Receptor-biased pegylated il-2 variants combinations and uses thereof

Abstract

The present invention relates to the field of immunotherapy, in particular, the present invention relates to site-directed modified PEGylated IL-2 variants to provide a combination of PEGylated IL-2 variants with different receptor bias. The combination inhibits the expansion of immunosuppressive lymphocytes and reduces the terminal differentiation and exhaustion of effector lymphocytes by synergistic effects, has synergistic anti-tumor effects, and can be used for enhancing immune response, treating proliferative diseases such as tumors. In addition, the combination can also be used for in vitro culture of immune cells in adoptive cell immunotherapy, reducing the aging and exhaustion of immune cells, so that the fate of immune cells in adoptive cell immunotherapy is directed to long-term immunity. Therefore, the present invention also provides a combination therapy for enhancing immune response, treating proliferative diseases such as tumors, and an in vitro method for preparing or culturing immune cells for adoptive cell therapy.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunotherapy, in particular, the present application relates to site-directed modified PEGylated IL-2 variants to provide a combination of PEGylated IL-2 variants with different receptor bias. The present application also provides combination therapies for enhancing immune responses, treating proliferative diseases such as tumors, and in vitro methods for preparing or culturing immune cells for adoptive cell therapy. BACKGROUND

[0002] Interleukin-2 (IL2) is an important class of cytokines, and is a cytokine necessary for the development and function of B cells, T cells and NK cells. The receptor of IL2 is divided into IL2-Ra monomer, IL2-Rb y dimer and IL2-Ra b y trimer, in which the affinity of IL2 to a monomer is weak, 10 -8 M, the affinity to b y dimer is moderate, 10 -9 M, and the affinity to a b y trimer is the highest, 10 -11 M. Due to the low affinity, the dimer IL-2R or endogenous IL-2 needs to be induced at a high level to produce a "dimer IL-2R:IL-2" reactivity, which is contrary to the trimer IL-2R, which allows the host cell to directly react to low concentration of IL-2. Therefore, it is currently believed that the a chain of the trimer IL-2R does not participate in signal transduction, but initiates the binding to IL-2 (KD≈10-8M), and then the binding to the b y complex with an increased affinity by 1000 times.

[0003] The distribution of the receptor of IL2 on different immune cells also has differences: the IL2-Ra b y trimer is long-term expressed on the surface of regulatory T cells (Treg), and therefore has the highest affinity to IL2; the IL2-Rb y dimer is expressed on the surface of resting effector T cells (Teff), killer T cells (CTL) and NK cells, and has general affinity to IL2, only after the activation of effector T cells and NK cells, the IL2-Ra b y trimer is expressed, and the affinity to IL2 is enhanced. There is evidence that during the initiation of CD8+T cells, the expression of a chain is quite dynamic - a part of T cells up-regulate the expression of a chain and perceive strong IL-2 signal, resulting in faster T cell proliferation but eventually differentiation, which in turn limits their effectiveness. In contrast, low-a T cells that are less sensitive to IL-2 preferentially up-regulate CD127 and CD62L, thereby generating functional long-lived memory cells. Without changing the basic process of a chain up-regulation, it becomes valuable to have more memory T cell populations in vivo, but it is challenging for clinical applications. Therefore, in tumor treatment, the regulation of the imbalance ratio of Teff / Treg, CTL / Treg has gradually become a new research and development direction of immunotherapy.

[0004] Currently marketed IL2-targeted drugs are all non-receptor selective. In the treatment of tumors, high doses of IL2 are needed to activate Teff and CTL, which also brings serious systemic toxicity, which is the main source of side effects. In addition, the interaction of IL2 and IL2-Ra in lung endothelial cells also induces vascular leakage syndrome, and the immune suppression caused by the activation of Treg cells also limits the drug response.

[0005] Therefore, solving the problem of the lack of selectivity of existing IL2 anti-tumor drugs for receptors is crucial for the clinical application of IL2-based tumor immunotherapy. SUMMARY

[0006] As described above, although IL-2 has been widely used to enhance immune cells in the body, the accompanying properties of promoting the expansion of immunosuppressive lymphocytes and driving the terminal differentiation and exhaustion of effector lymphocytes hinder the effectiveness of IL-2 as an anti-tumor agent.

[0007] The inventors report here a strategy that explores the dynamic expression pattern of IL-2 receptors by two classes of receptor-biased PEGylated IL-2 variants to enable the priming of lymphocytes with persistent anti-tumor immune properties. Specifically, the first class of receptor-biased PEGylated IL-2 variants, non-aPEGylate, can activate and expand CD8+ T cells, CD4+ cells, and even NK cells, but not Treg cells, due to its biased affinity for dimeric, but not trimeric, IL-2R, and in addition, it induces reduced terminal differentiation and exhaustion of CD8+ cells. The second class of receptor-biased PEGylated IL-2 variants, non-βPEGylate, neither activates CD8+ T nor Treg cells, but it synergizes with the first class of non-aPEGylate to further expand CD4+ and CD8+ T cells and reduce Treg and differentiated and exhausted CD8+ T cell populations, due to its moderate affinity for the a chain of the trimeric IL-2R, exhibiting superior local and systemic anti-tumor responses.

[0008] The inventors subsequently demonstrated in a model of CAR-T therapy targeting CD19 that the first class of receptor-biased PEGylated IL-2 variants (non- aPEGylate) and the second class of receptor-biased PEGylated IL-2 variants (non- bPEGylate) synergistically direct the fate of lymphocytes towards long-term immunity. The experimental evidence supports a putative mechanism by which the synergistic receptor-biased PEGylated IL-2 variants induce a sequential shift in the IL-2:IL-2R interaction that not only circumvents the pleiotropic effects of IL-2 but also optimizes the differential memory program of T cells, pointing the way for next-generation IL-2 in T lymphocytes and adoptive T cell transfer therapies.

[0009] Based on the first class of receptor-biased PEGylated IL-2 variants (non- aPEGylate) and the second class of receptor-biased PEGylated IL-2 variants (non- bPEGylate), the present application provides a combination of PEGylated IL-2 variants with different receptor bias. The combination synergistically inhibits the expansion of immunosuppressive lymphocytes and reduces the terminal differentiation and exhaustion of effector lymphocytes, increases the proportion and number of central memory cells, has a synergistic anti-tumor effect, and thus can be used to enhance immune response, treat proliferative diseases such as tumors. In addition, the combination can also be used for in vitro culture of immune cells in adoptive cell immunotherapy, reduce the aging and exhaustion of immune cells, enhance the proliferation of T cells, reduce the proliferation of Treg cells, maintain the proliferation of Tscm and T effector cells, reduce the degree of exhaustion of T effector cells, and avoid the excessive activation of T cells by endogenous IL-2, so that the fate of immune cells in adoptive cell immunotherapy is directed towards long-term immunity. Therefore, the present application also provides a combination therapy for enhancing immune response, treating proliferative diseases such as tumors, and an in vitro method for preparing or culturing immune cells for adoptive cell therapy.

[0010] Compositions

[0011] In a first aspect, the present application provides a composition comprising:

[0012] (1) a first site-directed modified IL-2 comprising a PEG group modification at a residue of a first amino acid position compared to wild-type IL-2, wherein the first site- directed modified IL-2 does not bind to or binds to the IL-2 receptor a (IL-2Ra) subunit with a KD value greater than 1E-8 M (e.g., 1E-7 to 1E-6 M in magnitude); and

[0013] (2) a second site-directed modified IL-2 comprising a PEG group modification at a residue in a second amino acid position as compared to wild-type IL-2, wherein the second site- directed modified IL-2 does not bind to the IL-2 receptor beta (IL-2Rβ) subunit.

[0014] It is understood by one skilled in the art that a composition comprising a first site- directed modified IL-2 and a second site-directed modified IL-2 does not mean that both must be administered simultaneously and / or formulated for delivery together, although these methods of delivery are within the scope described herein. In some embodiments, the first site-directed modified IL-2 and the second site-directed modified IL-2 can be administered together in a single formulation. In some embodiments, the first site-directed modified IL-2 and the second site-directed modified IL-2 can be administered separately in different formulations. The first site-directed modified IL-2 in the combination can be administered with the second site-directed modified IL-2 in any order, e.g., simultaneously, before, or after.

[0015] In certain embodiments, the composition comprises multiple compositions or dosage forms. In certain embodiments, the first site-directed modified IL-2 and the second site- directed modified IL-2 are in separate compositions or dosage forms.

[0016] In certain embodiments, the composition comprises one composition or dosage form. In certain embodiments, the first site-directed modified IL-2 and the second site- directed modified IL-2 are in the same composition or dosage form.

[0017] In certain embodiments, the wild-type IL-2 has the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the IL-2 related amino acid positions described herein are positions in SEQ ID NO: 1.

[0018] In certain embodiments, “does not bind” described herein means that no binding is detected by surface plasmon resonance technology (SPR). In certain embodiments, the KD values described herein are determined by surface plasmon resonance technology (SPR).

[0019] Receptor bias of first site-directed modified IL-2

[0020] The first site-directed modified IL-2 described herein has a bias towards non-α receptors.

[0021] In certain embodiments, the bias towards non-α includes: (i) binding to IL2-Rβγ dimer with a KD value of less than 1E-8 M (e.g., in the order of 1E-9 to 1E-8 M), and / or, (ii) no binding or binding to IL2-Rαβγ trimer with a KD value of greater than 1E-8 (e.g., in the order of 1E-7 to 1E-6).

[0022] I. Binding properties to IL-2Rα

[0023] In certain embodiments, the first site-directed modified IL-2 does not bind to the IL-2 receptor alpha (IL-2Rα) subunit. In certain embodiments, the first amino acid position of such first site-directed modified IL-2 is selected from F42, Y45, E62, K64, P65, E68.

[0024] In certain embodiments, the first site-directed modified IL-2 only has low affinity for the IL-2 receptor alpha (IL-2Rα) subunit, e.g., binds to the IL-2 receptor alpha (IL-2Rα) subunit with a KD value of greater than 1E-8 M (e.g., greater than 9E-7 M, 8E-7 M, 7E-7 M, 6E-7 M, 5E-7 M, 4E-7 M, 3E-7 M, 2E-7 M, 1E-7 M, 9E-6 M, 8E-6 M, 7E-6 M, 6E-6 M, 5E-6 M, 4E-6 M, 3E-6 M, 2E-6 M, 1E-6 M, or greater). In certain embodiments, the first site-directed modified IL-2 binds to the IL-2 receptor alpha (IL-2Rα) subunit with a KD value in the order of 1E-7 to 1E-6 M. In certain embodiments, the first amino acid position of such first site-directed modified IL-2 is selected from K35, T37, R38, T41, K48, K49.

[0025] II. Binding properties to IL2-Rβγ dimer

[0026] In certain embodiments, the first site-directed modified IL-2 binds to IL2-Rβγ dimer with a KD value of less than 1E-8 M (e.g., less than 2E-8 M, 3E-8 M, 4E-8 M, 5E-8 M, 6E-8 M, 7E-8 M, 8E-8 M, 9E-8 M, 1E-9 M, or less). In certain embodiments, the first site-directed modified IL-2 binds to IL2-Rβγ dimer with a KD value in the order of 1E-9 to 1E-8 M. In certain embodiments, the first amino acid position of such first site-directed modified IL-2 is selected from F42, Y45, E62, K64, P65, E68, K35, T37, R38, T41, K48, K49.

[0027] III. Binding properties to IL2-Rαβγ trimer

[0028] In certain embodiments, the first site-directed modified IL-2 does not bind to the IL2-Raβγ trimer. In certain embodiments, the first amino acid position of such first site- directed modified IL-2 is selected from F42, Y45, E62, K64, P65, E68.

[0029] In certain embodiments, the first site-directed modified IL-2 has only low affinity for the IL2-Raβγ trimer, e.g., binds the IL2-Raβγ trimer with a KD value greater than 1E-8 M (e.g., greater than 9E-7 M, 8E-7 M, 7E-7 M, 6E-7 M, 5E-7 M, 4E-7 M, 3E-7 M, 2E-7 M, 1E-7 M, 9E-6 M, 8E-6 M, 7E-6 M, 6E-6 M, 5E-6 M, 4E-6 M, 3E-6 M, 2E-6 M, 1E-6 M, or greater). In certain embodiments, the first site-directed modified IL-2 binds the IL2-Raβγ trimer with a KD value in the order of 1E-7 to 1E-6 M. In certain embodiments, the first amino acid position of such first site-directed modified IL-2 is selected from K35, T37, R38, T41, K48, K49.

[0030] In certain embodiments, when the first site-directed modified IL-2 has low affinity for the IL2-Raβγ trimer, the first site-directed modified IL-2 has a first affinity for the IL2-Rβγ dimer that is higher than a second affinity for the IL2-Raβγ trimer (i.e., a first KD value for binding the IL2-Rβγ dimer is lower than a second KD value for binding the IL2-Raβγ trimer). In certain embodiments, the first affinity of the first site-directed modified IL-2 for the IL2-Rβγ dimer is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold greater than the second affinity for the IL2-Raβγ trimer. In certain embodiments, the first amino acid position of such first site-directed modified IL-2 is selected from K35, T37, T41, K48.

[0031] In certain embodiments, when the first site-directed modified IL-2 has a low affinity for the IL2-RaPγ trimer, the first site-directed modified IL-2 can also have a first affinity for the IL2-RPγ dimer that is not higher (e.g., lower) than a second affinity for the IL2-RaPγ trimer (i.e., the first KD value for binding the IL2-RPγ dimer is not lower (e.g., higher) than the second KD value for binding the IL2-RaPγ trimer), but the difference in affinity (e.g., the ratio of the two KD values) of the first site-directed modified IL-2 for the IL2-RPγ dimer and the IL2-RaPγ trimer is less than the difference in affinity (e.g., the ratio of the two KD values) of native IL-2 for the IL2-RPγ dimer and the IL2-RaPγ trimer. In such embodiments, the first KD value of the first site-directed modified IL-2 for binding the IL2-RPγ dimer is not more than 10-fold, e.g., not more than 9-fold, not more than 8-fold, not more than 7-fold, not more than 6-fold, not more than 5-fold, not more than 4-fold, not more than 3-fold, or not more than 2-fold, than the second KD value for binding the IL2-RaPγ trimer. In certain embodiments, the first KD value of the first site-directed modified IL-2 for binding the IL2-RPγ dimer is between 8-fold and 3-fold, e.g., between 7-fold and 3-fold, than the second KD value for binding the IL2-RaPγ trimer. In certain embodiments, native IL-2 binds the IL2-RPγ dimer with a first KD value of the order of 1E-9 M. In certain embodiments, native IL-2 binds the IL2-RaPγ trimer with a second KD value of the order of 1E-11 M. In certain embodiments, the first KD value of native IL-2 for binding the IL2-RPγ dimer is about 40-fold than the second KD value for binding the IL2-RaPγ trimer. In certain embodiments, the first amino acid position of such first site-directed modified IL-2 is selected from R38, K49.

[0032] Receptor bias of second site-directed modified IL-2

[0033] The second site-directed modified IL-2 described herein has a bias for non-β receptors.

[0034] In certain embodiments, the bias for non-β receptors includes (i) binding the IL2-RaPγ trimer with a KD value of less than 1E-8 (e.g., of the order of 1E-9 to 1E-8), and / or (ii) not binding to the IL2-RPγ dimer.

[0035] In certain embodiments, the second site-directed modified IL-2 does not bind to the IL-2 receptor β (IL-2Rβ) subunit.

[0036] In certain embodiments, the second site-directed modified IL-2 binds to the IL2-Raβγ trimer with a KD value of less than 1E-8 M (e.g., less than 2E-8 M, 3E-8 M, 4E-8 M, 5E-8 M, 6E-8 M, 7E-8 M, 8E-8 M, 9E-8 M, 1E-9 M, or less). In certain embodiments, the second site-directed modified IL-2 binds to the IL2-Raβγ trimer with a KD value of 1E-9 to 1E-8 M in magnitude.

[0037] In certain embodiments, the second site-directed modified IL-2 does not bind to the IL2-Rβγ dimer.

[0038] In certain embodiments, the first amino acid position of the first site-directed modified IL-2 is selected from F42, Y45, E62, K64, P65, E68, K35, T37, R38, T41, K48, K49. In certain embodiments, the first amino acid position is selected from F42, Y45, E62, K64, P65, E68. In certain embodiments, the first amino acid position is F42, Y45, E62, P65, or E68.

[0039] In certain embodiments, the second amino acid position of the second site-directed modified IL-2 is selected from H16, D20, A73, H79. In certain embodiments, the second amino acid position of the second site-directed modified IL-2 is selected from D20. In certain embodiments, the first amino acid position is F42, Y45, E62, P65, or E68, and the second amino acid position is D20.

[0040] In certain embodiments, the first amino acid position is Y45, and the second amino acid position is D20-20K, H16-20K, A73-20K, H79-20K. In certain embodiments, the first amino acid position is Y45, and the second amino acid position is D20.

[0041] In certain embodiments, the first site-directed modified IL-2 comprises a PEG modification group having an average molecular weight of 5-60 kDa, e.g., 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, or 60 kDa. In certain embodiments, the first site-directed modified IL-2 comprises a PEG modification group having an average molecular weight of 5-40 kDa, e.g., 5-30 kDa, 5-25 kDa, 5-20 kDa, 10-40 kDa, 10-30 kDa, 15-30 kDa, 10-25 kDa, or 15-25 kDa, e.g., 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, or 40 kDa. In certain embodiments, the first site-directed modified IL-2 comprises a PEG modification group having an average molecular weight of 5 kDa, 10 kDa, or 20 kDa. In certain embodiments, the first site-directed modified IL-2 comprises a PEG modification group having an average molecular weight of 20 kDa.

[0042] In certain embodiments, the second site-directed modified IL-2 comprises a PEG modification group having an average molecular weight of 5-60 kDa, e.g., 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, or 60 kDa. In certain embodiments, the second site-directed modified IL-2 comprises a PEG modification group having an average molecular weight of 5-40 kDa, e.g., 5-30 kDa, 5-25 kDa, 5-20 kDa, 10-40 kDa, 10-30 kDa, 15-30 kDa, 10-25 kDa, or 15-25 kDa, e.g., 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, or 40 kDa. In certain embodiments, the second site-directed modified IL-2 comprises a PEG modification group having an average molecular weight of 5 kDa, 10 kDa, or 20 kDa. In certain embodiments, the second site-directed modified IL-2 comprises a PEG modification group having an average molecular weight of 20 kDa.

[0043] In certain embodiments, the PEG modification group comprised by the first site- directed modified IL-2 and the PEG modification group comprised by the second site- directed modified IL-2 have substantially the same average molecular weight.

[0044] In certain embodiments, the first site-directed modified IL-2 has a residue at a first amino acid position (e.g., amino acid position F42, Y45, E62, P65, or E68) mutated to a non-natural amino acid to which the PEG group is attached, as compared to wild-type IL-2 (e.g., SEQ ID NO: 1).

[0045] In certain embodiments, the second site-directed modified IL-2 has a residue at a second amino acid position (e.g., amino acid position D20, H16, A73, or H79) mutated to a non-natural amino acid to which the PEG group is attached, as compared to wild-type IL-2 (e.g., SEQ ID NO: 1).

[0046] In certain embodiments, the non-natural amino acid contains a chemical functional group, e.g., a carbonyl, alkyne, and azido group, etc., which are generally capable of efficiently and selectively forming stable covalent bonds; and the PEG group comprises a tag group that is chemically reactive with the chemical functional group to form a covalent bond, such that the PEG group is attached to the non-natural amino acid.

[0047] In certain embodiments, the non-natural amino acid contains an azido group, and the PEG group comprises a tag group that is click chemically reactive with the azido group, such that the PEG group is attached to the non-natural amino acid.

[0048] In certain embodiments, the non-natural amino acid is a lysine derivative containing an azido group. In certain embodiments, the non-natural amino acid is Nε-2-azidoethyloxycarbonyl-L-lysine (NAEK).

[0049] In certain embodiments, the non-natural amino acid is a tyrosine derivative containing an azido group. In certain embodiments, the non-natural amino acid is 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid.

[0050] In certain embodiments, the tag group that is click chemically reactive with the azido group is a chemical moiety comprising a dibenzocyclooctyne group, e.g., dibenzocyclooctyne (DBCO), 4-dibenzocyclooctynol (DIBO), or BCN (bicyclo[6.1.0]nonyne).

[0051] In certain embodiments, the labeling group capable of click chemistry with an azido group is DBCO, the first site-directed modified IL-2 has a residue at a first amino acid position (e.g., amino acid position F42, Y45, E62, P65, or E68) replaced with a structure of Formula la as compared to wild-type IL-2, and the second site-directed modified IL-2 has a residue at a second amino acid position (e.g., amino acid position D20, H16, A73, or H79) replaced with a structure of Formula la as compared to wild-type IL-2:

[0052]

[0053] wherein the direction from R1to R2is the N-terminal to C-terminal direction of the amino acid sequence, wherein the Nthamino acid is the residue at the first amino acid position (e.g., the F42, Y45, E62, P65, or E68amino acid) or the second amino acid position (e.g., the D20, H16, A73, or H79amino acid), R1is the 1stto (N-1)thamino acid residue of the IL-2 amino acid sequence, R2is the (N+1)thto C-terminal amino acid residue of the IL-2 amino acid sequence, and R3is a PEG group.

[0054] In certain embodiments, the labeling group capable of click chemistry with an azido group is DIBO, the first site-directed modified IL-2 has a residue at a first amino acid position (e.g., amino acid position F42, Y45, E62, P65, or E68) replaced with a structure of Formula lb as compared to wild-type IL-2, and the second site-directed modified IL-2 has a residue at a second amino acid position (e.g., amino acid position D20, H16, A73, or H79) replaced with a structure of Formula lb as compared to wild-type IL-2:

[0055]

[0056] wherein the direction from R1to R2is the N-terminal to C-terminal direction of the amino acid sequence, wherein the Nthamino acid is the residue at the first amino acid position (e.g., the F42, Y45, E62, P65, or E68amino acid) or the second amino acid position (e.g., the D20, H16, A73, or H79amino acid), R1is the 1stto (N-1)thamino acid residue of the IL-2 amino acid sequence, R2is the (N+1)thto C-terminal amino acid residue of the IL-2 amino acid sequence, and R3is a PEG group.

[0057] In certain embodiments, the labeling group capable of click chemistry reaction with an azido group is BCN, the first site-directed modified IL-2 has a residue at a first amino acid position (e.g., amino acid position F42, Y45, E62, P65, or E68) replaced by a structure of Formula Ic, and the second site-directed modified IL-2 has a residue at a second amino acid position (e.g., amino acid position D20, H16, A73, or H79) replaced by a structure of Formula Ic:

[0058]

[0059] wherein the direction from R1to R2is the N-terminal to C-terminal direction of the amino acid sequence, wherein the amino acid at position N is the residue at the first amino acid position (e.g., the amino acid at position F42, Y45, E62, P65, or E68) or the second amino acid position (e.g., the amino acid at position D20, H16, A73, or H79), R1is the amino acid residue at position 1 to position N-1 of the IL-2 amino acid sequence, R2is the amino acid residue at position N+1 to the C-terminal of the IL-2 amino acid sequence, and R3is a PEG group.

[0060] In certain embodiments, the PEGylated IL-2 variants referred to in this application are named as follows: [position of mutation to unnatural amino acid compared to SEQ ID NO: 1] - [average molecular weight of the attached PEG group].

[0061] In certain exemplary embodiments, the first site-directed modified IL-2 is selected from F42-20K, Y45-20K, E62-20K, P65-20K, or E68-20K. In certain exemplary embodiments, the second site-directed modified IL-2 is D20-20K, H16-20K, A73-20K, or H79-20K. For example, Y45-20K refers to a PEGylated IL-2 variant whose amino acid sequence differs from SEQ ID NO: 1 in that Y45 is replaced with the unnatural amino acid NAEK, and the position is further attached with a PEG group having an average molecular weight of 20 kDa.

[0062] Preparation of compositions

[0063] The first site-directed modified IL-2 and the second site-directed modified IL-2 comprised in the composition provided in the first aspect of the application can be prepared by any method known in the art.

[0064] In certain embodiments, the unnatural amino acid can be site- directed inserted by unnatural amino acid orthogonal translation technology, followed by attaching a PEG group to the unnatural amino acid.

[0065] Non-natural amino acid orthogonal translation technology is well known to those skilled in the art. Non-natural amino acid orthogonal translation technology utilizes a stop codon to insert a non-natural amino acid into the amino acid sequence of a protein during protein translation, effectively expanding the number of amino acid codons, and thus non-natural amino acid orthogonal translation technology is also referred to as genetic code expansion technology. Typically, a non-natural amino acid orthogonal translation system involves a tRNA, an aminoacyl tRNA synthetase, and a nucleic acid sequence of interest having one or more stop codons. The above system is introduced into a host cell and cultured in a medium containing appropriate nutrients and one or more non-natural amino acids to be inserted. The host cell is then maintained under conditions that allow the expression of the protein of interest. In response to the non-natural codon, one or more non-natural amino acids are incorporated into the polypeptide chain.

[0066] In certain embodiments, a non-natural amino acid can be inserted by non-natural amino acid orthogonal translation technology, which can contain a chemical functional group, e.g., a carbonyl group, an alkyne group, an azido group, etc., which is generally capable of efficiently and selectively forming a stable covalent bond, followed by site-directed modification of the PEG group by chemical reaction to form a covalent bond.

[0067] In certain embodiments, a non-natural amino acid can be inserted by non-natural amino acid orthogonal translation technology, followed by site-directed modification of the PEG group by click chemistry reaction. In certain embodiments, the non-natural amino acid and the PEG group each contain a chemical group capable of undergoing click chemistry reaction.

[0068] In a second aspect, the present application provides a method for preparing the composition of the first aspect, comprising preparing the first site-directed modified IL-2 and preparing the second site-directed modified IL-2, wherein,

[0069] Preparation of the first site-directed modified IL-2 comprises:

[0070] - providing: (al) a first site-mutated IL-2, which has a residue at a first amino acid position (e.g., amino acid position F42, Y45, E62, P65, or E68) mutated to a non-natural amino acid compared to wild-type IL-2; (bl) a PEG group modified by a labeling group, which can form a covalent bond with the non-natural amino acid;

[0071] - co-incubating (al) with (bl) to couple the non-natural amino acid with the PEG group by chemical reaction;

[0072] Preparation of the second site-directed modified IL-2 comprises:

[0073] - providing: (a2) a second site-directed mutant IL-2 having a residue at a second amino acid position (e.g., amino acid position D20, H16, A73, or H79) mutated to a non-natural amino acid as compared to wild-type IL-2; (b2) a PEG group modified with a labeling group capable of forming a covalent bond with the non-natural amino acid;

[0074] - co-incubating (a2) with (b2) to couple the non-natural amino acid to the PEG group via a chemical reaction.

[0075] In certain embodiments, the non-natural amino acid in the first site-directed mutant IL-2 comprises a chemical functional group, e.g., a carbonyl group, an alkyne group, an azido group, etc., which are generally capable of efficiently and selectively forming stable covalent bonds; and the labeling group in the PEG group is capable of chemically reacting with the chemical functional group to form a covalent bond.

[0076] In certain embodiments, the non-natural amino acid in the second site-directed mutant IL-2 comprises a chemical functional group, e.g., a carbonyl group, an alkyne group, an azido group, etc., which are generally capable of efficiently and selectively forming stable covalent bonds; and the labeling group in the PEG group is capable of chemically reacting with the chemical functional group to form a covalent bond.

[0077] In certain embodiments, the non-natural amino acids in the first and second site- directed mutant IL-2s comprise the same chemical functional group.

[0078] In certain embodiments, the non-natural amino acids in the first and second site- directed mutant IL-2s comprise an azido group.

[0079] In certain embodiments, the non-natural amino acids in the first and second site- directed mutant IL-2s are the same.

[0080] In certain embodiments, the non-natural amino acid is a lysine derivative comprising an azido group. In certain embodiments, the non-natural amino acid is Nε-2-azidoethyloxycarbonyl-L-lysine (NAEK).

[0081] In certain embodiments, the non-natural amino acid is a tyrosine derivative comprising an azido group. In certain embodiments, the non-natural amino acid is 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid.

[0082] In certain embodiments, preparing the first site-directed mutant IL-2 comprises:

[0083] - providing: (a1) a first site-mutated IL-2, which has a residue at a first amino acid position (such as amino acid position F42, Y45, E62, P65, or E68) mutated to a non-natural amino acid containing an azido group compared to wild-type IL-2; (b1) a PEG group modified with a labeling group that is capable of click chemistry with an azido group;

[0084] - co-incubating (a1) with (b1) to couple the non-natural amino acid to the PEG group via click reaction.

[0085] In certain embodiments, preparing the second site-modified IL-2 comprises:

[0086] - providing: (a2) a second site-mutated IL-2, which has a residue at a second amino acid position (such as amino acid position D20, H16, A73, or H79) mutated to a non-natural amino acid containing an azido group compared to wild-type IL-2; (b2) a PEG group modified with a labeling group that is capable of click chemistry with an azido group;

[0087] - co-incubating (a2) with (b2) to couple the non-natural amino acid to the PEG group via click reaction.

[0088] In certain embodiments, the click chemistry reaction is a copper-free click chemistry reaction. Copper-free click chemistry reactions are click reactions that are enabled by the introduction of cyclooctynes, where the strain of the eight-membered ring allows for reaction with azides without a catalyst. One of these reagents consists of so-called DBCO compounds. Azide-modified macromolecules can now be labeled without a metal catalyst, which not only allows for studies in living cells, but also prevents damage to the protein.

[0089] In certain embodiments, the labeling group that is capable of click chemistry with an azido group is a chemical moiety comprising an alkyne group. In certain embodiments, the labeling group that is capable of click chemistry with an azido group is a chemical moiety comprising a dibenzocyclooctyne group. In certain embodiments, the labeling group that is capable of click chemistry with an azido group is dibenzocyclooctyne (DBCO), 4-dibenzocyclooctynol (DIBO), or BCN (bicyclo[6.1.0]nonyne). In certain embodiments, the labeling group that is capable of click chemistry with an azido group is DBCO.

[0090] In certain embodiments, the DBCO-tagged PEG group has the structure shown in Formula IIa, wherein R3 is a PEG group.

[0091]

[0092] In certain embodiments, the DIBO-tagged PEG group has the structure shown in Formula IIb, wherein R3 is a PEG group.

[0093]

[0094] In certain embodiments, the first site-mutated IL-2 and the second site-mutated IL-2 are provided by an orthogonal translation technology of unnatural amino acids.

[0095] In certain embodiments, the orthogonal translation technology of unnatural amino acids comprises the following steps:

[0096] obtaining a nucleic acid sequence encoding a site-mutated IL-2, wherein the codon corresponding to the amino acid position to be mutated is mutated to TAG;

[0097] operably linking the nucleic acid sequence encoding the site-mutated IL-2 to a vector to obtain a site-mutated sequence expression vector;

[0098] co-transfecting the site-mutated sequence expression vector with a vector encoding a suppressor tRNA for amber codon and an aminoacyl tRNA synthetase specific for the unnatural amino acid into a host cell, culturing in a medium containing the unnatural amino acid and inducing expression to obtain IL-2 site-mutated to the unnatural amino acid.

[0099] In certain embodiments, the unnatural amino acid is Nε-2-azidoethyloxycarbonyl-L-lysine (NAEK). In certain embodiments, the aminoacyl tRNA synthetase specific for the unnatural amino acid is NAEK-specific aminoacyl tRNA synthetase.

[0100] In certain embodiments, the vector encoding a suppressor tRNA for amber codon and an aminoacyl tRNA synthetase specific for the unnatural amino acid is pSURAR-YAV (also known as pSUPAR-YAV-tRNA / PylRS), which is obtained from Escherichia coli containing plasmid pSUPAR-YAV-tRNA / PylRS, which is deposited in the China General Microbiological Culture Collection Center (No. 1 Huayuancang, Beijing, China), which is located in the Institute of Microbiology, Chinese Academy of Sciences, on April 8, 2013, and has the accession number CGMCC No: 7432 and the classification name Escherichia coli.

[0101] In certain embodiments, the first site-directed mutated IL-2 has a residue at a first amino acid position (e.g., amino acid position F42, Y45, E62, P65, or E68) replaced by the structure of Formula III compared to wild-type IL-2 and the second site-directed mutated IL-2 has a residue at a second amino acid position (e.g., amino acid position D20, H16, A73, or H79) replaced by the structure of Formula III compared to wild-type IL-2:

[0102]

[0103] The direction from R1 to R2 is the N-terminal to C-terminal direction of the amino acid sequence, wherein the amino acid at position N is the residue at the first amino acid position (e.g., the amino acid at position F42, Y45, E62, P65, or E68) or the residue at the second amino acid position (e.g., the amino acid at position D20, H16, A73, or H79), R1 is the amino acid residue at position 1 to N-1 of the IL-2 amino acid sequence, and R2 is the amino acid residue at position N+1 to the C-terminal of the IL-2 amino acid sequence.

[0104] Kit

[0105] The combination of the first site-directed modified IL-2 and the second site-directed modified IL-2 provided herein can reduce the senescence and exhaustion of immune cells, enhance the proliferation of T cells, reduce the proliferation of Treg cells, maintain the proliferation of Tscm and T effector cells, reduce the degree of exhaustion of T effector cells, and avoid the over-activation of T cells by endogenous IL-2, leading to the fate of immune cells in adoptive cellular immunotherapy to long-term immunity. Based on this, the present application also provides a kit as described below, an in vitro method for preparing or culturing immune cells for adoptive cellular therapy, and immune cells for adoptive cellular therapy obtained by the method.

[0106] In a third aspect, the present application provides a kit comprising the composition of the first aspect. In certain embodiments, the kit further comprises a package insert comprising instructions for using the composition to prepare and / or culture immune cells for adoptive cellular therapy in vitro. The present application also relates to the use of the composition of the first aspect or the kit of the third aspect for preparing or culturing immune cells for adoptive cellular therapy in vitro.

[0107] In the present context, adoptive cell therapy can include tumor infiltrating T cell (TIL) therapy, chimeric antigen receptor T cell therapy (CAR-T), T cell receptor therapy (TCR), NK cell therapy, etc. In the present context, the engineered immune cells for adoptive cell therapy can be any cell known in the art for use in adoptive cell therapy. In certain embodiments, the engineered immune cells for adoptive cell therapy comprise lymphocytes, such as T cells, NK cells, or combinations thereof.

[0108] In certain embodiments, the immune cells for adoptive cell therapy are engineered immune cells expressing a chimeric antigen receptor and / or comprising a nucleic acid molecule encoding the chimeric antigen receptor. In certain embodiments, the engineered immune cells comprise lymphocytes, such as T cells, NK cells, or combinations thereof, expressing an IL2-RaP3 trimer.

[0109] In certain embodiments, the immune cells for adoptive cell therapy are tumor infiltrating lymphocytes (TILs).

[0110] In a fourth aspect, the present application provides a kit comprising the composition of the first aspect and a nucleic acid molecule encoding a chimeric antigen receptor. In certain embodiments, the kit further comprises a package insert comprising instructions for using the composition and the nucleic acid molecule to prepare in vitro engineered immune cells for adoptive cell therapy, the engineered immune cells expressing a chimeric antigen receptor and / or comprising a nucleic acid molecule encoding the chimeric antigen receptor.

[0111] In certain embodiments, the nucleic acid molecule encoding a chimeric antigen receptor is present in an expression vector.

[0112] In certain embodiments, the expression vector is a viral (e.g., lentiviral, retroviral, or adenoviral) vector. In certain embodiments, the expression vector is a non-viral vector.

[0113] The present application also relates to the use of the composition of the first aspect and, optionally, the nucleic acid molecule encoding a chimeric antigen receptor or the kit of the fourth aspect for preparing in vitro engineered immune cells for adoptive cell therapy, the engineered immune cells expressing a chimeric antigen receptor and / or comprising a nucleic acid molecule encoding the chimeric antigen receptor.

[0114] The present application also provides a kit comprising the composition of the first aspect and immune cells for adoptive cell therapy. In certain embodiments, the kit further comprises a package insert comprising instructions for using the composition to culture the immune cells in vitro for adoptive cell therapy. The present application also relates to the composition of the first aspect and optionally immune cells for adoptive cell therapy for use in the in vitro culture of immune cells for adoptive cell therapy.

[0115] In certain embodiments, the immune cells for adoptive cell therapy are engineered immune cells expressing a chimeric antigen receptor and / or comprising a nucleic acid molecule encoding the chimeric antigen receptor. In certain embodiments, the engineered immune cells comprise lymphocytes, such as T cells, NK cells, or a combination thereof, expressing an IL2-RaP3 trimer.

[0116] In certain embodiments, the immune cells for adoptive cell therapy are tumor infiltrating lymphocytes (TILs).

[0117] In certain embodiments, the chimeric antigen receptors described herein possess the meaning well known to the person skilled in the art, which typically comprises an extracellular antigen binding domain, optionally a spacer domain, a transmembrane domain, and one or more intracellular signaling domains. In certain embodiments, the intracellular signaling domains are selected from a primary signaling domain and / or a costimulatory signaling domain. In certain embodiments, the extracellular antigen binding domain comprises an antibody or an antigen binding fragment, such as a scFv, that specifically binds to a tumor associated antigen, such as CD19.

[0118] Culture and preparation of immune cells for adoptive cell therapy

[0119] In a fifth aspect, the present application provides a method of culturing immune cells for adoptive cell therapy, the method comprising culturing the cells in a cell culture medium comprising a first site-directed modified IL-2 and a second site-directed modified IL-2, wherein the first and second site-directed modified IL-2 are as defined in the first aspect.

[0120] The cell culture medium can be any medium capable of supporting cell growth, typically comprising inorganic salts, vitamins, glucose, a buffer system and essential amino acids, and typically having an osmolarity of about 280-330 mOsmol. In certain embodiments, the cell culture medium is a medium capable of supporting growth of immune cells, such as lymphocytes, such as T cells and / or NK cells. In certain embodiments, the cell culture medium is a complete medium. In certain embodiments, the cell culture medium comprises a basal medium (such as RPMI 1640), serum (such as FBS), sodium pyruvate, non-essential amino acids. In certain embodiments, the cell culture medium does not comprise serum.

[0121] In certain embodiments, the method further comprises harvesting the cells for storage (e.g. reformulation in a cryopreservation medium) or administration (e.g. for adoptive cell therapy).

[0122] In certain embodiments, the provided culture conditions in the presence of the first site-directed modified IL-2 and the second site-directed modified IL-2 reduce senescence and exhaustion of the immune cells, preserve their stemness state, enhance T proliferation of the cells, reduce proliferation of Treg cells, maintain the proliferative state of Tscm and T effector cells, reduce the degree of exhaustion of T effector cells, avoid excessive activation of T cells by endogenous IL-2 produced upon T cell activation.

[0123] In certain embodiments, the immune cells for adoptive cell therapy are engineered immune cells expressing a chimeric antigen receptor and / or comprising a nucleic acid molecule encoding the chimeric antigen receptor. In certain embodiments, the engineered immune cells comprise lymphocytes, such as T cells, NK cells or a combination thereof, expressing an IL2-RaP3 trimer.

[0124] In certain embodiments, the immune cells for adoptive cell therapy are tumor infiltrating lymphocytes (TILs).

[0125] In a sixth aspect, the present application provides a method of preparing immune cells for adoptive cell therapy, comprising using a first site-directed modified IL-2 and a second site- directed modified IL-2 as defined in the first aspect.

[0126] In certain embodiments of the sixth aspect, the present application provides a method of preparing immune cells for adoptive cell therapy, the immune cells for adoptive cell therapy being engineered immune cells expressing a chimeric antigen receptor and / or comprising a nucleic acid molecule encoding the chimeric antigen receptor, wherein the method comprises:

[0127] (1) providing immune cells from a patient or a healthy donor;

[0128] (2) introducing a nucleic acid molecule encoding a chimeric antigen receptor into the immune cell of step (1) in the presence of the first site-directed modified IL-2 and the second site- directed modified IL-2, thereby providing the engineered immune cell; wherein the first site- directed modified IL-2 and the second site-directed modified IL-2 are as defined in the first aspect.

[0129] In certain embodiments, step (2) is performed in a cell culture medium comprising the first site-directed modified IL-2 and the second site-directed modified IL-2. The cell culture medium can be any medium capable of supporting cell growth, typically comprising inorganic salts, vitamins, glucose, a buffer system and essential amino acids, and typically having an osmolarity of about 280-330 mOsmol. In certain embodiments, the cell culture medium is a medium capable of supporting the growth of immune cells, such as lymphocytes, such as T cells and / or NK cells. In certain embodiments, the cell culture medium is a complete medium. In certain embodiments, the cell culture medium comprises a basal medium (such as RPMI 1640), serum (such as FBS), sodium pyruvate, non-essential amino acids. In certain embodiments, the cell culture medium does not comprise serum.

[0130] In certain embodiments, the nucleic acid molecule encoding a chimeric antigen receptor in step (2) is present in an expression vector.

[0131] In certain embodiments, the nucleic acid molecule encoding a chimeric antigen receptor in step (2) is introduced into the cell by infection with a viral (e.g., lentiviral, retroviral or adenoviral) vector. In certain embodiments, the nucleic acid molecule encoding a chimeric antigen receptor in step (2) is introduced into the cell by infection with a lentiviral vector.

[0132] In certain embodiments, the nucleic acid molecule encoding a chimeric antigen receptor in step (2) is introduced into the cell by a non-viral vector.

[0133] In certain embodiments, in step (1), the immune cell is pre-treated, the pre-treatment comprising sorting, activating and / or proliferating the immune cell. In certain embodiments, the pre-treatment comprises contacting the immune cell with an anti-CD3 antibody and an anti-CD28 antibody, thereby stimulating the immune cell and inducing its proliferation, thereby generating the pre-treated immune cell. In certain embodiments, the pre-treatment comprises isolating T cells from peripheral blood mononuclear cells (PBMCs).

[0134] In certain embodiments, the method further comprises, after step (2): (3) continuing culturing the immune cells obtained in step (2) in a cell culture medium comprising the first site- directed modified IL-2 and the second site-directed modified IL-2.

[0135] In certain embodiments, the method further comprises harvesting the cells for storage (e.g., reformulation in a cryopreservation medium) or administration (e.g., for adoptive cell therapy).

[0136] In certain embodiments, the immune cells comprise lymphocytes, such as T cells, NK cells, or any combination thereof, expressing IL2-RaP3trimer. In certain embodiments, the immune cells are T cells.

[0137] In certain embodiments, the provided conditions for the presence of the first site- directed modified IL-2 and the second site-directed modified IL-2 reduce over-activation and terminal differentiation of the immune cells.

[0138] In certain embodiments of the sixth aspect, the present application further provides a method of preparing immune cells for adoptive cell therapy, the immune cells for adoptive cell therapy being tumor infiltrating lymphocytes (TILs), wherein the method comprises: isolating infiltrating lymphocytes from tumor tissue and culturing in a cell culture medium comprising the first site-directed modified IL-2 and the second site-directed modified IL-2, wherein the first site-directed modified IL-2 and the second site-directed modified IL-2 are as defined in the first aspect, and the cell culture medium is as defined above.

[0139] In a seventh aspect, the present application provides immune cells for adoptive cell therapy, prepared or cultured by the method of any one of the above aspects.

[0140] In certain embodiments, the immune cells of the seventh aspect have reduced senescence and exhaustion, enhanced T cell proliferation, reduced Treg cell proliferation, maintained Tscm and T effector cell proliferation, reduced degree of Teffector cell exhaustion, and / or reduced over-activation by endogenous IL-2.

[0141] In an eighth aspect, the present application provides a population of immune cells comprising the immune cells of the seventh aspect, and optionally unmodified and / or unsuccessfully modified immune cells. In certain embodiments, the immune cells of the seventh aspect comprise about 10-100%, preferably 40-80%, of the total number of cells in the population of immune cells.

[0142] Use in synergistically enhancing an immune response

[0143] The first site-directed modified IL-2 and the second site-directed modified IL-2 provided herein have a significant synergistic effect of enhancing immune responses, and thus the combination of the first site-directed modified IL-2 and the second site-directed modified IL-2 provided herein can be used to treat disease situations in which the immune system of the host is stimulated for benefit, particularly conditions in which an enhanced cellular immune response is desired, which can include disease situations in which the host immune response is inadequate or defective, such as in a tumor.

[0144] In a tenth aspect, the present application provides a pharmaceutical composition comprising the composition of the first aspect and a pharmaceutically acceptable carrier and / or excipient.

[0145] In certain embodiments, the pharmaceutical composition comprises multiple compositions or dosage forms. In certain embodiments, the first site-directed modified IL-2 and the second site-directed modified IL-2 are in separate compositions or dosage forms. In certain embodiments, the pharmaceutical composition comprises a first composition of the first site-directed modified IL-2 and a pharmaceutically acceptable carrier and / or excipient, and a second composition of the second site-directed modified IL-2 and a pharmaceutically acceptable carrier and / or excipient.

[0146] In certain embodiments, the pharmaceutical composition comprises one composition or dosage form. In certain embodiments, the first site-directed modified IL-2 and the second site-directed modified IL-2 are in the same composition or dosage form. In certain embodiments, the pharmaceutical composition comprises a single composition of the first site-directed modified IL-2, the second site-directed modified IL-2, and a pharmaceutically acceptable carrier and / or excipient.

[0147] The first site-directed modified IL-2 and the second site-directed modified IL-2, their combinations, and the composition of the first aspect or the pharmaceutical composition of the tenth aspect described herein can be formulated in dosage forms compatible with their intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The formulations for parenteral administration can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0148] Pharmaceutical compositions adapted for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, polyoxyethylated castor oil ELTM, or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by the inclusion of agents which delay absorption, for example, aluminum monostearate and gelatin.

[0149] In an eleventh aspect, the present application provides a method for enhancing an immune response, preventing and / or treating a proliferative disease (e.g., a tumor), comprising administering to a subject in need thereof the composition of the first aspect or the pharmaceutical composition of the tenth aspect. In certain embodiments, the method comprises administering to the subject the first site-directed modified IL-2 and the second site-directed modified IL-2 in combination.

[0150] In a twelfth aspect, the present application also provides the use of the composition of the first aspect or the pharmaceutical composition of the tenth aspect for enhancing an immune response, preventing and / or treating a proliferative disease (e.g., a tumor), or the use of the combination of the first site-directed modified IL-2 and the second site-directed modified IL-2 for enhancing an immune response, preventing and / or treating a proliferative disease (e.g., a tumor), or the use of the combination of the first site-directed modified IL-2 and the second site-directed modified IL-2 in the manufacture of a medicament for enhancing an immune response, preventing and / or treating a proliferative disease (e.g., a tumor).

[0151] In certain embodiments, the first site-directed modified IL-2 and the second site- directed modified IL-2 are formulated separately into two or more compositions (e.g., a kit comprising each component) in combination with one another. The separate components administered in combination with one another can be administered simultaneously, separately, or sequentially. In certain embodiments, the separate components administered in combination with one another can be administered to the subject at a different time than the administration of the other component; for example, each administration can be given non-simultaneously (e.g., separately or sequentially) at intervals of a given time period as part of a treatment regimen. In certain embodiments, the separate components administered in combination with one another can also be administered sequentially, but substantially simultaneously, during the same administration period. Furthermore, the separate components administered in combination with one another can be administered to the subject by the same or different routes.

[0152] In certain embodiments, the first site-directed modified IL-2 and the second site- directed modified IL-2 are formulated together into a single composition, e.g., for simultaneous delivery.

[0153] In another aspect, the present application provides a kit comprising a medicament comprising the first site-directed modified IL-2 and optionally a pharmaceutically acceptable carrier and / or excipient, and a package insert comprising instructions for administering the medicament in combination with a composition comprising the second site-directed modified IL-2 and optionally a pharmaceutically acceptable carrier and / or excipient to enhance an immune response, prevent, and / or treat a proliferative disease (e.g., a tumor) in a subject.

[0154] In another aspect, the present application provides a kit comprising a medicament comprising the second site-directed modified IL-2 and optionally a pharmaceutically acceptable carrier and / or excipient, and a package insert comprising instructions for administering the medicament in combination with a composition comprising the first site-directed modified IL-2 and optionally a pharmaceutically acceptable carrier and / or excipient to enhance an immune response, prevent, and / or treat a proliferative disease (e.g., a tumor) in a subject.

[0155] In another aspect, the present application provides a kit comprising a first medicament comprising the first site-directed modified IL-2 and optionally a pharmaceutically acceptable carrier and / or excipient, and a second medicament comprising the second site- directed modified IL-2 and optionally a pharmaceutically acceptable carrier and / or excipient. In certain embodiments, the kit further comprises a package insert comprising instructions for administering the first medicament and the second medicament to enhance an immune response, prevent, and / or treat a proliferative disease (e.g., a tumor) in a subject.

[0156] In certain embodiments of the methods, uses and kits as described above, the immune response is a cellular immune response. In certain embodiments, the immune response is a T cell- mediated immune response, in particular an effector T cell (Teff)-mediated immune response. In certain embodiments, the enhancing the immune response further comprises reducing or inhibiting Treg cell function.

[0157] In certain embodiments of the methods, uses and kits as described above, the proliferative disease is a tumor, including a solid tumor or a hematological tumor, also including metastatic cancer, relapsed or refractory cancer. In other embodiments, the proliferative disease is hypergammaglobulinemia, a lymphoproliferative disorder, a paraproteinemias, purpura, sarcoidosis, Sezary Syndrome, Waldenstron's macroglobulinemia, Gaucher's Disease, histiocytosis, and any other cellular proliferation disease outside of neoplasia in an organ system.

[0158] In certain embodiments of the methods, uses and kits as described above, the tumor is a solid tumor. In certain embodiments, the solid tumor is a metastatic cancer, relapsed or refractory cancer.

[0159] In certain embodiments of the methods, uses and kits as described above, the tumor is a hematological tumor, such as a leukemia, lymphoma or myeloma. In certain embodiments, the hematological tumor is a metastatic cancer, relapsed or refractory cancer.

[0160] In certain embodiments of the methods, uses and kits as described above, the tumor is selected from melanoma, renal cell carcinoma, non-small cell lung cancer, lymphoma, head and neck squamous cell carcinoma, urothelial carcinoma, ovarian cancer, gastric cancer and breast cancer.

[0161] In certain embodiments of the methods, uses and kits as described above, the subject is a mammal, such as a human.

[0162] In certain embodiments of the methods, uses and kits as described above, the first site-directed modified IL-2 and the second site-directed modified IL-2 in the composition are administered simultaneously, separately or sequentially.

[0163] In certain embodiments of the methods, uses and kits as described above, the first site- directed modified IL-2 and the second site-directed modified IL-2, combinations thereof, and the compositions or pharmaceutical compositions of the application can be formulated into any dosage form known in the medical arts, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injectable solutions, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, and the like. The preferred dosage form will depend on the intended mode of administration and therapeutic use. The compositions or pharmaceutical compositions of the application should be sterile and stable under the conditions of manufacture and storage. A preferred dosage form is an injection. Such injections can be sterile injection solutions. For example, sterile injection solutions can be prepared by incorporating the active ingredients into an appropriate solvent with one or more of the other ingredients, as described above, followed by filtered sterilization. In addition, sterile solutions can be prepared as sterile powders (e.g., by vacuum drying or freeze-drying), followed by the addition of the appropriate aqueous carrier prior to use. Such sterile powders can be reconstituted into solutions or suspensions with an appropriate carrier prior to use.

[0164] In certain embodiments of the methods, uses and kits as described above, the first site- directed modified IL-2 and the second site-directed modified IL-2, combinations thereof, and the compositions or pharmaceutical compositions of the application can be administered by any suitable method known in the art, including but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intraleaflet, intracisternal, inguinal, intravesical, local (e.g., powders, ointments or drops), or nasal routes. However, for many therapeutic uses, the preferred route / means of administration is parenteral administration (e.g., intravenous injection or bolus, subcutaneous injection, intraperitoneal injection, intramuscular injection). The skilled artisan will appreciate that the route and / or means of administration will vary depending on the intended purpose. In certain embodiments, the first site-directed modified IL-2 and the second site-directed modified IL-2, combinations thereof, and the compositions or pharmaceutical compositions of the application are administered by intravenous injection or bolus.

[0165] In certain embodiments of the methods, uses and kits as described above, the first site- directed modified IL-2 and the second site-directed modified IL-2, combinations thereof, and the compositions or pharmaceutical compositions of the application can be formulated in dosage unit form for ease of administration. Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0166] In certain embodiments of the methods, uses and kits as described above, the first site- directed modified IL-2 and the second site-directed modified IL-2, combinations thereof, and the compositions or pharmaceutical compositions of the application can be administered alone or in combination with additional pharmaceutically active agents (e.g., anti-tumor agents) or additional therapies (e.g., anti-tumor therapies).

[0167] Use in adoptive cell therapy

[0168] The combination of the first site-directed modified IL-2 and the second site-directed modified IL-2 provided by the present application can allow the fate of the immune cells in adoptive cell immunotherapy to be directed towards long-term immunity. Thereby, the therapeutic application of the immune cells is further provided.

[0169] In a thirteenth aspect, the present application provides a pharmaceutical composition comprising the immune cell of the seventh aspect or the immune cell population of the eighth aspect and a pharmaceutically acceptable carrier and / or excipient.

[0170] The immune cell of the seventh aspect, the immune cell population of the eighth aspect, or the pharmaceutical composition of the thirteenth aspect can be formulated in dosage unit form appropriate for the intended route of administration. Examples of the route of administration include parenteral administration, e.g., intravenous administration, intradermal administration, subcutaneous administration, oral administration (e.g., inhalation administration), transdermal administration (i.e., topical administration), transmucosal administration, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, a saline solution, a fixed oil, a polyethylene glycol, glycerine, propylene glycol, or other synthetic solvents; an antibacterial agent such as benzyl alcohol or methyl parabens; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as ethylenediaminetetraacetic acid (EDTA); a buffer such as acetate, citrate, or phosphate, and an agent for adjusting tonicity such as sodium chloride or dextrose. The pH can be adjusted with an acid or a base, such as hydrochloric acid or sodium hydroxide. Formulations for parenteral administration can be enclosed in ampules, disposable syringes, or multiple-dose vials made of glass or plastic.

[0171] Pharmaceutical compositions adapted for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, polyoxyethylated castor oil ELTM, or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by the inclusion of agents which delay absorption, for example, aluminum monostearate and gelatin.

[0172] In a fourteenth aspect, the present application provides a method for enhancing an immune response, preventing and / or treating a proliferative disease (e.g., a tumor), comprising administering to a subject in need thereof the immune cell of the seventh aspect, the population of immune cells of the eighth aspect, or the pharmaceutical composition of the thirteenth aspect.

[0173] In certain embodiments, the method comprises the steps of: (1) obtaining an immune cell using the method of the fifth aspect and / or the method of the sixth aspect; (2) administering the immune cell obtained in step (1) or a population of cells comprising the same to the subject for treatment.

[0174] In certain embodiments, a chimeric antigen receptor cell therapy is administered to a subject, the method comprising: obtaining an engineered immune cell expressing a chimeric antigen receptor using the method of the fifth aspect and / or the method of the sixth aspect of the present application, followed by administering the engineered immune cell to the subject.

[0175] In certain embodiments, a TIL therapy is administered to a subject, the method comprising: obtaining a tumor infiltrating lymphocyte (TIL) using the method of the fifth aspect and / or the method of the sixth aspect of the present application, administering the tumor infiltrating lymphocyte (TIL) to the subject.

[0176] In a fifteenth aspect, the present application also provides the use of the immune cell of the seventh aspect, the immune cell population of the eighth aspect, or the pharmaceutical composition of the thirteenth aspect for enhancing immune response, preventing and / or treating a proliferative disease (e.g., a tumor), or in the manufacture of a medicament for enhancing immune response, preventing and / or treating a proliferative disease (e.g., a tumor).

[0177] In certain embodiments of the methods and uses as described above, the immune cell, immune cell population, or pharmaceutical composition can be administered in combination with, e.g., simultaneously, separately, or sequentially, the first site-directed modified IL-2 and the second site-directed modified IL-2 as defined in the first aspect.

[0178] In a sixteenth aspect, the present application also provides the use of the combination of the composition of the first aspect and immune cells for adoptive cell therapy for enhancing immune response, preventing and / or treating a proliferative disease (e.g., a tumor), or in the manufacture of a medicament for enhancing immune response, preventing and / or treating a proliferative disease (e.g., a tumor).

[0179] In a seventeenth aspect, the present application also provides a method for enhancing immune response, preventing and / or treating a proliferative disease, comprising administering to a subject in need thereof the combination of the composition of the first aspect and immune cells for adoptive cell therapy.

[0180] In certain embodiments of the sixteenth or seventeenth aspect, the immune cells for adoptive cell therapy are engineered immune cells expressing a chimeric antigen receptor and / or comprising a nucleic acid molecule encoding the chimeric antigen receptor. In certain embodiments, the engineered immune cells comprise lymphocytes, such as T cells, NK cells, or any combination thereof, expressing an IL2-RaP3 trimer.

[0181] In certain embodiments of the sixteenth or seventeenth aspect, the immune cells for adoptive cell therapy are tumor infiltrating lymphocytes (TILs).

[0182] In certain embodiments of the sixteenth or seventeenth aspect, the composition of the first aspect and the immune cells for adoptive cell therapy are present in the same composition or dosage form, and thus can be administered simultaneously.

[0183] In certain embodiments of the sixteenth or seventeenth aspect, the composition of the first aspect and the immune cells for adoptive cell therapy are present in separate compositions or dosage forms, and thus can be administered separately or sequentially.

[0184] In certain embodiments of the methods and uses described above, the immune response is a cellular immune response. In certain embodiments, the immune response is a T cell- mediated immune response, in particular an effector T cell (Teff)-mediated immune response. In certain embodiments, the enhancing immune response further comprises reducing or inhibiting Treg cell function.

[0185] In certain embodiments of the methods and uses described above, the proliferative disease is a tumor, including a solid tumor or a hematological tumor, also including metastatic cancer, relapsed or refractory cancer. In other embodiments, the proliferative disease is hypergammaglobulinemia, a lymphoproliferative disorder, a paraproteinemias, purpura, sarcoidosis, Sezary Syndrome, Waldenstron's macroglobulinemia, Gaucher's Disease, histiocytosis, and any other cellular proliferation disease outside of neoplasia in an organ system.

[0186] In certain embodiments of the methods and uses described above, the tumor is a solid tumor. In certain embodiments, the solid tumor is a metastatic cancer, relapsed or refractory cancer.

[0187] In certain embodiments of the methods and uses described above, the tumor is a hematological tumor, such as a leukemia, lymphoma or myeloma. In certain embodiments, the hematological tumor is a metastatic cancer, relapsed or refractory cancer.

[0188] In certain embodiments of the methods and uses described above, the tumor is selected from melanoma, renal cell carcinoma, non-small cell lung cancer, lymphoma, head and neck squamous cell carcinoma, urothelial carcinoma, ovarian cancer, gastric cancer and breast cancer.

[0189] In certain embodiments of the methods and uses described above, the tumor is a lymphoma.

[0190] In certain embodiments of the methods and uses described above, when referring to chimeric antigen receptor cell therapy, the tumor preferably comprises a hematological tumor, including metastatic cancer, relapsed or refractory cancer; for example the tumor is selected from a lymphoma.

[0191] In certain embodiments of the methods and uses as described above, when referring to tumor infiltrating lymphocyte (TIL) therapy, the tumor preferably comprises a solid tumor, including metastatic cancer, relapsed or refractory cancer; for example the tumor is selected from melanoma, renal cell carcinoma, non-small cell lung cancer, lymphoma, head and neck squamous cell carcinoma, urothelial carcinoma, ovarian cancer, gastric cancer, and breast cancer.

[0192] In certain embodiments of the methods and uses as described above, the subject is a mammal, for example a human.

[0193] In certain embodiments of the methods and uses as described above, the immune cell of the seventh aspect, the population of immune cells of the eighth aspect, or the pharmaceutical composition of the thirteenth aspect can be formulated into any dosage form known in the medical arts, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, troches, suppositories, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), inhalants, sprays, and the like. The preferred dosage form depends on the intended mode of administration and therapeutic use. The compositions or pharmaceutical compositions of the present application should be sterile and stable under the conditions of manufacture and storage. A preferred dosage form is an injection. Such injections can be sterile injection solutions. For example, sterile injection solutions can be prepared by incorporating the active ingredients into an appropriate solvent with one or more of the other ingredients, as described, and, if desired, filtering sterilizing the solution, followed by filling into a suitable container. In addition, sterile injection solutions can be prepared as sterile lyophilized powders (for example, by freeze-drying or vacuum drying) for reconstitution with a suitable solvent prior to use. Such lyophilized powders can be reconstituted with a suitable solvent such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (for example, 0.9% (w / v) NaCl), dextrose solution (for example, 5% dextrose), surfactant-containing solution (for example, 0.01% polysorbate 20), pH-buffered solution (for example, phosphate-buffered saline), Ringer's solution, and any combination thereof.

[0194] In certain embodiments of the methods and uses described above, the immune cells of the seventh aspect, the population of immune cells of the eighth aspect, or the pharmaceutical composition of the thirteenth aspect can be administered by any suitable method known in the art, including but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrafollicular, intracelluar webbing, inguinal, intravesical, local (e.g., powder, salve, or drops), or nasal routes. However, for many therapeutic uses, the preferred route / means of administration is parenteral administration (e.g., intravenous injection or bolus, subcutaneous injection, intraperitoneal injection, intramuscular injection). The skilled artisan will appreciate that the route and / or means of administration will vary depending on the intended purpose. In certain embodiments, the immune cells of the seventh aspect, the population of immune cells of the eighth aspect, or the pharmaceutical composition of the thirteenth aspect is administered by intravenous injection or bolus.

[0195] In certain embodiments of the methods and uses described above, the immune cells of the seventh aspect, the population of immune cells of the eighth aspect, or the pharmaceutical composition of the thirteenth aspect can be formulated in dosage unit form for ease of administration. Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0196] In certain embodiments of the methods and uses described above, the immune cells of the seventh aspect, the population of immune cells of the eighth aspect, or the pharmaceutical composition of the thirteenth aspect can be administered alone or in combination with another pharmaceutically active agent (e.g., an anti-tumor agent) or another therapy (e.g., an anti-tumor therapy).

[0197] Definitions of terms

[0198] In the present application, the scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art, unless otherwise indicated. Also, the molecular genetic, nucleic acid chemical, cell culture, biochemical, cellular biologic, and other techniques described herein are conventional techniques used by those skilled in the relevant art. In order to better understand the present application, the following definitions and explanations of terms are provided.

[0199] When the terms "for example," "for instance," "such as," "including," "containing," "consisting of," or variations thereof, are used herein, these terms are not to be interpreted in an exclusive or exhaustive sense, but rather are used merely for illustration.

[0200] The terms "a" and "an" and "the" and similar referents in the context of describing the application (especially in the context of the following claims) are to be interpreted in an inclusive rather than an exclusive utilization, unless otherwise noted or clearly contradicted by context. So, for example, a reference to "a compound" is a reference to one or more compounds and a reference to "the compound" is a reference to one or more compounds.

[0201] As used herein, the term "non-natural amino acid" refers to an amino acid other than the 20 amino acids that occur naturally in proteins. Non-limiting examples of non-natural amino acids include: Nε-2-azidoethyloxycarbonyl-L-lysine (NAEK), p-acetyl-L-phenylalanine, p-iodo-L-phenylalanine, p-methoxyphenylalanine, O-methyl-L-tyrosine, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcp-serine, L-dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-borono-phenylalanine, O-propargyltyrosine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-bromophenylalanine, selenocysteine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, non-natural analogs of tyrosine amino acids; non-natural analogs of glutamine amino acids; non-natural analogs of phenylalanine amino acids; non-natural analogs of serine amino acids; non-natural analogs of threonine amino acids; alkyl, aryl, acyl, azido, cyano, halogen, hydrazine, hydrazide, hydroxyl, alkenyl, alkynyl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate, phosphate, phosphonate, phosphine, heterocycle, enone, imine, aldehyde, hydroxylamine, keto, or amino substituted amino acids or combinations thereof; amino acids with photoactivatable cross-linkers; spin-labeled amino acids; fluorescent amino acids; metal binding amino acids; metal containing amino acids; radioactive amino acids; photocaged and / or photoisomerizable amino acids; biotin or biotin analog containing amino acids; keto containing amino acids; polyethylene glycol or polyether containing amino acids; heavy atom substituted amino acids; chemically or photo-cleavable amino acids; amino acids with extended side chains; toxic group containing amino acids; sugar substituted amino acids; carbon linked sugar containing amino acids; redox active amino acids; a-hydroxyl containing acids; aminothio acids; alpha, alpha disubstituted amino acids; beta-amino acids; cyclic amino acids other than proline or histidine; and aromatic amino acids other than phenylalanine, tyrosine, or tryptophan.

[0202] In some embodiments, the unnatural amino acid comprises a selective reactive group, or a reactive group for site-selective labeling of a target polypeptide. The chemical reaction can be a biocompatible and selective reaction, a Cu(I)-catalyzed or “copper-free” alkyne-azide triazole formation reaction, a Staudinger ligation, an inverse-electron-demand Diels-Alder (IEDDA) reaction, a “light-click” chemistry, or a metal-mediated process such as olefin metathesis and Suzuki-Miyaura or Sonogashira cross-coupling, among others.

[0203] As used herein, the term “vector” refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When the vector is capable of directing the expression of the inserted polynucleotide, the vector is referred to as an expression vector. The vector can be introduced into a host cell by transformation, transduction, or transfection, so that the host cell takes up (and can express) the genetic material carried by the vector. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1 -derived artificial chromosomes (PACs); bacteriophages, such as lambda phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to, promoter sequences, transcriptional initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, a vector can contain a replication origin.

[0204] As used herein, the term “host cell” refers to a cell that can be used to introduce a vector, including but not limited to, prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells. In certain embodiments, the host cell comprises E. coli.

[0205] As used herein, the term "chimeric antigen receptor (CAR)" refers to a recombinant polypeptide construct comprising at least one extracellular antigen binding domain, optionally a spacer domain, a transmembrane domain, and an intracellular signaling domain, which combines antibody-based specificity for an antigen of interest, e.g., a tumor antigen, with an activating intracellular domain of an immune effector cell to exhibit specific immunological activity against cells expressing the antigen of interest, such as tumor cells. In the present application, the expression "immune cell expressing a CAR" refers to an immune cell expressing a CAR and having the antigen specificity determined by the targeting domain of the CAR.

[0206] As used herein, the term "extracellular antigen binding domain" refers to a polypeptide that is capable of specifically binding an antigen or receptor of interest. The domain will be capable of interacting with a cell surface molecule. For example, the extracellular antigen binding domain can be selected to recognize an antigen that is a cell surface marker of target cells associated with a particular disease state. Typically, the extracellular antigen binding domain is an antibody-derived targeting domain.

[0207] As used herein, the term "intracellular signaling domain" refers to a protein moiety that transmits the effector function signal and directs the cell to perform a specialized function. Thus, the intracellular signaling domain has the ability to activate at least one normal effector function of the immune effector cell expressing the CAR. For example, an effector function of a T cell can be cytolytic activity or helper activity, including secretion of cytokines.

[0208] As used herein, the term "primary signaling domain" refers to a protein moiety that is capable of modulating the primary activation of the TCR complex either in a stimulatory manner or in an inhibitory manner. Primary signaling domains that act in a stimulatory manner typically contain signaling motifs known as immunoreceptor tyrosine-based activation motifs (ITAMs). Non-limiting examples of ITAMs containing primary signaling domains of particular use in the present application include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, CD22, DAP10, CD79a, CD79b, and CD66d.

[0209] As used herein, the term "co-stimulatory signaling domain" refers to an intracellular signaling domain of a co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for efficient activation and function of T lymphocytes following binding to antigen. Non-limiting examples of such co-stimulatory molecules include CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1) CD270 (HVEM), CD278 (ICOS), DAP10.

[0210] As used herein, the term "immune cell" refers to any cell of the immune system having one or more effector functions (e.g., cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and / or CDC). Typically, an immune cell is a cell that has hematopoietic origin and plays a role in the immune response. In certain embodiments, an immune cell refers to an immune effector cell. The term "effector function" refers to a specialized function of an immune effector cell, e.g., a function or response that enhances or promotes an immune attack on a target cell (e.g., killing of the target cell, or inhibition of its growth or proliferation). An effector function of a T cell, for example, can be cytolytic activity or an activity that assists or includes secretion of cytokines. Examples of immune effector cells include T cells (e.g., alpha / beta T cells and gamma / delta T cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived macrophages.

[0211] Exemplary immune effector cells useful in the CARs described herein include T lymphocytes. The term "T cell" or "T lymphocyte" is well understood in the art and is intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. A T cell can be a T helper (Th) cell, e.g., a T helper 1 (Thl) or T helper 2 (Th2) cell. A T cell can be a helper T cell (HTL; CD4 T cell), CD4 T cell, cytotoxic T cell (CTL; CD8 T cell), CD4 CD8 T cell, CD4 CD8 T cell, or any other T cell subset. In certain embodiments, T cells can include naive T cells and memory T cells.

[0212] The immune cells described herein can be self / autologous ("self") or non-self ("non-self," e.g., allogeneic, syngeneic, or xenogeneic). As used herein, "self" refers to a cell from the same subject; "allogeneic" refers to a cell from the same species as the comparison cell but with a different genetic makeup; "syngeneic" refers to a cell from a different subject that is genetically identical to the comparison cell; and "xenogeneic" refers to a cell from a different species than the comparison cell. In preferred embodiments, the cells of the present application are allogeneic.

[0213] As used herein, the term "antibody" refers to an immunoglobulin molecule that is generally comprised of two pairs of polypeptide chains (each pair having one light (LC) and one heavy (HC) chain). Antibody light chains can be assigned to a class, kappa (kappa) and lambda (lambda) light chains. Heavy chains can be assigned to a class, mu, delta, gamma, alpha, or epsilon, and define a type of antibody, IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 3 or more amino acids. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is comprised of one domain, CL. The constant domains exhibit less inter-chain variability than the variable domains, but still exhibit some inter-chain variability, particularly at the "hotspot" positions. The variable domains of the heavy and light chains, which are about 90-110 or more amino acids in size, are primarily responsible for binding the antibody to an antigen. The VHand VLregions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VHand VLis composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The assignment of amino acids to each variable domain can follow the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883. H and V L The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The assignment of amino acids to each variable domain can follow the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.

[0214] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion". See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of an antibody can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', (Fab')2, Fv, disulfide linked Fv, scFv, di-scFv, (scFv)2, and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.

[0215] As used herein, the term "Fab fragment" means an antibody fragment consisting of a VL, VH, CL and CH1 domain; the term "F(ab')2 fragment" means an antibody fragment comprising two Fab fragments connected by a disulfide bridge on the hinge region; the term "Fab' fragment" means a fragment obtained after reduction of the disulfide bond connecting the two heavy chain fragments in a F(ab')2 fragment, consisting of one complete light chain and a Fd fragment of a heavy chain (consisting of a VH and CH1 domain).

[0216] As used herein, the term "Fv" means an antibody fragment consisting of a VL and VH domain of a single arm of an antibody. An Fv fragment is generally considered to be the smallest antibody fragment that is capable of forming a complete antigen-binding site. It is generally believed that the six CDRs confer the antigen-binding specificity of an antibody. However, even a single variable region (e.g., an Fd fragment, which contains only three CDRs specific for an antigen) is capable of recognizing and binding antigen, although its affinity can be lower than that of a complete binding site.

[0217] As used herein, the term "scFv" refers to a single polypeptide chain comprising a VL and a VH domain, wherein the VL and VH are connected by a linker. Such scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeating GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used. In some cases, a disulfide bond can also exist between the VH and VL of the scFv. In certain embodiments of the application, the scFv can form a di-scFv, which refers to two or more individual scFv linked in series to form an antibody. In certain embodiments of the application, the scFv can form a (scFv)2, which refers to two or more individual scFv linked in parallel to form an antibody.

[0218] As used herein, the term "pharmaceutically acceptable carriers and / or excipients" refers to carriers and / or excipients that are compatible, in pharmacological and / or physiological terms, with the subject and active ingredient, which are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancers, diluents, agents to maintain osmotic pressure, agents to retard absorption, preservatives. For example, pH adjusting agents include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or non-ionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents to maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents to retard absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), and the like. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thiomersal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like. Stabilizers have the meaning commonly understood by those skilled in the art, which are capable of stabilizing the desired activity of the active ingredient in the pharmaceutical, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried lactose, albumin or casein) or their degradation products (such as lactalbumin hydrolysate), and the like.

[0219] As used herein, the term "prevention" refers to a method carried out in order to stop or delay the occurrence of a disease or disorder or symptoms in a subject. As used herein, the term "treatment" refers to a method carried out in order to obtain a beneficial or desired clinical result. For the purposes of this application, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Moreover, "treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0220] As used herein, the term "subject" refers to a mammal, for example a primate, e.g., a human. In certain embodiments, the subject (e.g., human) has a tumor.

[0221] Advantages of the invention

[0222] The present invention is a specific site selective PEG modification of IL2, to obtain a combination of first site modified IL-2 and second site modified IL-2, wherein the first site modified IL-2 has a blocking effect on IL2-Rα receptor and a selectivity on IL2-Rβγ receptor, can preferentially activate Teff, CTL and NK cells and reduce the activation of Treg, in addition, it also induces reduced terminal differentiation and exhaustion of CD8+ cells, and increases the proportion and number of central memory cells; the second site modified IL-2 has a blocking effect on IL2-Rβ but not on IL2-Rα, can compete with endogenous IL2 secreted by cells for IL2-Rα, and competitively inhibit the effect of endogenous IL2 on activating Treg. The combination of the two can make up for the disadvantage of the first site modified IL-2 in increasing the degree of exhaustion after cell activation, while retaining the advantage of the first site modified IL-2 in activating central memory cells, and better exert the anti-tumor therapeutic effect. The combination provided by the present invention has a significant synergistic effect of enhancing immune response and anti-tumor effect, in addition, it can also guide the fate of lymphocytes to long-term immunity in adoptive cell therapy, which has important clinical value.

[0223] Embodiments of the present application will be described in detail below with reference to the attached drawings and examples, but a person skilled in the art will understand that the following drawings and examples are only used to illustrate the present application, and are not a limitation on the scope of the present application. According to the following detailed description of the preferred embodiments and the drawings, various purposes and advantages of the present application will become apparent to those skilled in the art. BRIEF DESCRIPTION OF DRAWINGS

[0224] Figure 1: Coomassie blue staining results of pegylated IL-2 variants Y45-20K and D20-20K.

[0225] Figure 2 : Surface plasmon resonance (SPR) affinity assay results of pegylated IL-2 variants on different subunits of IL-2 receptor.

[0226] Figure 3 : Cell phosphorylated STAT5 (pSTAT5) level assay results after treatment with pegylated IL-2 variants Y45-20K and D20-20K.

[0227] Figure 4 : Analysis results of PK / PD properties of pegylated IL-2 variants Y45-20K and D20-20K.

[0228] Figure 5 : Effect of D20-20K on Y45-20K-mediated T cell proliferation and activation in vitro.

[0229] Figure 6 : Effect of D20-20K on Y45-20K-mediated T cell proliferation and activation in vivo in healthy mice.

[0230] Figure 7 : Anti-tumor effect of D20-20K in combination with Y45-20K in a mouse tumor model.

[0231] Figure 8 : Effect of D20-20K in combination with Y45-20K on T cell subsets in the lymph nodes of tumor-bearing mice.

[0232] Figure 9 : Effect of D20-20K in combination with Y45-20K on vascular leakage syndrome (VLS).

[0233] Figure 10 : Effect of D20-20K in combination with Y45-20K on the proliferation of CAR-T cells in vitro culture.

[0234] Figure 11 : Effect of D20-20K in combination with Y45-20K on the senescence and exhaustion of CAR-T cells in vitro culture.

[0235] Figure 12 : Effect of D20-20K in combination with Y45-20K on the senescence and exhaustion of T cells at different differentiation stages in CAR-T cell in vitro culture.

[0236] Figure 13 : Effect of none-α variant (Y45-20K) in combination with various none-β variants on CD8 and CD4 T cells. Figure 13a: expression of immune checkpoints PD-1 and TIM-3; Figure 13 b: expression of effector cytokines Perforin, Granzyme B and IFN-γ; Figure 13 c: expression of apoptosis-related biomarkers CD57, IL-10.

[0237] Figure 14 : none-β variant (D20-20K) in combination with various none-α variants on CD8 and CD4 T cells. Figure 14 a: expression of immune checkpoints PD-1 and TIM-3; Figure 14 b: expression of effector cytokines Perforin, Granzyme B and IFN-γ; Figure 14 c: expression of apoptosis-related biomarkers CD57, IL-10.

[0238] Sequence information

[0239] Information of part of sequences involved in the present application is provided in Table 1 below.

[0240] Table 1: Description of sequences

[0241]

[0242]

[0243] DETAILED DESCRIPTION

[0244] Embodiments of the present application will be described in detail below with reference to the examples, but those skilled in the art will understand that the following examples are only for illustration of the present application and should not be regarded as limiting the scope of the present application. The specific conditions not noted in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments not noted for the manufacturer are all conventional products that can be obtained by purchase in the market.

[0245] Synthesis of NAEK:

[0246]

[0247] To a solution of compound 2 (5.5 g, 63.2 mmol) in dichloromethane (120 ml) was added slowly at -3°C a suspension of N,N'-carbonyldiimidazole (15.36 g, 94.8 mmol) in dichloromethane (55 ml). The reaction was stirred for 12 hours. Then 200 mL of water was added and the organic layer was washed twice with brine, dried over Na2S04, filtered and concentrated under vacuum. The residue was further purified by silica gel chromatography eluting with PE / EtOAc (1 :1) to give compound 3 (10.7 g, 59 mmol) as a colorless oil in 93% yield.

[0248] To a solution of compound 3 (10.7 g, 59 mmol) in dichloromethane (100 ml) was added a solution of Boc-Lys-OH (12.2 g, 49.2 mmol) in 1 M aqueous NaOH (50 ml) at room temperature. Then TBAB (0.16 g, 0.01 eq) was added. The reaction mixture was stirred for 12 hours, cooled to 0°C and then the pH was adjusted to 2-3 with an ice-bath of 1 M aqueous HC1. The aqueous phase was extracted with DCM and the organic layer was washed twice with brine. Then the organic layer was dried over Na2S04, filtered and concentrated under vacuum. The residue was further purified by silica gel chromatography eluting with PE / EtOAc / HAc (100:100:1) to give compound 4 (15.1 g, 41.94 mmol) as a colorless oil in 85% yield.

[0249] To a solution of compound 4 (15.1 g, 41.94 mmol) in dichloromethane (80 ml) was added slowly trifluoroacetic acid (20 ml). The reaction was stirred for 0.5 hours at room temperature and then the solvent was evaporated under vacuum. The residue was re-dissolved in methanol (5 ml) and precipitated in diethyl ether. The precipitate was collected and dried under vacuum to give compound 5 (6.63 g, 25.58 mmol) as a white solid, i.e. NAEK, in 61% yield.

[0250]

[0251] Synthesis of DBCO-PEG:

[0252] ​

[0253] Compound 1, 10 g (48 mmol) hydroxylamine hydrochloride 16.8 g (240 mmol) were dissolved in anhydrous ethanol, 26 ml of pyridine was added, heated to reflux for 36 h, after cooling, 25 ml of ethyl acetate was added, 125 ml of 1 N HCl was added and stirred for 0.5 h, the organic layer was separated, the organic layer was washed with saturated NaCl solution, and after drying, the white solid compound 2 was obtained by concentration. Yield: 100%.

[0254] Compound 2, 10 g was dissolved in 50 ml of methanesulfonic acid, and 6.25 g of phosphorus pentoxide was added and stirred to dissolve. The above product was added to 10 g of compound 2, and stirred at 100 °C for 1.5 h. After the reaction solution was cooled, it was poured into 300 ml of water, and a solid was precipitated, which was filtered and dried to obtain a white solid compound 3. Yield: 100%.

[0255] 160 ml of ethyl ether was cooled to 0 °C, 5.5 g of lithium aluminum hydride was added portionwise, and then 4 g of compound 3 was added portionwise, and refluxed at 35 °C for 72 h. The reaction solution was cooled to 0 °C, and 14.8 ml of saturated sodium sulfate solution was added to quench, filtered, and concentrated to obtain a yellow solid compound 4. Yield: 98%.

[0256] 766 mg of succinic acid monomethyl ester was dissolved in 15 ml of dichloromethane, and after cooling to 0 °C, 610 ul of oxalyl chloride was added dropwise, and stirred at room temperature for 1 h. It was concentrated and used as it was. 1 g of compound 4 was dissolved in 15 ml of dichloromethane, 1.16 ml of pyridine was added, and after cooling to 0 °C, succinyl chloride monomethyl ester was added dropwise, and stirred at room temperature for 0.5 h. The organic phase was washed with 1 N HCl, 1 N NaOH, and NaCl, dried with anhydrous sodium sulfate, and concentrated to obtain a yellow solid compound 5. Yield: 99%.

[0257] Compound 5 was dissolved in methanol / water (volume ratio = 2:1), and 6 equivalents of lithium hydroxide was added at room temperature, and stirred at room temperature for 8 h. Methanol was distilled off, and water was added to dissolve, and the aqueous phase was washed with dichloromethane, and the aqueous phase was adjusted to pH 2-3 with 1 N HCl, and the aqueous phase was extracted with dichloromethane. The organic phase was washed with saturated NaCl solution, and dried to obtain a yellow oily compound 6. Yield: 98%.

[0258] 1.4 g of compound 6 was dissolved in 50 ml of dichloromethane, and cooled to 0 °C, and 2.2 g of lysine was dissolved in dichloromethane and added dropwise, and stirred at room temperature for 3 h. 50 ml of saturated sodium thiosulfate solution was added, the organic layer was separated, washed with saturated NaCl solution, dried with anhydrous sodium sulfate, and dried to obtain a light yellow solid compound 7. Yield: 95%.

[0259] Compound 8 was obtained as a white powder by dissolving 2.1 g of compound 7 in 40 ml of anhydrous tetrahydrofuran, cooling to -40°C, adding a 1M tetrahydrofuran solution of potassium tert-butoxide dropwise, stirring for 1 h, quenching with a 1M HCl solution to pH = 2-3, extracting with dichloromethane, concentrating the organic layer, and column chromatography. Yield: 80%.

[0260] Compound 9 was obtained as a yellowish solid by dissolving 1.1 g of compound 8 in 20 ml of dichloromethane, adding 495 mg of N-hydroxysuccinimide, 825 mg of EDCl, stirring for 1 h at room temperature, washing the reaction solution with water and saturated NaCl solution, drying over anhydrous sodium sulfate, and concentrating. Yield: 100%.

[0261] Compound 10 was obtained by dissolving 2 g of NH2-PEG 20k -OMe in 25 ml of dichloromethane, adding 40 mg of compound 9, stirring for 1 h at room temperature, drying the organic phase, washing the residue with diethyl ether, and column chromatography. Yield: 85%.

[0262] Example 1: Construction and expression of a site-directed mutant IL-2 protein expression plasmid

[0263] (1) Selection of mutation site

[0264] The following mutation sites were selected on the IL-2 protein (SEQ ID NO: 1), wherein the positions are positions in SEQ ID NO: 1.

[0265] Table 2: Mutation sites

[0266] Protein names Amino acid position Amino acid Pre-mutated codon Post-mutated codon Y45 45 Y TAC TAG D20 20 D GAT TAG

[0267] (2) Expression of site-directed mutant protein containing unnatural amino acid

[0268] The nucleic acid sequence comprising the codon substitution described above is linked to a nucleic acid sequence encoding a His tag and cloned into a pET-21a(+) (addgene: #69740-3) E. coli plasmid expression vector, which is co-transfected with a pSURAR-YAV plasmid into a TransB(DE3) strain (purchased from Novagen, Cat. No: CD811-02). Among them, the pSURAR-YAV plasmid encodes a suppressor codon tRNA and a NAEK-specific aminoacyl tRNA synthetase, and by co-transfecting with a nucleic acid sequence comprising a TAG codon, a non-natural amino acid NAEK can be introduced at a specific site; the pSURAR-YAV plasmid refers to the plasmid pSUPAR-YAV-tRNA / PylRS obtained from the E. coli containing plasmid pSUPAR-YAV-tRNA / PylRS classified as Escherichia coli with the preservation address of China General Microbiological Culture Collection Center (No. 1 Huiyuanli, Beichenxi Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), the preservation date of April 8, 2013, and the preservation number of CGMCC No: 7432. The transformed strain is inoculated into 2 ml of LB medium containing 100 ug / ml ampicillin and 34 ug / ml chloramphenicol, and then cultured at 37°C, 220 rpm. The overnight culture is diluted to an optical density in 2xYT medium, and the culture is incubated at 37°C until A600nm reaches about 1.5. UAA (NAEK, synthesized by the laboratory itself) is added at a final concentration of 1 mM, and then protein expression is induced by adding isopropyl beta-d-thiogalactopyranoside (IPTG) and L-arabinose at a final concentration of 0.5 mM and 0.1%, respectively, and after half an hour, the temperature is reduced to 20°C. After about 18 hours of expression, the cells are harvested by centrifugation and resuspended in His-Bind buffer (20 mM phosphate, pH 8.0, 500 mM NaCl, 20 mM imidazole). The protein is extracted by passing the cells through a Micofluidizer twice at 1200 bar and under cooling conditions. Then centrifuged at 20,000g for 20 minutes, the supernatant is collected and stored at -80°C until further processing. Thus, a site-directed mutant IL-2 protein replaced with a non-natural amino acid at position Y45 or D20 is obtained.

[0269] Example 2: PEG modification of the site-directed mutant IL-2 protein

[0270] The supernatant obtained in Example 1 was subjected to a preliminary purification by Ni-NTA agarose (R90101, Invitrogen) followed by a click reaction. To produce site-directed PEGylated IL-2 analogues, the His-tagged IL-2 in the supernatant was enriched using Ni-NTA His-Bind Resin (Invitrogen) and DBCO-PEG was synthesized and then added to the elution buffer (20 mM phosphate, pH 8.0, 500 mM NaCl, 500 mM imidazole) with a final concentration of 1 mM. The reaction was carried out at 4°C with gentle shaking for 2 hours. The PEGylated IL-2 was then purified by cation exchange chromatography (Resource S, GE Healthcare) and FPLC size exclusion chromatography (Superdex 200 increase 10 / 300 GL, GE Healthcare) to remove unreacted PEG and IL-2. The main elution peak was collected, concentrated and buffer exchanged into PBS buffer using a 3 kDa centrifugal filter unit (Millipore). The purity of the PEGylation reaction product was checked by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing conditions with Coomassie blue staining. Two PEGylated IL-2 derivatives were obtained, designated as Y45-20K and D20-20K, respectively. The electrophoresis results are shown in Figure 1. Figure 1 As shown, the purity of the PEGylated IL-2 variants was over 95%.

[0271] In addition, the following PEGylated IL-2 derivatives were also prepared by the method described in Examples 1-2: H16-20K, A73-20K, H79-20K, F42-20K, E62-20K, K64-20K, P65-20K, E68-20K, K35-20K, T37-20K, R38-20K, T41-20K, K48-20K, K49-20K. The naming of the PEGylated IL-2 variants involved in this application is as follows: [position of the non-natural amino acid compared with SEQ ID NO: 1]-[average molecular weight of the PEG group connected]. Taking Y45-20K as an example, the difference in its amino acid sequence compared with SEQ ID NO: 1 is that Y45 is replaced by the non-natural amino acid NAEK, and a PEG group with an average molecular weight of 20 kDa is further connected to this position.

[0272] Example 3: Assay of binding activity to IL-2 receptor

[0273] This example assesses the affinity of PEGylated IL-2 derivatives for IL-2R trimer and dimer complexes by surface plasmon resonance (SPR). Human IL-2Ra, IL-2Rb, and IL-2Ryc receptors were immobilized on CM5 or protein A chips for analysis on a Biacore 8K system (GE Healthcare). Extracellular domains of human IL-2Ra (C-terminal 6xHis), IL-2Rb (C-terminal Fc), and IL-2Ry (C-terminal 6xHis) were purchased from Sino Biological. All kinetic experiments were performed at 25 °C using 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Surfactant P, pH 7.4 (running buffer). Data were analyzed using Biacore 8K evaluation software and fitted using a 1:1 stable affinity model to determine KD values and other kinetic parameters. Protein concentrations were measured using the BCA method (Pierce). Native IL-2 protein (purchased from PeproTech, Cat# 96-200-02-1000) was used as a control. The concentration of all samples was the mass of IL-2 without PEG attached.

[0274] Results are shown in Table 1, where NA is no binding. Figure 2 As shown in Table 1, where NA is no binding. The results show that D20-20K, H16-20K, A73-20K, H79-20K have no affinity for the beta subunit, have significantly reduced binding affinity for the dimer IL-2R, but maintain binding to the alpha chain of the trimer IL-2R, with a bias for non-beta receptors, which can be referred to as non-beta PEGylate. Y45-20K, F42-20K, E62-20K, K64-20K, P65-20K, E68-20K have no binding to alpha, have completely eliminated binding affinity for the trimer IL-2R, maintain binding to the dimer IL-2R, with a bias for non-alpha receptors, which can be referred to as non-alpha PEGylate; K35-20K, T37-20K, R38-20K, T41-20K, K48-20K, K49-20K have binding to alpha in the order of 1e-7~1e-6, which is weak binding, but have significantly higher binding affinity for the dimer IL-2R than for the trimer IL-2R, or although the binding affinity for the dimer IL-2R is weaker than for the trimer IL-2R, the gap (e.g. ratio) between the two is significantly smaller than the gap in binding affinity of native IL-2 for the dimer IL-2R and the trimer IL-2R, with significantly improved bias for the dimer IL-2R, and thus also belong to having a bias for non-alpha receptors, which can be referred to as non-alpha PEGylate.

[0275] Example 4: Determination of the activation tendency of Y45-20K and D20-20K on CD8+ T cells and Treg cells

[0276] This embodiment determines whether PEGylated IL-2 variants Y45-20K or D20-20K preferentially activate CD8+ T cells rather than Treg cells by measuring the level of phosphorylated STAT5 (pSTAT5), a key downstream mediator of IL-2R signaling. Approximately 2 × 10⁻⁶ 5 YT-1 cells (expressing CD122 and CD132 receptors), CD25+YT-1 (a stable cell line of YT-1 cells transduced with lentivirus to stably express the CD25 receptor), and human PBMC cells were seeded in 96-well plates and resuspended in RPMI complete medium containing serially diluted natural human IL-2 or PEGylated IL-2. Cells were stimulated at 37°C for 15 min and then immediately fixed by adding formaldehyde to 2.0% and incubating at room temperature for 15 min. Cell permeabilization was achieved by resuspending in ice-cold 87% methanol at 4°C for 30 min. Fixed and permeabilized cells were washed twice with FACS buffer and stained with the following antibodies for flow cytometry analysis: anti-human CD3-APC / Cy7, CD4-PE / Cy7, CD8-FITC, CD25-APC, CD127-PE, CD56 / CD16-Brilliant Violet 605, and pSTAT5-Pacific Blue (all purchased from Biolegend). For the pSTAT5 assay in specific hPBMC subsets, the cell populations were isolated and purified using a magnetic separation kit (Miltenyi Biotech) according to the manufacturer's instructions to obtain memory CD4+ T cells (MPCD4), memory CD8+ T cells (MPCD8), NK cells, and Treg cells, respectively.

[0277] All FACS antibodies were used at a dilution of 1 :50. Cells were then washed twice in staining buffer and mean fluorescence intensity (MFI) was determined on a CytoFLEX flow cytometer (Beckman-Coulter). Data were plotted as background subtracted MFI normalized to the maximum signal for each cell type (IL-2, 1 pg ml1). Background was defined as pSTAT5 MFI in unstimulated cells. Treg cells were defined as CD3+CD8-CD4+CD25highCD127low; NK cells as CD3-CD16+CD56+; CD8+ T cells as CD3+CD4-CD8+; MPCD4 cells as CD3+CD4+CD8-CD45RO+; MPCD8 cells as CD3+CD4-CD8+CD56-CD57-CD45RA-. After subtracting MFI of unstimulated cells and normalizing to maximum signal strength, dose response curves were fitted to a logistic model using GraphPad Prism data analysis software and half maximal effective concentration (EC50 values) and corresponding 95% confidence intervals were calculated. Experiments were performed in triplicates and repeated three times with similar results.

[0278] Results of the testing using NK-derived YT-1 cells and CD25+ YT-1 cell model are shown in Figure 3 a, where Y45-20K showed a clear distinction on CD25 (IL-2Ra)- and CD25 (IL-2Ra)+ YT-1 cells, clearly different from wild-type IL-2; D20-20K showed only baseline levels of Treg stimulation activity (5-20% pSTAT5 activation) at very high doses, which is related to its inability to bind to the beta subunit.

[0279] In addition, the selective activation of Teff cells by PEGylated IL-2 variants was re-evaluated using various immune cell subpopulations isolated from PBMCs of healthy donors, and the results are shown in Figure 3 b. We found that IL-2 stimulated Tregs and memory CD4+ T, memory CD8+ T and NK cells at all tested doses, while Y45-20K preferentially induced STAT5 phosphorylation in memory CD4+, memory CD8+ and NK cells, but not in Treg cells. D20-20K did not induce STAT5 phosphorylation in memory CD4+, memory CD8+, NK and Treg cells.

[0280] The above results are consistent with the results of the binding affinity evaluation, indicating that the pleiotropism of IL-2 can be relieved by blocking the specific region of the receptor binding site, and Y45-20K has the ideal ability to completely lack the priority to stimulate Teffs rather than Treg cells for IL-2Rα binding, and D20-20K significantly reduces the activation of both CD8+T and Treg cells.

[0281] Example 5: Analysis of PK / PD characteristics of PEGylated IL-2 variants Y45-20K / D20-20K

[0282] This example investigates the pharmacokinetic characteristics of PEGylated IL-2 variants. Female C57BL / 6 mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) with an average body weight of about 20 g were randomly divided into different groups (n = 3) and injected subcutaneously with PBS, wild-type human IL-2 or PEGylated IL-2 variants (0.25 mg / kg, based on hIL-2). At selected time points (30 min, 2 h, 4 h, 8 h, 24 h, 48 h, 72 h and 96 h), blood (100 μL each time) was taken from the orbital venous plexus of the mice, then centrifuged at 4000 g for 15 minutes at 4°C. The plasma was separated and stored at -80°C. The concentration of IL-2 was determined using a human IL-2 ELISA kit (Sinobiological). Pharmacokinetic parameters were analyzed by Kinetica 5.1 software, and expressed as mean ± standard deviation.

[0283] The results are shown in Table 1. Figure 4 Compared with wild-type IL-2, the PEGylated IL-2 variants have increased clearance half-life (T1 / 2), enhanced maintenance of blood concentration (area under the drug concentration-time curve, AUC), and increased mean residence time (MRT), with a peak time 14 times longer than IL-2, indicating a significant reduction in clearance rate. The above results show that Y45-20K has superior pharmacokinetic characteristics. In addition, D20-20K also has superior pharmacokinetic characteristics.

[0284] Example 6: Synergistic anti-tumor effect of PEGylated IL-2 variants Y45-20K / D20-20K

[0285] The data of the above examples have demonstrated that Y45-20K has the activity of preferentially activating CD8+T cells, however, it still activates Treg cells at high dose. As demonstrated in the above examples, D20-20K maintains the selective affinity to IL-2Ra while significantly reducing the activation of CD25+YT-1, this unique property is likely related to its undetectable binding to IL-2Rβ, therefore, if D20-20K is combined with Y45-20K for treatment, D20-20K can spatially occupy IL-2Ra and competitively block its mild interaction with Y45-20K leading to the over-activation of CD8+T cells and terminal differentiation and activation of Tregs. In this regard, this example investigates the synergistic anti-tumor effect of D20-20K and Y45-20K.

[0286] 6.1 Effect of D20-20K on Y45-20K-mediated T cell proliferation and activation in vitro

[0287] Experimental procedure

[0288] This example investigates whether there is a dose-dependent effect of D20-20K on the activation bias of Y45-20K. 2x10 5 Human PBMC cells were resuspended in 96-well plates with 100ul RPMI1640+10%FBS, 50ul of 0.4ug / ml Y45-20K (final concentration 0.1ug / ml) was added to the experimental wells, and then 50ul of D20-20K at different concentration gradients was added to make the final concentrations 1ug / ml, 0.1ug / ml, 0.01ug / ml, 0.001ug / ml, respectively, and the experimental wells were set in triplicate from three different healthy individuals. Cells were incubated at 37°C for 72h, then washed twice with FACS buffer, and stained with the following antibodies for flow cytometry analysis: anti-human CD3-APC / Cy7, CD4-PE / Cy7, CD8-FITC, CD25-APC, Foxp3-Pacific Blue, CD69-PE. Treg cells were defined as CD3+CD8-CD4+CD25+Foxp3+, and CD8+T cells were defined as CD3+CD4-CD8+, and the number of Treg and CD8 T cells and the mean fluorescence intensity (MFI) of their respective activation markers (Treg-Foxp3, CD8+T-CD69) were counted by flow cytometry.

[0289] 2x10 5YT-1 cells, CD25+YT-1 were seeded in 96-well plates, resuspended in 96-well plates with 100 ul RPMI1640 + 10% FBS, 50 ul of 0.4 ug / ml Y45-20K (final concentration 0.1 ug / ml) was added to the experimental wells, and then 50 ul of different concentration gradient of D20-20K was added to make the final concentration 1 ug / ml, 0.1 ug / ml, 0.01 ug / ml, 0.001 ug / ml respectively. Cells were stimulated at 37°C for 15 minutes, then immediately fixed by adding formaldehyde to 2.0% and incubating at room temperature for 15 minutes. Permeabilization of cells was achieved by suspension in ice-cold 87% methanol for 30 minutes at 4°C. Fixed and permeabilized cells were washed twice with FACS buffer and stained with pSTAT5-Pacific Blue antibody for flow cytometry analysis. Antibody was used at a dilution of 1:50. Cells were then washed twice in staining buffer and mean fluorescence intensity (MFI) was determined on a CytoFLEX flow cytometer (Beckman-Coulter). Data were plotted as MFI minus background, normalized to the maximum signal for each cell type (Y45-20K, 0.1 ug / ml -1 ). Background was defined as pSTAT5 MFI in unstimulated cells. After subtracting the MFI of unstimulated cells and normalizing to the maximum signal intensity, dose response curves were fitted to a logistic model using GraphPad Prism data analysis software and half maximal effective concentration (EC50 values) and corresponding 95% confidence intervals were calculated. Experiments were performed in triplicate and repeated three times with similar results.

[0290] Results are shown in Figure 5 , D20-20K has a dose-dependent inhibitory effect on Y45-20K-mediated activation and proliferation of Treg cells but not CD8+ T cells Figure 5 a for the effect on Treg proliferation and activation, Figure 5 c for the effect on CD8+ T proliferation and activation). STAT5 phosphorylation in CD25+ YT-1 cells was inhibited by D20-20K in a dose-dependent manner with an EC50 of 0.39 ug / ml Figure 5 e), while this inhibition was not observed in naive YT cells Figure 5 f).

[0291] 6.2 Effect of D20-20K on Y45-20K-mediated T proliferation and activation in vivo in healthy mice

[0292] Experimental procedure

[0293] C57BL / 6 mice (6-8 weeks old, female) were administered 45-20K (0.25 mg / kg x 3, every other day), 20-20K (0.25 mg / kg x 3, every other day), 45-20K and 20-20K (0.25 mg / kg x 3, every other day) or IL-2 (0.25 mg / kg every day x 5) by subcutaneous injection at the back of the neck, with an equal volume of PBS as a blank control. Three days after the end of administration, flow cytometry was performed on the spleen and lymph nodes.

[0294] Results are shown in Figure 6 Compared with other groups of mice, the proportion of CD8+ T cells and MPCD8+ T in the spleen of mice treated with Y45-20K / D20-20K combination therapy was further increased, and the proliferation of Tregs was significantly reduced in mice treated with Y45-20K / D20-20K combination therapy compared with Y45-20K alone.

[0295] 6.3 Anti-tumor effect of D20-20K and Y45-20K in a mouse tumor model

[0296] C57BL / 6 mice (6-8 weeks old) were implanted with B16F10 melanoma cells (China Cell Bank (Shanghai, China), 5x 10 5 ) subcutaneously in the right flank of each animal. The mice were sacrificed when the tumor reached 1500 mm 3 in size. The tumor volume was calculated as follows: V = a2*b / 2, where a is the width and b is the length of the tumor (both in millimeters). When the tumor was measured as 100 mm 3 , the animals were administered 45-20K (0.25 mg / kg x 3, every other day), 20-20K (0.25 mg / kg x 3, every other day), 45-20K and 20-20K (0.25 mg / kg x 3, every other day) or IL-2 (0.25 mg / kg every day x 5). On day 14, the tumor was minced and digested in a buffer containing 2 mg / ml collagenase type II and IV (GIBCO BRL) and 0.5 mg / ml DNase (Sigma Aldrich) at 37°C for 13 minutes to form a single-cell suspension, after which the immune cell subtypes in the tumor microenvironment were determined by flow cytometry. Immunohistochemical analysis of T cell density was performed on frozen tumor sections 14 days after administration.

[0297] Results are shown in Figure 7 The combination of Y45-20K and D20-20K treatment showed a very prominent anti-tumor effect, with a significant reduction in tumor burden and a significant prolongation of mouse survival Figure 7a) With the decrease of tumor burden, the proportion of CD8+ T cells in the spleen, tumor draining lymph nodes (TDLN) and especially in tumor tissue increased in Y45-20K / D20-20K combination therapy mice compared with other mice, but the proportion of Treg cells did not change significantly Figure 7 b) The results were consistent with immunohistochemical staining, which showed that co-treatment of Y45-20K and D20-20K induced the largest amount of T cell infiltration in tumor tissue compared with native IL-2 and PBS treatment Figure 7 c).

[0298] Memory CD8 cells play an important role in tumor immunity. Analysis of T cell subsets in the lymph nodes of tumor-bearing mice showed that the number of Tcm and MPCD8 cells in the lymph nodes increased by about 10 times in the Y45-20K alone treatment group or the Y45-20K combined with D20-20K treatment group, and the number of Tcm and MPCD8 cells in the Y45-20K combined with D20-20K treatment group was further improved based on the Y45-20K alone treatment group. Correspondingly, the number of T and Tem in the two experimental groups was slightly reduced Figure 8 a), indicating that Y45-20K can significantly expand the number of central memory T lymphocytes in vivo, and D20-20K alone cannot expand central memory cells, but can further increase the proportion and number of central memory cells in the CD8 subset in combination with Y45-20K, which is beneficial to exert the anti-tumor immune efficacy. In addition, the expression of CD25 marker in Tcm cell subset was further reduced, and the expression of CD122 marker was further increased, indicating that Tcm in the lymph nodes of mice in the Y45-20K alone treatment group or the Y45-20K combined with D20-20K treatment group can avoid the excessive activation of T cells by endogenous IL-2, reduce the sensitivity to IL-2, and improve the response ability to foreign antigen response. Although the Y45-20K alone treatment group can activate and expand the proliferation of Tcm, the number of MPCD8, T and Tem cell subsets increased significantly compared with the IL-2 treatment group, and the combination of D20-20K can reduce the expression of immune checkpoints in the above cell subsets, indicating that D20-20K combined with Y45-20K can retain the activation advantage of central memory cells of Y45-20K, and also compensate for the disadvantage of increased exhaustion of Y45-20K after cell activation, and better exert the anti-tumor treatment effect.

[0299] 6.4 Effect of D20-20K combined with Y45-20K on vascular leakage syndrome (VLS)

[0300] Conventional IL-2 therapy has a high risk of vascular leakage syndrome (VLS), which is generally believed to be caused by the binding of IL-2 to CD25+ lung endothelial cells, and accordingly this embodiment also evaluates the effect of combination therapy on pulmonary edema. A similar treatment regimen as described above is adopted, except that the administration of the PEGylated IL-2 variant is changed to a high administration frequency (every day, a total of five times). After the mice are sacrificed, the lung tissue is weighed, a lung tissue cell suspension is obtained for flow detection, and lung tissue sections are prepared for immunohistochemical analysis.

[0301] The results are shown in Figure 9 The lung cells expressing high levels of CD31 but not other immune cell lineage markers (Lin) were defined as endothelial cells (Ly5.2-B220-CD3-NK1.1-CD11b-CD11c-CD31+). It was found that the Y45-20K single administration group caused significant pulmonary edema, manifested as an increase in lung wet weight Figure 9 a), a decrease in the proportion of lung endothelial cells Figure 9 b), and an increase in lung lymphocyte infiltration; however, this side effect was significantly reduced after co-administration with D20-20K, possibly due to the spatial occupation of IL-2Rα on lung endothelial cells by D20-20K.

[0302] The above data show that Y45-20K and D20-20K exhibit significant synergy in anti-tumor therapy, selectively inducing CD8+ T cells with minimal effect on Treg cells, and are able to alleviate VLS by protecting lung endothelial cells from activation by Y45-20K or endogenous IL-2, with a significantly advantageous technical effect.

[0303] Example 7: Advantage of PEGylated IL-2 variant Y45-20K / D20-20K for CAR-T cell in vitro culture

[0304] IL-2 is a key nutritional factor for CAR-T cell in vitro expansion culture, which greatly weakens the therapeutic effect of CAR-T cell therapy due to its inevitable activation of Treg cells in donor cells and inevitable over-activation of CD8+ T cells and its terminal differentiation affecting CAR-T cell activity, and accordingly this experiment evaluates the effect of Y45-20K combined with D20-20K instead of conventional IL-2 on CAR-T cell therapy.

[0305] 7.1 Effect of D20-20K combined with Y45-20K on CAR-T cell in vitro culture proliferation

[0306] Construction of CAR lentiviral vector:

[0307] The anti-CD19 CAR contains FMC63 anti-CD19 scFv (SEQ ID NO: 12), CD8a hinge region (SEQ ID NO: 14) and transmembrane region (SEQ ID NO: 16) and 4-1BB cytoplasmic domain (SEQ ID NO: 18) and CD3z cytoplasmic domain (SEQ ID NO: 20), the expression cassette was synthesized by Suzhou Eko Biotech Co., Ltd. and cloned into a lentivirus vector. HEK293T cells were transfected with anti-CD19 CAR, pspax2 (addgene, #12260) and pMD2.g (addgene, #12259) plasmids using Lipofectamine 3000 (Life Technologies). The medium was changed 6 hours after transfection, and the viral supernatant was collected 48 hours after transfection. The viral particles were concentrated 30-fold by ultracentrifugation at 25,000 rpm for 2 hours, and frozen at -80°C until ready for use.

[0308] Preparation of CAR-T:

[0309] Human T cells were purified from peripheral blood mononuclear cells using Dynabead Human T Cell Kit (Life Technologies) and activated with CD3 / cd28 magnetic beads (Life Technologies) for 24 hours before infection. Concentrated lentivirus (FMC63-AntiCD19-CAR Lentivirus) was applied to activated human T cells (10 6 cells / mL in 24-well plates, MOI = 1) with the addition of 10 mg / mL polybrene and 100 ng / mL IL-2 or 100 ng / mL 45-20K, 100 ng / mL D20-20K, 100 ng / mL D20-20K + 100 ng / mL Y45-20K centrifuged at 1000g for 2h at 32°C. The next day, the supernatant was replaced with fresh medium containing IL-2 and its analogs corresponding to the group, and the transduced T cells were placed in complete growth medium at 0.5x10 6 cells / mL, and the medium containing IL-2 was changed every 3 days. Cell number and apoptosis status were monitored in real time.

[0310] Results are shown in Figure 10 Figure 6, none of the replacement of IL2 with Y45-20K, D20-20K or Y45-20K combined with D20-20K affected the transduction efficiency of CAR Figure 10a) After 10 days of culture with IL-2 variants, it was found that D20-20K replaced IL-2 to culture CAR-T cells almost no proliferation, while Y45-20K replaced IL-2 to culture cells proliferated significantly faster than IL-2, Y45-20K combined with D20-20K to replace IL-2 to culture CAR-T cells Proliferation rate similar to Y45-20K alone Figure 10 b) Apoptosis detection found that IL-2 culture can cause CAR-T cell apoptosis, which is related to the overactivation of IL-2 to T cells and the induction of terminal differentiation, while Y45-20K combined with D20-20K hardly induced CAR-T cell apoptosis Figure 10 c).

[0311] 7.2 D20-20K combined with Y45-20K on CAR-T cell in vitro culture aging, exhaustion

[0312] Experimental steps

[0313] The above prepared CAR-T was cultured for 10-14 days in the corresponding IL-2, and the related detection of cell phenotype was carried out. Surface antibody was fluorescently stained with fluorescently labeled antibody (CD4-PE / Cy7, CD8-FITC, CD25-BV785, CD57-PB, CD45RA-BV510, CD62L-Percp / cy5.5, CCR7-PE, PD-1-BV650, LAG-3-BV421) Incubate at 4°C for 30 minutes. For intranuclear staining, CAR-T cells were fixed and permeated using Foxp3 / transcription factor staining buffer (eBioscience), and then stained with Foxp3 antibody in perm buffer. The sample was washed twice with PBS and suspended in flow cytometry staining buffer (eBioscience). For intracellular cytokine staining, 500x Cell Activation Cocktail (with Brefeldin A, eBioscience, 423303) was used, and after 6h 37°C culture, washing was performed. After the last washing, the cells were fixed and permeated and stained according to the manufacturer's instructions using eBioscience intracellular fixation and permeation buffer kit (Thermo Fisher Scientific). Then incubate with specific antibodies at 4°C for 30 minutes: IL-2-BV421, IL-10-APC, Granzy B-percp / cy5.5.

[0314] Results are as follows Figure 11As shown, the secretion of granzyme B and IL10 of CAR-T cells cultured in vitro with PMA stimulation was significantly lower in the Y45-20K combined with D20-20K group than in the IL-2 group, indicating that the terminal differentiation and aging degree of CAR-T cells were reduced Figure 11 a, b), and the secretion of IL-2 was significantly increased, also indicating that CAR-T cells were in a stem state with a lower degree of differentiation Figure 11 c). Second, flow cytometry showed that the surface exhaustion and aging-related receptors such as PD-1, LAG-3 and CD57 of CAR-T cells cultured with Y45-20K combined with D20-20K were significantly reduced Figure 11 d-f), further proving that Y45-20K combined with D20-20K can reverse the aging and exhaustion state of CAR-T cells caused by IL-2 for in vitro culture.

[0315] 7.3D20-20K combined with Y45-20K on the aging and exhaustion of T cells at different differentiation stages in CAR-T cell in vitro culture

[0316] Experimental steps

[0317] Human T cells were purified from peripheral blood mononuclear cells using Dynabead Human T Cell Kit (Life Technologies), and CAR-T was prepared using the above method. To further study the effect of different cytokines on the differentiation of CAR-T cells, we sorted CAR-T cells at different differentiation stages by flow cytometry (sorting type, Symphony S6, BD) for separate culture. T cells were defined as CD3+CD8+CD45RA+CD45RO-CCR7+CD62L-CD95-; Tscm was defined as CD3+CD8+CD45RA+CD45RO-CCR7+CD62L-CD95+; Tcm was defined as CD3+CD8+CD45RA-CD45RO+CCR7+CD62L+; Tem was defined as CD3+CD8+CD45RA-CD45RO+CCR7-CD62L-; T effector was defined as CD3+CD8+CD45RA+CD45RO0-CCR7-CD62L-; Treg was defined as CD3+CD4+CD25 high CD127 low . and cultured with different cytokines for 10-14 days, and the cell number and growth state were monitored in real time.

[0318] We studied the initial T cells (T The aging and exhaustion degree of T memory stem cells (Tscm), T effector cells (T effector) and regulatory T cells (Treg) were detected. CD62L is a characteristic marker of cell stemness, and D20-20K combined with Y45-20K can further improve the T and the expression amount of CD62L in Tscm cells Figure 12 a), which shows that D20-20K combined with Y45-20K can further retain the stemness state of T cells. The detection results of the proliferation of T cells at different differentiation stages show that D20-20K combined with Y45-20K can further enhance the proliferation of T cells, reduce the proliferation of Treg cells, and have no significant effect on the proliferation of Tscm and T effector cells Figure 12 b). PD-1 and TIM-3 are markers of T cell exhaustion. The detection of T effector cells in the terminal differentiation state shows that D20-20K combined with Y45-20K can further reduce the expression amount of PD-1 and TIM-3, and reduce the exhaustion degree of T effector cells Figure 12 c). Finally, the detection of the expression amount of CD25 on T cells induced by IL-2 at different differentiation stages shows that D20-20K combined with Y45-20K can significantly reduce the expression amount of CD25 on the surface of Tscm, T effector and Treg cells, especially relative to Y45-20K alone, further reducing the expression amount of CD25 on Treg cells Figure 12 d), the decrease of the expression amount of CD25 can avoid the excessive activation of T cells caused by endogenous IL-2 produced by T cell activation, showing the superiority of D20-20K combined with Y45-20K.

[0319] Example 8: Effect of combination of None-α variant and None-β variant on maintaining T cell stemness and reducing T cell exhaustion

[0320] In this embodiment, the Non-α variants (F42-20K, Y45-20K, E62-20K, P65-20K, E68-20K) and Non-β variants (D20-20K, H16-20K, A73-20K, H79-20K) prepared in Examples 1-2 were combined for in vitro activity verification.

[0321] Experimental steps:

[0322] Take the peripheral blood of healthy people, separate PBMC with peripheral blood human lymphocyte separation medium, separate CD3 positive T lymphocytes with magnetic bead sorting kit, add CD3 / CD28 stimulation magnetic beads at a ratio of 1:1, and control the inoculation density at 1x106 Flow cytometry was performed on 24-well plates containing 1 ml of different cytokines at the same concentration (100 ng / ml). The medium was changed every 72 hours, and the culture was repeated for two weeks. Antibodies used for detection included: APC / Cy7 anti-human CD3, PE / Cy7 anti-human CD4, FITC anti-human CD8, BV785 anti-human PD-1, BV605 anti-human TIM-3, Pacific Blue anti-human CD57, APC anti-human IL-10, PE anti-human Perforin, Percp / cy5.5 anti-human Granzyme B, and BV510 anti-human IFN-γ.

[0323] Experimental results:

[0324] Treatment with the none-α variant (Y45-20K) resulted in decreased expression of the immune checkpoints PD-1 and TIM-3 on the surface of CD8 and CD4 T cells compared to IL-2. Figure 13 a) The expression levels of effector cytokines Perforin, Granzyme B, and IFN-γ were significantly reduced. Figure 13 b) The expression levels of apoptosis-related biomarkers CD57 and IL-10 decreased. Figure 13 c) This indicates that none-α can reduce the exhaustion of T cells during in vitro culture to a certain extent and maintain the stem-cell-like characteristics of the cells. When the none-α variant (Y45-20K) is used in combination with the none-β variant (D20-20K, H16-20K, A73-20K, H79-20K), the expression of PD-1 and TIM-3 is further reduced, the expression levels of effector cytokines Perforin, Granzyme B, and IFN-γ are further decreased, and the expression levels of apoptosis-related biomarkers CD57 and IL-10 are further reduced. Figure 13 ac).

[0325] On the basis of the above screening results, the none-β variant (D20-20K) is preferred, and the effect of combination with different none-α variants (F42-20K, Y45-20K, E62-20K, P65-20K, E68-20K) on T cell activation is verified. The experimental results show that the combination of D20-20K with different none-α variants has different effects on improving the survival state of CD8 and CD4 T cells, but can further reduce the expression of immune checkpoints PD-1 and TIM-3, the expression of effector cytokines Perforin, Granzyme B and IFN-γ, and the expression of apoptosis-related biomarkers CD57 and IL-10 to a certain extent Figure 14 a-c). Therefore, the combination of None-β and None-α can further improve the survival state of T cells on the basis of the effect of None-α, such as reducing the degree of T cell growth exhaustion, reducing the apoptosis induced by excessive activation, and delaying the differentiation into terminal effector cells.

[0326] The above results show that the combination of non-α variants with a bias for non-α receptors and non-β variants with a bias for non-β receptors can produce obvious synergistic effect and bring significant beneficial therapeutic effect.

[0327] Although the specific embodiments of the present application have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details according to all the teachings disclosed herein, and these changes are within the scope of protection of the present application. The entire scope of the present application is given by the appended claims and any equivalents thereof.

Claims

1. A composition comprising: (1) The first site-modified IL-2, which, compared with wild-type IL-2, contains a PEG group modification at the first amino acid position, wherein, The first site-modified IL-2 does not bind to the IL-2 receptor α (IL-2Rα) subunit or binds with a KD value greater than 1E-8 M; and (2) The second site-modified IL-2, which contains a PEG group modification at the second amino acid position compared with wild-type IL-2, wherein the second site-modified IL-2 does not bind to the IL-2 receptor β (IL-2Rβ) subunit; Wherein, the first amino acid is at position Y45, and the second amino acid is at position D20; The amino acid sequence of the wild-type IL-2 is SEQ ID NO: 1; the residue at the first amino acid position is mutated to a non-natural amino acid, and the non-natural amino acid is attached to the PEG group; the residue at the second amino acid position is mutated to a non-natural amino acid, and the non-natural amino acid is attached to the PEG group.

2. The composition of claim 1, wherein: (a) The average molecular weight of the PEG-modifying groups contained in the first site-modified IL-2 is 5 to 60 kDa; and / or, (b) The average molecular weight of the PEG-modifying groups contained in the second site-modified IL-2 is 5 to 60 kDa.

3. The composition of claim 1, wherein: The average molecular weight of the PEG-modifying groups contained in the first site-modified IL-2 is 5~40 kDa, and / or the average molecular weight of the PEG-modifying groups contained in the second site-modified IL-2 is 5~40 kDa.

4. The composition of claim 1, wherein: The average molecular weight of the PEG-modifying groups contained in the first site-modified IL-2 is 5~20 kDa, and / or the average molecular weight of the PEG-modifying groups contained in the second site-modified IL-2 is 5~20 kDa.

5. The composition of claim 1, wherein: The average molecular weight of the PEG-modifying groups contained in the first site-modified IL-2 is 5 kDa, 10 kDa, or 20 kDa, and / or the average molecular weight of the PEG-modifying groups contained in the second site-modified IL-2 is 5 kDa, 10 kDa, or 20 kDa.

6. The composition of claim 1, wherein, The non-natural amino acid contains a chemical functional group; the PEG group contains a labeling group that can chemically react with the chemical functional group, thereby the PEG group is linked to the non-natural amino acid.

7. The composition of claim 6, wherein, The chemical functional group is a carbonyl group, an alkynyl group, or an azide group.

8. The composition of claim 6, wherein, The non-natural amino acid contains an azide group, and the PEG group contains a labeling group that can undergo a click chemical reaction with the azide group, thereby linking the PEG group to the non-natural amino acid.

9. The composition of claim 8, wherein, The labeling group that can undergo a click chemical reaction with the azide group is a chemical moiety containing a dibenzocyclooctyn group.

10. The composition of claim 8, wherein, The labeling group that can undergo a click chemical reaction with the azide group is DBCO, DIBO, or BCN.

11. The composition of claim 8, wherein, The non-natural amino acids are lysine derivatives or tyrosine derivatives containing azide groups.

12. The composition of claim 8, wherein, The non-natural amino acid is Nε-2-azidoethoxycarbonyl-L-lysine or 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid.

13. A method for preparing the composition according to any one of claims 1-12, comprising preparing the first site-modified IL-2 and preparing the second site-modified IL-2, wherein, The preparation of the first site-modified IL-2 includes: - Provides: (a1) a first site-directed mutated IL-2, wherein the residue at the first amino acid position is mutated to a non-natural amino acid compared to wild-type IL-2; (b1) a PEG group modified by a labeling group, the labeling group being capable of forming a covalent bond with the non-natural amino acid; - Co-incubate (a1) and (b1) to couple non-natural amino acids with PEG groups through a chemical reaction; The preparation of the second site-modified IL-2 includes: - Provides: (a2) a second site-directed mutated IL-2, wherein the residue at the second amino acid position is mutated to a non-natural amino acid compared to wild-type IL-2; (b2) a PEG group modified by a labeling group, the labeling group being capable of forming a covalent bond with the non-natural amino acid; - Co-incubate (a2) and (b2) to couple non-natural amino acids with PEG groups through a chemical reaction.

14. The method of claim 13, wherein, The non-natural amino acid described in (a1) contains a chemical functional group, and the labeling group contained in the PEG group described in (b1) can react with the chemical functional group to form a covalent bond; and / or, the non-natural amino acid described in (a2) contains a chemical functional group; and the labeling group contained in the PEG group described in (b2) can react with the chemical functional group to form a covalent bond.

15. The method of claim 14, wherein, The chemical functional group is a carbonyl group, an alkynyl group, or an azide group.

16. The method of claim 14, wherein, The non-natural amino acids described in (a1) and (a2) contain azide groups.

17. The method of claim 14, wherein, The non-natural amino acids mentioned in (a1) and (a2) are lysine derivatives or tyrosine derivatives containing an azide group.

18. The method of claim 14, wherein, The non-natural amino acids mentioned in (a1) and (a2) are Nε-2-azidoethoxycarbonyl-L-lysine or 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid.

19. The method of claim 13, wherein, The preparation of the first site-modified IL-2 includes: - Provides: (a1) a first site-directed mutated IL-2, wherein the residue at the first amino acid position of wild-type IL-2 is mutated to a non-natural amino acid containing an azide group; (b1) a PEG group modified by a labeling group, said labeling group being capable of a click chemical reaction with the azide group; - Co-incubate (a1) and (b1) to couple non-natural amino acids to PEG groups via a click reaction; The preparation of the second site-modified IL-2 includes: - Provides: (a2) a second site-directed mutated IL-2, wherein the residue at the second amino acid position of wild-type IL-2 is mutated to a non-natural amino acid containing an azide group; (b2) a PEG group modified by a labeling group, said labeling group being capable of a click chemical reaction with the azide group; - Co-incubate (a2) and (b2) to couple non-natural amino acids to PEG groups via a click reaction.

20. The method of claim 19, wherein, The click chemical reaction is a copper-free click chemical reaction.

21. The method of claim 19, wherein, The labeling group that can undergo a click chemical reaction with the azide group is a chemical moiety containing an alkynyl group.

22. The method of claim 21, wherein, The labeling group that can undergo a click chemical reaction with the azide group is a chemical moiety containing a dibenzocyclooctyn group.

23. The method of claim 21, wherein, The labeling group that can undergo a click chemical reaction with the azide group is DBCO, DIBO, or BCN.

24. The method according to any one of claims 13-23, wherein, The first site-directed mutation IL-2 and the second site-directed mutation IL-2 are provided by non-natural amino acid orthogonal translation technology.

25. The method of claim 24, wherein, The non-natural amino acid orthogonal translation technique includes the following steps: - Obtain the nucleic acid sequence encoding IL-2 with site-directed mutation, wherein the codon corresponding to the amino acid position to be mutated is mutated to TAG; - The nucleic acid sequence encoding the site-directed mutation of IL-2 is operatively ligated to a vector to obtain a site-directed mutation sequence expression vector; - The site-directed mutation sequence expression vector was co-transfected with a vector encoding amber codon-repressed tRNA and aminoacyl-tRNA synthetase specific to non-natural amino acids into host cells, and the cells were cultured and induced to express the expression in a medium containing non-natural amino acids to obtain IL-2 with site-directed mutations to non-natural amino acids.

26. The method of claim 25, wherein, The non-natural amino acid is Nε-2-azidoethoxycarbonyl-L-lysine (NAEK); the aminoacyl-tRNA synthetase specific to the non-natural amino acid is a NAEK-specific aminoacyl-tRNA synthetase.

27. A kit comprising: the composition according to any one of claims 1-12.

28. The kit of claim 27, wherein, The kit further includes a package insert containing instructions for using the composition to prepare and / or culture immune cells for adoptive cell therapy in vitro.

29. The kit of claim 28, wherein, The immune cells used for adoptive cell therapy are modified immune cells that express chimeric antigen receptors and / or contain nucleic acid molecules encoding said chimeric antigen receptors; or, the immune cells used for adoptive cell therapy are tumor-infiltrating lymphocytes.

30. A kit comprising: the composition of any one of claims 1-12 and a nucleic acid molecule encoding a chimeric antigen receptor.

31. The kit according to claim 30, wherein, The kit further includes a packing instruction that includes instructions for using the composition and nucleic acid molecules to prepare, in vitro, modified immune cells for adoptive cell therapy, the modified immune cells expressing a chimeric antigen receptor and / or containing nucleic acid molecules encoding the chimeric antigen receptor.

32. The kit according to claim 30, wherein, The nucleic acid molecule encoding the chimeric antigen receptor is contained in an expression vector; the expression vector is a viral vector.

33. The kit according to claim 32, wherein, The viral vector is a lentivirus, a retrovirus, or an adenovirus.

34. A kit comprising: the composition according to any one of claims 1-12 and immune cells for adoptive cell therapy.

35. The kit according to claim 34, wherein, The kit further includes a package insert containing instructions for using the composition to culture the immune cells in vitro for adoptive cell therapy.

36. The kit according to claim 34, wherein, The immune cells used for adoptive cell therapy are modified immune cells that express chimeric antigen receptors and / or contain nucleic acid molecules encoding the chimeric antigen receptors.

37. The kit of claim 36, wherein, The modified immune cells include T cells, NK cells, or any combination thereof.

38. The kit according to claim 34, wherein, The immune cells used for adoptive cell therapy are tumor-infiltrating lymphocytes.

39. A method for culturing immune cells for adoptive cell therapy, the method comprising culturing the cells in a cell culture medium containing a first site-modified IL-2 and a second site-modified IL-2, wherein, The first point-modified IL-2 and the second point-modified IL-2 are defined as in any one of claims 1-12.

40. The method of claim 39, wherein, The immune cells used for adoptive cell therapy are modified immune cells that express chimeric antigen receptors and / or contain nucleic acid molecules encoding the chimeric antigen receptors.

41. The method of claim 40, wherein, The modified immune cells include T cells, NK cells, or any combination thereof.

42. The method of claim 40, wherein, The immune cells used for adoptive cell therapy are tumor-infiltrating lymphocytes.

43. A method for preparing immune cells for adoptive cell therapy, wherein the immune cells for adoptive cell therapy are modified immune cells that express a chimeric antigen receptor and / or contain a nucleic acid molecule encoding the chimeric antigen receptor, wherein, The method includes: (1) Provide immune cells from patients or healthy donors; (2) In the presence of a first site-modified IL-2 and a second site-modified IL-2, a nucleic acid molecule encoding a chimeric antigen receptor is introduced into the immune cells of step (1) to provide the modified immune cells; wherein the first site-modified IL-2 and the second site-modified IL-2 are as defined in any one of claims 1-12.

44. The method of claim 43, wherein, Step (2) is performed in a cell culture medium containing the first site-modified IL-2 and the second site-modified IL-2.

45. The method of claim 43, wherein, The nucleic acid molecule encoding the chimeric antigen receptor described in step (2) is present in the expression vector.

46. ​​The method of claim 43, wherein, In step (2), the nucleic acid molecule encoding the chimeric antigen receptor is introduced into the cell by a viral vector through infection.

47. The method of claim 46, wherein, The viral vector is a lentivirus, a retrovirus, or an adenovirus.

48. The method of claim 43, wherein, In step (1), the immune cells are pretreated, the pretreatment including sorting, activation and / or proliferation of the immune cells.

49. The method of claim 48, wherein, The pretreatment involves contacting immune cells with anti-CD3 and anti-CD28 antibodies to stimulate the immune cells and induce their proliferation, thereby generating pretreated immune cells.

50. The method of claim 43, wherein, The method further includes, after step (2), step (3) culturing the immune cells obtained in step (2) in a cell culture medium containing the first site-modified IL-2 and the second site-modified IL-2.

51. The method of claim 43, wherein, The immune cells include T cells, NK cells, or any combination thereof.

52. A method for preparing immune cells for adoptive cell therapy, wherein the immune cells for adoptive cell therapy are tumor-infiltrating lymphocytes, wherein, The method includes: isolating infiltrating lymphocytes from tumor tissue and culturing them in a cell culture medium containing a first site-modified IL-2 and a second site-modified IL-2, wherein the first site-modified IL-2 and the second site-modified IL-2 are as defined in any one of claims 1-12.

53. Use of the composition according to any one of claims 1-12 for the in vitro preparation or culture of immune cells for adoptive cell therapy.

54. The use as described in claim 53, wherein, The immune cells used for adoptive cell therapy are modified immune cells that express chimeric antigen receptors and / or contain nucleic acid molecules encoding the chimeric antigen receptors.

55. The use as described in claim 54, wherein, The modified immune cells include T cells, NK cells, or any combination thereof.

56. The use as described in claim 53, wherein, The immune cells used for adoptive cell therapy are tumor-infiltrating lymphocytes.

57. A pharmaceutical composition comprising the composition according to any one of claims 1-12 and a pharmaceutically acceptable carrier and / or excipient; wherein, The first site-modified IL-2 and the second site-modified IL-2 are in separate compositions or dosage forms; or, the first site-modified IL-2 and the second site-modified IL-2 are in the same composition or dosage form.

58. Use of the composition according to any one of claims 1-12 or the pharmaceutical composition according to claim 57 in the preparation of a medicament for enhancing immune response, preventing and / or treating tumors; wherein, The tumor was selected from melanoma and renal cell carcinoma.

59. The use as described in claim 58, wherein, The first site-modified IL-2 and the second site-modified IL-2 in the composition are applied simultaneously, separately, or sequentially.

60. A medicine box, comprising: The kit comprises a first drug containing a first site-modified IL-2 and an optional pharmaceutically acceptable carrier and / or excipient in the composition of any one of claims 1-12, and a second drug containing a second site-modified IL-2 and an optional pharmaceutically acceptable carrier and / or excipient in the composition of any one of claims 1-12; the kit further comprises instructions for use, the instructions for use containing instructions for administering the first drug and the second drug to enhance an immune response, prevent and / or treat tumors in a subject; wherein, The tumor was selected from melanoma and renal cell carcinoma.

61. Use of the immune cells obtained by the method of any one of claims 39-52 in the preparation of medicaments for enhancing immune responses, preventing and / or treating tumors; wherein, The tumor was selected from melanoma and lymphoma.

62. The use as described in claim 61, wherein, The immune cells are administered in combination with the first site-modified IL-2 and the second site-modified IL-2 as defined in any one of claims 1-12.

63. Use of the composition of any one of claims 1-12 in combination with immune cells for adoptive cell therapy in the preparation of a medicament for enhancing immune response, preventing and / or treating tumors; wherein the tumor is selected from melanoma and lymphoma.

64. The use as described in claim 63, wherein, The immune cells used for adoptive cell therapy are modified immune cells that express chimeric antigen receptors and / or contain nucleic acid molecules encoding the chimeric antigen receptors.

65. The use as described in claim 63, wherein, The immune cells used for adoptive cell therapy are tumor-infiltrating lymphocytes.

Images