL-2 receptor subunits to form high-affinity signaling complexes, or (iii) increasing the number of generative intermediate-affinity and high-affinity signaling complexes on the cell surface.

[0008]

[0008] Cytokine engineering efforts aimed at modulating receptor signaling by focusing on altering the receptor binding affinity of IL-2 have been successful [2, 6], but these efforts have focused on direct amino acid alterations to cytokine sequences that disrupt or enhance binding affinity to specific receptor subunits. Such efforts often require laborious and time-consuming techniques such as directed evolution, X-ray crystallography, molecular dynamics simulations, and iterative cytokine-receptor interface engineering, even though they are essentially just affinity approaches [2, 6]. An example of these sequence-based manipulation efforts is the development of interleukin-2 (IL-2) variants referred to as “superkines” or super 2s [6].

[0009]

[0009] Therefore, in relation to increasing IL-2 receptor signaling without requiring sequence-based modifications to the cytokines themselves, solutions are needed that utilize the various approaches described above for inducing cytokine receptor signaling. Furthermore, such solutions should increase the affinity of cytokines to signaling receptors while also increasing the total number of cytokine-receptor signaling complexes per T cell. Moreover, such solutions should lead to increased activation and proliferation of human immune cells. [Overview of the Initiative] [Means for solving the problem]

[0010] overview

[0010] Disclosed is an improved cell signaling complex that can promote the activation and proliferation of immune cells. More specifically, disclosed is a cell signaling complex that can promote the activation and proliferation of immune cells (e.g., human donor peripheral blood T cells, human peripheral blood NK cells, etc.).

[0011]

[0011] In some embodiments, the chimeric cytokine complex comprises a protein cage polypeptide, a plurality of engineered Fc antibody domains bound to the protein cage polypeptide, and one or more cytokines linked to each of the plurality of engineered Fc antibody domains.

[0012]

[0012] Also disclosed is a method for activating and proliferating immune cells. This method may include adding a chimeric cytokine complex to a population of immune cells. The chimeric cytokine complex may include a protein cage polypeptide, a plurality of manipulated Fc antibody domains bound to the protein cage polypeptide, and one or more cytokines linked to each of the plurality of Fc antibody domains.

[0013]

[0013] In some embodiments, the population of immune cells can be activated and proliferated in vitro. In various embodiments, the population of immune cells can be peripheral blood immune cells from a human donor.

[0014]

[0014] In various embodiments, the population of immune cells can be live T cells.

[0015]

[0015] In various embodiments, the population of immune cells can be natural killer (NK) cells.

[0016]

[0016] In some embodiments, the method can further include activating the population of T cells with anti-CD3 and anti-CD28 T cell activation reagents before adding the chimeric cytokine complex.

[0017]

[0017] In various embodiments, the cytokine can be an interleukin. The interleukin can be at least one of interleukin-2 (IL-2), IL-7, and IL-15.

[0018]

[0018] In various embodiments, the plurality of engineered Fc antibody domains can include 6 to 12 engineered Fc antibody domains bound to a protein cage polypeptide. In some embodiments, a total of 12 to 24 cytokines can be linked to the plurality of engineered Fc antibody domains.

[0019]

[0019] In various embodiments, each of the one or more cytokines can be linked to one of the engineered Fc antibody domains via an engineered metalloprotease-resistant linker sequence. In some embodiments, the engineered metalloprotease-resistant linker sequence can be 7 to 13 amino acid residues in length.

[0020]

[0020] In various embodiments, at least one of the engineered Fc antibody domains can be C-terminally linked to at least one N-terminus of a cytokine.

[0021]

[0021] In some embodiments, one of the cytokines can be interleukin 2 (IL-2), and at least one of the engineered Fc antibody domains can be linked to IL-2 via an engineered metalloprotease-resistant linker sequence comprising the amino acid sequence SLSPGKAPTS (SEQ ID NO: 20).

[0022]

[0022] In some embodiments, one of the cytokines is interleukin 7 (IL-7), and at least one of the engineered Fc antibody domains can be linked to IL-7 via an engineered metalloprotease-resistant linker sequence comprising the amino acid sequence SLSPGKDCDIEGK (SEQ ID NO: 21).

[0023]

[0023] In some embodiments, one of the cytokines can be interleukin-15 (IL-15), and at least one of the engineered Fc antibody domains can be linked to IL-15 via an engineered metalloprotease-resistant linker sequence comprising the amino acid sequence SLSPGKN (SEQ ID NO: 22).

[0024]

[0024] In some embodiments, at least one of the engineered Fc antibody domains can be N-terminally linked to the C-terminus of at least one of the cytokines.

[0025]

[0025] In some embodiments, one of the cytokines can be interleukin 2 (IL-2), and at least one of the engineered Fc antibody domains can be linked to IL-2 via an engineered metalloprotease-resistant linker sequence comprising the amino acid sequence TPKSCDKTHT (SEQ ID NO: 23).

[0026]

[0026] In some embodiments, one of the cytokines can be interleukin 7 (IL-7), and at least one of the engineered Fc antibody domains can be linked to IL-7 via an engineered metalloprotease-resistant linker sequence comprising the amino acid sequence HPKSCDKTHT (SEQ ID NO: 24).

[0027]

[0027] In some embodiments, one of the cytokines may be interleukin-15 (IL-15), and at least one of the engineered Fc antibody domains may be linked to IL-15 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence TSPKSCDKTHT (SEQ ID NO: 25).

[0028]

[0028] In various embodiments, the manipulated Fc antibody domain may be a manipulated human Fc antibody domain.

[0029]

[0029] In some embodiments, the manipulated human Fc antibody domain may be a manipulated human IgG1 Fc antibody domain.

[0030]

[0030] In various embodiments, one of the manipulated human IgG1 Fc antibody domains may contain an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% amino acid identity with respect to the amino acid sequence shown in SEQ ID NO: 1.

[0031]

[0031] In some embodiments, the manipulated human IgG1 Fc antibody domain may contain at least one of the following point mutations relative to SEQ ID NO: P75L, R76W, Y80K, Y80P, Y80R, Y80G, and Y80A to reduce affinity for a specific Fcγ receptor and functionally reduce antibody-dependent cell-mediated cytotoxicity (ADCC).

[0032]

[0032] In some embodiments, the manipulated human IgG1 Fc antibody domain may contain the following point mutation:Y80W in SEQ ID NO: 1 to increase affinity for a specific Fcγ receptor and functionally enhance antibody-dependent cell-mediated cytotoxicity (ADCC).

[0033]

[0033] In some embodiments, the manipulated human IgG1 Fc antibody domain may contain at least one of the following point mutations relative to Sequence ID No. 1: S23A, E53A, E77A, Y80F, V87A, A111G, K122A, and D160A, such that it reduces affinity for a particular Fcγ receptor and has a neutral effect on other Fcγ receptors.

[0034]

[0034] In some embodiments, the manipulated human IgG1 Fc antibody domain may contain at least one of the following point mutations in SEQ ID NO: E117, K118A, and A123T to increase affinity for a specific Fcγ receptor and to have a neutral effect on other Fcγ receptors.

[0035]

[0035] In some embodiments, the manipulated human IgG1 Fc antibody domain may contain at least one of the following point mutations in Sequence ID No. 1: H52A, R85A, and K106A, to increase affinity for a specific Fcγ receptor and decrease affinity for a specific other Fcγ receptor.

[0036]

[0036] In some embodiments, the manipulated human IgG1 Fc antibody domain may contain at least one of the following point mutations to SEQ ID NO: D54A, Q79A, and A111S to reduce affinity for a particular Fcγ receptor.

[0037]

[0037] In some embodiments, the manipulated human IgG1 Fc antibody domain may contain at least one of the following point mutations: T40A and K74A relative to SEQ ID NO: 1, in order to increase affinity for a particular Fcγ receptor.

[0038]

[0038] In some embodiments, the Fc antibody domain may be an engineered rabbit Fc antibody domain.

[0039]

[0039] In various embodiments, the chimeric cytokine complex may further include a signal peptide ligated to the N-terminus of at least one of the manipulated Fc antibody domains or at least one of the cytokines.

[0040]

[0040] In some embodiments, the signal peptide may be a mouse Ig heavy signal peptide used for expression in Chinese hamster ovary (CHO) cells. In some embodiments, the signal peptide may include the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 26).

[0041]

[0041] In various embodiments, the protein cage polypeptide may include a polypeptide having an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% amino acid identity with respect to the amino acid sequence shown in any one of SEQ ID NOs: 8 to 15.

[0042]

[0042] In some embodiments, the amino acid sequence constituting the polypeptide of the protein cage polypeptide may include at least the following point mutation: Y294A to the amino acid sequence shown in any one of SEQ ID NOs: 8 to 15.

[0043]

[0043] In some embodiments, the protein cage polypeptide may comprise a polypeptide containing a binding site for one of the manipulated Fc antibody domains. The binding site may comprise the amino acid sequence RWGSGADCAWHLGELVWCTAGSGWE (SEQ ID NO: 16).

[0044]

[0044] In some embodiments, the protein cage polypeptide may comprise a polypeptide containing a binding site for one of the manipulated Fc antibody domains. The binding site may comprise the amino acid sequence GGRWGADCAWHLGELVWCTAGWEGG (SEQ ID NO: 17).

[0045]

[0045] In some embodiments, the protein cage polypeptide may comprise a polypeptide comprising a binding site for one of the manipulated Fc antibody domains. The binding site may comprise the amino acid sequence GADCAWHLGELVWCTAG (SEQ ID NO: 18).

[0046]

[0046] In some embodiments, the protein cage polypeptide may comprise a polypeptide containing a binding site for one of the manipulated Fc antibody domains. The binding site may comprise the amino acid sequence RWGSGCDCAWHLGELVWCTCGSGWE (SEQ ID NO: 19).

[0047]

[0047] In some embodiments, the protein cage polypeptide can self-assemble into a tetrahedral pyramidal structure.

[0048]

[0048] In some embodiments, an Fc-cytokine complex is disclosed that comprises an engineered Fc antibody domain and one or more cytokines linked to the engineered Fc antibody domain.

[0049]

[0049] Also disclosed is a method for activating and proliferating immune cells using an Fc-cytokine complex. This method may include adding an Fc-cytokine complex to a population of immune cells. The Fc-cytokine complex may comprise an Fc antibody domain and one or more cytokines linked to the Fc antibody domain.

[0050]

[0050] In some embodiments, the immune cell population can be activated and proliferated in vitro. In various embodiments, the immune cell population may be peripheral blood immune cells from a human donor. In various embodiments, the immune cell population may be living T cells.

[0051]

[0051] In some embodiments, the population of immune cells may be natural killer (NK) cells.

[0052]

[0052] In some embodiments, the method may further include activating a population of T cells with anti-CD3 and anti-CD28 T cell activating reagents before adding the Fc-cytokine complex.

[0053]

[0053] In various embodiments, the cytokine may be an interleukin.

[0054]

[0054] In some embodiments, the interleukin may be at least one of interleukin-2 (IL-2), IL-7, and IL-15.

[0055]

[0055] In various embodiments, each of one or more cytokines may be linked to an engineered Fc antibody domain via an engineered metalloproteinase resistance linker sequence. In some embodiments, the engineered metalloproteinase resistance linker sequence may be 7 to 13 amino acid residues long.

[0056]

[0056] In various embodiments, the manipulated Fc antibody domain may be C-terminally ligated to at least one N-terminus of the cytokine.

[0057]

[0057] In some embodiments, one of the cytokines may be interleukin-2 (IL-2), and the engineered Fc antibody domain may be linked to IL-2 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence SLSPGKAPTS (SEQ ID NO: 20).

[0058]

[0058] In some embodiments, one of the cytokines may be interleukin 7 (IL-7), and the engineered Fc antibody domain may be linked to IL-7 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence SLSPGKDCDIEGK (SEQ ID NO: 21).

[0059]

[0059] In some embodiments, one of the cytokines may be interleukin-15 (IL-15), and the engineered Fc antibody domain may be linked to IL-15 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence SLSPGKN (SEQ ID NO: 22).

[0060]

[0060] In various embodiments, the manipulated Fc antibody domain may be N-terminally ligated to at least one C-terminus of the cytokine.

[0061]

[0061] In some embodiments, one of the cytokines may be interleukin-2 (IL-2), and the engineered Fc antibody domain may be linked to IL-2 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence TPKSCDKTHT (SEQ ID NO: 23).

[0062]

[0062] In some embodiments, one of the cytokines may be interleukin 7 (IL-7), and the engineered Fc antibody domain may be linked to IL-7 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence HPKSCDKTHT (SEQ ID NO: 24).

[0063]

[0063] In some embodiments, one of the cytokines may be interleukin-15 (IL-15), and the engineered Fc antibody domain may be linked to IL-15 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence TSPKSCDKTHT (SEQ ID NO: 25).

[0064]

[0064] In various embodiments, the manipulated Fc antibody domain may be a manipulated human Fc antibody domain. In some embodiments, the manipulated human Fc antibody domain may be a manipulated human IgG1 Fc antibody domain.

[0065]

[0065] In some embodiments, the manipulated human IgG1 Fc antibody domain may contain an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% amino acid identity with respect to the amino acid sequence shown in SEQ ID NO: 1.

[0066]

[0066] In some embodiments, the manipulated human IgG1 Fc antibody domain may contain at least one of the following point mutations relative to SEQ ID NO: P75L, R76W, Y80K, Y80P, Y80R, Y80G, and Y80A to reduce affinity for a specific Fcγ receptor and functionally reduce antibody-dependent cell-mediated cytotoxicity (ADCC).

[0067]

[0067] In some embodiments, the manipulated human IgG1 Fc antibody domain may contain the following point mutation:Y80W in SEQ ID NO: 1 to increase affinity for a specific Fcγ receptor and functionally enhance antibody-dependent cell-mediated cytotoxicity (ADCC).

[0068]

[0068] In some embodiments, the manipulated human IgG1 Fc antibody domain may contain at least one of the following point mutations relative to Sequence ID No. 1: S23A, E53A, E77A, Y80F, V87A, A111G, K122A, and D160A, such that it reduces affinity for a particular Fcγ receptor and has a neutral effect on other Fcγ receptors.

[0069]

[0069] In some embodiments, the manipulated human IgG1 Fc antibody domain may contain at least one of the following point mutations in SEQ ID NO: E117, K118A, and A123T to increase affinity for a particular Fcγ receptor and to have a neutral effect on other Fcγ receptors.

[0070]

[0070] In some embodiments, the manipulated human IgG1 Fc antibody domain may contain at least one of the following point mutations in Sequence ID No. 1: H52A, R85A, and K106A, to increase affinity for a particular Fcγ receptor and decrease affinity for a particular other Fcγ receptor.

[0071]

[0071] In some embodiments, the manipulated human IgG1 Fc antibody domain may contain at least one of the following point mutations to SEQ ID NO: D54A, Q79A, and A111S to reduce affinity for a particular Fcγ receptor.

[0072]

[0072] In some embodiments, the manipulated human IgG1 Fc antibody domain may contain at least one of the following point mutations: T40A and K74A relative to SEQ ID NO: 1, in order to increase affinity for a particular Fcγ receptor.

[0073]

[0073] In some embodiments, the Fc antibody domain may be an engineered rabbit Fc antibody domain.

[0074]

[0074] In various embodiments, the Fc-cytokine complex may further include an engineered Fc antibody domain or a signal peptide ligated to at least one N-terminus of the cytokine. In some embodiments, the signal peptide may be a mouse Ig heavy signal peptide used for expression in Chinese hamster ovary (CHO) cells. In some embodiments, the signal peptide may include the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 26). [Brief explanation of the drawing]

[0075] Brief explanation of the drawing [Figure 1A]

[0075] Figure 1A shows a predicted structure representing one embodiment of an engineered Fc antibody domain linked to multiple cytokines. [Figure 1B]

[0075] Figure 1B shows a predicted structure representing one embodiment of an engineered Fc antibody domain linked to multiple cytokines. [Figure 1C]

[0076] Figure 1C shows the predicted structure of one embodiment of the manipulated self-assembled protein cage polypeptide. [Figure 1D]

[0077] Figure 1D shows the predicted structure of one embodiment of a cytokine (e.g., IL-2) and its receptor signaling complex. [Figure 1E]

[0078] Figure 1E shows a predicted structure of one embodiment of a CCC, which includes an Fc-cytokine bound to an engineered self-assembled protein cage polypeptide and a receptor signaling complex bound to the cytokine of the chimeric cytokine complex (CCC). [Figure 2]

[0079] Figure 2 shows that the manipulated Fc antibody domain can be C-terminally or N-terminally ligated to cytokines, thereby enhancing the function of activating and proliferating T cells. [Figure 3]

[0080] Figure 3 shows example linker sequences designed to link Fc and cytokine components of various CCCs. [Figure 4]

[0081] Figure 4 shows an example of an engineered Fc component of CCC stabilized by the Fcγ receptor (FcγR) of a neighboring cell. [Figure 5]

[0082] Figure 5 shows that the Fc component of CCC can be manipulated by mutations in key sites within its FcγR binding region. [Figure 6]

[0083] Figure 6 is a graph showing the results of an ELISA specific to the detection of IFN-γ produced by T cells. Phosphate-buffered saline (PBS) was used as a control. [Figure 7]

[0084] Figure 7 is a graph showing the population of living T cells as determined by flow cytometry on day 7. T cells were activated and proliferated using CCC, Fc-IL-2, and IL-2 alone. Phosphate-buffered saline (PBS) solution was used as a control. [Figure 8]

[0085] Figure 8 is a graph showing the population of living T cells as determined by flow cytometry on day 7. T cells were activated and proliferated using combinations of CCC and cytokines, as well as cytokines alone. Phosphate-buffered saline (PBS) solution was used as a control. [Figure 9A]

[0086] Figure 9A is a graph showing the number of CD4+ T cells on day 7 that were activated and proliferated by CCC and cytokines alone. Phosphate-buffered saline (PBS) solution was used as a control. [Figure 9B]

[0086] Figure 9B is a graph showing the number of CD8+ T cells on day 7 that were activated and proliferated by CCC and cytokines alone. Phosphate-buffered saline (PBS) solution was used as a control. [Figure 9C]

[0086] Figure 9C is a graph showing the total number of living T cells on day 7 that were activated and proliferated by CCC and cytokines alone. Phosphate-buffered saline (PBS) solution was used as a control. [Figure 10]

[0087] Figure 10 is a graph showing that immediately after stimulation with anti-CD3 and anti-CD28 T cell activating reagents, peripheral blood donor T cells upregulated the expression of cytokines and chemokines that increase the expression of specific matrix metalloproteinases (MMPs) by T cells. [Figure 11A]

[0088] Figure 11A shows that NK cells on medium supplemented with CCC proliferated far more after 3 days of co-incubation than NK cells on medium supplemented with standard soluble cytokines alone or Fc-cytokines. [Figure 11B]

[0088] Figure 11B shows that NK cells on medium supplemented with CCC proliferated far more after 3 days of co-incubation than NK cells on medium supplemented with standard soluble cytokines alone or Fc-cytokines. [Figure 11C]

[0088] Figure 11C shows that NK cells on medium supplemented with CCC proliferated far more after 3 days of co-incubation than NK cells on medium supplemented with standard soluble cytokines alone or Fc-cytokines. [Figure 11D]

[0088] Figure 11D shows that NK cells on medium supplemented with CCC proliferated far more after 3 days of co-incubation than NK cells on medium supplemented with standard soluble cytokines alone or Fc-cytokines. [Modes for carrying out the invention]

[0076] Detailed explanation

[0089] Figures 1A and 1B show predicted structures representing one embodiment of an engineered Fc antibody domain linked to multiple cytokines. For example, Figure 1A shows the predicted structure of an engineered Fc antibody domain (e.g., an engineered IgG1 Fc heavy chain) C-terminally linked to two IL-2 cytokines. Similarly, Figure 1B shows the predicted structure of an engineered Fc antibody domain (e.g., an engineered IgG1 Fc heavy chain) N-terminally linked to two IL-2 cytokines.

[0077]

[0090] In some embodiments, the manipulated Fc antibody domain may be a manipulated human Fc antibody domain. For example, the Fc antibody domain may be a manipulated human IgG1 Fc antibody domain.

[0078]

[0091] In some embodiments, the manipulated human IgG1 Fc antibody domain may contain an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% amino acid identity with respect to the amino acid sequence shown in SEQ ID NO: 1 (see Table 1).

[0079]

[0092] In other embodiments, the Fc antibody domain may be an engineered rabbit Fc antibody domain. For example, the Fc antibody domain may be an engineered rabbit IgG Fc antibody domain.

[0080]

[0093] Figures 1A and 1B show cytokines as IL-2, but this disclosure intends to show that other interleukins such as IL-7 and IL-15 (e.g., Figures 3 and 6) can also act as cytokines. Furthermore, this disclosure intends to show that other receptor-engineered molecules (engineered or native) can be used as components of cellular signaling in CCCs.

[0081]

[0094] Figure 1C shows the predicted structure of the manipulated self-assembling protein cage polypeptide. As shown in Figure 1C, the protein cage polypeptide can self-assemble into a tetrahedral pyramidal structure. Alternatively, the protein cage polypeptide can self-assemble into a compact, asymmetrical multimer structure or a cage-cage multimer (including dimers).

[0082]

[0095] The protein cage polypeptide may be either a protein cage polypeptide or a scaffold protein, as discussed in U.S. Patent Application Publication No. 2022 / 0196655, the entirety of which is incorporated herein by reference.

[0083]

[0096] The engineered self-assembled protein cage polypeptide can function as a carrier or scaffold for multiple engineered Fc antibody domains, each linked to one or more cytokines. When multiple engineered Fc antibody domains (each linked to one or more cytokines) are bound to the protein cage polypeptide, such a structure is referred to herein as a chimeric cytokine complex (CCC).

[0084]

[0097] Figure 1D shows examples of cytokines (e.g., IL-2) and their receptor signaling complexes. In some embodiments, the receptor signaling complex may include IL-2Rα, IL-2Rβ, and γc. In other embodiments, the receptor complex may be a dimeric complex of only IL-2Rβ and γc.

[0085]

[0098] Figure 1E shows a predicted structure of a CCC that includes an Fc cytokine bound to an engineered self-assembled protein cage polypeptide, and a receptor signaling complex bound to a cytokine of the CCC (e.g., IL-2). The protein cage polypeptide may contain multiple potential binding sites for the engineered Fc antibody domain. For example, the protein cage polypeptide may contain up to 12 binding sites for the Fc antibody domain.

[0086]

[0099] Figure 1E shows a single Fc-cytokine chimera (e.g., Fc-IL-2) bound to a protein cage polypeptide, but this disclosure suggests that 6 to 12 manipulated Fc antibody domains can bind to a single protein cage polypeptide.

[0087]

[0100] Since each manipulated Fc antibody domain can contain up to two cytokines, each CCC can contain 12 to 24 cytokines.

[0088]

[0101] [Table 1]

[0089] [Table 2]

[0090] [Table 3]

[0091]

[0102] The protein cage polypeptide of CCC may contain polypeptides with a length of approximately 400 to 700 amino acid residues. In some embodiments, the protein cage polypeptide may contain polypeptides with a length of approximately 450 to 650 amino acid residues.

[0092]

[0103] In some embodiments, the protein cage polypeptide may consist of a polypeptide comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% amino acid identity with respect to the amino acid sequence shown in any one of SEQ ID NOs: 8-11 (see Table 2).

[0093]

[0104] The protein cage polypeptide shown in SEQ ID NO: 12 can be designed by the applicant to be a Y294A variant of the protein cage polypeptide shown in SEQ ID NO: 8 (see Table 2). The protein cage polypeptide shown in SEQ ID NO: 13 can be designed by the applicant to be a Y294A variant of the protein cage polypeptide shown in SEQ ID NO: 9 (see Table 2). The protein cage polypeptide shown in SEQ ID NO: 14 can be designed by the applicant to be a Y294A variant of the protein cage polypeptide shown in SEQ ID NO: 10 (see Table 2). The protein cage polypeptide shown in SEQ ID NO: 15 can be designed by the applicant to be a Y294A variant of the protein cage polypeptide shown in SEQ ID NO: 11 (see Table 2).

[0094]

[0105] Furthermore, the various protein cage polypeptides disclosed herein may include peptide sequences that can bind to the manipulated Fc antibody domain. For example, the protein cage polypeptides shown in SEQ ID NOs. 8 and 12 may include the amino acid sequence RWGSGADCAWHLGELVWCTAGSGWE (SEQ ID NOs. 16) (referred to herein as peptide sequence A, see Table 2) for binding to the manipulated Fc antibody domain.

[0095]

[0106] In addition, the protein cage polypeptides shown in SEQ ID NOs: 9 and 13 may contain the amino acid sequence GGRWGADCAWHLGELVWCTAGWEGG (SEQ ID NO: 17) (referred to herein as peptide sequence B, see Table 2) for binding to the manipulated Fc antibody domain.

[0096]

[0107] Furthermore, the protein cage polypeptides shown in SEQ ID NOs: 10 and 14 may contain the amino acid sequence GADCAWHLGELVWCTAG (SEQ ID NO: 18) (referred to herein as peptide sequence C, see Table 2) for binding to the manipulated Fc antibody domain.

[0097]

[0108] Furthermore, the protein cage polypeptides shown in SEQ ID NOs: 11 and 15 may contain the amino acid sequence RWGSGCDCAWHLGELVWCTCGSGWE (SEQ ID NO: 19) (referred to herein as peptide sequence D, see Table 2) for binding to the manipulated Fc antibody domain.

[0098]

[0109] [Table 4]

[0099] [Table 5]

[0100] [Table 6]

[0101] [Table 7]

[0102]

[0110] Figure 2 shows that the manipulated Fc antibody domain can be C-terminally or N-terminally ligated to cytokines (the Fc cytokines then being bound to protein cage polypeptides) to achieve enhanced function in activating and proliferating T cells beyond the capacity of soluble cytokines alone. While the applicant anticipates that C-terminally ligated Fc cytokines promote less stereorestricted FcγR binding, one unexpected finding by the applicant is that N-terminally ligated Fc cytokines (CCCs) also function well in activating and proliferating T cells beyond the capacity of soluble cytokines.

[0103]

[0111] As shown in Figure 2, the manipulated Fc antibody domain of CCC can be C-terminally ligated to at least one N-terminus of a cytokine. Table 1 lists representative sequences for several Fc-cytokine variants.

[0104]

[0112] For example, if one of the cytokines is interleukin-2 (IL-2), at least one of the engineered Fc antibody domains can be C-terminally ligated to the N-terminus of IL-2 via an engineered metalloproteinase resistance linker sequence (referred to herein as Fc-IL-2). In some embodiments, the engineered metalloproteinase resistance linker sequence may include the amino acid sequence SLSPGKAPTS (SEQ ID NO: 20) (see also Figure 3).

[0105]

[0113] Furthermore, for example, if one of the cytokines is interleukin-7 (IL-7), at least one of the manipulated Fc antibody domains may be C-terminally ligated to the N-terminus of IL-7 via a manipulated metalloproteinase resistance linker sequence (referred to herein as Fc-IL-7). In some embodiments, the manipulated metalloproteinase resistance linker sequence may include the amino acid sequence SLSPGKDCDIEGK (SEQ ID NO: 21) (see also Figure 3).

[0106]

[0114] As an additional example, if one of the cytokines is interleukin-15 (IL-15), at least one of the engineered Fc antibody domains may be C-terminally ligated to the N-terminus of IL-15 via an engineered metalloproteinase resistance linker sequence (referred to herein as Fc-IL-15). In some embodiments, the engineered metalloproteinase resistance linker sequence may include the amino acid sequence SLSPGKN (SEQ ID NO: 22) (see also Figure 3).

[0107]

[0115] Figure 2 also shows that the manipulated Fc antibody domain can be N-terminally ligated to at least one C-terminus of a cytokine (and subsequently bound to a protein cage polypeptide) to achieve enhanced T cell activation and proliferation beyond the capabilities of the soluble cytokine alone. Table 1 lists representative sequences for these Fc-cytokine variants.

[0108]

[0116] For example, if one of the cytokines is interleukin-2 (IL-2), at least one of the engineered Fc antibody domains can be N-terminally ligated to the C-terminus of IL-2 via an engineered metalloproteinase resistance linker sequence (referred to herein as IL-2-Fc). In some embodiments, the engineered metalloproteinase resistance linker may include the amino acid sequence TPKSCDKTHT (SEQ ID NO: 23) (see also Figure 3).

[0109]

[0117] Furthermore, for example, if one of the cytokines is interleukin-7 (IL-7), at least one of the manipulated Fc antibody domains may be N-terminally ligated to the C-terminus of IL-7 via a manipulated metalloproteinase resistance linker sequence (referred to herein as Fc-IL-7). In some embodiments, the manipulated metalloproteinase resistance linker sequence may include the amino acid sequence HPKSCDKTHT (SEQ ID NO: 24) (see also Figure 3).

[0110]

[0118] As an additional example, if one of the cytokines is interleukin-15 (IL-15), at least one of the engineered Fc antibody domains may be N-terminally ligated to the C-terminus of IL-15 via an engineered metalloproteinase resistance linker sequence (referred to herein as Fc-IL-15). In some embodiments, the engineered metalloproteinase resistance linker sequence may include the amino acid sequence TSPKSCDKTHT (SEQ ID NO: 25) (see also Figure 3).

[0111]

[0119] Furthermore, Figure 2 shows that the CCC may include a signal peptide or leader sequence ligated to the N-terminus of at least one manipulated Fc antibody domain or at least one cytokine.

[0112]

[0120] In some embodiments, the signal peptide may be a mouse Ig heavy signal peptide for expression in Chinese hamster ovary (CHO) cells. The signal peptide may improve the secretion efficiency of the manipulated molecule in CHO cells.

[0113]

[0121] As a more specific example, the signal peptide may contain the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 26).

[0114]

[0122] Figure 3 shows exemplary linker sequences designed to link the Fc components and cytokine components of the following CCCs: (1) Fc-IL-2 CCC, (2) IL-2-Fc CCC, (3) Fc-IL-7 CCC, (4) IL-7-Fc CCC, (5) Fc-IL-15 CCC, and (6) IL-15-Fc CCC (see also Table 1). The linkers are underlined in Figure 3.

[0115]

[0123] Furthermore, Figure 3 shows various cleavage sites (for example, aspartate proteases, cysteine ​​proteases, metalloproteases, serine proteases, and various protease superfamilies). As will be discussed in more detail with respect to Example 3, metalloproteases are known to mediate the detachment of cell surface receptors from cells or the cleavage of the functional domains of receptors and ligands. Figure 3 shows that, with the exception of serine proteases, none of the linkers are cleaved in the middle. This is less of a concern for serine proteases because they are often localized inside the cell itself.

[0116]

[0124] The amino acid sequences shown in Figure 3 (and listed below) are some of the amino acid sequences listed in Table 1 above:

[0125] Part of the manipulated Fc-IL-2: QQGNVFSCSVMHEALHNHYTQKSLSLSPGKAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML(Sequence ID 27).

[0126] Part of the manipulated IL-2-Fc: WITFCQSIISTLTPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP (Sequence ID 28).

[0127] Parts of the manipulated Fc-IL-7: SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNN (Sequence ID 29).

[0128] Part of the manipulated IL-7-Fc: DLCFLKRLLQEIKTCWNKILMGTKEHPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV(Sequence ID 30).

[0129] Part of the manipulated Fc-IL-15: WQQGNVFSCSVMHEALHNHYTQKSLSPGKNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKV (Sequence ID 31).

[0130] Part of the manipulated IL-15-Fc: FVHIVQMFINTSPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP(Sequence ID 32).

[0117]

[0131] Figure 4 shows an example of an engineered Fc component of CCC stabilized by the Fcγ receptor (FcγR) of a neighboring cell. The Fc component of CCC can be stabilized via trans-stabilization by a neighboring cell expressing FcγR.

[0118]

[0132] In some embodiments, the Fc component of CCC may be engineered to include one or more point mutations to modulate FcγR binding. For example, the Fc component may be engineered to be either FcγR-compliant (i.e., increased affinity for FcγR binding) or FcγR-incompetent (i.e., decreased affinity for FcγR binding) in order to avoid antibody-dependent cytotoxicity (ADCC) activation of NK cells.

[0119]

[0133] Three major classes of Fcγ receptors (FcγR) are present on leukocytes (FcγRI, FcγRII, and FcγRIII) and function in cellular processes such as ADCC, cytokine release, phagocytosis, and endocytosis [7]. ADCC is the primary signal for initiating NK cell-mediated killing activity, such as the release of perforin and granzymes, which requires clustering of FcγRIIIa receptors on the surface of NK cells [8]. Depending on the application, NK cell-mediated ADCC may or may not be desirable.

[0120]

[0134] Figure 5 shows that the Fc component of CCC (e.g., human IgG1 Fc) can be manipulated by mutations at key sites in its FcγR binding region. Various ways in which the FcγR binding region can be modified via point mutations are shown in Figure 5. Some of these point mutations are discussed in references [8, 9, and 10].

[0121]

[0135] In some embodiments, the manipulated human IgG1 Fc antibody domain may contain at least one of the following point mutations relative to Sequence ID No. 1 (see Table 1): P75L, R76W, Y80K, Y80P, Y80R, Y80G, and Y80A, to reduce affinity for a specific Fcγ receptor (e.g., FcγRIII) and functionally reduce ADCC.

[0122]

[0136] For example, the Y80K variant can significantly reduce binding to the FcγRIIIa receptor and decrease ADCC. Similarly, the Y80P variant can significantly reduce binding to FcγRIIIa and induce insufficient N-glycosylation.

[0123]

[0137] As another example, the Y80R variant can significantly reduce binding to the FcγRIIIa receptor. Similarly, the Y80G variant can significantly reduce binding to the FcγRIIIa receptor. Yet another example is the Y80A variant, which can significantly reduce binding to the FcγRIIIa receptor and also reduce ADCC.

[0124]

[0138] In some embodiments, the manipulated human IgG1 Fc antibody domain may contain at least one of the following point mutations: Y80W relative to Sequence ID No. 1 (see Table 1) to increase affinity for a specific Fcγ receptor and functionally enhance ADCC.

[0125]

[0139] In some embodiments, the manipulated human IgG1 Fc antibody domain may contain at least one of the following point mutations relative to Sequence ID No. 1 (see Table 1): S23A, E53A, E77A, Y80F, V87A, A111G, K122A, and D160A, such that it reduces affinity for a specific Fcγ receptor (e.g., FcγRIIIa) and has a neutral effect on other Fcγ receptors (e.g., FcγRII).

[0126]

[0140] In some embodiments, the manipulated human IgG1 Fc antibody domain may contain at least one of the following point mutations to Sequence ID No. 1 (see Table 1): E117, K118A, and A123T, to increase affinity for a specific Fcγ receptor (e.g., FcγRIIIa) and have a neutral effect on other Fcγ receptors (e.g., FcγRII).

[0127]

[0141] In some embodiments, the manipulated human IgG1 Fc antibody domain may contain at least one of the following point mutations to Sequence ID No. 1 (see Table 1): H52A, R85A, and K106A, to increase affinity for a specific Fcγ receptor (e.g., FcγRII) and decrease affinity for other Fcγ receptors (e.g., FcγRIIIa).

[0128]

[0142] In some embodiments, the manipulated human IgG1 Fc antibody domain may contain at least one of the following point mutations to SEQ ID NO: D54A, Q79A, and A111S to reduce its affinity for specific Fcγ receptors (e.g., FcγRII and FcγRIIIa).

[0129]

[0143] In some embodiments, the manipulated human IgG1 Fc antibody domain may contain at least one of the following point mutations: T40A and K74A, relative to SEQ ID NO: 1 (see Table 1) to increase affinity for specific Fcγ receptors (e.g., FcγRII and FcγRIIIa).

[0130]

[0144] As discussed above, the Fc component of CCC can be manipulated to either functionally enhance ADCC by becoming FcγR-binding competent (i.e., increasing affinity for FcγR binding), or functionally degrade ADCC by becoming FcγR-binding competent (i.e., decreasing affinity for FcγR binding). [Examples]

[0131] Examples

[0145] The following embodiments are given to illustrate various embodiments of the Disclosure. They are not intended to limit or define the entire scope of the Disclosure. The Disclosure is not limited to the specific embodiments described and illustrated herein, but should be understood to include all modifications and variations that fall within the scope of the Disclosure as defined in the appended embodiments.

[0132]

[0146] Example 1: Chimeric cytokine complexes (CCCs) enhance T cell activation and proliferation beyond the capacity of soluble cytokines.

[0147] One unexpected result derived from the experiments disclosed herein is that CCC enhances or significantly increases T cell activation and proliferation more than equivalent concentrations of soluble cytokines alone or even Fc-cytokines alone.

[0133]

[0148] Human peripheral blood CD3 + The T cells were thawed and the culture medium was changed to serum-free T cell proliferation medium. 1 × 10⁶ T cells 6 Cells were seeded at a concentration of cells / mL and placed in a 37°C, 5% CO2 incubator on day -1. On day 0, the cells were activated with anti-CD3 and anti-CD28 T cell activating reagents. In some embodiments, the T cell activating reagent may be a soluble T cell activator for in vitro use.

[0134]

[0149] In some embodiments, the anti-CD3 and anti-CD28 T cell activating reagents may comprise a plurality of self-assembled protein nanoparticles modified with anti-CD3 and anti-CD28 antibodies. The self-assembled protein nanoparticles may comprise protein cage polypeptides assembled into a three-dimensional structure.

[0135]

[0150] In some embodiments, the three-dimensional structure of the protein cage polypeptide may be a tetrahedral pyramid. Furthermore, the protein cage polypeptide can self-assemble into a compact, asymmetrical multimer structure or a cage-cage multimer (including a dimer).

[0136]

[0151] Three-dimensional structures (e.g., protein cage polypeptides formed in a tetrahedral pyramid) can function as scaffolds for anti-CD3 and anti-CD28 antibodies.

[0137]

[0152] In some embodiments, the protein cage polypeptide may consist of a polypeptide having an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% amino acid identity with respect to the amino acid sequence shown in any one of SEQ ID NOs: 8-10 (see Table 2).

[0138]

[0153] The protein cage polypeptide may contain polypeptides with a length of approximately 400 to 700 amino acid residues. In some embodiments, the protein cage polypeptide may contain polypeptides with a length of approximately 450 to 650 amino acid residues.

[0139]

[0154] In some embodiments, the anti-CD3 antibody may be an "OKT3" clone having multiple host isotypes, such as human, mouse, and rabbit. For example, any of the following anti-CD3 antibodies can be used: (i) anti-CD3 monoclonal antibody (OKT3) distributed by Takara Bio Inc., (ii) GMP monoclonal anti-human CD3 antibody (OKT3) distributed by ACROBiosystems, (iii) MACS® GMP CD3 pure antibody distributed by Miltenyi Biotec, or (iv) GMP Ultra-LEAF® purified anti-human CD3 SF antibody distributed by BioLegend.

[0140]

[0155] In some embodiments, the anti-CD28 antibody may be an agonist clone having multiple host isotypes, such as human, mouse, or rabbit. For example, any of the following anti-CD28 antibodies can be used: (i) anti-CD28[YTH913.12] antibody distributed by Absolute Antibody, (ii) anti-human CD28 antibody, clone 15E8, distributed by Miltenyi Biotec, (iii) Ultra-LEAF® purified anti-human CD28 antibody, clone cd28.2, distributed by BioLegend, or (iv) BD® purified mouse anti-human CD28 antibody, clone L293, distributed by BD Biosciences.

[0141]

[0156] Standard soluble cytokines were added to the culture medium at concentrations of 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, and 35 ng / mL. CCC was then added to the medium at concentrations equivalent to the standard cytokine concentrations (molar equivalents, me). Additionally, 10 ng / mL of Fc-IL-2 was added. The added CCC was identified as FC-IL-2 CCC. The added cytokine was identified as soluble IL-2. Next, the cells were returned to a 37°C, 5% CO2 incubator.

[0142]

[0157] On day 5, culture media were collected for an ELISA specific to the detection of interferon-gamma (IFN-γ). Figure 6 is a graph showing the results of the ELISA specific to the detection of IFN-γ. As shown in Figure 6, T cells on media supplemented with CCC (e.g., FC-IL-2 CCC) were activated far more significantly than T cells on media supplemented with cytokines (e.g., IL-2) alone or even Fc-cytokines (e.g., Fc-IL-2), as indicated by the level of IFN-γ produced by the T cells.

[0143]

[0158] Fresh cell culture medium supplemented with either CCC or standard cytokines was added to the cells at equivalent concentrations (e.g., 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, and 35 ng / mL). The cells were then returned to a 37°C, 5% CO2 incubator. On day 7, the cells were resuspended and collected for flow cytometry.

[0144]

[0159] Figure 7 is a graph showing the population of living T cells as determined by flow cytometry on day 7. Cells were stained with Zombie Aqua to determine the population of living T cells. As shown in Figure 7, the total number of living T cells on medium supplemented with CCC (e.g., Fc-IL-2 CCC) significantly exceeded the total number of living T cells on medium supplemented with cytokine (e.g., IL-2) alone or even with Fc-cytokines (e.g., Fc-IL-2) on day 7.

[0145]

[0160] One unexpected result derived from the experiments disclosed herein is that CCC at culture medium concentrations of 10 ng / mL to 50 ng / mL significantly enhances T cell proliferation more than equivalent and even higher concentrations of soluble cytokines (e.g., IL-2) alone.

[0146]

[0161] Another unexpected result derived from the experiments disclosed herein is that Fc-cytokine complexes of the type disclosed herein (e.g., Fc-IL-2) also enhance T cell proliferation over equivalent and even higher concentrations of soluble cytokine alone (e.g., IL-2).

[0147]

[0162] Figure 8 is a graph showing the population of live T cells determined using flow cytometry on day 7. Cells were stained with Zombie Aqua to determine the population of live T cells. As shown in Figure 8, the total live T cells on media supplemented with a combination of IL-7-Fc CCC (i.e., the engineered Fc antibody domain is N-terminally linked to the C-terminus of the IL-7 cytokine) and soluble cytokines (e.g., IL-15) significantly exceeded the total live T cells on media supplemented with only soluble cytokines (e.g., IL-7 and IL-15) on day 7.

[0148]

[0163] Example 2: Chimeric cytokine complexes (CCCs) exceed the ability of soluble cytokines to increase CD4 in proliferating T cells + Increase the number of T cells

[0164] Yet another unexpected result derived from the experiments disclosed herein is that the CCC significantly increases the CD4 + content in proliferating T cells. Figures 9A - 9C are graphs showing the number of CD4 + T cells, CD8 + T cells, and total live T cells, respectively, on day 7.

[0149]

[0165] Human peripheral blood T cells were thawed and media-exchanged into serum-free T cell growth media. T cells were seeded at a concentration of 1×10 6 cells / mL and then placed in a 37°C, 5% CO2 incubator on day -1. On day 0, the cells were activated with anti-CD3 and anti-CD28 T cell activation reagents.

[0150]

[0166] In some embodiments, the anti-CD3 and anti-CD28 T cell activating reagents may comprise a plurality of self-assembled protein nanoparticles modified with anti-CD3 and anti-CD28 antibodies. The self-assembled protein nanoparticles may comprise protein cage polypeptides assembled into a three-dimensional structure.

[0151]

[0167] In some embodiments, the three-dimensional structure of the protein cage polypeptide may be a tetrahedral pyramid. Furthermore, the protein cage polypeptide can self-assemble into a compact, asymmetrical multimer structure or a cage-cage multimer (including a dimer).

[0152]

[0168] Three-dimensional structures (e.g., protein cage polypeptides formed in a tetrahedral pyramid) can function as scaffolds for anti-CD3 and anti-CD28 antibodies.

[0153]

[0169] In some embodiments, the protein cage polypeptide may consist of a polypeptide having an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% amino acid identity with respect to the amino acid sequence shown in any one of SEQ ID NOs: 8-10 (see Table 2).

[0154]

[0170] The protein cage polypeptide may contain polypeptides with a length of approximately 400 to 700 amino acid residues. In some embodiments, the protein cage polypeptide may contain polypeptides with a length of approximately 450 to 650 amino acid residues.

[0155]

[0171] In some embodiments, the anti-CD3 antibody may be an "OKT3" clone having multiple host isotypes, such as human, mouse, and rabbit. For example, any of the following anti-CD3 antibodies can be used: (i) anti-CD3 monoclonal antibody (OKT3) distributed by Takara Bio Inc., (ii) GMP monoclonal anti-human CD3 antibody (OKT3) distributed by ACROBiosystems, (iii) MACS® GMP CD3 pure antibody distributed by Miltenyi Biotec, or (iv) GMP Ultra-LEAF® purified anti-human CD3 SF antibody distributed by BioLegend.

[0156]

[0172] In some embodiments, the anti-CD28 antibody may be an agonist clone having multiple host isotypes, such as human, mouse, or rabbit. For example, any of the following anti-CD28 antibodies can be used: (i) anti-CD28[YTH913.12] antibody distributed by Absolute Antibody, (ii) anti-human CD28 antibody, clone 15E8, distributed by Miltenyi Biotec, (iii) Ultra-LEAF® purified anti-human CD28 antibody, clone cd28.2, distributed by BioLegend, or (iv) BD® purified mouse anti-human CD28 antibody, clone L293, distributed by BD Biosciences.

[0157]

[0173] Standard soluble cytokines were added to the culture medium at a concentration of 20 ng / mL, and CCCs were added to the medium at a concentration (molar equivalent, me) equivalent to that of the standard cytokines. The added CCC was IL-2-Fc CCC. For comparison, soluble IL-2 was added. On day 7, the cells were resuspended and collected for flow cytometry. The cells were stained with Zombie Aqua to identify the population of viable cells. Next, T cells were run on a flow cytometer.

[0158]

[0174] Example 3: Multiplexing of cytokines and chemokines reveals increased expression of matrix metalloproteinases (MMPs) by T cells.

[0175] Human peripheral blood CD3 + The T cells were thawed and the culture medium was changed to serum-free T cell proliferation medium. 6 Cells were seeded in GREX® 24-well plates at a concentration of cells / mL, and then placed in a 37°C, 5% CO2 incubator on day -1. On day 0, T cells were activated with anti-CD3 and anti-CD28 T cell activation reagents.

[0159]

[0176] In some embodiments, the anti-CD3 and anti-CD28 T cell activating reagents may comprise a plurality of self-assembled protein nanoparticles modified with anti-CD3 and anti-CD28 antibodies. The self-assembled protein nanoparticles may comprise protein cage polypeptides assembled into a three-dimensional structure.

[0160]

[0177] In some embodiments, the three-dimensional structure of the protein cage polypeptide may be a tetrahedral pyramid. Furthermore, the protein cage polypeptide can self-assemble into a compact, asymmetrical multimer structure or a cage-cage multimer (including a dimer).

[0161]

[0178] Three-dimensional structures (e.g., protein cage polypeptides formed in a tetrahedral pyramid) can function as scaffolds for anti-CD3 and anti-CD28 antibodies.

[0162]

[0179] In some embodiments, the protein cage polypeptide may consist of a polypeptide having an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% amino acid identity with respect to the amino acid sequence shown in any one of SEQ ID NOs: 8-10 (see Table 2).

[0163]

[0180] The protein cage polypeptide may contain polypeptides with a length of approximately 400 to 700 amino acid residues. In some embodiments, the protein cage polypeptide may contain polypeptides with a length of approximately 450 to 650 amino acid residues.

[0164]

[0181] In some embodiments, the anti-CD3 antibody may be an "OKT3" clone having multiple host isotypes, such as human, mouse, and rabbit. For example, any of the following anti-CD3 antibodies can be used: (i) anti-CD3 monoclonal antibody (OKT3) distributed by Takara Bio Inc., (ii) GMP monoclonal anti-human CD3 antibody (OKT3) distributed by ACROBiosystems, (iii) MACS® GMP CD3 pure antibody distributed by Miltenyi Biotec, or (iv) GMP Ultra-LEAF® purified anti-human CD3 SF antibody distributed by BioLegend.

[0165]

[0182] In some embodiments, the anti-CD28 antibody may be an agonist clone having multiple host isotypes, such as human, mouse, or rabbit. For example, any of the following anti-CD28 antibodies can be used: (i) anti-CD28[YTH913.12] antibody distributed by Absolute Antibody, (ii) anti-human CD28 antibody, clone 15E8, distributed by Miltenyi Biotec, (iii) Ultra-LEAF® purified anti-human CD28 antibody, clone cd28.2, distributed by BioLegend, or (iv) BD® purified mouse anti-human CD28 antibody, clone L293, distributed by BD Biosciences.

[0166]

[0183] Cytokines (e.g., IL-2) were added to the culture medium to achieve a final concentration of 10 ng / mL. A 500 μL sample of IL-2-free medium was frozen and used as the control sample on day 0. Next, the T cells were returned to a 37°C, 5% CO2 incubator. On day 3, 500 μL of medium samples were collected from each sample well and frozen. Then, the used medium was replaced with fresh culture medium supplemented with IL-2 to achieve a final IL-2 concentration of 10 ng / mL. Next, the T cells were returned to a 37°C, 5% CO2 incubator. On day 7, 500 μL of medium samples were collected from each sample well and frozen. Then, the used medium was replaced with fresh culture medium supplemented with IL-2 to achieve a final IL-2 concentration of 10 ng / mL. Next, the T cells were returned to a 37°C, 5% CO2 incubator. Samples from day 0, day 3, and day 7 of freezing were subjected to a 71-human cytokine / chemokine multiplexing panel, in which analytes and fluorescent detection antibodies were captured using a combination of antibody-conjugated fluorescent beads to determine the concentrations of cytokines and chemokines in the culture medium.

[0167]

[0184] As shown in the graph in Figure 10, immediately after stimulation with anti-CD3 and anti-CD28 T cell activating reagents, peripheral blood donor T cells upregulated the expression of cytokines and chemokines that increase the expression of matrix metalloproteinases (MMPs) (also known as matrix metalloproteinases) by T cells.

[0168]

[0185] This is consistent with other studies showing that T cells and NK cells express MMPs and that the receptor CD100 can be “shed” from its surface by MMP proteolytic activity [11, 12]. Furthermore, chemokines and cytokines, TNF-alpha, IL-1, MIP-1-alpha, MIP-1-beta, and RANTES also express MMPs. + and CD4 +It has been shown that IL-2 stimulates upregulation of proMMP-9 secretion (a proprotease that precedes enzymatic cleavage of active MMP-9) by T cells [13, 14]. In addition, IL-2 stimulation increases MMP-9 production in T cells

[11] .

[0169]

[0186] When these research lines are linked to the pursuit of CCC-based and Fc-cytokine-based cellular agonisms, it is deemed extremely necessary to protect CCC chimeric Fc-cytokine linkers and Fc-cytokine complexes from metalloproteinase enzymes.

[0170]

[0187] Therefore, disclosed herein are chimeric Fc-cytokine linkers or CCC and Fc-cytokine complexes having linker sequences that are protected from metalloproteinase enzymes (see Figures 2 and 3). Such linkers were designed to contain no predicted metalloproteinase sites. This effort included the use of a protease site prediction tool called PROSPER

[15] .

[0171]

[0188] One technical challenge faced by the applicant is how to manipulate metalloproteinase-resistant linkers that promote agonism or cell receptor binding. One technical solution discovered and developed by the applicant is to manipulate metalloproteinase-resistant linkers so that the linker sequence contains 7–13 amino acid residues. This is supported by literature showing an inverse relationship between flexibility and agonism in the case of FcγR

[16] . The applicant predicts a similar relationship with Fc-cytokine chimeras resulting from similar types of receptor clustering mechanisms involved in both antibody [1] and cytokine agonism [2, 3].

[0172]

[0189] In some embodiments, the anti-CD3 and anti-CD28 T cell activating reagents may comprise a plurality of self-assembled protein nanoparticles modified with anti-CD3 and anti-CD28 antibodies. The self-assembled protein nanoparticles may comprise protein cage polypeptides assembled into a three-dimensional structure.

[0173]

[0190] In some embodiments, the three-dimensional structure of the protein cage polypeptide may be a tetrahedral pyramid. Furthermore, the protein cage polypeptide can self-assemble into a compact, asymmetrical multimer structure or a cage-cage multimer (including a dimer).

[0174]

[0191] Three-dimensional structures (for example, protein cage polypeptides formed in a tetrahedral pyramid) can function as scaffolds for anti-CD3 and anti-CD28 antibodies.

[0175]

[0192] In some embodiments, the protein cage polypeptide may consist of a polypeptide having an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% amino acid identity with respect to the amino acid sequence shown in any one of SEQ ID NOs: 8-10 (see Table 2).

[0176]

[0193] The protein cage polypeptide may contain polypeptides with a length of approximately 400 to 700 amino acid residues. In some embodiments, the protein cage polypeptide may contain polypeptides with a length of approximately 450 to 650 amino acid residues.

[0177]

[0194] Example 4: Chimeric cytokine complexes (CCCs) enhance the activation and proliferation of NK cells.

[0195] Another unexpected result derived from the experiments disclosed herein is that CCC enhances or significantly increases NK cell activation and proliferation compared to equivalent concentrations of soluble cytokines alone or Fc-cytokines alone.

[0178]

[0196] Day 0: Human peripheral blood CD56 + NK cells were thawed and replaced with serum-free NK cell proliferation medium. The medium was supplemented with 10% human platelet lysate and one of the following: (1) standard soluble cytokines (e.g., IL-2), (2) Fc-cytokine complexes (e.g., IL-2-Fc), (3) CCC (e.g., IL-2-Fc), and (4) CCC (e.g., IL-2-Fc) together with NK activators. For example, the NK activator could be a soluble NK activator used in vitro.

[0179]

[0197] NK cells 7 × 10 5 Cells were seeded at a concentration of cells / mL and then placed in a 37°C, 5% CO2 incubator. On the third day, the cells were imaged under a light microscope.

[0180]

[0198] Standard soluble cytokines were added to the culture medium at a concentration of 35 ng / mL, and CCCs were added to the medium at a concentration (molar equivalent, me) equivalent to that of the standard cytokines. The added CCCs were IL-2-Fc CCCs.

[0181]

[0199] Figures 11A to 11D show NK cells imaged under a light microscope. As shown in Figures 11A to 11D, donor peripheral blood CD56 cells on culture medium supplemented with CCCs (e.g., IL-2-Fc CCC, and IL-2-Fc CCC together with an NK activator) + NK cells proliferated far more after 3 days of co-incubation than NK cells on medium supplemented with standard soluble cytokines (e.g., IL-2) alone or Fc-cytokines alone (e.g., Fc-IL-2).

[0182]

[0200] Furthermore, this disclosure also includes the following provisions:

[0183]

[0201] Clause 1. A chimeric cytokine complex comprising a protein cage polypeptide; multiple engineered Fc antibody domains bound to the protein cage polypeptide; and one or more cytokines linked to each of the multiple engineered Fc antibody domains.

[0184]

[0202] Clause 2. The cytokine is an interleukin, a chimeric cytokine complex of Clause 1.

[0185]

[0203] Clause 3. The interleukin is at least one of interleukin-2 (IL-2), IL-7, and IL-15, a chimeric cytokine complex according to Clause 2.

[0186]

[0204] Clause 4. Multiple manipulated Fc antibody domains are chimeric cytokine complexes according to Clause 1, comprising 6 to 12 manipulated Fc antibody domains bound to a protein cage polypeptide.

[0187]

[0205] Clause 5. A chimeric cytokine complex according to Clause 4, wherein a total of 12 to 24 cytokines are linked to multiple manipulated Fc antibody domains.

[0188]

[0206] Clause 6. A chimeric cytokine complex according to Clause 1, wherein each of one or more cytokines is linked to one of the manipulated Fc antibody domains via a manipulated metalloproteinase resistance linker sequence.

[0189]

[0207] Clause 7. The manipulated metalloproteinase resistance linker sequence is a chimeric cytokine complex of Clause 6, having a length of 7-13 amino acid residues.

[0190]

[0208] Clause 8. A chimeric cytokine complex according to Clause 1, wherein at least one of the manipulated Fc antibody domains is C-terminally ligated to the N-terminus of at least one cytokine.

[0191]

[0209] Clause 9. A chimeric cytokine complex of Clause 8, wherein one of the cytokines is interleukin-2 (IL-2), and at least one of the engineered Fc antibody domains is linked to IL-2 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence SLSPGKAPTS (SEQ ID NO: 20).

[0192]

[0210] Clause 10. A chimeric cytokine complex of Clause 8, wherein one of the cytokines is interleukin-7 (IL-7), and at least one of the engineered Fc antibody domains is linked to IL-7 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence SLSPGKDCDIEGK (SEQ ID NO: 21).

[0193]

[0211] Clause 11. A chimeric cytokine complex of Clause 8, wherein one of the cytokines is interleukin-15 (IL-15), and at least one of the engineered Fc antibody domains is linked to IL-15 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence SLSPGKN (SEQ ID NO: 22).

[0194]

[0212] Clause 12. A chimeric cytokine complex according to Clause 1, wherein at least one of the manipulated Fc antibody domains is N-terminally ligated to the C-terminus of at least one cytokine.

[0195]

[0213] Clause 13. A chimeric cytokine complex of Clause 12, wherein one of the cytokines is interleukin-2 (IL-2), and at least one of the engineered Fc antibody domains is linked to IL-2 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence TPKSCDKTHT (SEQ ID NO: 23).

[0196]

[0214] Clause 14. A chimeric cytokine complex of Clause 12, wherein one of the cytokines is interleukin-7 (IL-7), and at least one of the engineered Fc antibody domains is linked to IL-7 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence HPKSCDKTHT (SEQ ID NO: 24).

[0197]

[0215] Clause 15. A chimeric cytokine complex of Clause 12, wherein one of the cytokines is interleukin-15 (IL-15), and at least one of the engineered Fc antibody domains is linked to IL-15 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence TSPKSCDKTHT (SEQ ID NO: 25).

[0198]

[0216] Clause 16. The manipulated Fc antibody domain is a chimeric cytokine complex of Clause 1, which is a manipulated human Fc antibody domain.

[0199]

[0217] Clause 17. The manipulated human Fc antibody domain is the manipulated human IgG1 Fc antibody domain of the chimeric cytokine complex of Clause 16.

[0200]

[0218] Clause 18. A chimeric cytokine complex according to Clause 17, wherein one of the manipulated human IgG1 Fc antibody domains comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% amino acid identity with respect to the amino acid sequence shown in SEQ ID NO: 1.

[0201]

[0219] Clause 19. The chimeric cytokine complex of Clause 18, comprising at least one of the following point mutations in SEQ ID NO: P75L, R76W, Y80K, Y80P, Y80R, Y80G, and Y80A, in the manipulated human IgG1 Fc antibody domain, to reduce affinity for a specific Fcγ receptor and functionally reduce antibody-dependent cell-mediated cytotoxicity (ADCC).

[0202]

[0220] Clause 20. The modified human IgG1 Fc antibody domain is a chimeric cytokine complex of Clause 18, comprising the following point mutation: Y80W in SEQ ID NO: 1, to increase affinity for a specific Fcγ receptor and functionally enhance antibody-dependent cell-mediated cytotoxicity (ADCC).

[0203]

[0221] Clause 21. The chimeric cytokine complex of Clause 18, wherein the manipulated human IgG1 Fc antibody domain includes at least one of the following point mutations in SEQ ID NO: S23A, E53A, E77A, Y80F, V87A, A111G, K122A, and D160A, such that it reduces affinity for a specific Fcγ receptor and has a neutral effect on other Fcγ receptors.

[0204]

[0222] Clause 22. The chimeric cytokine complex of Clause 18, wherein the manipulated human IgG1 Fc antibody domain includes at least one of the following point mutations in SEQ ID NO: E117, K118A, and A123T, such that it increases affinity for a specific Fcγ receptor and has a neutral effect on other Fcγ receptors.

[0205]

[0223] Clause 23. The chimeric cytokine complex of Clause 18, wherein the manipulated human IgG1 Fc antibody domain includes at least one of the following point mutations in SEQ ID NO: H52A, R85A, and K106A to increase affinity for a specific Fcγ receptor and decrease affinity for a specific other Fcγ receptor.

[0206]

[0224] Clause 24. The chimeric cytokine complex of Clause 18, wherein the manipulated human IgG1 Fc antibody domain includes at least one of the following point mutations to SEQ ID NO: D54A, Q79A, and A111S to reduce affinity for a specific Fcγ receptor.

[0207]

[0225] Clause 25. The modified human IgG1 Fc antibody domain comprises at least one of the following point mutations in SEQ ID NO: T40A and K74A to increase affinity for a specific Fcγ receptor, as a chimeric cytokine complex of Clause 18.

[0208]

[0226] Clause 26. The Fc antibody domain is an engineered rabbit Fc antibody domain, which is part of the chimeric cytokine complex of Clause 1.

[0209]

[0227] Clause 27. A chimeric cytokine complex according to Clause 1, further comprising a signal peptide ligated to the N-terminus of at least one manipulated Fc antibody domain or at least one cytokine.

[0210]

[0228] Clause 28. The signal peptide is a mouse Ig heavy signal peptide used for expression in Chinese hamster ovary (CHO) cells, a chimeric cytokine complex of Clause 27.

[0211]

[0229] Clause 29. The signal peptide is a chimeric cytokine complex of Clause 28, comprising the amino acid sequence MGWSCIILFLVATATATGVHS (SEQ ID NO: 26).

[0212]

[0230] Clause 30. The chimeric cytokine complex of Clause 1, comprising a polypeptide comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% amino acid identity with respect to the amino acid sequence shown in any one of SEQ ID NOs. 8-15.

[0213]

[0231] Clause 31. The chimeric cytokine complex of Clause 30, wherein the amino acid sequences constituting the polypeptide of the protein cage polypeptide include at least the following point mutation: Y294A to the amino acid sequence shown in any one of SEQ ID NOs: 8-15.

[0214]

[0232] Clause 32. The protein cage polypeptide comprises a polypeptide comprising a polypeptide containing a binding site for one of the engineered Fc antibody domains, wherein the binding site contains the amino acid sequence RWGSGADCAWHLGELVWCTAGSGWE (SEQ ID NO: 16), a chimeric cytokine complex of Clause 1.

[0215]

[0233] Clause 33. The protein cage polypeptide comprises a polypeptide containing a binding site for one of the engineered Fc antibody domains, the binding site comprising the amino acid sequence GGRWGADCAWHLGELVWCTAGWEGG (SEQ ID NO: 17), the chimeric cytokine complex of Clause 1.

[0216]

[0234] Clause 34. The protein cage polypeptide comprises a polypeptide containing a binding site for one of the engineered Fc antibody domains, the binding site containing the amino acid sequence GADCAWHLGELVWCTAG (SEQ ID NO: 18), a chimeric cytokine complex of Clause 1.

[0217]

[0235] Clause 35. The protein cage polypeptide comprises a polypeptide containing a binding site for one of the engineered Fc antibody domains, the binding site comprising the amino acid sequence RWGSGCDCAWHLGELVWCTCGSGWE (SEQ ID NO: 19), the chimeric cytokine complex of Clause 1.

[0218]

[0236] Clause 36. The protein cage polypeptide self-assembles into a tetrahedral pyramidal structure, a chimeric cytokine complex according to Clause 1.

[0219]

[0237] Clause 37. A method for activating and proliferating immune cells, comprising adding a chimeric cytokine complex to a population of immune cells, wherein the chimeric cytokine complex comprises a protein cage polypeptide, a plurality of manipulated Fc antibody domains bound to the protein cage polypeptide, and one or more cytokines linked to each of the plurality of Fc antibody domains.

[0220]

[0238] Clause 38. A population of immune cells is activated and proliferated in vitro, as described in Clause 37.

[0221]

[0239] Clause 39. The population of immune cells is human donor peripheral blood immune cells, as per the method of Clause 37.

[0222]

[0240] Clause 40. The method of claim 37, wherein the population of immune cells is living T cells.

[0223]

[0241] Clause 41. The population of immune cells is natural killer (NK) cells, as per Clause 37.

[0224]

[0242] Clause 42. The method of Clause 40, further comprising activating a population of T cells with anti-CD3 and anti-CD28 T cell activating reagents before adding the chimeric cytokine complex.

[0225]

[0243] Clause 43. The cytokine is an interleukin, as per Clause 37.

[0226]

[0244] Clause 44. The method of Clause 43, wherein the interleukin is at least one of interleukin-2 (IL-2), IL-7, and IL-15.

[0227]

[0245] Clause 45. The method of Clause 37, wherein the multiple manipulated Fc antibody domains comprise 6 to 12 manipulated Fc antibody domains bound to a protein cage polypeptide.

[0228]

[0246] Clause 46. The method of Clause 45, wherein a total of 12 to 24 cytokines are linked to multiple manipulated Fc antibody domains.

[0229]

[0247] The method of Clause 47, wherein each of one or more cytokines is linked to one of the manipulated Fc antibody domains via a manipulated metalloproteinase resistance linker sequence.

[0230]

[0248] Clause 48. The manipulated metalloproteinase-resistant linker sequence is 7-13 amino acid residues long, as per the method of Clause 47.

[0231]

[0249] Clause 49. The method of Clause 37, wherein at least one of the manipulated Fc antibody domains is C-terminally ligated to at least one N-terminus of a cytokine.

[0232]

[0250] Clause 50. The method of Clause 49, wherein one of the cytokines is interleukin-2 (IL-2), and at least one of the engineered Fc antibody domains is linked to IL-2 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence SLSPGKAPTS (SEQ ID NO: 20).

[0233]

[0251] Clause 51. The method of Clause 49, wherein one of the cytokines is interleukin-7 (IL-7), and at least one of the engineered Fc antibody domains is linked to IL-7 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence SLSPGKDCDIEGK (SEQ ID NO: 21).

[0234]

[0252] Clause 52. The method of Clause 49, wherein one of the cytokines is interleukin-15 (IL-15), and at least one of the engineered Fc antibody domains is linked to IL-15 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence SLSPGKN (SEQ ID NO: 22).

[0235]

[0253] Clause 53. The method of Clause 37, wherein at least one of the manipulated Fc antibody domains is N-terminally ligated to at least one C-terminus of a cytokine.

[0236]

[0254] Clause 54. The method of Clause 53, wherein one of the cytokines is interleukin-2 (IL-2), and at least one of the engineered Fc antibody domains is linked to IL-2 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence TPKSCDKTHT (SEQ ID NO: 23).

[0237]

[0255] Clause 55. The method of Clause 53, wherein one of the cytokines is interleukin-7 (IL-7), and at least one of the engineered Fc antibody domains is linked to IL-7 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence HPKSCDKTHT (SEQ ID NO: 24).

[0238]

[0256] Clause 56. The method of Clause 53, wherein one of the cytokines is interleukin-15 (IL-15), and at least one of the engineered Fc antibody domains is linked to IL-15 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence TSPKSCDKTHT (SEQ ID NO: 25).

[0239]

[0257] Clause 57. The manipulated Fc antibody domain is a manipulated human Fc antibody domain, as per the method of Clause 37.

[0240]

[0258] Clause 58. The method of Clause 57, wherein the manipulated human Fc antibody domain is a manipulated human IgG1 Fc antibody domain.

[0241]

[0259] Clause 59. The method of Clause 58, wherein one of the manipulated human IgG1 Fc antibody domains comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% amino acid identity with respect to the amino acid sequence shown in SEQ ID NO: 1.

[0242]

[0260] Clause 60. The method of Clause 59, wherein the manipulated human IgG1 Fc antibody domain comprises at least one of the following point mutations in SEQ ID NO: P75L, R76W, Y80K, Y80P, Y80R, Y80G, and Y80A to reduce affinity for a specific Fcγ receptor and functionally reduce antibody-dependent cell-mediated cytotoxicity (ADCC).

[0243]

[0261] Clause 61. The method of Clause 59, wherein the manipulated human IgG1 Fc antibody domain includes the following point mutation: Y80W in SEQ ID NO: 1, such that it increases affinity for a specific Fcγ receptor and functionally enhances antibody-dependent cell-mediated cytotoxicity (ADCC).

[0244]

[0262] Clause 62. The method of Clause 59, wherein the manipulated human IgG1 Fc antibody domain comprises at least one of the following point mutations in Sequence ID No. 1: S23A, E53A, E77A, Y80F, V87A, A111G, K122A, and D160A, such that it reduces affinity for a specific Fcγ receptor and has a neutral effect on other Fcγ receptors.

[0245]

[0263] Clause 63. The method of Clause 59, wherein the manipulated human IgG1 Fc antibody domain includes at least one of the following point mutations in SEQ ID NO: E117, K118A, and A123T, such that it increases affinity for a specific Fcγ receptor and has a neutral effect on other Fcγ receptors.

[0246]

[0264] Clause 64. The method of Clause 59, wherein the manipulated human IgG1 Fc antibody domain comprises at least one of the following point mutations in SEQ ID NO: H52A, R85A, and K106A to increase affinity to a specific Fcγ receptor and decrease affinity to a specific other Fcγ receptor.

[0247]

[0265] Clause 65. The method of Clause 59, wherein the manipulated human IgG1 Fc antibody domain comprises at least one of the following point mutations in SEQ ID NO: D54A, Q79A, and A111S to reduce affinity for a particular Fcγ receptor.

[0248]

[0266] Clause 66. The method of Clause 59, wherein the manipulated human IgG1 Fc antibody domain includes at least one of the following point mutations in SEQ ID NO: T40A and K74A to increase affinity for a particular Fcγ receptor.

[0249]

[0267] The method of clause 37, wherein the Fc antibody domain is an engineered rabbit Fc antibody domain.

[0250]

[0268] The method of clause 37, further comprising a signal peptide linked to at least one of the at least one engineered Fc antibody domain or at least one of the cytokines at the N-terminus.

[0251]

[0269] The method of clause 68, wherein the signal peptide is a mouse Ig heavy signal peptide used for expression in Chinese hamster ovary (CHO) cells.

[0252]

[0270] The method of clause 69, wherein the signal peptide comprises the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 26).

[0253]

[0271] The method of clause 37, wherein the protein cage polypeptide is composed of a polypeptide comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% amino acid identity to the amino acid sequence shown in any one of SEQ ID NOs: 8 - 15.

[0254]

[0272] The method of clause 71, wherein the amino acid sequence constituting the polypeptide of the protein cage polypeptide comprises at least the following point mutation with respect to the amino acid sequence shown in any one of SEQ ID NOs: 8 - 15: Y294A.

[0255]

[0273] The method of clause 37, wherein the protein cage polypeptide is composed of a polypeptide comprising a binding site for one of the engineered Fc antibody domains, and the binding site comprises the amino acid sequence RWGSGADCAWHLGELVWCTAGSGWE (SEQ ID NO: 16).

[0256]

[0274] Clause 74. The protein cage polypeptide comprises a polypeptide having a binding site for one of the manipulated Fc antibody domains, the binding site having the amino acid sequence GGRWGADCAWHLGELVWCTAGWEGG (SEQ ID NO: 17), as per the method of Clause 37.

[0257]

[0275] Clause 75. The protein cage polypeptide comprises a polypeptide having a binding site for one of the manipulated Fc antibody domains, the binding site having the amino acid sequence GADCAWHLGELVWCTAG (SEQ ID NO: 18), as per the method of Clause 37.

[0258]

[0276] Clause 76. The protein cage polypeptide comprises a polypeptide having a binding site for one of the manipulated Fc antibody domains, the binding site having the amino acid sequence RWGSGCDCAWHLGELVWCTCGSGWE (SEQ ID NO: 19), as per the method of Clause 37.

[0259]

[0277] Clause 77. The protein cage polypeptide self-assembles into a tetrahedral pyramidal structure, as in the method of Clause 37.

[0260]

[0278] An Fc-cytokine complex comprising Clause 78; an engineered Fc antibody domain; and one or more cytokines ligated to the engineered Fc antibody domain.

[0261]

[0279] Clause 79. The cytokine is an interleukin, Fc-cytokine complex of Clause 78.

[0262]

[0280] Clause 80. The interleukin is at least one of interleukin-2 (IL-2), IL-7, and IL-15, as per Clause 79, the Fc-cytokine complex.

[0263]

[0281] Clause 81. An Fc-cytokine complex according to Clause 78, wherein each of one or more cytokines is linked to an engineered Fc antibody domain via an engineered metalloproteinase resistance linker sequence.

[0264]

[0282] Clause 82. The manipulated metalloproteinase-resistant linker sequence is the Fc-cytokine complex of Clause 81, having a length of 7-13 amino acid residues.

[0265]

[0283] Clause 83. An Fc-cytokine complex according to Clause 78, wherein the manipulated Fc antibody domain is C-terminally ligated to at least one N-terminus of the cytokine.

[0266]

[0284] Clause 84. The Fc-cytokine complex of Clause 83, wherein one of the cytokines is interleukin-2 (IL-2), and the engineered Fc antibody domain is linked to IL-2 via an engineered metalloproteinase-resistant linker sequence containing the amino acid sequence SLSPGKAPTS (SEQ ID NO: 20).

[0267]

[0285] Clause 85. The Fc-cytokine complex of Clause 83, wherein one of the cytokines is interleukin-7 (IL-7), and the engineered Fc antibody domain is linked to IL-7 via an engineered metalloproteinase resistance linker sequence containing the amino acid sequence SLSPGKDCDIEGK (SEQ ID NO: 21).

[0268]

[0286] Clause 86. The Fc-cytokine complex of Clause 83, wherein one of the cytokines is interleukin-15 (IL-15), and the engineered Fc antibody domain is linked to IL-15 via an engineered metalloproteinase-resistant linker sequence containing the amino acid sequence SLSPGKN (SEQ ID NO: 22).

[0269]

[0287] Clause 87. An Fc-cytokine complex according to Clause 78, wherein the manipulated Fc antibody domain is N-terminally ligated to at least one C-terminus of the cytokine.

[0270]

[0288] Clause 88. The Fc-cytokine complex of Clause 87, wherein one of the cytokines is interleukin 2 (IL-2), and the engineered Fc antibody domain is linked to IL-2 via an engineered metalloprotease-resistant linker sequence comprising the amino acid sequence TPKSCDKTHT (SEQ ID NO: 23).

[0271]

[0289] Clause 89. The Fc-cytokine complex of Clause 87, wherein one of the cytokines is interleukin 7 (IL-7), and the engineered Fc antibody domain is linked to IL-7 via an engineered metalloprotease-resistant linker sequence comprising the amino acid sequence HPKSCDKTHT (SEQ ID NO: 24).

[0272]

[0290] Clause 90. The Fc-cytokine complex of Clause 87, wherein one of the cytokines is interleukin-15 (IL-15), and the engineered Fc antibody domain is linked to IL-15 via an engineered metalloprotease-resistant linker sequence comprising the amino acid sequence TSPKSCDKTHT (SEQ ID NO: 25).

[0273]

[0291] Clause 91. The Fc-cytokine complex of Clause 78, wherein the engineered Fc antibody domain is an engineered human Fc antibody domain.

[0274]

[0292] Clause 92. The Fc-cytokine complex of Clause 91, wherein the engineered human Fc antibody domain is an engineered human IgG1 Fc antibody domain.

[0275]

[0293] Clause 93. The Fc-cytokine complex of Clause 92, wherein the engineered human IgG1 Fc antibody domain comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% amino acid identity to the amino acid sequence shown in SEQ ID NO: 1.

[0276]

[0294] Clause 94. The Fc-cytokine complex of Clause 93, wherein the manipulated human IgG1 Fc antibody domain includes at least one of the following point mutations in SEQ ID NO: P75L, R76W, Y80K, Y80P, Y80R, Y80G, and Y80A in relation to SEQ ID NO: 1, to reduce affinity for a specific Fcγ receptor and functionally reduce antibody-dependent cell-mediated cytotoxicity (ADCC).

[0277]

[0295] Clause 95. The Fc-cytokine complex of Clause 93, comprising the following point mutation:Y80W in SEQ ID NO: 1, wherein the manipulated human IgG1 Fc antibody domain increases affinity for a specific Fcγ receptor and functionally enhances antibody-dependent cell-mediated cytotoxicity (ADCC).

[0278]

[0296] Clause 96. The Fc-cytokine complex of Clause 93, wherein the manipulated human IgG1 Fc antibody domain includes at least one of the following point mutations in SEQ ID NO: S23A, E53A, E77A, Y80F, V87A, A111G, K122A, and D160A in relation to SEQ ID NO: 1, such that it reduces affinity for a specific Fcγ receptor and has a neutral effect on other Fcγ receptors.

[0279]

[0297] Clause 97. The Fc-cytokine complex of Clause 93, wherein the manipulated human IgG1 Fc antibody domain includes at least one of the following point mutations in SEQ ID NO: E117, K118A, and A123T, such that it increases affinity for a specific Fcγ receptor and has a neutral effect on other Fcγ receptors.

[0280]

[0298] Clause 98. The Fc-cytokine complex of Clause 93, wherein the manipulated human IgG1 Fc antibody domain includes at least one of the following point mutations in SEQ ID NO: H52A, R85A, and K106A to increase affinity to a specific Fcγ receptor and decrease affinity to a specific other Fcγ receptor.

[0281]

[0299] Clause 99. The Fc-cytokine complex of Clause 93, wherein the manipulated human IgG1 Fc antibody domain includes at least one of the following point mutations in SEQ ID NO: D54A, Q79A, and A111S to reduce affinity for a specific Fcγ receptor.

[0282]

[0300] Clause 100. The Fc-cytokine complex of Clause 93, wherein the manipulated human IgG1 Fc antibody domain includes at least one of the following point mutations in SEQ ID NO: T40A and K74A to increase affinity for a specific Fcγ receptor.

[0283]

[0301] Clause 101. The Fc antibody domain is an engineered rabbit Fc antibody domain, as per the Fc-cytokine complex of Clause 78.

[0284]

[0302] Clause 102. The Fc-cytokine complex of Clause 78, further comprising an engineered Fc antibody domain or a signal peptide ligated to at least one N-terminus of a cytokine.

[0285]

[0303] Clause 103. The signal peptide is the mouse Ig heavy signal peptide used for expression in Chinese hamster ovary (CHO) cells, as per the Fc-cytokine complex of Clause 102.

[0286]

[0304] Clause 104. The signal peptide is the Fc-cytokine complex of Clause 103, comprising the amino acid sequence MGWSCIILFLVATATATGVHS (SEQ ID NO: 26).

[0287]

[0305] Clause 105. A method for activating and proliferating immune cells, comprising adding an Fc-cytokine complex to a population of immune cells, wherein the Fc-cytokine complex comprises: an Fc antibody domain and one or more cytokines ligated to the Fc antibody domain.

[0288]

[0306] Clause 106. The method of Clause 105, wherein a population of immune cells is activated and proliferated in vitro.

[0289]

[0307] Clause 107. The population of immune cells is the peripheral blood immune cells of a human donor, as per the method of Clause 105.

[0290]

[0308] Clause 108. The population of immune cells is living T cells, according to the method of Clause 105.

[0291]

[0309] Clause 109. The population of immune cells is natural killer (NK) cells, as per Clause 105.

[0292]

[0310] The method of Clause 105, further comprising activating a population of T cells with anti-CD3 and anti-CD28 T cell activating reagents before adding the Fc-cytokine complex.

[0293]

[0311] Clause 111. The cytokine is an interleukin, as per Clause 105.

[0294]

[0312] Clause 112. The method of Clause 111, wherein the interleukin is at least one of interleukin-2 (IL-2), IL-7, and IL-15.

[0295]

[0313] The method of Clause 113. The method of Clause 105, wherein each of one or more cytokines is linked to an engineered Fc antibody domain via an engineered metalloproteinase resistance linker sequence.

[0296]

[0314] Clause 114. The manipulated metalloproteinase-resistant linker sequence is 7-13 amino acid residues long, as described in Clause 113.

[0297]

[0315] Clause 115. The method of Clause 105, wherein the manipulated Fc antibody domain is C-terminally ligated to at least one N-terminus of a cytokine.

[0298]

[0316] Clause 116. The method of Clause 115, wherein one of the cytokines is interleukin-2 (IL-2), and the engineered Fc antibody domain is linked to IL-2 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence SLSPGKAPTS (SEQ ID NO: 20).

[0299]

[0317] Clause 117. The method of Clause 115, wherein one of the cytokines is interleukin-7 (IL-7), and the engineered Fc antibody domain is linked to IL-7 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence SLSPGKDCDIEGK (SEQ ID NO: 21).

[0300]

[0318] Clause 118. The method of Clause 115, wherein one of the cytokines is interleukin-15 (IL-15), and the engineered Fc antibody domain is linked to IL-15 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence SLSPGKN (SEQ ID NO: 22).

[0301]

[0319] Clause 119. The method of Clause 105, wherein the manipulated Fc antibody domain is N-terminally ligated to at least one C-terminus of a cytokine.

[0302]

[0320] Clause 120. The method of Clause 119, wherein one of the cytokines is interleukin-2 (IL-2), and the engineered Fc antibody domain is linked to IL-2 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence TPKSCDKTHT (SEQ ID NO: 23).

[0303]

[0321] Clause 121. The method of Clause 119, wherein one of the cytokines is interleukin-7 (IL-7), and the engineered Fc antibody domain is linked to IL-7 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence HPKSCDKTHT (SEQ ID NO: 24).

[0304]

[0322] Clause 122. The method of Clause 119, wherein one of the cytokines is interleukin-15 (IL-15), and the engineered Fc antibody domain is linked to IL-15 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence TSPKSCDKTHT (SEQ ID NO: 25).

[0305]

[0323] Clause 123. The manipulated Fc antibody domain is a manipulated human Fc antibody domain, as per the method of Clause 105.

[0306]

[0324] Clause 124. The manipulated human Fc antibody domain is the manipulated human IgG1 Fc antibody domain, as per the method of Clause 123.

[0307]

[0325] Clause 125. The method of Clause 124, wherein the manipulated human IgG1 Fc antibody domain comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% amino acid identity with respect to the amino acid sequence shown in SEQ ID NO: 1.

[0308]

[0326] Clause 126. The method of Clause 125, wherein the manipulated human IgG1 Fc antibody domain comprises at least one of the following point mutations in SEQ ID NO: P75L, R76W, Y80K, Y80P, Y80R, Y80G, and Y80A to reduce affinity for a specific Fcγ receptor and functionally reduce antibody-dependent cell-mediated cytotoxicity (ADCC).

[0309]

[0327] Clause 127. The method of Clause 125, wherein the manipulated human IgG1 Fc antibody domain includes the following point mutation: Y80W in SEQ ID NO: 1, such that it increases affinity for a specific Fcγ receptor and functionally enhances antibody-dependent cell-mediated cytotoxicity (ADCC).

[0310]

[0328] Clause 128. The method of Clause 125, wherein the manipulated human IgG1 Fc antibody domain comprises at least one of the following point mutations in Sequence ID No. 1: S23A, E53A, E77A, Y80F, V87A, A111G, K122A, and D160A, such that it reduces affinity for a specific Fcγ receptor and has a neutral effect on other Fcγ receptors.

[0311]

[0329] Clause 129. The method of Clause 125, wherein the manipulated human IgG1 Fc antibody domain includes at least one of the following point mutations in SEQ ID NO: E117, K118A, and A123T, such that it increases affinity for a specific Fcγ receptor and has a neutral effect on other Fcγ receptors.

[0312]

[0330] Clause 130. The method of Clause 125, wherein the manipulated human IgG1 Fc antibody domain includes at least one of the following point mutations in SEQ ID NO: H52A, R85A, and K106A to increase affinity to a specific Fcγ receptor and decrease affinity to a specific other Fcγ receptor.

[0313]

[0331] Clause 131. The method of Clause 125, wherein the manipulated human IgG1 Fc antibody domain comprises at least one of the following point mutations in SEQ ID NO: D54A, Q79A, and A111S to reduce affinity for a particular Fcγ receptor.

[0314]

[0332] Clause 132. The method of Clause 125, wherein the manipulated human IgG1 Fc antibody domain includes at least one of the following point mutations in SEQ ID NO: T40A and K74A to increase affinity for a particular Fcγ receptor.

[0315]

[0333] Clause 133. The Fc antibody domain is an engineered rabbit Fc antibody domain, as per the method of Clause 105.

[0316]

[0334] Clause 134. The method of Clause 105, further comprising a manipulated Fc antibody domain or a signal peptide ligated to at least one N-terminus of a cytokine.

[0317]

[0335] Clause 135. The signal peptide is a mouse Ig heavy signal peptide used for expression in Chinese hamster ovary (CHO) cells, as described in Clause 134.

[0318]

[0336] Clause 136. The signal peptide comprises the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 26) as described in Clause 135.

[0319]

[0337] Several embodiments have been described. Nevertheless, those skilled in the art will understand that various changes and modifications can be made to this disclosure without departing from the spirit and scope of the embodiments. Elements of systems, devices, apparatus, and methods shown in any embodiment are illustrative for a particular embodiment and can be used in combination with other embodiments in this disclosure or in other ways in other embodiments in this disclosure. For example, the steps of any method shown in the drawings or described in this disclosure do not require a particular order or sequence shown or described in order to achieve the desired result. In addition, other step operations may be provided to achieve the desired result, and steps or operations may be excluded or omitted from the described method or process. Furthermore, any component or part of any apparatus or system described in this disclosure or shown in the drawings may be removed, excluded or omitted to achieve the desired result. In addition, certain components or parts of systems, devices, or apparatus shown or described herein have been omitted for the sake of brevity and clarity.

[0320]

[0338] Therefore, other embodiments are within the scope of the following claims, and this specification and / or drawings may be considered illustrative rather than restrictive.

[0321]

[0339] Each of the individual variations or embodiments described and illustrated herein has distinct components and features that can be readily separated from or combined with any of the features of other variations or embodiments. Specific situations, materials, substance compositions, processes, process actions, or steps can be modified to suit the purpose, spirit, or scope of the invention.

[0322]

[0340] The methods enumerated herein can be carried out in any logically possible order of the enumerated events, and in the order in which the events are enumerated. Furthermore, additional steps or operations may be provided or omitted to achieve the desired result.

[0323]

[0341] Furthermore, where a range of values ​​is provided, all intervening values ​​between the upper and lower limits of that range, and all other values ​​described or intervening within that range, are included within the present invention. In addition, any optional feature of the described inventive variation may be described and claimed independently or in combination with one or more of the features described herein. For example, a description of the range 1 to 5 should be considered to disclose partial ranges such as 1 to 3, 1 to 4, 2 to 4, 2 to 5, 3 to 5, and individual values ​​within those ranges, such as 1.5, 2.5, etc., as well as any whole or partial increments between them.

[0324]

[0342] All existing subject matter referenced herein (e.g., publications, patents, patent applications, and academic papers) is incorporated herein by reference in its entirety, except where such subject matter may conflict with that of the present invention (in which case the material present herein shall prevail). The referenced material is provided only for disclosure prior to the filing date of this application. Nothing herein should be construed as an acknowledgment that the present invention has no prior rights to such material by prior invention.

[0325]

[0343] References to singular items include the possibility of multiple identical items existing. More specifically, the singular forms “a,” “an,” “the,” and “the” used herein and in the appended claims include multiple referents unless the context clearly indicates otherwise. It should also be noted that the claims may be drafted to exclude any optional elements. Thus, this statement is intended to function as an antecedent for using exclusive terms such as “solely,” “only,” or for using “negative” limitations in relation to the enumeration of elements of the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art to which the invention pertains.

[0326]

[0344] A reference to the phrase "at least one of" means any combination of one or more items or components (or an enumerated list of items or components) when such a phrase modifies multiple items or components. For example, the phrase "at least one of A, B, and C" means: (i) A; (ii) B; (iii) C; (iv) A, B, and C; (v) A and B; (vi) B and C; or (vii) A and C.

[0327]

[0345] In understanding the scope of this disclosure, the terms “comprising” and its derivatives as used herein are intended to be open-ended terms that specifically indicate the existence of specified features, elements, components, groups, integers, and / or steps, but not exclude the existence of other unspecified features, elements, components, groups, integers, and / or steps. The same applies to similar terms such as “including,” “having,” and their derivatives. Furthermore, the terms “part,” “section,” “part,” “member,” “element,” or “component” may have a dual meaning of one part or multiple parts when used in the singular. The following directional terms used herein—“forward,” “backward,” “upward,” “downward,” “vertical,” “horizontal,” “downward,” “lateral,” and “vertical”—and any other similar directional terms refer to the location of a part of a device or apparatus, or the direction of a part of a device or apparatus being translated or moved.

[0328]

[0346] Finally, as used herein, terms of degree such as “substantially,” “about,” and “approximately” mean a specific value, or a reasonable amount of deviation from a specific value (e.g., a maximum deviation of ±0.1%, ±1%, ±5%, or ±10%, such that the final result does not change significantly or greatly). For example, “about 1.0 cm” can be interpreted as “1.0 cm” or “0.9 cm to 1.1 cm.” When terms of degree such as “about” or “approximately” are used to refer to a number or value that is part of a range, the term may be used to modify both the minimum and maximum number or value.

[0329]

[0347] Structures in the figures may be shown as separate and communicating only with a few specific structures, and not with other structures. Structures may be merged with each other, perform overlapping functions, and communicate with other structures not shown as connected in the figures. Accordingly, this specification and / or drawings may be considered illustrative rather than restrictive.

[0330]

[0348] All cited references are incorporated herein by reference in their entirety.

[0331]

[0349] This disclosure is not intended to be limited to any specific form expressed herein, but rather to encompass substitutes, modifications, and equivalents of the variations or embodiments described herein. Furthermore, the scope of this disclosure fully encompasses other variations or embodiments that may become apparent to those skilled in the art in consideration of this disclosure.

[0332] References [1] Mayes, PA, Hance, KW & Hoos, A. The promise and challenges of immune agonist antibody development in cancer. Nat. Rev. Drug Discov. 17, 509-527 (2018). [2] Spangler, JB, Moraga, I., Mendoza, JL & Garcia, KC Insights into cytokine-receptor interactions from cytokine engineering. Annu. Rev. Immunol. 33, 139-167 (2015). [3] Brooks, AJ et al. Mechanism of activation of protein kinase JAK2 by the growth hormone receptor. Science 344, 1249783 (2014). [4] O'Shea, JJ & Plenge, R. JAK and STAT signaling molecules in immunoregulation and immune-mediated disease. Immunity 36, 542-550 (2012). [5] Liao, W., Lin, J.X. & Leonard, W.J. Interleukin-2 at the crossroads of effector responses, tolerance, and immunotherapy. Immunity 38, 13-25 (2013). [6] Levin, A.M. et al. Exploiting a natural conformational switch to engineer an interleukin-2’superkine’. Nature 484, 529-533 (2012). [7] Powell, M.S. & Hogarth, P.M. Fc receptors. Adv. Exp. Med. Biol. 640, 22-34 (2008). [8] de Taeye, S.W. et al. FcgammaR Binding and ADCC Activity of Human IgG Allotypes. Front. Immunol. 11, 740 (2020). [9] Shields, R.L. et al. High resolution mapping of the binding site on human IgG1 for Fc gamma RI, Fc gamma RII, Fc gamma RIII, and FcRn and design of IgG1 variants with improved binding to the Fc gamma R. J. Biol. Chem. 276, 6591-6604 (2001).

[10] Isoda, Y. et al. Importance of the Side Chain at Position 296 of Antibody Fc in Interactions with FcgammaRIIIa and Other Fcgamma Receptors. PLOS One 10, e0140120 (2015).

[11] Edsparr, K., Basse, P.H., Goldfarb, R.H. & Albertsson, P. Matrix metalloproteinases in cytotoxic lymphocytes impact on tumour infiltration and immunomodulation. Cancer Microenviron 4, 351-360 (2011).

[12] Benson, H.L. et al. Endogenous matrix metalloproteinases 2 and 9 regulate activation of CD4+ and CD8+ T cells. Am J Respir Cell Mol Biol 44, 700-708 (2011).

[13] Johnatty, R.N. et al. Cytokine and chemokine regulation of proMMP-9 and TIMP-1 production by human peripheral blood lymphocytes. J. Immunol. 158, 2327-2333 (1997).

[14] de Almeida, L.G.N. et al. Matrix Metalloproteinases:From Molecular Mechanisms to Physiology, Pathophysiology, and Pharmacology. Pharmacol. Rev. 74, 712-768 (2022).

[15] Song, J. et al. PROSPER:an integrated feature-based tool for predicting protease substrate cleavage sites. PLOS One 7, e50300 (2012).

[16] Liu, X. et al. Human immunoglobulin G hinge regulates agonistic anti-CD40 immunostimulatory and antitumour activities through biophysical flexibility. Nature Communications 10, 4206 (2019).

Claims

1. Protein cage polypeptide and; Multiple manipulated Fc antibody domains bound to the protein cage polypeptide; One or more cytokines linked to each of the multiple manipulated Fc antibody domains and A chimeric cytokine complex containing these cytokines.

2. The chimeric cytokine complex according to claim 1, wherein the cytokine is an interleukin.

3. The chimeric cytokine complex according to claim 2, wherein the interleukin is at least one of interleukin-2 (IL-2), IL-7, and IL-15.

4. The chimeric cytokine complex according to claim 1, wherein the plurality of manipulated Fc antibody domains comprises 6 to 12 manipulated Fc antibody domains bound to the protein cage polypeptide.

5. The chimeric cytokine complex according to claim 4, wherein a total of 12 to 24 cytokines are linked to the plurality of manipulated Fc antibody domains.

6. The chimeric cytokine complex according to claim 1, wherein each of the one or more cytokines is linked to one of the manipulated Fc antibody domains via a manipulated metalloproteinase resistance linker sequence.

7. The chimeric cytokine complex according to claim 6, wherein the manipulated metalloproteinase-resistant linker sequence has a length of 7 to 13 amino acid residues.

8. The chimeric cytokine complex according to claim 1, wherein at least one of the manipulated Fc antibody domains is C-terminally ligated to the N-terminus of at least one of the cytokines.

9. The chimeric cytokine complex according to claim 8, wherein one of the cytokines is interleukin-2 (IL-2), and at least one of the manipulated Fc antibody domains is linked to IL-2 via a manipulated metalloproteinase resistance linker sequence comprising the amino acid sequence SLSPPGKAPTS (SEQ ID NO: 20).

10. The chimeric cytokine complex according to claim 8, wherein one of the cytokines is interleukin-7 (IL-7), and at least one of the manipulated Fc antibody domains is linked to IL-7 via a manipulated metalloproteinase resistance linker sequence comprising the amino acid sequence SLSPGKDCDIEGK (SEQ ID NO: 21).

11. The chimeric cytokine complex according to claim 8, wherein one of the cytokines is interleukin-15 (IL-15), and at least one of the manipulated Fc antibody domains is linked to IL-15 via a manipulated metalloproteinase resistance linker sequence comprising the amino acid sequence SLSPGKN (SEQ ID NO: 22).

12. The chimeric cytokine complex according to claim 1, wherein at least one of the manipulated Fc antibody domains is N-terminally ligated to the C-terminus of at least one of the cytokines.

13. The chimeric cytokine complex according to claim 12, wherein one of the cytokines is interleukin-2 (IL-2), and at least one of the manipulated Fc antibody domains is linked to IL-2 via a manipulated metalloproteinase-resistant linker sequence comprising the amino acid sequence TPKSCDKTHT (SEQ ID NO: 23).

14. The chimeric cytokine complex according to claim 12, wherein one of the cytokines is interleukin-7 (IL-7), and at least one of the manipulated Fc antibody domains is linked to IL-7 via a manipulated metalloproteinase resistance linker sequence comprising the amino acid sequence HPKSCDKTHT (SEQ ID NO: 24).

15. The chimeric cytokine complex according to claim 12, wherein one of the cytokines is interleukin-15 (IL-15), and at least one of the manipulated Fc antibody domains is linked to IL-15 via a manipulated metalloproteinase resistance linker sequence comprising the amino acid sequence TSPKSCDKTHT (SEQ ID NO: 25).

16. The chimeric cytokine complex according to claim 1, wherein the manipulated Fc antibody domain is a manipulated human Fc antibody domain.

17. The chimeric cytokine complex according to claim 16, wherein the manipulated human Fc antibody domain is a manipulated human IgG1 Fc antibody domain.

18. A method for activating and proliferating immune cells, The process involves adding a chimeric cytokine complex to a population of immune cells, wherein the chimeric cytokine complex is: Protein cage polypeptide and; Multiple manipulated Fc antibody domains bound to the protein cage polypeptide; One or more cytokines linked to each of the plurality of Fc antibody domains and A method that includes this.

19. With the manipulated Fc antibody domain; One or more cytokines linked to the manipulated Fc antibody domain and An Fc-cytokine complex containing this compound.

20. A method for activating and proliferating immune cells, The process involves adding an Fc-cytokine complex to a population of immune cells, wherein the Fc-cytokine complex is: Fc antibody domain and; One or more cytokines linked to the Fc antibody domain and A method that includes this.

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