CN116036243A

CN116036243A - Receptor-biased PEGylated IL-2 variant combinations and uses thereof

Info

Publication number: CN116036243A
Application number: CN202211376914.9A
Authority: CN
Inventors: 周德敏; 纪德重; 孙家琦; 张博
Original assignee: Ningbo Institute Of Marine Medicine Peking University; Peking University
Current assignee: Ningbo Institute Of Marine Medicine Peking University; Peking University
Priority date: 2022-04-20
Filing date: 2022-11-04
Publication date: 2023-05-02
Also published as: WO2023202035A1

Abstract

The present invention relates to the field of immunotherapy, in particular, the present invention relates to site-directed modification of pegylated IL-2 variants to provide combinations of pegylated IL-2 variants with different receptor bias. The composition can inhibit immunosuppression lymphocyte proliferation and reduce terminal differentiation and depletion of effector lymphocyte by synergistic effect, has synergistic antitumor effect, and can be used for enhancing immune response and treating proliferation diseases such as tumor. In addition, the combination can also be used for in vitro culture of immune cells in adoptive cell immunotherapy, reducing aging and depletion of immune cells, so that fate of immune cells in adoptive cell immunotherapy leads to long-term immunity. Thus, the invention also provides combination therapies for enhancing immune responses, treating proliferative diseases such as tumors, and in vitro methods of preparing or culturing immune cells for adoptive cell therapy.

Description

Receptor-biased PEGylated IL-2 variant combinations and uses thereof

Technical Field

The present invention relates to the field of immunotherapy, in particular, the present invention relates to site-directed modification of pegylated IL-2 variants to provide combinations of pegylated IL-2 variants with different receptor bias. The invention also provides combination therapies for enhancing immune responses, treating proliferative diseases such as tumors, and in vitro methods of preparing or culturing immune cells for adoptive cell therapy.

Background

Interleukin-2 (IL 2) is an important class of cytokines, B cells, T cells and NK cellsCytokines necessary for cell development and function. Receptors for IL2 are classified into IL2-Rα monomer, IL2-Rβγ dimer and IL2-Rαβγ trimer, wherein IL2 has a weak affinity for α monomer of 10 ^-8 M, moderate affinity for βγ dimer of 10 ^-9 M has an affinity with αβγ trimer of at most 10 ^-11 M. Because of the low affinity, either dimeric IL-2R or endogenous IL-2 needs to be induced at high levels to produce "dimeric IL-2R: IL-2" responsiveness, which in contrast to trimeric IL-2R, allows host cells to react directly to low concentrations of IL-2. Thus, it is currently believed that the α chain of trimeric IL-2R does not participate in signaling, but rather initiates binding to IL-2 (KD. Apprxeq.10-8M) and subsequently 1000-fold increased affinity to the βγ complex.

The distribution of the receptors for IL2 across different immune cells also varies: IL2-Rαβγ trimer is expressed on the surface of regulatory T cells (Treg) for a long period of time, and therefore has the highest affinity for IL 2; IL2-Rβγ dimer is expressed on resting effector T cells (Teff), killer T Cells (CTL) and NK cells, and generally has an affinity for IL2, and only after activation of effector T cells and NK cells, the IL2-Rαβγ trimer is expressed and has an increased affinity for IL 2. Evidence has shown that during cd8+ T cell priming, alpha chain expression is rather dynamic—a portion of T cells up-regulate alpha chain expression and sense strong IL-2 signals, leading to faster T cell proliferation but eventually differentiation, which in turn limits their effectiveness. In contrast, low alpha T cells that are less sensitive to IL-2 preferentially up-regulate CD127 and CD62L, resulting in functional long-life memory cells. Having more memory T cell populations in vivo becomes valuable without altering the fundamental process of alpha chain upregulation, but is challenging for clinical applications. Thus, in tumor therapy, the imbalance ratio adjustment of Teff/Treg and CTL/Treg is gradually becoming a new development direction of immunotherapy.

The related drugs of the IL2 target spot on the market at present have no receptor selectivity. In the treatment of tumors, higher doses of IL2 are required when Teff and CTL are to be activated, thereby also bringing about serious systemic toxicity, which is currently the main source of side effects. In addition, the interaction of IL2 with IL 2-ra in lung endothelial cells also induces vascular leakage syndrome, and immunosuppression caused by activation of Treg cells also leads to limited drug response.

Therefore, solving the problem that the existing IL2 antitumor drugs have no selectivity to the receptor is of great importance for the clinical application of IL 2-based tumor immunotherapy.

Disclosure of Invention

As described above, although IL-2 has been widely used to enhance immune cells in the body, the attendant properties of promoting expansion of immunosuppressive lymphocytes and driving terminal differentiation and depletion of effector lymphocytes have hampered the effectiveness of IL-2 as an antitumor agent.

The inventors herein report a strategy to explore the dynamic expression pattern of IL-2 receptors by two classes of receptor-biased PEGylated IL-2 variants, such that the fate of initiating lymphocytes has persistent anti-tumor immunity properties. Specifically, the first class of receptor-biased PEGylated IL-2 variants are non-alpha PEGylates, which can activate and expand CD8+ T cells, CD4+ cells, and even NK cells, rather than Treg cells, due to their biased affinity for dimer rather than trimer IL-2R, and which in addition induce reduced terminal differentiation and depletion of CD8+ cells. The second class of receptor-biased pegylated IL-2 variants are non-beta peylates which activate neither cd8+ T nor Treg cells, but which act synergistically with the first class of non-alpha peylates to further expand cd4+ and cd8+ T cells and reduce Treg and differentiated and depleted cd8+ T cell populations due to their maintenance of moderate affinity for the a chain of trimeric IL-2R, exhibiting excellent local and systemic anti-tumor responses.

The inventors subsequently demonstrated in a CD 19-targeted CAR-T therapy model that the above-described first class of receptor-biased pegylated IL-2 variants (non-alpha pegylated) and second class of receptor-biased pegylated IL-2 variants (non-beta pegylated) direct the fate of lymphocytes to long-term immunity through synergy. The experimental evidence supports a putative mechanism by which the sequence of IL-2:IL-2R interactions is altered by co-receptor biased PEGylated IL-2 variants, which not only circumvents the pleiotropic effects of IL-2, but also optimizes the differential memory program of T cells, indicating direction for the next generation of IL-2 in T lymphocytes and adoptive T cell transfer therapies.

Based on the above-described first class of receptor-biased PEGylated IL-2 variants (non-alpha PEGylates) and second class of receptor-biased PEGylated IL-2 variants (non-beta PEGylates), the present invention provides combinations of PEGylated IL-2 variants with different receptor bias. The composition has synergistic effect in inhibiting proliferation of immunosuppressive lymphocyte, reducing terminal differentiation and depletion of effector lymphocyte, increasing proportion and number of central memory cells, and synergistic antitumor effect, and can be used for enhancing immune response and treating hyperplasia diseases such as tumor. In addition, the combination can also be used for in vitro culture of immune cells in adoptive cell immunotherapy, reducing aging and depletion of immune cells, enhancing T

Cell proliferation, reduced proliferation of Treg cells, maintenance of proliferation of Tscm and T effector cells, reduced degree of depletion of T effector cells, and avoidance of excessive activation of T cells by endogenous IL-2, leading to fate-directed long-term immunity of immune cells in adoptive cell immunotherapy. Thus, the invention also provides combination therapies for enhancing immune responses, treating proliferative diseases such as tumors, and in vitro methods of preparing or culturing immune cells for adoptive cell therapy.

Composition and method for producing the same

In a first aspect, the present invention provides a composition comprising:

(1) A first site-modified IL-2 comprising a PEG group modification at a residue at a first amino acid position compared to wild-type IL-2, wherein the first site-modified IL-2 does not bind to an IL-2 receptor alpha (IL-2rα) subunit or binds with a KD value greater than 1E-8M (e.g., on the order of 1E-7 to 1E-6M); and

(2) A second site-specific modified IL-2 comprising a PEG group modification at a residue at a second amino acid position compared to wild-type IL-2, wherein the second site-specific modified IL-2 does not bind to an IL-2 receptor β (IL-2rβ) subunit.

Those of skill in the art understand that compositions comprising a first site-directed modified IL-2 and a second site-directed modified IL-2 do not mean that the two must be administered and/or formulated simultaneously 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 may 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 may 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.

In certain embodiments, the composition comprises a plurality of 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 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 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 those in SEQ ID NO. 1.

In certain embodiments, "unbound" as described herein means that no binding is detected by Surface Plasmon Resonance (SPR). In certain embodiments, KD values described herein are determined by Surface Plasmon Resonance (SPR).

Receptor bias for first site-directed modification of IL-2

The first site-modified IL-2 described herein has a bias towards non-alpha receptors (non-alpha).

In certain embodiments, the bias towards non-alpha receptors (non-alpha) comprises: (i) Binding to IL2-Rβγ dimer with a KD value of less than 1E-8M (e.g., on the order of 1E-9 to 1E-8M), and/or (ii) not binding to IL2-Rαβγ trimer or binding with a KD value of greater than 1E-8 (e.g., on the order of 1E-7 to 1E-6).

I. Binding properties to IL-2Rα

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

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

Binding Properties to IL2-Rβγ dimer

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

Binding Properties to IL2-Rαβγ trimer

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

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

In certain embodiments, when the first site-modified IL-2 has a low affinity for an IL2-rαβγ trimer, the first site-modified IL-2 has a first affinity for an IL2-rβγ dimer that is higher than a second affinity for an IL2-rαβγ trimer (i.e., the first KD value for binding to an IL2-rβγ dimer is lower than the second KD value for binding to an IL2-rαβγ trimer). In certain embodiments, the first affinity of the first site-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-rαβγ trimer. In certain embodiments, the first amino acid position of such first site-modified IL-2 is selected from K35, T37, T41, K48.

In certain embodiments, when the first-site modified IL-2 has a low affinity for IL2-rαβγ trimer, the first-site modified IL-2 may also have a first affinity for IL2-rβγ dimer that is no higher (e.g., lower) than a second affinity for IL2-rβγ trimer (i.e., a first KD value that binds IL2-rβγ dimer that is no lower (e.g., higher) than a second KD value that binds IL2-rαβγ trimer), but the difference in affinity of the first-site modified IL-2 for IL2-rβγ dimer and IL2-rαβγ trimer (e.g., the ratio of the KD values) is less than the difference in affinity of the native IL-2 for IL2-rβγ dimer and IL2-rβγ trimer (e.g., the ratio of the KD values). In such embodiments, the first KD value for the first site-modified IL-2 binding IL2-rβγ dimer is not greater than 10-fold, e.g., not greater than 9-fold, not greater than 8-fold, not greater than 7-fold, not greater than 6-fold, not greater than 5-fold, not greater than 4-fold, not greater than 3-fold, or not greater than 2-fold, of the second KD value for binding IL2-rβγ trimer. In certain embodiments, the first site-modified IL-2 binds to IL2-rβγ dimer with a first KD value that is 8-3 times, e.g., 7-3 times, that of the second KD value that binds to IL2-rαβγ trimer. In certain embodiments, native IL-2 binds IL2-Rβγ dimer with a first KD value on the order of 1E-9M. In certain embodiments, native IL-2 binds IL2-Rαβγ trimer with a second KD value on the order of 1E-11M. In certain embodiments, the first KD value for binding of native IL-2 to IL2-Rβγ dimer is about 40 times the second KD value for binding to IL2-Rαβγ trimer. In certain embodiments, the first amino acid position of such first site-modified IL-2 is selected from R38, K49.

Receptor bias for second site-directed modification of IL-2

The second site-directed modification described herein of IL-2 has a bias towards non-beta receptors (non-beta).

In certain embodiments, the bias towards non- β receptors (non- β) comprises: (i) Binding to the IL2-Rαβγ trimer with a KD value of less than 1E-8 (e.g., on the order of 1E-9 to 1E-8), and/or (ii) not binding to the IL2-Rβγ dimer.

In certain embodiments, the second site-directed modified IL-2 does not bind to an IL-2 receptor beta (IL-2 Rbeta) subunit.

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

In certain embodiments, the second site-directed modified IL-2 does not bind to IL2-rβγ dimer.

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

In certain embodiments, the second amino acid position of the second site-directed modified IL-2 is selected from the group consisting of 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.

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.

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

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

In certain embodiments, the first site-specific modified IL-2 comprises a PEG-modifying group and the second site-specific modified IL-2 comprises a PEG-modifying group having substantially the same average molecular weight.

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

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

In certain embodiments, the unnatural amino acid contains chemical functional groups, e.g., carbonyl, alkynyl, and azide groups, etc., that are generally effective and selective for forming stable covalent bonds; the PEG group comprises a labeling group capable of chemically reacting with the chemical functional group to form a covalent bond, whereby the PEG group is attached to the unnatural amino acid.

In certain embodiments, the unnatural amino acid contains an azide group, and the PEG group comprises a label group that is capable of click chemistry with the azide group, such that the PEG group is attached to the unnatural amino acid.

In certain embodiments, the unnatural amino acid is a lysine derivative that contains an azide group. In certain embodiments, the unnatural amino acid is N ε -2-azidoethoxycarbonyl-L-lysine (NAEK).

In certain embodiments, the unnatural amino acid is a tyrosine derivative that contains an azide group. In certain embodiments, the unnatural amino acid is 2-amino-3- (4- (azidomethyl) phenyl) propanic acid.

In certain embodiments, the labeling group that is capable of click chemistry with an azide group is a chemical moiety comprising a dibenzocyclooctyne group, such as Dibenzocyclooctyne (DBCO), 4-Dibenzocyclooctynol (DIBO), or BCN (bicyclo [6.1.0] none).

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

wherein the direction from R1 to R2 is the N-terminal to C-terminal direction of the amino acid sequence, wherein the amino acid at the N-position is the residue at the first amino acid position (such as the amino acid at the F42, Y45, E62, P65 or E68) or the residue at the second amino acid position (such as the amino acid at the D20, H16, A73 or H79), R1 is the amino acid residue at the 1-N-1 position of the amino acid sequence of IL-2, R2 is the amino acid residue at the N+1-C-terminal of the amino acid sequence of IL-2, and R3 is a PEG group.

In certain embodiments, the tag group that is capable of click chemistry with an azide group is DIBO, the residue of the first site-specific modified IL-2 at a first amino acid position (e.g., amino acid position F42, Y45, E62, P65, or E68) as compared to wild-type IL-2 and the residue of the second site-specific modified IL-2 at a second amino acid position (e.g., amino acid position D20, H16, A73, or H79) as compared to wild-type IL-2 is replaced by a structure of formula Ib:

In certain embodiments, the labeling group that is capable of click chemistry with an azide group is BCN, the residue of the first site-specific modified IL-2 at a first amino acid position (e.g., amino acid position F42, Y45, E62, P65, or E68) as compared to wild-type IL-2 and the residue of the second site-specific modified IL-2 at a second amino acid position (e.g., amino acid position D20, H16, a73, or H79) as compared to wild-type IL-2 is replaced by a structure of formula Ic:

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

In certain exemplary embodiments, the first site-directed modification 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, A-20K or H79-20K. Taking Y45-20K as an example, this refers to the following PEGylated IL-2 variant: the amino acid sequence of this variant differs from that of SEQ ID NO. 1 in that Y45 is replaced by the unnatural amino acid NAEK and that position is further linked to a PEG group of average molecular weight 20 kDa.

Preparation of the composition

The first site-specific modified IL-2 and the second site-specific modified IL-2 comprised in the composition provided by the first aspect of the invention may be prepared by any method known in the art.

In certain embodiments, the unnatural amino acid can be inserted site-specifically by orthogonal translation techniques of the unnatural amino acid, followed by attachment of a PEG group to the unnatural amino acid.

Techniques for orthogonal translation of unnatural amino acids are well known to those skilled in the art. The unnatural amino acid orthogonal translation technique inserts unnatural amino acids into the amino acid sequence of a protein during translation of the protein using stop codons, effectively expanding the number of amino acid codons, and thus the unnatural amino acid orthogonal translation technique is also referred to as a genetic codon expansion technique. Typically, an unnatural amino acid orthogonal translation system involves a tRNA, an aminoacyl tRNA synthetase, and a nucleic acid sequence of interest with 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 unnatural amino acids to be inserted. The host cell is then maintained under conditions that allow expression of the protein of interest. One or more unnatural amino acids are incorporated into the polypeptide chain in response to the unnatural codon.

In certain embodiments, unnatural amino acids can be site-directed inserted by orthogonal translation techniques of unnatural amino acids that can contain chemically functional groups, e.g., carbonyl groups, alkynyl groups, azide groups, etc., that are generally capable of efficiently and selectively forming stable covalent bonds, followed by site-directed modification of PEG groups by chemical reaction to form covalent bonds.

In certain embodiments, the unnatural amino acid can be site-directed inserted by an unnatural amino acid orthogonal translation technique, followed by site-directed modification of the PEG group by a click chemistry reaction. In certain embodiments, the unnatural amino acid and PEG group, respectively, comprise a chemical group that is capable of undergoing a click chemistry reaction.

In a second aspect, the invention provides a method of preparing the composition of the first aspect, comprising preparing the first site-specific modified IL-2 and preparing the second site-specific modified IL-2, wherein,

preparing the first site-modified IL-2 comprises:

-providing: (a1) A first site-directed mutant IL-2 that is mutated to an unnatural amino acid at a residue at a first amino acid position (e.g., amino acid position F42, Y45, E62, P65, or E68) as compared to wild-type IL-2; (b1) A PEG group modified with a labeling group, the labeling group being capable of forming a covalent bond with the unnatural amino acid;

-co-incubating (a 1) with (b 1) to couple the unnatural amino acid with the PEG group by chemical reaction;

preparing the second site-directed modified IL-2 comprises:

-providing: (a2) A second site-directed mutant IL-2 that is mutated to an unnatural amino acid at a residue at a second amino acid position (e.g., amino acid position D20, H16, a73, or H79) as compared to wild-type IL-2; (b2) A PEG group modified with a labeling group, the labeling group being capable of forming a covalent bond with the unnatural amino acid;

-co-incubating (a 2) with (b 2) and coupling the unnatural amino acid to the PEG group by chemical reaction.

In certain embodiments, the first site-directed mutant IL-2 comprises an unnatural amino acid that contains a chemical functional group, e.g., a carbonyl group, an alkynyl group, an azide group, etc., that is generally capable of efficiently and selectively forming a stable covalent bond; the PEG group comprises a labeling group capable of chemically reacting with the chemical functional group to form a covalent bond.

In certain embodiments, the second site-directed mutated IL-2 comprises an unnatural amino acid that has a chemical functional group, e.g., a carbonyl group, an alkynyl group, an azide group, etc., that is generally effective and selective for forming a stable covalent bond; the PEG group comprises a labeling group capable of chemically reacting with the chemical functional group to form a covalent bond.

In certain embodiments, the unnatural amino acid in the first site-directed mutated IL-2 and the second site-directed mutated IL-2 comprise the same chemical functional group.

In certain embodiments, the unnatural amino acid in the first site-directed mutated IL-2 and the second site-directed mutated IL-2 comprises an azide group.

In certain embodiments, the unnatural amino acid in the first site-directed mutated IL-2 and the second site-directed mutated IL-2 are the same.

In certain embodiments, preparing the first site-modified IL-2 comprises:

-providing: (a1) A first site-directed mutant IL-2 that is mutated to an unnatural amino acid that contains an azide group at a residue at a first amino acid position (e.g., amino acid position F42, Y45, E62, P65, or E68) as compared to wild-type IL-2; (b1) A PEG group modified with a labeling group that is capable of click chemistry with an azide group;

-co-incubating (a 1) with (b 1) and coupling the unnatural amino acid to the PEG group by a click reaction.

In certain embodiments, preparing the second site-directed modified IL-2 comprises:

-providing: (a2) A second site-directed mutant IL-2 that is mutated to an unnatural amino acid that contains an azide group at a residue at a second amino acid position (e.g., amino acid position D20, H16, a73, or H79) as compared to wild-type IL-2; (b2) A PEG group modified with a labeling group that is capable of click chemistry with an azide group;

-co-incubating (a 2) with (b 2) and coupling the unnatural amino acid to the PEG group by a click reaction.

In certain embodiments, the click chemistry is a copper-free click chemistry. Copper-free click chemistry is a click reaction that is accomplished by introducing cyclooctyne to maintain cell activity, where the tension of the octatomic ring allows for reaction with azide in the absence of a catalyst. One of these agents consists of a so-called DBCO compound. Azide-modified macromolecules can now be labeled without a metal catalyst, which not only can be used for living cell studies, but also prevents damage to the protein.

In certain embodiments, the labeling group that is capable of click chemistry with an azide group is a chemical moiety comprising an alkyne group. In certain embodiments, the labeling group that is capable of click chemistry with an azide group is a chemical moiety comprising a dibenzocyclooctyne group. In certain embodiments, the labeling group that is capable of click chemistry with an azide group is Dibenzocyclooctyne (DBCO), 4-Dibenzocyclooctynol (DIBO), or BCN (bicyclo [6.1.0] none). In certain embodiments, the labeling group that is capable of click chemistry with an azide group is DBCO.

In certain embodiments, the DBCO-labeled PEG group has a structure according to formula IIa, wherein R3 is a PEG group.

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

In certain embodiments, the first site-directed mutated IL-2 and the second site-directed mutated IL-2 are provided by unnatural amino acid orthogonal translation techniques.

In certain embodiments, the unnatural amino acid orthogonal translation technique comprises the steps of:

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

-operably linking said nucleic acid sequence encoding site-directed mutated IL-2 with a vector to obtain a site-directed mutated sequence expression vector;

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

In certain embodiments, the unnatural amino acid is N ε -2-azidoethoxycarbonyl-L-lysine (NAEK). In certain embodiments, the aminoacyl-tRNA synthetase specific to an unnatural amino acid is a NAEK-specific aminoacyl-tRNA synthetase.

In certain embodiments, the vector encoding the amber codon-suppressing tRNA and the aminoacyl tRNA synthetase that is specific for an unnatural amino acid is pSURAR-YAV (also referred to as pSUPAR-YAV-tRNA/PyleRS), which is obtained from Escherichia coli harboring the plasmid pSUPAR-YAV-tRNA/PyleRS, deposited with the China general microbiological culture Collection center (national institute of microorganisms, national institute of sciences of China, having a collection number of CGMCC No:7432 the classification is designated as Escherichia coli.

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

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

Kit for detecting a substance in a sample

The combination of the first site-specific modified IL-2 and the second site-specific modified IL-2 provided by the invention can reduce the aging and the exhaustion of immune cells in the in vitro culture of the immune cells, and strengthen T

Cell proliferation, reduced proliferation of Treg cells, maintenance of proliferation of Tscm and T effector cells, reduced degree of depletion of T effector cells, and avoidance of excessive activation of T cells by endogenous IL-2, leading to fate-directed long-term immunity of immune cells in adoptive cell immunotherapy. Based on this, the invention also provides kits as described below, in vitro methods of preparing or culturing immune cells for adoptive cell therapy, and immune cells for adoptive cell therapy obtained by the methods.

In a third aspect, the invention provides a kit comprising a composition according to the first aspect. In certain embodiments, the kit further comprises package insert comprising instructions for using the composition to prepare and/or culture immune cells for adoptive cell therapy in vitro. The invention also relates to the use of a composition according to the first aspect or a kit according to the third aspect for in vitro preparation or culture of immune cells for adoptive cell therapy.

Herein, adoptive cell therapy (adoptive cell therapy) may include tumor infiltrating T cell (TIL) therapy, chimeric antigen receptor T cell therapy (CAR-T), T cell receptor Therapy (TCR), NK cell therapy, and the like. In this context, the engineered immune cells for adoptive cell therapy may be any cells known in the art for adoptive cell therapy. In certain embodiments, the engineered immune cells for adoptive cell therapy include lymphocytes, such as T cells, NK cells, or combinations thereof.

In certain embodiments, the immune cell for adoptive cell therapy is an engineered immune cell that expresses a chimeric antigen receptor and/or comprises a nucleic acid molecule encoding the chimeric antigen receptor. In certain embodiments, the engineered immune cells include lymphocytes that express IL2-rαβγ trimers, such as T cells, NK cells, or a combination thereof.

In certain embodiments, the immune cells used for adoptive cell therapy are Tumor Infiltrating Lymphocytes (TILs).

In a fourth aspect, the invention provides a kit comprising a composition according to the first aspect and a nucleic acid molecule encoding a chimeric antigen receptor. In certain embodiments, the kit further comprises packaging instructions comprising instructions for using the composition and the nucleic acid molecule to prepare an engineered immune cell for adoptive cell therapy in vitro, the engineered immune cell expressing a chimeric antigen receptor and/or comprising a nucleic acid molecule encoding the chimeric antigen receptor.

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

In certain embodiments, the expression vector is a viral (e.g., lentiviral, retrovirus, or adenovirus) vector. In certain embodiments, the expression vector is a non-viral vector.

The invention also relates to the use of a composition according to the first aspect and optionally a nucleic acid molecule encoding a chimeric antigen receptor or a kit according to the fourth aspect for in vitro preparation of an engineered immune cell expressing a chimeric antigen receptor and/or comprising a nucleic acid molecule encoding said chimeric antigen receptor for adoptive cell therapy.

The invention also provides a kit comprising a composition according to the first aspect and an immune cell for adoptive cell therapy. In certain embodiments, the kit further comprises package insert comprising instructions for using the composition to culture the immune cells in vitro for adoptive cell therapy. The invention also relates to a composition according to the first aspect and optionally an immune cell for adoptive cell therapy for use in vitro culturing an immune cell for adoptive cell therapy.

In certain embodiments, the chimeric antigen receptors described herein have a meaning well known to those of skill in the art, typically comprising an extracellular antigen binding domain, an optional spacer domain, a transmembrane domain, and one or more intracellular signaling domains. In certain embodiments, the intracellular signaling domain is selected from a primary signaling domain and/or a co-stimulatory signaling domain. In certain embodiments, the extracellular antigen-binding domain comprises an antibody or antigen-binding fragment (e.g., scFv) that specifically binds a tumor-associated antigen (e.g., CD 19).

Culture and preparation of immune cells for adoptive cell therapy

In a fifth aspect, the invention 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-specific modified IL-2 and a second site-specific modified IL-2, wherein the first site-specific modified IL-2 and the second site-specific modified IL-2 are as defined in the first aspect.

The cell culture medium may be any medium capable of supporting cell growth, typically contains inorganic salts, vitamins, glucose, buffer systems, and essential amino acids, and typically has an osmolality of about 280-330 mOsmol. In certain embodiments, the cell culture medium is a medium capable of supporting the growth of immune cells (e.g., 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 basal medium (e.g., RPMI 1640), serum (e.g., FBS), sodium pyruvate, non-essential amino acids. In certain embodiments, the cell culture medium does not comprise serum.

In certain embodiments, the methods further comprise harvesting the cells for storage (e.g., reconstitution in cryopreservation medium) or administration (e.g., for adoptive cell therapy).

In certain embodiments, provided culture conditions in the presence of a first site-specific modified IL-2 and a second site-specific modified IL-2 reduce aging and depletion of immune cells, preserve their dry state, and enhance T

The proliferation of the cells is reduced, the proliferation of the Treg cells is reduced, the proliferation states of the Tscm and T effector cells are maintained, the depletion degree of the T effector cells is reduced, and the excessive activation of the T cells by endogenous IL-2 generated by the activation of the T cells is avoided. />

In a sixth aspect, the invention provides a method of preparing an immune cell for adoptive cell therapy comprising using a first site-modified IL-2 and a second site-modified IL-2 as defined in the first aspect.

In certain embodiments of the sixth aspect, the present invention provides a method of preparing an immune cell for adoptive cell therapy, the immune cell for adoptive cell therapy being an engineered immune cell that expresses a chimeric antigen receptor and/or comprises a nucleic acid molecule encoding the chimeric antigen receptor, wherein the method comprises:

(1) Providing immune cells from a patient or healthy donor;

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

In certain embodiments, step (2) is performed in a cell culture medium comprising the first site-specific modified IL-2 and the second site-specific modified IL-2. The cell culture medium may be any medium capable of supporting cell growth, typically contains inorganic salts, vitamins, glucose, buffer systems, and essential amino acids, and typically has an osmolality of about 280-330 mOsmol. In certain embodiments, the cell culture medium is a medium capable of supporting the growth of immune cells (e.g., 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 basal medium (e.g., RPMI 1640), serum (e.g., FBS), sodium pyruvate, non-essential amino acids. In certain embodiments, the cell culture medium does not comprise serum.

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

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

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

In certain embodiments, in step (1), the immune cells are pre-treated, including sorting, activation and/or proliferation of immune cells. In certain embodiments, the pretreatment comprises contacting the immune cells with an anti-CD 3 antibody and an anti-CD 28 antibody, thereby stimulating and inducing proliferation of the immune cells, thereby generating pretreated immune cells. In certain embodiments, the pretreatment comprises isolating T cells from Peripheral Blood Mononuclear Cells (PBMCs).

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

In certain embodiments, the immune cells include lymphocytes that express IL2-rαβγ trimer, such as T cells, NK cells, or any combination thereof. In certain embodiments, the immune cell is a T cell.

In certain embodiments, provided conditions for the preparation of IL-2 in the presence of a first site-specific modification and a second site-specific modification reduce over-activation and terminal differentiation of immune cells.

In certain embodiments of the sixth aspect, the invention also provides a method of preparing an immune cell for adoptive cell therapy, the immune cell for adoptive cell therapy being a Tumor Infiltrating Lymphocyte (TIL), wherein the method comprises: infiltrating lymphocytes are isolated from tumor tissue and cultured in a cell culture medium comprising a first site-specific modified IL-2 and a second site-specific modified IL-2, wherein the first site-specific modified IL-2 and the second site-specific modified IL-2 are as defined in the first aspect, and the cell culture medium is as defined above.

In a seventh aspect, the invention provides an immune cell for adoptive cell therapy prepared or cultured by the method of any one of the above aspects.

In certain embodiments, the immune cells of the seventh aspect have reduced aging and depletion, enhanced T

Cell proliferation, reduced proliferation of Treg cells, sustained proliferation of Tscm and T effector cells, reduced degree of depletion of teactor cells, and/or reduced overactivation by endogenous IL-2.

In an eighth aspect, the invention 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% to 100%, preferably 40% to 80% of the total cell number of the immune cell population.

Use in synergistically enhancing immune responses

The first and second site-directed modified IL-2 provided herein have a significant synergistic effect in enhancing an immune response, and thus the combination of the first and second site-directed modified IL-2 provided herein may be used to treat disease conditions in which stimulation of the immune system of a host would be beneficial, particularly conditions in which it is desirable to enhance a cellular immune response, which may include disease conditions in which the immune response of the host is inadequate or defective, such as a tumor.

In a tenth aspect, the present invention provides a pharmaceutical composition comprising a composition according to the first aspect and a pharmaceutically acceptable carrier and/or excipient.

In certain embodiments, the pharmaceutical composition comprises a plurality of 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: the first composition of the first site-directed modified IL-2 and a pharmaceutically acceptable carrier and/or excipient, and the second composition of the second site-directed modified IL-2 and a pharmaceutically acceptable carrier and/or excipient.

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: the first site-directed modified IL-2, the second site-directed modified IL-2, and a pharmaceutically acceptable carrier and/or excipient.

The first site-directed modified IL-2 and the second site-directed modified IL-2, combinations thereof, and the composition of the first aspect or the pharmaceutical composition of the tenth aspect of the invention may be formulated into a dosage form compatible with its 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. Solutions or suspensions for parenteral, intradermal or subcutaneous administration applications may include the following components: sterile diluents such as water for injection, saline solutions, fixed oils, polyethylene glycols, glycerol, propylene glycol or other synthetic solvents; antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulphite; chelating agents such as ethylenediamine tetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for adjusting tonicity such as sodium chloride or dextrose. The pH may be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. Formulations for parenteral administration may be packaged in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (wherein water is 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 syringability 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 may be a solvent or dispersion medium including, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycols, and the like), and suitable mixtures thereof. The 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 including in the composition agents which delay absorption, for example, aluminum monostearate and gelatin.

In an eleventh aspect, the present invention 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 a composition according to the first aspect or a pharmaceutical composition according to the tenth aspect. In certain embodiments, the methods comprise co-administering the first site-specific modified IL-2 and the second site-specific modified IL-2 to a subject.

In a twelfth aspect, the present invention also provides the use of a composition according to the first aspect or a pharmaceutical composition according to the tenth aspect for enhancing an immune response, preventing and/or treating a proliferative disease (e.g. a tumour), or for the manufacture of a medicament for enhancing an immune response, preventing and/or treating a proliferative disease (e.g. a tumour). The invention also provides the use of a combination of a first site-specific modified IL-2 and a second site-specific modified IL-2 for enhancing an immune response, preventing and/or treating a proliferative disease (e.g. a tumor), or for the manufacture of a medicament for enhancing an immune response, preventing and/or treating a proliferative disease (e.g. a tumor).

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). The individual components administered in combination with each other may be administered simultaneously, separately or sequentially. In certain embodiments, the separate components administered in combination with each other may be administered to the subject at a different time than the time at which the other components are administered; for example, as part of a treatment regimen, each administration may be administered at intervals of a given period of time non-simultaneously (e.g., separately or sequentially). In certain embodiments, the individual components administered in combination with each other may also be administered sequentially during the same administration period, but substantially simultaneously. Furthermore, separate components administered in combination with each other may be administered to a subject by the same or different routes.

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.

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

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

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

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

In certain embodiments of the methods, uses and kits described above, the proliferative disease is a tumor, including solid or hematological tumors, and also metastatic, recurrent or refractory cancers. In other embodiments, the proliferative Disease is hypergammaglobulinemia (hypergammaglobulinemia), lymphoproliferative disorders, opathies (paraproteinemia), purpura (purura), sarcoidosis, szechuan's macroglobulinemia, gaucher's Disease, histiocytosis (histiocytosis), and any other cell proliferation Disease that is outside of neoplasia (neoplasias) in the organ system.

In certain embodiments of the methods, uses and kits described above, the tumor is a solid tumor. In certain embodiments, the solid tumor is a metastatic cancer, a recurrent or refractory cancer.

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

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

In certain embodiments of the methods, uses and kits described above, the subject is a mammal, e.g., a human.

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

In certain embodiments of the methods, uses and kits described above, the first site-modified IL-2 and second site-modified IL-2 described herein, combinations thereof, and compositions or pharmaceutical compositions of the invention, may be formulated into any dosage form known in the medical arts, e.g., tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injectable solutions, sterile powders for injection, and injectable concentrated solutions), 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 invention should be sterile and stable under the conditions of manufacture and storage. One preferred dosage form is an injection. Such injections may be sterile injectable solutions. For example, sterile injectable solutions can be prepared by the following methods: the necessary amount of the active ingredient is incorporated in a suitable solvent, and optionally, other desired ingredients (including, but not limited to, pH modifiers, surfactants, adjuvants, ionic strength enhancers, isotonicity agents, preservatives, diluents, or any combination thereof) are incorporated simultaneously, followed by filter sterilization. In addition, the sterile injectable solutions may be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze-drying) for convenient storage and use. Such sterile lyophilized powders may be dispersed in a suitable carrier prior to use, such as water for injection (WFI), water for bacteriostatic injection (BWFI), sodium chloride solutions (e.g., 0.9% (w/v) NaCl), dextrose solutions (e.g., 5% dextrose), surfactant-containing solutions (e.g., 0.01% polysorbate 20), pH buffered solutions (e.g., phosphate buffered solutions), ringer's solution, and any combination thereof.

In certain embodiments of the methods, uses and kits described above, the first site-modified IL-2 and the second site-modified IL-2 described herein, combinations thereof, and compositions or pharmaceutical compositions of the invention, may be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic, intrainguinal, intravesical, topical (e.g., powder, ointment or drops), or nasal routes. However, for many therapeutic uses, the preferred route/mode of administration is parenteral (e.g., intravenous injection or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). The skilled artisan will appreciate that the route and/or mode of administration will vary depending on the intended purpose. In certain embodiments, the first site-specific modified IL-2 and the second site-specific modified IL-2, combinations thereof, and the compositions or pharmaceutical compositions of the invention are administered by intravenous injection or bolus injection.

In certain embodiments of the methods, uses and kits described above, the first site-modified IL-2 and second site-modified IL-2, combinations thereof, and the compositions or pharmaceutical compositions of the invention, as described herein, can be formulated in dosage unit form for ease of administration. Dosage unit form refers to physically discrete units suitable as unitary dosages for subjects to be treated; each unit contains a predetermined amount of active ingredient calculated to produce the desired therapeutic effect in combination with the required pharmaceutical carrier.

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

Use in adoptive cell therapy

The combination of the first site-directed modified IL-2 and the second site-directed modified IL-2 provided herein can lead to the fate of immune cells in adoptive cellular immunotherapy to long-term immunity. Thereby further providing therapeutic uses of said immune cells.

In a thirteenth aspect, the invention provides a pharmaceutical composition comprising an immune cell according to the seventh aspect or an immune cell population according to the eighth aspect, and a pharmaceutically acceptable carrier and/or excipient.

The immune cell of the seventh aspect, the population of immune cells of the eighth aspect or the pharmaceutical composition of the thirteenth aspect may be formulated in a dosage form compatible with its 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. Solutions or suspensions for parenteral, intradermal or subcutaneous administration applications may include the following components: sterile diluents such as water for injection, saline solutions, fixed oils, polyethylene glycols, glycerol, propylene glycol or other synthetic solvents; antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulphite; chelating agents such as ethylenediamine tetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for adjusting tonicity such as sodium chloride or dextrose. The pH may be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. Formulations for parenteral administration may be packaged in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

In a fourteenth aspect, the present invention 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 an immune cell according to the seventh aspect, an immune cell population according to the eighth aspect or a pharmaceutical composition according to the thirteenth aspect.

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 cells obtained in step (1) or a population of cells comprising the same to the subject for treatment.

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 of the invention and/or the method of the sixth aspect, and subsequently administering the engineered immune cell to the subject.

In certain embodiments, a TIL therapy is administered to a subject, the method comprising: obtaining tumor-infiltrating lymphocytes (TILs) using the method of the fifth aspect of the invention and/or the method of the sixth aspect, and administering the tumor-infiltrating lymphocytes (TILs) to the subject.

In a fifteenth aspect, the present invention also provides the use of an immune cell according to the seventh aspect, an immune cell population according to the eighth aspect or a pharmaceutical composition according to the thirteenth aspect for enhancing an immune response, preventing and/or treating a proliferative disease (e.g. a tumour), or for the manufacture of a medicament for enhancing an immune response, preventing and/or treating a proliferative disease (e.g. a tumour).

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

In a sixteenth aspect, the invention also provides the use of a composition according to the first aspect in combination with an immune cell for adoptive cell therapy for enhancing an immune response, preventing and/or treating a proliferative disease (e.g. a tumour) or in the manufacture of a medicament for enhancing an immune response, preventing and/or treating a proliferative disease (e.g. a tumour).

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

In certain embodiments of the sixteenth or seventeenth aspect, the immune cells for adoptive cell therapy are engineered immune cells that express a chimeric antigen receptor and/or comprise a nucleic acid molecule encoding the chimeric antigen receptor. In certain embodiments, the engineered immune cells include lymphocytes that express IL2-rαβγ trimer, such as T cells, NK cells, or any combination thereof.

In certain embodiments of the sixteenth or seventeenth aspect, the immune cells used for adoptive cell therapy are Tumor Infiltrating Lymphocytes (TILs).

In certain embodiments of the sixteenth or seventeenth aspect, the composition of the first aspect is present in the same composition or dosage form as the immune cells used for adoptive cell therapy, and thus may be administered simultaneously.

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 may be administered separately or sequentially.

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, particularly an effector T cell (Teff) mediated immune response. In certain embodiments, the enhancing an immune response further comprises reducing or inhibiting Treg cell function.

In certain embodiments of the methods and uses described above, the proliferative disease is a tumor, including solid or hematological tumors, and also metastatic cancers, recurrent or refractory cancers. In other embodiments, the proliferative Disease is hypergammaglobulinemia (hypergammaglobulinemia), lymphoproliferative disorders, opathies (paraproteinemia), purpura (purura), sarcoidosis, szechuan's macroglobulinemia, gaucher's Disease, histiocytosis (histiocytosis), and any other cell proliferation Disease that is outside of neoplasia (neoplasias) in the organ system.

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, a recurrent or refractory cancer.

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

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

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

In certain embodiments of the methods and uses described above, when chimeric antigen receptor cell therapies are involved, the tumor preferably comprises a hematological tumor, including metastatic cancer, recurrent or refractory cancer; for example, the tumor is selected from lymphomas.

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

In certain embodiments of the methods and uses described above, the subject is a mammal, e.g., a human.

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 may be formulated into any dosage form known in the medical arts, e.g., tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injectable solutions, injectable sterile powders and injectable concentrated solutions), 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 invention should be sterile and stable under the conditions of manufacture and storage. One preferred dosage form is an injection. Such injections may be sterile injectable solutions. For example, sterile injectable solutions can be prepared by the following methods: the necessary amount of the active ingredient is incorporated in a suitable solvent, and optionally, other desired ingredients (including, but not limited to, pH modifiers, surfactants, adjuvants, ionic strength enhancers, isotonicity agents, preservatives, diluents, or any combination thereof) are incorporated simultaneously, followed by filter sterilization. In addition, the sterile injectable solutions may be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze-drying) for convenient storage and use. Such sterile lyophilized powders may be dispersed in a suitable carrier prior to use, such as water for injection (WFI), water for bacteriostatic injection (BWFI), sodium chloride solutions (e.g., 0.9% (w/v) NaCl), dextrose solutions (e.g., 5% dextrose), surfactant-containing solutions (e.g., 0.01% polysorbate 20), pH buffered solutions (e.g., phosphate buffered solutions), ringer's solution, and any combination thereof.

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 may be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic, inguinal, intravesical, topical (e.g., powder, ointment, or drops), or nasal routes. However, for many therapeutic uses, the preferred route/mode of administration is parenteral (e.g., intravenous injection or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). The skilled artisan will appreciate that the route and/or mode of administration will vary depending on the intended purpose. In certain embodiments, the immune cell 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 injection.

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 may be formulated in dosage unit form for ease of administration. Dosage unit form refers to physically discrete units suitable as unitary dosages for subjects to be treated; each unit contains a predetermined amount of active ingredient calculated to produce the desired therapeutic effect in combination with the required pharmaceutical carrier.

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 may be administered alone or in combination with another pharmaceutically active agent (e.g., an antineoplastic agent) or another therapy (e.g., an antineoplastic therapy).

Definition of terms

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Moreover, the procedures of molecular genetics, nucleic acid chemistry, cell culture, biochemistry, cell biology and the like as used herein are all conventional procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

When used herein, the terms "for example," such as, "" including, "" comprising, "or variations thereof, are not to be construed as limiting terms, but rather as meaning" but not limited to "or" not limited to.

The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

As used herein, the term "unnatural amino acid" refers to an amino acid that is other than the 20 amino acids naturally occurring in a protein. Non-limiting examples of unnatural amino acids include: non-natural analogs of N epsilon-2-azidoethoxycarbonyl-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-borophenylalanine, O-propargyl tyrosine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-bromophenylalanine, selenocysteine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, tyrosine; non-natural analogs of glutamine amino acids; a non-natural analog of a phenylalanine amino acid; unnatural analogues of serine amino acids; a non-natural analog of a threonine amino acid; alkyl, aryl, acyl, azido, cyano, halogen, hydrazine, hydrazide, hydroxy, alkenyl, alkynyl, ether, thiol, sulfonyl, seleno, ester, thio acid, borate (borate), borate (boronate), phosphate, phosphonyl, phosphine, heterocycle, ketene, imine, aldehyde, hydroxylamine, keto, or amino substituted amino acid, or a combination thereof; amino acids with photoactivatable cross-linking agents; spin-labeled amino acids; fluorescent amino acids; metal binding amino acids; metal-containing amino acids; a radioactive amino acid; photo-caged and/or photo-isomerisable amino acids; an amino acid comprising biotin or a biotin analogue; amino acids containing keto groups; amino acids comprising polyethylene glycol or polyether; heavy atom substituted amino acids; chemically cleavable or photocleavable amino acids; amino acids having extended side chains; amino acids containing toxic groups; sugar-substituted amino acids; a carbon-linked sugar-containing amino acid; a redox active amino acid; an acid containing a-hydroxy; an aminothiopropionic acid; alpha, alpha disubstituted amino acids; a beta-amino acid; cyclic amino acids other than proline or histidine; and aromatic amino acids other than phenylalanine, tyrosine or tryptophan.

In some embodiments, the unnatural amino acid comprises a selectively reactive group, or a reactive group for site-selectively labeling a target polypeptide. The chemical reaction may be a biorthogonal reaction (e.g., a biocompatible and selective reaction), a Cu (I) catalyzed or "copper-free" alkyne-azidotriazole formation reaction, a staudinger ligation (Staudinger ligation), an inverse electron-demanding Diels-Alder (IEDDA) reaction, a "photo-click" chemistry or metal-mediated process (e.g., olefin metathesis and Suzuki-Miyaura or Sonogashira cross-coupling), and the like.

As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes, such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC), or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage, animal viruses, etc. Animal viruses that may be used as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus, papilloma vacuolation virus (e.g., SV 40). A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication origin.

As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to, a prokaryotic cell such as e.g. escherichia coli or bacillus subtilis, a fungal cell such as e.g. yeast cells or aspergillus, an insect cell such as e.g. S2 drosophila cells or Sf9, or an animal cell such as e.g. fibroblasts, 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.

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 that combines antibody-based specificity for an antigen of interest (e.g., a tumor antigen) with an immune effector cell activating intracellular domain to exhibit specific immune activity against cells expressing the antigen of interest (e.g., tumor cells). In the present invention, the expression "CAR-expressing immune cell" refers to an immune cell that expresses a CAR and has antigen specificity determined by the targeting domain of the CAR.

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

As used herein, the term "intracellular signaling domain" refers to the portion of a protein that signals effector signaling functions and directs cells to perform a specific function. Thus, the intracellular signaling domain has the ability to activate at least one normal effector function of an immune effector cell expressing the CAR. For example, the effector function of a T cell may be cytolytic activity or helper activity, including secretion of cytokines.

As used herein, the term "primary signaling domain" refers to a protein moiety capable of modulating primary activation of a TCR complex, either in a stimulatory manner or in an inhibitory manner. The primary signaling domain acting in a stimulatory manner typically contains a signaling motif known as an immune receptor tyrosine-based activation motif (ITAM). Non-limiting examples of ITAMs containing primary signaling domains particularly useful in the present invention include those derived from TCR ζ, fcrγ, fcrβ, cd3γ, cd3δ, cd3ε, cd3ζ, CD22, DAP10, CD79a, CD79b, and CD66 d.

As used herein, the term "costimulatory signaling domain" refers to the intracellular signaling domain of a costimulatory molecule. Costimulatory 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 upon binding to an antigen. Non-limiting examples of such co-stimulatory molecules include CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX 40), CD137 (4-1 BB), CD150 (SLAMF 1) CD270 (HVEM), CD278 (ICOS), DAP10.

As used herein, the term "immune cell" refers to any cell of the immune system that has one or more effector functions (e.g., cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and/or CDC). Typically, immune cells are cells that have hematopoietic origin and play a role in the immune response. In certain embodiments, immune cells refer to immune effector cells. The term "effector function" refers to a specialized function of immune effector cells, such as a function or response that enhances or promotes immune attack on (e.g., killing or inhibiting growth or proliferation of) target cells. The effector function of T cells may be, for example, cytolytic activity or activity that aids or includes secretion of cytokines. Examples of immune effector cells include T cells (e.g., α/β T cells and γ/δ T cells), B cells, natural Killer (NK) cells, natural Killer T (NKT) cells, mast cells, and bone marrow-derived macrophages.

Exemplary immune effector cells that can be used in the CARs described herein include T lymphocytes. The term "T cell" or "T lymphocyte" is well known in the art and is intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes or activated T lymphocytes. The T cells may be T helper (Th) cells, such as T helper 1 (Th 1) or T helper 2 (Th 2) cells. The T cells may be helper T cells (HTL; CD4T cells) CD4T cells, cytotoxic T cells (CTL; CD8T cells), CD4CD8T cells or any other T cell subset. In certain embodiments, T cells may include naive T cells and memory T cells.

The immune cells of the invention may be autologous/autologous ("self") or non-autologous ("non-self", e.g., allogeneic, syngeneic or allogeneic). As used herein, "autologous" refers to cells from the same subject; "allogeneic" refers to cells of the same species that are genetically distinct from the comparison cells; "isogenic" refers to cells from a different subject that are genetically identical to the comparison cells; "allogeneic" refers to cells from a different species than the comparison cells. In a preferred embodiment, the cells of the invention are allogeneic.

As used herein, the term "antibody" refers to an immunoglobulin molecule that is typically composed of two pairs of polypeptide chains, each pair having one Light Chain (LC) and one Heavy Chain (HC). Antibody light chains can be classified as kappa (kappa) and lambda (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the isotypes of antibodies are defined as IgM, igD, igG, igA and IgE, respectively. Within the light and heavy chains, the variable and constant regions are linked by a "J" region of about 12 or more amino acids, the heavy chain also comprisingA "D" region containing about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH 1, CH2 and CH 3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The constant domains are not directly involved in binding of antibodies to antigens, but exhibit a variety of effector functions, such as may mediate binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q). VH and VL regions can also be subdivided into regions of high variability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FR). Each V is _H And V _L By the following sequence: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 consist of 3 CDRs and 4 FRs arranged from amino-terminus to carboxy-terminus. The variable regions (VH and VL) of each heavy/light chain pair form antigen binding sites, respectively. The amino acid assignment in each region or domain can be followed by 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.

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 to which the full-length antibody binds, and/or competes with the full-length antibody for specific binding to an antigen, also referred to as an "antigen-binding portion. See generally Fundamental Immunology, ch.7 (Paul, W., ed., 2 nd edition, raven Press, N.Y. (1989), which is incorporated herein by reference in its entirety for all purposes, antigen binding fragments of antibodies may be generated 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 capacity to the polypeptide.

As used hereinAs used herein, the term "Fab fragment" means an antibody fragment consisting of VL, VH, CL and CH1 domains; the term "F (ab') ₂ Fragment "means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; the term "Fab 'fragment" means a reduction-linked F (ab') ₂ The resulting fragment after disulfide bonding of the two heavy chain fragments in the fragment consists of one complete light and heavy chain Fd fragment (consisting of VH and CH1 domains).

As used herein, the term "Fv" means an antibody fragment consisting of VL and VH domains of a single arm of an antibody. Fv fragments are generally considered to be the smallest antibody fragment that forms the complete antigen binding site. It is believed that the six CDRs confer antigen binding specificity to the antibody. However, even one variable region (e.g., fd fragment, which contains only three CDRs specific for an antigen) is able to recognize and bind antigen, although its affinity may be lower than the complete binding site.

As used herein, the term "scFv" refers to a single polypeptide chain comprising VL and VH domains, wherein the VL and VH are connected by a linker (linker). Such scFv molecules may have the general structure: NH (NH) ₂ -VL-linker-VH-COOH or NH ₂ -VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a polypeptide having an amino acid sequence (GGGGS) can be used ₄ Is a joint of a metal wire. In some cases, disulfide bonds may also exist between VH and VL of scFv. In certain embodiments of the invention, an scFv may form a di-scFv, which refers to two or more individual scFv in tandem to form an antibody. In certain embodiments of the invention, scFv may be formed (scFv) ₂ It refers to the formation of antibodies from two or more individual scfvs in parallel.

As used herein, the term "pharmaceutically acceptable carrier and/or excipient" refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and active ingredient, which is well known in the art (see, e.g., remington's Pharmaceutical sciences. Mediated by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995), and includes, but is not limited to: pH modifiers, surfactants, adjuvants, ionic strength enhancers, diluents, agents to maintain osmotic pressure, agents to delay absorption, preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or nonionic 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 that maintain osmotic pressure include, but are not limited to, sugar, naCl, and the like. Agents that delay absorption include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (e.g., buffered saline), alcohols and polyols (e.g., glycerol), and the like. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like. Stabilizers have the meaning commonly understood by those skilled in the art and are capable of stabilizing the desired activity of the active ingredient in a medicament, including but not limited to sodium glutamate, gelatin, SPGA, saccharides (e.g., sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (e.g., glutamic acid, glycine), proteins (e.g., dried whey, albumin or casein) or degradation products thereof (e.g., lactalbumin hydrolysate), and the like.

As used herein, the term "preventing" refers to a method that is performed in order to prevent or delay the occurrence of a disease or disorder or symptom in a subject. As used herein, the term "treatment" refers to a method that is performed in order to obtain beneficial or desired clinical results. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., no longer worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and diminishment of symptoms (whether partial or total), whether detectable or undetectable. Furthermore, "treatment" may also refer to an extension of survival compared to the expected survival (if not treated).

As used herein, the term "subject" refers to a mammal, such as a primate mammal, e.g., a human. In certain embodiments, the subject (e.g., human) has a tumor.

Advantageous effects of the invention

The invention carries out specific site selective polyethylene glycol (PEG) modification on IL2 to obtain a combination comprising IL-2 modified by a first site and IL-2 modified by a second site, wherein the IL-2 modified by the first site has a blocking effect on IL2-Rα receptor and is selective on IL2-Rβγ receptor, and can preferentially activate Teff, CTL and NK cells to reduce activation Treg, and in addition, the invention induces reduced terminal differentiation and depletion of CD8+ cells to improve the proportion and quantity of central memory cells; the second site-specific modified IL-2 has the effect of blocking IL-2-Rβ but not IL-2-Rα, and can compete with endogenous IL-2 secreted by cells for IL-2-Rα, and compete for inhibition of endogenous IL-2 activation of tregs. The combination of the two can make up the disadvantage that the depletion degree of the IL-2 modified by the first fixed point is increased after the activation of the cells under the condition of keeping the activation advantage of the central memory cell of the IL-2 modified by the first fixed point, and better plays the anti-tumor treatment effect. The combination provided by the invention has obvious synergistic effects of enhancing immune response and resisting tumor, can guide the fate of lymphocytes to long-term immunity in adoptive cell therapy, and has important clinical value.

Embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are only for illustrating the present invention and are not to be construed as limiting the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the following detailed description of the preferred embodiments and the accompanying drawings.

Drawings

Fig. 1: coomassie blue staining results for PEGylated IL-2 variants Y45-20K and D20-20K.

Fig. 2: surface Plasmon Resonance (SPR) affinity assay results for different subunits of the IL-2 receptor for PEGylated IL-2 variants.

Fig. 3: results of cell phosphorylation STAT5 (pSTAT 5) level assays following treatment with pegylated IL-2 variants Y45-20K and D20-20K.

Fig. 4: PK/PD characterization of pegylated IL-2 variants Y45-20K and D20-20K.

Fig. 5: effects of D20-20K on Y45-20K mediated proliferation and activation of T cells in vitro.

Fig. 6: effects of D20-20K on Y45-20K mediated T proliferation and activation in healthy mice.

Fig. 7: anti-tumor effects of D20-20K in combination with Y45-20K in mouse tumor models.

Fig. 8: effects of D20-20K in combination with Y45-20K on T cell subsets in lymph nodes of tumor-bearing mice.

Fig. 9: effects of D20-20K in combination with Y45-20K on Vascular Leak Syndrome (VLS).

Fig. 10: effect of D20-20K in combination with Y45-20K on CAR-T cell proliferation in vitro culture.

Fig. 11: effects of D20-20K in combination with Y45-20K on CAR-T cell aging and depletion in vitro culture.

Fig. 12: effects of D20-20K in combination with Y45-20K on senescence and depletion of T cells at different differentiation stages in CAR-T cell in vitro culture.

Fig. 13: effects of none- α variants (Y45-20K) in combination with various none- β variants on CD8 and CD 4T cells. Fig. 13a: expression of immune checkpoints PD-1 and TIM-3; fig. 13b: the expression of effector cytokine performin, granzyme B and IFN- γ; fig. 13c: expression of apoptosis-related biomarkers CD57, IL-10 expression.

Fig. 14: effects of the none- β variant (D20-20K) in combination with various none- α variants on CD8 and CD 4T cells. Fig. 14a: expression of immune checkpoints PD-1 and TIM-3; fig. 14b: the expression of effector cytokine performin, granzyme B and IFN- γ; fig. 14c: expression of apoptosis-related biomarkers CD57, IL-10 expression.

Sequence information

The information of the partial sequences to which the present invention relates is provided in table 1 below.

Table 1: description of the sequence

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Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Synthesis of NAEK:

2-Bromoethanol (8 g,64 mmol) and sodium azide (6.24 g,96 mmol) were added to acetone (60 ml) and water (30 ml) at room temperature. The reaction mixture was refluxed at 60 ℃ for 10h, cooled to room temperature and the acetone was removed by evaporation in vacuo. The residue was extracted with diethyl ether. The organic layer was washed twice with brine, then with Na ₂ SO ₄ Drying, filtration and evaporation gave 2-azidoethanol (compound 2) in 99% yield (5.5 g,63.2 mmol) without further purification.

A solution of compound 2 (5.5 g,63.2 mmol) in dichloromethane (120 ml) was slowly added to a suspension of N, N' -carbonyldiimidazole (15.36 g,94.8 mmol) in dichloromethane (55 ml) at-3 ℃. The reaction was carried out for 12 hours with stirring. Then 200mL of water was added and the organic layer was washed twice with brine, then with Na ₂ SO ₄ Dried, filtered and concentrated under vacuum. The residue was further purified by silica gel chromatography,PE/EtOAc (1:1) eluted affording compound 3 (10.7 g,59 mmol) as a colorless oil in 93% yield.

A solution of compound 3 (10.7 g,59 mmol) in dichloromethane (100 ml) was added at room temperature to a solution of Boc-Lys-OH (12.2 g,49.2 mmol) in 1M NaOH (50 ml) in water. TBAB (0.16 g,0.01 eq) was then added. The reaction mixture was allowed to react for 12 hours with stirring, cooled to 0℃and then adjusted to pH 2-3 with ice-bath 1M aqueous HCl. The aqueous phase was extracted with DCM and the organic layer was washed twice with brine. Then the organic layer was taken up with Na ₂ SO ₄ Dried, 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.

Compound 4 (15.1 g,41.94 mmol) was dissolved in dichloromethane (80 ml) and trifluoroacetic acid (20 ml) was then slowly added. The reaction solution was stirred at room temperature for 0.5 hours, and then the solvent was removed by evaporation under vacuum. The residue was redissolved 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, NAEK in 61% yield.

Synthesis of DBCO-PEG:

compound 1, 10g (48 mmol) hydroxylamine hydrochloride (16.8 g (240 mmol)) are dissolved in absolute ethanol, 26ml pyridine is added, heating reflux is carried out for 36h, after cold cutting, 25ml ethyl acetate is added, 125ml1NHCl is added and stirred for 0.5h, the organic layer is separated, the organic layer is washed with saturated NaCl solution, several layers are dried and concentrated to obtain white solid compound 2, yield: 100%.

6.25g of phosphorus pentoxide was dissolved in 50ml of methanesulfonic acid and stirred for further use. The above product was added to 10g of compound 2 and stirred at 100℃for 1.5h. After the reaction liquid is cooled, pouring the cooled reaction liquid into 300ml of water, precipitating solid, carrying out suction filtration and drying, pulping the dried solid by diethyl ether, carrying out suction filtration and drying to obtain a white solid compound 3, and obtaining the yield: 100%.

160ml of diethyl ether was cooled to 0℃and 5.5g of lithium aluminum hydride were added in portions, followed by 4g of Compound 3 in portions and reflux at 35℃for 72h. The reaction solution was cooled to 0℃and quenched by addition of 14.8ml of saturated sodium sulfate solution, filtered and concentrated to give Compound 4 as a yellow solid in 98% yield.

766mg of monomethyl succinate is dissolved in 15ml of dichloromethane, cooled to 0 ℃, and then 610ul of oxalyl chloride is added dropwise, and stirred at room temperature for 1h. Concentrating for use. 1g of compound 4 was dissolved in 15ml of methylene chloride, 1.16ml of pyridine was added, cooled to 0℃and then added dropwise to monomethyl succinyl chloride, stirred at room temperature for 0.5h, the organic phase was washed with 1N HCl, 1N NaOH, and then dried over anhydrous sodium sulfate, and concentrated to give compound 5 as a yellow solid. Yield: 99%.

Dissolving the compound 5 in methanol/water (volume ratio=2:1), adding 6 equivalents of lithium hydroxide at room temperature, stirring at room temperature for 8 hours, spin-drying methanol, adding water for dissolution, washing an aqueous phase by dichloromethane, adjusting the pH of the aqueous phase by 1NHCl to be=2-3, extracting the aqueous phase by dichloromethane, washing an organic phase by saturated NaCl solution, and spin-drying to obtain yellow oily compound 6, wherein the yield: 98%.

1.4g of compound 6 is dissolved in 50ml of dichloromethane, cooled to 0 ℃, 2.2g of liquid element is dissolved in dichloromethane and then is dripped into the dichloromethane, stirring is carried out for 3 hours at room temperature, 50ml of saturated sodium thiosulfate solution is added, the separated organic layer is washed by saturated NaCl solution, dried by anhydrous sodium sulfate, and light yellow solid compound 7 is obtained after spin drying, the yield is: 95%.

2.1g of compound 7 is dissolved in 40ml of anhydrous tetrahydrofuran, cooled to-40 ℃, and the prepared tetrahydrofuran solution of 1M potassium tert-butoxide is dripped into the solution, stirred for 1h, quenched by adding 1M HCl solution until the pH value is=2-3, extracted by dichloromethane, and the organic layer is concentrated and separated by column chromatography to obtain pink solid compound 8, the yield is: 80%.

1.1g of compound 8 was dissolved in 20ml of methylene chloride, 495mg of N-hydroxysuccinimide, 825mg of EDCl was added, and the mixture was stirred at room temperature for 1 hour, the reaction solution was washed with water, saturated NaCl solution, dried over anhydrous sodium sulfate, and concentrated to give compound 9 as a pale yellow solid, yield: 100%.

2g of NH2-PEG _20k OMe was dissolved in 25ml dichloromethane, 40mg of Compound 9 was added, stirred at room temperature for 1h, the organic phase was dried by spinning, and the residue was washed with diethyl ether and separated by column chromatography to give Compound 10 in 85% yield.

Example 1: construction and expression of site-directed mutagenesis IL-2 protein expression plasmid

(1) Selection of mutation sites

A mutation site shown below was selected on the IL-2 protein (SEQ ID NO: 1), wherein the position is that in SEQ ID NO: 1.

Table 2: mutation site

Protein name	Amino acid position	Amino acids	Pre-mutation codons	Post-mutation codons
					Y45	45	Y	TAC	TAG
D20	20	D	GAT	TAG

(2) Expression of site-directed muteins comprising unnatural amino acids

The above nucleic acid sequence comprising a codon substitution was synthesized and ligated to a nucleic acid sequence encoding a His tag and cloned into the pET-21a (+) (adedge: # 69740-3) E.coli plasmid expression vector, which was co-transfected with the pSURAR-YAV plasmid into the TransB (DE 3) strain (purchased from full gold, catalog number: CD 811-02). Wherein the pSURAR-YAV plasmid encodes an amber codon-suppressing tRNA and an NAEK-specific aminoacyltRNA synthetase, and an unnatural amino acid NAEK can be introduced at a specific site by co-transfection with a nucleic acid sequence comprising a TAG codon; the pSURAR-YAV plasmid is prepared from the general microbiological culture Collection center of China general microbiological culture Collection center (national institute of microbiology, national academy of sciences of China, the Korean area, north Chen West Lu 1) with a collection date of 2013, 4 months and 8 days with a collection number of CGMCCNo:7432 Classification the plasmid pSUPAR-YAV-tRNA/PyleRS obtained from Escherichia coli harboring the plasmid pSUPAR-YAV-tRNA/PyleRS was designated as Escherichia coli. The transformed strain was inoculated into 2ml of LB medium containing 100ug/ml ampicillin and 34ug/ml chloramphenicol, and then cultured at 37℃and 220 rpm. The overnight cultures were diluted to optical density in 2 XYT medium and the cultures were incubated at 37℃until A600nm reached about 1.5. UAA (NAEK, laboratory self-synthesis) was added at a final concentration of 1mM, and protein expression was induced by adding isopropyl beta-d-thiogalactopyranoside (IPTG) and L-arabinose at a final concentration of 0.5mM and 0.1%, respectively, and after half an hour, the temperature was lowered to 20 ℃. After about 18 hours of expression, cells were harvested by centrifugation and resuspended in His-Bind buffer (20 mM phosphate, pH 8.0, 500mM NaCl,20mM imidazole). The protein was extracted by passing the cells through a Micofluidizer twice under 1200bar and cooling conditions. The supernatant was then collected by centrifugation at 20,000g for 20 minutes and stored frozen at-80℃until further processing. Thereby obtaining the site-directed mutant IL-2 protein with the unnatural amino acid substituted at position Y45 or position D20.

Example 2: PEG modification of site-directed mutagenesis IL-2 protein

The supernatant obtained in example 1 was initially purified by Ni-NTA agarose (R90101, invitrogen) and then subjected to a click reaction. To produce site-directed PEGylated IL-2 analogs, the supernatant was enriched for His-tagged IL-2 using Ni-NTAHis-Bind Resin (Invitrogen), and DBCO-PEG was synthesized and then added to elution buffer (20 mM phosphate, pH 8.0, 500mM NaCl,500mM imidazole) at a final concentration of 1mM. The reaction was carried out at 4℃while gently 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 200in create 10/300GL,GE Healthcare) to remove unreacted PEG and IL-2. The main elution peak was collected using 3kDa centrifugal filter unit (Millipore), concentrated and buffer exchanged into PBS buffer. The purity of the PEGylated 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 Y45-20K and D20-20K, respectively. The electrophoresis results are shown in FIG. 1, and the purity of PEGylated IL-2 variants is more than 95%.

In addition, the following PEGylated IL-2 derivatives were also prepared by the methods described in examples 1-2: H16-20K, A73-20K, H79-20K, F-20K, E62-20K, K64-20K, P65-20K, E68-20K, K-20K, T37-20K, R-20K, T41-20K, K48-20K, K49-20K. The nomenclature of the PEGylated IL-2 variants referred to in this application is: [ position of mutation to unnatural amino acid compared to SEQ ID NO:1 ] - [ average molecular weight of attached PEG group ]. Taking Y45-20K as an example, the amino acid sequence differs from SEQ ID NO. 1 in that Y45 is replaced by the unnatural amino acid NAEK, and this position is further linked to a PEG group of average molecular weight 20 kDa.

Example 3: determination of binding Activity to IL-2 receptor

This example evaluates the affinity of PEGylated IL-2 derivatives for IL-2R trimer and dimer complexes by Surface Plasmon Resonance (SPR). Human IL-2Rα, IL-2Rβ and IL-2Ryc receptors were immobilized on CM5 or protein A chips for analysis on the Biacore 8K system (GE Healthcare). The extracellular domains of human IL-2Rα (C-terminal 6 XHis), IL-2Rβ (C-terminal Fc) and IL-2Rγ (C-terminal 6 XHis) were purchased from Yinqiao China. All kinetic experiments were performed at 25℃using 10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05%Surfactant P, pH 7.4 (running buffer). The 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 concentration was measured using BCA method (Pierce). Among them, a natural IL-2 protein (available from PeproTech under the trade name 96-200-02-1000) was used as a control. The concentration of all samples was the mass of IL-2 without PEG attached.

The results are shown in FIG. 2, where NA is not binding. The results show that D20-20K, H16-20K, A73-20K, H79-20K has no affinity for the beta subunit and significantly reduced binding affinity for dimeric IL-2R, but maintains binding to the alpha chain of trimeric IL-2R, with a bias towards non-beta receptors (non-beta), which may be referred to as non-beta PEGylates. Y45-20K, F42-20K, E62-20K, K64-20K, P65-20K, E68-20K does not bind to alpha, binding affinity for trimeric IL-2R is completely eliminated, binding to dimeric IL-2R is maintained, and there is a bias towards non-alpha receptors (non-alpha), which may be referred to as non-alpha PEGylates; K35-20K, T37-20K, R38-20K, T41-20K, K48-20K, K49-20K binds alpha in the order of magnitude of 1 e-7-1 e-6, is weakly binding, but binds to dimer IL-2R with significantly higher affinity than trimer IL-2R, or while binding to dimer IL-2R is weaker than trimer IL-2R, the difference (e.g., ratio) between the two is significantly smaller than the difference between the binding affinities of native IL-2 to dimer IL-2R and trimer IL-2R, has significantly improved bias towards dimer IL-2R, and therefore also has a bias towards non-alpha receptors (non-alpha), which may be referred to as non-alpha PEGylates.

Example 4: determination of activation tendency of Y45-20K and D20-20K on CD8+ T cells and Treg cells

This example determines whether PEGylated IL-2 variant Y45-20K or D20-20K preferentially activates CD8+ T cells over Treg cells by measuring the level of phosphorylated STAT5 (pSTAT 5), a key downstream mediator of IL-2R signaling. Will be about 2 x 10 ⁵ YT-1 cells (expressing CD122 and CD132 receptors), CD25+YT-1 (stably transformed cell line of YT-1 cells stably expressing CD25 receptor by lentiviral transduction), human PBMC cells were individually seeded in 96-well plates and resuspended in RPMI completelyIn the culture medium, the culture medium contains serial dilution of natural human IL-2 or PEGylated IL-2. Cells were stimulated for 15 min at 37 ℃ and then immediately fixed by adding formaldehyde to 2.0% and incubating for 15 min at room temperature. Permeabilization of the cells was achieved by suspending in ice-cold 87% methanol at 4℃for 30 min. The 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, pSTAT5-Pacific Blue (all available from biolegend). For pSTAT5 assay in hPBMC specific subpopulations, purified cell populations were isolated using a magnetic separation kit (MiltenyiBiotech) according to manufacturer's instructions to obtain memory cd4+ T cells (MPCD 4), memory cd8+ T cells (MPCD 8), NK cells and Treg cells, respectively.

All FACS antibodies were used at a dilution of 1:50. Cells were then washed twice in staining buffer and the Mean Fluorescence Intensity (MFI) was determined on a CytoFLEX flow cytometer (Beckman-Coulter). The data are plotted as background-subtracted MFI normalized to the maximum signal (IL-2, 1 μg ml-1) for each cell type. The background was defined as pSTAT5 MFI in unstimulated cells. Treg cells are defined as cd3+cd8-cd4+cd25highcd127low; NK cells are defined as CD3-CD16+CD56+; cd8+ T cells are defined as cd3+cd4-cd8+; MPCD4 cells are defined as CD3+CD4+CD8-CD45RO+; MPCD8 cells are defined as CD3+CD4-CD8+CD56-CD57-CD45RA-. After subtracting the MFI of the unstimulated cells and normalizing to maximum signal intensity, dose response curves were fitted to logistic models using GraphPad Prism data analysis software and half maximal effective concentrations (EC 50 values) and corresponding 95% confidence intervals were calculated. Experiments were performed in triplicate and repeated three times with similar results.

The results of the assays using NK-derived YT-1 cells and CD25+YT-1 cell models are shown in FIG. 3a, where Y45-20K showed a significant difference in CD25 (IL-2Rα) -and CD25 (IL-2Rα) +YT-1 cells, which is significantly different from wild type IL-2; d20-20K still showed only baseline levels of Treg stimulatory activity (5-20% of pSTAT5 activation) at very high doses, which is associated with its inability to bind to the β subunit.

Furthermore, the selective activation of Teff cells by pegylated IL-2 variants was reevaluated using various immune cell subsets isolated from healthy donor PBMCs, the results are shown in figure 3b. We found that IL-2 stimulated Treg and memory cd4+ T, memory cd8+ T and NK cells at all doses tested, whereas Y45-20K preferentially induced STAT5 phosphorylation in memory cd4+, memory cd8+ and NK cells, but not Treg cells. d20-20K did not induce STAT5 phosphorylation in memory cd4+, memory cd8+, NK and Treg cells.

The above results are consistent with the results of binding affinity assessment, indicating that pleiotropic effects of IL-2 can be relieved by blocking specific regions of the receptor binding site, and that Y45-20K has the desirable ability to preferentially stimulate Teffs rather than Treg cells for IL-2rα binding due to complete lack, whereas D20-20K has significantly reduced activation for both cd8+ T and Treg cells.

Example 5: PK/PD profiling of PEGylated IL-2 variants Y45-20K/D20-20K

This example examines the pharmacokinetic properties of the PEGylated IL-2 variants. Female C57BL/6 mice (purchased from beijing villi laboratory animal technologies limited) with an average body weight of about 20g were selected, randomly divided into different groups (n=3) and injected with PBS, wild-type human IL-2 or pegylated IL-2 variant (0.25 mg/kg, based on hll-2) by subcutaneous injection. At selected time points (30 min, 2h, 4h, 8h, 24h, 48h, 72h and 96 h), blood was taken from the mouse orbital veins (100 μl each) and then centrifuged at 4000g for 15 min at 4 ℃. Plasma was isolated and stored at-80 ℃. IL-2 concentration was determined using a human IL-2ELISA kit (Sinobiology). Pharmacokinetic parameters were analyzed by Kinetica 5.1 software and expressed as mean ± standard deviation.

The results are shown in FIG. 4, in which the PEGylated IL-2 variants have an increased clearance half-life (T1/2), enhanced maintenance of blood concentration (area under drug concentration versus time curve, AUC), and increased Mean Residence Time (MRT), 14 times longer peak time than IL-2, indicating a substantial reduction in clearance, compared to wild-type IL-2. The above results indicate that Y45-20K has superior pharmacokinetic properties. In addition, D20-20K also has superior pharmacokinetic properties.

Example 6: antitumor synergistic effect of PEGylated IL-2 variant Y45-20K/D20-20K

The data of the above examples have demonstrated that Y45-20K has the activity of preferentially activating cd8+ T cells, yet it still activates Treg cells at high doses. As demonstrated in the examples above, D20-20K maintains a selective affinity for IL-2rα while significantly reducing activation of cd25+yt-1, a unique property that may be associated with its undetectable binding to IL-2rβ, and thus, if D20-20K is treated in combination with Y45-20K, D20-20K may spatially occupy IL-2rα and competitively block its mild interaction with Y45-20K resulting in excessive activation of cd8+ T cells and terminal differentiation and activation of Treg. Accordingly, this example examined the synergistic antitumor effect of D20-20K and Y45-20K.

6.1D20-20K Effect on Y45-20K mediated proliferation and activation of T cells in vitro

Experimental procedure

This example examined whether the activation bias of D20-20K for Y45-20K had a dose dependent effect. Will be 2X 10 ⁵ Human PBMC cells were resuspended in 96-well plates using 100ul RPMI1640+10%FBS, 50ul of 0.4ug/ml Y45-20K (final concentration 0.1 ug/ml) was added to the wells, and 50ul of different gradients of D20-20K were added to the wells to give final concentrations of 1ug/ml,0.1ug/ml,0.01ug/ml,0.001ug/ml, respectively, and duplicate wells were set from three different healthy individuals, respectively. Cells were cultured at 37 ℃ 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 are defined as CD3+CD8-CD4+CD25+Foxp3+; cd8+ T cells were defined as cd3+cd4-cd8+, and the average fluorescence intensity (MFI) of Treg and CD 8T cell numbers and the respective activation markers (Treg-Foxp 3, cd8+t-CD 69) were counted with a flow cytometer.

Will be 2X 10 ⁵ YT-1 cells and CD25+YT-1 cells were inoculated into 96-well plates, resuspended in 96-well plates by 100ul RPMI1640+10%FBS, and 50ul of 0.4ug/ml Y45-20K (final concentration 0.1 ug/ml) was added to the experimental wells, followed by 50ul of different concentration gradients of D20-20K to give final concentrations of 1ug/ml,0.1ug/ml,0.01ug/ml, and 0.001ug/ml, respectively. Cells were stimulated for 15 min at 37 ℃ and then immediately fixed by adding formaldehyde to 2.0% and incubating for 15 min at room temperature. Permeabilization of the cells was achieved by suspending in ice-cold 87% methanol at 4℃for 30 min. The fixed and permeabilized cells were washed twice with FACS buffer and stained with pSTAT5-Pacific Blue antibody for flow cytometry analysis. Antibodies were used at a dilution of 1:50. Cells were then washed twice in staining buffer and the Mean Fluorescence Intensity (MFI) was determined on a CytoFLEX flow cytometer (Beckman-Coulter). The data are plotted as background-subtracted MFI normalized to the maximum signal for each cell type (Y45-20K, 0.1 μg ml ^-1 ). The background was defined as pSTAT5 MFI in unstimulated cells. After subtracting the MFI of the unstimulated cells and normalizing to maximum signal intensity, dose response curves were fitted to logistic models using GraphPad Prism data analysis software and half maximal effective concentrations (EC 50 values) and corresponding 95% confidence intervals were calculated. Experiments were performed in triplicate and repeated three times with similar results.

The results are shown in fig. 5, where D20-20K has a dose-dependent inhibition of Y45-20K mediated activation and proliferation of Treg cells, but not cd8+ T cells (fig. 5a is the effect on Treg proliferation and activation and fig. 5c is 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 0.39ug/ml (fig. 5 e), whereas no such inhibition was observed in native YT cells (fig. 5 f).

6.2D20-20K Effect on Y45-20K mediated T proliferation and activation in healthy mice

Experimental procedure

C57BL/6 mice (6 to 8 week old, females) were given subcutaneously 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 x 5 daily) to the posterior cervical portion, and an equal volume of PBS was injected as a placebo. Flow assays for spleen and lymph nodes were performed three days after dosing was completed.

The results are shown in FIG. 6, where the CD8+ T cell and MPCD8+ T ratios were further increased in spleens of mice treated with the Y45-20K/D20-20K combination compared to other groups of mice, and the Y45-20K/D20-20K combination treatment significantly reduced proliferation of tregs compared to Y45-20K alone.

Anti-tumor effects of 6.3D20-20K and Y45-20K in mouse tumor model

B16F10 melanoma cells (China academy of sciences cell bank (Shanghai, china) were subcutaneously implanted on the right side (right rank) of C57BL/6 mice (6 to 8 weeks old), 5X 10 per animal ⁵ ). When the tumor reaches 1500mm ³ Mice were sacrificed at size. Tumor volumes were calculated as follows: v=a2×b/2, where a is the width and b is the tumor length (all in millimeters). 7 days after implantation, when the tumor measured 100mm ³ At this time, animals were administered 45-20K (0.25 mg/kg x3 every other day), 20-20K (0.25 mg/kg x3 every other day), 45-20K and 20-20K (0.25 mg/kg x3 every other day), or IL-2 (0.25 mg/kg x 5 every day). On day 14, tumors were minced and digested in buffer containing 2mg/ml collagenase type II and IV (GIBCO BRL) and 0.5mg/ml DNase (Sigma Aldrich) for 13 min at 37℃to form a single cell suspension, followed by flow cytometry to determine the immune cell subtype in the tumor microenvironment. Immunohistochemical analysis of T cell density was performed on frozen tumor sections 14 days post-dose.

The results are shown in FIG. 7, and the combination of Y45-20K and D20-20K treatments showed very prominent antitumor effect, significantly reduced tumor burden and significantly prolonged survival in mice (FIG. 7 a). With decreasing tumor burden, the proportion of cd8+ T cells in spleen, tumor Draining Lymph Nodes (TDLN) and especially tumor tissue was increased in Y45-20K/D20-20K combination treated mice compared to other mice, but no significant change in Treg cell proportion (fig. 7 b). The results are consistent with immunohistochemical staining, indicating that co-treatment of Y45-20K and D20-20K induced the greatest amount of T cell infiltration in tumor tissue compared to native IL-2 and PBS treatment (FIG. 7 c).

The memory CD8 cells play an important role in tumor immunity, and analysis of T cell subsets in lymph nodes of tumor-bearing mice shows that the number of Tcm and MPCD8 cells in the lymph nodes is obviously increased by about 10 times in a Y45-20K single treatment group or a Y45-20K combined D20-20K treatment group, and the Y45-20K combined D20-20K treatment group is based on the Y45-20K single treatment group,the number of Tcm and MPCD8 is further increased. Correspondingly, T in both experimental groups

And Tem was slightly decreased (fig. 8 a), indicating that Y45-20K was able to significantly expand a large number of central memory T lymphocytes in vivo, and D20-20K alone did not expand central memory cells, but was able to combine with Y45-20K to further increase the proportion and number of central memory cells in the CD8 subpopulation, contributing to the anti-tumor immune efficacy. In addition, the expression level of the CD25 marker in the Tcm cell subset is further reduced, and the expression level of the CD122 marker is further increased, which means that the Tcm of the lymph nodes of the mice in the Y45-20K single treatment group or the Y45-20K combined D20-20K treatment group can avoid the excessive activation of endogenous IL-2 to T cells, reduce the sensitivity to IL-2 and improve the response capacity to the re-response of the external antigen. While the Y45-20K alone treatment group was able to largely activate proliferation of amplified Tcm, MPCD8, T +. >

And LAG-3 and PD-1 markers in the Tem cell subset are obviously improved compared with the IL-2 treatment group, the combination of D20-20K can reduce the expression of immune checkpoints in the cell subset, which means that the combination of D20-20K and Y45-20K can retain the central memory cell activation advantage of Y45-20K, and can make up the disadvantage that the depletion degree of Y45-20K after cell activation is increased, and better play an anti-tumor treatment effect.

Effects of 6.4D20-20K in combination with Y45-20K on Vascular Leak Syndrome (VLS)

The high risk of Vascular Leak Syndrome (VLS) in conventional IL-2 treatment is generally thought to be caused by the binding of IL-2 to cd25+ lung endothelial cells, whereby this example also evaluates the effect of combination therapy on pulmonary edema. A similar treatment regimen as described above was employed, wherein administration of the pegylated IL-2 variant was changed to a high dosing frequency (five times per day). After mice were sacrificed, lung tissues were weighed to obtain a lung tissue cell suspension for flow detection, and lung tissue sections were prepared for immunohistochemical analysis.

As a result, as shown in FIG. 9, lung cells expressing high levels of CD31 but not other immune cell lineage markers (Lin) were defined as endothelial cells (Ly5.2-B220-CD 3-NK1.1-CD11B-CD11 c-CD31+). As a result, it was found that Y45-20K single administration group resulted in significant pulmonary edema, which was manifested by an increase in lung wet weight (fig. 9 a), a decrease in lung endothelial cell proportion (fig. 9 b), and an increase in intra-pulmonary lymphocyte infiltration; however, this side effect was significantly reduced after co-administration with D20-20K, probably due to IL-2Rα spatially occupying lung endothelial cells on D20-20K.

The data show that Y45-20K and D20-20K show significant synergy in anti-tumor therapy, selectively induce CD8+ T cells, have minimal impact on Treg cells, and can alleviate VLS by protecting lung endothelial cells from activation by Y45-20K or endogenous IL-2, with significant beneficial technical effects.

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

IL-2 is a key trophic factor for in vitro expansion culture of CAR-T cells, and greatly weakens the curative effect of CAR-T cell therapy due to unavoidable activation of Treg cells in donor cells and unavoidable overactivation of CD8+ T cells and resulting terminal differentiation thereof, which affect the activity of CAR-T cells, so that the experiment Y45-20K is combined with D20-20K to replace the influence of traditional IL-2 on CAR-T cell therapy.

Effects of 7.1D20-20K in combination with Y45-20K on CAR-T cell proliferation in vitro culture

Construction of CAR lentiviral vector:

the anti-CD19 CAR comprises 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 cassettes of which were delegated to Aikandeli Biotechnology (Suzhou) Inc. for synthesis and cloning into lentiviral vectors. HEK293T cells were transfected with anti-CD19 CAR, pspax2 (adedge, # 12260) and pmd2.G (adedge, # 12259) plasmids using Lipofectamine 3000 (Life Technologies). The medium was changed 6 hours after transfection and virus supernatant was collected 48 hours after transfection. The virus particles were concentrated 30-fold by ultracentrifugation at 25,000rpm for 2 hours and frozen at-80 ℃ until ready for use.

Preparation of CAR-T:

human T cells were purified from peripheral blood mononuclear cells using the Dynabead human T cell kit (Life Technologies) and activated with CD3/CD28 magnetic beads (Life Technologies) for 24 hours prior to infection. Concentrated lentiviruses (FMC 63-AntiCD19-CAR Lentivirus) were applied to activated human T cells (10 in 24 well plates ⁶ Per well, MOI=1), 10mg/mL polybrene and 100ng/mL IL-2 or 100ng/mL 45-20K,100ng/mL D20-20K+100ng/mL Y45-20K were added and centrifuged at 1000g at 32℃for 2h. The next day, the supernatant was replaced with fresh medium containing the corresponding group of IL-2 and its analogues, and the transduced T cells were placed in complete growth medium to maintain 0.5x10 ⁶ The culture medium containing IL-2 was changed every 3 days at cells/mL. Cell numbers and apoptotic status were monitored in real time.

As shown in FIG. 10, neither Y45-20K, D20-20K, nor Y45-20K in combination with D20-20K, did not affect the transduction efficiency of the CAR (FIG. 10 a), and it was found that after 10 days of CAR-T cell culture with IL-2 variant, the CAR-T cells cultured with D20-20K in combination with IL-2 showed little proliferation, whereas the proliferation rate of the cells cultured with Y45-20K in combination with IL-2 was significantly higher than that of the CAR-T cells cultured with IL-2, and the proliferation rate of the CAR-T cells cultured with Y45-20K in combination with D20-20K in combination with IL-2 was similar to that of the CAR-T cells cultured with Y45-20K alone (FIG. 10 b). Apoptosis assays found that incubation with IL-2 resulted in apoptosis of CAR-T cells, which was associated with excessive activation of T cells by IL-2 and induction of terminal differentiation, whereas Y45-20K combined with D20-20K induced little apoptosis of CAR-T cells (fig. 10 c).

Effects of 7.2D20-20K in combination with Y45-20K on CAR-T cell aging and depletion in vitro culture

Experimental procedure

The CAR-T prepared as described above was cultured in the respective IL-2 for 10 to 14 days, and the cell phenotype was examined in association with each other. The surface antibodies were fluorescent stained with fluorescent labeled antibodies (CD 4-PE/Cy7, CD8-FITC, CD25-BV785, CD57-PB, CD45RA-BV510, CD62L-Percp/Cy5.5, CCR7-PE, PD-1-BV650, LAG-3-BV 421) and incubated at 4℃for 30 minutes. For intranuclear staining, CAR-T cells were fixed and permeabilized using Foxp 3/transcription factor staining buffer (eBioscience) and then stained with Foxp3 antibody in perm buffer. Samples were washed twice with PBS and suspended in flow cytometer staining buffer (eBioscience). For intracellular cytokine staining, 500x Cell Activation Cocktail (with Brefeldin a, daceae 423303) was used, and washing was performed after 6h 37 ℃ incubation. After the last wash, the cells were fixed and permeabilized using the eBioscience intracellular fixation and permeabilization buffer kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Then incubated with specific antibodies for 30 minutes at 4 ℃): IL-2-BV421, IL-10-APC, granzy B-percp/cy5.5.

As shown in FIG. 11, after PMA stimulation of in vitro cultured CAR-T cells, Y45-20K in combination with D20-20K secreted granzyme B and IL10 significantly lower than in the IL-2 group, indicating a decrease in the final differentiation and senescence levels of CAR-T cells (FIGS. 11a, B) and a significant increase in the secretion of IL-2, as well as a lower differentiation level of CAR-T cells in a dry state (FIG. 11 c). Second, flow assays showed that surface depletion of CAR-T cells cultured with Y45-20K in combination with D20-20K was significantly reduced with senescence-associated receptors such as PD-1, LAG-3, and CD57 (fig. 11D-f), again demonstrating that Y45-20K in combination with D20-20K could reverse the senescence and depletion state caused by IL-2 use in CAR-T cell culture in vitro.

Effects of 7.3D20-20K in combination with Y45-20K on senescence and depletion of T cells at different differentiation stages in CAR-T cell in vitro culture

Experimental procedure

Human T cells were purified from peripheral blood mononuclear cells using Dynabead human T cell kit (Life Technologies), and CAR-T was prepared as described above. To further investigate the effect of different cytokines on CAR-T cell differentiation, we selected CAR-T cells in different differentiation states by flow cytometry (sortation, symphony s6, BD) for individual culture. T (T)

Cells were defined as CD3+CD8+CD45RA+CD45RO-CCR7+CD62L-CD95-; tscm is defined as CD3+CD8+CD45RA+CD45RO-CCR7+CD62L-CD95+; tcm is defined as cd3+cd8+cd45ra-cd45ro+ccr7+cd62l+; tem is defined as CD3+CD8+CD45RA-CD45RO+CCR7-CD62L-; t effector is defined as CD3+CD8+CD45RA+CD45RO0-CCR7-CD62L-; treg is defined as CD3+CD4+CD25 ^high CD127 ^low And culturing and amplifying for 10-14 days by using different cytokines, and monitoring the cell number and the growth state in real time.

We performed on naive T cells at different differentiation stages in CAR-T cells (T

) The degree of senescence, depletion of T memory stem cells (Tsccm), effector T cells (T effector) and regulatory T cells (Treg) was examined. CD62L is a characteristic marker of cell stem property, and the combination of D20-20K and Y45-20K can further promote T +.>

And the amount of CD62L expressed in Tscm cells (FIG. 12 a), demonstrating that D20-20K in combination with Y45-20K can further preserve the T cell stem state. The detection results of T cell proliferation conditions at different differentiation stages show that the combination of D20-20K and Y45-20K can further enhance T +.>

Proliferation of cells reduced proliferation of Treg cells without significant effect on the proliferation status of Tscm and T effector cells (fig. 12 b). PD-1 and TIM-3 are markers of T cell depletion, and detection of T effector cells in a terminally differentiated state shows that the combination of D20-20K and Y45-20K can further reduce the expression level of PD-1 and TIM-3 and reduce the depletion degree of the T effector cells (FIG. 12 c). Finally, the detection of the expression quantity of CD25 on T cells in different differentiation states induced by IL-2 shows that the combination of D20-20K and Y45-20K can obviously reduce the expression quantity of CD25 on the surfaces of Tscm, T effector and Treg cells, in particular, the further reduction of CD25 expression on Treg cells relative to Y45-20K alone (FIG. 12D), the reduced CD25 expression avoids the excessive activation of T cells by endogenous IL-2 produced by T cell activation, and shows the superiority of D20-20K in combination with Y45-20K.

Example 8: combination of None-alpha variants and None-beta variants has effects on maintaining T cell stem properties and reducing T cell depletion

This example demonstrates in vitro activity of the Non-alpha variant (F42-20K, Y45-20K, E62-20K, P65-20K, E68-20K) prepared in example 1-2 in combination with the Non-beta variant (D20-20K, H16-20K, A-20K, H79-20K).

The experimental steps are as follows:

peripheral blood of healthy people is taken, PBMC is separated by using peripheral blood human lymphocyte separation liquid, CD3 positive T lymphocytes are separated by using a magnetic bead separation kit, and bead is added: cell=1: 1, the inoculation density is controlled to be 1x10 ⁶ Per ml,24 well plates, 1ml per well, and incubated with different species of cytokines (100 ng/ml) at the same concentration, with one fluid change every 72 hours, followed by two weeks of continuous incubation. The detection antibodies include: 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, BV510 anti-human IFN-gamma.

Experimental results:

treatment with none- α variants (Y45-20K) showed a significantly reduced expression levels of effector cytokines, performin, granzyme B and IFN- γ (FIG. 13B) compared to reduced expression of immune checkpoints PD-1 and TIM-3 on the surfaces of IL-2, CD8 and CD 4T cells (FIG. 13 a), and reduced expression levels of apoptosis-related biomarkers, CD57 and IL-10 (FIG. 13 c), indicating that none- α was able to reduce the degree of T cell depletion during in vitro culture, maintaining stem-cell-like characteristics of the cells. When the none- α variant (Y45-20K) was combined with the none- β variant (D20-20K, H-20K, A-20K, H79-20K), the expression of PD-1 and TIM-3 exhibited a further decrease in the expression levels of effector cytokines, perforin, granzyme B and IFN- γ, and the expression levels of apoptosis-related biomarkers CD57, IL-10 were further decreased (FIGS. 13 a-c).

Based on the above screening results, none- β variants (D20-20K) are preferred, and the effect on T cell activation by combination with different none- α variants (F42-20K, Y45-20K, E-20K, P65-20K, E-20K) was verified, and experimental results show that the effect on CD8 and CD 4T cell survival was improved to a different extent by combination of D20-20K with different none- α variants, but the expression of immune checkpoints PD-1 and TIM-3, the expression of effector cytokines, granzyme B, IFN- γ, and the expression of apoptosis-related biomarkers CD57 and IL-10 were reduced to some extent (FIGS. 14 a-c). Therefore, the combination of None-beta and None-alpha can further improve the survival state of T cells, such as reducing the degree of the growth and exhaustion of T cells, reducing the apoptosis induced by overactivation and delaying the differentiation to terminal effector cells on the basis of the synergism of the None-alpha.

The above results demonstrate that combining non-alpha variants with a bias towards non-alpha receptors with non-beta variants with a bias towards non-beta receptors produces a significant synergy, leading to a significant beneficial therapeutic effect.

Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate that: many modifications and variations of details may be made to adapt to a particular situation and the invention is intended to be within the scope of the invention. The full scope of the invention is given by the appended claims together with any equivalents thereof.

Claims

1. A composition comprising:

2. The composition of claim 1, wherein the first site-modified IL-2 (i) binds to IL2-rβγ dimer with a KD value less than 1E-8M (e.g., on the order of 1E-9 to 1E-8M), and/or (ii) does not bind to IL2-rαβγ trimer or binds with a KD value greater than 1E-8 (e.g., on the order of 1E-7 to 1E-6).

3. The composition of claim 1 or 2, wherein the second site-specific modified IL-2 (i) binds to IL2-rαβγ trimer with a KD value less than 1E-8 (e.g., on the order of 1E-9-1E-8), and/or (ii) does not bind to IL2-rβγ dimer.

4. A composition according to any one of claims 1 to 3, wherein the first amino acid position is selected from F42, Y45, E62, K64, P65, E68, K35, T37, R38, T41, K48, K49;

Preferably, the first amino acid position is selected from the group consisting of F42, Y45, E62, K64, P65, E68.

5. The composition of any one of claims 1-4, wherein the second amino acid position is selected from H16, D20, a73, H79;

preferably, the second amino acid position is selected from D20.

6. The composition of any one of claims 1-5, wherein the first amino acid position is F42, Y45, E62, K64, P65, or E68 and the second amino acid position is D20; alternatively, the first amino acid position is Y45 and the second amino acid position is H16, D20, a73, H79;

preferably, the first amino acid position is Y45 and the second amino acid position is D20.

7. The composition of any one of claims 1-6, wherein:

(a) The first site-modified IL-2 comprises a PEG-modifying group having an average molecular weight of 5-60 kDa, e.g. 5kDa, 10kDa, 15kDa, 20kDa, 25kDa, 30kDa, 35kDa, 40kDa, 45kDa, 50kDa or 60kDa; preferably, the first site-modified IL-2 comprises PEG-modifying groups 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. 5kDa, 10kDa, 15kDa, 20kDa, 25kDa, 30kDa or 40kDa;

And/or the number of the groups of groups,

(b) The second site-directed modified IL-2 comprises PEG-modifying groups having an average molecular weight of 5-60 kDa, e.g. 5kDa, 10kDa, 15kDa, 20kDa, 25kDa, 30kDa, 35kDa, 40kDa, 45kDa, 50kDa or 60kDa; preferably, the second site-directed modified IL-2 comprises PEG-modifying groups 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. 5kDa, 10kDa, 15kDa, 20kDa, 25kDa, 30kDa or 40kDa.

8. The composition of any one of claims 1-7, wherein the residue at the first amino acid position is mutated to an unnatural amino acid to which the PEG group is attached; and/or the residue at the second amino acid position is mutated to an unnatural amino acid to which the PEG group is attached.

9. The composition of claim 8, wherein the unnatural amino acid contains a chemical functional group (e.g., carbonyl, alkynyl, or azide group); the PEG group comprises a labeling group capable of chemically reacting with the chemical functional group, whereby the PEG group is attached to an unnatural amino acid;

Preferably, the unnatural amino acid contains an azide group, and the PEG group comprises a label group that is capable of click chemistry with the azide group, such that the PEG group is attached to the unnatural amino acid;

preferably, the labelling group capable of click chemical reaction with an azide group is a chemical moiety comprising a dibenzocyclooctynyl group, such as DBCO, DIBO or BCN;

preferably, the unnatural amino acid is a lysine derivative or a tyrosine derivative containing an azide group, such as N ε -2-azidoethoxycarbonyl-L-lysine (NAEK) or 2-amino-3- (4- (azidomethyl) phenyl) propanic acid.

10. A method of preparing the composition of any one of claims 1-9, comprising preparing the first site-directed modified IL-2 and preparing the second site-directed modified IL-2, wherein,

preparing the first site-modified IL-2 comprises:

-providing: (a1) A first site-directed mutated IL-2 having a residue at a first amino acid position mutated to an unnatural amino acid as compared to wild-type IL-2; (b1) A PEG group modified with a labeling group, the labeling group being capable of forming a covalent bond with the unnatural amino acid;

Preparing the second site-directed modified IL-2 comprises:

-providing: (a2) A second site-directed mutant IL-2 having a residue at a second amino acid position mutated to an unnatural amino acid as compared to wild-type IL-2; (b2) A PEG group modified with a labeling group, the labeling group being capable of forming a covalent bond with the unnatural amino acid;

11. The method of claim 10, wherein the unnatural amino acid of (a 1) comprises a chemical functional group (e.g., carbonyl, alkynyl, azide group), and the PEG group of (b 1) comprises a labeling group that is capable of chemically reacting with the chemical functional group to form a covalent bond; and/or the unnatural amino acid of (a 2) contains a chemical functional group (e.g., carbonyl, alkynyl, azide group); (b2) Wherein the PEG group comprises a labeling group capable of chemically reacting with the chemical functional group to form a covalent bond;

preferably, the unnatural amino acids of (a 1) and (a 2) comprise an azide group;

preferably, the unnatural amino acid described in (a 1) and (a 2) is a lysine derivative or a tyrosine derivative comprising an azide group, e.g. N ε -2-azidoethoxycarbonyl-L-lysine (NAEK) or 2-amino-3- (4- (azidomethyl) phenyl) propanoic acid.

12. The method of claim 10, wherein the step of,

the preparing of the first site-modified IL-2 comprises:

-providing: (a1) A first site-directed mutant IL-2 having a residue at a first amino acid position that is mutated to an unnatural amino acid that contains an azide group as compared to wild-type IL-2; (b1) A PEG group modified with a labeling group that is capable of click chemistry with an azide group;

-co-incubating (a 1) with (b 1) coupling the unnatural amino acid to the PEG group by a click reaction;

the preparing of the second site-directed modified IL-2 comprises:

-providing: (a2) A second site-directed mutant IL-2 having a residue at a second amino acid position that is mutated to an unnatural amino acid that contains an azide group as compared to wild-type IL-2; (b2) A PEG group modified with a labeling group that is capable of click chemistry with an azide group;

-co-incubating (a 2) with (b 2) to couple the unnatural amino acid to the PEG group by a click reaction;

preferably, the click chemistry is a copper-free click chemistry;

preferably, the labelling group capable of click chemical reaction with an azide group is an alkynyl containing chemical moiety, for example a dibenzocyclooctynyl containing chemical moiety, for example DBCO, DIBO or BCN;

Preferably, the labelling group capable of click chemical reaction with an azide group is DBCO.

13. The method of any one of claims 10-12, wherein the first site-directed mutated IL-2 and the second site-directed mutated IL-2 are provided by an unnatural amino acid orthogonal translation technique;

preferably, the non-natural amino acid orthogonal translation technique comprises the steps of:

co-transfecting the site-directed mutant sequence expression vector with a vector encoding an amber codon suppression tRNA and an aminoacyl tRNA synthetase specific for an unnatural amino acid into a host cell, culturing and inducing expression in a medium containing the unnatural amino acid to obtain IL-2 site-directed mutant to the unnatural amino acid;

preferably, the unnatural amino acid is N [ epsilon ] -2-azidoethoxycarbonyl-L-lysine (NAEK); preferably, the aminoacyl-tRNA synthetase that is specific for an unnatural amino acid is a NAEK-specific aminoacyl-tRNA synthetase.

14. A kit comprising: the composition of any one of claims 1-9;

preferably, the kit further comprises package insert comprising instructions for using the composition to prepare and/or culture immune cells for adoptive cell therapy in vitro;

preferably, the immune cell for adoptive cell therapy is an engineered immune cell that expresses a chimeric antigen receptor and/or comprises a nucleic acid molecule encoding the chimeric antigen receptor; preferably, the chimeric antigen receptor comprises an extracellular antigen binding domain, a transmembrane domain, and one or more intracellular signaling domains; preferably, the extracellular antigen-binding domain comprises an antibody or antigen-binding fragment (e.g., scFv) that specifically binds a tumor-associated antigen;

preferably, the immune cells used for adoptive cell therapy are Tumor Infiltrating Lymphocytes (TILs).

15. A kit comprising: the composition of any one of claims 1-9, a nucleic acid molecule encoding a chimeric antigen receptor;

preferably, the kit further comprises packaging instructions comprising instructions for using the composition and the nucleic acid molecule to prepare an engineered immune cell for adoptive cell therapy in vitro, the engineered immune cell expressing a chimeric antigen receptor and/or comprising a nucleic acid molecule encoding the chimeric antigen receptor;

Preferably, the chimeric antigen receptor comprises an extracellular antigen binding domain, a transmembrane domain, and one or more intracellular signaling domains; preferably, the extracellular antigen-binding domain comprises an antibody or antigen-binding fragment (e.g., scFv) that specifically binds a tumor-associated antigen;

preferably, the nucleic acid molecule encoding a chimeric antigen receptor is present in an expression vector;

preferably, the expression vector is a viral (e.g., lentiviral, retroviral, or adenoviral) vector.

16. A kit comprising: the composition of any one of claims 1-9, an immune cell for adoptive cell therapy;

preferably, the kit further comprises packaging instructions comprising instructions for using the composition to culture the immune cells in vitro for adoptive cell therapy;

Preferably, the engineered immune cells include lymphocytes that express IL2-rαβγ trimer, such as T cells, NK cells, or any combination thereof;

17. A method of culturing an immune cell for adoptive cell therapy, said method comprising culturing said cell in a cell culture medium comprising a first site-specific modified IL-2 and a second site-specific modified IL-2, wherein said first site-specific modified IL-2 and second site-specific modified IL-2 are as defined in any one of claims 1-9;

18. A method of preparing an immune cell for adoptive cell therapy, the immune cell for adoptive cell therapy being an engineered immune cell that expresses a chimeric antigen receptor and/or comprises a nucleic acid molecule encoding the chimeric antigen receptor, wherein the method comprises:

(1) Providing immune cells from a patient or healthy donor;

(2) Introducing a nucleic acid molecule encoding a chimeric antigen receptor into the immune cell of step (1) in the presence of a first site-specific modified IL-2 and a second site-specific modified IL-2, thereby providing the engineered immune cell; wherein the first site-specific modified IL-2 and the second site-specific modified IL-2 are as defined in any one of claims 1-9;

preferably, step (2) is performed in a cell culture medium comprising the first site-specific modified IL-2 and the second site-specific modified IL-2;

preferably, in step (2) the nucleic acid molecule encoding the chimeric antigen receptor is present in an expression vector;

preferably, in step (2) the nucleic acid molecule encoding the chimeric antigen receptor is introduced into the cell by infection with a viral (e.g., lentiviral, retroviral or adenoviral) vector;

Preferably, in step (1), the immune cells are pre-treated, the pre-treatment comprising sorting, activation and/or proliferation of immune cells;

preferably, the pretreatment comprises contacting immune cells with an anti-CD 3 antibody and an anti-CD 28 antibody, thereby stimulating and inducing proliferation of the immune cells, thereby generating pretreated immune cells;

preferably, after step (2), the method further comprises: (3) Continuing the step of culturing the immune cells obtained in step (2) in a cell culture medium comprising the first site-specific modified IL-2 and the second site-specific modified IL-2;

preferably, the immune cells include lymphocytes that express IL2-rαβγ trimers, such as T cells, NK cells, or any combination thereof;

preferably, the chimeric antigen receptor comprises an extracellular antigen binding domain, a transmembrane domain, and one or more intracellular signaling domains; preferably, the extracellular antigen-binding domain comprises an antibody or antigen-binding fragment (e.g., scFv) that specifically binds a tumor-associated antigen.

19. A method of preparing an immune cell for adoptive cell therapy, the immune cell for adoptive cell therapy being a Tumor Infiltrating Lymphocyte (TIL), wherein the method comprises: isolating infiltrating lymphocytes from the tumor tissue and culturing in a cell culture medium comprising a first site-specific modified IL-2 and a second site-specific modified IL-2, said first site-specific modified IL-2 and second site-specific modified IL-2 being as defined in any one of claims 1-9.

20. An immune cell for adoptive cell therapy obtained by the method of any one of claims 17-19.

21. An immune cell population comprising the immune cell of claim 20.

22. Use of a composition according to any one of claims 1-9 for in vitro preparation or culture of immune cells for adoptive cell therapy;

23. A pharmaceutical composition comprising the composition of any one of claims 1-9, a pharmaceutically acceptable carrier and/or excipient;

For example, the first site-directed modified IL-2 and the second site-directed modified IL-2 are in separate compositions or dosage forms;

for example, the first site-directed modified IL-2 and the second site-directed modified IL-2 are in the same composition or dosage form.

24. Use of a composition according to any one of claims 1 to 9 or a pharmaceutical composition according to claim 23 for the manufacture of a medicament for enhancing an immune response, preventing and/or treating a proliferative disease;

for example, the first site-directed modified IL-2 and the second site-directed modified IL-2 in the composition are administered simultaneously, separately or sequentially;

preferably, the proliferative disease is a tumor;

preferably, the tumor comprises a solid tumor or a hematological tumor;

preferably, the tumor comprises a metastatic cancer, a recurrent or refractory cancer;

preferably, the tumor is selected from the group consisting of melanoma, renal cell carcinoma, non-small cell lung carcinoma, lymphoma, head and neck squamous cell carcinoma, urothelial carcinoma, ovarian carcinoma, gastric carcinoma, and breast carcinoma.

25. A kit comprising:

(i) A medicament comprising a first site-specific modified IL-2 in a composition according to any one of claims 1-9 and optionally a pharmaceutically acceptable carrier and/or excipient, and package instructions comprising instructions for administering the medicament in combination with a composition comprising a second site-specific modified IL-2 in a composition according to any one of claims 1-9 and optionally a pharmaceutically acceptable carrier and/or excipient to enhance an immune response, prevent and/or treat a proliferative disorder (e.g. a tumor) in a subject; or alternatively, the process may be performed,

(ii) A medicament comprising the second site-specific modified IL-2 in the composition of any one of claims 1-9 and optionally a pharmaceutically acceptable carrier and/or excipient, and package instructions comprising instructions for administering the medicament in combination with a composition comprising the first site-specific modified IL-2 in the composition of any one of claims 1-9 and optionally a pharmaceutically acceptable carrier and/or excipient to enhance an immune response, prevent and/or treat a proliferative disorder (e.g., a tumor) in a subject; or alternatively, the process may be performed,

(iii) A first medicament comprising a first site-specific modified IL-2 in a composition according to any one of claims 1-9 and optionally a pharmaceutically acceptable carrier and/or excipient, a second medicament comprising a second site-specific modified IL-2 in a composition according to any one of claims 1-9 and optionally a pharmaceutically acceptable carrier and/or excipient; preferably, the kit further comprises package insert comprising instructions for administering the first and second medicaments to enhance an immune response, prevent and/or treat a proliferative disease (e.g., a tumor) in a subject;

preferably, the proliferative disease is a tumor;

Preferably, the tumor comprises a solid tumor or a hematological tumor;

26. A pharmaceutical composition comprising the immune cell of claim 20 or the population of immune cells of claim 21, and a pharmaceutically acceptable carrier and/or excipient.

27. Use of an immune cell of claim 20, an immune cell population of claim 21 or a pharmaceutical composition of claim 26 in the manufacture of a medicament for enhancing an immune response, preventing and/or treating a proliferative disorder;

preferably, the immune cell, population of immune cells or pharmaceutical composition is administered in combination with a first site-specific modified IL-2 and a second site-specific modified IL-2 as defined in any one of claims 1-9, e.g. simultaneously, separately or sequentially;

preferably, the proliferative disease is a tumor;

preferably, the tumor comprises a solid tumor or a hematological tumor;

28. Use of a composition according to any one of claims 1-9 in combination with immune cells for adoptive cell therapy in the manufacture of a medicament for enhancing immune responses, preventing and/or treating proliferative diseases;

preferably, the immune cell for adoptive cell therapy is an engineered immune cell that expresses a chimeric antigen receptor and/or comprises a nucleic acid molecule encoding the chimeric antigen receptor;

preferably, the immune cells for adoptive cell therapy are Tumor Infiltrating Lymphocytes (TILs);

preferably, the proliferative disease is a tumor;

preferably, the tumor comprises a solid tumor or a hematological tumor;