WO2023202035A1 - 受体偏向的peg化il-2变体组合及其应用 - Google Patents

受体偏向的peg化il-2变体组合及其应用 Download PDF

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WO2023202035A1
WO2023202035A1 PCT/CN2022/129975 CN2022129975W WO2023202035A1 WO 2023202035 A1 WO2023202035 A1 WO 2023202035A1 CN 2022129975 W CN2022129975 W CN 2022129975W WO 2023202035 A1 WO2023202035 A1 WO 2023202035A1
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周德敏
纪德重
孙家琦
张博
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北京大学宁波海洋药物研究院
北京大学
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Definitions

  • the present invention relates to the field of immunotherapy. Specifically, the present invention relates to site-directed modified PEGylated IL-2 variants to provide a combination of PEGylated IL-2 variants with different receptor biases. The present invention also provides combination therapies for enhancing immune responses, treating proliferative diseases such as tumors, and in vitro methods for preparing or culturing immune cells for adoptive cell therapy.
  • Interleukin-2 is an important cytokine that is necessary for the development and function of B cells, T cells and NK cells.
  • IL2 receptors are divided into IL2-R ⁇ monomers, IL2-R ⁇ dimers and IL2-R ⁇ trimers.
  • IL2 has a weak affinity with ⁇ monomers of 10 -8 M and a moderate affinity with ⁇ dimers of 10 -9 M, and the highest affinity to ⁇ trimer is 10 -11 M.
  • Due to low affinity either dimeric IL-2R or endogenous IL-2 needs to be induced at high levels to produce a “dimeric IL-2R:IL-2” reactivity, as opposed to trimeric IL-2R, This allows host cells to respond directly to low concentrations of IL-2. Therefore, it is currently believed that the ⁇ chain of the trimeric IL-2R does not participate in signaling, but rather initiates binding to IL-2 (KD ⁇ 10-8M) and subsequent binding to the ⁇ complex with a 1000-fold increased affinity.
  • IL2-R ⁇ trimers are long-term expressed on the surface of regulatory T cells (Treg), so they have the highest affinity for IL2; IL2-R ⁇ dimers are expressed on resting cells
  • Treg regulatory T cells
  • IL2-R ⁇ dimers are expressed on resting cells
  • the surface of effector T cells (Teff), killer T cells (CTL) and NK cells has a general affinity for IL2. Only after the activation of effector T cells and NK cells will they express IL2-R ⁇ trimers and increase their affinity for IL2.
  • ⁇ chain expression is quite dynamic—a subset of T cells upregulate ⁇ chain expression and sense strong IL-2 signals, leading to faster T cell proliferation but eventual differentiation. This in turn limits their effectiveness.
  • low-alpha T cells that are less sensitive to IL-2 preferentially upregulate CD127 and CD62L, thereby generating functional long-lived memory cells.
  • having a larger population of memory T cells in the body becomes valuable but challenging for clinical application. Therefore, in tumor treatment, regulating the unbalanced ratio of Teff/Treg and CTL/Treg has gradually become a new research and development direction of immunotherapy.
  • IL2 target-related drugs currently on the market has receptor selectivity.
  • Teff and CTL need to be activated, higher doses of IL2 are required, which also brings serious systemic toxicity and is the main source of side effects at present.
  • the interaction between IL2 and IL2-R ⁇ in pulmonary endothelial cells can also induce vascular leakage syndrome, and immunosuppression caused by activation of Treg cells can also limit drug response.
  • IL-2 has been widely used to enhance the body's immune cells, its accompanying properties of promoting the expansion of immunosuppressive lymphocytes and driving terminal differentiation and exhaustion of effector lymphocytes hinder IL-2's use as an anti-tumor agent. effectiveness.
  • the first class of receptor-biased PEGylated IL-2 variant is non- ⁇ PEGylate, which can activate and expand CD8+ T cells, CD4+ cells, and even NK cells, but not Treg cells, due to its Preferential affinity for dimeric rather than trimeric IL-2R, in addition it induces terminal differentiation and exhaustion of reduced CD8+ cells.
  • a second class of receptor-biased PEGylated IL-2 variant is non- ⁇ PEGylate, which activates neither CD8+ T nor Treg cells, but it synergizes with class I non- ⁇ PEGylate to further expand CD4+ and CD8+ T cells and reduced Tregs as well as differentiated and exhausted CD8+ T cell populations, which exhibit excellent local and systemic antitumor effects due to their maintenance of moderate affinity for the ⁇ chain of the trimeric IL-2R. reaction.
  • the inventor subsequently confirmed the above-mentioned first-type receptor-biased PEGylated IL-2 variant (non- ⁇ PEGylate) and second-type receptor-biased PEGylated IL-2 in a CD19-targeted CAR-T treatment model.
  • the variant (non- ⁇ PEGylate) synergistically directs lymphocyte fate toward long-term immunity.
  • the present invention provides different Combinations of receptor-biased PEGylated IL-2 variants.
  • This combination synergistically inhibits the expansion of immunosuppressive lymphocytes, reduces the terminal differentiation and exhaustion of effector lymphocytes, increases the proportion and number of central memory cells, and has a synergistic anti-tumor effect, thus it can be used to enhance immune responses and treat Proliferative diseases such as tumors.
  • this combination can also be used for the in vitro culture of immune cells in adoptive cellular immunotherapy, reducing the aging and exhaustion of immune cells and enhancing T cell proliferation, reduce the proliferation of Treg cells, maintain the proliferation 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, making the immune system more effective in adoptive cell immunotherapy.
  • Cell fate directs long-term immunity.
  • the present invention also provides combination therapies for enhancing immune responses, treating proliferative diseases such as tumors, and in vitro methods for preparing or culturing immune cells for adoptive cell therapy.
  • the invention provides a composition comprising:
  • a first site-specifically modified IL-2 which contains a PEG group modification at the residue at the first amino acid position compared to wild-type IL-2, wherein the first site-specifically modified IL-2 is
  • the IL-2 receptor alpha (IL-2R ⁇ ) subunit does not bind or binds with a KD value greater than 1E-8M (e.g., on the order of 1E-7 to 1E-6M); and
  • the second site-directed modified IL-2 which contains a PEG group modification at the residue at the second amino acid position compared with wild-type IL-2, wherein the second site-directed modified IL-2 has a negative effect on IL-2 2 receptor beta (IL-2R ⁇ ) subunit does not bind.
  • IL-2R ⁇ IL-2 2 receptor beta
  • compositions comprising a first site-directed modified IL-2 and a second site-directed modified IL-2 does not mean that both must be administered simultaneously and/or formulated for delivery together, notwithstanding these delivery methods. range described here.
  • the first site-directed modified IL-2 and the second site-directed modified IL-2 can be administered together in a single formulation.
  • the first site-directed modified IL-2 and the second site-directed modified IL-2 can be administered separately in different formulations.
  • the first site-directed modified IL-2 in the combination can be administered in any order with the second site-directed modified IL-2, for example simultaneously, before or after.
  • compositions comprise multiple compositions or dosage forms.
  • first site-directed modified IL-2 and the second site-directed modified IL-2 are in separate compositions or dosage forms.
  • compositions comprise one composition or dosage form.
  • first site-directed modified IL-2 and the second site-directed modified IL-2 are in the same composition or dosage form.
  • the wild-type IL-2 has the amino acid sequence set forth in SEQ ID NO: 1.
  • an IL-2-related amino acid position described herein is the position in SEQ ID NO: 1.
  • not binding means that no binding is detected by surface plasmon resonance technology (SPR).
  • the KD values described herein are determined by surface plasmon resonance (SPR) technology.
  • the first site-specific modified IL-2 described herein has a preference for non-alpha receptors (non-alpha).
  • the bias toward non-alpha receptors includes: (i) binding IL2- with a KD value less than 1E-8M (eg, on the order of 1E-9 to 1E-8M) R ⁇ dimer, and/or, (ii) does not bind to IL2-R ⁇ trimer or binds with a KD value greater than 1E-8 (eg, on the order of 1E-7 to 1E-6).
  • the first site-specific modified IL-2 does not bind to the IL-2 receptor alpha (IL-2R ⁇ ) subunit.
  • the first amino acid position of such first site-specifically modified IL-2 is selected from the group consisting of F42, Y45, E62, K64, P65, E68.
  • the first site-specific modified IL-2 only has a low affinity for the IL-2 receptor alpha (IL-2R ⁇ ) subunit, for example, greater than 1E-8M (eg, 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) with a KD value that binds to the IL-2 receptor alpha (IL-2R ⁇ ) subunit.
  • 1E-8M eg, greater than 9E-7M, 8E-7M, 7E-7M, 6E-7M, 5E-7M, 4E-7M, 3E-7M, 2E-7M, 1E-6M or greater
  • the first site-specific modified IL-2 binds to the IL-2 receptor alpha (IL-2R ⁇ ) subunit with a KD value on the order of 1E-7 to 1E-6M.
  • the first amino acid position of such first site-specifically modified IL-2 is selected from the group consisting of K35, T37, R38, T41, K48, K49.
  • the first site-specific modified IL-2 is 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) binds to IL2-R ⁇ dimer. In certain embodiments, the first site-specific modified IL-2 binds to the IL2-R ⁇ dimer with a KD value in the order of 1E-9 to 1E-8M.
  • the first amino acid position of such first site-specifically modified IL-2 is selected from the group consisting of F42, Y45, E62, K64, P65, E68, K35, T37, R38, T41, K48, K49.
  • the first site-specific modified IL-2 does not bind to the IL2-R ⁇ trimer.
  • the first amino acid position of such first site-specifically modified IL-2 is selected from the group consisting of F42, Y45, E62, K64, P65, E68.
  • the first site-specific modified IL-2 has only a low affinity for the IL2-R ⁇ trimer, for example, with greater than 1E-8M (eg, 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) binds to the IL2-R ⁇ trimer.
  • 1E-8M eg, greater than 9E-7M, 8E-7M, 7E-7M , 6E-7M, 5E-7M, 4E-7M, 3E-7M, 2E-7M, 1E-6M or greater
  • the first site-specific modified IL-2 binds to the IL2-R ⁇ trimer with a KD value in the order of 1E-7 to 1E-6M.
  • the first amino acid position of such first site-specifically modified IL-2 is selected from the group consisting of K35, T37, R38, T41, K48, K49.
  • the affinity of the first site-specific modified IL-2 for the IL2-R ⁇ dimer is The first affinity is higher than the second affinity for the IL2-R ⁇ trimer (ie, the first KD value for binding the IL2-R ⁇ dimer is lower than the second KD value for binding the IL2-R ⁇ trimer).
  • the first affinity of the first site-specific modified IL-2 for IL2-R ⁇ dimers is at least 2-fold, 3-fold, 4-fold greater than the second affinity for IL2-R ⁇ trimers. times, 5x, 6x, 7x, 8x, 9x or 10x.
  • the first amino acid position of such first site-specifically modified IL-2 is selected from the group consisting of K35, T37, T41, K48.
  • the affinity of the first site-specific modified IL-2 for the IL2-R ⁇ dimer is The first affinity may also be no higher (e.g., lower) than the second affinity for the IL2-R ⁇ trimer (i.e., the first KD value for binding the IL2-R ⁇ dimer is no lower (e.g., higher than) binding the IL2- The second KD value of R ⁇ trimer), but the difference in the affinity of the first site-specific modified IL-2 to IL2-R ⁇ dimer and IL2-R ⁇ trimer (for example, the ratio of the KD values of the two) It is smaller than the difference in the affinity of natural IL-2 for IL2-R ⁇ dimer and IL2-R ⁇ trimer (for example, the ratio of the KD values of the two).
  • the first KD value of the first site-specific modified IL-2 for binding to the IL2-R ⁇ dimer is no more than 10 times the second KD value for binding to the IL2-R ⁇ trimer, e.g. Not higher than 9 times, not higher than 8 times, not higher than 7 times, not higher than 6 times, not higher than 5 times, not higher than 4 times, not higher than 3 times or not higher than 2 times.
  • the first KD value of the first site-specific modified IL-2 binding to the IL2-R ⁇ dimer is 8 to 3 times the second KD value of binding to the IL2-R ⁇ trimer, For example, 7 times to 3 times.
  • native IL-2 binds the IL2-R ⁇ dimer with a first KD value on the order of 1E-9M. In certain embodiments, native IL-2 binds the IL2-R ⁇ trimer with a second KD value on the order of 1E-11M. In certain embodiments, the first KD value of native IL-2 for binding to the IL2-R ⁇ dimer is about 40 times greater than the second KD value for binding to the IL2-R ⁇ trimer. In certain embodiments, the first amino acid position of such first site-specifically modified IL-2 is selected from the group consisting of R38, K49.
  • the second site-directed modified IL-2 described herein has a preference for non-beta receptors (non-beta).
  • the bias toward non-beta receptors includes: (i) binding IL2- with a KD value less than 1E-8 (eg, on the order of 1E-9 to 1E-8) R ⁇ trimers, and/or, (ii) do not bind to IL2-R ⁇ dimers.
  • the second site-directed modified IL-2 does not bind to the IL-2 receptor beta (IL-2R ⁇ ) subunit.
  • the second site-directed modified IL-2 is produced in an amount 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) binds to the IL2-R ⁇ trimer.
  • the second site-directed modified IL-2 binds to the IL2-R ⁇ trimer with a KD value in the order of 1E-9 to 1E-8M.
  • the second site-directed modified IL-2 does not bind IL2-R ⁇ dimers.
  • the first amino acid position of the first site-specific modified IL-2 is selected from the group consisting of F42, Y45, E62, K64, P65, E68, K35, T37, R38, T41, K48, K49. In certain embodiments, the first amino acid position is selected from F42, Y45, E62, K64, P65, E68. In certain embodiments, the first amino acid position is F42, Y45, E62, P65, or E68.
  • the second amino acid position of the second site-directed modified IL-2 is selected from the group consisting of H16, D20, A73, and 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.
  • 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.
  • the average molecular weight of the PEG modification group included in the first site-specific modified IL-2 is 5 to 60 kDa, such as 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45kDa, 50kDa or 60kDa.
  • the average molecular weight of the PEG modification group included in the first site-specific modified IL-2 is 5-40 kDa, such as 5-30 kDa, 5-25 kDa, 5-20 kDa, 10-40 kDa, 10-30kDa, 15-30kDa, 10-25kDa or 15-25kDa, for example, 5kDa, 10kDa, 15kDa, 20kDa, 25kDa, 30kDa or 40kDa.
  • the first site-specific modified IL-2 includes a PEG modification group with an average molecular weight of 5 kDa, 10 kDa, or 20 kDa.
  • the first site-specific modified IL-2 includes a PEG modification group with an average molecular weight of 20 kDa.
  • the second site-directed modified IL-2 contains a PEG modification group with an average molecular weight of 5 to 60 kDa, such as 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa. , 50kDa or 60kDa.
  • the second site-directed modified IL-2 contains a PEG modification group with an average molecular weight of 5 to 40 kDa, such as 5 to 30 kDa, 5 to 25 kDa, 5 to 20 kDa, 10 to 40 kDa, 10 ⁇ 30kDa, 15-30kDa, 10-25kDa or 15-25kDa, for example, 5kDa, 10kDa, 15kDa, 20kDa, 25kDa, 30kDa or 40kDa.
  • the second site-directed modified IL-2 comprises a PEG modification group with an average molecular weight of 5 kDa, 10 kDa, or 20 kDa.
  • the second site-directed modified IL-2 comprises a PEG modification group with an average molecular weight of 20 kDa.
  • 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.
  • the first site-specific modified IL-2 is at a first amino acid position (e.g., amino acid positions F42, Y45, E62, P65) compared to wild-type IL-2 (e.g., SEQ ID NO: 1).
  • E68 residue is mutated into a non-natural amino acid, and the non-natural amino acid is connected to the PEG group.
  • the second site-directed modified IL-2 is at a second amino acid position (e.g., amino acid position D20, H16, A73, or H79) compared to wild-type IL-2 (e.g., SEQ ID NO: 1)
  • the residues are mutated into unnatural amino acids to which the PEG group is attached.
  • the unnatural amino acids contain chemical functional groups, such as carbonyl, alkynyl, and azide groups, etc., which are generally capable of effectively and selectively forming stable covalent bonds; so
  • the PEG group includes a labeling group capable of chemically reacting with the chemical functionality to form a covalent bond whereby the PEG group is attached to the unnatural amino acid.
  • the unnatural amino acid contains an azide group
  • the PEG group includes a labeling group capable of a click chemical reaction with the azide group, whereby the PEG group is attached to the unnatural amino acid .
  • the unnatural amino acid is a lysine derivative containing an azide group. In certain embodiments, the unnatural amino acid is N ⁇ -2-azidoethoxycarbonyl-L-lysine (NAEK).
  • the unnatural amino acid is a tyrosine derivative containing an azide group. In certain embodiments, the unnatural amino acid is 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid.
  • the labeling group capable of click chemical reaction with the azide group is a chemical moiety containing a dibenzocyclooctyne group, such as dibenzocyclooctyne (DBCO), 4-dibenzocyclooctynol (DIBO), or BCN(bicyclo[6.1.0]nonyne).
  • DBCO dibenzocyclooctyne
  • DIBO 4-dibenzocyclooctynol
  • BCN bicyclo[6.1.0]nonyne
  • the labeling group capable of click chemical reaction with the azide group is DBCO
  • the first site-specific modified IL-2 is at the first amino acid position compared with wild-type IL-2. (such as amino acid position F42, Y45, E62, P65 or E68) and the second site-directed modified IL-2 has a second amino acid position (such as amino acid position D20, H16, A73) compared with wild-type IL-2 or H79) residues are replaced by the structure of formula Ia:
  • the direction from R1 to R2 is the N-terminal to C-terminal direction of the amino acid sequence, wherein the amino acid at position N is the residue at the first amino acid position (such as amino acid at position F42, Y45, E62, P65 or E68) Or the residue at the second amino acid position (such as amino acid position D20, H16, A73 or H79), R1 is the 1st to N-1st amino acid residues of the IL-2 amino acid sequence, and R2 is the 1st to N-1th amino acid residue of the IL-2 amino acid sequence. From the amino acid residues from position N+1 to the C terminus, R3 is a PEG group.
  • the labeling group capable of click chemical reaction with the azide group is DIBO
  • the direction from R1 to R2 is the N-terminal to C-terminal direction of the amino acid sequence, wherein the amino acid at position N is the residue at the first amino acid position (such as amino acid at position F42, Y45, E62, P65 or E68) Or the residue at the second amino acid position (such as amino acid position D20, H16, A73 or H79), R1 is the 1st to N-1st amino acid residues of the IL-2 amino acid sequence, and R2 is the 1st to N-1th amino acid residue of the IL-2 amino acid sequence. From the amino acid residues from position N+1 to the C terminus, R3 is a PEG group.
  • the labeling group capable of click chemical reaction with the azide group is BCN
  • the direction from R1 to R2 is the N-terminal to C-terminal direction of the amino acid sequence, wherein the amino acid at position N is the residue at the first amino acid position (such as amino acid at position F42, Y45, E62, P65 or E68) Or the residue at the second amino acid position (such as amino acid position D20, H16, A73 or H79), R1 is the 1st to N-1st amino acid residues of the IL-2 amino acid sequence, and R2 is the 1st to N-1th amino acid residue of the IL-2 amino acid sequence. From the amino acid residues from position N+1 to the C terminus, R3 is a PEG group.
  • the PEGylated IL-2 variants contemplated by this application are named as follows: [position mutated to a non-natural amino acid compared to SEQ ID NO: 1]-[attached PEG group average molecular weight].
  • the first site-specific modified IL-2 is selected from F42-20K, Y45-20K, E62-20K, P65-20K, or E68-20K.
  • the second site-directed modified IL-2 is D20-20K, H16-20K, A73-20K, or H79-20K.
  • Y45-20K refers to the following PEGylated IL-2 variant: The difference in the amino acid sequence of this variant compared with SEQ ID NO:1 is that Y45 is replaced with the unnatural amino acid NAEK, and this position is further connected to the average PEG group with a molecular weight of 20 kDa.
  • the first site-directed modified IL-2 and the second site-directed modified IL-2 contained in the composition provided by the first aspect of the present invention can be prepared by any method known in the art.
  • a non-natural amino acid can be inserted site-directed through non-natural amino acid orthogonal translation techniques, followed by attaching a PEG group to the non-natural amino acid.
  • Non-natural amino acid orthogonal translation techniques are well known to those skilled in the art.
  • Non-natural amino acid orthogonal translation technology uses stop codons to insert non-natural amino acids into the amino acid sequence of the protein during protein translation, which actually expands the number of amino acid codons. Therefore, non-natural amino acid orthogonal translation technology is also called Genetic code expansion technology.
  • non-natural amino acid orthogonal translation systems involve tRNA, an aminoacyl tRNA synthetase, and a nucleic acid sequence of interest with one or more stop codons. The system described above is introduced into a host cell and cultured in a medium containing appropriate nutrients and the unnatural amino acid(s) to be inserted. The host cell is then maintained under conditions that allow expression of the protein of interest.
  • One or more unnatural amino acids are incorporated into a polypeptide chain in response to an unnatural codon.
  • non-natural amino acids can be site-directedly inserted through 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 Generally, it can effectively and selectively form stable covalent bonds, and then form covalent bonds through chemical reactions to modify the PEG group in a targeted manner.
  • non-natural amino acids can be inserted into the site-specifically via non-natural amino acid orthogonal translation technology, followed by site-directed modification of the PEG group through click chemistry reactions.
  • the unnatural amino acid and PEG group each comprise a chemical group capable of click chemistry reactions.
  • the present invention provides a method for preparing the composition of the first aspect, which includes preparing the first site-directed modified IL-2 and preparing the second site-directed modified IL-2, wherein,
  • Preparing the first site-specific modified IL-2 includes:
  • a1) a first site-mutated IL-2, in which the residue at the first amino acid position (such as amino acid position F42, Y45, E62, P65 or E68) compared to wild-type IL-2 is mutated to Non-natural amino acids;
  • a PEG group modified by a labeling group the labeling group can form a covalent bond with the non-natural amino acid;
  • Preparing the second site-directed modified IL-2 includes:
  • a2) a second site-directed mutated IL-2, in which the residue at the second amino acid position (such as amino acid position D20, H16, A73 or H79) compared to wild-type IL-2 is mutated to a non-natural amino acid ;
  • the first site-specifically mutated IL-2 comprises a non-natural amino acid containing chemical functional groups, such as carbonyl, alkynyl, azide groups, etc., which are generally capable of effective and selective
  • the labeling group contained in the PEG group can chemically react with the chemical functional group to form a covalent bond.
  • the second site-directed mutated IL-2 comprises a non-natural amino acid containing chemical functional groups, such as carbonyl, alkynyl, azide groups, etc., which are generally effective and selective. to form 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.
  • chemical functional groups such as carbonyl, alkynyl, azide groups, etc.
  • the unnatural amino acids in the first site-directed mutated IL-2 and the second site-directed mutated IL-2 comprise the same chemical functional group.
  • the unnatural amino acids in the first site-directed mutated IL-2 and the second site-directed mutated IL-2 comprise an azide group.
  • the non-natural amino acids in the first site-directed mutated IL-2 and the second site-directed mutated IL-2 are the same.
  • the unnatural amino acid is a lysine derivative containing an azide group. In certain embodiments, the unnatural amino acid is N ⁇ -2-azidoethoxycarbonyl-L-lysine (NAEK).
  • the unnatural amino acid is a tyrosine derivative containing an azide group. In certain embodiments, the unnatural amino acid is 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid.
  • preparing the first site-specific modified IL-2 includes:
  • a1) a first site-mutated IL-2, in which the residue at the first amino acid position (such as amino acid position F42, Y45, E62, P65 or E68) compared to wild-type IL-2 is mutated to Unnatural amino acids containing an azide group;
  • a1 a PEG group modified by a labeling group, which can undergo a click chemical reaction with the azide group;
  • preparing the second site-directed modified IL-2 includes:
  • a2) a second site-directed mutated IL-2 in which a residue at a second amino acid position (such as amino acid position D20, H16, A73 or H79) compared to wild-type IL-2 is mutated to contain an azide group of unnatural amino acids;
  • a second site-directed mutated IL-2 in which a residue at a second amino acid position (such as amino acid position D20, H16, A73 or H79) compared to wild-type IL-2 is mutated to contain an azide group of unnatural amino acids;
  • the click chemistry is 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 without a catalyst.
  • One of these reagents consists of the so-called DBCO compound. Azide-modified macromolecules can now be labeled without metal catalysts, allowing them to be studied in living cells while preventing protein damage.
  • the labeling group capable of click chemical reaction with the azide group is a chemical moiety comprising an alkynyl group. In certain embodiments, the labeling group capable of click chemical reaction with an azide group is a chemical moiety comprising a dibenzocyclooctyne group. In certain embodiments, the labeling group capable of click chemical reaction with the azide group is dibenzocyclooctyne (DBCO), 4-dibenzocyclooctynol (DIBO) or BCN (bicyclo[6.1.0]nonyne). In certain embodiments, the labeling group capable of click chemical reaction with the azide group is DBCO.
  • the DBCO-labeled PEG group has the structure of Formula IIa, wherein R3 is a PEG group.
  • the DIBO-tagged PEG group has the structure of Formula IIb, wherein R3 is a PEG group.
  • 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.
  • the non-natural amino acid orthogonal translation technology includes the following steps:
  • the unnatural amino acid is N ⁇ -2-azidoethoxycarbonyl-L-lysine (NAEK).
  • the aminoacyl-tRNA synthetase specific for an unnatural amino acid is a NAEK-specific aminoacyl-tRNA synthetase.
  • the vector encoding an amber codon suppressor tRNA and an aminoacyl tRNA synthetase specific for unnatural amino acids is pSURAR-YAV (also known as pSUPAR-YAV-tRNA/PylRS), which is derived from The strain was obtained from Escherichia coli containing the plasmid pSUPAR-YAV-tRNA/PylRS. The strain was deposited in the General Microbiology Center of the China Committee for the Collection of Microorganisms (No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences). The preservation date is April 8, 2013, the preservation number is CGMCC No: 7432, and the classification is named Escherichia coli.
  • the first site-mutated IL-2 is a residue at a first amino acid position (e.g., amino acid position F42, Y45, E62, P65, or E68) as compared to wild-type IL-2 and the Compared with wild-type IL-2, 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 is replaced by the structure of Formula III:
  • the direction from R1 to R2 is the N-terminal to C-terminal direction of the amino acid sequence, where the amino acid at position N is the residue at the first amino acid position (such as amino acid at position F42, Y45, E62, P65 or E68) or the amino acid at position E68. Residues at two amino acid positions (such as amino acid position D20, H16, A73 or H79), R1 is the 1st to N-1st amino acid residues of the IL-2 amino acid sequence, and R2 is the Nth amino acid residue of the IL-2 amino acid sequence. Amino acid residues from position +1 to the C terminus.
  • the combination of the first site-modified IL-2 and the second site-modified IL-2 provided by the present invention can reduce the aging and exhaustion of immune cells and enhance T cell proliferation, reduce the proliferation of Treg cells, maintain the proliferation 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, making the immune system more effective in adoptive cell immunotherapy.
  • Cell fate directs long-term immunity.
  • the present invention also provides kits as described below, in vitro methods for preparing or cultivating immune cells for adoptive cell therapy, and immune cells for adoptive cell therapy obtained by this method.
  • the present invention provides a kit comprising the composition described in the first aspect.
  • the kit further comprises a package insert containing instructions for using the composition to prepare and/or culture immune cells in vitro for adoptive cell therapy.
  • the present invention also relates to the use of the composition described in the first aspect or the kit described in the third aspect for in vitro preparation or culture of immune cells for adoptive cell therapy.
  • 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 wait.
  • TIL tumor-infiltrating T cells
  • CAR-T chimeric antigen receptor T cell therapy
  • TCR T cell receptor therapy
  • NK cell therapy wait.
  • the engineered immune cells used for adoptive cell therapy may be any cells known in the art for use in adoptive cell therapy.
  • the engineered immune cells for adoptive cell therapy include lymphocytes, such as T cells, NK cells, or combinations thereof.
  • the immune cells used for adoptive cell therapy are immune cells engineered to express a chimeric antigen receptor and/or comprise a nucleic acid molecule encoding the chimeric antigen receptor.
  • the engineered immune cells include lymphocytes expressing IL2-R ⁇ trimers, such as T cells, NK cells, or combinations thereof.
  • the immune cells used in adoptive cell therapy are tumor-infiltrating lymphocytes (TIL).
  • TIL tumor-infiltrating lymphocytes
  • the present invention provides a kit comprising the composition described in the first aspect and a nucleic acid molecule encoding a chimeric antigen receptor.
  • the kit further comprises package inserts containing instructions for using the nucleic acid molecules in the composition to prepare engineered immune cells in vitro for adoptive cell therapy, wherein The engineered immune cells express a chimeric antigen receptor and/or comprise a nucleic acid molecule encoding the chimeric antigen receptor.
  • the nucleic acid molecule encoding a chimeric antigen receptor is present in an expression vector.
  • 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 composition described in the first aspect and optionally the nucleic acid molecule encoding a chimeric antigen receptor or the kit described in the fourth aspect for use in vitro preparation of modified immune cells for adoptive cell therapy.
  • the use of the modified immune cell expresses a chimeric antigen receptor and/or contains 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.
  • the kit further comprises a package insert containing 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 optionally immune cells for adoptive cell therapy for culturing immune cells for adoptive cell therapy in vitro.
  • the immune cells used for adoptive cell therapy are immune cells engineered to express a chimeric antigen receptor and/or comprise a nucleic acid molecule encoding the chimeric antigen receptor.
  • the engineered immune cells include lymphocytes expressing IL2-R ⁇ trimers, such as T cells, NK cells, or combinations thereof.
  • the immune cells used in adoptive cell therapy are tumor-infiltrating lymphocytes (TIL).
  • TIL tumor-infiltrating lymphocytes
  • chimeric antigen receptors as described herein have meanings known to those skilled in the art, which typically include an extracellular antigen binding domain, an optional spacer domain, a transmembrane domain, and One or more intracellular signaling domains.
  • the intracellular signaling domain is selected from primary signaling domains and/or costimulatory signaling domains.
  • the extracellular antigen-binding domain comprises an antibody or antigen-binding fragment (eg, scFv) that specifically binds a tumor-associated antigen (eg, CD19).
  • the invention provides a method of culturing immune cells for adoptive cell therapy, said method comprising in a cell culture medium comprising a first site-specific modified IL-2 and a second site-directed modified IL-2 The cells are cultured, wherein the first site-directed modified IL-2 and the second site-directed modified IL-2 are as defined in the first aspect.
  • the cell culture medium can be any medium that can support cell growth, usually contains inorganic salts, vitamins, glucose, buffer systems and essential amino acids, and usually has an osmotic pressure of about 280-330 mOsmol.
  • 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.
  • the cell culture medium is complete culture medium.
  • the cell culture medium includes 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 contain serum.
  • the methods further include harvesting the cells for storage (eg, reconstitution in cryopreservation medium) or administration (eg, for adoptive cell therapy).
  • the culture conditions provided in the presence of the first site-directed modified IL-2 and the second site-directed modified IL-2 can reduce the senescence and exhaustion of immune cells, retain their stemness state, enhance T Cell proliferation, reducing the proliferation of Treg cells, maintaining the proliferation state of Tscm and T effector cells, reducing the exhaustion of T effector cells, and avoiding the excessive activation of T cells by endogenous IL-2 produced by T cell activation.
  • the immune cells used for adoptive cell therapy are immune cells engineered to express a chimeric antigen receptor and/or comprise a nucleic acid molecule encoding the chimeric antigen receptor.
  • the engineered immune cells include lymphocytes expressing IL2-R ⁇ trimers, such as T cells, NK cells, or combinations thereof.
  • the immune cells used in adoptive cell therapy are tumor-infiltrating lymphocytes (TILs).
  • TILs tumor-infiltrating lymphocytes
  • the present invention provides a method of preparing immune cells for adoptive cell therapy, comprising using a first site-directed modified IL-2 and a second site-directed modified IL-2 as defined in the first aspect.
  • the invention provides methods of preparing immune cells for adoptive cell therapy, the immune cells for adoptive cell therapy being engineered immune cells that express chimeric Antigen receptors and/or nucleic acid molecules encoding said chimeric antigen receptors, wherein the method includes:
  • step (2) is performed in a cell culture medium comprising the first site-directed modified IL-2 and the second site-directed modified IL-2.
  • the cell culture medium can be any medium that can support cell growth, usually contains inorganic salts, vitamins, glucose, buffer systems and essential amino acids, and usually has an osmotic pressure of about 280-330 mOsmol.
  • 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.
  • the cell culture medium is complete culture medium.
  • the cell culture medium includes basal medium (such as RPMI 1640), serum (such as FBS), sodium pyruvate, non-essential amino acids.
  • the cell culture medium does not contain serum.
  • the nucleic acid molecule encoding the chimeric antigen receptor described in step (2) is present in an expression vector.
  • the nucleic acid molecule encoding the chimeric antigen receptor described in step (2) is introduced into the cell by means of infection by a viral (eg, lentivirus, retrovirus, or adenovirus) vector. In certain embodiments, the nucleic acid molecule encoding the chimeric antigen receptor described in step (2) is introduced into the cell by infection with a lentiviral vector.
  • a viral eg, lentivirus, retrovirus, or adenovirus
  • the nucleic acid molecule encoding the chimeric antigen receptor described in step (2) is introduced into the cell by a non-viral vector.
  • the immune cells are pretreated, and the pretreatment includes sorting, activation and/or proliferation of immune cells.
  • the pretreating includes contacting the immune cells with an anti-CD3 antibody and an anti-CD28 antibody, thereby stimulating the immune cells and inducing their proliferation, thereby generating pretreated immune cells.
  • the pretreatment includes isolating T cells from peripheral blood mononuclear cells (PBMC).
  • PBMC peripheral blood mononuclear cells
  • the method further includes: (3) continuing in a cell culture medium comprising the first site-directed modified IL-2 and the second site-directed modified IL-2.
  • the step of culturing the immune cells obtained in step (2) is not limited to:
  • the methods further include harvesting the cells for storage (eg, reconstitution in cryopreservation medium) or administration (eg, for adoptive cell therapy).
  • the immune cells include lymphocytes expressing IL2-R ⁇ trimers, such as T cells, NK cells, or any combination thereof. In certain embodiments, the immune cells are T cells.
  • the production conditions provided in the presence of the first site-directed modified IL-2 and the second site-directed modified IL-2 can reduce excessive activation and terminal differentiation of immune cells.
  • the invention also provides methods of preparing immune cells for adoptive cell therapy, the immune cells for adoptive cell therapy being tumor-infiltrating lymphocytes (TIL), wherein , the method includes: isolating infiltrating lymphocytes from tumor tissue and culturing them in a cell culture medium containing a first site-specific modified IL-2 and a second site-specific modified IL-2, wherein the first site-specific modified IL-2
  • TIL tumor-infiltrating lymphocytes
  • the method includes: isolating infiltrating lymphocytes from tumor tissue and culturing them in a cell culture medium containing a first site-specific modified IL-2 and a second site-specific modified IL-2, wherein the first site-specific modified IL-2
  • the site-modified IL-2 and the second site-modified IL-2 are as defined in the first aspect, and the cell culture medium is as defined above.
  • 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.
  • the immune cells of the seventh aspect have reduced senescence and exhaustion, enhanced T Cell proliferation, reduced Treg cell proliferation, maintained Tscm and T effector cell proliferation, reduced T effector cell exhaustion, and/or reduced overactivation by endogenous IL-2.
  • the present invention provides a population of immune cells, which includes the immune cells described in the seventh aspect, and optionally unmodified and/or unsuccessfully modified immune cells.
  • the immune cells of the seventh aspect account for approximately 10%-100% of the total cell number of the immune cell population, preferably 40%-80%.
  • the first site-specific modified IL-2 and the second site-directed modified IL-2 provided by the present invention have a significant synergistic effect in enhancing the immune response. Therefore, the first site-specific modified IL-2 and the second site-directed modified IL-2 provided by the present invention
  • the combination of two site-directed modified IL-2s may be used to treat disease conditions in which stimulation of the host's immune system is beneficial, particularly conditions where enhanced cellular immune response is desired, which may include disease conditions in which the host immune response is insufficient or defective, such as tumors.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the composition described in the first aspect and a pharmaceutically acceptable carrier and/or excipient.
  • the pharmaceutical compositions comprise multiple compositions or dosage forms.
  • the first site-directed modified IL-2 and the second site-directed modified IL-2 are in separate compositions or dosage forms.
  • the pharmaceutical composition comprises: a first composition formed by the first site-specific modified IL-2 and a pharmaceutically acceptable carrier and/or excipient, and the second A second composition formed by site-specific modified IL-2 and pharmaceutically acceptable carriers and/or excipients.
  • the pharmaceutical composition comprises a composition or dosage form.
  • the first site-directed modified IL-2 and the second site-directed modified IL-2 are in the same composition or dosage form.
  • the pharmaceutical composition comprises: the first site-directed modified IL-2, the second site-directed modified IL-2 and a pharmaceutically acceptable carrier and/or excipient to form a single composition. combination.
  • the first site-directed modified IL-2 and the second site-directed modified IL-2 of the present invention, their combinations, and the compositions described in the first aspect or the pharmaceutical compositions described in the tenth aspect can be formulated into A dosage form compatible with its intended route of administration.
  • routes of administration include parenteral administration, e.g., intravenous administration, intradermal administration, subcutaneous administration, oral administration (e.g., inhalation administration), transdermal administration (i.e., topical administration), transdermal administration (i.e., topical administration), Mucosal and rectal administration.
  • Solutions or suspensions for parenteral, intradermal or subcutaneous administration applications may include the following components: sterile diluents such as water for injection, saline solution, 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 acetate, citrate or Phosphates, and agents used to adjust tonicity, such as sodium chloride or glucose.
  • the pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide.
  • Formulations for parenteral administration may be enclosed in ampoules, disposable syringes or multi-dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the ready preparation of sterile injectable solutions or dispersions.
  • suitable carriers include physiological saline, bacteriostatic water, polyoxyethylene castor oil ELTM, or phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the composition must be sterile and should be fluid to the extent that syringability exists. It must be stable under the conditions of manufacture and storage, and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier may be a solvent or dispersion medium including, for example, water, ethanol, polyols (eg, glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof. Protection against microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases it will be preferable to include isotonic agents such as sugars, polyols such as mannitol, sorbitol, sodium chloride in the composition. 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.
  • the present invention provides a method for enhancing immune response, preventing and/or treating proliferative diseases (such as tumors), comprising administering the composition of the first aspect to a subject in need thereof Or the pharmaceutical composition described in the tenth aspect.
  • the method includes co-administering the first site-directed modified IL-2 and the second site-directed modified IL-2 to the subject.
  • the present invention also 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 , use in the preparation of medicaments for enhancing immune response, preventing and/or treating proliferative diseases (such as tumors).
  • the present invention also provides the use of the combination of the first site-directed modified IL-2 and the second site-directed modified IL-2 for enhancing immune response, preventing and/or treating proliferative diseases (such as tumors), or in the preparation of Use in drugs for enhancing immune responses, preventing and/or treating proliferative diseases (e.g. tumors).
  • the first site-directed modified IL-2 and the second site-directed modified IL-2 are formulated separately into two or more compositions (e.g., agents containing each component box).
  • the individual components administered in conjunction with each other can be administered simultaneously, separately or sequentially.
  • individual components administered in conjunction with each other can be administered to the subject at a different time than the other components are administered; for example, as part of a treatment regimen, each administration can be administered for a given time period are given at intervals other than simultaneously (e.g., individually or sequentially).
  • individual components administered in conjunction with one another can also be administered sequentially, but substantially simultaneously, during the same administration period.
  • the individual components administered in combination with each other may be administered to a subject by the same or different routes.
  • the first site-directed modified IL-2 and the second site-directed modified IL-2 are formulated together into a single composition, for example, for simultaneous delivery.
  • the invention provides a pharmaceutical kit comprising a medicament containing the first site-specific modified IL-2 and optional pharmaceutically acceptable carriers and/or excipients, and package instructions.
  • the package insert includes administering the drug in combination with a composition containing the second site-directed modified IL-2 and optionally a pharmaceutically acceptable carrier and/or excipient to enhance efficacy in a subject Instructions for immune response, prevention and/or treatment of proliferative diseases (e.g. tumors).
  • the invention provides a pharmaceutical kit comprising a medicament containing the second site-directed modified IL-2 and optional pharmaceutically acceptable carriers and/or excipients, and package instructions,
  • the package insert includes administering the drug in combination with a composition containing the first site-specific modified IL-2 and optionally a pharmaceutically acceptable carrier and/or excipient to enhance potency in a subject.
  • Instructions for immune response, prevention and/or treatment of proliferative diseases e.g. tumors).
  • the invention provides a kit comprising a first medicament containing the first site-specific modified IL-2 and an optional pharmaceutically acceptable carrier and/or excipient, containing The second site-directed modified IL-2 and optionally a second drug with a pharmaceutically acceptable carrier and/or excipient.
  • the kit further comprises instructions for administering the first agent and the second agent to enhance an immune response, prevent and/or treat a proliferative disease (e.g., tumor) in a subject packaging instructions.
  • a proliferative disease e.g., tumor
  • the immune response is a cellular immune response.
  • the immune response is a T cell-mediated immune response, particularly an effector T cell (Teff)-mediated immune response.
  • enhancing the immune response further includes reducing or inhibiting Treg cell function.
  • the proliferative disease is a tumor, including a solid tumor or a hematological tumor, including metastatic cancer, relapsed or refractory cancer.
  • the proliferative disease is hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias, purpura, sarcoidosis, Sézary syndrome (Sezary Syndrome), Waldenstron's macroglobulinemia, Gaucher's Disease, histiocytosis, and any other cell proliferation disease other than neoplasia in an organ system.
  • the tumor is a solid tumor.
  • the solid tumor is a metastatic cancer, a relapsed or refractory cancer.
  • the tumor is a hematological tumor, such as leukemia, lymphoma, or myeloma.
  • the hematological cancer is metastatic cancer, relapsed or refractory cancer.
  • the tumor is selected from the group consisting of 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.
  • the subject is a mammal, such as a human.
  • the first site-specific modified IL-2 and the second site-specific modified IL-2 in the composition are administered simultaneously, separately, or sequentially.
  • the first site-directed modified IL-2 and the second site-directed modified IL-2 of the invention, combinations thereof, and combinations of the invention may 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 invention should be sterile and stable under the conditions of production and storage.
  • One preferred dosage form is an injection.
  • Such injections may be sterile injectable solutions.
  • sterile injectable solutions may be prepared by incorporating the requisite dosage of the active ingredient in an appropriate solvent and, optionally, other desired ingredients (including, but not limited to, pH adjusters, surfactants, etc.). active agent, adjuvant, ionic strength enhancer, isotonic agent, preservative, diluent, or any combination thereof) followed by filtered sterilization.
  • sterile injectable solutions may be prepared as sterile lyophilized powders (for example, by vacuum drying or freeze drying) for ease of storage and use.
  • Such sterile lyophilized powder can be dispersed in a suitable carrier before use, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (such as 0.9% (w/v) NaCl), Glucose solutions (eg 5% glucose), surfactant containing solutions (eg 0.01% polysorbate 20), pH buffer solutions (eg phosphate buffer solution), Ringer's solution and any combination thereof.
  • WFI water for injection
  • BWFI bacteriostatic water for injection
  • sodium chloride solution such as 0.9% (w/v) NaCl
  • Glucose solutions eg 5% glucose
  • surfactant containing solutions eg 0.01% polysorbate 20
  • pH buffer solutions eg phosphate buffer solution
  • Ringer's solution any combination thereof.
  • the first site-directed modified IL-2 and the second site-directed modified IL-2 of the invention, combinations thereof, and combinations of the invention may be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, eyeball, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum, Inguinal, intravesical, topical (eg, powder, ointment, or drops), or nasal route.
  • the preferred route/mode of administration is parenteral (eg intravenous or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection).
  • the route and/or mode of administration will vary depending on the intended purpose.
  • 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 are administered by intravenous injection or bolus injection give.
  • the first site-directed modified IL-2 and the second site-directed modified IL-2 of the invention, combinations thereof, and combinations of the invention may be formulated in dosage unit form for ease of administration.
  • Dosage unit form refers to physically discrete units suitable as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the first site-directed modified IL-2 and the second site-directed modified IL-2 of the invention, combinations thereof, and combinations of the invention may be administered alone or in combination with another pharmaceutically active agent (eg, an anti-tumor agent) or additional therapy (eg, an anti-tumor therapy).
  • another pharmaceutically active agent eg, an anti-tumor agent
  • additional therapy eg, an anti-tumor 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 guide the fate of immune cells in adoptive cellular immunotherapy to long-term immunity. This further provides therapeutic applications of the immune cells.
  • 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 of the seventh aspect, the immune cell population of the eighth aspect, or the pharmaceutical composition of the thirteenth aspect can be formulated into a dosage form compatible with its intended route of administration.
  • routes of administration include parenteral administration, e.g., intravenous administration, intradermal administration, subcutaneous administration, oral administration (e.g., inhalation administration), transdermal administration (i.e., topical administration), transdermal administration (i.e., topical administration), Mucosal and rectal administration.
  • Solutions or suspensions for parenteral, intradermal or subcutaneous administration applications may include the following components: sterile diluents such as water for injection, saline solution, 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 acetate, citrate or Phosphates, and agents used to adjust tonicity, such as sodium chloride or glucose.
  • the pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide.
  • Formulations for parenteral administration may be enclosed in ampoules, disposable syringes or multi-dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the ready preparation of sterile injectable solutions or dispersions.
  • suitable carriers include physiological saline, bacteriostatic water, polyoxyethylene castor oil ELTM, or phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the composition must be sterile and should be fluid to the extent that syringability exists. It must be stable under the conditions of manufacture and storage, and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier may be a solvent or dispersion medium including, for example, water, ethanol, polyols (eg, glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof. Protection against microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases it will be preferable to include isotonic agents such as sugars, polyols such as mannitol, sorbitol, sodium chloride in the composition. 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.
  • the present invention provides a method for enhancing immune response, preventing and/or treating proliferative diseases (such as tumors), which comprises administering the immune cells of the seventh aspect to a subject in need , the immune cell group described in the eighth aspect or the pharmaceutical composition described in the thirteenth aspect.
  • the method includes the following steps: (1) using the method described in the fifth aspect and/or the method described in the sixth aspect to obtain immune cells; (2) converting the immune cells obtained in step (1) Immune cells or a population of cells comprising the same are administered to the subject for treatment.
  • chimeric antigen receptor cell therapy is administered to a subject, the method comprising: using the method described in the fifth aspect of the present invention and/or the method described in the sixth aspect to obtain expression of the chimeric antigen
  • the modified immune cells of the recipient are then administered to the subject.
  • 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, to The subject is administered the tumor-infiltrating lymphocytes (TIL).
  • TIL tumor infiltrating lymphocytes
  • the present invention also provides 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 for enhancing immune response, prevention and/or treatment.
  • the immune cells, immune cell populations, or pharmaceutical compositions may be combined with a first site-specific modified IL-2 as defined in the first aspect and a second site-specific modified IL-2 as defined in the first aspect.
  • the modified IL-2 is administered in combination, for example simultaneously, separately or sequentially.
  • the present invention also provides the use of a combination of the composition of the first aspect and immune cells for adoptive cell therapy to enhance immune response, prevent and/or treat proliferative diseases (such as tumors) , or use in the preparation of medicaments for enhancing immune response, preventing and/or treating proliferative diseases (such as tumors).
  • the present invention also provides a method for enhancing immune response, preventing and/or treating proliferative diseases, which includes administering to a subject in need the composition described in the first aspect and a method for adoptive Combination of immune cells for cell therapy.
  • the immune cells used for adoptive cell therapy are engineered immune cells that express chimeric antigen receptors and/or contain genes encoding the chimeric antigen receptors. Nucleic acid molecules that bind antigen receptors.
  • the engineered immune cells include lymphocytes expressing IL2-R ⁇ trimers, such as T cells, NK cells, or any combination thereof.
  • the immune cells used for adoptive cell therapy are tumor-infiltrating lymphocytes (TIL).
  • TIL tumor-infiltrating lymphocytes
  • composition of the first aspect and the immune cells used for adoptive cell therapy are present in the same composition or dosage form and can be administered simultaneously.
  • composition of the first aspect and the immune cells for adoptive cell therapy are present in separate compositions or dosage forms, and thus can be separated or sequential Apply.
  • the immune response is a cellular immune response.
  • the immune response is a T cell-mediated immune response, particularly an effector T cell (Teff)-mediated immune response.
  • enhancing the immune response further includes reducing or inhibiting Treg cell function.
  • the proliferative disease is a tumor, including a solid tumor or a hematological tumor, including metastatic cancer, relapsed or refractory cancer.
  • the proliferative disease is hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias, purpura, sarcoidosis, Sézary syndrome (Sezary Syndrome), Waldenstron's macroglobulinemia, Gaucher's Disease, histiocytosis, and any other cell proliferation disease other than neoplasia in an organ system.
  • the tumor is a solid tumor.
  • the solid tumor is a metastatic cancer, a relapsed or refractory cancer.
  • the tumor is a hematological tumor, such as leukemia, lymphoma, or myeloma.
  • the hematological cancer is metastatic cancer, relapsed or refractory cancer.
  • the tumor is selected from the group consisting of melanoma, renal cell carcinoma, non-small cell lung cancer, lymphoma, head and neck squamous cell carcinoma, urothelial carcinoma, ovarian cancer , stomach cancer and breast cancer.
  • the tumor is lymphoma.
  • the tumors when involving chimeric antigen receptor cell therapy, preferably include hematological tumors, including metastatic cancers, relapsed or refractory cancers; e.g. The tumor is selected from lymphoma.
  • the tumor when tumor-infiltrating lymphocyte (TIL) therapy is involved, the tumor preferably includes a solid tumor, including metastatic cancer, relapsed or refractory cancer; e.g.
  • the tumor is selected from the group consisting of 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.
  • the subject is a mammal, such as a human.
  • 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 a formula that has been established in the medical field.
  • Any known dosage form such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile injections Powder and concentrated solution 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 invention should be sterile and stable under the conditions of production and storage.
  • One preferred dosage form is an injection.
  • Such injections may be sterile injectable solutions.
  • sterile injectable solutions may be prepared by incorporating the requisite dosage of the active ingredient in an appropriate solvent and, optionally, other desired ingredients (including, but not limited to, pH adjusters, surfactants, etc.). active agent, adjuvant, ionic strength enhancer, isotonic agent, preservative, diluent, or any combination thereof) followed by filtered sterilization.
  • sterile injectable solutions may be prepared as sterile lyophilized powders (for example, by vacuum drying or freeze drying) for ease of storage and use.
  • Such sterile lyophilized powder can be dispersed in a suitable carrier before use, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (such as 0.9% (w/v) NaCl), Glucose solutions (eg 5% glucose), surfactant containing solutions (eg 0.01% polysorbate 20), pH buffer solutions (eg phosphate buffer solution), Ringer's solution and any combination thereof.
  • WFI water for injection
  • BWFI bacteriostatic water for injection
  • sodium chloride solution such as 0.9% (w/v) NaCl
  • Glucose solutions eg 5% glucose
  • surfactant containing solutions eg 0.01% polysorbate 20
  • pH buffer solutions eg phosphate buffer solution
  • Ringer's solution any combination thereof.
  • 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 obtained by known methods in the art. Any suitable method of administration, including, but not limited to, oral, buccal, sublingual, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum, inguinal, intravesical, topical (e.g., powder, ointments or drops), or the nasal route.
  • the preferred route/mode of administration is parenteral (eg intravenous or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). The skilled artisan will understand that the route and/or mode of administration will vary depending on the intended purpose.
  • the immune cells of the seventh aspect, the immune cell population of the eighth aspect, or the pharmaceutical composition of the thirteenth aspect are administered by intravenous injection or bolus injection.
  • the immune cells of the seventh aspect, the immune cell population of the eighth aspect, or the pharmaceutical composition of the thirteenth aspect may be formulated in dosage unit form.
  • dosage unit form refers to physically discrete units suitable as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • 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 can Administered in conjunction with another pharmaceutically active agent (e.g., an antineoplastic agent) or additional therapy (e.g., an antineoplastic therapy).
  • another pharmaceutically active agent e.g., an antineoplastic agent
  • additional therapy e.g., an antineoplastic therapy
  • unnatural amino acid refers to amino acids other than the 20 amino acids naturally found in proteins.
  • unnatural amino acids include: N ⁇ -2-azidoethoxycarbonyl-L-lysine (NAEK), p-acetyl-L-phenylalanine, p-iodo-L-phenylalanine Acid, p-methoxyphenylalanine, O-methyl-L-tyrosine, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, L-3-( 2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl -GlcNAcp-serine, L-dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p
  • the unnatural amino acid contains a selective reactive group, or a reactive group for site-selective labeling of a target polypeptide.
  • Chemical reactions can be bioorthogonal reactions (e.g., biocompatibility and selectivity reactions), Cu(I)-catalyzed or "copper-free" alkyne-azidotriazole formation reactions, Staudinger ligation, Inverse-electron-demand Diels-Alder (IEDDA) reactions, “light-click” chemistry, or metal-mediated processes (such as olefin metathesis and Suzuki-Miyaura or Sonogashira cross-coupling) wait.
  • the term "vector” refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into the host cell through transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell.
  • Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC) ; Phages 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, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, Polyomavacuolating viruses (such as SV40).
  • retroviruses including lentiviruses
  • adenoviruses such as herpes simplex virus
  • poxviruses poxviruses
  • baculoviruses papillomaviruses
  • papillomaviruses papillomaviruses
  • Polyomavacuolating viruses such as SV40.
  • a vector can contain a variety of expression-controlling elements, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes
  • the term "host cell” refers to a cell that can be used to introduce a vector, which includes, but is not limited to, prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, etc. 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 E. coli.
  • chimeric antigen receptor refers to a recombinant polypeptide comprising at least one extracellular antigen-binding domain, optionally a spacer domain, a transmembrane domain, and an intracellular signaling domain Constructs that combine antibody-based specificity for an antigen of interest (eg, a tumor antigen) with an immune effector cell-activating intracellular domain to exhibit specific immune activity against cells expressing the antigen of interest (eg, tumor cells).
  • the expression "CAR-expressing immune cell” refers to an immune cell that expresses a CAR and has an antigen specificity determined by the targeting domain of the CAR.
  • extracellular antigen-binding domain refers to a polypeptide capable of specifically binding to an antigen or receptor of interest. This domain will be able to interact with cell surface molecules.
  • the extracellular antigen-binding domain can be selected to recognize an antigen that is a cell surface marker on a target cell associated with a particular disease state.
  • the extracellular antigen binding domain is an antibody-derived targeting domain.
  • intracellular signaling domain refers to the portion of a protein that conducts effector signaling functions and directs the cell to perform specialized functions. Therefore, the intracellular signaling domain has the ability to activate at least one normal effector function of the CAR-expressing immune effector cell.
  • the effector function of T cells can be cytolytic activity or auxiliary activity, including the secretion of cytokines.
  • primary signaling domain refers to a portion of a protein capable of modulating primary activation of a TCR complex in a stimulatory manner or in an inhibitory manner.
  • Primary signaling domains that act in a stimulatory manner often contain signaling motifs known as immunoreceptor tyrosine-based activation motifs (ITAMs).
  • ITAMs containing primary signaling domains particularly useful in the present invention include those derived from TCR ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD22, DAP10, CD79a, CD79b, and CD66d.
  • costimulatory signaling domain refers to the intracellular signaling domain of a costimulatory molecule.
  • Costimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for efficient activation and function of T lymphocytes after binding to an antigen.
  • Non-limiting examples of costimulatory molecules include CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMFl) CD270 ( HVEM), CD278(ICOS), DAP10.
  • immune cell refers to any cell of the immune system having one or more effector functions (eg, cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and/or CDC).
  • effector functions eg, cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and/or CDC.
  • immune cells are cells that are of hematopoietic origin and play a role in immune responses.
  • immune cells refer to immune effector cells.
  • effector function refers to a specialized function of an immune effector cell, such as a function or response that enhances or promotes an immune attack on a target cell (eg, kills the target cell, or inhibits its growth or proliferation).
  • the effector function of a T cell may, for example, be cytolytic activity or auxiliary or activity including secretion of cytokines.
  • immune effector cells include T cells (eg, alpha/beta T cells and gamma/delta T cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived macrophages.
  • T lymphocytes include T lymphocytes.
  • T cell or "T lymphocyte” is art-recognized and is intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes.
  • the T cells may be T helper (Th) cells, such as T helper 1 (Th1) or T helper 2 (Th2) cells.
  • T cells can be helper T cells (HTL; CD4 T cells) CD4 T cells, cytotoxic T cells (CTL; CD8 T cells), CD4CD8 T cells, CD4CD8 T cells, or any other subset of T cells.
  • T cells can include naive T cells and memory T cells.
  • Immune cells of the invention may be self/autologous ("self") or non-self ("non-self", eg allogeneic, syngeneic or allogeneic).
  • autologous refers to cells from the same subject;
  • allogeneic refers to cells of the same species that are genetically different from the comparison cells;
  • isogenic means cells that are genetically different from the comparison cells.
  • allogeneic means cells from a different species than the comparison cells.
  • the cells of the invention are allogeneic.
  • antibody refers to an immunoglobulin molecule typically 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 into kappa (kappa) and lambda (lambda) light chains.
  • Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are connected by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids.
  • Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region consists of 3 domains (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 interaction of immunoglobulins with host tissues or factors, including various cells of the immune system (e.g., effector cells) and classical complement. Binding of the first component of the system (C1q).
  • VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs), interspersed with more conservative regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL consists of 3 CDRs and 4 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 respectively form the antigen-binding site.
  • the assignment of amino acids to each region or domain can follow 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; Definition of Chothia et al. (1989) Nature 342:878-883.
  • 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 that the full-length antibody binds, and/or competes with the full-length antibody for binding Specific binding of antigen, which is also called the "antigen-binding moiety".
  • antigen-binding moiety which is also called the "antigen-binding moiety.
  • Non-limiting examples of antigen-binding fragments include Fab, Fab', (Fab') 2 , Fv, disulfide-linked Fv, scFv, di- scFv, (scFv) 2 , and polypeptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.
  • Fab fragment means an antibody fragment consisting of the VL, VH, CL and CH1 domains
  • F(ab') 2 fragment means an antibody fragment consisting of a fragment that passes through a disulfide bridge on the hinge region An antibody fragment that connects two Fab fragments
  • Fab'fragment means the fragment obtained by reducing the disulfide bond connecting the two heavy chain fragments in the F(ab') 2 fragment, consisting of a complete light chain and heavy chain. The Fd fragment of the chain (consisting of VH and CH1 domains).
  • Fv means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. Fv fragments are generally considered to be the smallest antibody fragments that can form a complete antigen-binding site. It is generally believed that six CDRs confer the antigen-binding specificity of an antibody. However, even a variable region (such as an Fd fragment, which contains only three CDRs specific for the antigen) can recognize and bind the antigen, although its affinity may be lower than that of the intact binding site.
  • scFv refers to a single polypeptide chain comprising VL and VH domains connected by a linker.
  • Such scFv molecules may have the 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.
  • GGGGS linker with the amino acid sequence
  • a disulfide bond may also exist between VH and VL of scFv.
  • scFv can form di-scFv, which refers to two or more individual scFvs connected in series to form an antibody.
  • scFv can form (scFv) 2 , which refers to two or more individual scFvs joining in parallel to form an antibody.
  • the term "pharmaceutically acceptable carrier and/or excipient” means a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, They are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers Agents, diluents, agents to maintain osmotic pressure, agents to delay absorption, preservatives.
  • pH adjusting agents include, but are not limited to, phosphate buffer.
  • 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, etc.
  • Agents that maintain osmotic pressure include, but are not limited to, sugar, NaCl, and the like.
  • Agents that delay absorption include, but are not limited to, monostearate and gelatin.
  • Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), and the like.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc.
  • Stabilizers have the meaning commonly understood by those skilled in the art, which can stabilize the desired activity of active ingredients in medicines, 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 dry whey, albumin or casein) or their degradation products (such as lactalbumin hydrolyzate), etc.
  • sugars such as sorbitol, mannitol, starch, sucrose
  • lactose lactose
  • dextran or glucose
  • amino acids such as glutamic acid, glycine
  • proteins such as dry whey, albumin or casein
  • degradation products such as lactalbumin hydrolyzate
  • prevention refers to a method performed to prevent or delay the occurrence of a disease or condition or symptom in a subject.
  • treatment refers to a method performed to obtain a beneficial or desired clinical result.
  • beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction of the extent of the disease, stabilization (i.e., no worsening) of the state of the disease, delaying or slowing the progression of the disease, ameliorating or alleviating the disease. status, and relief of symptoms (whether partial or complete), whether detectable or undetectable.
  • treatment may also refer to prolonging survival compared to expected survival if not receiving treatment.
  • the term "subject” refers to a mammal, such as a primate mammal, such as a human.
  • the subject eg, human
  • the present invention performs specific site-selective polyethylene glycol (PEG) modification on IL2 to obtain a combination of IL-2 with the first site-specific modification and IL-2 with the second site-specific modification, wherein the first site-specific modification IL-2 has a blocking effect on IL2-R ⁇ receptors and is selective for IL2-R ⁇ receptors. It can preferentially activate Teff, CTL and NK cells and reduce the activation of Treg. In addition, it also induces the terminal of reduced CD8+ cells.
  • PEG polyethylene glycol
  • the second site-specific modified IL-2 has the effect of blocking IL2-R ⁇ but not IL2-R ⁇ , and can compete with the endogenous IL2 secreted by cells for IL2-R ⁇ , competitively inhibits the role of endogenous IL2 in activating Treg.
  • the combination of the two while retaining the central memory cell activation advantage of the first point-modified IL-2, can also compensate for the disadvantage of the first point-modified IL-2 in increasing the degree of depletion after cell activation, and is better. exert anti-tumor therapeutic effects.
  • the combination provided by the invention has significant synergistic enhancement of immune response and anti-tumor effects. In addition, it can also direct the fate of lymphocytes to long-term immunity in adoptive cell therapy, which has important clinical value.
  • Figure 1 Coomassie blue staining results of PEGylated IL-2 variants Y45-20K and D20-20K.
  • Figure 2 Results of surface plasmon resonance (SPR) affinity determination of PEGylated IL-2 variants for different subunits of the IL-2 receptor.
  • SPR surface plasmon resonance
  • Figure 3 Determination of phosphorylated STAT5 (pSTAT5) levels in cells after treatment with PEGylated IL-2 variants Y45-20K and D20-20K.
  • Figure 4 Results of PK/PD characterization of PEGylated IL-2 variants Y45-20K and D20-20K.
  • Figure 5 Effect of D20-20K on Y45-20K-mediated T cell proliferation and activation in vitro.
  • Figure 6 Effect of D20-20K on Y45-20K-mediated T proliferation and activation in healthy mice.
  • Figure 7 Anti-tumor effect of D20-20K combined with Y45-20K in mouse tumor model.
  • Figure 8 Effect of D20-20K combined with Y45-20K on T cell subsets in lymph nodes of tumor-bearing mice.
  • FIG. 9 Effect of D20-20K combined with 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 Effect of the combination of D20-20K and Y45-20K on senescence and exhaustion of CAR-T cells cultured in vitro.
  • Figure 12 Effect of the combination of D20-20K and Y45-20K on the aging and exhaustion of T cells at different differentiation stages in CAR-T cell culture in vitro.
  • Figure 13 Effects of none- ⁇ variant (Y45-20K) in combination with various none- ⁇ 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- ⁇ ;
  • Figure 13c Expression of apoptosis-related biomarkers CD57, IL- 10 Express the situation.
  • Figure 14 Effects of none- ⁇ variant (D20-20K) in combination with various none- ⁇ 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- ⁇ ;
  • Figure 14c Expression of apoptosis-related biomarkers CD57, IL- 10 Express the situation.
  • the mutation site shown below was selected on the IL-2 protein (SEQ ID NO:1), where the position described is the position in SEQ ID NO:1.
  • the above-mentioned nucleic acid sequence containing codon replacement is synthesized, connected to the nucleic acid sequence encoding the His tag and cloned into the pET-21a(+) (addgene: #69740-3) E. coli plasmid expression vector, which is co-transfected with the pSURAR-YAV plasmid. into the TransB(DE3) strain (purchased from Quanjin, catalog number: CD811-02).
  • the pSURAR-YAV plasmid encodes an amber codon suppressor tRNA and a NAEK-specific aminoacyl tRNA synthetase.
  • the non-natural amino acid NAEK can be introduced at a specific site; pSURAR-YAV
  • the plasmid refers to the plasmid from the General Microbiology Center of the Chinese Microbial Culture Collection Committee (Institute of Microbiology, Chinese Academy of Sciences, No. 1, Beichen West Road, Chaoyang District, Beijing), with a deposit date of April 8, 2013, and a deposit number of CGMCC No.
  • the transformed strain was inoculated into 2 ml LB medium containing 100ug/ml ampicillin and 34ug/ml chloramphenicol, and then cultured at 37°C and 220rpm. Dilute the overnight culture to optical density in 2 ⁇ YT medium and incubate the culture at 37°C until A600nm reaches approximately 1.5.
  • UAA (NAEK, self-synthesized in the laboratory) was added at a final concentration of 1mM, followed by isopropyl ⁇ -d-thiogalactopyranoside (IPTG) and L-arabinose at final concentrations of 0.5mM and 0.1%, respectively.
  • IPTG isopropyl ⁇ -d-thiogalactopyranoside
  • L-arabinose L-arabinose
  • To induce protein expression half an hour later, the temperature was lowered to 20°C.
  • cells were harvested by centrifugation and resuspended in His-Bind buffer (20mM phosphate, pH 8.0, 500mM NaCl, 20mM imidazole). Protein was extracted by passing the cells through a Micofluidizer twice at 1200 bar and cooling.
  • Example 2 PEG modification of site-directed mutagenesis of IL-2 protein
  • the supernatant obtained in Example 1 was initially purified by Ni-NTA agarose (R90101, Invitrogen), and then click reaction was performed.
  • His-tagged IL-2 in the supernatant was enriched using Ni-NTA His-Bind Resin (Invitrogen) and DBCO-PEG was synthesized and then added to the elution buffer solution (20mM phosphate, pH 8.0, 500mM NaCl, 500mM imidazole), the final concentration is 1mM.
  • the reaction was carried out at 4°C with gentle shaking for 2 hours.
  • PEGylated IL-2 was then purified by cation exchange chromatography (Resource S, GE Healthcare) and FPLC size exclusion chromatography (Superdex 200 increase 10/300 GL, GE Healthcare) to remove unreacted PEG and IL-2 .
  • the main elution peak was collected using a 3 kDa centrifugal filter unit (Millipore), concentrated and buffer exchanged into PBS buffer.
  • 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 exceeds 95%.
  • PEGylated IL-2 derivatives were also prepared by the method described in Example 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 nomenclature of the PEGylated IL-2 variants involved in this application is: [position of mutation to unnatural amino acid compared to SEQ ID NO: 1] - [average molecular weight of the attached PEG group].
  • Y45-20K the difference between its amino acid sequence and SEQ ID NO:1 is that Y45 is replaced with the unnatural amino acid NAEK, and a PEG group with an average molecular weight of 20kDa is further connected to this position.
  • Example 3 Determination of binding activity to IL-2 receptor
  • This example evaluates the affinity of PEGylated IL-2 derivatives for IL-2R trimer and dimer complexes by surface plasmon resonance (SPR).
  • SPR surface plasmon resonance
  • Human IL-2R ⁇ , IL-2R ⁇ , and IL-2R ⁇ c receptors were immobilized on CM5 or Protein A chips for analysis on a Biacore 8K system (GE Healthcare).
  • the extracellular domains of human IL-2R ⁇ (C-terminal 6xHis), IL-2R ⁇ (C-terminal Fc) and IL-2R ⁇ (C-terminal 6xHis) were purchased from Yiqiao Shenzhou.
  • Binding has a preference for non- ⁇ receptors (non- ⁇ ), which can be called non- ⁇ PEGylate; K35-20K, T37-20K, R38-20K, T41-20K, K48-20K, K49-20K are specific to ⁇
  • the binding force is on the order of 1e-7 ⁇ 1e-6, which is a weak binding, but its binding affinity to dimeric IL-2R is significantly higher than that of trimeric IL-2R, or although its binding affinity to dimeric IL-2R Weaker than trimeric IL-2R, but the gap (such as ratio) between the two is significantly smaller than the gap in the binding affinity of natural IL-2 to dimeric IL-2R and trimeric IL-2R, with significantly improved It is biased towards dimeric IL-2R, and therefore also has a bias towards non- ⁇ receptors (non- ⁇ ), and can be called non- ⁇ PEGylate.
  • Example 4 Determination of the tendency of Y45-20K and D20-20K to activate CD8+ T cells and Treg cells
  • This example determines whether PEGylated IL-2 variants Y45-20K or D20-20K preferentially activate CD8+ T cells rather than Tregs by measuring levels of phosphorylated STAT5 (pSTAT5), a key downstream mediator of IL-2R signaling. cell.
  • pSTAT5 phosphorylated STAT5
  • a magnetic separation kit (MiltenyiBiotech) was used to separate the purified cell populations according to the manufacturer's instructions to obtain memory CD4 + T cells (MPCD4), memory CD8 + T cells (MPCD8), and NK cells and Treg cells.
  • Treg cells are defined as CD3+CD8-CD4+CD25highCD127low; NK cells are defined as CD3-CD16+CD56+; CD8+T cells are defined as CD3+CD4-CD8+; MPCD4 cells are defined as CD3+CD4+CD8-CD45RO+; MPCD8 cells are defined as CD3+CD4-CD8+CD56-CD57-CD45RA-.
  • FIG. 3a The results of detection using NK-derived YT-1 cells and CD25+YT-1 cell models are shown in Figure 3a, in which Y45-20K is expressed in CD25(IL-2R ⁇ )- and CD25(IL-2R ⁇ )+YT-1 cells. showed a significant difference from wild-type IL-2; D20-20K still only showed baseline levels of Treg stimulating activity (5-20% of pSTAT5 activation) at very high doses, which was different from that of the ⁇ subunit. Cannot be combined related.
  • Example 5 Analysis of PK/PD characteristics of PEGylated IL-2 variant Y45-20K/D20-20K
  • This example examines the pharmacokinetic properties of PEGylated IL-2 variants.
  • Human IL-2 or PEGylated IL-2 variants (0.25 mg/kg based on hIL-2).
  • blood 100 ⁇ L each time was taken from the mouse orbital vein and then centrifuged at 4000 g for 15 min 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.
  • Y45-20K has the activity of preferentially activating CD8+ T cells, but it still activates Treg cells at high doses.
  • D20-20K maintains selective affinity for IL-2R ⁇ while significantly reducing activation of CD25+YT-1. This unique property may be related to its undetectable binding to IL-2R ⁇ . Therefore, If D20-20K is combined with Y45-20K for treatment, D20-20K may spatially occupy IL-2R ⁇ and competitively block its mild interaction with Y45-20K, leading to the over-activation and eventual death of CD8+ T cells. Terminal differentiation and activation of Tregs. Accordingly, this example examined the synergistic anti-tumor effect of D20-20K and Y45-20K.
  • This example examines whether D20-20K has a dose-dependent effect on the activation bias of Y45-20K. Resuspend 2 ⁇ 10 5 human PBMC cells in a 96-well plate with 100ul RPMI1640+10% FBS, add 50ul0.4ug/ml Y45-20K (final concentration 0.1ug/ml) into the experimental well, and then add 50ul different concentration gradients The final concentrations of D20-20K were 1ug/ml, 0.1ug/ml, 0.01ug/ml, and 0.001ug/ml, and the repeated experimental wells were from three different healthy individuals.
  • Treg cells were cultured at 37°C for 72 h, then washed twice with FACS buffer and stained with the following antibodies for flow cytometric analysis: anti-human CD3-APC/Cy7, CD4-PE/Cy7, CD8-FITC, CD25-APC, Foxp3-Pacific Blue, CD69-PE.
  • Treg cells are defined as CD3+CD8-CD4+CD25+Foxp3+;
  • CD8+T cells are defined as CD3+CD4-CD8+.
  • the number of Treg and CD8 T cells and their respective activation markers mean fluorescence intensity (MFI).
  • Permeabilize cells by suspending in ice-cold 87% methanol for 30 min at 4°C. Fixed and permeabilized cells were washed twice with FACS buffer and stained with pSTAT5-Pacific Blue antibody for flow cytometric analysis. 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 are plotted as background-subtracted MFI, normalized to the maximum signal for each cell type (Y45-20K, 0.1 ⁇ g ml ⁇ 1 ). Background is defined as pSTAT5MFI in unstimulated cells.
  • the dose-response curve was fit to a logistic model using GraphPad Prism data analysis software and the half-maximum effective concentration (EC50 value) and corresponding 95% were calculated Confidence interval. The experiments were performed in triplicate and repeated three times with similar results.
  • 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, b shows the effect on Treg proliferation and activation
  • Figure 5c, d are the effects on CD8+T proliferation and activation.
  • STAT5 phosphorylation in CD25+YT-1 cells was inhibited by D20-20K in a dose-dependent manner with an EC50 of 0.39ug/ml (Fig. 5e), whereas this inhibition was not observed in native YT cells (Fig. 5f).
  • mice C57BL/6 mice (6 to 8 weeks old, female) were administered subcutaneously to the back of the neck 45-20K (0.25mg/kg x 3, every other day), 20-20K (0.25mg/kg x 3, every other day) ), 45-20K and 20-20K (each 0.25mg/kg x 3, every other day), or IL-2 (0.25mg/kg per day x 5), equal volume PBS injection was the blank control group.
  • Flow cytometry of spleen and lymph nodes was performed three days after the end of dosing.
  • tumors were minced and digested in buffer containing 2 mg/ml type II and IV collagenase (GIBCO BRL) and 0.5 mg/ml DNase (Sigma Aldrich) at 37°C for 13 min to form single cell suspensions. fluid, followed by flow cytometry to identify immune cell subtypes in the tumor microenvironment. Immunohistochemical analysis of T cell density was performed on frozen tumor sections 14 days after dosing.
  • mice treated with Y45-20K/D20-20K combination increased the proportion of CD8+ T cells in the spleen, tumor-draining lymph nodes (TDLN) and especially tumor tissue compared with other mice, but There was no significant change in the proportion of Treg cells (Fig. 7b). This result was consistent with immunohistochemical staining, indicating that co-treatment of Y45-20K and D20-20K induced the largest amount of T cell infiltration in tumor tissue compared with native IL-2 and PBS treatment (Fig. 7c).
  • Analysis of T cell subsets in the lymph nodes of tumor-bearing mice showed that in the Y45-20K alone treatment group or Y45-20K combined with D20-20K treatment group, Tcm in the lymph nodes, The number of MPCD8 cells increased significantly by about 10 times, and the numbers of Tcm and MPCD8 in the Y45-20K combined with D20-20K treatment group were further improved based on the Y45-20K alone treatment group.
  • T in the two experimental groups and Tem were slightly reduced ( Figure 8a), indicating that Y45-20K can significantly expand central memory T lymphocytes in vivo.
  • D20-20K alone will not expand central memory cells, but it can be combined with Y45-20K to further increase CD8 subtypes.
  • the proportion and number of central memory cells in the group are conducive to the anti-tumor immune effect.
  • the CD25 marker expression in the Tcm cell subpopulation further decreased, and the CD122 marker expression further increased, indicating that Tcm in the lymph nodes of mice in the Y45-20K alone treatment group or Y45-20K combined with D20-20K treatment group can avoid Endogenous IL-2 overactivates T cells, reducing their sensitivity to IL-2 and improving their ability to respond to foreign antigens again.
  • Y45-20K treatment group alone can significantly activate the proliferation of amplified Tcm, MPCD8, T
  • the LAG-3 and PD-1 markers in the Tem cell subpopulation were significantly increased compared with the IL-2 treatment group.
  • the combination of D20-20K can reduce the expression of immune checkpoints in the above cell subpopulations, indicating that the D20-20K combination
  • the use of Y45-20K can retain the central memory cell activation advantage of Y45-20K, and can also compensate for the disadvantage of Y45-20K in increasing the degree of depletion of cells after activation, and better exert the anti-tumor therapeutic effect.
  • VLS vascular leak syndrome
  • VLS vascular leak syndrome
  • Y45-20K displays significant synergy with D20-20K in anti-tumor therapy, which selectively induces CD8+ T cells, has minimal impact on Treg cells, and is able to protect lung endothelial cells from Y45-20K or activation of endogenous IL-2 to alleviate VLS, with significant beneficial 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 CAR-T cell expansion and culture in vitro. Because it inevitably activates Treg cells in donor cells and inevitably overactivates CD8+ T cells and causes their terminal differentiation, it affects CAR-T cells. T cell activity significantly weakens the therapeutic effect of CAR-T cell therapy. According to this experiment, Y45-20K combined with D20-20K replaced traditional IL-2 on CAR-T cell therapy.
  • Anti-CD19 CAR contains FMC63anti-CD19scFv (SEQ ID NO:12), CD8a hinge region (SEQ ID NO:14) and transmembrane region (SEQ ID NO:16), and 4-1BB cytoplasmic domain (SEQ ID NO:18) and CD3z cytoplasmic domain (SEQ ID NO:20), whose expression cassette was synthesized and cloned into a lentiviral vector by Iconde Biotechnology (Suzhou) Co., Ltd.
  • HEK293T cells were transfected with anti-CD19CAR, pspax2 (addgene, #12260) and pMD2.g (addgene, #12259) plasmids using Lipofectamine 3000 (Life Technologies). The culture medium was replaced 6 hours after transfection, and the viral supernatant was collected 48 hours after transfection. Viral particles were concentrated 30-fold by ultracentrifugation at 25,000 rpm for 2 hours and frozen at -80°C until ready for use.
  • 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 h prior to infection.
  • Concentrated lentivirus FMC63-AntiCD19-CAR Lentivirus
  • the CAR-T prepared above were cultured in their corresponding IL-2 for 10-14 days, and related detection of cell phenotype was performed.
  • Surface antibodies use fluorescently 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) for fluorescent staining and incubation at 4°C for 30 minutes.
  • CAR-T cells were fixed and permeabilized using Foxp3/transcription factor staining buffer (eBioscience) and then stained with Foxp3 antibody in perm buffer.
  • Human T cells were purified from peripheral blood mononuclear cells using Dynabead Human T Cell Kit (Life Technologies), and CAR-T was prepared using the above method.
  • CAR-T was prepared using the above method.
  • T Cells are defined as CD3+CD8+CD45RA+CD45RO-CCR7+CD62L-CD95-; Tscm is defined as CD3+CD8+CD45RA+CD45RO-CCR7+CD62L-CD95+; Tcm is defined as CD3+CD8+CD45RA-CD45RO+CCR7+CD62L+; Tem is defined as CD3+CD8+CD45RA-CD45RO+CCR7-CD62L-; T effector is defined as CD3+CD8+CD45RA+CD45RO0-CCR7-CD62L-; Treg is defined as CD3+CD4+CD25 high CD127 low . And cultured with different cytokines Expand for 10-14 days, and monitor cell number and growth status in real time.
  • T cells T memory stem cells
  • T effector T effector
  • Treg regulatory T cells
  • CD62L is a characteristic marker of cell stemness.
  • the combination of D20-20K and Y45-20K can further improve T and the expression of CD62L in Tscm cells ( Figure 12a), indicating that the combination of D20-20K and Y45-20K can further preserve the stemness state of T cells.
  • the results of testing the proliferation of T cells at different differentiation stages show that the combination of D20-20K and Y45-20K can further enhance T cell proliferation.
  • PD-1 and TIM-3 are markers of T cell exhaustion. Testing of T effector cells in a terminally differentiated state shows that the combination of D20-20K and Y45-20K can further reduce the expression of PD-1 and TIM-3. , reducing the degree of exhaustion of T effector cells (Fig. 12c).
  • CD25 expression on T cells with 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 compared with Y45 -20K alone further reduced the expression of CD25 on Treg cells ( Figure 12d).
  • the reduced expression of CD25 can avoid the excessive activation of T cells by endogenous IL-2 produced by T cell activation, showing that D20-20K is closely related to The superiority of the Y45-20K joint.
  • Example 8 Effect of the combination of None- ⁇ variant and None- ⁇ variant on maintaining T cell stemness and reducing T cell exhaustion
  • This example compares the Non- ⁇ variants (F42-20K, Y45-20K, E62-20K, P65-20K, E68-20K) and Non- ⁇ variants (D20-20K, H16) prepared in Example 1-2. -20K, A73-20K, H79-20K) combination for in vitro activity verification.
  • the density is controlled at 1x10 6 /ml, 24-well plate, 1ml per well, and cultured with cytokines containing different types but with the same concentration (100ng/ml), the medium is changed every 72 hours, and flow cytometry is performed after two weeks of continuous culture. detection.
  • Detection antibodies include: APC/Cy7 anti-human CD3, PE/Cy7 anti-human CD4, FITC anti-human CD8, BV785 anti-human PD-1, BV605 anti-human TIM-3, Pacific Blue anti-human CD57, APC anti-human IL-10, PE anti-human Perforin, Percp/cy5.5 anti-human Granzyme B, BV510 anti-human IFN- ⁇ .
  • the combination of None- ⁇ and None- ⁇ will further improve the survival status of T cells based on the synergistic effect of none- ⁇ , such as reducing the degree of T cell growth exhaustion, reducing apoptosis induced by over-activation, and delaying Differentiation into terminal effector cells.

Abstract

提供具备不同受体偏向性的PEG化IL-2变体的组合。该组合通过协同作用抑制免疫抑制淋巴细胞扩增并减少效应淋巴细胞的终末分化和耗竭，具备协同增效的抗肿瘤作用，从而可用于增强免疫应答、治疗增生性疾病如肿瘤。此外，该组合还能够用于过继细胞免疫治疗中的免疫细胞的体外培养，减少免疫细胞的衰老和耗竭，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。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。

Description

受体偏向的PEG化IL-2变体组合及其应用 技术领域
本发明涉及免疫治疗领域，具体地，本发明涉及定点修饰的PEG化IL-2变体，以提供具备不同受体偏向性的PEG化IL-2变体的组合。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。
背景技术
白细胞介素-2(IL2)是一类重要的细胞因子，是B细胞、T细胞及NK细胞发育和功能所必须的细胞因子。IL2的受体分为IL2-Rα单体，IL2-Rβγ二聚体和IL2-Rαβγ三聚体，其中IL2与α单体亲和力较弱为10 -8M，与βγ二聚体亲和力中等为10 -9M，与αβγ三聚体亲和力最高为10 -11M。由于亲和力低，二聚体IL-2R或内源性IL-2都需要以高水平诱导才能产生“二聚体IL-2R:IL-2”反应性，这与三聚体IL-2R相反，其允许宿主细胞直接对低浓度的IL-2产生反应。因此，目前认为三聚体IL-2R的α链不参与信号传导，而是启动与IL-2的结合(KD≈10-8M)，以及随后与βγ复合物的亲和力增加1000倍的结合。
IL2的受体在不同免疫细胞上的分布也存在差异：IL2-Rαβγ三聚体长期表达于调节性T细胞(Treg)表面，因此对IL2亲和力最高；IL2-Rβγ二聚体表达在静息的效应T细胞(Teff)、杀伤性T细胞(CTL)及NK细胞表面，对IL2亲和力一般，只有在效应T细胞及NK细胞激活以后，才会表达IL2-Rαβγ三聚体，对IL2亲和力增强。已有证据表明，在CD8+T细胞启动期间，α链的表达是相当动态的——一部分T细胞上调α链表达并感知强烈的IL-2信号，导致T细胞增殖更快但最终分化，这反过来又限制了它们的有效性。相比之下，对IL-2不太敏感的低α的T细胞优先上调CD127和CD62L，从而产生功能性长寿命记忆细胞。在不改变α链上调的基本过程的情况下，在体内拥有更多的记忆T细胞群变得有价值，但对临床应用具有挑战性。因此在肿瘤治疗中，对Teff/Treg，CTL/Treg的失衡比例调节目前逐渐成为免疫治疗的一个新的研发方向。
目前上市的IL2靶点相关药物均无受体选择性。在用于肿瘤的治疗中，当需要激活Teff及CTL时需要较高剂量IL2，由此也带来严重的系统毒性，是目前副作用的主要来 源。此外，IL2与IL2-Rα在肺内皮细胞的相互作用还会诱导血管渗漏综合征，激活Treg细胞导致的免疫抑制也会使得药物应答受限。
因此，解决现有IL2抗肿瘤药物对受体没有选择性的问题对于基于IL2的肿瘤免疫疗法的临床应用至关重要。
发明内容
如上所述的，虽然IL-2已被广泛用于增强机体免疫细胞，然而促进免疫抑制淋巴细胞扩增以及驱动效应淋巴细胞的终末分化和耗竭的附带特性阻碍了IL-2作为抗肿瘤剂的有效性。
发明人在此报告了一种策略，该策略通过两类受体偏向的PEG化IL-2变体来探索IL-2受体的动态表达模式，使得启动淋巴细胞的命运具有持久抗肿瘤免疫特性。具体地，第一类受体偏向的PEG化IL-2变体为non-α PEGylate，它可以激活和扩增CD8+T细胞、CD4+细胞甚至NK细胞，而不是Treg细胞，这归因于其对二聚体而不是三聚体IL-2R的偏向亲和力，此外它还诱导减少的CD8+细胞的终末分化和耗竭。第二类受体偏向的PEG化IL-2变体为non-β PEGylate，其既不激活CD8+T也不激活Treg细胞，但它与第一类non-α PEGylate协同作用以进一步扩增CD4+和CD8+T细胞并减少Treg以及分化和耗尽的CD8+T细胞群，这归因于其保持对三聚体IL-2R的a链的中等亲和力，表现出优异的局部和系统性抗肿瘤反应。
发明人随后在靶向CD19的CAR-T治疗模型中证实了上述第一类受体偏向的PEG化IL-2变体(non-α PEGylate)和第二类受体偏向的PEG化IL-2变体(non-β PEGylate)通过协同作用将淋巴细胞的命运导向长期免疫。上述实验证据支持一种推定的机制，通过协同受体偏向的PEG化IL-2变体使得IL-2:IL-2R相互作用产生顺序改变，这不仅规避了IL-2的多效性作用，而且优化了T细胞的差异记忆程序，为T淋巴细胞和过继性T细胞转移疗法中的下一代IL-2指明了方向。
基于上述第一类受体偏向的PEG化IL-2变体(non-α PEGylate)和第二类受体偏向的PEG化IL-2变体(non-β PEGylate)，本发明提供了具备不同受体偏向性的PEG化IL-2变体的组合。该组合通过协同作用抑制免疫抑制淋巴细胞扩增并减少效应淋巴细胞的终末分化和耗竭，提高中央记忆细胞的比例和数量，具备协同增效的抗肿瘤作用，从而可用于增强免疫应答、治疗增生性疾病如肿瘤。此外，该组合还能够用于过继细胞免疫治疗中的免疫细胞的体外培养，减少免疫细胞的衰老和耗竭，增强T
Figure PCTCN2022129975-appb-000001
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。
组合物
第一方面，本发明提供了组合物，其包含：
(1)第一定点修饰的IL-2，其与野生型IL-2相比在第一氨基酸位置的残基包含PEG基团修饰，其中，所述第一定点修饰的IL-2对IL-2受体α(IL-2Rα)亚基不结合或以大于1E-8M(例如数量级为1E-7～1E-6M)的KD值结合；以及
(2)第二定点修饰的IL-2，其与野生型IL-2相比在第二氨基酸位置的残基包含PEG基团修饰，其中，所述第二定点修饰的IL-2对IL-2受体β(IL-2Rβ)亚基不结合。
本领域技术人员理解，包含第一定点修饰的IL-2和第二定点修饰的IL-2的组合物并不意味着两者必须同时给予和/或配制用于一起递送，尽管这些递送方法在此处描述的范围。在一些实施方案中，第一定点修饰的IL-2和第二定点修饰的IL-2可以在单一制剂中一起施用。在一些实施方案中，第一定点修饰的IL-2和第二定点修饰的IL-2可以在不同制剂中分开施用。组合中的第一定点修饰的IL-2可以与第二定点修饰的IL-2可以任何顺序施用，例如同时、之前或之后施用。
在某些实施方案中，所述组合物包含多个组合物或剂型。在某些实施方案中，所述第一定点修饰的IL-2和第二定点修饰的IL-2在分开的组合物或剂型中。
在某些实施方案中，所述组合物包含一个组合物或剂型。在某些实施方案中，所述第一定点修饰的IL-2和第二定点修饰的IL-2在相同的组合物或剂型中。
在某些实施方案中，所述野生型IL-2具有SEQ ID NO:1所示的氨基酸序列。在某些实施方案中，本文中所述的IL-2相关的氨基酸位置是SEQ ID NO:1中的位置。
在某些实施方案中，本文中所述的“不结合”是指通过表面等离子共振技术(SPR)未检测到结合。在某些实施方案中，本文中所述的KD值通过表面等离子共振技术(SPR)测定。
第一定点修饰的IL-2的受体偏向性
本文所述的第一定点修饰的IL-2具有对非α受体(non-α)的偏向性。
在某些实施方案中，所述对非α受体(non-α)的偏向性包括：(i)以小于1E-8M(例如数量级为1E-9～1E-8M)的KD值结合IL2-Rβγ二聚体，和/或，(ii)对IL2-Rαβγ三聚体不结合或以大于1E-8(例如数量级为1E-7～1E-6)的KD值结合。
I.对IL-2Rα的结合特性
在某些实施方案中，所述第一定点修饰的IL-2对IL-2受体α(IL-2Rα)亚基不结合。在某些实施方案中，此类第一定点修饰的IL-2的第一氨基酸位置选自F42、Y45、E62、K64、P65、E68。
在某些实施方案中，所述第一定点修饰的IL-2对IL-2受体α(IL-2Rα)亚基仅具备低亲和力，例如以大于1E-8M(例如大于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或更大)的KD值结合IL-2受体α(IL-2Rα)亚基。在某些实施方案中，所述第一定点修饰的IL-2以数量级为1E-7～1E-6M的KD值结合IL-2受体α(IL-2Rα)亚基。在某些实施方案中，此类第一定点修饰的IL-2的第一氨基酸位置选自K35、T37、R38、T41、K48、K49。
II.对IL2-Rβγ二聚体的结合特性
在某些实施方案中，所述第一定点修饰的IL-2以小于1E-8M(例如小于2E-8M、3E-8M、4E-8M、5E-8M、6E-8M、7E-8M、8E-8M、9E-8M、1E-9M或更小)的KD值结合IL2-Rβγ二聚体。在某些实施方案中，所述第一定点修饰的IL-2以数量级为1E-9～1E-8M的KD值结合IL2-Rβγ二聚体。在某些实施方案中，此类第一定点修饰的IL-2的第一氨基酸位置选自F42、Y45、E62、K64、P65、E68、K35、T37、R38、T41、K48、K49。
III.对IL2-Rαβγ三聚体的结合特性
在某些实施方案中，所述第一定点修饰的IL-2对IL2-Rαβγ三聚体不结合。在某些实施方案中，此类第一定点修饰的IL-2的第一氨基酸位置选自F42、Y45、E62、K64、P65、E68。
在某些实施方案中，所述第一定点修饰的IL-2对IL2-Rαβγ三聚体仅具备低亲和力，例如以大于1E-8M(例如大于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或更大)的KD值结合IL2-Rαβγ三聚体。在某些实施方案中，所述第一定点修饰的IL-2以数量级为1E-7～1E-6M的KD值结合IL2-Rαβγ三聚 体。在某些实施方案中，此类第一定点修饰的IL-2的第一氨基酸位置选自K35、T37、R38、T41、K48、K49。
在某些实施方案中，当所述第一定点修饰的IL-2对IL2-Rαβγ三聚体具备低亲和力时，所述第一定点修饰的IL-2对IL2-Rβγ二聚体的第一亲和力高于对IL2-Rαβγ三聚体的第二亲和力(即，结合IL2-Rβγ二聚体的第一KD值低于结合IL2-Rαβγ三聚体的第二KD值)。在某些实施方案中，所述第一定点修饰的IL-2对IL2-Rβγ二聚体的第一亲和力是对IL2-Rαβγ三聚体的第二亲和力的至少2倍、3倍、4倍、5倍、6倍、7倍、8倍、9倍或10倍。在某些实施方案中，此类第一定点修饰的IL-2的第一氨基酸位置选自K35、T37、T41、K48。
在某些实施方案中，当所述第一定点修饰的IL-2对IL2-Rαβγ三聚体具备低亲和力时，所述第一定点修饰的IL-2对IL2-Rβγ二聚体的第一亲和力也可以不高于(例如低于)对IL2-Rαβγ三聚体的第二亲和力(即，结合IL2-Rβγ二聚体的第一KD值不低于(例如高于)结合IL2-Rαβγ三聚体的第二KD值)，但是所述第一定点修饰的IL-2对IL2-Rβγ二聚体和IL2-Rαβγ三聚体的亲和力的差距(例如两者KD值的比值)小于天然IL-2对IL2-Rβγ二聚体和IL2-Rαβγ三聚体的亲和力的差距(例如两者KD值的比值)。在此类实施方案中，所述第一定点修饰的IL-2结合IL2-Rβγ二聚体的第一KD值不高于结合IL2-Rαβγ三聚体的第二KD值的10倍，例如不高于9倍，不高于8倍，不高于7倍，不高于6倍，不高于5倍，不高于4倍，不高于3倍或不高于2倍。在某些实施方案中，所述第一定点修饰的IL-2结合IL2-Rβγ二聚体的第一KD值为结合IL2-Rαβγ三聚体的第二KD值的8倍～3倍，例如7倍～3倍。在某些实施方案中，天然IL-2以数量级为1E-9M的第一KD值结合IL2-Rβγ二聚体。在某些实施方案中，天然IL-2以数量级为1E-11M的第二KD值结合IL2-Rαβγ三聚体。在某些实施方案中，天然IL-2结合IL2-Rβγ二聚体的第一KD值为结合IL2-Rαβγ三聚体的第二KD值的约40倍。在某些实施方案中，此类第一定点修饰的IL-2的第一氨基酸位置选自R38、K49。
第二定点修饰的IL-2的受体偏向性
本文所述的第二定点修饰的IL-2具有对非β受体(non-β)的偏向性。
在某些实施方案中，所述对非β受体(non-β)的偏向性包括：(i)以小于1E-8(例如数量级为1E-9～1E-8)的KD值结合IL2-Rαβγ三聚体，和/或，(ii)对IL2-Rβγ二聚体不结合。
在某些实施方案中，所述第二定点修饰的IL-2对IL-2受体β(IL-2Rβ)亚基不结合。
在某些实施方案中，所述第二定点修饰的IL-2以小于1E-8M(例如小于2E-8M、3E-8M、4E-8M、5E-8M、6E-8M、7E-8M、8E-8M、9E-8M、1E-9M或更小)的KD值结合IL2-Rαβγ三聚体。在某些实施方案中，所述第二定点修饰的IL-2以数量级为1E-9～1E-8M的KD值结合IL2-Rαβγ三聚体。
在某些实施方案中，所述第二定点修饰的IL-2对IL2-Rβγ二聚体不结合。
在某些实施方案中，所述第一定点修饰的IL-2的第一氨基酸位置选自F42、Y45、E62、K64、P65、E68、K35、T37、R38、T41、K48、K49。在某些实施方案中，所述第一氨基酸位置选自F42、Y45、E62、K64、P65、E68。在某些实施方案中，所述第一氨基酸位置为F42、Y45、E62、P65或E68。
在某些实施方案中，所述第二定点修饰的IL-2的第二氨基酸位置选自H16、D20、A73、H79。在某些实施方案中，所述第二定点修饰的IL-2的第二氨基酸位置选自D20。在某些实施方案中，所述第一氨基酸位置为F42、Y45、E62、P65或E68，所述第二氨基酸位置为D20。
在某些实施方案中，所述第一氨基酸位置为Y45，所述第二氨基酸位置为D20-20K、H16-20K、A73-20K、H79-20K。在某些实施方案中，所述第一氨基酸位置为Y45，所述第二氨基酸位置为D20。
在某些实施方案中，所述第一定点修饰的IL-2包含的PEG修饰基团的平均分子量为5～60kDa，例如为5kDa、10kDa、15kDa、20kDa、25kDa、30kDa、35kDa、40kDa、45kDa、50kDa或60kDa。在某些实施方案中，所述第一定点修饰的IL-2包含的PEG修饰基团的平均分子量为5～40kDa，例如为5～30kDa、5～25kDa、5～20kDa、10～40kDa、10～30kDa、15～30kDa、10～25kDa或15～25kDa，例如为5kDa、10kDa、15kDa、20kDa、25kDa、30kDa或40kDa。在某些实施方案中，所述第一定点修饰的IL-2包含的PEG修饰基团的平均分子量为5kDa、10kDa或20kDa。在某些实施方案中，所述第一定点修饰的IL-2包含的PEG修饰基团的平均分子量为20kDa。
在某些实施方案中，所述第二定点修饰的IL-2包含的PEG修饰基团的平均分子量为5～60kDa，例如为5kDa、10kDa、15kDa、20kDa、25kDa、30kDa、35kDa、 40kDa、45kDa、50kDa或60kDa。在某些实施方案中，所述第二定点修饰的IL-2包含的PEG修饰基团的平均分子量为5～40kDa，例如为5～30kDa、5～25kDa、5～20kDa、10～40kDa、10～30kDa、15～30kDa、10～25kDa或15～25kDa，例如为5kDa、10kDa、15kDa、20kDa、25kDa、30kDa或40kDa。在某些实施方案中，所述第二定点修饰的IL-2包含的PEG修饰基团的平均分子量为5kDa、10kDa或20kDa。在某些实施方案中，所述第二定点修饰的IL-2包含的PEG修饰基团的平均分子量为20kDa。
在某些实施方案中，所述第一定点修饰的IL-2包含的PEG修饰基团和所述第二定点修饰的IL-2包含的PEG修饰基团具备基本相同的平均分子量。
在某些实施方案中，所述第一定点修饰的IL-2与野生型IL-2(例如SEQ ID NO:1)相比在第一氨基酸位置(如氨基酸位置F42、Y45、E62、P65或E68)的残基被突变为非天然氨基酸，所述非天然氨基酸上连接有所述PEG基团。
在某些实施方案中，所述第二定点修饰的IL-2与野生型IL-2(例如SEQ ID NO:1)相比在第二氨基酸位置(如氨基酸位置D20、H16、A73或H79)的残基被突变为非天然氨基酸，所述非天然氨基酸上连接有所述PEG基团。
在某些实施方案中，所述非天然氨基酸含有化学官能基团，例如，羰基、炔基、和叠氮基团等，这些基团一般能够有效且选择性地形成稳定的共价键；所述PEG基团包含能与该化学官能基团发生化学反应以形成共价键的标记基团，从而所述PEG基团连接至非天然氨基酸。
在某些实施方案中，所述非天然氨基酸含有叠氮基团，所述PEG基团包含能与叠氮基团发生click化学反应的标记基团，从而所述PEG基团连接至非天然氨基酸。
在某些实施方案中，所述非天然氨基酸为含有叠氮基团的赖氨酸衍生物。在某些实施方案中，所述非天然氨基酸为Nε-2-叠氮乙氧羰基-L-赖氨酸(NAEK)。
在某些实施方案中，所述非天然氨基酸为含有叠氮基团的酪氨酸衍生物。在某些实施方案中，所述非天然氨基酸为2-amino-3-(4-(azidomethyl)phenyl)propanoic acid。
在某些实施方案中，所述能与叠氮基团发生click化学反应的标记基团为包含二苯并环辛炔基团的化学部分，例如dibenzocyclooctyne(DBCO)、4-dibenzocyclooctynol(DIBO)或BCN(bicyclo[6.1.0]nonyne)。
在某些实施方案中，所述能与叠氮基团发生click化学反应的标记基团为DBCO，所述第一定点修饰的IL-2与野生型IL-2相比在第一氨基酸位置(如氨基酸位置F42、Y45、E62、P65或E68)的残基以及所述第二定点修饰的IL-2与野生型IL-2相比在第 二氨基酸位置(如氨基酸位置D20、H16、A73或H79)的残基被式Ia的结构替代：
Figure PCTCN2022129975-appb-000002
其中，由R1到R2的方向为氨基酸序列的N末端到C末端方向，其中第N位的氨基酸为所述第一氨基酸位置的残基(如第F42、Y45、E62、P65或E68位氨基酸)或第二氨基酸位置的残基(如第D20、H16、A73或H79位氨基酸)，R1为IL-2氨基酸序列的第1至第N-1位氨基酸残基，R2为IL-2氨基酸序列的第N+1位至C末端的氨基酸残基，R3为PEG基团。
在某些实施方案中，所述能与叠氮基团发生click化学反应的标记基团为DIBO，所述第一定点修饰的IL-2与野生型IL-2相比在第一氨基酸位置(如氨基酸位置F42、Y45、E62、P65或E68)的残基以及所述第二定点修饰的IL-2与野生型IL-2相比在第二氨基酸位置(如氨基酸位置D20、H16、A73或H79)的残基被式Ib的结构替代：
Figure PCTCN2022129975-appb-000003
其中，由R1到R2的方向为氨基酸序列的N末端到C末端方向，其中第N位的氨基酸为所述第一氨基酸位置的残基(如第F42、Y45、E62、P65或E68位氨基酸)或第二氨基酸位置的残基(如第D20、H16、A73或H79位氨基酸)，R1为IL-2氨基酸序列的第1至第N-1位氨基酸残基，R2为IL-2氨基酸序列的第N+1位至C末端的氨基酸残基，R3为PEG基团。
在某些实施方案中，所述能与叠氮基团发生click化学反应的标记基团为BCN，所述第一定点修饰的IL-2与野生型IL-2相比在第一氨基酸位置(如氨基酸位置F42、Y45、E62、P65或E68)的残基以及所述第二定点修饰的IL-2与野生型IL-2相比在第二氨基酸位置(如氨基酸位置D20、H16、A73或H79)的残基被式Ic的结构替代：
Figure PCTCN2022129975-appb-000004
其中，由R1到R2的方向为氨基酸序列的N末端到C末端方向，其中第N位的氨基酸为所述第一氨基酸位置的残基(如第F42、Y45、E62、P65或E68位氨基酸)或第二氨基酸位置的残基(如第D20、H16、A73或H79位氨基酸)，R1为IL-2氨基酸序列的第1至第N-1位氨基酸残基，R2为IL-2氨基酸序列的第N+1位至C末端的氨基酸残基，R3为PEG基团。
在某些实施方案中，本申请中所涉及的PEG化IL-2变体的命名方式为：[与SEQ ID NO:1相比突变为非天然氨基酸的位置]-[所连接的PEG基团的平均分子量]。
在某些示例性实施方案中，所述第一定点修饰的IL-2选自F42-20K、Y45-20K、E62-20K、P65-20K或E68-20K。在某些示例性实施方案中，所述第二定点修饰的IL-2为D20-20K、H16-20K、A73-20K或H79-20K。以Y45-20K为例，其是指以下PEG化IL-2变体：该变体的氨基酸序列与SEQ ID NO:1相比差异在于Y45被置换为非天然氨基酸NAEK，并且该位置进一步连接平均分子量为20kDa的PEG基团。
组合物的制备
本发明第一方面所提供的组合物中包含的第一定点修饰的IL-2和第二定点修饰的IL-2可以通过本领域已知的任何方法制备。
在某些实施方案中，可以通过非天然氨基酸正交翻译技术定点插入非天然氨基酸，随后将PEG基团与该非天然氨基酸连接。
非天然氨基酸正交翻译技术是本领域技术人员熟知的。非天然氨基酸正交翻译技术利用终止密码子在蛋白翻译过程中将非天然氨基酸插入到蛋白质的氨基酸序列中，实际上扩展了氨基酸密码子的数量，因此非天然氨基酸正交翻译技术也被称为遗传密码子扩展技术。典型地，非天然氨基酸正交翻译系统涉及tRNA、氨酰基tRNA合成酶以及具有一个或多个终止密码子的目的核酸序列。将上述系统引入宿主细胞，并在含有适当营养素以及待插入的一种或多种非天然氨基酸的培养基中培养。然后将宿主细胞维持在允许目的蛋白质表达的条件下。响应于非天然密码子，将一个或多个非天然氨基酸掺入多肽链中。
在某些实施方案中，可以通过非天然氨基酸正交翻译技术定点插入非天然氨基酸，所述非天然氨基酸可以含有化学官能基团，例如，羰基、炔基、叠氮基团等，这些基团一般能够有效且选择性地形成稳定的共价键，随后通过化学反应形成共价键来定点修饰PEG基团。
在某些实施方案中，可以通过非天然氨基酸正交翻译技术定点插入非天然氨基酸，随后通过点击化学反应来定点修饰PEG基团。在某些实施方案中，所述非天然氨基酸和PEG基团分别包含能够发生点击化学反应的化学基团。
第二方面，本发明提供了制备第一方面所述的组合物的方法，其包括制备所述第一定点修饰的IL-2和制备所述第二定点修饰的IL-2，其中，
制备所述第一定点修饰的IL-2包括：
-提供：(a1)第一定点突变的IL-2，其与野生型IL-2相比在第一氨基酸位置(如氨基酸位置F42、Y45、E62、P65或E68)的残基被突变为非天然氨基酸；(b1)由标记基团修饰的PEG基团，所述标记基团能与所述非天然氨基酸形成共价键；
-将(a1)与(b1)共孵育，通过化学反应将非天然氨基酸与PEG基团偶联；
制备所述第二定点修饰的IL-2包括：
-提供：(a2)第二定点突变的IL-2，其与野生型IL-2相比在第二氨基酸位置(如氨基酸位置D20、H16、A73或H79)的残基被突变为非天然氨基酸；(b2)由标记基团修饰的PEG基团，所述标记基团能与所述非天然氨基酸形成共价键；
-将(a2)与(b2)共孵育，通过化学反应将非天然氨基酸与PEG基团偶联。
在某些实施方案中，所述第一定点突变的IL-2包含的非天然氨基酸含有化学官能基团，例如，羰基、炔基、叠氮基团等，这些基团一般能够有效且选择性地形成稳定的共价键；所述PEG基团包含的标记基团能与该化学官能团发生化学反应形成共价键。
在某些实施方案中，所述第二定点突变的IL-2包含的非天然氨基酸含有化学官能基团，例如，羰基、炔基、叠氮基团等，这些基团一般能够有效且选择性地形成稳定的共价键；所述PEG基团包含的标记基团能与该化学官能团发生化学反应形成共价键。
在某些实施方案中，所述第一定点突变的IL-2和第二定点突变的IL-2中的非天然氨基酸包含相同的化学官能基团。
在某些实施方案中，所述第一定点突变的IL-2和第二定点突变的IL-2中的非天然氨基酸包含叠氮基团。
在某些实施方案中，所述第一定点突变的IL-2和第二定点突变的IL-2中的非天然氨 基酸相同。
在某些实施方案中，所述非天然氨基酸为含有叠氮基团的赖氨酸衍生物。在某些实施方案中，所述非天然氨基酸为Nε-2-叠氮乙氧羰基-L-赖氨酸(NAEK)。
在某些实施方案中，所述非天然氨基酸为含有叠氮基团的酪氨酸衍生物。在某些实施方案中，所述非天然氨基酸为2-amino-3-(4-(azidomethyl)phenyl)propanoic acid。
在某些实施方案中，制备所述第一定点修饰的IL-2包括：
-提供：(a1)第一定点突变的IL-2，其与野生型IL-2相比在第一氨基酸位置(如氨基酸位置F42、Y45、E62、P65或E68)的残基被突变为含有叠氮基团的非天然氨基酸；(b1)由标记基团修饰的PEG基团，所述标记基团能与叠氮基团发生click化学反应；
-将(a1)与(b1)共孵育，通过click反应将非天然氨基酸与PEG基团偶联。
在某些实施方案中，制备所述第二定点修饰的IL-2包括：
-提供：(a2)第二定点突变的IL-2，其与野生型IL-2相比在第二氨基酸位置(如氨基酸位置D20、H16、A73或H79)的残基被突变为含有叠氮基团的非天然氨基酸；(b2)由标记基团修饰的PEG基团，所述标记基团能与叠氮基团发生click化学反应；
-将(a2)与(b2)共孵育，通过click反应将非天然氨基酸与PEG基团偶联。
在某些实施方案中，所述click化学反应为无铜的click化学反应。无铜click化学反应是通过引入环辛炔来维持细胞活性而实现的click反应，其中八元环的张力允许在没有催化剂的条件下与叠氮发生反应。这些试剂中的一种由所谓的DBCO化合物组成。叠氮修饰的大分子现在可以在没有金属催化剂的条件下进行标记，这样不仅可用于活细胞的研究，而且阻止了对蛋白的损伤。
在某些实施方案中，所述能与叠氮基团发生click化学反应的标记基团为包含炔基的化学部分。在某些实施方案中，所述能与叠氮基团发生click化学反应的标记基团为包含二苯并环辛炔基团的化学部分。在某些实施方案中，所述能与叠氮基团发生click化学反应的标记基团为dibenzocyclooctyne(DBCO)、4-dibenzocyclooctynol(DIBO)或BCN(bicyclo[6.1.0]nonyne)。在某些实施方案中，所述能与叠氮基团发生click化学反应的标记基团为DBCO。
在某些实施方案中，DBCO标记的PEG基团具有式IIa所示的结构，其中R3为PEG基团。
Figure PCTCN2022129975-appb-000005
在某些实施方案中，DIBO标记的PEG基团具有式IIb所示的结构，其中R3为PEG基团。
Figure PCTCN2022129975-appb-000006
在某些实施方案中，通过非天然氨基酸正交翻译技术提供所述第一定点突变的IL-2和所述第二定点突变的IL-2。
在某些实施方案中，所述非天然氨基酸正交翻译技术包括以下步骤：
-获得编码定点突变的IL-2的核酸序列，其中，待突变氨基酸位置对应的密码子被突变为TAG；
-将所述编码定点突变的IL-2的核酸序列与载体可操作地连接，得到定点突变序列表达载体；
-将所述定点突变序列表达载体与编码琥珀密码子抑制型tRNA和对非天然氨基酸特异性的氨酰tRNA合成酶的载体共转染宿主细胞，在含有非天然氨基酸的培养基中培养并诱导表达，得到定点突变为非天然氨基酸的IL-2。
在某些实施方案中，所述非天然氨基酸为Nε-2-叠氮乙氧羰基-L-赖氨酸(NAEK)。在某些实施方案中，所述对非天然氨基酸特异性的氨酰tRNA合成酶为NAEK特异性氨酰tRNA合成酶。
在某些实施方案中，所述编码琥珀密码子抑制型tRNA和对非天然氨基酸特异性的氨酰tRNA合成酶的载体为pSURAR-YAV(也称为pSUPAR-YAV-tRNA/PylRS)，其从含有质粒pSUPAR-YAV-tRNA/PylRS的大肠埃希氏菌中获取，该菌株保藏在中国微生物菌种保藏委员会普通微生物中心(北京市朝阳区北辰西路1号院，中国科学院微生物研究所)，保藏日期为2013年4月8日，保藏号为CGMCC No：7432，分类命名为大肠 埃希氏菌。
在某些实施方案中，所述第一定点突变的IL-2与野生型IL-2相比在第一氨基酸位置(如氨基酸位置F42、Y45、E62、P65或E68)的残基以及所述第二定点突变的IL-2与野生型IL-2相比在第二氨基酸位置(如氨基酸位置D20、H16、A73或H79)的残基被式III的结构替代：
Figure PCTCN2022129975-appb-000007
由R1到R2的方向为氨基酸序列的N末端到C末端方向，其中第N位的氨基酸为所述第一氨基酸位置的残基(如第F42、Y45、E62、P65或E68位氨基酸)或第二氨基酸位置的残基(如第D20、H16、A73或H79位氨基酸)，R1为IL-2氨基酸序列的第1至第N-1位氨基酸残基，R2为IL-2氨基酸序列的第N+1位至C末端的氨基酸残基。
试剂盒
本发明提供的第一定点修饰的IL-2和第二定点修饰的IL-2的组合可以在免疫细胞的体外培养中减少免疫细胞的衰老和耗竭，增强T
Figure PCTCN2022129975-appb-000008
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。
第三方面，本发明提供了试剂盒，其包含第一方面所述的组合物。在某些实施方案中，所述试剂盒进一步包含包装说明书，所述包装说明书包含使用所述组合物来在体外制备和/或培养用于过继性细胞治疗的免疫细胞的说明。本发明还涉及第一方面所述的组合物或第三方面所述的试剂盒用于体外制备或培养用于过继性细胞治疗的免疫细胞的用途。
在本文中，过继性细胞治疗(adoptive cell therapy)可以包括肿瘤浸润T细胞(TIL)疗法、嵌合抗原受体T细胞疗法(CAR-T)、T细胞受体疗法(TCR)、NK细胞疗法等。在本文中，所述用于过继性细胞治疗的经改造的免疫细胞可以本领域已知用于过继性细胞疗法的任意细胞。在某些实施方案中，所述用于过继性细胞治疗的经改造的免疫细胞包括淋巴细胞，例如T细胞、NK细胞或其组合。
在某些实施方案中，所述用于过继性细胞治疗的免疫细胞是经改造的免疫细胞，其表达嵌合抗原受体和/或包含编码所述嵌合抗原受体的核酸分子。在某些实施方案中，所述经改造的免疫细胞包括表达IL2-Rαβγ三聚体的淋巴细胞，例如T细胞、NK细胞或其组合。
在某些实施方案中，所述用于过继性细胞治疗的免疫细胞是肿瘤浸润淋巴细胞(TIL)。
第四方面，本发明提供了试剂盒，其包含第一方面所述的组合物以及编码嵌合抗原受体的核酸分子。在某些实施方案中，所述试剂盒进一步包含包装说明书，所述包装说明书包含使用所述组合物中以及核酸分子来在体外制备用于过继性细胞治疗的经改造的免疫细胞的说明，所述经改造的免疫细胞表达嵌合抗原受体和/或包含编码所述嵌合抗原受体的核酸分子。
在某些实施方案中，所述编码嵌合抗原受体的核酸分子存在于表达载体中。
在某些实施方案中，所述表达载体为病毒(例如，慢病毒、逆转录病毒或腺病毒)载体。在某些实施方案中，所述表达载体为非病毒载体。
本发明还涉及第一方面所述的组合物以及任选的编码嵌合抗原受体的核酸分子或者第四方面所述的试剂盒用于体外制备用于过继性细胞治疗的经改造的免疫细胞的用途，所述经改造的免疫细胞表达嵌合抗原受体和/或包含编码所述嵌合抗原受体的核酸分子。
本发明还提供了包含第一方面所述的组合物以及用于过继性细胞治疗的免疫细胞的试剂盒。在某些实施方案中，所述试剂盒进一步包含包装说明书，所述包装说明书包含使用所述组合物来在体外培养所述免疫细胞以用于过继性细胞治疗的说明。本发明还涉及第一方面所述的组合物以及任选的用于过继性细胞治疗的免疫细胞用于体外培养用于过继性细胞治疗的免疫细胞。
在某些实施方案中，所述用于过继性细胞治疗的免疫细胞是经改造的免疫细胞，其表达嵌合抗原受体和/或包含编码所述嵌合抗原受体的核酸分子。在某些实施方案中，所述经改造的免疫细胞包括表达IL2-Rαβγ三聚体的淋巴细胞，例如T细胞、NK细胞或其 组合。
在某些实施方案中，所述用于过继性细胞治疗的免疫细胞是肿瘤浸润淋巴细胞(TIL)。
在某些实施方案中，本文中所述的嵌合抗原受体具备本领域技术人员公知的含义，其典型地包括细胞外抗原结合结构域、任选的间隔结构域、跨膜结构域、以及一个或多个胞内信号传导结构域。在某些实施方案中，所述胞内信号传导结构域选自初级信号传导结构域和/或共刺激信号传导结构域。在某些实施方案中，所述细胞外抗原结合结构域包含特异性结合肿瘤相关抗原(例如CD19)的抗体或抗原结合片段(例如scFv)。
用于过继性细胞治疗的免疫细胞的培养与制备
第五方面，本发明提供了培养用于过继性细胞治疗的免疫细胞的方法，所述方法包括在包含第一定点修饰的IL-2和第二定点修饰的IL-2的细胞培养基中培养所述细胞，其中，所述第一定点修饰的IL-2和第二定点修饰的IL-2如第一方面中定义。
细胞培养基可以是能够支持细胞生长的任何培养基，通常包含无机盐、维生素、葡萄糖、缓冲体系和必需氨基酸，并且通常具有约280-330mOsmol的渗透压。在某些实施方案中，所述细胞培养基是能够支持免疫细胞(如淋巴细胞，如T细胞和/或NK细胞)生长的培养基。在某些实施方案中，所述细胞培养基为完全培养基。在某些实施方案中，所述细胞培养基包含基础培养基(如RPMI 1640)、血清(如FBS)、丙酮酸钠、非必需氨基酸。在某些实施方案中，所述细胞培养基不包含血清。
在某些实施方案中，所述方法还包括收获细胞用于储存(例如在冷冻保存培养基中重新配制)或施用(例如用于过继性细胞治疗)。
在某些实施方案中，所提供的存在第一定点修饰的IL-2和第二定点修饰的IL-2的培养条件可减少免疫细胞的衰老和耗竭，保留其干性状态，增强T
Figure PCTCN2022129975-appb-000009
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。
在某些实施方案中，所述用于过继性细胞治疗的免疫细胞是经改造的免疫细胞，其表达嵌合抗原受体和/或包含编码所述嵌合抗原受体的核酸分子。在某些实施方案中，所述经改造的免疫细胞包括表达IL2-Rαβγ三聚体的淋巴细胞，例如T细胞、NK细胞或其组合。
在某些实施方案中，所述用于过继性细胞治疗的免疫细胞是肿瘤浸润淋巴细胞 (TIL)。
第六方面，本发明提供了制备用于过继性细胞治疗的免疫细胞的方法，其包括使用如第一方面中定义的第一定点修饰的IL-2和第二定点修饰的IL-2。
在第六方面的某些实施方案中，本发明提供了制备用于过继性细胞治疗的免疫细胞的方法，所述用于过继性细胞治疗的免疫细胞是经改造的免疫细胞，其表达嵌合抗原受体和/或包含编码所述嵌合抗原受体的核酸分子，其中，所述方法包括：
(1)提供来自患者或者健康供体的免疫细胞；
(2)在存在第一定点修饰的IL-2和第二定点修饰的IL-2的条件下，将编码嵌合抗原受体的核酸分子引入步骤(1)所述的免疫细胞，从而提供所述经改造的免疫细胞；其中，所述第一定点修饰的IL-2和第二定点修饰的IL-2如第一方面中定义。
在某些实施方案中，步骤(2)在包含所述第一定点修饰的IL-2和第二定点修饰的IL-2的细胞培养基中进行。细胞培养基可以是能够支持细胞生长的任何培养基，通常包含无机盐、维生素、葡萄糖、缓冲体系和必需氨基酸，并且通常具有约280-330mOsmol的渗透压。在某些实施方案中，所述细胞培养基是能够支持免疫细胞(如淋巴细胞，如T细胞和/或NK细胞)生长的培养基。在某些实施方案中，所述细胞培养基为完全培养基。在某些实施方案中，所述细胞培养基包含基础培养基(如RPMI 1640)、血清(如FBS)、丙酮酸钠、非必需氨基酸。在某些实施方案中，所述细胞培养基不包含血清。
在某些实施方案中，在步骤(2)中所述编码嵌合抗原受体的核酸分子存在于表达载体中。
在某些实施方案中，在步骤(2)中所述编码嵌合抗原受体的核酸分子由病毒(例如，慢病毒、逆转录病毒或腺病毒)载体通过感染的方式引入细胞。在某些实施方案中，在步骤(2)中所述编码嵌合抗原受体的核酸分子由慢病毒载体通过感染的方式引入细胞。
在某些实施方案中，在步骤(2)中所述编码嵌合抗原受体的核酸分子由非病毒载体引入细胞。
在某些实施方案中，在步骤(1)中，所述免疫细胞经预处理，所述预处理包括免疫细胞的分选、激活和/或增殖。在某些实施方案中，所述预处理包括将免疫细胞与抗CD3抗体和抗CD28抗体接触，从而刺激所述免疫细胞并诱导其增殖，由此生成经预处理的免疫细胞。在某些实施方案中，所述预处理包括从外周血单核细胞(PBMC)分离T细胞。
在某些实施方案中，在步骤(2)之后所述方法还包括：(3)在包含所述第一定点修饰的 IL-2和第二定点修饰的IL-2的细胞培养基中继续培养步骤(2)获得的免疫细胞的步骤。
在某些实施方案中，所述方法还包括收获细胞用于储存(例如在冷冻保存培养基中重新配制)或施用(例如用于过继性细胞治疗)。
在某些实施方案中，所述免疫细胞包括表达IL2-Rαβγ三聚体的淋巴细胞，如T细胞、NK细胞或其任意组合。在某些实施方案中，所述免疫细胞是T细胞。
在某些实施方案中，所提供的存在第一定点修饰的IL-2和第二定点修饰的IL-2的制备条件可减少免疫细胞的过度激活及终末分化。
在第六方面的某些实施方案中，本发明还提供了制备用于过继性细胞治疗的免疫细胞的方法，所述用于过继性细胞治疗的免疫细胞是肿瘤浸润淋巴细胞(TIL)，其中，所述方法包括：从肿瘤组织中分离浸润淋巴细胞并在包含第一定点修饰的IL-2和第二定点修饰的IL-2的细胞培养基中进行培养，其中，所述第一定点修饰的IL-2和第二定点修饰的IL-2如第一方面中定义，所述细胞培养基如上文中定义。
第七方面，本发明提供了用于过继性细胞治疗的免疫细胞，其由如上任一方面所述的方法制备或培养。
在某些实施方案中，第七方面所述的免疫细胞具备减少的衰老和耗竭，增强的T
Figure PCTCN2022129975-appb-000010
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T effector细胞耗竭程度，和/或降低的由内源性IL-2导致的过度激活。
第八方面，本发明提供了免疫细胞群，其包括第七方面所述的免疫细胞，以及可选的未改造和/或未成功改造的免疫细胞。在某些实施方案中，第七方面所述的免疫细胞占免疫细胞群总细胞数的大约10％-100％，优选地40％-80％。
在协同增强免疫应答中的应用
本发明提供的第一定点修饰的IL-2和第二定点修饰的IL-2具有显著的协同增强免疫应答的作用，因此本发明提供的所述第一定点修饰的IL-2和第二定点修饰的IL-2的组合可用于治疗刺激宿主的免疫系统以获益的疾病情形，特别是期望增强细胞免疫应答的状况，可以包括宿主免疫应答不足或缺陷性的疾病情形，例如肿瘤。
第十方面，本发明提供了药物组合物，其包含第一方面所述的组合物以及药学上可接受的载体和/或赋形剂。
在某些实施方案中，所述药物组合物包含多个组合物或剂型。在某些实施方案中， 所述第一定点修饰的IL-2和第二定点修饰的IL-2在分开的组合物或剂型中。在某些实施方案中，所述药物组合物包含：所述第一定点修饰的IL-2和药学上可接受的载体和/或赋形剂形成的第一组合物，以及所述第二定点修饰的IL-2和药学上可接受的载体和/或赋形剂形成的第二组合物。
在某些实施方案中，所述药物组合物包含一个组合物或剂型。在某些实施方案中，所述第一定点修饰的IL-2和第二定点修饰的IL-2在相同的组合物或剂型中。在某些实施方案中，所述药物组合物包含：所述第一定点修饰的IL-2、第二定点修饰的IL-2以及药学上可接受的载体和/或赋形剂形成的单一组合物。
本发明所述的第一定点修饰的IL-2和第二定点修饰的IL-2、它们的组合以及第一方面所述的组合物或第十方面所述的药物组合物可以被配制成与其预期施用途径相兼容的剂型。施用途径的实例包括肠胃外给药，例如，静脉内给药、皮内给药、皮下给药、口服给药(例如，吸入给药)、经皮给药(即，局部给药)、经粘膜给药和直肠给药。用于肠胃外给药、皮内给药或皮下给药应用的溶液或悬浮液可包括以下组分：无菌稀释剂诸如注射用水、盐水溶液、固定油、聚乙二醇、甘油、丙二醇或其它合成溶剂；抗菌剂诸如苄醇或对羟基苯甲酸甲酯；抗氧化剂诸如抗坏血酸或亚硫酸氢钠；螯合剂诸如乙二胺四乙酸(EDTA)；缓冲剂诸如醋酸盐、柠檬酸盐或磷酸盐，和用于调整张力的试剂，诸如氯化钠或葡萄糖。pH可以用酸或碱，诸如盐酸或氢氧化钠调节。肠胃外给药的制剂可以封装在由玻璃或塑料制成的安瓿、一次性注射器或多剂量小瓶中。
适于注射使用的药物组合物包括无菌水溶液(其中水可溶)或分散体和用于随时制备无菌注射溶液或分散体的无菌粉末。对于静脉内施用，合适的载体包括生理盐水、抑菌水、聚氧乙烯蓖麻油ELTM或磷酸缓冲盐水(PBS)。在所有情况下，组合物必须是无菌的，并且应该是达到存在易注射性程度的流体。它必须在制造和储存条件下是稳定的，并且必须防止微生物诸如细菌和真菌的污染作用下保存。载体可以是包括例如水、乙醇、多元醇(例如，甘油、丙二醇和液体聚乙二醇等)，及其合适的混合物的溶剂或分散介质。防止微生物的作用可以通过各种抗细菌和抗真菌剂来实现，例如对羟基苯甲酸酯类、氯丁醇、苯酚、抗坏血酸、硫柳汞等。在许多情况下，在组合物中将优选包括等渗剂，例如糖类，多元醇诸如甘露醇、山梨醇，氯化钠。可注射组合物的延长吸收可以通过在组合物中包括延迟吸收的试剂，例如，单硬脂酸铝和明胶来实现。
第十一方面，本发明提供了一种用于增强免疫应答、预防和/或治疗增生性疾病(例如肿瘤)的方法，其包括向有需要的受试者施用第一方面所述的组合物或第十方面所述 的药物组合物。在某些实施方案中，所述方法包括向受试者联合施用所述第一定点修饰的IL-2和第二定点修饰的IL-2。
第十二方面，本发明还提供了第一方面所述的组合物或第十方面所述的药物组合物用于增强免疫应答、预防和/或治疗增生性疾病(例如肿瘤)的用途，或者，在制备用于增强免疫应答、预防和/或治疗增生性疾病(例如肿瘤)的药物中的用途。本发明还提供了第一定点修饰的IL-2和第二定点修饰的IL-2的组合用于增强免疫应答、预防和/或治疗增生性疾病(例如肿瘤)的用途，或者，在制备用于增强免疫应答、预防和/或治疗增生性疾病(例如肿瘤)的药物中的用途。
在某些实施方案中，所述第一定点修饰的IL-2和第二定点修饰的IL-2单独地调配成两种或两种以上的组合物(例如，包含每种组分的试剂盒)。彼此联合施用的单独组分可以同时、分开或相继施用。在某些实施方案中，彼此联合施用的单独组分可以在与施用其它组分的时间不同的时间施用于受试者；例如，作为治疗方案的一部分，每次施用可以以给定的时间段的间隔非同时(例如，单独地或顺序地)给予。在某些实施方案中，彼此联合施用的单独组分还可以在同一施用时段期间顺序地施用，但基本上同时施用。此外，可以通过相同或不同的途径向受试者施用彼此联合施用的单独组分。
在某些实施方案中，所述第一定点修饰的IL-2和第二定点修饰的IL-2一起调配成单个组合物，例如以用于同时递送。
在另一方面，本发明提供了一种药盒，其包含含有所述第一定点修饰的IL-2和可选的药学上可接受的载体和/或赋形剂的药物，和包装说明书，所述包装说明书包含将所述药物与含有所述第二定点修饰的IL-2和可选的药学上可接受的载体和/或赋形剂的组合物组合施用来在受试者中增强免疫应答、预防和/或治疗增生性疾病(例如肿瘤)的说明。
在另一方面，本发明提供了一种药盒，其包含含有所述第二定点修饰的IL-2和可选的药学上可接受的载体和/或赋形剂的药物，和包装说明书，所述包装说明书包含将所述药物与含有所述第一定点修饰的IL-2和可选的药学上可接受的载体和/或赋形剂的组合物组合施用来在受试者中增强免疫应答、预防和/或治疗增生性疾病(例如肿瘤)的说明。
在另一方面，本发明提供了一种药盒，其包含含有所述第一定点修饰的IL-2和可选的药学上可接受的载体和/或赋形剂的第一药物，含有所述第二定点修饰的IL-2和可选的药学上可接受的载体和/或赋形剂的第二药物。在某些实施方案中，所述药盒进一步包含含有施用所述第一药物和所述第二药物来在受试者中增强免疫应答、预防和/或治疗增生性疾病(例如肿瘤)的说明的包装说明书。
在如上所述的方法、用途和药盒的某些实施方案中，所述免疫应答是细胞免疫应答。在某些实施方案中，所述免疫应答是T细胞介导的免疫应答，特别是效应T细胞(Teff)介导的免疫应答。在某些实施方案中，所述增强免疫应答还包括降低或抑制Treg细胞功能。
在如上所述的方法、用途和药盒的某些实施方案中，所述增生性疾病是肿瘤，包括实体瘤或血液肿瘤，也包括转移性癌症、复发性或难治性癌症。在另一些实施方式中，所述增生性疾病是高丙种球蛋白血症(hypergammaglobulinemia)、淋巴增生性病症、病变蛋白血症(paraproteinemias)、紫癜(purpura)、结节病、塞扎里综合征(Sezary Syndrome)、Waldenstron's巨球蛋白血症、高歇氏病(Gaucher's Disease)、组织细胞增多病(histiocytosis)以及任何其它位于器官系统中的瘤形成(neoplasia)外的细胞增殖疾病。
在如上所述的方法、用途和药盒的某些实施方案中，所述肿瘤是实体瘤。在某些实施方案中，所述实体瘤是转移性癌症，复发性或难治性癌症。
在如上所述的方法、用途和药盒的某些实施方案中，所述肿瘤是血液肿瘤，例如白血病、淋巴瘤或骨髓瘤。在某些实施方案中，所述血液肿瘤是转移性癌症，复发性或难治性癌症。
在如上所述的方法、用途和药盒的某些实施方案中，所述肿瘤选自黑色素瘤、肾细胞癌、非小细胞肺癌、淋巴瘤、头颈部鳞状细胞癌、尿路上皮癌、卵巢癌、胃癌和乳腺癌。
在如上所述的方法、用途和药盒的某些实施方案中，所述受试者为哺乳动物，例如人。
在如上所述的方法、用途和药盒的某些实施方案中，所述组合物中的第一定点修饰的IL-2和第二定点修饰的IL-2同时、分开或相继施用。
在如上所述的方法、用途和药盒的某些实施方案中，本发明所述的第一定点修饰的IL-2和第二定点修饰的IL-2、它们的组合以及本发明的组合物或药物组合物可以配制成医学领域已知的任何剂型，例如，片剂、丸剂、混悬剂、乳剂、溶液、凝胶剂、胶囊剂、粉剂、颗粒剂、酏剂、锭剂、栓剂、注射剂(包括注射液、注射用无菌粉末与注射用浓溶液)、吸入剂、喷雾剂等。优选剂型取决于预期的给药方式和治疗用途。本发明的组合物或药物组合物应当是无菌的并在生产和储存条件下稳定。一种优选的剂型是注射剂。此类注射剂可以是无菌注射溶液。例如，可通过下述方法来制备无菌注射溶液：在适当的溶剂中掺入必需剂量的活性成分，以及任选地，同时掺入其他期望的成分(包括 但不限于，pH调节剂，表面活性剂，佐剂，离子强度增强剂，等渗剂、防腐剂、稀释剂，或其任何组合)，随后过滤除菌。此外，可以将无菌注射溶液制备为无菌冻干粉剂(例如，通过真空干燥或冷冻干燥)以便于储存和使用。此类无菌冻干粉剂可在使用前分散于合适的载体中，例如注射用水(WFI)、抑菌性注射用水(BWFI)、氯化钠溶液(例如0.9％(w/v)NaCl)、葡萄糖溶液(例如5％葡萄糖)、含有表面活性剂的溶液(例如0.01％聚山梨醇20)、pH缓冲溶液(例如磷酸盐缓冲溶液)、Ringer氏溶液及其任意组合。
在如上所述的方法、用途和药盒的某些实施方案中，本发明所述的第一定点修饰的IL-2和第二定点修饰的IL-2、它们的组合以及本发明的组合物或药物组合物可以通过本领域已知的任何合适的方法来施用，包括但不限于，口服、口腔、舌下、眼球、局部、肠胃外、直肠、叶鞘内、内胞浆网槽内、腹股沟、膀胱内、局部(如，粉剂、药膏或滴剂)，或鼻腔途径。但是，对于许多治疗用途而言，优选的给药途径/方式是胃肠外给药(例如静脉注射或推注，皮下注射，腹膜内注射，肌内注射)。技术人员应理解，给药途径和/或方式将根据预期目的而发生变化。在某些实施方案中，本发明所述的第一定点修饰的IL-2和第二定点修饰的IL-2、它们的组合以及本发明的组合物或药物组合物通过静脉注射或推注给予。
在如上所述的方法、用途和药盒的某些实施方案中，本发明所述的第一定点修饰的IL-2和第二定点修饰的IL-2、它们的组合以及本发明的组合物或药物组合物可以以剂量单位形式配制以易于施用。剂量单位形式是指适合作为单一剂量用于待治疗对象的物理上离散的单位；每个单位含有预定量的经计算与所需的药物载体联合以产生的期望的治疗效果的活性成分。
在如上所述的方法、用途和药盒的某些实施方案中，本发明所述的第一定点修饰的IL-2和第二定点修饰的IL-2、它们的组合以及本发明的组合物或药物组合物可以单独施用，也可以与另外的药学活性剂(例如抗肿瘤剂)或另外的疗法(例如抗肿瘤疗法)联合施用。
在过继性细胞治疗中的应用
本发明提供的第一定点修饰的IL-2和第二定点修饰的IL-2的组合可以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。由此进一步提供所述免疫细胞的治疗应用。
第十三方面，本发明提供了药物组合物，其包含第七方面所述的免疫细胞或第八方 面所述的免疫细胞群以及药学上可接受的载体和/或赋形剂。
第七方面所述的免疫细胞、第八方面所述的免疫细胞群或第十三方面所述的药物组合物可以被配制成与其预期施用途径相兼容的剂型。施用途径的实例包括肠胃外给药，例如，静脉内给药、皮内给药、皮下给药、口服给药(例如，吸入给药)、经皮给药(即，局部给药)、经粘膜给药和直肠给药。用于肠胃外给药、皮内给药或皮下给药应用的溶液或悬浮液可包括以下组分：无菌稀释剂诸如注射用水、盐水溶液、固定油、聚乙二醇、甘油、丙二醇或其它合成溶剂；抗菌剂诸如苄醇或对羟基苯甲酸甲酯；抗氧化剂诸如抗坏血酸或亚硫酸氢钠；螯合剂诸如乙二胺四乙酸(EDTA)；缓冲剂诸如醋酸盐、柠檬酸盐或磷酸盐，和用于调整张力的试剂，诸如氯化钠或葡萄糖。pH可以用酸或碱，诸如盐酸或氢氧化钠调节。肠胃外给药的制剂可以封装在由玻璃或塑料制成的安瓿、一次性注射器或多剂量小瓶中。
适于注射使用的药物组合物包括无菌水溶液(其中水可溶)或分散体和用于随时制备无菌注射溶液或分散体的无菌粉末。对于静脉内施用，合适的载体包括生理盐水、抑菌水、聚氧乙烯蓖麻油ELTM或磷酸缓冲盐水(PBS)。在所有情况下，组合物必须是无菌的，并且应该是达到存在易注射性程度的流体。它必须在制造和储存条件下是稳定的，并且必须防止微生物诸如细菌和真菌的污染作用下保存。载体可以是包括例如水、乙醇、多元醇(例如，甘油、丙二醇和液体聚乙二醇等)，及其合适的混合物的溶剂或分散介质。防止微生物的作用可以通过各种抗细菌和抗真菌剂来实现，例如对羟基苯甲酸酯类、氯丁醇、苯酚、抗坏血酸、硫柳汞等。在许多情况下，在组合物中将优选包括等渗剂，例如糖类，多元醇诸如甘露醇、山梨醇，氯化钠。可注射组合物的延长吸收可以通过在组合物中包括延迟吸收的试剂，例如，单硬脂酸铝和明胶来实现。
第十四方面，本发明提供了一种用于增强免疫应答、预防和/或治疗增生性疾病(例如肿瘤)的方法，其包括向有需要的受试者施用第七方面所述的免疫细胞、第八方面所述的免疫细胞群或第十三方面所述的药物组合物。
在某些实施方案中，所述方法包括以下步骤：(1)使用第五方面所述的方法和/或第六方面所述的方法获得免疫细胞；(2)将步骤(1)中获得的免疫细胞或包含其的细胞群体施用至所述受试者以进行治疗。
在某些实施方案中，向受试者施用嵌合抗原受体细胞疗法，所述方法包括：使用本发明第五方面所述的方法和/或第六方面所述的方法获得表达嵌合抗原受体的经改造的免疫细胞，随后向所述受试者施用所述经改造的免疫细胞。
在某些实施方案中，向受试者施用TIL疗法，所述方法包括：使用本发明第五方面所述的方法和/或第六方面所述的方法获得肿瘤浸润淋巴细胞(TIL)，向所述受试者施用所述肿瘤浸润淋巴细胞(TIL)。
第十五方面，本发明还提供了第七方面所述的免疫细胞、第八方面所述的免疫细胞群或第十三方面所述的药物组合物用于增强免疫应答、预防和/或治疗增生性疾病(例如肿瘤)的用途，或者，在制备用于增强免疫应答、预防和/或治疗增生性疾病(例如肿瘤)的药物中的用途。
在如上所述的方法和用途的某些实施方案中，所述免疫细胞、免疫细胞群或药物组合物可以与第一方面中所述定义的第一定点修饰的IL-2和第二定点修饰的IL-2联合施用，例如同时、分开或相继施用。
第十六方面，本发明还提供了第一方面所述的组合物与用于过继性细胞治疗的免疫细胞的组合用于增强免疫应答、预防和/或治疗增生性疾病(例如肿瘤)的用途，或者，在制备用于增强免疫应答、预防和/或治疗增生性疾病(例如肿瘤)的药物中的用途。
第十七方面，本发明还提供了用于增强免疫应答、预防和/或治疗增生性疾病的方法，其包括向有需要的受试者施用第一方面所述的组合物与用于过继性细胞治疗的免疫细胞的组合。
在第十六方面或第十七方面的某些实施方案中，所述用于过继性细胞治疗的免疫细胞是经改造的免疫细胞，其表达嵌合抗原受体和/或包含编码所述嵌合抗原受体的核酸分子。在某些实施方案中，所述经改造的免疫细胞包括表达IL2-Rαβγ三聚体的淋巴细胞，如T细胞、NK细胞或其任意组合。
在第十六方面或第十七方面的某些实施方案中，所述用于过继性细胞治疗的免疫细胞是肿瘤浸润淋巴细胞(TIL)。
在第十六方面或第十七方面的某些实施方案中，第一方面所述的组合物与用于过继性细胞治疗的免疫细胞存在于相同的组合物或剂型中，从而可同时施用。
在第十六方面或第十七方面的某些实施方案中，第一方面所述的组合物与用于过继性细胞治疗的免疫细胞存在于分开的组合物或剂型中，从而可分开或相继施用。
在如上所述的方法和用途的某些实施方案中，所述免疫应答是细胞免疫应答。在某些实施方案中，所述免疫应答是T细胞介导的免疫应答，特别是效应T细胞(Teff)介导的 免疫应答。在某些实施方案中，所述增强免疫应答还包括降低或抑制Treg细胞功能。
在如上所述的方法和用途的某些实施方案中，所述增生性疾病是肿瘤，包括实体瘤或血液肿瘤，也包括转移性癌症、复发性或难治性癌症。在另一些实施方式中，所述增生性疾病是高丙种球蛋白血症(hypergammaglobulinemia)、淋巴增生性病症、病变蛋白血症(paraproteinemias)、紫癜(purpura)、结节病、塞扎里综合征(Sezary Syndrome)、Waldenstron's巨球蛋白血症、高歇氏病(Gaucher's Disease)、组织细胞增多病(histiocytosis)以及任何其它位于器官系统中的瘤形成(neoplasia)外的细胞增殖疾病。
在如上所述的方法和用途的某些实施方案中，所述肿瘤是实体瘤。在某些实施方案中，所述实体瘤是转移性癌症，复发性或难治性癌症。
在如上所述的方法和用途的某些实施方案中，所述肿瘤是血液肿瘤，例如白血病、淋巴瘤或骨髓瘤。在某些实施方案中，所述血液肿瘤是转移性癌症，复发性或难治性癌症。
在如上所述的方法和用途的某些实施方案中，所述肿瘤选自黑色素瘤、肾细胞癌、非小细胞肺癌、淋巴瘤、头颈部鳞状细胞癌、尿路上皮癌、卵巢癌、胃癌和乳腺癌。
在如上所述的方法和用途的某些实施方案中，所述肿瘤是淋巴瘤。
在如上所述的方法和用途的某些实施方案中，当涉及嵌合抗原受体细胞疗法时，所述肿瘤优选地包括血液肿瘤，包括转移性癌症、复发性或难治性癌症；例如所述肿瘤选自淋巴瘤。
在如上所述的方法和用途的某些实施方案中，当涉及肿瘤浸润淋巴细胞(TIL)疗法时，所述肿瘤优选地包括实体瘤，包括转移性癌症、复发性或难治性癌症；例如所述肿瘤选自黑色素瘤、肾细胞癌、非小细胞肺癌、淋巴瘤、头颈部鳞状细胞癌、尿路上皮癌、卵巢癌、胃癌和乳腺癌。
在如上所述的方法和用途的某些实施方案中，所述受试者为哺乳动物，例如人。
在如上所述的方法和用途的某些实施方案中，第七方面所述的免疫细胞、第八方面所述的免疫细胞群或第十三方面所述的药物组合物可以配制成医学领域已知的任何剂型，例如，片剂、丸剂、混悬剂、乳剂、溶液、凝胶剂、胶囊剂、粉剂、颗粒剂、酏剂、锭剂、栓剂、注射剂(包括注射液、注射用无菌粉末与注射用浓溶液)、吸入剂、喷雾剂等。优选剂型取决于预期的给药方式和治疗用途。本发明的组合物或药物组合物应当是无菌的并在生产和储存条件下稳定。一种优选的剂型是注射剂。此类注射剂可以是无菌注射溶液。例如，可通过下述方法来制备无菌注射溶液：在适当的溶剂中掺入必 需剂量的活性成分，以及任选地，同时掺入其他期望的成分(包括但不限于，pH调节剂，表面活性剂，佐剂，离子强度增强剂，等渗剂、防腐剂、稀释剂，或其任何组合)，随后过滤除菌。此外，可以将无菌注射溶液制备为无菌冻干粉剂(例如，通过真空干燥或冷冻干燥)以便于储存和使用。此类无菌冻干粉剂可在使用前分散于合适的载体中，例如注射用水(WFI)、抑菌性注射用水(BWFI)、氯化钠溶液(例如0.9％(w/v)NaCl)、葡萄糖溶液(例如5％葡萄糖)、含有表面活性剂的溶液(例如0.01％聚山梨醇20)、pH缓冲溶液(例如磷酸盐缓冲溶液)、Ringer氏溶液及其任意组合。
在如上所述的方法和用途的某些实施方案中，第七方面所述的免疫细胞、第八方面所述的免疫细胞群或第十三方面所述的药物组合物可以通过本领域已知的任何合适的方法来施用，包括但不限于，口服、口腔、舌下、眼球、局部、肠胃外、直肠、叶鞘内、内胞浆网槽内、腹股沟、膀胱内、局部(如，粉剂、药膏或滴剂)，或鼻腔途径。但是，对于许多治疗用途而言，优选的给药途径/方式是胃肠外给药(例如静脉注射或推注，皮下注射，腹膜内注射，肌内注射)。技术人员应理解，给药途径和/或方式将根据预期目的而发生变化。在某些实施方案中，第七方面所述的免疫细胞、第八方面所述的免疫细胞群或第十三方面所述的药物组合物通过静脉注射或推注给予。
在如上所述的方法和用途的某些实施方案中，第七方面所述的免疫细胞、第八方面所述的免疫细胞群或第十三方面所述的药物组合物可以以剂量单位形式配制以易于施用。剂量单位形式是指适合作为单一剂量用于待治疗对象的物理上离散的单位；每个单位含有预定量的经计算与所需的药物载体联合以产生的期望的治疗效果的活性成分。
在如上所述的方法和用途的某些实施方案中，第七方面所述的免疫细胞、第八方面所述的免疫细胞群或第十三方面所述的药物组合物可以单独施用，也可以与另外的药学活性剂(例如抗肿瘤剂)或另外的疗法(例如抗肿瘤疗法)联合施用。
术语定义
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的分子遗传学、核酸化学、细胞培养、生物化学、细胞生物学等操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
当本文使用术语“例如”、“如”、“诸如”、“包括”、“包含”或其变体时，这些术语将不被认为是限制性术语，而将被解释为表示“但不限于”或“不限于”。
除非本文另外指明或根据上下文明显矛盾，否则术语“一个”和“一种”以及“该”和类似指称物在描述本发明的上下文中(尤其在以下权利要求的上下文中)应被解释成覆盖单数和复数。
如本文中所使用的，术语“非天然氨基酸”是指除了天然存在于蛋白质中的20种氨基酸以外的氨基酸。非天然氨基酸的非限制性例子包括：Nε-2-叠氮乙氧羰基-L-赖氨酸(NAEK)、对-乙酰基-L-苯丙氨酸、对-碘-L-苯丙氨酸、对-甲氧基苯丙氨酸、O-甲基-L-酪氨酸、对-炔丙氧基苯丙氨酸、对-炔丙基-苯丙氨酸、L-3-(2-萘基)丙氨酸、3-甲基-苯丙氨酸、O-4-烯丙基-L-酪氨酸、4-丙基-L-酪氨酸、三-O-乙酰基-GlcNAcp-丝氨酸、L-多巴,氟化苯丙氨酸、异丙基-L-苯丙氨酸、对-叠氮基-L-苯丙氨酸、对-酰基-L-苯丙氨酸、对-苯甲酰基-L-苯丙氨酸、对-硼苯丙氨酸、O-炔丙基酪氨酸、L-磷酸丝氨酸、膦酰丝氨酸、膦酰酪氨酸、对-溴苯丙氨酸、硒代半胱氨酸、对-氨基-L-苯丙氨酸、异丙基-L-苯丙氨酸、酪氨酸氨基酸的非天然类似物；谷氨酰胺氨基酸的非天然类似物；苯丙氨酸氨基酸的非天然类似物；丝氨酸氨基酸的非天然类似物；苏氨酸氨基酸的非天然类似物；烷基、芳基、酰基、叠氮基、氰基、卤素、肼、酰肼、羟基、烯基、炔基、醚、硫醇、磺酰基、硒基、酯、硫代酸、硼酸酯(borate)、硼酸酯(boronate)、磷酸基、膦酰基、膦、杂环、烯酮、亚胺、醛、羟胺、酮基或氨基取代的氨基酸或其组合；具有光可激活的交联剂的氨基酸；自旋标记的氨基酸；荧光氨基酸；金属结合氨基酸；含有金属的氨基酸；放射性氨基酸；光笼化和/或光可异构的氨基酸；含有生物素或生物素类似物的氨基酸；含有酮基的氨基酸；包含聚乙二醇或聚醚的氨基酸；重原子取代的氨基酸；化学可裂解或光可裂解的氨基酸；具有延长侧链的氨基酸；含有毒性基团的氨基酸；糖取代的氨基酸；碳连接的含糖氨基酸；氧化还原活性氨基酸；含有a-羟基的酸；氨基硫代酸；α,α二取代的氨基酸；β-氨基酸；除了脯氨酸或组氨酸以外的环状氨基酸；以及除了苯丙氨酸、酪氨酸或色氨酸以外的芳香族氨基酸。
在一些实施方案中，非天然氨基酸包含选择性反应基团，或用于位点选择性标记靶多肽的反应基团。化学反应可以是双正交反应(例如，生物相容性和选择性反应)，Cu(I)催化或“无铜”炔-叠氮三唑形成反应、施陶丁格连接(Staudinger ligation)、反电子需求的迪尔斯-阿尔德(inverse-electron-demand Diels-Alder，IEDDA)反应、“光-点击”化学或金属介导的过程(如烯烃复分解和Suzuki-Miyaura或Sonogashira交叉偶联)等。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载 体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞。在某些实施方案中，所述宿主细胞包括大肠杆菌。
如本文中使用的，术语“嵌合抗原受体(CAR)”是指包含至少一个细胞外抗原结合结构域、任选的间隔结构域、跨膜结构域和胞内信号传导结构域的重组多肽构建体，其将针对目的抗原(例如肿瘤抗原)的基于抗体的特异性与免疫效应细胞活化胞内结构域组合以展现针对表达该目的抗原细胞(如肿瘤细胞)的特异性免疫活性。在本发明中，表述“表达CAR的免疫细胞”是指表达CAR并且具有由该CAR的靶向结构域决定的抗原特异性的免疫细胞。
如本文中使用的，术语“胞外抗原结合结构域”是指能够特异性结合目的抗原或受体的多肽。该结构域将能够与细胞表面分子相互作用。例如，可以选择胞外抗原结合结构域来识别作为与特定疾病状态相关的靶细胞细胞表面标志物的抗原。典型地，所述胞外抗原结合结构域是抗体衍生的靶向结构域。
如本文中使用的，术语“胞内信号传导结构域”是指传导效应信号功能信号并引导细胞进行专门的功能的蛋白质部分。因此，胞内信号传导结构域具有激活表达CAR的免疫效应细胞的至少一种正常效应子功能的能力。例如，T细胞的效应子功能可以是细胞溶解活性或辅助活性，包括细胞因子的分泌。
如本文中使用的，术语“初级信号传导结构域”是指能够以刺激方式或以抑制方式调节TCR复合物的初级活化的蛋白质部分。以刺激方式作用的初级信号传导结构域通常含有已知为基于免疫受体酪氨酸的活化基序(ITAM)的信号传导基序。含有特别用于本 发明中的初级信号传导结构域的ITAM的非限制性实例包括衍生自TCRζ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD3ζ、CD22、DAP10、CD79a、CD79b和CD66d的那些。
如本文中使用的，术语“共刺激信号传导结构域”是指共刺激分子的胞内信号传导结构域。共刺激分子是除抗原受体或Fc受体以外的在结合到抗原后提供T淋巴细胞的高效活化和功能所需的第二信号的细胞表面分子。所述共刺激分子的非限制性实例包括CARD11、CD2、CD7、CD27、CD28、CD30、CD40、CD54(ICAM)、CD83、CD134(OX40)、CD137(4-1BB)、CD150(SLAMF1)CD270(HVEM)、CD278(ICOS)、DAP10。
如本文中使用的，术语“免疫细胞”是指具有一种或多种效应功能(例如，细胞毒性细胞杀伤活性、细胞因子的分泌、ADCC和/或CDC的诱导)的免疫系统的任何细胞。典型地，免疫细胞是具有造血的起源并在免疫应答中起作用的细胞。在某些实施方案中，免疫细胞是指免疫效应细胞。术语“效应功能”指免疫效应细胞的特化功能，例如增强或促进对靶细胞的免疫攻击(例如对靶细胞的杀伤，或者抑制其生长或增殖)的功能或反应。T细胞的效应功能，例如，可以是细胞溶解活性或者辅助或者包括细胞因子的分泌在内的活性。免疫效应细胞的实例包括T细胞(例如α/βT细胞和γ/δT细胞)、B细胞、天然杀伤(NK)细胞、天然杀伤T(NKT)细胞、肥大细胞和骨髓来源巨噬细胞。
可用于本文所述的CAR的示例性免疫效应细胞包括T淋巴细胞。术语“T细胞”或“T淋巴细胞”是本领域公知的并且意图包括胸腺细胞、未成熟的T淋巴细胞、成熟T淋巴细胞、静息T淋巴细胞或活化的T淋巴细胞。T细胞可以是T辅助(Th)细胞，例如T辅助1(Th1)或T辅助2(Th2)细胞。T细胞可以是辅助T细胞(HTL；CD4T细胞)CD4T细胞、细胞毒性T细胞(CTL；CD8T细胞)、CD4CD8T细胞、CD4CD8T细胞或任何其它T细胞子组。在某些实施方案中，T细胞可以包括原初T细胞和记忆T细胞。
本发明所述的免疫细胞可以是自身的/自体的(“自我”)或非自身的(“非自我”，例如同种异体的、同基因的或异基因的)。如本文中使用的，“自身的”是指来自同一受试者的细胞；“同种异体的”是指与比较细胞遗传不同的同一物种的细胞；“同基因的”是指与比较细胞遗传相同的来自不同受试者的细胞；“异基因的”是指与比较细胞来自不同物种的细胞。在优选实施例中，本发明的细胞是同种异体的。
如本文中使用的，术语“抗体”是指，通常由两对多肽链(每对具有一条轻链(LC)和一条重链(HC))组成的免疫球蛋白分子。抗体轻链可分类为κ(kappa)和λ (lambda)轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。恒定结构域不直接参与抗体与抗原的结合，但展现出多种效应子功能，如可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各V H和V L由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗原结合部位。氨基酸在各区域或结构域的分配可遵循Kabat,Sequences of Proteins of Immunological Interest(National Institutes of Health,Bethesda,Md.(1987and 1991))，或Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883的定义。
如本文中使用的，术语抗体的“抗原结合片段”是指包含全长抗体的片段的多肽，其保持特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合，其也被称为“抗原结合部分”。通常参见，Fundamental Immunology,Ch.7(Paul,W.,ed.,第2版，Raven Press,N.Y.(1989)，其以其全文通过引用合并入本文，用于所有目的。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗体的抗原结合片段。抗原结合片段的非限制性实例包括Fab、Fab’、(Fab’) 2、Fv、二硫键连接的Fv、scFv、di-scFv、(scFv) 2和这样的多肽，其包含足以赋予多肽特异性抗原结合能力的抗体的至少一部分。
如本文中所使用的，术语“Fab片段”意指由VL、VH、CL和CH1结构域组成的抗体片段；术语“F(ab’) 2片段”意指包含通过铰链区上的二硫桥连接的两个Fab片段的抗体片段；术语“Fab’片段”意指还原连接F(ab’) 2片段中两个重链片段的二硫键后所获片段，由一条完整的轻链和重链的Fd片段(由VH和CH1结构域组成)组成。
如本文中所使用的，术语“Fv”意指由抗体的单臂的VL和VH结构域组成的抗体片段。Fv片段通常被认为是，能形成完整的抗原结合位点的最小抗体片段。一般认为，六个CDR赋予抗体的抗原结合特异性。然而，即便是一个可变区(例如Fd片段，其仅仅含有三个对抗原特异的CDR)也能够识别并结合抗原，尽管其亲和力可能低于完整的 结合位点。
如本文中所使用的，术语“scFv”是指，包含VL和VH结构域的单个多肽链，其中所述VL和VH通过接头(linker)相连。此类scFv分子可具有一般结构：NH 2-VL-接头-VH-COOH或NH 2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(GGGGS) 4的接头。在一些情况下，scFv的VH与VL之间还可以存在二硫键。在本发明的某些实施方案中，scFv可形成di-scFv，其指的是两个或两个以上单个scFv串联而形成抗体。在本发明的某些实施方案中，scFv可形成(scFv) 2，其指的是两个或两个以上单个scFv并联而形成抗体。
如本文中所使用的，术语“药学上可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂，稀释剂，维持渗透压的试剂，延迟吸收的试剂，防腐剂。例如，pH调节剂包括但不限于磷酸盐缓冲液。表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80。离子强度增强剂包括但不限于氯化钠。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。维持渗透压的试剂包括但不限于糖、NaCl及其类似物。延迟吸收的试剂包括但不限于单硬脂酸盐和明胶。稀释剂包括但不限于水，水性缓冲液(如缓冲盐水)，醇和多元醇(如甘油)等。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如硫柳汞，2-苯氧乙醇，对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。稳定剂具有本领域技术人员通常理解的含义，其能够稳定药物中的活性成分的期望活性，包括但不限于谷氨酸钠，明胶，SPGA，糖类(如山梨醇，甘露醇，淀粉，蔗糖，乳糖，葡聚糖，或葡萄糖)，氨基酸(如谷氨酸，甘氨酸)，蛋白质(如干燥乳清，白蛋白或酪蛋白)或其降解产物(如乳白蛋白水解物)等。
如本文中所使用的，术语“预防”是指，为了阻止或延迟疾病或病症或症状在受试者体内的发生而实施的方法。如本文中所使用的，术语“治疗”是指，为了获得有益或所需临床结果而实施的方法。为了本发明的目的，有益或所需的临床结果包括(但不限于)减轻症状、缩小疾病的范围、稳定(即，不再恶化)疾病的状态，延迟或减缓疾病的发展、改善或减轻疾病的状态、和缓解症状(无论部分或全部)，无论是可检测或是不可检测的。此外，“治疗”还可以指，与期望的存活期相比(如果未接受治疗)，延长存活期。
如本文中使用的，术语“受试者”是指哺乳动物，例如灵长类哺乳动物，例如人。在某些实施方案中，所述受试者(例如人)患有肿瘤。
发明的有益效果
本发明对IL2进行特异位点选择性的聚乙二醇(PEG)修饰，获得了包含第一定点修饰的IL-2和第二定点修饰的IL-2的组合，其中第一定点修饰的IL-2对IL2-Rα受体具有阻断作用，对IL2-Rβγ受体具有选择性，可以优先激活Teff、CTL及NK细胞而降低激活Treg，此外它还诱导减少的CD8+细胞的终末分化和耗竭，提高中央记忆细胞的比例和数量；第二定点修饰的IL-2具有阻断IL2-Rβ但不阻断IL2-Rα的作用，可以与细胞分泌的内源性IL2竞争IL2-Rα，竞争抑制内源性IL2激活Treg的作用。两者的联合在保留第一定点修饰的IL-2的中央记忆细胞激活优势的情况下，又能弥补第一定点修饰的IL-2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。本发明提供的组合具有显著的协同的增强免疫应答和抗肿瘤作用，此外还可以在过继性细胞治疗中将淋巴细胞的命运导向长期免疫，具有重要临床价值。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
附图说明
图1：PEG化IL-2变体Y45-20K和D20-20K的考马斯蓝染色结果。
图2：PEG化IL-2变体对IL-2受体不同亚基的表面等离子共振(SPR)亲和力测定结果。
图3：PEG化IL-2变体Y45-20K和D20-20K处理后细胞磷酸化STAT5(pSTAT5)水平测定结果。
图4：PEG化IL-2变体Y45-20K和D20-20K的PK/PD特性分析结果。
图5：D20-20K对Y45-20K介导的体外T细胞增殖、活化的影响。
图6：D20-20K对Y45-20K介导的健康小鼠体内T增殖、活化的影响。
图7：D20-20K与Y45-20K联合在小鼠肿瘤模型中的抗肿瘤作用。
图8：D20-20K与Y45-20K联合对荷瘤小鼠淋巴结中T细胞亚群的影响。
图9：D20-20K与Y45-20K联合对血管渗漏综合征(VLS)的影响。
图10：D20-20K与Y45-20K联合对CAR-T细胞体外培养增殖的影响。
图11：D20-20K与Y45-20K联合对CAR-T细胞体外培养衰老、耗竭的影响。
图12：D20-20K与Y45-20K联合对CAR-T细胞体外培养中不同分化阶段的T细胞的衰老、耗竭的影响。
图13：none-α变体(Y45-20K)与多种none-β变体联用对CD8和CD4 T细胞的影响。图13a：免疫检查点PD-1和TIM-3的表达情况；图13b：效应细胞因子Perforin，Granzyme B以及IFN-γ的表达情况；图13c：凋亡相关生物标志物的表达CD57、IL-10表达情况。
图14：none-β变体(D20-20K)与多种none-α变体联用对CD8和CD4 T细胞的影响。图14a：免疫检查点PD-1和TIM-3的表达情况；图14b：效应细胞因子Perforin，Granzyme B以及IFN-γ的表达情况；图14c：凋亡相关生物标志物的表达CD57、IL-10表达情况。
序列信息
本发明涉及的部分序列的信息提供于下面的表1中。
表1：序列的描述
Figure PCTCN2022129975-appb-000011
Figure PCTCN2022129975-appb-000012
Figure PCTCN2022129975-appb-000013
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
NAEK的合成：
Figure PCTCN2022129975-appb-000014
将2-溴乙醇(8g,64mmol)和叠氮钠(6.24g,96mmol)室温下加入丙酮(60ml)和水(30ml)中。反应混合液60℃回流10h，冷却至室温，真空蒸发除去丙酮。残留物用二乙基乙醚提取。有机层用盐水洗两次，然后用Na 2SO 4干燥，过滤，蒸发，得到2-叠氮乙醇(化合物2)，产率99％(5.5g,63.2mmol)，不用进一步纯化。
化合物2(5.5g,63.2mmol)溶于二氯甲烷(120ml)得到的溶液在-3℃条件下缓慢加入溶于二氯甲烷(55ml)的N,N’-羰基二咪唑(15.36g,94.8mmol)悬浮液中。搅拌条件下反应12小时。然后加入200mL水，有机层用盐水先后洗两次，然后用Na 2SO 4干燥，过滤并在真空条件下浓缩。残留物进一步用二氧化硅凝胶层析纯化，PE/EtOAc(1:1)洗脱，得到呈无色油状物的化合物3(10.7g,59mmol)，产率93％。
化合物3(10.7g,59mmol)溶于二氯甲烷(100ml)得到的溶液在室温下加入溶于1M NaOH(50ml)水溶液的Boc-Lys-OH(12.2g,49.2mmol)溶液中。接着加入TBAB(0.16g,0.01eq)。反应混合液在搅拌条件下反应12小时，冷却至0℃，然后用冰浴的1M HCl水溶液调pH值至2-3。水相用DCM提取，有机层用盐水先后洗两次。然后有机层用Na 2SO 4干燥，过滤并在真空条件下浓缩。残留物进一步用二氧化硅凝胶层析纯化，PE/EtOAc/HAc(100:100:1)洗脱，得到呈无色油状物的化合物4(15.1g,41.94mmol)，产 率85％。
化合物4(15.1g,41.94mmol)溶于二氯甲烷(80ml)中，然后缓慢加入三氟乙酸(20ml)。反应液在室温下搅拌反应0.5小时，然后在真空条件下蒸发除去溶剂。残留物重溶于甲醇(5ml)中，并在乙醚中沉淀。收集沉淀物在真空条件下干燥，得到呈白色固体的化合物5(6.63g,25.58mmol)，即NAEK，产率61％。
DBCO-PEG的合成：
Figure PCTCN2022129975-appb-000015
将化合物1，10g(48mmol)盐酸羟胺16.8g(240mmol)溶于无水乙醇中，加入26ml吡啶，加热回流36h，冷切后，加入25ml乙酸乙酯，加入125ml1NHCl搅拌0.5h，分离有机层，用饱和NaCl溶液洗涤有机层，有几层干燥后浓缩得白色固体化合物2，收率：100％。
将6.25g五氧化二磷溶于50ml甲烷磺酸中，搅拌溶解备用。将将上述产物加到10g化合物2中，100度搅拌1.5h。反应液冷却后，倒入300ml水中，析出固体，抽滤烘干，干燥后固体用乙醚打浆，抽滤，烘干得白色固体化合物3，收率：100％。
160ml乙醚冷却到0度，分批加入5.5g氢化铝锂，之后分批加入4g化合物3，35度回流72h。反应液冷却至0度，加入14.8ml饱和硫酸钠溶液猝灭，过滤，浓缩得黄色固体化合物4，收率98％。
766mg琥珀酸单甲酯溶于15ml二氯甲烷，冷却至0度后，滴入610ul草酰氯，室温搅拌1h。浓缩备用。将1g化合物4溶于15ml二氯甲烷中，加入1.16ml吡啶，冷却至0度后滴入琥珀酰氯单甲酯，室温搅拌拌0.5h，有机相经1NHCl洗，1NNaOH洗，NaCl洗涤后，用无水硫酸钠干燥，浓缩得黄色固体化合物5。收率：99％。
将化合物5溶于甲醇/水(体积比＝2：1)中，室温下加入6当量氢氧化锂，室温搅拌8h，旋干甲醇，加入水溶解，水相经二氯甲烷洗涤，水相用1NHCl调PH＝2-3，水相 再经二氯甲烷萃取萃取，有机相用饱和NaCl溶液洗涤，旋干得黄色油状物化合物6，收率：98％。
将1.4g化合物6溶于50ml二氯甲烷中，冷却至0度，将2.2g液素溶于二氯甲烷后滴入，室温搅拌3h，加入50ml饱和硫代硫酸钠溶液，分离有机层用饱和NaCl溶液洗涤，无水硫酸钠干燥，旋干得淡黄固体化合物7，收率：95％。
将2.1g化合物7溶于40ml无水四氢呋喃中，冷却至-40度，将制成1M叔丁醇钾的四氢呋喃溶液滴入，搅拌1h，加入1MHCl溶液淬灭，至pH＝2-3，用二氯甲烷萃取，有机层浓缩后经柱层析分离，得粉白色固体化合物8，收收率：80％。
将1.1g化合物8溶于20ml二氯甲烷，加入495mg N-羟基琥珀酰亚胺，825mg EDCl，室温搅拌1h，反应液经水洗涤，饱和NaCl溶液洗涤，无水硫酸钠干燥，浓缩后得淡黄色固体化合物9，收率：100％。
将2g NH2-PEG 20k-OMe溶于25ml二氯甲烷中，加入40mg化合物9，室温搅拌1h，旋干有机相，残留物经乙醚洗涤后经柱层析分离得化合物10，收率85％。
实施例1：定点突变IL-2蛋白表达质粒的构建及表达
(1)突变位点的选择
在IL-2蛋白(SEQ ID NO:1)上选择了如下所示的突变位点，其中所述的位置是在SEQ ID NO:1中的位置。
表2：突变位点
蛋白名称 氨基酸位置 氨基酸 突变前密码子 突变后密码子
Y45 45 Y TAC TAG
D20 20 D GAT TAG
(2)包含非天然氨基酸的定点突变蛋白的表达
合成上述包含密码子置换的核酸序列连接编码His标签的核酸序列并克隆到pET-21a(+)(addgene：#69740-3)大肠杆菌质粒表达载体中，该载体与pSURAR-YAV质粒共转染到TransB(DE3)菌株(购自全式金，目录号：CD811-02)中。其中，pSURAR-YAV质粒编码琥珀密码子抑制型tRNA和NAEK特异性氨酰tRNA合成酶，通过与包含TAG密码子的核酸序列共转染，可以在特定位点引入非天然氨基酸NAEK；pSURAR-YAV质粒是指从保藏地址为中国微生物菌种保藏委员会普通微生物中心(北京市朝阳区北辰西路1号院，中国科学院微生物研究所)、保藏日期为2013年4月8日、保藏号为CGMCC No：7432的分类命名为大肠埃希氏菌的含有质粒pSUPAR-YAV-tRNA/PylRS 的大肠埃希氏菌中获取的质粒pSUPAR-YAV-tRNA/PylRS。将转化后的菌株接种到含有100ug/ml氨苄青霉素、34ug/ml氯霉素的2ml LB培养基中，然后在37℃、220rpm培养。将过夜培养物在2×YT培养基中稀释至光密度，并在37℃下培养培养物，直至A600nm达到约1.5。添加最终浓度为1mM的UAA(NAEK，实验室自行合成)，然后通过添加最终浓度分别为0.5mM和0.1％的异丙基β-d-硫代吡喃半乳糖苷(IPTG)和L阿拉伯糖诱导蛋白质表达，半小时后，温度降至20℃。表达约18小时后，通过离心收获细胞并重悬于His-Bind缓冲液(20mM磷酸盐，pH 8.0，500mM NaCl，20mM咪唑)中。通过使细胞在1200bar和冷却的条件下通过Micofluidizer两次来提取蛋白。然后在20,000g下离心20分钟，收集上清液并在-80℃下冷冻保存直至进一步处理。由此获得在第Y45位或第D20位被替换为非天然氨基酸的定点突变IL-2蛋白。
实施例2：定点突变IL-2蛋白的PEG修饰
实施例1中获得的上清液通过Ni-NTA琼脂糖(R90101,Invitrogen)进行初步纯化，然后进行点击反应。为了生产定点PEG化的IL-2类似物，使用Ni-NTA His-Bind Resin(Invitrogen)富集上清液中带His标签的IL-2，并合成DBCO-PEG然后将其添加到洗脱缓冲液(20mM磷酸盐，pH 8.0，500mM NaCl，500mM咪唑)中，最终浓度为1mM。反应在4℃下进行，同时轻轻摇动2小时。然后通过阳离子交换色谱法(Resource S，GE Healthcare)和FPLC尺寸排阻色谱法(Superdex 200 increase 10/300 GL，GE Healthcare)纯化PEG化的IL-2，以去除未反应的PEG和IL-2。使用3kDa centrifugal filter unit(Millipore)收集主洗脱峰、浓缩并缓冲液交换到PBS缓冲液中。通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)在变性条件下用考马斯蓝染色检查PEG化反应产物纯度。分别获得两种PEG化IL-2衍生物，分别命名为Y45-20K和D20-20K。电泳结果如图1所示，PEG化的IL-2变体纯度超过95％。
此外，通过实施例1-2所述的方法还制备获得了下述PEG化IL-2衍生物：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。本申请中所涉及的PEG化IL-2变体的命名方式为：[与SEQ ID NO:1相比突变为非天然氨基酸的位置]-[所连接的PEG基团的平均分子量]。以Y45-20K为例，其氨基酸序列与SEQ ID NO:1相比差异在于Y45被置换为非天然氨基酸NAEK，并且该位置进一步连接平均分子量为20kDa的PEG基团。
实施例3：与IL-2受体的结合活性测定
本实施例通过表面等离子共振(SPR)评估PEG化IL-2衍生物对IL-2R三聚体和二聚体复合物的亲和力。人IL-2Rα、IL-2Rβ和IL-2Rγc受体被固定在CM5或蛋白A芯片上，用于在Biacore 8K系统(GE Healthcare)上进行分析。人IL-2Rα(C-末端6xHis)、IL-2Rβ(C-末端Fc)和IL-2Rγ(C-末端6xHis)的胞外结构域购自义翘神州。所有动力学实验均在25℃下使用10mM HEPES、150mM NaCl、3mM EDTA、0.05％Surfactant P、pH 7.4(运行缓冲液)进行。使用Biacore 8K评估软件分析数据，并使用1:1稳定亲和力模型拟合数据以确定KD值和其他动力学参数。使用BCA法(Pierce)测量蛋白浓度。其中，以天然IL-2蛋白(买自PeproTech货号：96-200-02-1000)作为对照。所有样品的浓度均为没有连接PEG的IL-2的质量。
结果如图2所示，其中NA为没有结合力。结果显示，D20-20K、H16-20K、A73-20K、H79-20K对β亚基无亲和力，对二聚体IL-2R的结合亲和力显著降低，但维持对三聚体IL-2R的α链的结合，具有对非β受体(non-β)的偏向性，可称为non-βPEGylate。Y45-20K、F42-20K、E62-20K、K64-20K、P65-20K、E68-20K与α无结合，对三聚体IL-2R的结合亲和力完全消除，维持对二聚体IL-2R的结合，具有对非α受体(non-α)的偏向性，可称为non-α PEGylate；K35-20K、T37-20K、R38-20K、T41-20K、K48-20K、K49-20K对α的结合力数量级在1e-7～1e-6，为弱结合，但对二聚体IL-2R的结合亲和力明显高于三聚体IL-2R，或者虽然对二聚体IL-2R的结合亲和力弱于三聚体IL-2R，但两者之间的差距(例如比值)显著小于天然IL-2对二聚体IL-2R和三聚体IL-2R的结合亲和力的差距，具有明显改善的对二聚体IL-2R的偏向性，因此也属于具有对非α受体(non-α)的偏向性，可称为non-α PEGylate。
实施例4：Y45-20K及D20-20K对CD8+T细胞和Treg细胞激活倾向性测定
本实施例通过测定磷酸化STAT5(pSTAT5)的水平(IL-2R信号传导的关键下游介质)来确定PEG化IL-2变体Y45-20K或D20-20K是否优先激活CD8+T细胞而不是Treg细胞。将大约2×10 5YT-1细胞(表达CD122和CD132受体)、CD25+YT-1(YT-1细胞经慢病毒转导稳定表达CD25受体的稳转细胞系)、人PBMC细胞分别接种在96孔板中，并重悬在RPMI完全培养基中，所述培养基包含连续稀释的天然人IL-2或PEG化IL-2。细胞在37℃下刺激15分钟，然后立即通过加入甲醛至2.0％并在室温下 孵育15分钟来固定。通过在4℃下在冰冷的87％甲醇中悬浮30分钟来实现细胞的透化。固定和透化的细胞用FACS缓冲液洗涤两次，并用以下抗体染色用于流式细胞术分析：抗人CD3-APC/Cy7、CD4-PE/Cy7、CD8-FITC、CD25-APC、CD127-PE、CD56/CD16-Brilliant Violet 605、pSTAT5-Pacific Blue(均购自biolegend)。对于hPBMC特定亚群中的pSTAT5测定，根据制造商的说明，使用磁分离试剂盒(MiltenyiBiotech)分离纯化的细胞群，分别获得记忆CD4+T细胞(MPCD4)、记忆CD8+T细胞(MPCD8)、NK细胞和Treg细胞。
所有FACS抗体均以1:50的稀释度使用。然后将细胞在染色缓冲液中洗涤两次，并在CytoFLEX流式细胞仪(Beckman-Coulter)上测定平均荧光强度(MFI)。数据绘制为减去背景的MFI，归一化为每种细胞类型的最大信号(IL-2,1μg ml-1)。背景定义为未经刺激的细胞中的pSTAT5MFI。Treg细胞定义为CD3+CD8-CD4+CD25highCD127low；NK细胞定义为CD3-CD16+CD56+；CD8+T细胞定义为CD3+CD4-CD8+；MPCD4细胞定义为CD3+CD4+CD8-CD45RO+；MPCD8细胞定义为CD3+CD4-CD8+CD56-CD57-CD45RA-。在减去未经刺激细胞的MFI并归一化为最大信号强度后，使用GraphPad Prism数据分析软件将剂量反应曲线拟合为logistic模型，并计算半数最大有效浓度(EC50值)和相应的95％置信区间。实验按照一式三份进行，并重复三次，所得结果相似。
使用NK衍生的YT-1细胞及CD25+YT-1细胞模型进行检测的结果如图3a所示，其中Y45-20K在CD25(IL-2Rα)-和CD25(IL-2Rα)+YT-1细胞上表现出显著的区别，与野生型IL-2明显不同；D20-20K在非常高的剂量下仍仅显示基线水平的Treg刺激活性(pSTAT5激活的5-20％)，这与其与β亚基不能结合有关。
此外，使用从健康供体PBMC中分离的各种免疫细胞亚群中重新评估了PEG化IL-2变体对Teff细胞的选择性激活，结果如图3b。我们发现IL-2在所有测试剂量下都刺激Treg和记忆CD4+T、记忆CD8+T和NK细胞，而Y45-20K优先在记忆CD4+、记忆CD8+和NK细胞中诱导STAT5磷酸化，但不是Treg细胞。D20-20K在记忆CD4+、记忆CD8+、NK和Treg细胞中均不诱导STAT5磷酸化。
上述结果与结合亲和力评估的结果一致，表明通过阻断受体结合位点的特定区域可以解除IL-2的多效性，并且Y45-20K由于完全缺乏对IL-2Rα结合具有优先刺激Teffs而不是Treg细胞的理想能力，而D20-20K对于CD8+T和Treg细胞的活化均显著降低。
实施例5：PEG化IL-2变体Y45-20K/D20-20K的PK/PD特性分析
本实施例考察PEG化IL-2变体的药代动力学特性。选择平均体重约为20g的雌性C57BL/6小鼠(购自北京维通利华实验动物技术有限公司)，将小鼠随机分为不同组(n＝3)并通过皮下注射注射PBS、野生型人IL-2或PEG化IL-2变体(0.25mg/kg，基于hIL-2)。在选定的时间点(30min、2h、4h、8h、24h、48h、72h和96h)，从小鼠眼窝静脉中取血(每次100μL)，然后在4℃以4000g离心15分钟。分离血浆并储存在-80℃。使用人IL-2ELISA试剂盒(Sinobiological)测定IL-2的浓度。药代动力学参数通过Kinetica 5.1软件进行分析，以平均值±标准差表示。
结果如图4所示，与野生型IL-2相比，PEG化IL-2变体具有增加的清除半衰期(T1/2)、增强的血液浓度维持(药物浓度下面积与时间曲线，AUC)，并且平均停留时间(MRT)增加，峰值时间比IL-2长14倍，表明清除率大幅降低。上述结果表明，Y45-20K具有优越的药代动力学特性。此外，D20-20K也具有优越的药代动力学特性。
实施例6：PEG化IL-2变体Y45-20K/D20-20K的抗肿瘤协同增效作用
以上实施例的数据已经证实Y45-20K具有优先激活CD8+T细胞的活性，然而其在高剂量下仍然激活Treg细胞。如上实施例中已证实的，D20-20K保持对IL-2Rα的选择性亲和力同时显著降低了对CD25+YT-1的激活，该独特特性可能与其与IL-2Rβ的不可检测结合有关，因此，如果将D20-20K与Y45-20K组合进行治疗，D20-20K可能在空间上占据IL-2Rα并竞争性地阻断其与Y45-20K的轻度相互作用导致CD8+T细胞的过度激活及终末分化和对Treg的激活。据此，本实施例考察了D20-20K与Y45-20K的协同抗肿瘤作用。
6.1 D20-20K对Y45-20K介导的体外T细胞增殖、活化的影响
实验步骤
本实施例考察D20-20K对于Y45-20K的激活偏倚是否存在剂量依赖的影响。将2×10 5人PBMC细胞用100ul RPMI1640+10％FBS重悬在96孔板中，实验孔中加入50ul0.4ug/ml Y45-20K(终浓度0.1ug/ml)，再加入50ul不同浓度梯度的D20-20K，使其终浓度分别为1ug/ml，0.1ug/ml，0.01ug/ml，0.001ug/ml，设置的重复实验孔分别来自三个不同的健康个体。细胞在37℃下培养72h，然后FACS缓冲液洗涤两次，并用以下抗体染色用于流式细胞术分析：抗人CD3-APC/Cy7、CD4-PE/Cy7、CD8-FITC、CD25- APC、Foxp3-Pacific Blue，CD69-PE。Treg细胞定义为CD3+CD8-CD4+CD25+Foxp3+；CD8+T细胞定义为CD3+CD4-CD8+,用流式细胞仪统计Treg和CD8T细胞数量以及各自激活标志(Treg-Foxp3,CD8+T-CD69)的平均荧光强度(MFI)。
将2×10 5YT-1细胞、CD25+YT-1分别接种在96孔板中，用100ul RPMI1640+10％FBS重悬在96孔板中，实验孔中加入50ul 0.4ug/ml Y45-20K(终浓度0.1ug/ml)，再加入50ul不同浓度梯度的D20-20K，使其终浓度分别为1ug/ml，0.1ug/ml，0.01ug/ml，0.001ug/ml。细胞在37℃下刺激15分钟，然后立即通过加入甲醛至2.0％并在室温下孵育15分钟来固定。通过在4℃下在冰冷的87％甲醇中悬浮30分钟来实现细胞的透化。固定和透化的细胞用FACS缓冲液洗涤两次，并用pSTAT5-Pacific Blue抗体染色用于流式细胞术分析。抗体以1:50的稀释度使用。然后将细胞在染色缓冲液中洗涤两次，并在CytoFLEX流式细胞仪(Beckman-Coulter)上测定平均荧光强度(MFI)。数据绘制为减去背景的MFI，归一化为每种细胞类型的最大信号(Y45-20K,0.1μg ml -1)。背景定义为未经刺激的细胞中的pSTAT5MFI。在减去未经刺激细胞的MFI并归一化为最大信号强度后，使用GraphPad Prism数据分析软件将剂量反应曲线拟合为logistic模型，并计算半数最大有效浓度(EC50值)和相应的95％置信区间。实验按照一式三份进行，并重复三次，所得结果相似。
结果如图5所示，D20-20K对Y45-20K介导的Treg细胞而非CD8+T细胞的活化和增殖具有剂量依赖性抑制作用(图5a，b为对Treg增殖与活化的影响，图5c，d为对CD8+T增殖与活化的影响)。CD25+YT-1细胞中的STAT5磷酸化被D20-20K以剂量依赖性方式抑制，EC50为0.39ug/ml(图5e)，而在天然YT细胞中未观察到这种抑制(图5f)。
6.2 D20-20K对Y45-20K介导的健康小鼠体内T增殖、活化的影响
实验步骤
C57BL/6小鼠(6至8周龄，雌性)后颈部皮下给药45-20K(0.25mg/kg x 3，每隔一天)、20-20K(0.25mg/kg x 3，每隔一天)、45-20K和20-20K(各0.25mg/kg x 3，每隔一天)、或IL-2(0.25mg/kg每天x 5)，等体积PBS注射为空白对照组。在给药结束后三天进行脾脏和淋巴结的流式检测。
结果如图6所示，与其他组小鼠相比，Y45-20K/D20-20K联合治疗的小鼠脾脏中CD8+T细胞和MPCD8+T比例近一步升高，相比于Y45-20K单独使用，Y45-20K/D20- 20K联合治疗显著性降低Treg的增殖。
6.3 D20-20K与Y45-20K在小鼠肿瘤模型中的抗肿瘤作用
C57BL/6小鼠(6至8周龄)右侧(right flank)皮下植入B16F10黑色素瘤细胞(中国科学院细胞库(中国上海)，每只动物5 x 10 5)。当肿瘤达到1500mm 3大小时处死小鼠。肿瘤体积计算如下：V＝a2*b/2，其中a是宽度，b是肿瘤长度(均以毫米为单位)。植入后7天，当肿瘤测量为100mm 3时，给动物施用45-20K(0.25mg/kg x 3，每隔一天)、20-20K(0.25mg/kg x 3，每隔一天)、45-20K和20-20K(各0.25mg/kg x 3，每隔一天)、或IL-2(0.25mg/kg每天x 5)。第14天，切碎肿瘤并在含有2mg/ml II型和IV型胶原酶(GIBCO BRL)和0.5mg/ml DNase(Sigma Aldrich)的缓冲液中在37℃下消化13分钟，形成单细胞悬液，随后通过流式细胞术确定肿瘤微环境中免疫细胞亚型。在给药后14天对冷冻肿瘤切片进行T细胞密度的免疫组织化学分析。
结果如图7所示，Y45-20K与D20-20K治疗组合表现出非常突出的抗肿瘤作用，肿瘤负荷明显减少，小鼠存活率显著延长(图7a)。随着肿瘤负荷的降低，与其他小鼠相比，Y45-20K/D20-20K联合治疗的小鼠在脾脏、肿瘤引流淋巴结(TDLN)和特别是肿瘤组织中的CD8+T细胞比例增加，但Treg细胞比例无明显变化(图7b)。该结果与免疫组织化学染色一致，表明与天然IL-2和PBS处理相比，Y45-20K和D20-20K的共同处理诱导肿瘤组织中最大量的T细胞浸润(图7c)。
记忆性CD8细胞在肿瘤免疫中发挥着重要的作用，对荷瘤小鼠淋巴结中T细胞亚群分析表明，Y45-20K单独处理组或Y45-20K联合D20-20K治疗组中，淋巴结中Tcm，MPCD8细胞数量显著提高10倍左右，而Y45-20K联合D20-20K治疗组在Y45-20K单独处理组的基础上，Tcm和MPCD8的数量又有进一步的提高。与之对应的，两实验组中的T
Figure PCTCN2022129975-appb-000016
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
Figure PCTCN2022129975-appb-000017
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。
6.4 D20-20K与Y45-20K联合对血管渗漏综合征(VLS)的影响
传统IL-2治疗存在血管渗漏综合征(VLS)的高风险，通常认为这是由IL-2与CD25+肺内皮细胞结合引起的，据此本实施例还评估了联合治疗对肺水肿的影响。采用与上述类似的治疗方案，其中PEG化IL-2变体的施用改为高给药频率(每天，共五次)。处死小鼠后，对肺组织进行称重，获得肺组织细胞悬液进行流式检测，制备肺组织切片进行免疫组织化学分析。
结果如图9所示，表达高水平CD31而非其他免疫细胞谱系标志物(Lin)的肺细胞被定义为内皮细胞(Ly5.2-B220-CD3-NK1.1-CD11b-CD11c-CD31+)。结果发现Y45-20K单给药组导致明显的肺水肿，表现为肺湿重增加(图9a)，肺内皮细胞比例降低(图9b)，肺内淋巴细胞浸润增加(图9c)；然而，在与D20-20K共同给药后这种副作用明显减轻，可能是由于D20-20K空间上占据肺内皮细胞的IL-2Rα。
上述数据表明Y45-20K与D20-20K在抗肿瘤治疗中显示出显著的协同作用，其选择性诱导CD8+T细胞，对Treg细胞的影响最小，并且能够通过保护肺内皮细胞免受Y45-20K或内源性IL-2的激活来减轻VLS，具有显著有利的技术效果。
实施例7：PEG化IL-2变体Y45-20K/D20-20K用于CAR-T细胞体外培养的优势
IL-2为CAR-T细胞体外扩增培养的关键营养因子，由于其本身不可避免的激活供体细胞中的Treg细胞以及不可避免的过度激活CD8+T细胞以及导致其终末分化影响CAR-T细胞活性而大幅削弱CAR-T细胞疗法的疗效果，据此本实验Y45-20K联合D20-20K替代传统IL-2对CAR-T细胞疗法的影响。
7.1 D20-20K与Y45-20K联合对CAR-T细胞体外培养增殖的影响
CAR慢病毒载体的构建：
抗CD19CAR包含FMC63anti-CD19scFv(SEQ ID NO:12)、CD8a铰链区(SEQ ID NO:14)和跨膜区(SEQ ID NO:16)以及4-1BB胞质结构域(SEQ ID NO:18)和CD3z胞质结构域(SEQ ID NO:20)，其表达盒委托爱康得生物科技(苏州)有限公司合成并克隆到慢病毒载体中。使用Lipofectamine 3000(Life Technologies)用抗CD19CAR、pspax2(addgene，#12260)和pMD2.g(addgene，#12259)质粒转染HEK293T细胞。转染后6小时更换培养基，转染后48小时收集病毒上清液。以25,000rpm超速离 心2小时将病毒颗粒浓缩30倍，并在-80℃冷冻直至准备使用。
CAR-T的制备：
使用Dynabead人T细胞试剂盒(Life Technologies)从外周血单个核细胞中纯化人T细胞，并在感染前用CD3/cd28磁珠(Life Technologies)激活24小时。将浓缩慢病毒(FMC63-AntiCD19-CAR Lentivirus)应用于活化的人T细胞(24孔培养板中10 6/孔，MOI＝1)，加入10mg/mL polybrene和100ng/mL IL-2或100ng/mL 45-20K，100ng/mL D20-20K,100ng/mL D20-20K+100ng/mL Y45-20K在1000g条件下32℃离心2h。次日，用含有相对应组别的IL-2及其类似物的新鲜培养基替换上清液，将转导后的T细胞置于完全生长培养基中保持0.5 x10 6cells/mL，每3天更换一次含有IL-2的培养基。实时监测细胞数量和凋亡状态。
结果如图10所示，无论用Y45-20K，D20-20K还是Y45-20K联合D20-20K替代IL2均不影响CAR的转导效率(图10a)，用IL-2变体对CAR-T细胞培养10天后发现，D20-20K替代IL-2进行培养CAR-T细胞几乎不增殖，而Y45-20K替代IL-2进行培养细胞增殖速度显著高于用IL-2培养，Y45-20K联合D20-20K替代IL-2进行培养CAR-T细胞增殖速度与单用Y45-20K相似(图10b)。细胞凋亡检测发现用IL-2培养会引起CAR-T细胞的凋亡，这与IL-2对T细胞的过度激活及诱导终末分化有关，而Y45-20K联合D20-20K几乎不诱导CAR-T细胞的凋亡(图10c)。
7.2 D20-20K与Y45-20K联合对CAR-T细胞体外培养衰老、耗竭的影响
实验步骤
将上述制备的CAR-T在各自对应的IL-2培养10-14天，进行细胞表型的相关检测。表面抗体用荧光标记抗体(CD4-PE/Cy7、CD8-FITC、CD25-BV785、CD57-PB、CD45RA-BV510、CD62L-Percp/cy5.5、CCR7-PE、PD-1-BV650、LAG-3-BV421)进行荧光染色，在4℃孵育30分钟。对于核内染色，CAR-T细胞使用Foxp3/转录因子染色缓冲液(eBioscience)固定和渗透，然后用perm缓冲液中的Foxp3抗体染色。样品用PBS洗涤两次，悬浮于流式细胞仪染色缓冲液(eBioscience)中。对于细胞内细胞因子染色，使用500x Cell Activation Cocktail(with Brefeldin A，达科为，423303)，6h 37℃培养后进行洗涤。最后一次洗涤后，固定细胞，根据制造商的说明，使用eBioscience细胞内固定和渗透缓冲套件(Thermo Fisher Scientific)进行渗透和染色。然后用特异性抗体4℃孵育30分钟：IL-2-BV421、IL-10-APC、Granzy B-percp/cy5.5。
结果如图11所示，体外培养的CAR-T细胞经PMA刺激后，Y45-20K联合D20- 20K培养的CAR-T细胞分泌颗粒酶B和IL10的明显低于IL-2组，说明CAR-T细胞的终末分化及衰老程度降低(图11a，b)，且IL-2的分泌量明显增多，同样说明CAR-T细胞处于一个分化程度较低的干性状态(图11c)。其次，流式检测显示Y45-20K联合D20-20K培养的CAR-T细胞表面耗竭与衰老相关受体如PD-1、LAG-3和CD57明显降低(图11d-f)，再次证明Y45-20K联合D20-20K可以逆转IL-2用于CAR-T细胞体外培养引起的衰老、耗竭状态。
7.3 D20-20K与Y45-20K联合对CAR-T细胞体外培养中不同分化阶段的T细胞的衰老、耗竭的影响
实验步骤
使用Dynabead人T细胞试剂盒(Life Technologies)从外周血单个核细胞中纯化人T细胞，并用上述方法进行CAR-T的制备。为进一步研究不同细胞因子对于CAR-T细胞分化的影响，我们通过流式细胞仪(分选型，SymphonyS6，BD)分选出处在不同分化状态的CAR-T细胞进行单独培养。T
Figure PCTCN2022129975-appb-000018
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。
我们对CAR-T细胞中处于不同分化阶段的初始T细胞(T
Figure PCTCN2022129975-appb-000019
)、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
Figure PCTCN2022129975-appb-000020
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
Figure PCTCN2022129975-appb-000021
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T effector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。
实施例8：None-α变体与None-β变体联用对维持T细胞干性，降低T细胞耗竭的影响
本实施例对实施例1-2中制备的Non-α变体(F42-20K、Y45-20K、E62-20K、P65-20K、E68-20K)与Non-β变体(D20-20K、H16-20K、A73-20K、H79-20K)组合进行体外活性验证。
实验步骤：
取健康人外周血，用外周血人淋巴细胞分离液分离出PBMC，用磁珠分选试剂盒分离出CD3阳性T淋巴细胞，加入bead：cell＝1：1的CD3/CD28刺激磁珠，接种密度控制在1x10 6/ml，24孔板，每孔1ml，并且用含有不同种类但浓度相同的细胞因子进行培养(100ng/ml)，每72小时换一次液，连续培养两周后进行流式检测。检测抗体包括：APC/Cy7 anti-human CD3,PE/Cy7 anti-human CD4,FITC anti-human CD8,BV785 anti-human PD-1,BV605 anti-human TIM-3,Pacific Blue anti-human CD57,APC anti-human IL-10,PE anti-human Perforin,Percp/cy5.5 anti-human Granzyme B,BV510 anti-human IFN-γ.
实验结果：
用none-α变体(Y45-20K)处理，相比于IL-2，CD8和CD4 T细胞表面的免疫检查点PD-1和TIM-3的表达有所降低(图13a)，效应细胞因子Perforin，Granzyme B以及IFN-γ表达水平显著降低(图13b)，凋亡相关生物标志物的表达CD57、IL-10表达水平下降(图13c)，表明none-α能够一定程度降低T细胞在体外培养过程中的耗竭程度，维持细胞的stem-cell-like干细胞样特性。当none-α变体(Y45-20K)与none-β变体(D20-20K、H16-20K、A73-20K、H79-20K)联用后，PD-1和TIM-3的表达呈现出近一步降低，效应细胞因子Perforin，Granzyme B以及IFN-γ表达水平近一步下降，凋亡相关生物标志物CD57、IL-10表达水平近一步下降(图13a-c)。
在上述筛选结果基础上，优选none-β变体(D20-20K)，验证与不同none-α变体(F42-20K、Y45-20K、E62-20K、P65-20K、E68-20K)联用对T细胞激活作用的影响，实验结果表明，D20-20K与不同none-α变体联用后，对改善CD8和CD4 T细胞生 存状态的影响程度不同，但均可在一定程度上近一步降低免疫检查点PD-1和TIM-3的表达、效应细胞因子Perforin，Granzyme B、IFN-γ表达以及减少凋亡相关生物标志物的表达CD57、IL-10表达水平(图14a-c)。因此，None-β联用None-α方案，会协同none-α作用的基础上，进一步改善T细胞的生存状态，如降低T细胞生长耗竭程度，减少因过度激活诱导的细胞凋亡，以及延缓向终端效应细胞分化。
上述结果表明，将具有对非α受体偏向性的non-α变体与具有对非β受体偏向性的non-β变体联用会产生明显的协同作用，带来显著有利的治疗效果。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部分为由所附权利要求及其任何等同物给出。

Claims (76)

  1. 组合物，其包含：
    (1)第一定点修饰的IL-2，其与野生型IL-2相比在第一氨基酸位置的残基包含PEG基团修饰，其中，所述第一定点修饰的IL-2对IL-2受体α(IL-2Rα)亚基不结合或以大于1E-8M(例如数量级为1E-7～1E-6M)的KD值结合；以及
    (2)第二定点修饰的IL-2，其与野生型IL-2相比在第二氨基酸位置的残基包含PEG基团修饰，其中，所述第二定点修饰的IL-2对IL-2受体β(IL-2Rβ)亚基不结合。
  2. 权利要求1所述的组合物，其中，所述第一定点修饰的IL-2(i)以小于1E-8M(例如数量级为1E-9～1E-8M)的KD值结合IL2-Rβγ二聚体，和/或，(ii)对IL2-Rαβγ三聚体不结合或以大于1E-8(例如数量级为1E-7～1E-6)的KD值结合。
  3. 权利要求1或2所述的组合物，其中，所述第二定点修饰的IL-2(i)以小于1E-8(例如数量级为1E-9～1E-8)的KD值结合IL2-Rαβγ三聚体，和/或，(ii)对IL2-Rβγ二聚体不结合。
  4. 权利要求1-3任一项所述的组合物，其中，所述第一氨基酸位置选自F42、Y45、E62、K64、P65、E68、K35、T37、R38、T41、K48、K49。
  5. 权利要求1-3任一项所述的组合物，其中，所述第一氨基酸位置选自F42、Y45、E62、K64、P65、E68。
  6. 权利要求1-5任一项所述的组合物，其中，所述第二氨基酸位置选自H16、D20、A73、H79；
    优选地，所述第二氨基酸位置选自D20。
  7. 权利要求1-6任一项所述的组合物，其中，所述第一氨基酸位置为F42、Y45、E62、K64、P65或E68，所述第二氨基酸位置为D20；或者，所述第一氨基酸位置为 Y45，所述第二氨基酸位置为H16、D20、A73、H79；
    优选地，所述第一氨基酸位置为Y45，所述第二氨基酸位置为D20。
  8. 权利要求1-7任一项所述的组合物，其中，所述第一定点修饰的IL-2包含的PEG修饰基团的平均分子量为5～60kDa，例如为5kDa、10kDa、15kDa、20kDa、25kDa、30kDa、35kDa、40kDa、45kDa、50kDa或60kDa；
    优选地，所述第一定点修饰的IL-2包含的PEG修饰基团的平均分子量为5～40kDa，例如为5～30kDa、5～25kDa、5～20kDa、10～40kDa、10～30kDa、15～30kDa、10～25kDa或15～25kDa，例如为5kDa、10kDa、15kDa、20kDa、25kDa、30kDa或40kDa。
  9. 权利要求1-8任一项所述的组合物，其中，所述第二定点修饰的IL-2包含的PEG修饰基团的平均分子量为5～60kDa，例如为5kDa、10kDa、15kDa、20kDa、25kDa、30kDa、35kDa、40kDa、45kDa、50kDa或60kDa；
    优选地，所述第二定点修饰的IL-2包含的PEG修饰基团的平均分子量为5～40kDa，例如为5～30kDa、5～25kDa、5～20kDa、10～40kDa、10～30kDa、15～30kDa、10～25kDa或15～25kDa，例如为5kDa、10kDa、15kDa、20kDa、25kDa、30kDa或40kDa。
  10. 权利要求1-9任一项所述的组合物，其中，所述第一氨基酸位置的残基被突变为非天然氨基酸，所述非天然氨基酸上连接有所述PEG基团。
  11. 权利要求1-10任一项所述的组合物，其中，所述第二氨基酸位置的残基被突变为非天然氨基酸，所述非天然氨基酸上连接有所述PEG基团。
  12. 权利要求10或11所述的组合物，其中，所述非天然氨基酸含有化学官能基团(例如，羰基、炔基或叠氮基团)；所述PEG基团包含能与该化学官能基团发生化学反应的标记基团，从而所述PEG基团连接至非天然氨基酸。
  13. 权利要求10或11所述的组合物，其中，所述非天然氨基酸含有叠氮基团，所述 PEG基团包含能与叠氮基团发生click化学反应的标记基团，从而所述PEG基团连接至非天然氨基酸。
  14. 权利要求13所述的组合物，其中，所述能与叠氮基团发生click化学反应的标记基团为包含二苯并环辛炔基团的化学部分，例如DBCO、DIBO或BCN。
  15. 权利要求10-14任一项所述的组合物，其中，所述非天然氨基酸为含有叠氮基团的赖氨酸衍生物或酪氨酸衍生物，例如Nε-2-叠氮乙氧羰基-L-赖氨酸(NAEK)或2-amino-3-(4-(azidomethyl)phenyl)propanoic acid。
  16. 制备权利要求1-15任一项所述的组合物的方法，其包括制备所述第一定点修饰的IL-2和制备所述第二定点修饰的IL-2，其中，
    制备所述第一定点修饰的IL-2包括：
    -提供：(a1)第一定点突变的IL-2，其与野生型IL-2相比在第一氨基酸位置的残基被突变为非天然氨基酸；(b1)由标记基团修饰的PEG基团，所述标记基团能与所述非天然氨基酸形成共价键；
    -将(a1)与(b1)共孵育，通过化学反应将非天然氨基酸与PEG基团偶联；
    制备所述第二定点修饰的IL-2包括：
    -提供：(a2)第二定点突变的IL-2，其与野生型IL-2相比在第二氨基酸位置的残基被突变为非天然氨基酸；(b2)由标记基团修饰的PEG基团，所述标记基团能与所述非天然氨基酸形成共价键；
    -将(a2)与(b2)共孵育，通过化学反应将非天然氨基酸与PEG基团偶联。
  17. 权利要求16所述的方法，其中，(a1)中所述的非天然氨基酸含有化学官能基团(例如，羰基、炔基、叠氮基团)，(b1)中所述PEG基团包含的标记基团能与该化学官能团发生化学反应形成共价键；和/或，(a2)中所述的非天然氨基酸含有化学官能基团(例如，羰基、炔基、叠氮基团)；(b2)中所述PEG基团包含的标记基团能与该化学官能团发生化学反应形成共价键。
  18. 权利要求16所述的方法，其中，(a1)和(a2)中所述的非天然氨基酸包含叠 氮基团。
  19. 权利要求16所述的方法，其中，(a1)和(a2)中所述的非天然氨基酸为包含叠氮基团的赖氨酸衍生物或酪氨酸衍生物，例如Nε-2-叠氮乙氧羰基-L-赖氨酸(NAEK)或2-amino-3-(4-(azidomethyl)phenyl)propanoic acid。
  20. 权利要求16-19任一项所述的方法，其中，
    所述制备所述第一定点修饰的IL-2包括：
    -提供：(a1)第一定点突变的IL-2，其与野生型IL-2相比在第一氨基酸位置的残基被突变为含有叠氮基团的非天然氨基酸；(b1)由标记基团修饰的PEG基团，所述标记基团能与叠氮基团发生click化学反应；
    -将(a1)与(b1)共孵育，通过click反应将非天然氨基酸与PEG基团偶联；
    所述制备所述第二定点修饰的IL-2包括：
    -提供：(a2)第二定点突变的IL-2，其与野生型IL-2相比在第二氨基酸位置的残基被突变为含有叠氮基团的非天然氨基酸；(b2)由标记基团修饰的PEG基团，所述标记基团能与叠氮基团发生click化学反应；
    -将(a2)与(b2)共孵育，通过click反应将非天然氨基酸与PEG基团偶联。
  21. 权利要求20所述的方法，其中，所述click化学反应为无铜的click化学反应。
  22. 权利要求20或21所述的方法，其中，所述能与叠氮基团发生click化学反应的标记基团为包含炔基的化学部分，例如包含二苯并环辛炔基团的化学部分，例如DBCO、DIBO或BCN。
  23. 权利要求20或21所述的方法，其中，所述能与叠氮基团发生click化学反应的标记基团为DBCO。
  24. 权利要求16-23任一项所述的方法，其中，通过非天然氨基酸正交翻译技术提供所述第一定点突变的IL-2和所述第二定点突变的IL-2。
  25. 权利要求24所述的方法，所述非天然氨基酸正交翻译技术包括以下步骤：
    -获得编码定点突变的IL-2的核酸序列，其中，待突变氨基酸位置对应的密码子被突变为TAG；
    -将所述编码定点突变的IL-2的核酸序列与载体可操作地连接，得到定点突变序列表达载体；
    -将所述定点突变序列表达载体与编码琥珀密码子抑制型tRNA和对非天然氨基酸特异性的氨酰tRNA合成酶的载体共转染宿主细胞，在含有非天然氨基酸的培养基中培养并诱导表达，得到定点突变为非天然氨基酸的IL-2。
  26. 权利要求25所述的方法，其中，所述非天然氨基酸为Nε-2-叠氮乙氧羰基-L-赖氨酸(NAEK)；优选地，所述对非天然氨基酸特异性的氨酰tRNA合成酶为NAEK特异性氨酰tRNA合成酶。
  27. 试剂盒，其包含：权利要求1-15任一项所述的组合物；
    优选地，所述试剂盒进一步包含包装说明书，所述包装说明书包含使用所述组合物来在体外制备和/或培养用于过继性细胞治疗的免疫细胞的说明。
  28. 权利要求27所述的试剂盒，其中，所述用于过继性细胞治疗的免疫细胞是经改造的免疫细胞，其表达嵌合抗原受体和/或包含编码所述嵌合抗原受体的核酸分子。
  29. 权利要求27所述的试剂盒，其中，所述用于过继性细胞治疗的免疫细胞是肿瘤浸润淋巴细胞(TIL)。
  30. 试剂盒，其包含：权利要求1-15任一项所述的组合物以及编码嵌合抗原受体的核酸分子；
    优选地，所述试剂盒进一步包含包装说明书，所述包装说明书包含使用所述组合物中以及核酸分子来在体外制备用于过继性细胞治疗的经改造的免疫细胞的说明，所述经改造的免疫细胞表达嵌合抗原受体和/或包含编码所述嵌合抗原受体的核酸分子。
  31. 权利要求30所述的试剂盒，其中，所述编码嵌合抗原受体的核酸分子存在于表 达载体中。
  32. 权利要求31所述的试剂盒，其中所述表达载体为病毒(例如，慢病毒、逆转录病毒或腺病毒)载体。
  33. 试剂盒，其包含：权利要求1-15任一项所述的组合物以及用于过继性细胞治疗的免疫细胞；
    优选地，所述试剂盒进一步包含包装说明书，所述包装说明书包含使用所述组合物来在体外培养所述免疫细胞以用于过继性细胞治疗的说明。
  34. 权利要求33所述的试剂盒，其中，用于过继性细胞治疗的免疫细胞是经改造的免疫细胞，其表达嵌合抗原受体和/或包含编码所述嵌合抗原受体的核酸分子。
  35. 权利要求34所述的试剂盒，其中，所述经改造的免疫细胞包括表达IL2-Rαβγ三聚体的淋巴细胞，如T细胞、NK细胞或其任意组合。
  36. 权利要求33所述的试剂盒，其中，用于过继性细胞治疗的免疫细胞是肿瘤浸润淋巴细胞(TIL)。
  37. 权利要求28、30-32、34、35任一项所述的试剂盒，其中，所述嵌合抗原受体包括细胞外抗原结合结构域、跨膜结构域、以及一个或多个胞内信号传导结构域。
  38. 权利要求37所述的试剂盒，其中，所述细胞外抗原结合结构域包含特异性结合肿瘤相关抗原的抗体或抗原结合片段(例如scFv)。
  39. 权利要求37所述的试剂盒，其中，所述细胞外抗原结合结构域包含特异性结合CD19的抗体或抗原结合片段(例如scFv)。
  40. 培养用于过继性细胞治疗的免疫细胞的方法，所述方法包括在包含第一定点修饰的IL-2和第二定点修饰的IL-2的细胞培养基中培养所述细胞，其中，所述第一定点修饰 的IL-2和第二定点修饰的IL-2如权利要求1-15任一项中定义。
  41. 权利要求40所述的方法，其中，所述用于过继性细胞治疗的免疫细胞是经改造的免疫细胞，其表达嵌合抗原受体和/或包含编码所述嵌合抗原受体的核酸分子；
    优选地，所述嵌合抗原受体如权利要求37-39任一项中定义。
  42. 权利要求41所述的方法，其中，所述经改造的免疫细胞包括表达IL2-Rαβγ三聚体的淋巴细胞，如T细胞、NK细胞或其任意组合。
  43. 权利要求40所述的方法，其中，所述用于过继性细胞治疗的免疫细胞是肿瘤浸润淋巴细胞(TIL)。
  44. 制备用于过继性细胞治疗的免疫细胞的方法，所述用于过继性细胞治疗的免疫细胞是经改造的免疫细胞，其表达嵌合抗原受体和/或包含编码所述嵌合抗原受体的核酸分子，其中，所述方法包括：
    (1)提供来自患者或者健康供体的免疫细胞；
    (2)在存在第一定点修饰的IL-2和第二定点修饰的IL-2的条件下，将编码嵌合抗原受体的核酸分子引入步骤(1)所述的免疫细胞，从而提供所述经改造的免疫细胞；其中，所述第一定点修饰的IL-2和第二定点修饰的IL-2如权利要求1-15任一项中定义。
  45. 权利要求44所述的方法，其中步骤(2)在包含所述第一定点修饰的IL-2和第二定点修饰的IL-2的细胞培养基中进行。
  46. 权利要求44或45所述的方法，其中，在步骤(2)中所述编码嵌合抗原受体的核酸分子存在于表达载体中。
  47. 权利要求44-46任一项所述的方法，其中，在步骤(2)中所述编码嵌合抗原受体的核酸分子由病毒(例如，慢病毒、逆转录病毒或腺病毒)载体通过感染的方式引入细胞。
  48. 权利要求44-47任一项所述的方法，其中，在步骤(1)中，所述免疫细胞经预处理，所述预处理包括免疫细胞的分选、激活和/或增殖。
  49. 权利要求48所述的方法，其中，所述预处理包括将免疫细胞与抗CD3抗体和抗CD28抗体接触，从而刺激所述免疫细胞并诱导其增殖，由此生成经预处理的免疫细胞。
  50. 权利要求44-49任一项所述的方法，其中，在步骤(2)之后所述方法还包括：(3)在包含所述第一定点修饰的IL-2和第二定点修饰的IL-2的细胞培养基中继续培养步骤(2)获得的免疫细胞的步骤。
  51. 权利要求44-50任一项所述的方法，其中，所述免疫细胞包括表达IL2-Rαβγ三聚体的淋巴细胞，如T细胞、NK细胞或其任意组合。
  52. 权利要求36-43任一项所述的方法，其中，所述嵌合抗原受体如权利要求37-39任一项中定义。
  53. 制备用于过继性细胞治疗的免疫细胞的方法，所述用于过继性细胞治疗的免疫细胞是肿瘤浸润淋巴细胞(TIL)，其中，所述方法包括：从肿瘤组织中分离浸润淋巴细胞并在包含第一定点修饰的IL-2和第二定点修饰的IL-2的细胞培养基中进行培养，所述第一定点修饰的IL-2和第二定点修饰的IL-2如权利要求1-15任一项中定义。
  54. 用于过继性细胞治疗的免疫细胞，其由权利要求40-53任一项所述的方法获得。
  55. 免疫细胞群，其包括权利要求54所述的免疫细胞。
  56. 权利要求1-15任一项所述的组合物用于体外制备或培养用于过继性细胞治疗的免疫细胞的用途。
  57. 权利要求56所述的用途，其中，所述用于过继性细胞治疗的免疫细胞为经改造的免疫细胞，其表达嵌合抗原受体和/或包含编码所述嵌合抗原受体的核酸分子；
    优选地，所述嵌合抗原受体如权利要求37-39任一项中定义。
  58. 权利要求57所述的用途，其中，所述经改造的免疫细胞包括表达IL2-Rαβγ三聚体的淋巴细胞，如T细胞、NK细胞或其任意组合。
  59. 权利要求56所述的用途，其中，所述用于过继性细胞治疗的免疫细胞是肿瘤浸润淋巴细胞(TIL)。
  60. 药物组合物，其包含权利要求1-15任一项所述的组合物以及药学上可接受的载体和/或赋形剂；
    例如，所述第一定点修饰的IL-2和第二定点修饰的IL-2在分开的组合物或剂型中；
    例如，所述第一定点修饰的IL-2和第二定点修饰的IL-2在相同的组合物或剂型中。
  61. 权利要求1-15任一项所述的组合物或权利要求60所述的药物组合物用于增强免疫应答、预防和/或治疗增生性疾病的用途，或者，在制备用于增强免疫应答、预防和/或治疗增生性疾病的药物中的用途；
    例如，所述组合物中的第一定点修饰的IL-2和第二定点修饰的IL-2同时、分开或相继施用。
  62. 用于增强免疫应答、预防和/或治疗增生性疾病的方法，其包括向有需要的受试者施用权利要求1-15任一项所述的组合物或权利要求60所述的药物组合物；
    优选地，所述受试者为哺乳动物，例如人；
    优选地，所述组合物中的第一定点修饰的IL-2和第二定点修饰的IL-2同时、分开或相继施用。
  63. 药盒，其包含含有权利要求1-15任一项所述的组合物中的第一定点修饰的IL-2和可选的药学上可接受的载体和/或赋形剂的药物，和包装说明书，所述包装说明书包含将所述药物与含有权利要求1-15任一项所述的组合物中的第二定点修饰的IL-2和可选的药学上可接受的载体和/或赋形剂的组合物组合施用来在受试者中增强免疫应答、预防和/或治疗增生性疾病(例如肿瘤)的说明。
  64. 药盒，其包含含有权利要求1-15任一项所述的组合物中的第二定点修饰的IL-2和可选的药学上可接受的载体和/或赋形剂的药物，和包装说明书，所述包装说明书包含将所述药物与含有权利要求1-15任一项所述的组合物中的第一定点修饰的IL-2和可选的药学上可接受的载体和/或赋形剂的组合物组合施用来在受试者中增强免疫应答、预防和/或治疗增生性疾病(例如肿瘤)的说明。
  65. 药盒，其包含含有权利要求1-15任一项所述的组合物中的第一定点修饰的IL-2和可选的药学上可接受的载体和/或赋形剂的第一药物，含有权利要求1-15任一项所述的组合物中的第二定点修饰的IL-2和可选的药学上可接受的载体和/或赋形剂的第二药物；
    优选地，所述药盒进一步包含包装说明书，所述包装说明书包含施用所述第一药物和所述第二药物来在受试者中增强免疫应答、预防和/或治疗增生性疾病(例如肿瘤)的说明。
  66. 权利要求61所述的用途、权利要求62所述的方法或权利要求63-65任一项所述的药盒，其中，所述增生性疾病是肿瘤；
    优选地，所述肿瘤包括实体瘤或血液肿瘤；
    优选地，所述肿瘤包括转移性癌症、复发性或难治性癌症；
    优选地，所述肿瘤选自黑色素瘤、肾细胞癌、非小细胞肺癌、淋巴瘤、头颈部鳞状细胞癌、尿路上皮癌、卵巢癌、胃癌和乳腺癌。
  67. 药物组合物，其包含权利要求54所述的免疫细胞或权利要求55所述的免疫细胞群以及药学上可接受的载体和/或赋形剂。
  68. 权利要求54所述的免疫细胞、权利要求55所述的免疫细胞群或权利要求67所述的药物组合物用于增强免疫应答、预防和/或治疗增生性疾病的用途，或者，在制备用于增强免疫应答、预防和/或治疗增生性疾病的药物中的用途。
  69. 权利要求68所述的用途，其中，所述免疫细胞、免疫细胞群或药物组合物与权利要求1-15任一项中定义的第一定点修饰的IL-2和第二定点修饰的IL-2联合施用，例 如同时、分开或相继施用。
  70. 用于增强免疫应答、预防和/或治疗增生性疾病的方法，其包括向有需要的受试者施用权利要求54所述的免疫细胞、权利要求55所述的免疫细胞群或权利要求67所述的药物组合物；
    优选地，所述受试者为哺乳动物，例如人。
  71. 权利要求70所述的方法，其中，所述方法包括：使用权利要求42所述的方法或权利要求44-52所述的方法获得经改造的免疫细胞，向所述受试者施用所述经改造的免疫细胞。
  72. 权利要求70所述的方法，其中，所述方法包括：使用权利要求43所述的方法或权利要求53所述的方法获得肿瘤浸润淋巴细胞(TIL)，向所述受试者施用所述肿瘤浸润淋巴细胞(TIL)。
  73. 权利要求70-72任一项所述的方法，其中，所述免疫细胞、免疫细胞群或药物组合物与权利要求1-15任一项中定义的第一定点修饰的IL-2和第二定点修饰的IL-2联合施用，例如同时、分开或相继施用。
  74. 权利要求1-15任一项所述的组合物与用于过继性细胞治疗的免疫细胞的组合用于增强免疫应答、预防和/或治疗增生性疾病的用途，或者，在制备用于增强免疫应答、预防和/或治疗增生性疾病的药物中的用途；
    优选地，所述用于过继性细胞治疗的免疫细胞是经改造的免疫细胞，其表达嵌合抗原受体和/或包含编码所述嵌合抗原受体的核酸分子；
    优选地，所述用于过继性细胞治疗的免疫细胞是肿瘤浸润淋巴细胞(TIL)。
  75. 用于增强免疫应答、预防和/或治疗增生性疾病的方法，其包括向有需要的受试者施用权利要求1-15任一项所述的组合物与用于过继性细胞治疗的免疫细胞的组合；
    优选地，所述受试者为哺乳动物，例如人；
    优选地，所述用于过继性细胞治疗的免疫细胞是经改造的免疫细胞，其表达嵌合抗 原受体和/或包含编码所述嵌合抗原受体的核酸分子；
    优选地，所述用于过继性细胞治疗的免疫细胞是肿瘤浸润淋巴细胞(TIL)。
  76. 权利要求68或69所述的用途、权利要求70-73任一项所述的方法、权利要求74所述的用途或权利要求75所述的方法，其中，所述增生性疾病是肿瘤；
    优选地，所述肿瘤包括实体瘤或血液肿瘤；
    优选地，所述肿瘤包括转移性癌症、复发性或难治性癌症；
    优选地，所述肿瘤选自黑色素瘤、肾细胞癌、非小细胞肺癌、淋巴瘤、头颈部鳞状细胞癌、尿路上皮癌、卵巢癌、胃癌和乳腺癌。
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