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

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 biases. The combination inhibits the expansion of immunosuppressive lymphocytes and reduces the terminal differentiation and exhaustion of effector lymphocytes through synergistic effect, and has a synergistic anti-tumor effect, so it can be used to enhance immune response and treat proliferative diseases such as tumors. In addition, the combination can also be used in the in vitro culture of immune cells in adoptive cell immunotherapy, reducing the aging and exhaustion of immune cells, so that the fate of immune cells in adoptive cell immunotherapy can be directed to long-term immunity. Accordingly, the present invention also provides in vitro methods for enhancing immune responses, combination therapy for treating proliferative diseases such as tumors, and producing or culturing immune cells for adoptive cell therapy.

Description

Combinations of receptor-biased PEGylated IL-2 variants and their applications

Technical Field

The present invention relates to the field of immunotherapy, and in particular, the present invention relates to site-specifically modified PEGylated IL-2 variants to provide a combination of PEGylated IL-2 variants with different receptor biases. The present invention also provides a combination therapy for enhancing immune response, treating proliferative diseases such as tumors, and an in vitro method for preparing or culturing immune cells for adoptive cell therapy.

Background Art

Interleukin-2 (IL2) is an important class of cytokines that are essential for the development and function of B cells, T cells, and NK cells. The receptors of IL2 are divided into IL2-Rα monomers, IL2-Rβγ dimers, and IL2-Rαβγ trimers. Among them, IL2 has a weak affinity with α monomers of ^10-8 M, a medium affinity with βγ dimers of ^10-9 M, and the highest affinity with αβγ trimers of ^10-11 M. Due to the low affinity, both dimeric IL-2R and endogenous IL-2 need to be induced at high levels to produce "dimeric IL-2R:IL-2" reactivity, which is the opposite of trimeric IL-2R, which allows host cells to respond directly to low concentrations of IL-2. Therefore, it is currently believed that the α chain of the trimeric IL-2R is not involved in signaling, but rather initiates binding to IL-2 (KD ≈ 10-8 M) and subsequent binding to the βγ complex with a 1000-fold increased affinity.

The distribution of IL2 receptors on different immune cells also varies: IL2-Rαβγ trimers are long-term expressed on the surface of regulatory T cells (Treg), so they have the highest affinity for IL2; IL2-Rβγ dimers are expressed on the surface of resting effector T cells (Teff), cytotoxic T cells (CTL) and NK cells, and have a general affinity for IL2. Only after the activation of effector T cells and NK cells will IL2-Rαβγ trimers be expressed, and the affinity for IL2 is enhanced. There is evidence that during CD8+ T cell priming, the expression of the α chain is quite dynamic - a portion of T cells upregulates α chain expression and perceives strong IL-2 signals, resulting in faster T cell proliferation but eventual differentiation, which in turn limits their effectiveness. In contrast, low-α T cells that are less sensitive to IL-2 preferentially upregulate CD127 and CD62L, resulting in functional long-lived memory cells. Without changing the basic process of α chain upregulation, it becomes valuable to have a larger population of memory T cells in vivo, but it is challenging for clinical application. Therefore, in tumor treatment, regulating the unbalanced ratio of Teff/Treg and CTL/Treg is gradually becoming a new research and development direction of immunotherapy.

Currently available IL2 target-related drugs have no receptor selectivity. In the treatment of tumors, when Teff and CTL need to be activated, a higher dose of IL2 is required, which also brings serious systemic toxicity and is the main source of side effects. In addition, the interaction between IL2 and IL2-Rα in lung endothelial cells can also induce vascular leakage syndrome, and the immunosuppression caused by the activation of Treg cells can also limit drug response.

Therefore, solving the problem that existing IL2 anti-tumor drugs have no selectivity for receptors is crucial for the clinical application of IL2-based tumor immunotherapy.

Summary of the invention

As mentioned above, although IL-2 has been widely used to enhance the body's immune cells, the attendant properties of promoting the expansion of immunosuppressive lymphocytes and driving the terminal differentiation and exhaustion of effector lymphocytes have hampered the effectiveness of IL-2 as an anti-tumor agent.

The inventors report here a strategy that explores the dynamic expression pattern of the IL-2 receptor through two types of receptor-biased PEGylated IL-2 variants, so that the fate of the initiated lymphocytes has durable anti-tumor immune properties. Specifically, the first type of receptor-biased PEGylated IL-2 variant is non-αPEGylate, which can activate and expand CD8+T cells, CD4+ cells and even NK cells, but not Treg cells, due to its biased affinity for dimeric rather than trimeric IL-2R, and it also induces terminal differentiation and exhaustion of reduced CD8+ cells. The second type of receptor-biased PEGylated IL-2 variant is non-βPEGylate, which neither activates CD8+T nor Treg cells, but it synergizes with the first type of non-αPEGylate to further expand CD4+ and CD8+T cells and reduce Treg and differentiated and exhausted CD8+T cell populations, due to its moderate affinity for the α chain of trimeric IL-2R, showing excellent local and systemic anti-tumor responses.

The inventors subsequently confirmed in the CD19-targeted CAR-T therapy model that the above-mentioned first class receptor-biased PEGylated IL-2 variant (non-αPEGylate) and the second class receptor-biased PEGylated IL-2 variant (non-βPEGylate) directed the fate of lymphocytes to long-term immunity through synergistic action. The above experimental evidence supports a putative mechanism that the PEGylated IL-2 variants biased by co-receptors cause sequential changes in the IL-2:IL-2R interaction, which not only circumvents the pleiotropic effects of IL-2, but also optimizes the differential memory program of T cells, indicating the direction for the next generation of IL-2 in T lymphocyte and adoptive T cell transfer therapy.

细胞的增殖，降低Treg细胞的增殖，维持Tscm和T effector细胞的增殖，降低T effector细胞的耗竭程度，并避免内源性IL-2对于T细胞的过度激活，使得过继细胞免疫治疗中的免疫细胞的命运导向长期免疫。因此，本发明还提供了用于增强免疫应答、治疗增生性疾病如肿瘤的组合疗法，以及制备或培养用于过继性细胞治疗的免疫细胞的体外方法。Based on the above-mentioned first type receptor-biased PEGylated IL-2 variant (non-αPEGylate) and second type receptor-biased PEGylated IL-2 variant (non-βPEGylate), the present invention provides a combination of PEGylated IL-2 variants with different receptor biases. The combination inhibits the proliferation of immunosuppressive lymphocytes and reduces the terminal differentiation and exhaustion of effector lymphocytes through synergistic action, increases the proportion and number of central memory cells, and has a synergistic anti-tumor effect, so it can be used to enhance immune response and treat proliferative diseases such as tumors. In addition, the combination can also be used for the in vitro culture of immune cells in adoptive cell immunotherapy, reduce the aging and exhaustion of immune cells, and enhance T

The invention also provides a method for enhancing immune response and treating proliferative diseases such as tumors, and an in vitro method for preparing or culturing immune cells for adoptive cell therapy.

Composition

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

(1) a first site-directed modified IL-2, which comprises a PEG group modification at the first amino acid position of the residue compared to wild-type IL-2, wherein the first site-directed modified IL-2 does not bind to the IL-2 receptor α (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-directed modified IL-2, which comprises a PEG group modification at the residue at the second amino acid position compared to wild-type IL-2, wherein the second site-directed modified IL-2 does not bind to the IL-2 receptor β (IL-2Rβ) subunit.

It is understood by those skilled in the art that a composition comprising a first site-modified IL-2 and a second site-modified IL-2 does not mean that the two must be administered simultaneously and/or formulated for delivery together, despite the scope of these delivery methods described herein. In some embodiments, the first site-modified IL-2 and the second site-modified IL-2 can be administered together in a single formulation. In some embodiments, the first site-modified IL-2 and the second site-modified IL-2 can be administered separately in different formulations. The first site-modified IL-2 in the combination can be administered in any order with the second site-modified IL-2, for example, simultaneously, before or after administration.

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 shown in SEQ ID NO: 1. In certain embodiments, the amino acid positions associated with IL-2 described herein are the positions in SEQ ID NO: 1.

In certain embodiments, "no binding" as described herein refers to no binding detected by surface plasmon resonance (SPR). In certain embodiments, the KD values described herein are determined by surface plasmon resonance (SPR).

The first site-directed modification of IL-2 receptor tropism

The first site-directed modified IL-2 described herein has a preference for non-α receptors.

In certain embodiments, the preference for non-α receptors includes: (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 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-directed modified IL-2 does not bind to the IL-2 receptor α (IL-2Rα) subunit. In certain embodiments, the first amino acid position of such first site-directed modified IL-2 is selected from F42, Y45, E62, K64, P65, E68.

In certain embodiments, the first site-directed modified IL-2 has only a low affinity for the IL-2 receptor α (IL-2Rα) subunit, for example, with a KD value 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) to bind to the IL-2 receptor α (IL-2Rα) subunit. In certain embodiments, the first site-directed modified IL-2 binds to the IL-2 receptor α (IL-2Rα) subunit with a KD value of the order of 1E-7 to 1E-6M. In certain embodiments, the first amino acid position of such first site-directed modified IL-2 is selected from K35, T37, R38, T41, K48, K49.

II. Binding properties to IL2-Rβγ dimer

In certain embodiments, the first site-modified IL-2 binds to the 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 the IL2-Rβγ dimer with a KD value of 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.

III. Binding properties to IL2-Rαβγ trimer

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

In certain embodiments, the first site-directed modified IL-2 has only low affinity for the IL2-Rαβγ trimer, for example, with a KD value 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) to bind to the IL2-Rαβγ trimer. In certain embodiments, the first site-directed modified IL-2 binds to the IL2-Rαβγ trimer with a KD value of the order of 1E-7 to 1E-6M. In certain embodiments, the first amino acid position of such first site-directed modified IL-2 is selected from K35, T37, R38, T41, K48, K49.

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

In certain embodiments, when the first site-directed modified IL-2 has a low affinity for the IL2-Rαβγ trimer, the first affinity of the first site-directed modified IL-2 for the IL2-Rβγ dimer may also be no higher than (e.g., lower than) the second affinity for the IL2-Rαβγ trimer (i.e., the first KD value for binding to the IL2-Rβγ dimer is no lower than (e.g., higher than) the second KD value for binding to the IL2-Rαβγ trimer), but the difference in affinities of the first site-directed modified IL-2 for the IL2-Rβγ dimer and the IL2-Rαβγ trimer (e.g., the ratio of the two KD values) is smaller than the difference in affinities of natural IL-2 for the IL2-Rβγ dimer and the IL2-Rαβγ trimer (e.g., the ratio of the two KD values). In such embodiments, the first KD value of the first site-directed modified IL-2 binding to the IL2-Rβγ dimer is no higher than 10 times the second KD value of binding to the IL2-Rαβγ trimer, for example, no higher than 9 times, no higher than 8 times, no higher than 7 times, no higher than 6 times, no higher than 5 times, no higher than 4 times, no higher than 3 times or no higher than 2 times. In certain embodiments, the first KD value of the first site-directed modified IL-2 binding to the IL2-Rβγ dimer is 8 to 3 times the second KD value of binding to the IL2-Rαβγ trimer, for example, 7 to 3 times. In certain embodiments, natural IL-2 binds to the IL2-Rβγ dimer with a first KD value of the order of 1E-9M. In certain embodiments, natural IL-2 binds to the IL2-Rαβγ trimer with a second KD value of the order of 1E-11M. In certain embodiments, the first KD value of natural IL-2 binding to IL2-Rβγ dimer is about 40 times the second KD value of binding to IL2-Rαβγ trimer.In certain embodiments, the first amino acid position of such first site-directed modified IL-2 is selected from R38, K49.

Second site-directed modification of IL-2 receptor preference

The second site-directed modified IL-2 described herein has a preference for non-β receptors.

In certain embodiments, the preference for non-β receptors includes: (i) binding to 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 IL2-Rβγ dimer.

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

In certain embodiments, the second site-directed modified IL-2 binds to the 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 the IL2-Rαβγ trimer with a KD value of the order of 1E-9 to 1E-8M.

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

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

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

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 average molecular weight of the PEG modification group included in the first site-directed modified IL-2 is 5 to 60 kDa, for example, 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa or 60 kDa. In certain embodiments, the average molecular weight of the PEG modification group included in the first site-directed modified IL-2 is 5 to 40 kDa, for example, 5 to 30 kDa, 5 to 25 kDa, 5 to 20 kDa, 10 to 40 kDa, 10 to 30 kDa, 15 to 30 kDa, 10 to 25 kDa or 15 to 25 kDa, for example, 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa or 40 kDa. In certain embodiments, the average molecular weight of the PEG modification group included in the first site-directed modified IL-2 is 5 kDa, 10 kDa or 20 kDa. In certain embodiments, the first site-directed modified IL-2 comprises a PEG modification group with an average molecular weight of 20 kDa.

In certain embodiments, the average molecular weight of the PEG modification group included in the second site-directed modified IL-2 is 5 to 60 kDa, for example, 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa or 60 kDa. In certain embodiments, the average molecular weight of the PEG modification group included in the second site-directed modified IL-2 is 5 to 40 kDa, for example, 5 to 30 kDa, 5 to 25 kDa, 5 to 20 kDa, 10 to 40 kDa, 10 to 30 kDa, 15 to 30 kDa, 10 to 25 kDa or 15 to 25 kDa, for example, 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa or 40 kDa. In certain embodiments, the average molecular weight of the PEG modification group included in the second site-directed modified IL-2 is 5 kDa, 10 kDa or 20 kDa. In certain embodiments, the average molecular weight of the PEG modification group included in the second site-directed modified IL-2 is 20 kDa.

In certain embodiments, the PEG modification group included in the first site-directed modified IL-2 and the PEG modification group included in the second site-directed modified IL-2 have substantially the same average molecular weight.

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

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

In certain embodiments, the non-natural amino acid contains a chemical functional group, such as a carbonyl group, an alkynyl group, and an azide group, which are generally capable of effectively and selectively forming a stable covalent bond; the PEG group comprises a labeling group that can chemically react with the chemical functional group to form a covalent bond, so that the PEG group is connected to the non-natural amino acid.

In certain embodiments, the non-natural amino acid contains an azide group, and the PEG group comprises a labeling group that can undergo click chemistry reaction with the azide group, thereby attaching the PEG group to the non-natural amino acid.

In certain embodiments, the non-natural amino acid is a lysine derivative containing an azide group. In certain embodiments, the non-natural amino acid is Nε-2-azidoethoxycarbonyl-L-lysine (NAEK).

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

In certain embodiments, the labeling group capable of undergoing click chemical reaction 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]nonyne).

In certain embodiments, the labeling group capable of undergoing click chemical reaction with an azide group is DBCO, and the residue at the first amino acid position (such as amino acid position F42, Y45, E62, P65 or E68) of the first site-directed modified IL-2 compared to wild-type IL-2 and the residue at the second amino acid position (such as amino acid position D20, H16, A73 or H79) of the second site-directed modified IL-2 compared to wild-type IL-2 are replaced by the structure of Formula Ia:

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

In certain embodiments, the labeling group capable of undergoing click chemical reaction with an azide group is DIBO, and the residue at the first amino acid position (such as amino acid position F42, Y45, E62, P65 or E68) of the first site-directed modified IL-2 compared to wild-type IL-2 and the residue at the second amino acid position (such as amino acid position D20, H16, A73 or H79) of the second site-directed modified IL-2 compared to wild-type IL-2 are replaced by the structure of Formula Ib:

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

In certain embodiments, the labeling group capable of undergoing click chemical reaction with an azide group is BCN, and the residue at the first amino acid position (such as amino acid position F42, Y45, E62, P65 or E68) of the first site-directed modified IL-2 compared to wild-type IL-2 and the residue at the second amino acid position (such as amino acid position D20, H16, A73 or H79) of the second site-directed modified IL-2 compared to wild-type IL-2 are replaced by the structure of Formula Ic:

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

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

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

Preparation of composition

The first site-directed modified IL-2 and the second site-directed modified IL-2 contained in the composition provided in the first aspect of the present invention can be prepared by any method known in the art.

In certain embodiments, the non-natural amino acid can be site-specifically inserted by non-natural amino acid orthogonal translation technology, and the PEG group can be subsequently attached to the non-natural amino acid.

Non-natural amino acid orthogonal translation technology is well known to those skilled in the art. Non-natural amino acid orthogonal translation technology utilizes stop codons to insert non-natural amino acids into the amino acid sequence of protein during protein translation, which actually expands the number of amino acid codons, so non-natural amino acid orthogonal translation technology is also referred to as genetic codon expansion technology. Typically, non-natural amino acid orthogonal translation systems involve tRNA, aminoacyl tRNA synthetases, and target nucleic acid sequences with one or more stop codons. The above system is introduced into a host cell and cultivated in a culture medium containing appropriate nutrients and one or more non-natural amino acids to be inserted. The host cell is then maintained under conditions that allow expression of the target protein. In response to non-natural codons, one or more non-natural amino acids are incorporated into a polypeptide chain.

In certain embodiments, non-natural amino acids can be site-specifically inserted by non-natural amino acid orthogonal translation technology. The non-natural amino acids can contain chemical functional groups, such as carbonyl groups, alkynyl groups, azide groups, etc. These groups are generally able to effectively and selectively form stable covalent bonds, and then the PEG group is site-specifically modified by forming a covalent bond through a chemical reaction.

In certain embodiments, the non-natural amino acid can be inserted into the non-natural amino acid by orthogonal translation technology, and then the PEG group is modified by click chemistry. In certain embodiments, the non-natural amino acid and the PEG group respectively include chemical groups capable of click chemistry reaction.

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

The preparation of the first site-directed modified IL-2 comprises:

- providing: (a1) a first site-directed mutant IL-2, wherein the residue at the first amino acid position (such as amino acid position F42, Y45, E62, P65 or E68) is mutated to a non-natural amino acid compared to wild-type IL-2; (b1) a PEG group modified by a labeling group, wherein the labeling group is capable of forming a covalent bond with the non-natural amino acid;

- co-incubating (a1) and (b1) to couple the unnatural amino acid to the PEG group through a chemical reaction;

The preparation of the second site-directed modified IL-2 comprises:

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

- (a2) is incubated with (b2) to couple the unnatural amino acid to the PEG group through a chemical reaction.

In certain embodiments, the non-natural amino acid contained in the first site-directed mutagenesis IL-2 contains a chemical functional group, for example, a carbonyl group, an alkynyl group, an azide group, etc., which are generally capable of effectively and selectively forming a stable covalent bond; the labeling group contained in the PEG group can chemically react with the chemical functional group to form a covalent bond.

In certain embodiments, the non-natural amino acid contained in the second site-directed mutagenesis IL-2 contains a chemical functional group, such as a carbonyl group, an alkynyl group, an azide group, etc., which are generally capable of effectively and selectively forming a stable covalent bond; the labeling group contained in the PEG group can chemically react with the chemical functional group to form a covalent bond.

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

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

In certain embodiments, the non-natural amino acid is a lysine derivative containing an azide group. In certain embodiments, the non-natural amino acid is Nε-2-azidoethoxycarbonyl-L-lysine (NAEK).

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

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

- providing: (a1) a first site-directed mutant IL-2, wherein the residue at the first amino acid position (such as amino acid position F42, Y45, E62, P65 or E68) is mutated to a non-natural amino acid containing an azide group compared to wild-type IL-2; (b1) a PEG group modified by a labeling group, wherein the labeling group can undergo a click chemical reaction with the azide group;

- (a1) is incubated with (b1) to couple the unnatural amino acid to the PEG group via a click reaction.

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

- providing: (a2) a second site-directed mutant IL-2, wherein the residue at the second amino acid position (such as amino acid position D20, H16, A73 or H79) is mutated to a non-natural amino acid containing an azide group compared to wild-type IL-2; (b2) a PEG group modified by a labeling group, wherein the labeling group can undergo a click chemical reaction with the azide group;

- (a2) is incubated with (b2) to couple the unnatural amino acid to the PEG group via a click reaction.

In certain embodiments, the click chemistry is a copper-free click chemistry. Copper-free click chemistry is a click reaction achieved by introducing cyclooctyne to maintain cell activity, where the strain of the eight-membered ring allows reaction with azide in the absence of a catalyst. One of these reagents consists of so-called DBCO compounds. Azide-modified macromolecules can now be labeled in the absence of metal catalysts, which can not only be used for the study of living cells, but also prevent damage to proteins.

In certain embodiments, the labeling group capable of undergoing a click chemical reaction with an azide group is a chemical moiety comprising an alkynyl group. In certain embodiments, the labeling group capable of undergoing a click chemical reaction with an azide group is a chemical moiety comprising a dibenzocyclooctyne group. In certain embodiments, the labeling group capable of undergoing a click chemical reaction with an azide group is dibenzocyclooctyne (DBCO), 4-dibenzocyclooctynol (DIBO) or BCN (bicyclo[6.1.0]nonyne). In certain embodiments, the labeling group capable of undergoing a click chemical reaction with an azide group is DBCO.

In certain embodiments, the DBCO-labeled PEG group has a structure as shown in Formula IIa, wherein R3 is a PEG group.

In certain embodiments, the DIBO-labeled PEG group has a structure as 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 non-natural amino acid orthogonal translation technology.

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

- 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 the nucleic acid sequence encoding the site-directed mutated IL-2 to a vector to obtain a site-directed mutated sequence expression vector;

- The site-directed mutation sequence expression vector is co-transfected into a host cell with a vector encoding an amber codon suppressor tRNA and an aminoacyl-tRNA synthetase specific for a non-natural amino acid, and the cell is cultured and induced to express in a medium containing a non-natural amino acid to obtain IL-2 with a site-directed mutation to a non-natural amino acid.

In certain embodiments, the unnatural amino acid is Nε-2-azidoethoxycarbonyl-L-lysine (NAEK). In certain embodiments, the aminoacyl-tRNA synthetase specific for the unnatural amino acid is a NAEK-specific aminoacyl-tRNA synthetase.

In certain embodiments, the vector encoding an amber codon suppressor tRNA and an aminoacyl-tRNA synthetase specific for an unnatural amino acid is pSURAR-YAV (also known as pSUPAR-YAV-tRNA/PylRS), which is obtained from Escherichia coli containing the plasmid pSUPAR-YAV-tRNA/PylRS, which is deposited in the General Microbiology Center of the China Culture Collection of Microorganisms (No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences) on April 8, 2013, with a deposit number of CGMCC No: 7432, and is classified as Escherichia coli.

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

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

Reagent test kit

细胞的增殖，降低Treg细胞的增殖，维持Tscm和T effector细胞的增殖，降低T effector细胞的耗竭程度，并避免内源性IL-2对于T细胞的过度激活，使得过继细胞免疫治疗中的免疫细胞的命运导向长期免疫。基于此，本发明还提供了如下所述的试剂盒，制备或培养用于过继性细胞治疗的免疫细胞的体外方法，以及由此方法获得的用于过继性细胞治疗的免疫细胞。The combination of the first site-specific modified IL-2 and the second site-specific modified IL-2 provided by the present invention can reduce the aging and exhaustion of immune cells in the in vitro culture of immune cells, enhance T

The invention also provides a kit as described below, an in vitro method for preparing or culturing immune cells for adoptive cell therapy, and immune cells for adoptive cell therapy obtained by the method.

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

In this article, adoptive cell therapy can include tumor infiltrating T cells (TIL) therapy, chimeric antigen receptor T cell therapy (CAR-T), T cell receptor therapy (TCR), NK cell therapy, etc. In this article, the modified immune cells for adoptive cell therapy can be any cells known in the art for adoptive cell therapy. In certain embodiments, the modified immune cells for adoptive cell therapy include lymphocytes, such as T cells, NK cells, or a combination thereof.

In certain embodiments, the immune cells used for adoptive cell therapy are modified immune cells that express chimeric antigen receptors and/or contain nucleic acid molecules encoding the chimeric antigen receptors. In certain embodiments, the modified immune cells include lymphocytes expressing 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 present invention provides a kit comprising the composition of the first aspect and a nucleic acid molecule encoding a chimeric antigen receptor. In certain embodiments, the kit further comprises a package insert, the package insert comprising instructions for using the composition and the nucleic acid molecule to prepare in vitro engineered immune cells for adoptive cell therapy, the engineered immune cells expressing a chimeric antigen receptor and/or comprising a nucleic acid molecule encoding the chimeric antigen receptor.

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

In certain embodiments, the expression vector is a viral (eg, lentiviral, retroviral or adenoviral) vector. In certain embodiments, the expression vector is a non-viral vector.

The present invention also relates to the use of the composition described in the first aspect and optionally a nucleic acid molecule encoding a chimeric antigen receptor or the kit described in the fourth aspect for preparing in vitro modified immune cells for adoptive cell therapy, wherein the modified immune cells express a chimeric antigen receptor and/or contain a nucleic acid molecule encoding the chimeric antigen receptor.

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

In certain embodiments, the immune cells used for adoptive cell therapy are modified immune cells that express chimeric antigen receptors and/or contain nucleic acid molecules encoding the chimeric antigen receptors. In certain embodiments, the modified immune cells include lymphocytes expressing 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 certain embodiments, the chimeric antigen receptor described herein has a meaning known to those skilled in the art, which typically includes 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 to a tumor-associated antigen (e.g., CD19).

Cultivation and preparation of immune cells for adoptive cell therapy

In a fifth aspect, the present invention provides a method for 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.

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

In certain embodiments, the method further comprises harvesting the cells for storage (eg, reconstitution in cryopreservation medium) or administration (eg, for adoptive cell therapy).

细胞的增殖，降低Treg细胞的增殖，维持Tscm和T effector细胞的增殖状态，降低T effector细胞的耗竭程度，避免T细胞激活产生的内源性IL-2对于T细胞的过度激活。In certain embodiments, the culture conditions provided by the presence of the first site-directed modified IL-2 and the second site-directed modified IL-2 can reduce senescence and exhaustion of immune cells, preserve their stemness, and enhance T

T cells, reduce the proliferation of Treg cells, maintain the proliferation state of Tscm and T effector cells, reduce the exhaustion of T effector cells, and avoid the excessive activation of T cells by endogenous IL-2 produced by T cell activation.

In certain embodiments, the immune cells used for adoptive cell therapy are modified immune cells that express chimeric antigen receptors and/or contain nucleic acid molecules encoding the chimeric antigen receptors. In certain embodiments, the modified immune cells include lymphocytes expressing 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 sixth aspect, the present invention provides a method for preparing immune cells for adoptive cell therapy, which comprises using the first site-directed modified IL-2 and the second site-directed modified IL-2 as defined in the first aspect.

In certain embodiments of the sixth aspect, the present invention provides a method for preparing an immune cell for adoptive cell therapy, wherein the immune cell for adoptive cell therapy is a modified 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 patients or healthy donors;

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

In certain embodiments, step (2) is carried out in a cell culture medium comprising the IL-2 modified at the first fixed point and the IL-2 modified at the second fixed point. The cell culture medium can be any culture medium capable of supporting cell growth, generally comprising inorganic salts, vitamins, glucose, a buffer system and essential amino acids, and generally having an osmotic pressure of about 280-330mOsmol. In certain embodiments, the cell culture medium is a culture medium capable of supporting the growth of immune cells (such as lymphocytes, such as T cells and/or NK cells). In certain embodiments, the cell culture medium is a complete culture medium. In certain embodiments, the cell culture medium comprises a basal culture medium (such as RPMI 1640), serum (such as FBS), sodium pyruvate, and 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 the 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 via a non-viral vector.

In certain embodiments, in step (1), the immune cells are pretreated, and the pretreatment includes sorting, activation and/or proliferation of immune cells. In certain embodiments, the pretreatment includes contacting the immune cells with anti-CD3 antibodies and anti-CD28 antibodies, thereby stimulating the immune cells and inducing their proliferation, thereby generating pretreated immune cells. In certain embodiments, the pretreatment includes separating T cells from peripheral blood mononuclear cells (PBMC).

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

In certain embodiments, the method further comprises harvesting the cells for storage (eg, reconstitution in cryopreservation medium) or administration (eg, for adoptive cell therapy).

In certain embodiments, the immune cell comprises a lymphocyte expressing IL2-Rαβγ trimer, such as a T cell, a NK cell, or any combination thereof. In certain embodiments, the immune cell is a T cell.

In certain embodiments, the production conditions provided in which the first site-directed modified IL-2 and the second site-directed modified IL-2 are present can reduce over-activation and terminal differentiation of immune cells.

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

In a seventh aspect, the present invention provides immune cells for adoptive cell therapy, which are prepared or cultured by the method described in any of the above aspects.

细胞增殖，降低的Treg细胞增殖，维持的Tscm和T effector细胞增殖，降低的Teffector细胞耗竭程度，和/或降低的由内源性IL-2导致的过度激活。In certain embodiments, the immune cells of the seventh aspect have reduced aging and exhaustion, enhanced T

cell proliferation, reduced Treg cell proliferation, maintained Tscm and T effector cell proliferation, reduced Teffector cell exhaustion, and/or reduced over-activation by endogenous IL-2.

In an eighth aspect, the present invention provides an immune cell population comprising the immune cells of the seventh aspect, and optional unmodified and/or unsuccessfully modified immune cells. In certain embodiments, the immune cells of the seventh aspect account for about 10%-100%, preferably 40%-80% of the total number of cells in the immune cell population.

Application in synergistically enhancing immune response

The first site-specific modified IL-2 and the second site-specific modified IL-2 provided by the present invention have a significant synergistic effect of enhancing the immune response. Therefore, the combination of the first site-specific modified IL-2 and the second site-specific modified IL-2 provided by the present invention can be used to treat disease conditions that stimulate the host's immune system to benefit, especially conditions that expect to enhance cellular immune responses, which may include disease conditions in which the host's immune response is insufficient or defective, such as tumors.

In a tenth aspect, the present invention provides a pharmaceutical composition comprising the composition described in 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-modified IL-2 and the second site-modified IL-2 are in separate compositions or dosage forms. In certain embodiments, the pharmaceutical composition comprises: a first composition formed by the first site-modified IL-2 and a pharmaceutically acceptable carrier and/or excipient, and a second composition formed by the second site-modified IL-2 and a pharmaceutically acceptable carrier and/or excipient.

In certain embodiments, the pharmaceutical composition comprises a composition or dosage form. In certain embodiments, the first site-modified IL-2 and the second site-modified IL-2 are in the same composition or dosage form. In certain embodiments, the pharmaceutical composition comprises: a single composition formed by the first site-modified IL-2, the second site-modified IL-2, and a pharmaceutically acceptable carrier and/or excipient.

The first site-modified IL-2 and the second site-modified IL-2 described in the present invention, their combination, and the composition described in the first aspect or the pharmaceutical composition described in the tenth aspect can be formulated into a dosage form compatible with its intended route of administration. Examples of routes of administration include parenteral administration, for example, intravenous administration, intradermal administration, subcutaneous administration, oral administration (e.g., inhalation administration), transdermal administration (i.e., topical administration), transmucosal administration, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous administration may include the following components: sterile diluents such as water for injection, saline solutions, fixed oils, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates, or phosphates, and agents for adjusting tension, such as sodium chloride or glucose. pH can be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. The preparation for parenteral administration can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

The pharmaceutical composition suitable for injection includes sterile aqueous solution (wherein water soluble) or dispersion and sterile powder for preparing sterile injection solution or dispersion at any time. For intravenous administration, suitable carriers include physiological saline, antibacterial water, polyoxyethylene castor oil ELTM or phosphate buffered saline (PBS). In all cases, the composition must be sterile, and should be a fluid that reaches the degree of easy injection. It must be stable under manufacturing and storage conditions, and must prevent microorganisms such as bacteria and fungi from contaminating and preserving. The carrier can be a solvent or dispersion medium including, for example, water, ethanol, polyols (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Preventing the effect of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, isotonic agents will preferably be included in the composition, such as sugars, polyols such as mannitol, sorbitol, sodium chloride. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

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

In the twelfth aspect, the present invention further provides the use of the composition described in the first aspect or the pharmaceutical composition described in the tenth aspect for enhancing immune response, preventing and/or treating proliferative diseases (such as tumors), or, in the preparation of a medicament for enhancing immune response, preventing and/or treating proliferative diseases (such as tumors). The present invention also provides the use of a combination of a first site-directed modified IL-2 and a second site-directed modified IL-2 for enhancing immune response, preventing and/or treating proliferative diseases (such as tumors), or, in the preparation of a medicament for enhancing immune response, preventing and/or treating proliferative diseases (such as tumors).

In certain embodiments, the first site-modified IL-2 and the second site-modified IL-2 are separately formulated into two or more compositions (e.g., a kit comprising each component). The individual components administered in combination with each other can be administered simultaneously, separately or sequentially. In certain embodiments, the individual components administered in combination with each other can be administered to the subject at a time different from the time of administering the other components; for example, as part of a treatment regimen, each administration can be given non-simultaneously (e.g., individually or sequentially) at intervals of a given time period. In certain embodiments, the individual components administered in combination with each other can also be administered sequentially during the same administration period, but are administered substantially simultaneously. In addition, the individual components administered in combination with each other can be administered to the 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 present invention provides a kit comprising a drug containing the first site-directed modified IL-2 and an optional pharmaceutically acceptable carrier and/or excipient, and a package insert comprising instructions for administering the drug in combination with a composition containing the second site-directed modified IL-2 and an optional pharmaceutically acceptable carrier and/or excipient to enhance an immune response, prevent and/or treat a proliferative disease (e.g., a tumor) in a subject.

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

In another aspect, the present invention provides a kit comprising a first drug comprising the first site-directed modified IL-2 and an optional pharmaceutically acceptable carrier and/or excipient, a second drug comprising the second site-directed modified IL-2 and an optional pharmaceutically acceptable carrier and/or excipient. In certain embodiments, the kit further comprises a package insert containing instructions for administering the first drug and the second drug 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, purposes and kits as described above, the immune response is a cellular immune response. In certain embodiments, the immune response is a T cell-mediated immune response, particularly an effector T cell (Teff)-mediated immune response. In certain embodiments, the enhanced immune response also includes reducing or suppressing Treg cell function.

In certain embodiments of the methods, uses and kits described above, the proliferative disease is a tumor, including solid tumors or hematological tumors, including metastatic cancers, recurrent or refractory cancers. In other embodiments, the proliferative disease is hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias, purpura, sarcoidosis, Sezary Syndrome, Waldenstron's macroglobulinemia, Gaucher's Disease, histiocytosis, and any other cell proliferation disease outside of neoplasia in an 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 blood tumor, such as a leukemia, lymphoma or myeloma. In certain embodiments, the blood tumor is a metastatic cancer, a relapsed or refractory cancer.

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

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

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

In certain embodiments of the methods, uses and kits described above, the first site-directed modified IL-2 and the second site-directed modified IL-2, their combinations and the compositions or pharmaceutical compositions of the present invention can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use. The composition or pharmaceutical composition of the present invention should be sterile and stable under production and storage conditions. A preferred dosage form is an injection. Such an injection can be a sterile injection solution. For example, a sterile injection solution can be prepared by the following method: incorporating the necessary dose of the active ingredient in an appropriate solvent, and optionally, simultaneously incorporating other desired ingredients (including but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by filtration and sterilization. In addition, sterile injectable solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze drying) for storage and use. Such sterile lyophilized powders can be dispersed in a suitable carrier before use, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w/v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffer solution (e.g., phosphate buffer solution), 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 in the present invention, their combinations, and the compositions or pharmaceutical compositions of the present invention can be administered by any suitable method known in the art, including but not limited to, oral, oral, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic, inguinal, intravesical, topical (e.g., powders, ointments or drops), or nasal routes. However, for many therapeutic uses, the preferred route/mode of administration is parenteral administration (e.g., intravenous or bolus, subcutaneous, intraperitoneal, intramuscular). The skilled person will appreciate that the route and/or mode of administration will vary depending on the intended purpose. In certain embodiments, the first site-modified IL-2 and the second site-modified IL-2 described in the present invention, their combinations, and the compositions or pharmaceutical compositions of the present invention are administered by intravenous or bolus injection.

In certain embodiments of the methods, uses and kits described above, the first site-directed modified IL-2 and the second site-directed modified IL-2, their combinations and the compositions or pharmaceutical compositions of the present invention can be formulated in dosage unit form for ease of administration. Dosage unit form refers to physically discrete units suitable as single doses for the subject 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-directed modified IL-2 and the second site-directed modified IL-2 described in the present invention, their combination, and the composition or pharmaceutical composition of the present invention can be administered alone or in combination with another pharmaceutically active agent (e.g., an anti-tumor agent) or another therapy (e.g., an anti-tumor therapy).

Application in adoptive cell therapy

The combination of the first site-specific modified IL-2 and the second site-specific modified IL-2 provided by the present invention can direct the fate of immune cells in adoptive cell immunotherapy to long-term immunity, thereby further providing the therapeutic application of the immune cells.

In a thirteenth aspect, the present invention provides a pharmaceutical composition comprising the immune cells described in the seventh aspect or the immune cell population described in the eighth aspect and a pharmaceutically acceptable carrier and/or excipient.

The immune cells described in the seventh aspect, the immune cell group described in the eighth aspect, or the pharmaceutical composition described in the thirteenth aspect can be formulated into a dosage form compatible with its intended route of administration. Examples of routes of administration include parenteral administration, for example, intravenous administration, intradermal administration, subcutaneous administration, oral administration (e.g., inhalation administration), transdermal administration (i.e., topical administration), transmucosal administration, and rectal administration. Solutions or suspensions for parenteral administration, intradermal administration, or subcutaneous administration may include the following components: sterile diluents such as water for injection, saline solutions, fixed oils, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates, or phosphates, and agents for adjusting tension, such as sodium chloride or glucose. pH can be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. The preparation for parenteral administration can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

The pharmaceutical composition suitable for injection includes sterile aqueous solution (wherein water soluble) or dispersion and sterile powder for preparing sterile injection solution or dispersion at any time. For intravenous administration, suitable carriers include physiological saline, antibacterial water, polyoxyethylene castor oil ELTM or phosphate buffered saline (PBS). In all cases, the composition must be sterile, and should be a fluid that reaches the degree of easy injection. It must be stable under manufacturing and storage conditions, and must prevent microorganisms such as bacteria and fungi from contaminating and preserving. The carrier can be a solvent or dispersion medium including, for example, water, ethanol, polyols (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Preventing the effect of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, isotonic agents will preferably be included in the composition, such as sugars, polyols such as mannitol, sorbitol, sodium chloride. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

In a fourteenth aspect, the present invention provides a method for enhancing immune response, preventing and/or treating proliferative diseases (such as tumors), comprising administering the immune cells described in the seventh aspect, the immune cell population described in the eighth aspect, or the pharmaceutical composition described in the thirteenth aspect to a subject in need.

In certain embodiments, the method comprises the following steps: (1) obtaining immune cells using the method described in aspect 5 and/or aspect 6; (2) administering the immune cells obtained in step (1) or a cell population comprising the same to the subject for treatment.

In certain embodiments, chimeric antigen receptor cell therapy is administered to a subject, the method comprising: obtaining modified immune cells expressing a chimeric antigen receptor using the method described in the fifth aspect of the invention and/or the method described in the sixth aspect, and then administering the modified immune cells to the subject.

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

In the fifteenth aspect, the present invention also provides the use of the immune cells described in the seventh aspect, the immune cell population described in the eighth aspect or the pharmaceutical composition described in the thirteenth aspect for enhancing immune response, preventing and/or treating proliferative diseases (such as tumors), or in the preparation of drugs for enhancing immune response, preventing and/or treating proliferative diseases (such as tumors).

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

In the sixteenth aspect, the present invention also provides the use of the combination of the composition described in the first aspect and immune cells for adoptive cell therapy for enhancing immune response, preventing and/or treating proliferative diseases (such as tumors), or in the preparation of a medicament for enhancing immune response, preventing and/or treating proliferative diseases (such as tumors).

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

In certain embodiments of the sixteenth or seventeenth aspects, the immune cells used for adoptive cell therapy are modified immune cells that express chimeric antigen receptors and/or contain nucleic acid molecules encoding the chimeric antigen receptors. In certain embodiments, the modified immune cells include lymphocytes expressing IL2-Rαβγ trimers, such as T cells, NK cells, or any combination thereof.

In certain embodiments of the sixteenth aspect or the seventeenth aspect, the immune cells used for adoptive cell therapy are tumor infiltrating lymphocytes (TIL).

In certain embodiments of the sixteenth or seventeenth aspect, the composition of the first aspect and the immune cells for adoptive cell therapy are present in the same composition or dosage form, so that they can 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 can be administered separately or sequentially.

In certain embodiments of the methods and uses as 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 enhanced immune response also includes reducing or suppressing Treg cell function.

In certain embodiments of the methods and uses described above, the proliferative disease is a tumor, including solid tumors or hematological tumors, including metastatic cancers, recurrent or refractory cancers. In other embodiments, the proliferative disease is hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias, purpura, sarcoidosis, Sezary Syndrome, Waldenstron's macroglobulinemia, Gaucher's Disease, histiocytosis, and any other cell proliferation disease outside of neoplasia located in an 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 blood tumor, such as a leukemia, lymphoma or myeloma. In certain embodiments, the blood tumor is a metastatic cancer, a relapsed or refractory cancer.

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

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

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

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

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

In certain embodiments of the methods and uses described above, the immune cells described in the seventh aspect, the immune cell population described in the eighth aspect, or the pharmaceutical composition described in the thirteenth aspect can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use. The composition or pharmaceutical composition of the present invention should be sterile and stable under production and storage conditions. A preferred dosage form is an injection. Such an injection can be a sterile injection solution. For example, a sterile injection solution can be prepared by the following method: incorporating the necessary dose of the active ingredient in an appropriate solvent, and optionally, simultaneously incorporating other desired ingredients (including but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by filtration and sterilization. In addition, sterile injectable solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze drying) for storage and use. Such sterile lyophilized powders can be dispersed in a suitable carrier before use, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w/v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

In certain embodiments of the methods and uses as described above, the immune cells described in the seventh aspect, the immune cell groups described in the eighth aspect, or the pharmaceutical composition described in the thirteenth aspect can be administered by any suitable method known in the art, including but not limited to, oral, oral, sublingual, eyeball, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum, inguinal, intravesical, topical (e.g., powders, ointments or drops), or nasal routes. However, for many therapeutic uses, the preferred route of administration/mode is parenteral administration (e.g., intravenous injection or push injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). Technicians should understand that the route of administration and/or mode will change according to the intended purpose. In certain embodiments, the immune cells described in the seventh aspect, the immune cell groups described in the eighth aspect, or the pharmaceutical composition described in the thirteenth aspect are administered by intravenous injection or push injection.

In certain embodiments of the methods and uses described above, the immune cells of the seventh aspect, the immune cell population of the eighth aspect, or the pharmaceutical composition of the thirteenth aspect can be formulated in dosage unit form for ease of administration. Dosage unit form refers to physically discrete units suitable as single doses for the subject 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 described in the seventh aspect, the immune cell population described in the eighth aspect, or the pharmaceutical composition described in the thirteenth aspect can be administered alone or in combination with another pharmaceutically active agent (e.g., an anti-tumor agent) or another therapy (e.g., an anti-tumor therapy).

Definition of terms

In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the molecular genetics, nucleic acid chemistry, cell culture, biochemistry, cell biology and other operating steps used herein are conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

When the terms "for example," "such as," "including," "including," "comprising," or variations thereof are used herein, these terms will not be considered as limiting terms, but will be interpreted to mean "but not limited to" or "not limited to."

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) should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

As used herein, the term "non-natural amino acid" refers to an amino acid other than the 20 amino acids naturally present in proteins. Non-limiting examples of non-natural amino acids include: Nε-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 non-natural analogs of tyrosine amino acids; non-natural analogs of glutamine amino acids; non-natural analogs of phenylalanine amino acids; non-natural analogs of serine amino acids; non-natural analogs of threonine amino acids analogs; alkyl, aryl, acyl, azido, cyano, halogen, hydrazine, hydrazide, hydroxyl, alkenyl, alkynyl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate, phosphate, phosphono, phosphine, heterocycle, enone, imine, aldehyde, hydroxylamine, keto or amino substituted amino acids or combinations thereof; amino acids with photoactivatable cross-linkers; spin-labeled amino acids; fluorescent amino acids; metal binding amino acids; metal containing amino acids; radioactive amino acids; photocaged and/or photoisomerizable amino acids; Amino acids containing biotin or biotin analogs; amino acids containing a keto group; amino acids containing polyethylene glycol or polyether; heavy atom substituted amino acids; chemically cleavable or photocleavable amino acids; amino acids with extended side chains; amino acids containing toxic groups; sugar substituted amino acids; carbon-linked sugar-containing amino acids; redox-active amino acids; acids containing an α-hydroxyl group; amino thioacids; α,α disubstituted amino acids; β-amino acids; cyclic amino acids other than proline or histidine; and aromatic amino acids other than phenylalanine, tyrosine or tryptophan.

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

As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). A vector can 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 initiation site.

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

As used herein, the term "chimeric antigen receptor (CAR)" refers to a recombinant polypeptide construct comprising at least one extracellular antigen binding domain, an optional spacer domain, a transmembrane domain, and an intracellular signaling domain, which combines the antibody-based specificity for a target antigen (e.g., a tumor antigen) with an immune effector cell activation intracellular domain to exhibit specific immune activity for cells expressing the target antigen (e.g., tumor cells). In the present invention, the expression "CAR-expressing immune cells" refers to immune cells expressing CAR and having 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. This domain will be able to interact with cell surface molecules. For example, an extracellular antigen binding domain can 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 a protein portion that conducts effector signaling function signals and guides cells to perform specialized functions. Therefore, the intracellular signaling domain has the ability to activate at least one normal effector function of an immune effector cell expressing a CAR. For example, the effector function of a T cell can be a cytolytic activity or an auxiliary activity, including the secretion of cytokines.

As used herein, the term "primary signaling domain" refers to a portion of a protein that is capable of regulating the primary activation of a TCR complex in a stimulatory or inhibitory manner. Primary signaling domains that act in a stimulatory manner typically contain a signaling motif known as an immunoreceptor 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, FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ε, CD3 ζ, CD22, DAP10, CD79a, CD79b, and CD66d.

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

As used herein, the term "immune cell" refers to any cell of the immune system with one or more effector functions (e.g., cytotoxic cell killing activity, secretion of cytokines, ADCC and/or CDC induction). Typically, immune cells are cells with hematopoietic origin and play a role in immune response. In certain embodiments, immune cells refer to immune effector cells. The term "effector function" refers to the specialized functions of immune effector cells, such as enhancing or promoting the function or reaction of immune attack on target cells (e.g., killing of target cells, or inhibiting their growth or proliferation). The effector function of T cells, for example, can be cytolytic activity or auxiliary or including the activity of secretion of cytokines. Examples of immune effector cells include T cells (e.g., α/βT cells and γ/δT 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 for CAR 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. T cells can be T helper (Th) cells, such as T helper 1 (Th1) or T helper 2 (Th2) cells. T cells can be helper T cells (HTL; CD4T cells) CD4T cells, cytotoxic T cells (CTL; CD8T cells), CD4CD8T cells, CD4CD8T cells or any other T cell subsets. In certain embodiments, T cells may include primary T cells and memory T cells.

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

As used herein, the term "antibody" refers to an immunoglobulin molecule that is generally composed of two pairs of polypeptide chains, each pair having a light chain (LC) and a heavy chain (HC). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the isotype of the antibody is defined as IgM, IgD, IgG, IgA and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of 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 three domains (CH1, CH2 and CH3). 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 domain is not directly involved in the binding of antibodies to antigens, but exhibits a variety of effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can also be subdivided into regions with high variability (called complementary determining regions (CDRs)), interspersed with more conservative regions called framework regions (FRs). Each _VH and _VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain/light chain pair form antigen binding sites, respectively. The distribution of amino acids in various regions or domains can follow the definition of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.

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 the antigen, which is also referred to as an "antigen-binding portion". See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd edition, Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be produced by recombinant DNA technology 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 that contain at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.

As 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 the fragment obtained after reducing the disulfide bonds linking the two heavy chain fragments in the F(ab') ₂ fragment, consisting of a complete light chain and the Fd fragment (consisting of the VH and CH1 domains) of the heavy chain.

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

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

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 the active ingredient, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, and preservatives. For example, pH regulators 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, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meanings generally understood by those skilled in the art, which can stabilize the desired activity of the active ingredient in the drug, including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin or casein) or their degradation products (such as lactalbumin hydrolysate), etc.

As used herein, the term "prevention" refers to a method implemented 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 implemented in order to obtain a beneficial or desired clinical result. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partially or completely), whether detectable or undetectable. In addition, "treatment" can also refer to prolonging survival compared to the expected survival (if not receiving treatment).

As used herein, the term "subject" refers to a mammal, such as a primate mammal, such as a human. In certain embodiments, the subject (eg, human) suffers from a tumor.

Advantageous Effects of the Invention

The present invention performs site-specific selective polyethylene glycol (PEG) modification on IL2, and obtains a combination of a first site-modified IL-2 and a second site-modified IL-2, wherein the first site-modified IL-2 has a blocking effect on the IL2-Rα receptor, is selective for the IL2-Rβγ receptor, can preferentially activate Teff, CTL and NK cells and reduce the activation of Treg, and further induces terminal differentiation and exhaustion of reduced CD8+ cells, and increases the proportion and number of central memory cells; the second site-modified IL-2 has the effect of blocking IL2-Rβ but not IL2-Rα, and can compete with endogenous IL2 secreted by cells for IL2-Rα, and competitively inhibit the effect of endogenous IL2 activating Treg. The combination of the two can make up for the disadvantage of the first site-modified IL-2 for the increased degree of exhaustion after cell activation, while retaining the central memory cell activation advantage of the first site-modified IL-2, and better exert the anti-tumor therapeutic effect. The combination provided by the present invention has significant synergistic effects of enhancing immune response and anti-tumor effects. In addition, it can also direct 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 in conjunction with the accompanying drawings and examples, but it will be appreciated by those skilled in the art that the following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Coomassie blue staining results of PEGylated IL-2 variants Y45-20K and D20-20K.

Figure 2: Surface plasmon resonance (SPR) affinity determination results of PEGylated IL-2 variants for different subunits of IL-2 receptor.

FIG3 : Results of determination of phosphorylated STAT5 (pSTAT5) levels in cells after treatment with PEGylated IL-2 variants Y45-20K and D20-20K.

Figure 4: PK/PD properties analysis results of PEGylated IL-2 variants Y45-20K and D20-20K.

Figure 5: Effects of D20-20K on Y45-20K-mediated T cell proliferation and activation in vitro.

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

Figure 7: Anti-tumor effect of the combination of D20-20K and Y45-20K in a mouse tumor model.

Figure 8: Effects of the combination of D20-20K and Y45-20K on T cell subsets in the lymph nodes of tumor-bearing mice.

Figure 9: Effect of the combination of D20-20K and Y45-20K on vascular leak syndrome (VLS).

Figure 10: Effect of the combination of D20-20K and Y45-20K on the proliferation of CAR-T cells in vitro.

Figure 11: Effects of the combination of D20-20K and Y45-20K on aging and exhaustion of CAR-T cells cultured in vitro.

Figure 12: Effects of the combination of D20-20K and Y45-20K on the aging and exhaustion of T cells at different differentiation stages during in vitro culture of CAR-T cells.

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

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

Sequence information

The information of the partial sequences involved in the present invention is provided in Table 1 below.

Table 1: Description of sequences

DETAILED DESCRIPTION

The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

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 ° C for 10 h, cooled to room temperature, and acetone was removed by vacuum evaporation. The residue was extracted with diethyl ether. The organic layer was washed twice with brine, then dried over Na ₂ SO ₄ , filtered, and evaporated to obtain 2-azidoethanol (Compound 2) with a yield of 99% (5.5 g, 63.2 mmol) without further purification.

The solution obtained by dissolving 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) dissolved in dichloromethane (55 ml) at -3°C. The mixture was reacted for 12 hours under stirring. Then 200 mL of water was added, and the organic layer was washed twice with brine, then dried over Na ₂ SO ₄ , filtered and concentrated under vacuum. The residue was further purified by silica gel chromatography, eluted with PE/EtOAc (1:1), and compound 3 (10.7 g, 59 mmol) was obtained as a colorless oil with a yield of 93%.

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

Compound 4 (15.1 g, 41.94 mmol) was dissolved in dichloromethane (80 ml), and trifluoroacetic acid (20 ml) was slowly added. The reaction solution was stirred at room temperature for 0.5 hours, and then the solvent was evaporated under vacuum. The residue was redissolved in methanol (5 ml) and precipitated in ether. The precipitate was collected and dried under vacuum to obtain compound 5 (6.63 g, 25.58 mmol) as a white solid, namely NAEK, with a yield of 61%.

Synthesis of DBCO-PEG:

Dissolve compound 1, 10 g (48 mmol) and 16.8 g (240 mmol) of hydroxylamine hydrochloride in anhydrous ethanol, add 26 ml of pyridine, heat under reflux for 36 h, cool and add 25 ml of ethyl acetate, add 125 ml of 1N HCl and stir for 0.5 h, separate the organic layer, wash the organic layer with saturated NaCl solution, dry the layers and concentrate to obtain white solid compound 2, yield: 100%.

Dissolve 6.25g of phosphorus pentoxide in 50ml of methanesulfonic acid, stir and dissolve for later use. Add the above product to 10g of compound 2 and stir at 100 degrees for 1.5h. After the reaction solution is cooled, pour it into 300ml of water, precipitate solid, filter and dry it, and then slurry the solid with ether, filter and dry it to obtain white solid compound 3, yield: 100%.

160 ml of ether was cooled to 0 degrees, 5.5 g of lithium aluminum hydride was added in batches, and then 4 g of compound 3 was added in batches, and refluxed at 35 degrees for 72 hours. The reaction solution was cooled to 0 degrees, quenched by adding 14.8 ml of saturated sodium sulfate solution, filtered, and concentrated to obtain yellow solid compound 4 with a yield of 98%.

766 mg of monomethyl succinate was dissolved in 15 ml of dichloromethane, cooled to 0 degrees, and 610 ul of oxalyl chloride was added dropwise, and stirred at room temperature for 1 hour. Concentrate for later use. 1 g of compound 4 was dissolved in 15 ml of dichloromethane, 1.16 ml of pyridine was added, cooled to 0 degrees, and monomethyl succinate was added dropwise, and stirred at room temperature for 0.5 hours. The organic phase was washed with 1N HCl, 1N NaOH, and NaCl, and then dried over anhydrous sodium sulfate and concentrated to obtain yellow solid compound 5. Yield: 99%.

Compound 5 was dissolved in methanol/water (volume ratio = 2:1), 6 equivalents of lithium hydroxide were added at room temperature, and the mixture was stirred at room temperature for 8 h. The methanol was dried by spin drying, and water was added to dissolve the mixture. The aqueous phase was washed with dichloromethane, and the pH of the aqueous phase was adjusted to 2-3 with 1N HCl. The aqueous phase was extracted with dichloromethane again, and the organic phase was washed with saturated NaCl solution and dried by spin drying to obtain compound 6 as a yellow oil. The yield was 98%.

Dissolve 1.4 g of compound 6 in 50 ml of dichloromethane, cool to 0 degrees, dissolve 2.2 g of liquid in dichloromethane and add dropwise, stir at room temperature for 3 hours, add 50 ml of saturated sodium thiosulfate solution, separate the organic layer, wash with saturated NaCl solution, dry over anhydrous sodium sulfate, and spin dry to obtain light yellow solid compound 7, yield: 95%.

2.1 g of compound 7 was dissolved in 40 ml of anhydrous tetrahydrofuran, cooled to -40 degrees, 1 M potassium tert-butoxide tetrahydrofuran solution was added dropwise, stirred for 1 hour, quenched by adding 1 M HCl solution to pH = 2-3, extracted with dichloromethane, and the organic layer was concentrated and separated by column chromatography to obtain a powdery white solid compound 8. The yield was 80%.

1.1 g of compound 8 was dissolved in 20 ml of dichloromethane, and 495 mg of N-hydroxysuccinimide and 825 mg of EDCl were added. The mixture was stirred at room temperature for 1 h. The reaction solution was washed with water, washed with saturated NaCl solution, dried over anhydrous sodium sulfate, and concentrated to obtain a light yellow solid compound 9. The yield was 100%.

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

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

(1) Selection of mutation sites

The mutation sites shown below were selected on the IL-2 protein (SEQ ID NO: 1), wherein the positions described are the positions in SEQ ID NO: 1.

Table 2: Mutation sites

Protein name Amino acid position Amino Acids Codon before mutation Mutated codon Y45 45 Y TAC TAG D20 20 D GAT TAG

(2) Expression of site-directed mutant proteins containing unnatural amino acids

The synthesized nucleic acid sequence containing the codon substitution was connected to the nucleic acid sequence encoding the His tag and cloned into the pET-21a(+) (addgene: #69740-3) Escherichia coli plasmid expression vector, which was co-transfected with the pSURAR-YAV plasmid into the TransB (DE3) strain (purchased from Quanshijin, catalog number: CD811-02). Among them, the pSURAR-YAV plasmid encodes amber codon suppressor tRNA and NAEK-specific aminoacyl tRNA synthetase, and can introduce non-natural amino acid NAEK at a specific site by co-transfection with a nucleic acid sequence containing a TAG codon; the pSURAR-YAV plasmid refers to the plasmid pSUPAR-YAV-tRNA/PylRS obtained from the Escherichia coli containing the plasmid pSUPAR-YAV-tRNA/PylRS, which is deposited at the General Microbiological Center of the China Microbiological Culture Collection Committee (No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), on April 8, 2013, and with a deposit number of CGMCC No: 7432. The transformed strain was inoculated into 2 ml LB medium containing 100 ug/ml ampicillin and 34 ug/ml chloramphenicol, and then cultured at 37° C. and 220 rpm. The overnight culture was diluted to optical density in 2×YT medium, and the culture was cultured at 37°C until A600nm reached about 1.5. UAA (NAEK, synthesized in the laboratory) was added at a final concentration of 1 mM, and protein expression was then induced by adding isopropyl β-d-thiogalactopyranoside (IPTG) and L-arabinose at final concentrations of 0.5 mM and 0.1%, respectively. Half an hour later, the temperature was lowered to 20°C. After about 18 hours of expression, the cells were harvested by centrifugation and resuspended in His-Bind buffer (20 mM phosphate, pH 8.0, 500 mM NaCl, 20 mM imidazole). Proteins were extracted by passing the cells through a Micofluidizer twice under 1200 bar and cooling conditions. The supernatant was then collected and stored frozen at -80°C until further processing after centrifugation at 20,000 g for 20 minutes. Thus, a site-directed mutant IL-2 protein in which position Y45 or position D20 is replaced with a non-natural amino acid is obtained.

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

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

Example 3: Determination of binding activity to IL-2 receptor

This example evaluates the affinity of PEGylated IL-2 derivatives to IL-2R trimer and dimer complexes by surface plasmon resonance (SPR). Human IL-2Rα, IL-2Rβ and IL-2Rγc 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 6xHis), IL-2Rβ (C-terminal Fc) and IL-2Rγ (C-terminal 6xHis) were purchased from Yiqiao Shenzhou. All kinetic experiments were performed at 25°C 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 the data were fitted using a 1:1 stable affinity model to determine the KD value and other kinetic parameters. Protein concentration was measured using the BCA method (Pierce). The natural IL-2 protein (purchased from PeproTech, catalog number: 96-200-02-1000) was used as a control. The concentrations of all samples were the mass of IL-2 without PEG.

The results are shown in Figure 2, where NA means no binding. The results showed that D20-20K, H16-20K, A73-20K, and H79-20K had no affinity for the β subunit, and their binding affinity for the dimeric IL-2R was significantly reduced, but they maintained binding to the α chain of the trimeric IL-2R, and had a preference for non-β receptors (non-β), and can be called non-βPEGylate. Y45-20K, F42-20K, E62-20K, K64-20K, P65-20K, and E68-20K have no binding to α, completely eliminate the binding affinity to trimeric IL-2R, maintain the binding to dimeric IL-2R, and have a preference for non-α receptors (non-α), so they can be called non-αPEGylate; K35-20K, T37-20K, R38-20K, T41-20K, K48-20K, and K49-20K have a binding affinity to α of 1e- 7～1e-6, which is weak binding, but the binding affinity to dimeric IL-2R is significantly higher than that to trimeric IL-2R, or although the binding affinity to dimeric IL-2R is weaker than that to trimeric IL-2R, the difference between the two (such as the ratio) is significantly smaller than the difference in the binding affinity of natural IL-2 to dimeric IL-2R and trimeric IL-2R, and it has significantly improved preference for dimeric IL-2R, so it also has preference for non-α receptors (non-α) and can be called non-αPEGylate.

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

This example determines whether the PEGylated IL-2 variants Y45-20K or D20-20K preferentially activate CD8+T cells rather than Treg cells by measuring the level of phosphorylated STAT5 (pSTAT5), a key downstream mediator of IL-2R signaling. Approximately 2×10 ⁵ YT-1 cells (expressing CD122 and CD132 receptors), CD25+YT-1 (a stable cell line in which YT-1 cells are transduced with lentiviral transduction to stably express the CD25 receptor), and human PBMC cells were seeded in 96-well plates and resuspended in RPMI complete medium containing serially diluted natural human IL-2 or PEGylated IL-2. The cells were stimulated at 37°C for 15 minutes and then immediately fixed by adding formaldehyde to 2.0% and incubating at room temperature for 15 minutes. Permeabilization of the cells was achieved by suspending in ice-cold 87% methanol at 4°C for 30 minutes. 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 purchased from biolegend). For pSTAT5 determination in specific hPBMC subsets, the purified cell populations were separated using a magnetic separation kit (Miltenyi Biotech) according to the manufacturer's instructions to obtain memory CD4+T cells (MPCD4), memory CD8+T cells (MPCD8), NK cells, and Treg cells, respectively.

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

The results of the detection using NK-derived YT-1 cells and CD25+YT-1 cell models are shown in Figure 3a, where Y45-20K showed significant differences on CD25 (IL-2Rα)- and CD25 (IL-2Rα)+YT-1 cells, which was significantly different from wild-type IL-2; D20-20K still only showed baseline levels of Treg stimulation activity (5-20% of pSTAT5 activation) at very high doses, which was related to its inability to bind to the β subunit.

In addition, the selective activation of Teff cells by PEGylated IL-2 variants was re-evaluated using various immune cell subsets isolated from PBMCs of healthy donors, and 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 tested doses, while 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 the pleiotropic effects of IL-2 can be relieved by blocking specific regions of the receptor binding site, and that Y45-20K has the ideal ability to preferentially stimulate Teffs rather than Treg cells due to its complete lack of IL-2Rα binding, while D20-20K significantly reduced the activation of both CD8+ T and Treg cells.

Example 5: PK/PD Characterization Analysis of PEGylated IL-2 Variant Y45-20K/D20-20K

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

The results are shown in Figure 4. Compared with wild-type IL-2, the PEGylated IL-2 variant has an increased elimination half-life (T1/2), enhanced blood concentration maintenance (area under the drug concentration and time curve, AUC), and an increased mean residence time (MRT), and the peak time is 14 times longer than IL-2, indicating that the clearance rate is greatly reduced. The above results show that Y45-20K has superior pharmacokinetic properties. In addition, D20-20K also has superior pharmacokinetic properties.

Example 6: Anti-tumor synergistic effect of PEGylated IL-2 variants Y45-20K/D20-20K

The data of the above examples have confirmed that Y45-20K has the activity of preferentially activating CD8+T cells, but it still activates Treg cells at high doses. As confirmed in the above examples, D20-20K maintains selective affinity for IL-2Rα while significantly reducing the activation of CD25+YT-1. This unique property may be related to its undetectable binding to IL-2Rβ. Therefore, if D20-20K is combined with Y45-20K for treatment, D20-20K may occupy IL-2Rα spatially 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 investigates the synergistic anti-tumor effect of D20-20K and Y45-20K.

6.1 Effects of D20-20K on Y45-20K-mediated T cell proliferation and activation in vitro

Experimental procedures

This example investigates whether D20-20K has a dose-dependent effect on the activation bias of Y45-20K. 2×10 ⁵ human PBMC cells were resuspended in 96-well plates with 100ul RPMI1640+10% FBS, 50ul0.4ug/mlY45-20K (final concentration 0.1ug/ml) was added to the experimental wells, and then 50ul of D20-20K with different concentration gradients was added to make the final concentrations 1ug/ml, 0.1ug/ml, 0.01ug/ml, 0.001ug/ml, respectively. The repeated experimental wells were set up from three different healthy individuals. The cells were cultured at 37°C for 72h, then washed twice with FACS buffer, and stained with the following antibodies for flow cytometry analysis: anti-human CD3-APC/Cy7, CD4-PE/Cy7, CD8-FITC, CD25-APC, Foxp3-Pacific Blue, CD69-PE. Treg cells were defined as CD3+CD8-CD4+CD25+Foxp3+; CD8+T cells were defined as CD3+CD4-CD8+. Flow cytometry was used to count the number of Treg and CD8 T cells and the mean fluorescence intensity (MFI) of their respective activation markers (Treg-Foxp3, CD8+T-CD69).

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

The results are shown in Figure 5. D20-20K has a dose-dependent inhibitory effect on the activation and proliferation of Treg cells but not CD8+T cells mediated by Y45-20K (Figure 5a shows the effect on Treg proliferation and activation, and Figure 5c shows 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 (Figure 5e), while such inhibition was not observed in natural YT cells (Figure 5f).

6.2 Effects of D20-20K on Y45-20K-mediated T cell proliferation and activation in healthy mice

Experimental procedures

C57BL/6 mice (6 to 8 weeks old, female) were subcutaneously administered 45-20K (0.25 mg/kg x 3, every other day), 20-20K (0.25 mg/kg x 3, every other day), 45-20K and 20-20K (each 0.25 mg/kg x 3, every other day), or IL-2 (0.25 mg/kg per day x 5) at the back of the neck, and an equal volume of PBS was injected as a blank control group. Flow cytometry of spleen and lymph nodes was performed three days after the end of administration.

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

6. Anti-tumor effects of 3D20-20K and Y45-20K in mouse tumor models

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

As shown in Figure 7, the combination of Y45-20K and D20-20K treatment showed a very prominent anti-tumor effect, with a significant reduction in tumor burden and a significant extension of mouse survival (Figure 7a). As tumor burden decreased, the proportion of CD8+ T cells in the spleen, tumor draining lymph nodes (TDLN), and especially tumor tissues increased in mice treated with Y45-20K/D20-20K combined treatment compared with other mice, but there was no significant change in the proportion of Treg cells (Figure 7b). This result is consistent with immunohistochemical staining, indicating that co-treatment with Y45-20K and D20-20K induced the largest amount of T cell infiltration in tumor tissues compared with natural IL-2 and PBS treatment (Figure 7c).

和Tem有轻微降低(图8a)，表示Y45-20K在体内能够显著性大量扩增中央记忆T淋巴细胞，单独D20-20K不会扩增中央记忆细胞，但能够联合Y45-20K进一步提高CD8亚群中中央记忆细胞的比例和数量，有利于发挥抗肿瘤免疫功效。此外，Tcm细胞亚群中CD25标志物表达量近一步降低，CD122标志物表达量近一步升高，表示Y45-20K单独处理组或Y45-20K联合D20-20K治疗组中小鼠淋巴结的Tcm能够避免内源性IL-2对于T细胞的过度激活，降低对IL-2的敏感性，提高对外来抗原再次应答的响应能力。Y45-20K单独处理组虽然能够大量激活扩增Tcm的增殖，但是MPCD8，T

和Tem细胞亚群中LAG-3和PD-1标志物相比于IL-2治疗组有显著提高，联用D20-20K能够降低上述细胞亚群中免疫检查点的表达，表示D20-20K联用Y45-20K能够保留Y45-20K的中央记忆细胞激活优势，又能弥补Y45-20K对于细胞激活之后耗竭程度增加的劣势，更好地发挥抗肿瘤治疗效果。Memory CD8 cells play an important role in tumor immunity. Analysis of T cell subsets in lymph nodes of tumor-bearing mice showed that the number of Tcm and MPCD8 cells in lymph nodes increased significantly by about 10 times in the Y45-20K alone treatment group or the Y45-20K combined with D20-20K treatment group. The number of Tcm and MPCD8 in the Y45-20K combined with D20-20K treatment group was further increased compared with the Y45-20K alone treatment group.

There was a slight decrease in Tem (Figure 8a), indicating that Y45-20K can significantly amplify central memory T lymphocytes in vivo. D20-20K alone will not amplify central memory cells, but it can be combined with Y45-20K to further increase the proportion and number of central memory cells in the CD8 subset, which is beneficial to the anti-tumor immune effect. In addition, the expression of CD25 markers in the Tcm cell subsets further decreased, and the expression of CD122 markers further increased, indicating that the Tcm in the lymph nodes of mice in the Y45-20K alone treatment group or the Y45-20K combined with D20-20K treatment group can avoid the excessive activation of T cells by endogenous IL-2, reduce sensitivity to IL-2, and improve the ability to respond to foreign antigens again. Although the Y45-20K alone treatment group can activate and amplify the proliferation of Tcm in large quantities, MPCD8, T

The LAG-3 and PD-1 markers in the TEM cell subsets were significantly increased compared with the IL-2 treatment group. The combined use of D20-20K can reduce the expression of immune checkpoints in the above cell subsets, indicating that the combination of D20-20K and Y45-20K can retain the central memory cell activation advantage of Y45-20K, and make up for the disadvantage of Y45-20K for increased exhaustion after cell activation, thereby better exerting the anti-tumor treatment effect.

6.4 Effect of the combination of D20-20K and Y45-20K on vascular leak syndrome (VLS)

Traditional IL-2 treatment has a high risk of vascular leakage syndrome (VLS), which is generally believed to be caused by the binding of IL-2 to CD25+ pulmonary endothelial cells, and the present embodiment also evaluates the effect of combined treatment on pulmonary edema. A similar treatment regimen as described above was adopted, in which the administration of PEGylated IL-2 variants was changed to a high dosing frequency (five times a day). After killing mice, lung tissue was weighed, lung tissue cell suspension was obtained for flow cytometry, and lung tissue sections were prepared for immunohistochemical analysis.

The results are shown in Figure 9. Lung cells expressing high levels of CD31 but not other immune cell lineage markers (Lin) are defined as endothelial cells (Ly5.2-B220-CD3-NK1.1-CD11b-CD11c-CD31+). The results showed that the Y45-20K monotherapy group caused significant pulmonary edema, as shown by increased lung wet weight (Figure 9a), decreased proportion of lung endothelial cells (Figure 9b), and increased lymphocyte infiltration in the lungs; however, this side effect was significantly reduced after co-administration with D20-20K, which may be due to the spatial occupancy of IL-2Rα of lung endothelial cells by D20-20K.

The above data indicate that Y45-20K and D20-20K show significant synergistic effects in anti-tumor therapy, selectively induce CD8+T cells with minimal effect on Treg cells, and are able to alleviate VLS by protecting lung endothelial cells from activation by Y45-20K or endogenous IL-2, with significant favorable technical effects.

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

IL-2 is a key nutritional factor for the in vitro expansion and culture of CAR-T cells. It inevitably activates Treg cells in donor cells and inevitably over-activates CD8+T cells, leading to their terminal differentiation and affecting the activity of CAR-T cells, which greatly weakens the therapeutic effect of CAR-T cell therapy. Based on this, this experiment investigated the effect of Y45-20K combined with D20-20K to replace traditional IL-2 on CAR-T cell therapy.

7.1 Effect of the combination of D20-20K and Y45-20K on the proliferation of CAR-T cells in vitro

Construction of CAR lentiviral vector:

The anti-CD19 CAR contains FMC63 anti-CD19 scFv (SEQ ID NO: 12), CD8a hinge region (SEQ ID NO: 14) and transmembrane region (SEQ ID NO: 16), as well as 4-1BB cytoplasmic domain (SEQ ID NO: 18) and CD3z cytoplasmic domain (SEQ ID NO: 20), and its expression cassette was commissioned to be synthesized by Aikangde Biotechnology (Suzhou) Co., Ltd. and cloned into a lentiviral vector. HEK293T cells were transfected with anti-CD19CAR, pspax2 (addgene, #12260) and pMD2.g (addgene, #12259) plasmids using Lipofectamine 3000 (Life Technologies). The medium was replaced 6 hours after transfection, and the viral supernatant was collected 48 hours after transfection. The viral particles were concentrated 30 times by ultracentrifugation at 25,000 rpm for 2 hours and frozen at -80°C 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 before infection. Concentrated lentivirus (FMC63-AntiCD19-CAR Lentivirus) was applied to activated human T cells (10 ⁶ /well in a 24-well culture plate, MOI=1), and 10 mg/mL polybrene and 100 ng/mL IL-2 or 100 ng/mL 45-20K, 100 ng/mL D20-20K, 100 ng/mL D20-20K+100 ng/mL Y45-20K were added and centrifuged at 1000g for 2 h at 32°C. The next day, the supernatant was replaced with fresh medium containing IL-2 and its analogs of the corresponding group, and the transduced T cells were placed in complete growth medium to maintain 0.5x10 ⁶ cells/mL, and the medium containing IL-2 was replaced every 3 days. Monitor cell number and apoptosis status in real time.

The results are shown in Figure 10. Whether Y45-20K, D20-20K or Y45-20K combined with D20-20K replaces IL2, the transduction efficiency of CAR is not affected (Figure 10a). After 10 days of culturing CAR-T cells with IL-2 variants, it was found that CAR-T cells hardly proliferated when D20-20K replaced IL-2, while the proliferation rate of cells cultured with Y45-20K replacing IL-2 was significantly higher than that of IL-2 culture. The proliferation rate of CAR-T cells cultured with Y45-20K combined with D20-20K replacing IL-2 was similar to that of Y45-20K alone (Figure 10b). Cell apoptosis detection found that IL-2 culture caused apoptosis of CAR-T cells, which was related to the excessive activation of T cells by IL-2 and the induction of terminal differentiation, while Y45-20K combined with D20-20K hardly induced apoptosis of CAR-T cells (Figure 10c).

7.2 Effects of the combination of D20-20K and Y45-20K on senescence and exhaustion of CAR-T cells cultured in vitro

Experimental procedures

The prepared CAR-T cells were cultured in the corresponding IL-2 for 10-14 days, and the cell phenotype was detected. The surface antibodies were fluorescently stained with fluorescent labeled antibodies (CD4-PE/Cy7, CD8-FITC, CD25-BV785, CD57-PB, CD45RA-BV510, CD62L-Percp/cy5.5, CCR7-PE, PD-1-BV650, LAG-3-BV421) and incubated at 4°C for 30 minutes. For intranuclear staining, CAR-T cells were fixed and permeabilized with Foxp3/transcription factor staining buffer (eBioscience) and then stained with Foxp3 antibody in perm buffer. The samples were washed twice with PBS and suspended in flow cytometry staining buffer (eBioscience). For intracellular cytokine staining, 500x Cell Activation Cocktail (with Brefeldin A, Dako, 423303) was used and washed after 6h 37°C culture. After the final wash, cells were fixed, permeabilized and stained using the eBioscience Intracellular Fixation and Permeabilization Buffer Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions, and then incubated with specific antibodies for 30 min at 4°C: IL-2-BV421, IL-10-APC, Granzy B-percp/cy5.5.

The results are shown in Figure 11. After the CAR-T cells cultured in vitro were stimulated with PMA, the secretion of granzyme B and IL10 by the CAR-T cells cultured with Y45-20K combined with D20-20K was significantly lower than that of the IL-2 group, indicating that the terminal differentiation and senescence of CAR-T cells were reduced (Figure 11a, b), and the secretion of IL-2 was significantly increased, which also indicated that CAR-T cells were in a stem state with a low degree of differentiation (Figure 11c). Secondly, flow cytometry showed that the surface exhaustion and senescence-related receptors such as PD-1, LAG-3 and CD57 of CAR-T cells cultured with Y45-20K combined with D20-20K were significantly reduced (Figure 11d-f), which once again proved that Y45-20K combined with D20-20K can reverse the senescence and exhaustion caused by IL-2 for CAR-T cells cultured in vitro.

7. Effects of 3D20-20K and Y45-20K combination on senescence and exhaustion of T cells at different differentiation stages in in vitro culture of CAR-T cells

Experimental procedures

细胞定义为CD3+CD8+CD45RA+CD45RO-CCR7+CD62L-CD95-；Tscm定义为CD3+CD8+CD45RA+CD45RO-CCR7+CD62L-CD95+；Tcm定义为CD3+CD8+CD45RA-CD45RO+CCR7+CD62L+；Tem定义为CD3+CD8+CD45RA-CD45RO+CCR7-CD62L-；T effector定义为CD3+CD8+CD45RA+CD45RO0-CCR7-CD62L-；Treg定义为CD3+CD4+CD25^highCD127^low.并用不同细胞因子培养扩增10-14天，实时监测细胞数量和生长状态。Human T cells were purified from peripheral blood mononuclear cells using the Dynabead human T cell kit (Life Technologies), and CAR-T was prepared using the above method. To further study the effects of different cytokines on CAR-T cell differentiation, we sorted out CAR-T cells in different differentiation states by flow cytometry (sorting type, Symphony S6, BD) for separate culture.

Cells were defined as CD3+CD8+CD45RA+CD45RO-CCR7+CD62L-CD95-; Tscm was defined as CD3+CD8+CD45RA+CD45RO-CCR7+CD62L-CD95+; Tcm was defined as CD3+CD8+CD45RA-CD45RO+CCR7+CD62L+; Tem was defined as CD3+CD8+CD45RA-CD45RO+CCR7-CD62L-; T effector was defined as CD3+CD8+CD45RA+CD45RO0-CCR7-CD62L-; Treg was defined as CD3+CD4+CD25 ^high CD127 ^low . They were cultured and expanded with different cytokines for 10-14 days, and the cell number and growth status were monitored in real time.

)、T记忆干细胞(Tscm)、效应T细胞(T effector)和调节性T细胞(Treg)的衰老、耗竭程度进行了检测。CD62L为细胞干性的特征标志物，D20-20K与Y45-20K联合可以近一步提升T

和Tscm细胞中CD62L的表达量(图12a)，说明D20-20K与Y45-20K联合可以近一步保留T细胞的干性状态。对不同分化阶段的T细胞增殖情况检测结果显示，D20-20K与Y45-20K联合可以近一步增强T

细胞的增殖，降低Treg细胞的增殖，而对Tscm和T effector细胞的增殖状态无显著影响(图12b)。PD-1和TIM-3为T细胞耗竭的标志物，对处于终末分化状态的T effector细胞检测显示，D20-20K与Y45-20K联合可以近一步降低PD-1和TIM-3的表达量，降低Teffector细胞的耗竭程度(图12c)。最后，对IL-2诱导不同分化状态的T细胞上CD25的表达量检测显示，D20-20K与Y45-20K联合可以明显降低Tscm、T effector和Treg细胞表面CD25的表达量，尤其是相对于Y45-20K单独使用近一步降低Treg细胞上CD25的表达量(图12d)，CD25的表达量下降可以避免T细胞激活产生的内源性IL-2对于T细胞的过度激活，显示出D20-20K与Y45-20K联合的优越性。We investigated the initial T cells (T cells) at different differentiation stages in CAR-T cells.

The aging and exhaustion of T cells (Tscm), T effector cells (T effector) and regulatory T cells (Treg) were detected. CD62L is a characteristic marker of cell stemness. The combination of D20-20K and Y45-20K can further enhance T

The expression of CD62L in Tscm cells and Tscm cells (Figure 12a) indicated that the combination of D20-20K and Y45-20K could further preserve the stemness of T cells. The results of the detection of T cell proliferation at different differentiation stages showed that the combination of D20-20K and Y45-20K could further enhance the proliferation of T cells.

The proliferation of T cells was inhibited, and the proliferation of Treg cells was reduced, while the proliferation state of Tscm and T effector cells was not significantly affected (Figure 12b). PD-1 and TIM-3 are markers of T cell exhaustion. The detection of T effector cells in a terminally differentiated state showed that the combination of D20-20K and Y45-20K can further reduce the expression of PD-1 and TIM-3 and reduce the degree of exhaustion of Teffector cells (Figure 12c). Finally, the detection of CD25 expression on T cells in different differentiation states induced by IL-2 showed that the combination of D20-20K and Y45-20K can significantly reduce the expression of CD25 on the surface of Tscm, T effector and Treg cells, especially further reducing the expression of CD25 on Treg cells compared with the use of Y45-20K alone (Figure 12d). The decrease in CD25 expression can avoid the excessive activation of T cells by endogenous IL-2 produced by T cell activation, showing the superiority of the combination of D20-20K and Y45-20K.

Example 8: Effects of the combination of None-α variant and None-β variant on maintaining T cell stemness and reducing T cell exhaustion

In this example, the in vitro activity of the combination of the Non-α variants (F42-20K, Y45-20K, E62-20K, P65-20K, E68-20K) and the Non-β variants (D20-20K, H16-20K, A73-20K, H79-20K) prepared in Example 1-2 was verified.

Experimental steps:

Peripheral blood was collected from healthy individuals, and PBMCs were separated using peripheral blood human lymphocyte separation fluid. CD3-positive T lymphocytes were separated using a magnetic bead sorting kit. CD3/CD28 stimulating magnetic beads were added at a ratio of bead:cell=1:1. The inoculation density was controlled at 1x10 ⁶ /ml, 24-well plates, 1ml per well, and cultured with different types of cytokines but at the same concentration (100ng/ml). The medium was changed every 72 hours, and flow cytometry was performed after two weeks of continuous culture. The detection antibodies included: APC/Cy7 anti-human CD3, PE/Cy7 anti-human CD4, FITC anti-human CD8, BV785 anti-human PD-1, BV605 anti-human TIM-3, Pacific Blue anti-human CD57, APC anti-human IL-10, PEanti-human Perforin, Percp/cy5.5 anti-human Granzyme B, BV510 anti-human IFN-γ.

Experimental results:

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

Based on the above screening results, the none-β variant (D20-20K) was preferred to verify the effect of the combination with different none-α variants (F42-20K, Y45-20K, E62-20K, P65-20K, E68-20K) on T cell activation. The experimental results showed that after D20-20K was combined with different none-α variants, the degree of effect on improving the survival status of CD8 and CD4 T cells was different, but both could further reduce the expression of immune checkpoints PD-1 and TIM-3, effector cytokines Perforin, Granzyme B, IFN-γ expression, and reduce the expression of apoptosis-related biomarkers CD57 and IL-10 expression levels to a certain extent (Figure 14a-c). Therefore, the combination of None-β and None-α will further improve the survival status of T cells on the basis of the synergistic effect of none-α, such as reducing the degree of T cell growth exhaustion, reducing cell apoptosis induced by excessive activation, and delaying differentiation into terminal effector cells.

The above results indicate that the combination of a non-α variant having a preference for non-α receptors and a non-β variant having a preference for non-β receptors can produce a significant synergistic effect and bring about a significantly beneficial therapeutic effect.

Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

Claims (28)

1. 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.

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;

-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 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;

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

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:

-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;

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

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

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;

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

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

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;

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

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

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,

(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,

(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;

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.

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;

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.

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;

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.

Images