TW202115115A - Immunoconjugates - Google Patents

Abstract

The present invention generally relates to immunoconjugates, particularly immunoconjugates comprising a mutant interleukin-2 polypeptide and an antibody that binds to CD8. In addition, the invention relates to polynucleotide molecules encoding the immunoconjugates, and vectors and host cells comprising such polynucleotide molecules. The invention further relates to methods for producing the mutant immunoconjugates, pharmaceutical compositions comprising the same, and uses thereof.

Description

Immunoconjugate

The present invention generally relates to immunoconjugates, specifically immunoconjugates comprising mutant interleukin-2 polypeptides and antibodies that bind to CD8. These immunoconjugates only have strong cis-targeting ^{for CD8 + cells} effect. In addition, the present invention relates to polynucleotide molecules encoding these immunoconjugates, as well as vectors and host cells containing such polynucleotide molecules. The present invention also relates to methods for producing these mutant immunoconjugates, pharmaceutical compositions containing them, and uses thereof.

Interleukin-2 (IL-2), also known as T cell growth factor (TCGF), is a 15.5 kDa globular glycoprotein that plays a key role in lymphocyte production, survival and homeostasis. It has a length of 133 amino acids and is composed of four antiparallel amphoteric alpha helices that form a quaternary structure essential for its function (Smith, Science 240, 1169-76 (1988); Bazan, Science 257, 410- 413 (1992)). The sequence of IL-2 from different species is found in NCBI RefSeq number NP000577 (human), NP032392 (mouse), NP446288 (rat) or NP517425 (chimpanzee).

IL-2 mediates its effects by binding to IL-2 receptors (IL-2R), which are composed of up to three individual subunits, and their different binding can produce different affinities for IL-2 Receptor form. The combination of α (CD25), β (CD122) and γ (γ _{c ,} CD132) subunits produces a high-affinity trimeric receptor for IL-2. The dipolymerized IL-2 receptor composed of β and γ subunits is called medium-affinity IL-2R. The alpha subunit forms the low-affinity monomer IL-2 receptor. Although the medium-affinity dimerized IL-2 receptor binds IL-2 with about 100 times lower affinity than the high-affinity trimerized receptor, both dimerized and trimerized IL-2 receptor variants can bind to IL-2 after binding. Transmission signal (Minami et al., Annu Rev Immunol 11, 245-268 (1993)). Therefore, the α subunit CD25 is not necessary for IL-2 signaling. It confers high affinity binding to its receptor, although the β subunit CD122 and the γ subunit are critical for signal transduction (Krieg et al., Proc Natl Acad Sci 107, 11906-11 (2010)). Trimeric IL-2 receptors (including CD25) are ^{expressed by (resting) CD4 +} forkhead box P3 (FoxP3) ⁺ regulatory T (T _reg ) cells. It is also transiently induced on conventionally activated T cells, and these cells only exhibit dimerized IL-2 receptors in the resting state. T _reg cells consistently express CD25 in the highest amount in vivo (Fontenot et al., Nature Immunol 6, 1142-51 (2005)).

IL-2 is mainly synthesized by activated T cells (specifically, CD4 ⁺ helper T cells). It stimulates the proliferation and differentiation of T cells, induces the production of cytotoxic T lymphocytes (CTL) and differentiation of peripheral blood lymphocytes into cytotoxic cells and lymphoid mediator activated killer (LAK) cells, promotes T cells to express cytokines and cytolytic molecules, Promote the proliferation and differentiation of B cells and the synthesis of immunoglobulin by B cells, and stimulate the production, proliferation and activation of natural killer (NK) cells (reviewed in, for example, Waldmann, Nat Rev Immunol 6, 595-601 (2009); Olejniczak and Kasprzak, Med Sci Monit 14, RA179-89 (2008); Malek, Annu Rev Immunol 26, 453-79 (2008)).

Its ability to expand lymphocyte populations in vivo and enhance these cell effector functions confers IL-2 anti-tumor effects, making IL-2 immunotherapy an attractive treatment option for certain metastatic cancers. Therefore, high-dose IL-2 therapy has been approved for patients with metastatic renal cell carcinoma and malignant melanoma.

However, IL-2 has a dual function in immune response: it not only mediates the expansion and activity of effector cells, but also mainly involves the maintenance of peripheral immune tolerance.

The main mechanism underlying self-tolerance is IL-2 induced activation-induced cell death (AICD) in T cells. AICD is a process by which fully activated T cells undergo programmed cell death through engagement with death receptors (such as CD95 (also known as Fas) or TNF receptors) expressed on the cell surface. When antigens that exhibit high-affinity IL-2 receptors (after pre-exposure to IL-2) during proliferation activate antigens for T cells, and are re-stimulated via T cell receptor (TCR)/CD3 complexes, Fas ligands are induced ( The expression of FasL) and/or tumor necrosis factor (TNF) makes these cells prone to Fas-mediated apoptosis. This process is IL-2 dependent (Lenardo, Nature 353, 858-61 (1991)) and is mediated by STAT5. Through the AICD process in T lymphocytes, not only tolerance to self-antigens but also tolerance to persistent antigens can be established, which are definitely not part of the host, such as tumor antigens.

In addition, IL-2 is also involved ^{in the maintenance of peripheral CD4 +} CD25 ⁺ regulatory T (T _reg ) cells (Fontenot et al., Nature Immunol 6, 1142-51 (2005); D'Cruz and Klein, Nature Immunol 6, 1152-59 (2005); Maloy and Powrie, Nature Immunol 6, 1171-72 (2005), which are also called suppressor T cells. They are assisted and activated by suppressor T cells via cell-cell contact, or via immunosuppressive cytokines (Such as IL-10 or TGF-β) to inhibit effector T cells from destroying their (self) targets. _{Depletion of T reg} cells has been shown to enhance IL-2 induced anti-tumor immunity (Imai et al., Cancer Sci 98, 416-23 (2007)).

Therefore, IL-2 is not the best option to inhibit tumor growth. The reason is that in the presence of IL-2, the generated CTL may recognize the tumor as itself and undergo AICD, or IL-2-dependent T _reg cells may inhibit immune response.

Another concern about IL-2 immunotherapy is the side effects of recombinant human IL-2 therapy. Patients receiving high-dose IL-2 treatment frequently experience severe cardiovascular, lung, kidney, liver, gastrointestinal, neurological, skin, blood, and systemic adverse events, which require intensive monitoring and hospitalization. Most of these side effects can be explained as: the development of the so-called vascular (or capillary) leak syndrome (VLS); pathological enhancement of vascular permeability, causing fluid extravasation in various organs (leading to lung and skin edema and Liver cell damage) and fluid depletion in blood vessels (leading to a decrease in blood pressure and a compensatory increase in heart rate). Except for discontinuation of IL-2, there is no treatment for VLS. To avoid VLS, low-dose IL-2 regimens have been tested in patients, but at the cost of sub-optimal treatment results. It is believed that VLS is caused by the release of pro-inflammatory cytokines, such as IL-2 activated NK cells to release tumor necrosis factor (TNF)-α. However, it has recently been shown that IL-2 induced pulmonary edema is caused by IL-2. Binding directly to lung endothelial cells, these lung endothelial cells exhibit low to moderate levels of functional αβγ IL-2 receptors (Krieg et al., Proc Nat Acad Sci USA 107, 11906-11 (2010)).

Several approaches have been taken to overcome these problems associated with IL-2 immunotherapy. For example, it has been found that the combination of IL-2 and certain anti-IL-2 monoclonal antibodies enhances the therapeutic effect of IL-2 in vivo (Kamimura et al., J Immunol 177, 306-14 (2006); Boyman et al. , Science 311, 1924-27 (2006)). In an alternative approach, IL-2 has been mutated in various ways to reduce its toxicity and/or enhance its efficacy. Hu et al. (Blood 101, 4853-4861 (2003); U.S. Patent Publication No. 2003/0124678) have replaced the arginine residue at position 38 of IL-2 with tryptophan to eliminate IL-2 Vascular permeability activity. Shanafelt et al. (Nature Biotechnol 18, 1197-1202 (2000)) have mutated aspartamide 88 to arginine to enhance the selectivity of T cells relative to NK cells. Heaton et al. (Cancer Res 53, 2597-602 (1993); US Patent No. 5,229,109) have introduced two mutations, Arg38Ala and Phe42Lys, to reduce the secretion of pro-inflammatory cytokines by NK cells. Gillies et al. (U.S. Patent Publication No. 2007/0036752) have replaced three residues in IL-2 (Asp20Thr, Asn88Arg, and Gln126Asp), which contribute to the affinity for medium-affinity IL-2 receptors, thereby reducing VLS. Gillies et al. (WO 2008/0034473) have also replaced Arg38Trp and Phe42Lys with amino acids to mutate the interface between IL-2 and CD25 to reduce the interaction with CD25 and the _{activation of T reg} cells, thereby enhancing the efficacy. For the same purpose, Wittrup et al. (WO 2009/061853) have produced IL-2 mutants that have increased affinity for CD25, but do not activate the receptor, thereby acting as an antagonist. The introduced mutation is intended to interrupt the interaction with the β-subunit and/or the γ-subunit of the receptor.

WO 2012/107417 describes a specific mutant IL-2 polypeptide designed to overcome the above-mentioned problems related to IL-2 immunotherapy (toxicity caused by VLS induction, tumor caused by AICD induction Tolerance, and _{immunosuppression caused by T reg} cell activation). The substitution of the phenylalanine residue at position 42 of IL-2 with alanine, the substitution of the tyrosine residue at position 45 with alanine and the substitution of the leucine residue at position 72 with glycine essentially eliminated this. The binding of mutant IL-2 polypeptide to the α-subunit of IL-2 receptor (CD25).

In addition to the methods mentioned above, IL-2 immunotherapy can be improved by selectively targeting IL-2 to tumors, for example, in the form of immunoconjugates containing antibodies that bind to antigens expressed on tumor cells. Several such immunoconjugates have been described (see, for example, Ko et al., J Immunother (2004) 27, 232-239; Klein et al., Oncoimmunology (2017) 6(3), e1277306).

However, tumors can evade such targeting by excreting, mutating or down-regulating the target antigen of the antibody. In addition, tumor-targeted IL-2 may not reach optimal contact with effector cells in the tumor microenvironment that actively exclude lymphocytes, such as cytotoxic T lymphocytes (CTL).

Therefore, there is still a need to further improve IL-2 immunotherapy. One way to circumvent the problem of tumor targeting is to target IL-2 directly to effector cells, specifically, CTLs.

Ghasemi et al. have described a fusion protein of IL-2 and NKG2D binding protein (Ghashemi et al., Nat Comm (2016) 7, 12878), which is used to target IL-2 to cells carrying NKG2D, such as natural killer (NK) cell.

The characteristics of the number, type and spatial distribution of immune cells in tumor tissues can provide key information about cancer diagnosis, prognosis, treatment options and treatment response. Specifically, CD8 ⁺ cytotoxic lymphocytes have always been reported as having diagnostic and prognostic significance in various cancers. Antibodies that bind to CD8 are described in, for example, WO 2019/033043 A2.

The present invention provides a novel method for direct targeting of mutant IL-2 with advantageous properties of immunotherapy to immune effector cells (such as cytotoxic T lymphocytes) rather than tumor cells. Targeting immune effector cells is achieved by binding mutant IL-2 molecules to antibodies that bind to CD8.

The IL-2 mutant used in the present invention has been designed to overcome the problems associated with IL-2 immunotherapy, in particular, to induce toxicity caused by VLS, induce tumor tolerance caused by AICD, and activate T _reg cells The immunosuppression caused. In addition to preventing tumors from evading tumor targeting as mentioned above, IL-2 mutant targeting immune effector cells can further enhance the preferential activation of CTL _{relative to immunosuppressive T reg cells.} Antibodies that bind to CD8 as disclosed herein have a strong cis-targeting effect ^{on CD8 + cells.} Therefore, it does not interfere with the interaction between the T cell antigen receptor (TCR) and the peptide-bound major histocompatibility complex (pMHC). In this regard, antibodies are non-functional.

In a first aspect, the present invention provides an immunoconjugate comprising a mutant interleukin-2 (IL-2) polypeptide and an antibody that binds to CD8, wherein the IL-2 polypeptide contains amino acid substitutions Mutant IL-2 polypeptides of F42A, Y45A and L72G (numbering relative to the human IL-2 sequence SEQ ID NO: 13).

In another aspect, the present invention provides an immunoconjugate comprising a mutant interleukin-2 (IL-2) polypeptide and an antibody that binds to CD8, wherein the mutant IL-2 polypeptide contains an amino acid Substitute the human IL-2 molecules of F42A, Y45A and L72G (numbered relative to the human IL-2 sequence SEQ ID NO: 13); and wherein the antibody comprises: (a) a heavy chain variable region (VH), which comprises SEQ ID NO: The heavy chain complementarity determining region (HCDR) of the amino acid sequence of ID NO: 1 1. HCDR containing the amino acid sequence of SEQ ID NO: 2 2. HCDR 3 containing the amino acid sequence of SEQ ID NO: 3; And (b) a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) containing the amino acid sequence of SEQ ID NO: 4 1, an LCDR containing the amino acid sequence of SEQ ID NO: 5 2 and LCDR 3 containing the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 28.

In another aspect, the present invention provides an immunoconjugate comprising a mutant interleukin-2 (IL-2) polypeptide and an antibody that binds to CD8, wherein the mutant IL-2 polypeptide contains an amino acid Substituting the human IL-2 molecule of F42A, Y45A and L72G (numbered relative to the human IL-2 sequence SEQ ID NO: 13); and wherein the antibody comprises: (a) a heavy chain variable region (VH), which comprises the same as SEQ ID NO: 13 The amino acid sequence of ID NO: 7 is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence; and (b) the light chain variable region (VL), which comprises An amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 29.

In some embodiments of the immunoconjugate of the present invention, the mutant IL-2 polypeptide further comprises an amino acid substituted for T3A and/or an amino acid substituted for C125A.

In some embodiments, the mutant IL-2 polypeptide comprises the sequence of SEQ ID NO: 14.

In some embodiments, the immunoconjugate contains no more than one mutant IL-2 polypeptide. In some such embodiments, the antibody comprises an Fc domain composed of a first subunit and a second subunit. In some such embodiments, the Fc domain is an IgG class, especially an IgG1 subclass Fc domain, and/or the Fc domain is a human Fc domain. In some embodiments, the antibody is of the IgG class, especially an immunoglobulin of the IgG1 subclass.

In some embodiments, wherein the immunoconjugate comprises an Fc domain, the Fc domain comprises a modification that promotes the binding of the first unit and the second unit of the Fc domain. In some embodiments, in the CH3 domain of the first unit of the Fc domain, the amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby being in the CH3 domain of the first unit Generates a bulge that can be located in the cavity in the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain, the amino acid residue is passed through an amino acid with a smaller side chain volume. Residue replacement, thereby creating a cavity in the CH3 domain of the second unit where the protuberances in the CH3 domain of the first unit can be positioned. In some embodiments, in the first subunit of the Fc domain, the threonine residue at position 366 is replaced by a tryptophan residue (T366W), and in the second subunit of the Fc domain, position 407 The tyrosine residue is replaced by valine residue (Y407V), and optionally the threonine residue at position 366 is replaced by serine residue (T366S) and the leucine residue at position 368 is replaced by Alanine residue substitution (L368A) (numbered according to Kabat EU index). In some such embodiments, in the first unit of the Fc domain, in addition, the serine residue at position 354 is replaced by a cysteine residue (S354C) or the glutamine residue at position 356 is replaced by Cysteine residue replacement (E356C), and in the second unit of the Fc domain, in addition, the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C) (numbering according to Kabat EU index ). In some embodiments, the mutant IL-2 polypeptide is fused to the carboxy terminal amino acid of one of the secondary units of the Fc domain (specifically, the first unit of the Fc domain) at its amino terminal amino acid, Optionally fused via a connecting peptide. In some of these embodiments, the connecting peptide has the amino acid sequence of SEQ ID NO:15.

In some embodiments, wherein the immunoconjugate comprises an Fc domain, the Fc domain comprises reducing binding to Fc receptors (specifically Fcy receptors) and/or effector functions (specifically, antibody-dependent cell-mediated cytotoxicity) (ADCC) One or more amino acid substitutions. In some such embodiments, the one or more amino acid substitutions are located at one or more positions selected from the group of L234, L235, and P329 (Kabat EU index number ). In some embodiments, each subunit in the Fc domain includes amino acid substitutions L234A, L235A, and P329G (Kabat EU index number).

In some embodiments according to the present invention, the immunoconjugate comprises: a polypeptide comprising at least about 80%, 85%, 90%, 95%, 96%, sequence of SEQ ID NO: 9 or SEQ ID NO: 30, An amino acid sequence that is 97%, 98%, 99% or 100% identical; a polypeptide comprising at least about 80%, 85%, 90%, 95%, 96%, 97%, and the sequence of SEQ ID NO: 10, An amino acid sequence that is 98%, 99%, or 100% identical; and a polypeptide comprising at least about 80%, 85%, 90%, 95%, 96% with the sequence of SEQ ID NO: 11 or SEQ ID NO: 12 , 97%, 98%, 99% or 100% identical amino acid sequence.

In some embodiments, the immunoconjugate comprises: a polypeptide comprising the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 29; a polypeptide comprising the amino acid sequence of SEQ ID NO: 10; and a polypeptide, It includes the amino acid sequence of SEQ ID NO:11.

In some embodiments, the immunoconjugate comprises: a polypeptide comprising the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 29; a polypeptide comprising the amino acid sequence of SEQ ID NO: 10; and a polypeptide, It contains the amino acid sequence of SEQ ID NO: 12.

In some embodiments, the immunoconjugate consists essentially of a mutant IL-2 polypeptide and an IgG1 immunoglobulin molecule joined by a linker sequence.

The present invention further provides one or more isolated polynucleotides encoding the immunoconjugates of the present invention as described herein, one or more vectors (specifically expression vectors) containing the polynucleotides, and The polynucleotide or the host cell of the vector.

A method for producing an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to CD8 is also provided, the method comprising (a) culturing the host cell of the present invention under conditions suitable for expressing the immunoconjugate, and visually Case (b) recover the immunoconjugate. The present invention also provides an immunoconjugate produced by the method, which comprises a mutant IL-2 polypeptide and an antibody that binds to CD8.

The present invention further provides a pharmaceutical composition comprising the immunoconjugate of the present invention and a pharmaceutically acceptable carrier; and a method of using the immunoconjugate of the present invention.

In particular, the present invention covers the immunoconjugates according to the present invention, which are used as medicaments, and for the treatment of diseases. In a specific embodiment, the disease is cancer.

The present invention also covers the use of an immunoconjugate according to the present invention for the manufacture of a medicament for the treatment of diseases. In a specific embodiment, the disease is cancer.

There is further provided a method of treating a disease in an individual, which comprises administering to the individual a therapeutically effective amount of a composition comprising the immunoconjugate according to the present invention in a pharmaceutically acceptable form. In a specific embodiment, the disease is cancer.

There is further provided a method of stimulating the immune system of an individual, which comprises administering to the individual an effective amount of a composition comprising the immunoconjugate according to the present invention in a pharmaceutically acceptable form.

Definitions Unless otherwise defined below, terms are used herein as they are generally used in this technology.

Unless otherwise indicated, as used herein, the term "interleukin-2" or "IL-2" refers to any vertebrate source, including mammals, such as primates (e.g., humans) and rodents (e.g., small animals). Rats and rats) any native IL-2. The term encompasses unprocessed IL-2 as well as any form of IL-2 produced by processing in the cell. The term also encompasses naturally occurring IL-2 variants, such as splice variants or allelic variants. The amino acid sequence of an exemplary human IL-2 is shown in SEQ ID NO: 13. The unprocessed human IL-2 additionally contains the N-terminal 20 amino acid signal peptide with the sequence of SEQ ID NO: 19, which is not present in the mature IL-2 molecule.

As used herein, the term "IL-2 mutant" or "mutant IL-2 polypeptide" is intended to encompass any mutant form of IL-2 molecules in various forms, including full-length IL-2, truncated IL-2 forms, and IL-2 forms therein. -2 The form of linking with another molecule by fusion or chemical bonding. "Full length" when used in reference to IL-2, is intended to mean a mature, natural length IL-2 molecule. For example, full-length human IL-2 refers to a molecule with 133 amino acids (see, for example, SEQ ID NO: 13). The various forms of IL-2 mutants are characterized by having at least one amino acid mutation that affects the interaction of IL-2 with CD25. This mutation may involve substitution, deletion, truncation or modification of the wild-type amino acid residue normally located at this position. Preferably, it is a mutant obtained by amino acid substitution. Unless otherwise indicated, an IL-2 mutant may be referred to herein as a mutant IL-2 peptide sequence, a mutant IL-2 polypeptide, a mutant IL-2 protein, or a mutant IL-2 analog.

The various forms of IL-2 are identified herein according to the sequence shown in SEQ ID NO: 13. Different names may be used herein to indicate the same mutation. For example, the phenylalanine at position 42 was mutated to alanine may be 42A, A42, A _42, F42A, or Phe42Ala FIG.

As used herein, "human IL-2 molecule" means at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94% comprising the human IL-2 sequence of SEQ ID NO: 13 , IL-2 molecules with at least about 95% or at least about 96% identical amino acid sequence. Specifically, the sequence identity is at least about 95%, and more specifically at least about 96%. In a specific embodiment, the human IL-2 molecule is a full-length IL-2 molecule.

As used herein, the term "amino acid mutation" is intended to encompass amino acid substitutions, deletions, insertions, and modifications. Any combination of substitutions, deletions, insertions and modifications can be made to obtain the final construct, and the limitation is that the final construct possesses the required characteristics, such as reduced binding to CD25. The deletion and insertion of the amino acid sequence includes the deletion and insertion of the amino end and/or the carboxyl end of the amino acid. An example of a terminal deletion is the deletion of the alanine residue in position 1 of full-length human IL-2. Preferably the amino acid mutation is an amino acid substitution. For the purpose of changing the binding characteristics of, for example, IL-2 polypeptides, non-conservative amino acid substitutions, that is, replacement of one amino acid with another amino acid with different structure and/or chemical properties are particularly preferred. Preferred amino acid substitutions include the substitution of hydrophilic amino acids for hydrophobic amino acids. Amino acid substitution includes substitution by non-naturally-occurring amino acids or naturally-occurring amino acid derivatives of twenty kinds of standard amino acids (e.g. 4-hydroxyproline, 3-methylhistidine, bird Amino acid, homoserine, 5-hydroxylysine) substitution. Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis and similar methods. It is considered that methods of changing amino acid side chain groups by methods other than genetic engineering (such as chemical modification) may also be applicable.

As used herein, the "wild-type" form of IL-2 is a form of IL-2, which is otherwise the same as the mutant IL-2 polypeptide, but the difference is that the wild-type form is in the mutant IL-2 polypeptide Each amino acid position has a wild-type amino acid. For example, if the IL-2 mutant is full-length IL-2 (that is, IL-2 is not fused or bound to any other molecule), then the wild-type form of the mutant is full-length native IL-2. If the IL-2 mutant is a fusion between IL-2 and another polypeptide (such as an antibody chain) encoded downstream of IL-2, the wild-type form of this IL-2 mutant has a wild-type amino group The acid sequence of IL-2 is fused to the same downstream polypeptide. In addition, if the IL-2 mutant is a truncated form of IL-2 (mutation or modified sequence in the untruncated portion of IL-2), the wild-type form of the IL-2 mutant is similar to the wild-type sequence Truncated IL-2. For the purpose of comparing the binding affinity or biological activity of the various forms of IL-2 mutants with the corresponding wild-type IL-2 receptors of IL-2, the term wild-type encompasses as compared to naturally occurring native IL-2 A form of IL-2 that does not affect IL-2 receptor binding to one or more amino acid mutations, such as the substitution of alanine for cysteine at the position corresponding to residue 125 of human IL-2. In some embodiments, the wild-type IL-2 used for the purpose of the present invention contains the amino acid substitution C125A (see SEQ ID NO: 20). In certain embodiments according to the present invention, the wild-type IL-2 polypeptide compared with the mutant IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 13. In other embodiments, the wild-type IL-2 polypeptide compared with the mutant IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 20.

Unless otherwise indicated, as used herein, the term "CD25" or "α-subunit of IL-2 receptor" refers to any vertebrate source (including mammals, such as primates (e.g., humans) and rodents). (E.g. mice and rats)) any native CD25. The term encompasses "full-length", unprocessed CD25, and any form of CD25 produced by processing in cells. The term also encompasses naturally occurring CD25 variants, such as splice variants or allelic variants. In certain embodiments, CD25 is human CD25. The amino acid sequence of human CD25 is found in, for example, UniProt accession number P01589 (type 185).

As used herein, the term "high-affinity IL-2 receptor" refers to the heterotrimeric form of IL-2 receptor consisting of: receptor γ-subunit (also known as common cytokine receptor γ-subunit Unit, γ _c or CD132, see UniProt accession number P14784 (type 192)), receptor β-subunit (also known as CD122 or p70, see UniProt accession number P31785 (style 197)) and receptor α-subunit ( Also known as CD25 or p55, see UniProt accession number P01589 (type 185)). In contrast, the term "medium affinity IL-2 receptor" refers to an IL-2 receptor that includes only γ-subunits and β-subunits without α-subunits (for a review, see, for example, Olejniczak and Kasprzak, Med Sci Monit 14, RA179-189 (2008)).

"Affinity" refers to the sum of non-covalent interaction forces between a single binding site of a molecule (such as a receptor) and its binding partner (such as a ligand). Unless otherwise specified, as used herein, "binding affinity" refers to the inherent binding affinity that reflects the 1:1 interaction between members of a binding pair (eg, an antigen-binding portion and an antigen, or a receptor and its ligand). The affinity of the molecule X to its partner Y can generally be represented by the dissociation constant (K _D ), which is the ratio of the dissociation rate constant to the association rate constant (k _off and k _{on, respectively} ). Therefore, the equivalent affinity can include different rate constants as long as the ratio of the rate constants remains the same. Affinity can be measured by well-established methods known in the art, including the methods described herein. One specific method for measuring affinity is surface plasmon resonance (SPR).

The affinity of mutant or wild-type IL-2 polypeptides for various forms of IL-2 receptors can be based on the method described in WO 2012/107417, by surface plasmon resonance (SPR), using standard instruments (such as BIAcore) (GE Healthcare)), and such as receptor subunits can be obtained by recombinant expression (see, for example, Shanafelt et al., Nature Biotechnol 18, 1197-1202 (2000)). Alternatively, the binding affinity of IL-2 mutants to different forms of IL-2 receptors can be evaluated using cell lines known to express one or other such forms of receptors. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

"Regulatory T cell" or "T _reg cell" means a specific type of CD4 ⁺ T cell that can inhibit the response of other T cells. T _reg cells are characterized by expressing the α-subunit of IL-2 receptor (CD25) and the transcription factor forkhead box P3 (FOXP3) (Sakaguchi, Annu Rev Immunol 22, 531-62 (2004)) and are inducing and maintaining It plays a key role in peripheral self-tolerance to antigens (including antigens expressed by tumors). T _reg cells require IL-2 to achieve their functions and the production and induction of its inhibitory characteristics.

As used herein, the term "effector cells" refers to a population of lymphocytes that mediate the cytotoxic effects of IL-2. Effector cells include effector T cells, such as CD8 ⁺ cytotoxic T cells, NK cells, lymphoid mediator activated killer (LAK) cells, and macrophages/monocytes.

"Specific binding" means that the binding is selective for the antigen and distinguishable from undesired or non-specific interactions. The ability of an antigen to bind to a specific antigen (such as CD8) can be achieved through enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those familiar with the technology, such as surface plasmon resonance (SPR) technology (for example, analysis on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)) and traditional combined analysis (Heeley, Endocr Res 28, 217-229 (2002)) measurement. In one embodiment, the degree of binding of an antibody to an unrelated protein is less than about 10% of the binding of the antibody to an antigen, as measured by, for example, SPR. The antibodies contained in the immunoconjugates described herein specifically bind to CD8.

As used herein, the term "polypeptide" refers to a molecule composed of monomers (amino acids) linearly connected via amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain of two or more amino acids, and does not refer to a specific length of the product. Therefore, the definition of "polypeptide" includes peptides, dipeptides, tripeptides, oligopeptides, "proteins", "amino acid chains" or any other terms used to refer to chains of two or more amino acids and The term "polypeptide" can be used in place of any of these terms, or the term "polypeptide" can be used interchangeably with any of these terms. The term "polypeptide" also refers to the post-modification products of the polypeptide, including (but not limited to) glycosylation, acetylation, phosphorylation, amination, derivatization with known protection/capping groups, and protein cleavage. Or modified by non-naturally occurring amino acids. Polypeptides can be derived from natural biological sources or produced by recombinant technology, but are not necessarily translated from a designated nucleic acid sequence. It can be produced in any way, including by chemical synthesis. A polypeptide may have a defined three-dimensional structure, but it does not necessarily have such a structure. Polypeptides with a defined three-dimensional structure are called folded, and polypeptides that do not have a defined three-dimensional structure but can adopt many different configurations are called unfolded.

An "isolated" polypeptide or a variant or derivative thereof means a polypeptide that is not in its natural environment. No specific level of purification is required. For example, the isolated polypeptide can be removed from its native or natural environment. For the purpose of the present invention, recombinantly produced polypeptides and proteins expressed in host cells are considered to be isolated, and native or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique are also considered After separation.

Relative to the reference polypeptide sequence, the "amino acid sequence identity percentage (%)" is defined as after aligning the reference polypeptide sequence with the candidate sequence and introducing gaps if necessary to achieve the maximum sequence identity percentage, and without any conservative substitutions The percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence when considered as part of the sequence identity. The alignment for the purpose of determining the percent identity of amino acid sequences can be achieved in various ways within the skill of this technology, for example, using publicly available computer software, such as BLAST, BLAST-2, Clustal W, Megalign ( DNASTAR) software or FASTA package. Those skilled in the art can determine the appropriate parameters for the alignment of sequences, including any algorithms required to achieve the maximum alignment over the entire length of the sequence being compared. However, for the purpose of this article, the% amino acid sequence identity value is generated using the BLOSUM50 comparison matrix using the ggsearch program in FASTA Suite 36.3.8c or later. The authors of the FASTA package are WR Pearson and DJ Lipman (1988), "Improved Tools for Biological Sequence Analysis", PNAS 85:2444-2448; WR Pearson (1996) "Effective protein sequence comparison" Meth. Enzymol. 266:227- 258; and Pearson et al. (1997) Genomics 46:24-36, and publicly available from http://fasta.bioch.virginia.edu/fasta_www2/fasta_down.shtml. Alternatively, a public server accessible at http://fasta.bioch.virginia.edu/fasta_www2/index.cgi can be used to compare sequences, using the ggsearch (global protein: protein) program and the default option (BLOSUM50 ; Beginning: -10; ext: -2; Ktup = 2) to ensure a global comparison rather than a local comparison. The percentage of amino acid identity is given in the title of the output comparison.

The term "polynucleotide" refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA), virus-derived RNA or plastid DNA (pDNA). Polynucleotides may contain conventional phosphodiester bonds or non- conventional bonds (e.g., amide bonds, such as those found in peptide nucleic acids (PNA)). The term "nucleic acid molecule" refers to any one or more nucleic acid segments present in polynucleotides, such as DNA or RNA fragments.

An "isolated" nucleic acid molecule or polynucleotide is intended to be a nucleic acid molecule, DNA or RNA that has been removed from the native environment. For example, for the purposes of the present invention, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered to be isolated. Other examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution. Isolated polynucleotides include polynucleotide molecules contained in cells that usually contain polynucleotide molecules, but polynucleotide molecules exist outside the chromosomes or at chromosomal locations that are different from their natural chromosomal locations. The isolated RNA molecules include the in vivo or in vitro RNA transcripts of the present invention, as well as positive and negative strand forms, and double strand forms. The isolated polynucleotide or nucleic acid of the present invention further includes such molecules produced synthetically. In addition, polynucleotides or nucleic acids may be or may include regulatory elements, such as promoters, ribosome binding sites, or transcription terminators.

"Isolated polynucleotide (or nucleic acid) encoding [such as the immunoconjugate of the present invention]" refers to one or more polynucleotides (or nucleic acids) encoding antibody heavy and light chains and/or IL-2 polypeptides (or fragments thereof). Nucleotide molecules include such polynucleotide molecules in a single vector or a separate vector, and such nucleic acid molecules present in one or more locations in a host cell.

The term "performance cassette" refers to a polynucleotide produced by recombinant or synthetic means, which has a series of specific nucleic acid elements that allow specific nucleic acid to be transcribed in the target cell. The recombination expression cassette can be incorporated into plastids, chromosomes, mitochondrial DNA, plastid DNA, viruses, or nucleic acid fragments. Generally, the recombinant performance cassette part of the expression vector includes the nucleic acid sequence to be transcribed, the promoter, and other sequences. In certain embodiments, the performance cassette comprises a polynucleotide sequence encoding the immunoconjugate of the present invention or a fragment thereof.

The term "vector" or "expression vector" refers to a DNA molecule used to introduce a specific gene and guide the expression of the specific gene, and the DNA molecule is operably combined with the specific gene in a cell. The term includes a vector in a self-replicating nucleic acid structure as well as a vector incorporated into the genome of a host cell into which it has been introduced. The performance carrier of the present invention includes a performance cassette. The expression vector allows transcription of large amounts of stable mRNA. Once the expression vector enters the target cell, the ribonucleic acid molecule or protein encoded by the gene is produced by the transcription and/or translation mechanism of the cell. In one embodiment, the expression vector of the present invention includes a performance cassette containing polynucleotide sequences that encode the immunoconjugate of the present invention or fragments thereof.

The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells", which include primary transformed cells and progeny derived therefrom (regardless of the number of generations). The nucleic acid content of the offspring may not be exactly the same as the parent cell, but it may contain mutations. This paper includes mutant progeny that have the same function or biological activity as screened or selected for the originally transformed cell. The host cell is any type of cell system that can be used to produce the immunoconjugate of the present invention. Host cells include cultured cells, such as mammalian cultured cells, such as HEK cells, CHO cells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or Fusion tumor cells, yeast cells, insect cells, and plant cells (to name a few), and also include cells contained in transgenic animals, transgenic plants, or cultured plants or animal tissues.

The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including (but not limited to) monoclonal antibodies, monovalent antibodies (such as one-arm antibodies) and antibody fragments, as long as they exhibit the desired antigen-binding activity ( That is, it can be combined with CD8 (such as human CD8, rhesus CD8 and/or rhesus CD8)).

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, that is, the individual antibodies contained in the population have the same and/or bind the same epitope, but there may be variant antibodies, such as containing Except for naturally occurring mutations or mutations that occur during the production of monoclonal antibody preparations, such variants are generally present in small amounts. In contrast to multiple antibody preparations which typically include different antibodies directed against different determinants (antigenic determinants), each monoclonal antibody system in a monoclonal antibody preparation is directed against a single determinant on the antigen. Therefore, the modifier "monoclonal" indicates that the characteristics of the antibody are obtained from a substantially homogeneous antibody population and should not be regarded as requiring the production of the antibody by any specific method. For example, the monoclonal antibodies used according to the present invention can be produced by a variety of techniques, including but not limited to fusion tumor methods, recombinant DNA methods, phage display methods, and the use of all or part of human immunoglobulin loci. Methods of transgenic animals, such methods of preparing monoclonal antibodies described herein, and other exemplary methods.

An "isolated" antibody is an antibody that has been separated from a component of its natural environment (ie, does not exist in its natural environment). No specific level of purification is required. For example, the isolated antibody can be removed from its native or natural environment. For the purpose of the present invention, recombinantly produced antibodies expressed in host cells are regarded as isolated, and natural or recombinant antibodies that have been separated, fractionated, or partially or substantially purified by any suitable technique are also regarded as isolated . Therefore, the immunoconjugates of the present invention are isolated. In some embodiments, the antibody is purified to a purity greater than 95% or 99%, such as by electrophoresis (such as SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (such as ion exchange or reverse phase). HPLC) method. For a review of methods used to assess antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

The terms "full-length antibody", "complete antibody" and "complete antibody" are used interchangeably herein, and refer to an antibody having a structure that is substantially similar to the structure of the native antibody.

"Antibody fragment" refers to a molecule that is different from an intact antibody and includes the part of an intact antibody that binds to the antigen to which an intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') ₂ , bifunctional antibodies, linear antibodies, single-chain antibody molecules (such as scFv), and single-domain antibodies. For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005). For a review of scFv fragments, see, for example, Plückthun, The Pharmacology of Monoclonal Antibodies, Volume 113, Rosenburg and Moore eds, Springer-Verlag, New York, pp. 269-315 (1994); see also WO 93/16185; and the United States Patent No. 5,571,894 and No. 5,587,458. _{For a discussion of Fab and F(ab') 2} fragments containing salvage receptor binding epitope residues and increased in vivo half-life, see US Patent No. 5,869,046. Bifunctional antibodies are antibody fragments in which two antigen binding sites can be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993). Trifunctional antibodies and tetrafunctional antibodies are also described in Hudson et al., Nat Med 9, 129-134 (2003). Single domain antibodies are antibody fragments that comprise all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, for example, US Patent No. 6,248,516 B1). Antibody fragments can be prepared by various techniques, including (but not limited to) proteolytic digestion of intact antibodies and production by recombinant host cells (such as E. coli or phage), as described herein.

The term "immunoglobulin molecule" refers to a protein with the structure of a naturally occurring antibody. For example, IgG immunoglobulins are heterotetrameric glycoproteins of about 150,000 daltons (daltons), which are composed of two light chains and two heavy chains bonded by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable domain (VH), also known as a variable heavy chain domain or a heavy chain variable region, followed by three constant domains (CH1, CH2, and CH3), also known as heavy Chain constant region. Similarly, from the N-terminus to the C-terminus, each light chain has a variable domain (VL), also known as a variable light chain domain or a light chain variable region, followed by a constant light chain domain (CL), also known as a light chain domain. Chain constant region. The heavy chain of immunoglobulin can be classified into one of five types, which are called α (IgA), δ (IgD), ε (IgE), γ (IgG) or μ (IgM), some of which can be further Divided into subtypes, such as γ ₁ (IgG ₁ ), γ ₂ (IgG ₂ ), γ ₃ (IgG ₃ ), γ ₄ (IgG ₄ ), α ₁ (IgA ₁ ), and α ₂ (IgA ₂ ). The light chain of immunoglobulin can be classified into one of two types based on the amino acid sequence of its constant domain. The two types are called kappa (κ) and lambda (λ). An immunoglobulin basically consists of two Fab molecules and an Fc domain connected via an immunoglobulin hinge region.

The term "antigen-binding domain" refers to a part of an antibody that contains a part or all of an antigen that specifically binds to and is complementary to a region. The antigen binding domain may be provided by, for example, one or more antibody variable domains (also referred to as antibody variable regions). Specifically, the antigen binding domain includes an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).

The term "variable region" or "variable domain" refers to the domain in the heavy or light chain of an antibody that participates in the binding of the antibody to the antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody usually have similar structures, and each domain contains four conserved framework regions (FR) and three hypervariable regions (HVR). See, for example, Kindt et al., Kuby Immunology, 6th edition, W.H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen binding specificity. As used herein with regard to variable region sequences, "Kabat numbering" refers to Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition. Public Health Service, National Institutes of Health , Bethesda, MD (1991) described the numbering system.

As used herein, all constant regions and amino acid positions in the constant regions of the heavy and light chains are based on Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Department of Public Health, National Institutes of Health, Bethesda , The Kabat numbering system described in MD (1991) is called "according to Kabat numbering" or "Kabat numbering" in this article. Specifically, the light chain constant domain CL of the kappa and lambda homotypes uses the Kabat numbering system (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, pages 647-660, Department of Public Health, National Institutes of Health , Bethesda, MD (1991)) and the heavy chain constant domains (CH1, hinge, CH2, and CH3) use the Kabat EU index numbering system (see pages 661-723), in this case, by reference to "according to Kabat EU index number" to further clarify.

As used herein, the term "hypervariable region" or "HVR" refers to each region in the variable domain of an antibody whose sequence is hypervariable ("complementarity determining region" or "CDR") and/or forms a structurally defined loop ( "Hypervariable loop") and/or contain antigen contact residues ("antigen contact"). Generally speaking, an antibody contains six HVRs; three in VH (H1, H2, H3), and three in VL (L1, L2, L3). In this article, exemplary HVRs include: (a) appearing in amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2) and the hypervariable ring at 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) appears in amino acid residues 24-34 ( L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2) and 95-102 (H3) CDRs (Kabat et al., Sequences of Proteins of Immunological Interest , 5th edition. Department of Public Health, National Institutes of Health, Bethesda, MD (1991)); (c) Appears in amino acid residues 27c-36 (L1), 46-55 (L2), 89- Antigen contacts at 96 (L3), 30-35b (H1), 47-58 (H2) and 93-101 (H3) (MacCallum et al . J. Mol. Biol. 262: 732-745 (1996)); And (d) a combination of (a), (b) and/or (c), including HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49- 56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3) and 94-102 (H3).

Unless otherwise indicated, herein, HVR residues and other residues (e.g., FR residues) in the variable domain are numbered according to the aforementioned literature by Kabat et al.

"Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, HVR and FR sequences generally appear in the following order in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

"Humanized" antibodies refer to chimeric antibodies containing amino acid residues from non-human HVR and amino acid residues from human FR. In certain embodiments, the humanized antibody will comprise substantially all of at least one and usually two variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to the HVR of the non-human antibody, and all or substantially all All FRs above correspond to the FRs of human antibodies. Such variable domains are referred to herein as "humanized variable regions". The humanized antibody may optionally comprise at least a part of the constant region of an antibody derived from a human antibody. In some embodiments, some FR residues in the humanized antibody are substituted with corresponding residues from a non-human antibody (eg, an antibody derived from HVR residues) to, for example, restore or improve antibody specificity or affinity. The "humanized form" of an antibody (such as a non-human antibody) refers to an antibody that has been humanized. Other forms of "humanized antibodies" covered by the present invention are those in which the constant region has been additionally modified or changed from the original antibody to produce the properties according to the present invention (especially in terms of C1q binding and/or Fc receptor (FcR) binding) Antibody.

A "human antibody" is an antibody whose amino acid sequence corresponds to the amino acid sequence of an antibody produced by human or human cells or derived from a non-human source that utilizes the human antibody lineage or other human antibody coding sequences. This definition of human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. In certain embodiments, the human antibody is derived from a non-human transgenic mammal, such as a mouse, rat, or rabbit. In certain embodiments, the human antibody is derived from a fusion tumor cell line. Antibodies or antibody fragments isolated from human antibody libraries are also considered human antibodies or human antibody fragments herein.

The "class" of an antibody or immunoglobulin refers to the type of constant domain or constant region possessed by its heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these antibodies can be further divided into subclasses (isotypes), such as IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ And IgA ₂ . The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

The term "Fc domain" or "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain containing at least a part of a constant region. The term includes native sequence Fc region and variant Fc region. Although the boundaries of the Fc region of an IgG heavy chain may vary slightly, the Fc region of a human IgG heavy chain is usually defined as extending from Cys226 or from Pro230 to the carboxyl end of the heavy chain. However, the antibody produced by the host cell may undergo the translation of one or more (specifically, one or two) amino acids at the C-terminus of the heavy chain and then divide. Therefore, an antibody produced by a host cell by expressing a specific nucleic acid molecule encoding a full-length heavy chain may include the full-length heavy chain, or it may include a split-type variant of the full-length heavy chain (also referred to herein as a "split-type variant"). Weight chain"). This can be the case when the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbered according to the Kabat EU index). Therefore, the C-terminal lysine (Lys447) or C-terminal glycine (Gly446) and lysine (K447) of the Fc region may or may not exist. If not otherwise indicated, the amino acid sequence of the heavy chain including the Fc domain (or a subunit of the Fc domain as defined herein) means herein no C-terminal glycine-lysine dipeptide. In one embodiment of the present invention, the heavy chain contained in the immunoconjugate of the present invention (the heavy chain includes the subunit of the Fc domain as described herein) includes an additional C-terminal glycine-lysine dipeptide ( G446 and K447, according to the Kabat EU index number). In one embodiment of the present invention, the heavy chain (including the subunit of the Fc domain as specified herein) contained in the immunoconjugate of the present invention contains another C-terminal glycine residue (G446, according to Kabat EU index number). The composition of the present invention, such as the pharmaceutical composition described herein, includes the population of immunoconjugates of the present invention. The population of immunoconjugates can include molecules with full-length heavy chains and molecules with split variant heavy chains. The population of immunoconjugates can be composed of a mixture of molecules with full-length heavy chains and molecules with split variant heavy chains, wherein at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of immunoconjugates have Split type variable weight chain. In one embodiment of the present invention, the composition comprising the immunoconjugate population of the present invention comprises an immunoconjugate comprising a heavy chain comprising a subunit of the Fc domain as specified herein, the heavy chain described having an additional C-terminus Glycine-lysine dipeptide (G446 and K447, numbered according to the Kabat EU index). In one embodiment of the present invention, the composition comprising the immunoconjugate population of the present invention comprises an immunoconjugate comprising a heavy chain including a subunit of the Fc domain as specified herein, the heavy chain described having an additional C-terminal glycoside Amino acid residue (G446, numbered according to Kabat EU index). In one embodiment of the present invention, such a composition comprises a population of immunoconjugates, and the population described comprises: a molecule comprising a heavy chain comprising a subunit of an Fc domain as specified herein; comprising a heavy chain comprising an Fc domain as specified herein The molecule of the heavy chain of the secondary unit, the heavy chain described has an additional C-terminal glycine residue (G446, according to the Kabat EU index number); and the heavy chain comprising the secondary unit of the Fc domain as specified herein The molecule, the heavy chain described has an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to the Kabat EU index). Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or the constant region is based on the EU numbering system, also known as the EU index, such as Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition. Department of Public Health, National Institutes of Health, Bethesda, MD, 1991 (see also above). As used herein, the "subunit" of the Fc domain refers to one of the two polypeptides forming the dimeric Fc domain, that is, the polypeptide comprising the C-terminal constant region of the immunoglobulin heavy chain, which is capable of stably self-binding. For example, the subunits of the IgG Fc domain include IgG CH2 and IgG CH3 constant domains.

"Modifications that promote the binding of the first and second subunits of the Fc domain" are manipulations of the peptide backbone or post-translational modification of the subunits of the Fc domain, thereby reducing or preventing the binding of the polypeptides containing the subunits of the Fc domain to the consistent polypeptide Dimer. As used herein, a modification that promotes binding specifically includes individual modifications to each of the two Fc domain subunits (ie, the first and second subunits of the Fc domain) that are desired to bind, wherein the Such modifications are complementary to each other in order to promote the binding of the two Fc domain subunits. For example, a modification that promotes binding can change the structure or charge of one or both of the Fc domain subunits in order to make it sterically or electrostatically advantageous, respectively. Therefore, (hetero)dimerization occurs between the polypeptide comprising the first Fc domain subunit and the polypeptide comprising the second Fc domain subunit, which is fused to each of these subunits (eg, antigen-binding portions) The meaning of the other components is not the same may not be the same. In some embodiments, the modification to promote binding includes amino acid mutations in the Fc domain, especially amino acid substitutions. In a specific embodiment, the modification that promotes binding comprises individual amino acid mutations, especially amino acid substitutions, in each of the two subunits of the Fc domain.

The term "effector function" when used in relation to antibodies refers to their biological activities attributable to the Fc region of the antibody, which vary by antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cell-mediated Antigen secretion, immune complex-mediated antigen-presenting cell uptake of antigens, down-regulation of cell surface receptors (such as B cell receptors) and B cell activation.

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism that prompts immune effector cells to lyse target cells coated with antibodies. The target cell is a cell that specifically binds to an antibody or a derivative thereof containing an Fc region (generally via a protein portion at the N-terminal of the Fc region). As used herein, the term "decreased ADCC" is defined as the decrease in the number of target cells lysed by the ADCC mechanism defined above in the medium surrounding the target cells at a predetermined antibody concentration within a specified time, and / Or in the medium surrounding the target cells, the antibody concentration required to achieve the lysis of a specified number of target cells by the ADCC mechanism within a specified time increases. ADCC reduction is relative to ADCC mediated by the same antibody that is produced by the same type of host cell using the same standard production, purification, formulation and storage methods (known to those skilled in the art), but has not been engineered. For example, the ADCC reduction mediated by an antibody containing an amino acid substitution that reduces ADCC in the Fc domain is relative to the ADCC mediated by the same antibody without the amino acid substitution in the Fc domain. Analysis suitable for measuring ADCC is well known in the art (see, for example, PCT Publication No. WO 2006/082515 or PCT Publication No. WO 2012/130831).

"Activated Fc receptor" is an Fc receptor that, after being engaged with the Fc domain of an antibody, triggers a signaling event that stimulates the receptor-bearing cells to perform effector functions. Human activated Fc receptors include FcyRIIIa (CD16a), FcyRI (CD64), FcyRIIa (CD32) and FcaRI (CD89).

As used herein, the term "engineered/engineered/engineering" is deemed to include any peptide backbone manipulation or post-translational modification of naturally occurring or recombinant polypeptides or fragments thereof. Engineering modification includes modification of amino acid sequence, glycosylation pattern, or side chain group of individual amino acid, and a combination of these methods.

"Reduced binding" (eg, decreased binding to Fc receptors or CD25) refers to a decrease in the affinity of the corresponding interaction, as measured by, for example, SPR. For the sake of clarity, the term also includes that the affinity is reduced to zero (or below the detection limit of the analytical method), that is, the interaction is completely removed. In contrast, "enhanced binding" refers to an increase in the binding affinity of individual interactions.

As used herein, the term "immunoconjugate" refers to a polypeptide molecule that includes at least one IL-2 molecule and at least one antibody. IL-2 molecules can engage with antibodies through a variety of interactions and a variety of configurations as described herein. In a specific embodiment, the IL-2 molecule is fused to the antibody via a peptide linker. The specific immunoconjugate according to the present invention basically consists of an IL-2 molecule and an antibody joined by one or more linking sequences.

"Fusion" means that components (such as antibodies and IL-2 molecules) are connected directly by peptide bonds or connected via one or more peptide linkers.

As used herein, when there are more than one type of parts, the terms "first" and "second" regarding the Fc domain subunits, etc. are used for the convenience of distinction. The use of these terms is not intended to confer a specific order or orientation of immunoconjugates, unless explicitly stated as such.

The "effective amount" of a drug refers to the amount required to produce physiological changes in the cells or tissues administered.

The "therapeutically effective amount" of a medicament (such as a pharmaceutical composition) refers to an amount that is effective to achieve the desired therapeutic or preventive results at the dosage and the necessary time period. For example, a therapeutically effective amount of an agent can eliminate, reduce, delay, minimize or prevent the side effects of the disease.

"Individual/subject" is a mammal. Mammals include, but are not limited to, domesticated animals (such as cows, sheep, cats, dogs, and horses), primates (such as humans and non-human primates, such as monkeys), rabbits, and rodents (such as mice) And rats). In particular, individuals (individual/subject) are humans.

The term "pharmaceutical composition" refers to a preparation in a form that allows the biological activity of the active ingredients contained therein to be effectively exerted, and it does not contain other components that have unacceptable toxicity to the individual to which the formulation will be administered.

"Pharmaceutically acceptable carrier" refers to ingredients in a pharmaceutical composition that are not toxic to an individual except for the active ingredients. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

As used herein, "treatment" (and its grammatical variants, such as "treat" or "treating") refers to clinical intervention that attempts to change the natural course of the individual's disease and can produce For prevention or during the clinical pathology process. The desired therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, alleviating any direct or indirect pathological results of the disease, preventing cancer metastasis, reducing the rate of disease progression, improving or alleviating the disease condition, and alleviating or improving the prognosis. In some embodiments, the immunoconjugates of the present invention are used to delay or slow down disease progression.

Detailed description of the embodiment

Mutant IL-2 Peptides The immunoconjugate of the present invention comprises a mutant IL-2 polypeptide with advantageous properties of immunotherapy. Specifically, the mutant IL-2 polypeptide excludes the pharmacological properties of IL-2 that cause toxicity but are not necessary for the efficacy of IL-2. Such mutant IL-2 polypeptides are described in detail in WO 2012/107417, which is incorporated herein by reference in its entirety. As discussed above, different forms of IL-2 receptors are composed of different subunits and exhibit different affinities for IL-2. Intermediate affinity IL-2 receptors composed of β and γ receptor subunits are expressed on resting effector cells and are sufficient for IL-2 signal transduction. In addition, the high-affinity IL-2 receptor containing the α-subunit of the receptor is mainly manifested in the regulation of T (T_reg ) On cells and on activated effector cells, where its engagement with IL-2 can promote T_reg Cell-mediated immunosuppression or activation-induced cell death (AICD). Therefore, without wishing to be bound by theory, the reduction or elimination of the affinity of IL-2 for the α-subunit of the IL-2 receptor should reduce the IL-2 induced down-regulation of effector cell functions by regulatory T cells and reduce AICD The process produces tumor resistance. On the other hand, maintaining the affinity for the medium-affinity IL-2 receptor should maintain the proliferation and activation of IL-2 induced effector cells (such as NK and T cells).

The mutant interleukin-2 (IL-2) polypeptide contained in the immunoconjugate of the present invention contains at least one amino acid mutation that eliminates or reduces the α of the mutant IL-2 polypeptide on the IL-2 receptor -The affinity of the subunit and maintain the affinity of the mutant IL-2 polypeptide to the medium-affinity IL-2 receptor, each compared to the wild-type IL-2 polypeptide.

A mutant of human IL-2 (hIL-2) with reduced affinity for CD25 can be, for example, substituted with an amino acid at positions 35, 38, 42, 43, 45 or 72 of the amino acid or a combination thereof (relative to human IL-2). -2 sequence SEQ ID NO: 13 numbering) was generated. Exemplary amino acid substitutions include K35E, K35A, R38A, R38E, R38N, R38F, R38S, R38L, R38G, R38Y, R38W, F42L, F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K , K43E, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R and L72K. Specific IL-2 mutants suitable for use in the immunoconjugates of the present invention include amino acid mutations corresponding to the amino acid positions of residue 42, 45 or 72 of human IL-2, or a combination thereof. In one embodiment, the amino acid mutation is an amino acid substitution selected from the group consisting of F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R and L72K, more specifically, selected from the group of F42A, Y45A and L72G Amino acid substitution. Compared with the wild-type form of IL-2 mutants, these mutants show substantially similar binding affinity to the medium-affinity IL-2 receptor, and to the α-subunit of the IL-2 receptor and high-affinity IL- 2 The affinity of the receptor is substantially reduced.

Other characteristics of applicable mutants may include the ability to induce the proliferation of T cells and/or NK cells with IL-2 receptors, and the induction of IL-2 signals in T cells and/or NK cells with IL-2 receptors The ability of conduction, the ability of NK cells to produce interferon (IFN)-γ as a secondary cytokine, and induce peripheral blood mononuclear cells (PBMC) to process secondary cytokines (specifically, IL-10 and TNF-α) ) Decreased ability, decreased ability to activate regulatory T cells, decreased ability to induce apoptosis of T cells, and decreased toxicity distribution in vivo.

The specific mutant IL-2 polypeptide suitable for the present invention contains three amino acid mutations. The amino acid mutations described eliminate or reduce the affinity of the mutant IL-2 polypeptide to the α-subunit of the IL-2 receptor, but maintain The affinity of the mutant IL-2 polypeptide to the medium-affinity IL-2 receptor. In one embodiment, the three amino acid mutations are located at positions corresponding to residues 42, 45 and 72 of human IL-2. In one embodiment, the three amino acids are mutated to amino acid substitutions. In one embodiment, the three amino acids are mutated into amino acid substitutions selected from the following group: F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S , Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R and L72K. In a specific embodiment, the three amino acids are mutated into amino acid substitutions F42A, Y45A and L72G (relative to the human IL-2 sequence numbering in SEQ ID NO: 13).

In certain embodiments, the amino acid mutation reduces the affinity of the mutant IL-2 polypeptide to the α-subunit of the IL-2 receptor by at least 5-fold, especially at least 10-fold, and more especially at least 25-fold. In embodiments where there is more than one amino acid mutation that reduces the affinity of the mutant IL-2 polypeptide to the α-subunit of the IL-2 receptor, the combination of these amino acid mutations can make the mutant IL- 2 The affinity of the polypeptide to the α-subunit of IL-2 receptor is reduced by at least 30-fold, at least 50-fold, or even at least 100-fold. In one embodiment, the amino acid mutation or the combination of amino acid mutation eliminates the affinity of the mutant IL-2 polypeptide to the α-subunit of the IL-2 receptor, so that the surface plasmon resonance cannot be detected Combine.

When the affinity of the IL-2 mutant to the medium-affinity IL-2 receptor is higher than that of the wild-type form of the IL-2 mutant by about 70%, a substantially similar binding to the medium-affinity receptor is achieved That is, the affinity of the mutant IL-2 polypeptide to the receptor is maintained. The IL-2 mutant of the present invention may exhibit an affinity higher than about 80% and even higher than about 90%.

The combination of the reduced affinity of IL-2 for the α-subunit of the IL-2 receptor and the elimination of O-glycosylation of IL-2 produces IL-2 proteins with improved properties. For example, when the mutant IL-2 polypeptide is expressed in mammalian cells (such as CHO or HEK cells), elimination of the O-glycosylation site results in a more homogeneous product.

Therefore, in certain embodiments, the mutant IL-2 polypeptide contains an additional amino acid mutation that eliminates the IL-2 O-glycosyl group at the position corresponding to residue 3 of human IL-2化site. In one embodiment, the elimination of this additional amino acid mutation corresponding to the IL-2 O-glycosylation site at the position of residue 3 of human IL-2 is an amino acid substitution. Exemplary amino acid substitutions include T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, and T3P. In a specific embodiment, the additional amino acid is mutated to an amino acid instead of T3A.

In certain embodiments, the mutant IL-2 polypeptide is substantially a full-length IL-2 molecule. In certain embodiments, the mutant IL-2 polypeptide is a human IL-2 molecule. In one embodiment, the mutant IL-2 polypeptide comprises the sequence of SEQ ID NO: 13 with at least one amino acid mutation, compared to the IL-2 polypeptide comprising SEQ ID NO: 13 without the mutation. At least one amino acid mutation eliminates or reduces the affinity of the mutant IL-2 polypeptide to the α-subunit of the IL-2 receptor, but maintains the affinity of the mutant IL-2 polypeptide to the medium-affinity IL-2 receptor. In another embodiment, the mutant IL-2 polypeptide comprises the sequence of SEQ ID NO: 13 with at least one amino acid mutation, compared to the IL-2 polypeptide comprising SEQ ID NO: 13 without the mutation, The at least one amino acid mutation eliminates or reduces the affinity of the mutant IL-2 polypeptide to the α-subunit of the IL-2 receptor, but maintains the affinity of the mutant IL-2 polypeptide to the medium-affinity IL-2 receptor.

In a specific embodiment, the mutant IL-2 polypeptide can trigger one or more cellular responses selected from the group consisting of: activated T lymphocyte proliferation, activated T lymphocyte differentiation, cytotoxic T cell (CTL) activity, activation B cell proliferation, activated B cell differentiation, natural killer (NK) cell proliferation, NK cell differentiation, activated T cell or NK cell to secrete cytokines, and NK/lymphocyte activated killer (LAK) anti-tumor cytotoxicity.

In one embodiment, the mutant IL-2 polypeptide has a reduced ability to induce and regulate IL-2 signaling in T cells compared to the wild-type IL-2 polypeptide. In one embodiment, the mutant IL-2 polypeptide induces less activation-induced cell death (AICD) of T cells than the wild-type IL-2 polypeptide. In one embodiment, the mutant IL-2 polypeptide has a reduced toxicity profile in vivo compared to the wild-type IL-2 polypeptide. In one embodiment, the mutant IL-2 polypeptide has an extended serum half-life compared to the wild-type IL-2 polypeptide.

The specific mutant IL-2 polypeptide suitable for use in the present invention contains four amino acid substitutions at positions corresponding to residues 3, 42, 45 and 72 of human IL-2. The specific amino acids are substituted with T3A, F42A, Y45A and L72G. As shown in WO 2012/107417, the quadruple mutant IL-2 polypeptide does not exhibit detectable binding to CD25, reduces the ability of T cells to induce apoptosis, and induces _{IL- in T reg} cells. 2 The ability of signal transmission is reduced, and the distribution of toxicity in vivo is reduced. However, it retains the ability to activate IL-2 signaling in effector cells, induce effector cell proliferation, and NK cells to produce IFN-γ as a secondary cytokine.

In addition, the mutant IL-2 polypeptide has other advantageous properties, such as reduced surface hydrophobicity, good stability, and good performance, as described in WO 2012/107417. Unexpectedly, the mutant IL-2 polypeptide also provides an extended serum half-life compared to wild-type IL-2.

In addition to mutations in the IL-2 region that forms the interface between IL-2 and CD25 or glycosylation sites, IL-2 mutants suitable for use in the present invention may also have a mutation in the amino acid sequence outside these regions. Or multiple mutations. Such additional mutations in human IL-2 may provide additional advantages, such as enhanced performance or stability. For example, the cysteine at position 125 can be replaced with a neutral amino acid, such as serine, alanine, threonine or valine, to produce C125S IL-2, C125A IL-2, C125T, respectively IL-2 or C125V IL-2, as described in U.S. Patent No. 4,518,584. As described therein, the N-terminal alanine residue of IL-2 can also be deleted to generate mutants such as des-A1 C125S or des-A1 C125A. Alternatively or in combination, IL-2 mutants may include mutations in which the methionine normally present at position 104 of wild-type human IL-2 is replaced with a neutral amino acid (such as alanine) (see U.S. Patent No. 5,206,344). The resulting mutants, such as des-A1 M104A IL-2, des-A1 M104A C125S IL-2, M104A IL-2, M104A C125A IL-2, des-A1 M104A C125A IL-2 or M104A C125S IL-2 (these And other mutants can be found in US Patent No. 5,116,943 and Weiger et al., Eur J Biochem 180, 295-300 (1989)), which can be used in combination with specific IL-2 mutations of the present invention.

Therefore, in certain embodiments, the mutant IL-2 polypeptide comprises an additional amino acid mutation at a position corresponding to residue 125 of human IL-2. In one embodiment, the additional amino acid is mutated to an amino acid substitution C125A.

Those skilled in the art will be able to determine which additional mutations can provide additional advantages for the purposes of the present invention. For example, it should be understood that amino acid mutations in the IL-2 sequence that reduce or eliminate the affinity of IL-2 for the medium-affinity IL-2 receptor, such as D20T, N88R or Q126D (see, for example, US 2007/0036752) , May not be suitable for inclusion in the mutant IL-2 polypeptide according to the present invention.

In one embodiment, compared to the corresponding wild-type IL-2 sequence, such as the human IL-2 sequence of SEQ ID NO: 13, the mutant IL-2 polypeptide contains no more than 12, no more than 11, and no More than 10, no more than 9, no more than 8, no more than 7, no more than 6, or no more than 5 amino acid mutations. In a specific embodiment, the mutant IL-2 polypeptide contains no more than 5 amino acid mutations compared to the corresponding wild-type IL-2 sequence, such as the human IL-2 sequence of SEQ ID NO: 13.

In one embodiment, the mutant IL-2 polypeptide comprises the sequence of SEQ ID NO: 14. In one embodiment, the mutant IL-2 polypeptide consists of the sequence of SEQ ID NO: 14.

Immunoconjugate The immunoconjugates as described herein include IL-2 molecules and antibodies. Such immunoconjugates significantly enhance the efficacy of IL-2 therapy by, for example, targeting IL-2 directly to the tumor microenvironment. According to the present invention, the antibody contained in the immunoconjugate may be a complete antibody or an immunoglobulin, or a part or variant thereof having a biological function (such as antigen-specific binding affinity).

The general benefits of immunoconjugate therapy are obvious. For example, the antibody contained in the immunoconjugate recognizes a tumor-specific epitope and causes the immunoconjugate molecule to target the tumor site. Therefore, the use of immunoconjugates at a much lower dose than unbound IL-2 can deliver high concentrations of IL-2 to the tumor microenvironment, thereby causing the various immune effector cells mentioned in this article. Activation and proliferation. In addition, since IL-2 is administered in the form of immunoconjugates to reduce the dose of cytokines themselves, the potential undesirable side effects of IL-2 are limited, and IL-2 is targeted to specific parts of the body with the help of immunoconjugates It can also reduce systemic exposure and thus reduce side effects (compared to those obtained with unbound IL-2). In addition, the circulating half-life of the immunoconjugate is longer than that of unbound IL-2 to promote the efficacy of the immunoconjugate. However, this feature of IL-2 immunoconjugates may once again aggravate the potential side effects of IL-2 molecules: because the circulating half-life of IL-2 immunoconjugates in the bloodstream is significantly longer than that of unbound IL-2, the fusion protein The IL-2 or other parts of the molecule have an increased chance of activating components normally present in blood vessels. There is the same concern with other fusion proteins containing IL-2 fused to other parts (such as Fc or albumin), which increases the half-life of IL-2 in the circulation. Therefore, immunoconjugates containing mutant IL-2 polypeptides as described herein and WO 2012/107417 are particularly advantageous, and their toxicity is reduced compared to the wild-type IL-2 form.

As described above, targeting IL-2 directly to immune effector cells rather than tumor cells can be beneficial for IL-2 immunotherapy.

Therefore, the present invention provides a mutant IL-2 polypeptide as described above and an antibody that binds to CD8. In one embodiment, the mutant IL-2 polypeptide and the antibody form a fusion protein, that is, the mutant IL-2 polypeptide and the antibody share a peptide bond. In some embodiments, the antibody comprises an Fc domain composed of first and second subunits. In a specific embodiment, the mutant IL-2 polypeptide is fused to the carboxy terminal amino acid of the primary unit of the Fc domain at its amino terminal amino acid, optionally via a linker peptide. In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is an immunoglobulin molecule, specifically, an IgG class immunoglobulin molecule, more specifically, an IgG ₁ subclass immunoglobulin molecule. In one such embodiment, the mutant IL-2 polypeptide shares an amino terminal peptide bond with one of the immunoglobulin heavy chains. In certain embodiments, the antibody is an antibody fragment. In some embodiments, the antibody is a Fab molecule or a scFv molecule. In one embodiment, the antibody is a Fab molecule. In another embodiment, the antibody is a scFv molecule. The immunoconjugate may also contain more than one antibody. When more than one antibody is included in the immunoconjugate, for example, a first antibody and a second antibody, each antibody can be independently selected from various forms of antibodies and antibody fragments. For example, the first antibody can be a Fab molecule and the second antibody can be a scFv molecule. In a specific embodiment, each of the first antibody and the second antibody is a scFv molecule or each of the first antibody and the second antibody is a Fab molecule. In a specific embodiment, each of the first antibody and the second antibody is a Fab molecule. In one embodiment, each of the first antibody and the second antibody binds to CD8.

Immunoconjugate patterns Exemplary immunoconjugate patterns are described in PCT Publication No. WO 2011/020783, which is incorporated herein by reference in its entirety. These immunoconjugates contain at least two antibodies. Therefore, in one embodiment, the immunoconjugate according to the present invention comprises a mutant IL-2 polypeptide as described herein and at least one first and second antibody. In a specific embodiment, the first and second antibody systems are independently selected from the group consisting of Fv molecules (specifically, scFv molecules) and Fab molecules. In a specific embodiment, the mutant IL-2 polypeptide and the first antibody share an amine group or carboxy-terminal peptide bond, and the second antibody shares an amine group with i) the mutant IL-2 polypeptide or ii) the first antibody. Group or carboxyl terminal peptide bond. In a specific embodiment, the immunoconjugate basically consists of the mutant IL-2 polypeptide and the first and second antibodies, especially Fab molecules connected by one or more linking sequences. The advantage of this type of formula is that it binds to the target antigen (CD8) with high affinity, but only the monomer binds to the IL-2 receptor, thus avoiding the carrying of the immune conjugate to other positions except the target site Immune cells with IL-2 receptors. In a specific embodiment, the mutant IL-2 polypeptide and the first antibody, specifically, the first Fab molecule shares the carboxy-terminal peptide bond, and further shares the amino group with the second antibody, specifically, the second Fab molecule Terminal peptide bond. In another embodiment, the first antibody, in particular, the first Fab molecule and the mutant IL-2 polypeptide share the carboxy terminal peptide bond, and further shares the amino group with the second antibody, in particular, the second Fab molecule Terminal peptide bond. In another embodiment, the first antibody, in particular, the first Fab molecule shares an amino terminal peptide bond with the first mutant IL-2 polypeptide, and is further connected to the second antibody, in particular, the second Fab molecule Shared carboxy terminal peptide. In a specific embodiment, the mutant IL-2 polypeptide shares the carboxy terminal peptide bond with the first heavy chain variable region and additionally shares the amino terminal peptide bond with the second heavy chain variable region. In another embodiment, the mutant IL-2 polypeptide shares the carboxy terminal peptide bond with the first light chain variable region and additionally shares the amino terminal peptide bond with the second light chain variable region. In another embodiment, the variable region of the first heavy or light chain is joined to the mutant IL-2 polypeptide by a carboxy-terminal peptide bond and is additionally variable with the second heavy or light chain via an amino-terminal peptide bond.区接。 Zone junction. In another embodiment, the variable region of the first heavy or light chain is joined to the mutant IL-2 polypeptide by an amino-terminal peptide bond and is additionally variable with the second heavy or light chain by a carboxy-terminal peptide bond.区接。 Zone junction. In one embodiment, the mutant IL-2 polypeptide shares the carboxy terminal peptide bond with the first Fab heavy chain or light chain and additionally shares the amino terminal peptide bond with the second Fab heavy chain or light chain. In another embodiment, the first Fab heavy chain or light chain shares the carboxy terminal peptide bond with the mutant IL-2 polypeptide and additionally shares the amino terminal peptide bond with the second Fab heavy chain or light chain. In other embodiments, the first Fab heavy chain or light chain shares an amino terminal peptide bond with the mutant IL-2 polypeptide and further shares a carboxy terminal peptide bond with the second Fab heavy chain or light chain. In one embodiment, the immunoconjugate comprises a mutant IL-2 polypeptide that shares an amino terminal peptide bond with one or more scFv molecules and additionally shares a carboxy terminal peptide bond with one or more scFv molecules.

However, a form particularly suitable for the immunoconjugate according to the present invention contains immunoglobulin molecules as antibodies. This type of immunoconjugate is described in WO 2012/146628, which is incorporated herein by reference in its entirety.

Thus, in certain embodiments, an immunoconjugate comprises a mutant IL-2 polypeptide as described herein, and the binding of the immunoglobulin molecules CD8, IgG molecules specific words, more specific _{words. 1} IgG molecule. In one embodiment, the immunoconjugate contains no more than one mutant IL-2 polypeptide. In one embodiment, the immunoglobulin molecule is a human immunoglobulin molecule. In one embodiment, the immunoglobulin molecule comprises human constant regions, such as human CH1, CH2, CH3, and/or CL domains. In one embodiment, the immunoglobulin comprises a human Fc domain, specifically a human IgG ₁ Fc domain. In one embodiment, the mutant IL-2 polypeptide shares an amine or carboxy terminal peptide bond with the immunoglobulin molecule. In one embodiment, immunoconjugates consisting essentially of mutant IL-2 polypeptide with an immunoglobulin molecule (IgG molecule specific words, words more particularly IgG _{molecules. 1)} or by a plurality of connection sequences bonding composition. In a specific embodiment, the mutant IL-2 polypeptide is fused to the carboxy terminal amino acid of one of the immunoglobulin heavy chains at the amino terminal amino acid, optionally via a linker peptide.

The mutant IL-2 polypeptide can be fused to the antibody either directly or via a linker peptide that contains one or more amino acids, typically about 2 to 20 amino acids. Connecting peptides are known in the art and are described herein. Suitable non-immunogenic linker peptides include, for example, (G ₄ S) _n , (SG ₄ ) _n , (G ₄ S) _n or G ₄ (SG ₄ ) _n linker peptides. "N" is generally 1 to 10, usually an integer of 2 to 4. In one embodiment, the connecting peptide has a length of at least 5 amino acids; in one embodiment, it has a length of 5 to 100 amino acids; in another embodiment, it has a length of 10 to 50 amino acids. The length. In a specific embodiment, the connecting peptide has a length of 15 amino acids. In one embodiment, the connecting peptide is (GxS) _n or (GxS) _n G _m , where G=glycine, S=serine, and (x=3, n=3, 4, 5, or 6, And m=0, 1, 2 or 3) or (x=4, n=2, 3, 4 or 5 and m=0, 1, 2 or 3), in one embodiment, x=4 and n= 2 or 3. In another embodiment, x=4 and n=3. In a specific embodiment, the connecting peptide is (G ₄ S) ₃ (SEQ ID NO: 15). In one embodiment, the connecting peptide has (or consists of) the amino acid sequence of SEQ ID NO: 15.

In a specific embodiment, the immunoconjugate comprises a mutant IL-2 molecule and an immunoglobulin molecule that binds to CD8, specifically, an immunoglobulin molecule of the IgG ₁ subclass, wherein the mutant IL-2 molecule is in its amino group. The terminal amino acid is fused to the carboxy terminal amino acid of one of the immunoglobulin heavy chains via the connecting peptide of SEQ ID NO: 15.

In a specific embodiment, the immunoconjugate includes a mutant IL-2 molecule and an antibody that binds to CD8, wherein the antibody includes an Fc domain composed of the first and second subunits, specifically, the human IgG ₁ Fc domain , And the mutant IL-2 molecule is fused to the carboxy terminal amino acid of one of the subunits of the Fc domain at its amino terminal amino acid via the connecting peptide of SEQ ID NO: 15.

CD8 Antibody The antibody contained in the immunoconjugate of the present invention binds to CD8 (specifically, human CD8), and can direct the mutant IL-2 polypeptide to the target site where CD8 is expressed, specifically, for example, the expression CD8 associated with tumors T cells.

Suitable CD8 antibodies that can be used in the immunoconjugates of the present invention are described in PCT Publication No. WO 2019/033043 A2, which is incorporated herein by reference in its entirety.

The immunoconjugate of the present invention may comprise two or more antibodies, and these antibodies may bind to the same or different antigens. However, in specific embodiments, each of these antibodies binds to CD8. In one embodiment, the antibody contained in the immunoconjugate of the present invention is monospecific. In a specific embodiment, the immunoconjugate comprises a single monospecific antibody, specifically a monospecific immunoglobulin molecule.

The antibody may be any type of antibody or fragment thereof that retains specific binding to CD8 (specifically, human CD8). Antibody fragments include (but are not limited to) Fv molecules, scFv molecules, Fab molecules and F(ab') ₂ molecules. However, in certain embodiments, the antibody is a full-length antibody. In some embodiments, the antibody comprises an Fc domain composed of first and second subunits. In some embodiments, the antibody is an immunoglobulin, specifically an IgG class, more specifically an IgG ₁ subclass immunoglobulin.

In some embodiments, the antibody is a monoclonal antibody.

Functional characteristics The anti-CD8 antibodies provided herein have one or more of the following characteristics: (a) The antibody does not inhibit or stimulate CD8⁺ Activation of T cells; (b) Antibody does not induce CD8⁺ T cell proliferation; (c) antibody does not induce IFNγ production; (d) antibody specifically binds to human CD8; (e) antibody specifically binds to rhesus CD8; (f) antibody specifically binds to rhesus CD8; (g) antibody does not Combine CD4⁺ Cell; (g) Antibody does not bind CD3^- Cell; and (h) antibody does not consume CD8 from the circulation⁺ T cells. Such characteristics can be assessed using well-known methods, such as the method used in PCT application WO 2019/033043 A2.

Anti-CD8 antibodies are antibodies that bind to CD8 with sufficient affinity and specificity. In certain embodiments, the anti-CD8 antibody binds to human CD8 with a Kn of about any of the following: 1 μM, 100 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 1 nM , 0.5 nM, 0.1 nM, 0.05 nM or 0.001 nM (e.g. 10 ^- nM ⁸ M or less, e.g. 10 ^-8 M to 10 ^-13 M, e.g. 10 ^-9 M to 10 ^-13 M), including these values Any range in between. In certain embodiments, the anti-CD8 antibody binds to rhesus CD8 with a Kn of about any of the following: 1 μM, 100 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM, or 0.001 nM (for example, 10 ^-8 M or less, for example, 10 ^-8 M to 10 ^-13 M, for example, 10 ^-9 M to 10 ^-13 M), including these Any range between values. In certain embodiments, the anti-CD8 antibody binds cynomolgus CD8 with the following Kn: 1 μM, 100 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM, or 0.001 nM (e.g. 10 ^-8 M or less, such as 10 ^-8 M to 10 ^-13 M, such as 10 ^-9 M to 10 ^-13 M), including any range between these values .

In certain embodiments, the anti-CD8 antibody binds to (a) human CD8 with the following Kn: about 1 μM, 100 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM or 0.001 nM (e.g. 10 ^-8 M or less, such as 10 ^-8 M to 10 ^-13 M, such as 10 ^-9 M to 10 ^-13 M), including those between these values Any range; bind with the following Kn (b) Rhesus monkey CD8: about 1 μM, 100 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 1 nM, 0.5 nM, 1.1 nM, 0.05 nM or 0.001 nM (e.g. 10 ^-8 M or less, such as 10 ^-8 M to 10 ^-13 M, such as 10 ^-9 M to 10 ^-13 M), including any range between these equivalent values; and The following Kn binding (c) Cynomolgus CD8: about 1 μM, 100 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM Or 0.001 nM (e.g. 10 ^-8 M or less, e.g. 10 ^-8 M to 10 ^-13 M, e.g. 10 ^-9 M to 10 ^-13 M), including any range between these equivalent values. The Kn of the anti-CD8 antibodies provided herein against human CD8, rhesus CD8 and/or rhesus CD8 can be determined by any method known in the art, including (but not limited to) such as ELISA, fluorescence activated cell sorting (FACS) analysis, radioimmunoprecipitation (RIA) and surface plasmon resonance (SPR). In certain embodiments, the Kn of the anti-CD8 antibodies provided herein against human CD8, rhesus CD8 and/or rhesus CD8 is determined by SPR. In certain embodiments, the Kn of the anti-CD8 antibodies provided herein against human CD8, rhesus CD8 and/or rhesus CD8 is determined by FACS.

In certain embodiments, the anti-CD8 antibodies provided herein do not bind (eg, specifically bind) mouse CD8. In certain embodiments, the anti-CD8 antibody does not bind (eg, specifically bind) to rat CD8. In certain embodiments, the anti-CD8 antibody does not bind (eg, specifically bind) mouse CD8 or rat CD8, for example, as determined by SPR and/or FACS.

In some embodiments, the antibody comprises: (a) HCDR1, which comprises the amino acid sequence of SEQ ID NO: 1; HCDR2, which comprises the amino acid sequence of SEQ ID NO: 2; HCDR3, which comprises SEQ ID NO: The amino acid sequence of 3; LCDR1, which includes the amino acid sequence of SEQ ID NO: 4; LCDR2, which includes the amino acid sequence of SEQ ID NO: 5; and LCDR3, which includes the amino acid sequence of SEQ ID NO: 6 Acid sequence.

In some embodiments, the antibody comprises: (a) a heavy chain variable region (VH), which comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 1 and HCDR2 containing the amino acid sequence of SEQ ID NO: 2 , HCDR3 containing the amino acid sequence of SEQ ID NO: 3; and (b) the light chain variable region (VL), which comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 4, and containing SEQ ID NO: 5 The amino acid sequence of LCDR2 and the LCDR3 containing the amino acid sequence of SEQ ID NO: 6 In some embodiments, the heavy chain and/or light chain variable regions are humanized variable regions. In some embodiments, the heavy chain and/or light chain variable regions comprise human framework regions (FR).

In some embodiments, the antibody comprises a variable heavy chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 7 District (VH). In some embodiments, the antibody comprises a variable light chain containing an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 8 Area (VL). In some embodiments, the antibody comprises: (a) a heavy chain variable region (VH) comprising at least about 95%, 96%, 97%, 98%, 99% of the amino acid sequence of SEQ ID NO: 7 Or 100% identical amino acid sequence; and (b) the light chain variable region (VL), which comprises at least about 95%, 96%, 97%, 98%, and the amino acid sequence of SEQ ID NO: 8 99% or 100% identical amino acid sequence.

In a specific embodiment, the antibody comprises: (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 7; and (b) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 8 Light chain variable region (VL).

In some embodiments, the antibody comprises: HCDR1, which comprises the amino acid sequence of SEQ ID NO: 1; HCDR2, which comprises the amino acid sequence of SEQ ID NO: 2; HCDR3, which comprises the amine of SEQ ID NO: 3 LCDR1, which includes the amino acid sequence of SEQ ID NO: 4; LCDR2, which includes the amino acid sequence of SEQ ID NO: 5; and LCDR3, which includes the amino acid sequence of SEQ ID NO: 28.

In some embodiments, the antibody comprises: (a) a heavy chain variable region (VH), which comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 1 and HCDR2 containing the amino acid sequence of SEQ ID NO: 2 , HCDR3 containing the amino acid sequence of SEQ ID NO: 3; and (b) the light chain variable region (VL), which comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 4, and containing SEQ ID NO: 5 The amino acid sequence of LCDR2 and the LCDR3 containing the amino acid sequence of SEQ ID NO:28. In some embodiments, the heavy chain and/or light chain variable regions are humanized variable regions. In some embodiments, the heavy chain and/or light chain variable regions comprise human framework regions (FR).

In some embodiments, the antibody comprises a variable heavy chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 7 District (VH). In some embodiments, the antibody comprises a variable light chain containing an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 29 Area (VL). In some embodiments, the antibody comprises: (a) a heavy chain variable region (VH) comprising at least about 95%, 96%, 97%, 98%, 99% of the amino acid sequence of SEQ ID NO: 7 Or a 100% identical amino acid sequence; and (b) the light chain variable region (VL), which comprises at least about 95%, 96%, 97%, 98%, and the amino acid sequence of SEQ ID NO: 29, 99% or 100% identical amino acid sequence.

In a specific embodiment, the antibody comprises: (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 7; and (b) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 29 Light chain variable region (VL).

In some embodiments, the antibody is a humanized antibody. In one embodiment, the antibody is an immunoglobulin molecule containing a human constant region, specifically, an IgG immunoglobulin molecule containing human CH1, CH2, CH3, and/or CL domains. Exemplary sequences of human constant domains are shown in SEQ ID NOs: 22 and 23 (human κ and λ CL domains, respectively) and SEQ ID NO: 24 (human IgG1 heavy chain constant domains CH1-CH2-CH3). In some embodiments, the antibody comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 23, in particular the amino acid sequence of SEQ ID NO: 24. In some embodiments, the antibody comprises a heavy chain constant region containing an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 24 . In particular, as described herein, the heavy chain constant region may include amino acid mutations in the Fc domain.

Fc domain In a specific embodiment, the antibody contained in the immunoconjugate according to the present invention comprises an Fc domain composed of first and second subunits. The Fc domain of an antibody consists of a pair of polypeptide chains containing the heavy chain domain of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, where each subunit includes CH2 and CH3 IgG heavy chain constant domains. The two subunits of the Fc domain can bind to each other stably. In one embodiment, the immunoconjugate of the present invention contains no more than one Fc domain.

In one embodiment, the Fc domain in the antibody contained in the immunoconjugate is an IgG Fc domain. In a specific embodiment, the Fc domain is an IgG ₁ Fc domain. In another embodiment, the Fc domain is an IgG ₄ Fc domain. _{In a more specific embodiment, the Fc domain is an IgG 4} Fc domain comprising an amino acid substitution (specifically, the amino acid substitution S228P) at position S228 (Kabat EU index number). This amino acid substitution reduces _{the Fab arm exchange of IgG 4} antibodies in vivo (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). In another specific embodiment, the Fc domain is a human Fc domain. In an even more specific embodiment, the Fc domain is a human IgG ₁ Fc domain. An exemplary sequence of the Fc region of human IgG ₁ is set forth in SEQ ID NO:21.

Modification of the Fc domain to promote heterodimerization The immunoconjugate according to the present invention comprises a mutant IL-2 polypeptide, specifically a single (not more than one) mutation fused to one or the other of the two subunits of the Fc domain Type IL-2 polypeptide, so the two subunits of the Fc domain are usually contained in two different polypeptide chains. Recombinant co-expression of these polypeptides and subsequent dimerization caused several possible combinations of the two polypeptides. In order to improve the yield and purity of immunoconjugates in recombinant manufacturing, it is therefore advantageous to introduce modifications that promote the binding of the desired polypeptide into the Fc domain of the antibody.

Therefore, in a specific embodiment, the Fc domain of the antibody contained in the immunoconjugate according to the present invention comprises a modification that promotes the binding of the first and second subunits of the Fc domain. The site of the most extensive protein-protein interaction between the two subunits in the human IgG Fc domain is located in the CH3 domain of the Fc domain. Therefore, in one embodiment, the modification is present in the CH3 domain of the Fc domain.

There are several methods for modifying the CH3 domain of the Fc domain to enhance heterodimerization, which are fully described in, for example, WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012058768, WO 2013157954, WO 2013096291. Generally, in all such methods, the CH3 domain of the first unit of the Fc domain and the CH3 domain of the second unit of the Fc domain are both engineered in a complementary manner such that each CH3 domain (or its heavy chain) It can no longer occur homodimerization itself, but is forced to heterodimerize with another CH3 domain that has been engineered in a complementary manner (so that the first and second CH3 domains are heterodimerized and the two first or second second No homodimer is formed between the two CH3 domains).

In a specific embodiment, the modification that promotes the binding of the first and second subunits of the Fc domain is a so-called "knob-into-hole" modification, which includes the two subunits of the Fc domain. The "knob" modification in one of the two subunits of the Fc domain and the "hole" modification in the other of the two subunits of the Fc domain.

The pestle-and-mortar technique is described in, for example, US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally speaking, the method includes introducing a bulge ("punch") at the interface of the first polypeptide and a corresponding cavity ("mortar") at the interface of the second polypeptide, so that the bulge can be positioned in the cavity In order to promote the formation of heterodimers and hinder the formation of homodimers. The bump is constructed by replacing the small amino acid side chain in the interface of the first polypeptide with a larger side chain (such as tyrosine or tryptophan). Compensating cavities with the same or similar size as the bulge are created in the interface of the second polypeptide by replacing the side chain of a large amino acid with a side chain of a smaller amino acid (for example, alanine or threonine).

Correspondingly, in a specific embodiment, in the CH3 domain of the first unit of the Fc domain of the antibody contained in the immunoconjugate, the amino acid residue is passed through the amino acid residue having a larger side chain volume. Replacement to produce a bulge in the CH3 domain of the first subunit that can be located in the cavity in the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain, amino acid residues The group is replaced by an amino acid residue with a smaller side chain volume, thereby creating a cavity in the CH3 domain of the second subunit for the protrusions in the CH3 domain of the first subunit to be located.

Preferably, the amino acid residue with larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W).

Preferably, the amino acid residue with a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V).

The bumps and cavities can be prepared by changing the nucleic acid encoding the polypeptide, for example, by site-specific mutagenesis or by peptide synthesis.

In a specific embodiment, in the CH3 domain of the first subunit ("knob" subunit) of the Fc domain, the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the Fc domain In the CH3 domain of the second subunit ("hole" subunit) of the domain, the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In one embodiment, in the second subunit of the Fc domain, in addition, the threonine residue at position 366 is replaced by serine residue (T366S) and the leucine residue at position 368 is replaced by alanine Residue substitution (L368A) (numbered according to Kabat EU index).

In yet another embodiment, in the first unit of the Fc domain, in addition, the serine residue at position 354 is replaced by a cysteine residue (S354C), or the glutamine residue at position 356 The group is replaced by a cysteine residue (E356C) (specifically, the serine residue at position 354 is replaced by a cysteine residue), and in the second subunit of the Fc domain, in addition, the position The tyrosine residue at 349 was replaced with a cysteine residue (Y349C) (numbering according to the Kabat EU index). The introduction of these two cysteine residues allows the formation of a disulfide bridge between the two subunits of the Fc domain, thereby further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

In a specific embodiment, the first unit of the Fc domain includes amino acid substitutions S354C and T366W, and the second unit of the Fc domain includes amino acid substitutions Y349C, T366S, L368A, and Y407V (numbered according to the Kabat EU index) .

In some embodiments, the second subunit of the Fc domain additionally includes amino acid substitutions H435R and Y436F (numbered according to the Kabat EU index).

In a specific embodiment, the mutant IL-2 polypeptide is fused to the first unit of the Fc domain (including the "knob" modification) (optionally via a linker peptide). Without wishing to be bound by theory, the fusion of the mutant IL-2 polypeptide with the knob-containing subunit of the Fc domain (further) minimizes the production of immunoconjugates containing two mutant IL-2 polypeptides (two knobs-containing The peptide is sterically hindered).

Other techniques for enhancing heterodimerization of CH3 modification are considered as alternatives according to the present invention, and are described, for example, in WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/ 089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012/058768, WO 2013/157954, WO 2013/096291.

In one embodiment, the heterodimerization method described in EP 1870459 is used instead. This method is based on the introduction of charged amino acids with opposite charges at specific amino acid positions in the CH3/CH3 domain interface between the two subunits of the Fc domain. A preferred embodiment for the antibody contained in the immunoconjugate is the amino acid mutation R409D, K370E present in one of the two CH3 domains (of the Fc domain) and the one present in the CH3 domain of the Fc domain. The amino acid mutations in the other were D399K and E357K (numbered according to the Kabat EU index).

In another embodiment, the antibody contained in the immunoconjugate of the present invention comprises: amino acid mutation T366W in the CH3 domain of the first unit of the Fc domain; and in the CH3 domain of the second unit of the Fc domain Amino acid mutations T366S, L368A, Y407V; and other amino acid mutations in the CH3 domain of the first unit of the Fc domain; R409D, K370E; and amino acid mutations in the CH3 domain of the second unit of the Fc domain D399K, E357K (according to Kabat EU index number).

In another embodiment, the antibody contained in the immunoconjugate of the present invention includes amino acid mutations S354C and T366W in the CH3 domain of the first unit of the Fc domain, and the CH3 domain of the second unit of the Fc domain The amino acid mutations in Y349C, T366S, L368A, Y407V; or the antibody contains amino acid mutations in the CH3 domain of the first unit of the Fc domain, Y349C, T366W, and CH3 domains of the second unit of the Fc domain Amino acid mutations S354C, T366S, L368A, Y407V, and other amino acid mutations in the CH3 domain of the first unit of the Fc domain R409D, K370E, and amino acids in the CH3 domain of the second unit of the Fc domain Mutations D399K, E357K (all are numbered according to the Kabat EU index).

In one embodiment, the heterodimerization method described in WO 2013/157953 is used instead. In one embodiment, the first CH3 domain contains the amino acid mutation T366K, and the second CH3 domain contains the amino acid mutation L351D (numbered according to the Kabat EU index). In yet another embodiment, the first CH3 domain further comprises an amino acid mutation L351K. In another embodiment, the second CH3 domain further comprises an amino acid mutation selected from Y349E, Y349D and L368E (preferably L368E) (numbered according to the Kabat EU index).

In one embodiment, the heterodimerization method described in WO 2012/058768 is used instead. In one embodiment, the first CH3 domain contains amino acid mutations L351Y, Y407A, and the second CH3 domain contains amino acid mutations T366A, K409F. In yet another embodiment, the second CH3 domain comprises another amino acid mutation at positions T411, D399, S400, F405, N390, or K392, such as amino acid mutations selected from: a) T411N, T411R, T411Q , T411K, T411D, T411E, or T411W; b) D399R, D399W, D399Y, or D399K; c) S400E, S400D, S400R, or S400K; d) F405I, F405M, F405T, F405S, F405V, or F405W; e) N390R, N390K, or N390D ; F) K392V, K392M, K392R, K392L, K392F or K392E (according to Kabat EU index number). In another embodiment, the first CH3 domain includes amino acid mutations L351Y, Y407A, and the second CH3 domain includes amino acid mutations T366V, K409F. In another embodiment, the first CH3 domain includes amino acid mutation Y407A, and the second CH3 domain includes amino acid mutations T366A, K409F. In another embodiment, the second CH3 domain further includes amino acid mutations K392E, T411E, D399R and S400R (numbered according to the Kabat EU index).

In one embodiment, the heterodimerization method described in WO 2011/143545 is used instead, for example having amino acid modifications (according to the Kabat EU index number) at positions selected from the group consisting of 368 and 409.

In one embodiment, the heterodimerization method described in WO 2011/090762 is used instead, which also uses the above-described pestle-and-mortar technique. In one embodiment, the first CH3 domain contains the amino acid mutation T366W, and the second CH3 domain contains the amino acid mutation Y407A. In one embodiment, the first CH3 domain contains the amino acid mutation T366Y, and the second CH3 domain contains the amino acid mutation Y407T (numbered according to the Kabat EU index).

In one embodiment, the antibody or its Fc domain contained in the immunoconjugate belongs to the IgG ₂ subclass, and the heterodimerization method described in WO 2010/129304 is used instead.

In an alternative embodiment, the modification that promotes the binding of the first and second subunits of the Fc domain includes a modification that mediates the electrostatic steering effect, for example, as described in PCT Publication WO 2009/089004. Generally speaking, this method involves replacing one or more amino acid residues at the interface of two Fc domain subunits with charged amino acid residues, so that the formation of homodimers is electrostatically unfavorable, but heterodimers It is advantageous to concentrate on static electricity. In one such embodiment, the first CH3 domain comprises a negatively charged amino acid (e.g., glutamine (E) or aspartic acid (D), preferably K392D or N392D) against K392 or N392. The second CH3 domain contains a positively charged amino acid (such as lysine (K) or arginine (R), preferably D399K, E356K, D356K or E357K, and more preferably D399K and E356K) Amino acid substitution of D399, E356, D356 or E357. In yet another embodiment, the first CH3 domain further comprises a negatively charged amino acid (such as glutamic acid (E) or aspartic acid (D), preferably K409D or R409D) for K409 or R409 Base acid substitution. In yet another embodiment, the first CH3 domain further or alternatively includes the amino acid of K439 and/or K370 with a negatively charged amino acid (such as glutamic acid (E) or aspartic acid (D)). Acid substitution (all are numbered according to the Kabat EU index).

In yet another embodiment, the heterodimerization method described in WO 2007/147901 is used instead. In one embodiment, the first CH3 domain includes amino acid mutations K253E, D282K, and K322D, and the second CH3 domain includes amino acid mutations D239K, E240K, and K292D (numbered according to the Kabat EU index).

In yet another embodiment, the heterodimerization method described in WO 2007/110205 may be used instead.

In one embodiment, the first unit of the Fc domain includes amino acid substitutions K392D and K409D, and the second unit of the Fc domain includes amino acid substitutions D356K and D399K (numbered according to the Kabat EU index).

Fc domain modification reduces Fc receptor binding and / or effector functions. The Fc domain confers favorable pharmacokinetic properties on immune conjugates, including a long serum half-life that promotes good accumulation in target tissues and a favorable tissue-blood distribution ratio. However, it may also cause undesired targeting of immune conjugates to cells expressing Fc receptors rather than the preferred antigen-carrying cells. In addition, the co-activation of the Fc receptor signaling pathway can cause the release of cytokines, which, in combination with the long half-life of IL-2 polypeptides and immune conjugates, cause excessive activation of cytokines receptors and cause serious side effects after systemic administration . Accordingly, conventional IgG-IL-2 immunoconjugates related to infusion reactions have been described (see, for example, King et al., J Clin Oncol 22, 4463-4473 (2004)).

Therefore, in a specific embodiment, the Fc domain of the antibody contained in the immunoconjugate according to the present invention exhibits reduced binding affinity for Fc receptors and/or reduced effector function _{compared to the native IgG 1 Fc domain.} In one such embodiment, the Fc domain (or antibody comprising the Fc domain) exhibits less than 50%, preferably less than 20%, and more than the _{native IgG 1} Fc domain (or _{antibody comprising the native IgG 1 Fc domain).} Preferably less than 10% and most preferably less than 5% of the binding affinity to the Fc receptor, and/or _{less than 50% compared to the native IgG 1} Fc domain (or _{antibody containing the native IgG 1} Fc domain), preferably less than 20% , More preferably less than 10% and most preferably less than 5% of the effector function. In one embodiment, the Fc domain (or an antibody comprising the Fc domain) does not substantially bind to Fc receptors and/or induce effector functions. In a specific embodiment, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activating Fc receptor. In a specific embodiment, the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, most specifically human FcγRIIIa. In one embodiment, the effector function is one or more selected from the group of CDC, ADCC, ADCP, and cytokine secretion. In a specific embodiment, the effector function is ADCC. In one embodiment, the Fc domain exhibits substantially similar binding affinity to the neonatal Fc receptor (FcRn) compared to the native IgG _{1 Fc domain.} When the Fc domain (or antibody containing the Fc domain) appears to be _{about 70% higher than the native IgG 1} Fc domain (or _{antibody containing the native IgG 1} Fc domain), specifically about 80% higher, and more specifically 90 higher % Of binding affinity to FcRn, a substantially similar binding to FcRn is achieved.

In certain embodiments, the Fc domain is engineered to have reduced binding affinity to Fc receptors and/or reduced effector functions compared to unengineered Fc domains. In a specific embodiment, the Fc domain of the antibody contained in the immunoconjugate includes one or more amino acid mutations that reduce the binding affinity of the Fc domain to Fc receptors and/or effector functions. Typically, the same one or more amino acid mutations are present in each of the two subunits of the Fc domain. In one embodiment, the amino acid mutation reduces the binding affinity of the Fc domain to the Fc receptor. In one embodiment, the amino acid mutation reduces the binding affinity of the Fc domain to the Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold. In embodiments where there is more than one amino acid mutation that reduces the binding affinity of the Fc domain to the Fc receptor, the combination of these amino acid mutations can reduce the binding affinity of the Fc domain to the Fc receptor by at least 10-fold, At least 20 times or even at least 50 times. In one embodiment, an antibody comprising an engineered Fc domain exhibits a ratio of less than 20%, specifically less than 10%, and more specifically less than 5% compared to an antibody comprising an unengineered Fc domain. Binding affinity of Fc receptor. In a specific embodiment, the Fc receptor is an Fcγ receptor. In some embodiments, the Fc receptor is a human Fc receptor. In some embodiments, the Fc receptor is an activating Fc receptor. In a specific embodiment, the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, most specifically human FcγRIIIa. Preferably, the binding to each of these receptors is reduced. In some embodiments, the binding affinity for complement components, specifically the binding affinity for C1q, is also reduced. In one embodiment, the binding affinity to neonatal Fc receptor (FcRn) is not reduced. When the Fc domain (or the antibody comprising the Fc domain) exhibits a binding affinity to FcRn that is about 70% higher than the unengineered form of the Fc domain (or the unengineered form of the antibody comprising the Fc domain) At the same time, a substantially similar binding to FcRn is achieved, that is, the binding affinity of the Fc domain to the receptor is maintained. The Fc domain or the antibody comprising the Fc domain contained in the immunoconjugate of the present invention may exhibit such an affinity of greater than about 80% and even greater than about 90%. In certain embodiments, the Fc domain of the antibody contained in the immunoconjugate is engineered to have reduced effector functions compared to the unengineered Fc domain. Reduced effector functions may include (but are not limited to) one or more of the following: reduction of complement-dependent cytotoxicity (CDC), reduction of antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis ( ADCP) decreased, decreased secretion of cytokines, decreased uptake of antigen by immune complex-mediated antigen-presenting cells, decreased binding to NK cells, decreased binding to macrophages, decreased binding to monocytes, and polymorphism Decreased nuclear cell binding, decreased direct signaling that induces apoptosis, decreased cross-linking of target-bound antibodies, decreased dendritic cell maturation, or decreased T cell activation. In one embodiment, the reduced effector function is one or more selected from the group consisting of reduced CDC, reduced ADCC, reduced ADCP, and reduced secretion of cytokines. In a specific embodiment, the reduced effector function is ADCC reduction. In one embodiment, the ADCC reduction is less than 20% of the ADCC induced by an unengineered Fc domain (or an antibody comprising an unengineered Fc domain).

In one embodiment, the amino acid that reduces the binding affinity and/or effector function of the Fc domain to the Fc receptor is mutated into an amino acid substitution. In one embodiment, the Fc domain comprises amino acid substitutions at positions selected from the group: E233, L234, L235, N297, P331, and P329 (numbered according to the Kabat EU index). In a more specific embodiment, the Fc domain comprises amino acid substitutions at positions selected from the group: L234, L235, and P329 (numbered according to the Kabat EU index). In some embodiments, the Fc domain contains amino acid substitutions L234A and L235A (numbered according to the Kabat EU index). In one such embodiment, the Fc domain is an IgG ₁ Fc domain, especially a human IgG ₁ Fc domain. In one embodiment, the Fc domain comprises an amino acid substitution at position P329. In a more specific embodiment, the amino acid is substituted with P329A or P329G, specifically P329G (according to the Kabat EU index number). In one embodiment, the Fc domain comprises an amino acid substitution at position P329 and another amino acid substitution at a position selected from E233, L234, L235, N297, and P331 (numbered according to the Kabat EU index). In a more specific embodiment, the other amino acid is substituted with E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In a specific embodiment, the Fc domain comprises amino acid substitutions at positions P329, L234, and L235 (numbered according to the Kabat EU index). In a more specific embodiment, the Fc domain includes amino acid mutations L234A, L235A, and P329G ("P329G LALA", "PGLALA" or "LALAPG"). Specifically, in a specific embodiment, each subunit in the Fc domain includes amino acid substitutions L234A, L235A, and P329G (Kabat EU index numbers), that is, in the first and second subunits of the Fc domain In each, the leucine residue at position 234 is replaced by an alanine residue (L234A), the leucine residue at position 235 is replaced by an alanine residue (L235A) and the proline at position 329 The residue was replaced with a glycine residue (P329G) (numbered according to the Kabat EU index). In one such embodiment, the Fc domain is an IgG ₁ Fc domain, especially a human IgG ₁ Fc domain. The "P329G LALA" amino acid substitution combination almost completely eliminates _{the binding of the human IgG 1} Fc domain to the Fcγ receptor (and complement), as described in PCT Publication No. WO 2012/130831, which is incorporated by reference in its entirety. Into this article. WO 2012/130831 also describes methods for preparing such mutant Fc domains and methods for determining their properties (such as Fc receptor binding or effector function).

Compared with IgG ₁ antibody, IgG ₄ antibody exhibits reduced Fc receptor binding affinity and reduced effector function. Therefore, in some embodiments, the Fc domain of the antibody contained in the immunoconjugate of the present invention is an IgG ₄ Fc domain, specifically, a human IgG ₄ Fc domain. In one embodiment, the IgG ₄ Fc domain contains an amino acid substitution at position S228, specifically the amino acid substitution S228P (numbered according to the Kabat EU index). In order to further reduce its binding affinity to the Fc receptor and/or its effector function, in one embodiment, the IgG ₄ Fc domain contains an amino acid substitution at position L235, specifically the amino acid substitution L235E (according to Kabat EU index number). In another embodiment, the IgG ₄ Fc domain contains an amino acid substitution at position P329, specifically the amino acid substitution P329G (numbered according to the Kabat EU index). In a specific embodiment, the IgG ₄ Fc domain contains amino acid substitutions at positions S228, L235 and P329, specifically amino acid substitutions S228P, L235E and P329G (numbered according to the Kabat EU index). Such IgG ₄ Fc domain mutants and their Fcγ receptor binding properties are described in PCT Publication No. WO 2012/130831, which is incorporated herein by reference in its entirety.

In a specific embodiment, the _{Fc domain that exhibits reduced binding affinity to Fc receptors and/or reduced effector functions compared to the native IgG 1} Fc domain is one that includes amino acid substitutions L234A, L235A, and optionally P329G. Human IgG _{1 Fc domain, or human IgG 4} Fc domain (numbered according to Kabat EU index) containing amino acid substitutions S228P, L235E, and optionally P329G.

In certain embodiments, N glycosylation of the Fc domain has been eliminated. In one such embodiment, the Fc domain comprises an amino acid mutation at position N297, specifically the amino acid substitution of alanine for asparagine (N297A) or aspartic acid for the amine of asparagine Base acid substitution (N297D) (numbered according to Kabat EU index).

In addition to the Fc domain described above and in PCT Publication No. WO 2012/130831, Fc domains with reduced Fc receptor binding and/or effector function also include Fc domain residues 238, 265, 269, 270 A substituted Fc domain of one or more of, 297, 327, and 329 (US Patent No. 6,737,056) (according to Kabat EU index number). Such Fc mutants include Fc mutants with substitutions of two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" in which residues 265 and 297 are substituted with alanine Fc mutant (US Patent No. 7,332,581).

The mutant Fc domain can be prepared by amino acid deletion, substitution, insertion or modification using genetic or chemical methods well known in the art. Genetic methods may include site-specific mutagenesis of coding DNA sequences, PCR, gene synthesis and similar methods. The correct nucleotide change can be verified by, for example, sequencing.

The binding to the Fc receptor can be easily determined, for example, by ELISA, or by surface plasmon resonance (SPR), using standard instruments, such as BIAcore instrument (GE Healthcare), and Fc can be obtained, for example, by recombinant expression. Receptor. Alternatively, the binding affinity of an Fc domain or an antibody containing an Fc domain to an Fc receptor can be evaluated using a cell line known to express a specific Fc receptor (such as human NK cells expressing an FcγIIIa receptor).

The effector function of an Fc domain or an antibody containing an Fc domain can be measured by methods known in the art. Examples of in vitro assays used to assess the ADCC activity of molecules of interest are described in U.S. Patent No. 5,500,362; Hellstrom et al. Proc Natl Acad Sci USA 83, 7059-7063 (1986); and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502 (1985); US Patent No. 5,821,337; Bruggemann et al., J Exp Med 166, 1351-1361 (1987). Alternatively, non-radioactive analysis methods can be used (see, for example, ACTI™ non-radioactive cytotoxicity analysis for flow cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox 96 ^® non-radioactive cytotoxicity analysis (Promega, Madison, WI)). Effector cells suitable for this type of analysis include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be assessed in vivo, for example in animal models such as those disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).

In some embodiments, the binding of the Fc domain to complement components (specifically to Clq) is reduced. Therefore, in some embodiments where the Fc domain is engineered to have reduced effector function, the reduced effector function includes reduced CDC. C1q binding analysis can be performed to determine whether the Fc domain or an Fc domain-containing antibody can bind to C1q and therefore have CDC activity. See, for example, C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, CDC analysis can be performed (see, for example, Gazzano-Santoro et al., J Immunol Methods 202, 163 (1996); Cragg et al., Blood 101, 1045-1052 (2003); and Cragg and Glennie, Blood 103, 2738-2743 (2004)).

Methods known in the art (see, for example, Petkova, SB, et al., Int'l. Immunol. 18(12): 1759-1769 (2006); WO 2013/120929) can also be used for FcRn binding and in vivo clearance/ Half-life determination.

Specific aspects of the invention In one aspect, the present invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to CD8, Wherein the mutant IL-2 polypeptide is a human IL-2 molecule, which contains amino acid substitutions F42A, Y45A and L72G (numbering relative to the human IL-2 sequence SEQ ID NO: 13); and Wherein the antibody comprises: (a) a heavy chain variable region (VH), which comprises the amino acid sequence of SEQ ID NO: 7; and (b) a light chain variable region (VL), which comprises SEQ ID NO: 8 The amino acid sequence.

In one aspect, the present invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to CD8, Wherein the mutant IL-2 polypeptide is a human IL-2 molecule, which contains amino acid substitutions T3A, F42A, Y45A, L72G and C125A (numbering relative to the human IL-2 sequence SEQ ID NO: 13); and Wherein the antibody comprises: (a) a heavy chain variable region (VH), which comprises the amino acid sequence of SEQ ID NO: 7; and (b) a light chain variable region (VL), which comprises SEQ ID NO: 8 The amino acid sequence.

In one aspect, the present invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to CD8, Wherein the mutant IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 14; and Wherein the antibody comprises: (a) a heavy chain variable region (VH), which comprises the amino acid sequence of SEQ ID NO: 7; and (b) a light chain variable region (VL), which comprises SEQ ID NO: 8 The amino acid sequence.

In one embodiment, according to any one of the above aspects of the present invention, the antibody is an IgG immunoglobulin comprising a human IgG ₁ Fc domain composed of first and second subunits, wherein the first time in the Fc domain In the unit, the threonine residue at position 366 is replaced by a tryptophan residue (T366W), and in the second unit of the Fc domain, the tyrosine residue at position 407 is replaced by a valine residue (Y407V) and optionally, the threonine residue at position 366 is replaced by serine residue (T366S) and the leucine residue at position 368 is replaced by alanine residue (L368A) (according to Kabat EU index Numbering), and wherein each subunit in the Fc domain further includes amino acid substitutions L234A, L235A and P329G (Kabat EU index numbering).

In this embodiment, the mutant IL-2 polypeptide can be fused to the carboxy terminal amino acid of the first unit of the Fc domain at its amino terminal amino acid, and fused via the connecting peptide of SEQ ID NO: 15.

In one aspect, the present invention provides an immunoconjugate comprising: a polypeptide comprising at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% of the sequence of SEQ ID NO: 9 %, 99%, or 100% identical amino acid sequence; polypeptide, which comprises at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the sequence of SEQ ID NO: 10 % Or 100% identical amino acid sequence; and a polypeptide comprising at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or the sequence of SEQ ID NO: 11 100% identical amino acid sequence.

In a preferred embodiment, the present invention provides an immunoconjugate comprising: a polypeptide comprising the amino acid sequence of SEQ ID NO: 9; a polypeptide comprising the amino acid sequence of SEQ ID NO: 10; and A polypeptide comprising the amino acid sequence of SEQ ID NO:11. In a more preferred embodiment, the present invention provides an immunoconjugate comprising: two polypeptides comprising the amino acid sequence of SEQ ID NO: 9; polypeptides comprising the amino acid sequence of SEQ ID NO: 10 ; And a polypeptide comprising the amino acid sequence of SEQ ID NO:11.

In one aspect, the present invention provides an immunoconjugate comprising: a polypeptide comprising at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% of the sequence of SEQ ID NO: 9 %, 99%, or 100% identical amino acid sequence; polypeptide, which comprises at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the sequence of SEQ ID NO: 10 % Or 100% identical amino acid sequence; and a polypeptide comprising at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or the sequence of SEQ ID NO: 12 100% identical amino acid sequence.

In a preferred embodiment, the present invention provides an immunoconjugate comprising: a polypeptide comprising the amino acid sequence of SEQ ID NO: 9; a polypeptide comprising the amino acid sequence of SEQ ID NO: 10; and A polypeptide comprising the amino acid sequence of SEQ ID NO: 12.

In another aspect, the present invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to CD8, wherein the mutant IL-2 polypeptide contains amino acid substitutions F42A, Y45A, and L72G ( Relative to the human IL-2 sequence SEQ ID NO: 13) human IL-2 molecule; and wherein the antibody comprises: (a) heavy chain variable region (VH), which comprises the amino acid of SEQ ID NO: 7 Sequence; and (b) the light chain variable region (VL), which includes the amino acid sequence of SEQ ID NO:29.

In one aspect, the present invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to CD8, wherein the mutant IL-2 polypeptide contains amino acid substitutions T3A, F42A, Y45A, L72G And C125A (numbered relative to the human IL-2 sequence SEQ ID NO: 13) human IL-2 molecule; and wherein the antibody comprises: (a) heavy chain variable region (VH), which comprises SEQ ID NO: 7 The amino acid sequence; and (b) the light chain variable region (VL), which includes the amino acid sequence of SEQ ID NO:29.

In one aspect, the present invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to CD8, wherein the mutant IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 14; and Wherein the antibody comprises: (a) a heavy chain variable region (VH), which comprises the amino acid sequence of SEQ ID NO: 7; and (b) a light chain variable region (VL), which comprises SEQ ID NO: 29 The amino acid sequence.

In one embodiment, according to any one of the above aspects of the present invention, the antibody is an IgG immunoglobulin comprising a human IgG ₁ Fc domain composed of first and second subunits, wherein the first time in the Fc domain In the unit, the threonine residue at position 366 is replaced by a tryptophan residue (T366W), and in the second unit of the Fc domain, the tyrosine residue at position 407 is replaced by a valine residue (Y407V) and optionally the threonine residue at position 366 is replaced by serine residue (T366S) and the leucine residue at position 368 is replaced by alanine residue (L368A) (according to Kabat EU index numbering) ), and each subunit of the Fc domain further includes amino acid substitutions L234A, L235A and P329G (Kabat EU index number).

In this embodiment, the mutant IL-2 polypeptide can be fused to the carboxy terminal amino acid of the first unit of the Fc domain at its amino terminal amino acid, and fused via the connecting peptide of SEQ ID NO: 15.

In one aspect, the present invention provides an immunoconjugate comprising: a polypeptide comprising at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% of the sequence of SEQ ID NO: 30 %, 99%, or 100% identical amino acid sequence; polypeptide, which comprises at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the sequence of SEQ ID NO: 10 % Or 100% identical amino acid sequence; and a polypeptide comprising at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or the sequence of SEQ ID NO: 11 100% identical amino acid sequence.

In a preferred embodiment, the present invention provides an immunoconjugate comprising: a polypeptide comprising the amino acid sequence of SEQ ID NO: 30; a polypeptide comprising the amino acid sequence of SEQ ID NO: 10; and A polypeptide comprising the amino acid sequence of SEQ ID NO:11. In a more preferred embodiment, the present invention provides an immunoconjugate comprising: two polypeptides comprising the amino acid sequence of SEQ ID NO: 30; polypeptides comprising the amino acid sequence of SEQ ID NO: 10 ; And a polypeptide comprising the amino acid sequence of SEQ ID NO:11.

In one aspect, the present invention provides an immunoconjugate comprising: a polypeptide comprising at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% of the sequence of SEQ ID NO: 30 %, 99%, or 100% identical amino acid sequence; polypeptide, which comprises at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the sequence of SEQ ID NO: 10 % Or 100% identical amino acid sequence; and a polypeptide comprising at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or the sequence of SEQ ID NO: 12 100% identical amino acid sequence.

In a preferred embodiment, the present invention provides an immunoconjugate comprising: a polypeptide comprising the amino acid sequence of SEQ ID NO: 30; a polypeptide comprising the amino acid sequence of SEQ ID NO: 10; and A polypeptide comprising the amino acid sequence of SEQ ID NO: 12.

Polynucleotides The present invention further provides isolated polynucleotides encoding the immunoconjugates or fragments thereof as described herein. In some embodiments, the fragment is an antigen binding fragment.

The polynucleotide encoding the immunoconjugate of the present invention can be expressed as a single polynucleotide encoding the complete immunoconjugate or as multiple (for example, two or more) polynucleotides co-expressing. Polypeptides encoded by co-expressed polynucleotides can be combined via, for example, disulfide bonds or other means to form functional immune conjugates. For example, the light chain portion of the antibody can be encoded by individual polynucleotides from the portion of the immunoconjugate comprising the heavy chain portion of the antibody and the mutant IL-2 polypeptide. When co-expression, heavy chain polypeptides and light chain polypeptides combine to form an immunoconjugate. In another example, the part of the immunoconjugate comprising one of the two Fc domain subunits and the mutant IL-2 polypeptide can be derived from the individual part of the immunoconjugate comprising the other of the two Fc domain subunits. Polynucleotide encoding. When co-expressed, the Fc domain subunits will combine to form an Fc domain.

In some embodiments, the isolated polynucleotide encodes a complete immunoconjugate according to the invention as described herein. In other embodiments, the isolated polynucleotide encodes the polypeptide contained in the immunoconjugate according to the invention as described herein.

In one embodiment, the isolated polynucleotide of the present invention encodes the heavy chain (e.g., immunoglobulin heavy chain) and mutant IL-2 polypeptide of the antibody contained in the immunoconjugate. In another embodiment, the isolated polynucleotide of the present invention encodes the light chain of the antibody contained in the immunoconjugate.

In certain embodiments, the polynucleotide or nucleic acid is DNA. In other embodiments, the polynucleotide of the present invention is RNA, for example, in the form of messenger RNA (mRNA). The RNA of the present invention can be single-stranded or double-stranded.

Recombinant methods The mutant IL-2 polypeptides suitable for use in the present invention can be prepared by deletion, substitution, insertion or modification using genetic or chemical methods well known in the art. Genetic methods may include site-specific mutagenesis of coding DNA sequences, PCR, gene synthesis and similar methods. The correct nucleotide change can be verified by, for example, sequencing. In this regard, the nucleotide sequence of native IL-2 has been described by Taniguchi et al. (Nature 302, 305-10 (1983)) and the nucleic acid encoding human IL-2 was obtained from public collection agencies, such as the American Type Culture Collection (American Type Culture Collection, Rockville MD). The sequence of native human IL-2 is shown in SEQ ID NO: 13. Substitutions or insertions may involve natural and non-natural amino acid residues. Amino acid modification includes well-known chemical modification methods, such as the addition of glycosylation sites or carbohydrate linkages and the like.

The immunoconjugate of the present invention can be obtained, for example, by solid-state peptide synthesis (for example, Merrifield solid phase synthesis) or recombinant production. For recombinant production, one or more polynucleotides (for example as described above) encoding immunoconjugates (fragments) are isolated and inserted into one or more vectors for further colonization in and/or in host cells which performed. Such polynucleotides can be easily separated and sequenced using conventional procedures. In one embodiment, a vector containing one or more polynucleotides of the present invention is provided, preferably an expression vector. The methods well known to those skilled in the art can be used to construct expression vectors containing coding sequences of immunoconjugates (fragments) and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, synthetic technology and in vivo recombination/gene recombination. See, for example, Maniatis et al., MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory, NY (1989); and Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and Wiley Interscience, NY (1989). . The expression vector can be a part of a plastid, a virus, or a nucleic acid fragment. The expression vector includes a performance cassette, and a polynucleotide (ie, a coding region) encoding an immunoconjugate (fragment) is operably combined with a promoter and/or other transcription or translation control elements to be colonized in the performance cassette. . As used herein, a "coding region" is a portion of a nucleic acid composed of codons that are translated into amino acids. Although the "stop codon" (TAG, TGA, or TAA) is not translated into an amino acid, it can be regarded as part of the coding region (if present), but any flanking sequence (e.g., promoter, ribosome binding site) , Transcription terminator, intron, 5'and 3'non-translated regions and similar sequences) are not part of the coding region. In a single polynucleotide construct, such as on a single vector, or in a separate polynucleotide construct, such as on a separate (different) vector, two or more coding regions may be present. In addition, any vector can contain a single coding region, or can contain two or more coding regions. For example, the vector of the present invention can encode one or more polypeptides, which are translated or co-translated into the final protein after protein cleavage. In addition, the vector, polynucleotide or nucleic acid of the present invention can encode heterologous coding regions, and these heterologous coding regions are fused or not fused with the polynucleotide encoding the immunoconjugate of the present invention or its variants or derivatives. . Heterologous coding regions include, but are not limited to, dedicated elements or motifs, such as secretory signal peptides or heterologous functional domains. The operative binding is when the coding region of a gene product (eg, a polypeptide) is combined with one or more regulatory sequences in such a way that the expression of the gene product is under the influence or control of the regulatory sequence. If the induced promoter function results in the transcription of the mRNA encoding the desired gene product and if the nature of the connection between the two DNA fragments does not interfere with the ability of the expression regulatory sequence to direct the expression of the gene product or does not interfere with the ability of the DNA template to be transcribed, then two DNA fragments (Such as the polypeptide coding region and the promoter that binds to it) is "operably bound". Therefore, if the promoter can affect the transcription of the nucleic acid, the promoter region is operably bound to the nucleic acid encoding the polypeptide. The promoter may be a cell-specific promoter that only directs substantial transcription of DNA in a predetermined cell. Other transcription control elements (such as enhancers, operons, repressors, and transcription termination signals) other than promoters can be operably combined with polynucleotides that direct cell-specific transcription. Suitable promoters and other transcription control regions are disclosed herein. Many transcription control regions are already known to those familiar with this technology. These regions include (but are not limited to) transcriptional control regions that function in vertebrate cells, such as (but not limited to) promoters and enhancer segments, which are derived from cytomegalovirus (such as immediate early promoters, together with internal Sub-A), simian virus 40 (e.g. early promoter) and retroviruses (such as Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes (such as actin, heat shock protein, bovine growth hormone, and rabbit β-hemoglobulin), and other sequences that can control gene expression in eukaryotic cells. Other suitable transcription control regions include tissue-specific promoters and enhancers, as well as inducible promoters (such as the promoter-inducible tetracyclin). Similarly, a variety of translation control elements are known to those skilled in the art. These elements include (but are not limited to) ribosome binding sites, translation start and stop codons, and elements derived from viral systems (specifically, internal ribosome entry sites or IRES, also known as CITE sequences) . Performance cassettes may also include other features, such as origins of replication, and/or chromosomal integration elements, such as retrovirus long terminal repeats (LTR), or adeno-associated virus (AAV) inverted terminal repeats (ITR).

The polynucleotide and nucleic acid coding region of the present invention may be connected to other coding regions encoding secretory peptides or signal peptides, thereby guiding the secretion of the polypeptide encoded by the polynucleotide of the present invention. According to the signal hypothesis, the protein secreted by mammalian cells has a signal peptide or secretory leader sequence. Once the output of the growing protein chain across the rough endoplasmic reticulum has started, the signal peptide or secretory leader sequence is cleaved from the mature protein. Those skilled in the art recognize that polypeptides secreted by vertebrate cells usually have a signal peptide fused to the N-terminus of the polypeptide, and the signal peptide is cleaved from the translated polypeptide to produce the polypeptide in a secreted or "mature" form. For example, human IL-2 is translated through a 20 amino acid signal sequence located at the N-terminus of the polypeptide, which is then cleaved to produce mature 133 amino acid human IL-2. In certain embodiments, native signal peptides are used, such as IL-2 signal peptide or immunoglobulin heavy chain or light chain signal peptide, or a functional derivation of that sequence that maintains the ability to guide the secretion of the polypeptide to which it is operably linked Things. Alternatively, a heterologous mammalian signal peptide, or a functional derivative thereof, can be used. For example, the wild-type leader sequence can be replaced with a human tissue plasminogen activator (TPA) or mouse β-glucuronidase leader sequence.

Within the polynucleotide encoding the immunoconjugate (fragment) or at the end of the polynucleotide, there may be a short code that can be used to facilitate subsequent purification (such as histidine tag) or to help label the immunoconjugate. DNA of protein sequence.

In yet another embodiment, a host cell is provided, which comprises one or more polynucleotides of the present invention. In certain embodiments, a host cell is provided, which comprises one or more vectors of the present invention. Polynucleotides and vectors may be used alone or in combination to incorporate any of the features described herein in relation to polynucleotides and vectors, respectively. In one such embodiment, the host cell contains one or more vectors (e.g., has been transformed or transfected with the vectors), and the vectors contain one or more polynucleotides encoding the immunoconjugates of the invention. As used herein, the term "host cell" refers to any kind of cell system, which can be engineered to produce the immunoconjugate of the present invention or fragments thereof. Host cells suitable for replicating and supporting the expression of immunoconjugates are well known in the art. Such cells can be transfected or transduced with specific expression vectors when appropriate, and cells containing a large number of vectors can be cultured for inoculation of large fermenters, so as to obtain sufficient immunoconjugates for clinical applications. Suitable host cells include prokaryotic microorganisms, such as Escherichia coli, or various eukaryotic cells, such as Chinese hamster ovary cells (CHO), insect cells or the like. For example, polypeptides can be produced in bacteria, especially when glycosylation is not required. After expression, the polypeptide can be isolated from the bacterial cytoplasm in the soluble fraction and the polypeptide can be further purified. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are suitable for selection or expression hosts for vectors encoding polypeptides, including glycosylation pathways that have been "humanized", resulting in partial or complete human glycosylation. Types of peptide fungi and yeast strains. See Gerngross, Nat Biotech 22, 1409-1414 (2004), and Li et al., Nat Biotech 24, 210-215 (2006). Host cells suitable for expressing (glycosylated) polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains that can be used in combination with insect cells have been identified, especially for the transfection of Spodoptera frugiperda cells. Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describes PLANTIBODIES ^™ technology for producing antibodies in transgenic plants). Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for growth in suspension may be suitable. Other examples of suitable mammalian host cell lines are monkey kidney CV1 cell lines (COS-7) transformed with SV40; human embryonic kidney cell lines (293 or 293T cells, such as Graham et al., J Gen Virol 36, 59 (1977) )); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells, as described in, for example, Mather, Biol Reprod 23, 243-251 (1980)); monkey Kidney Cells (CV1); African Green Monkey Kidney Cells (VERO-76); Human Cervical Cancer Cells (HELA); Canine Kidney Cells (MDCK); Buffalo Rat Hepatocytes (BRL 3A); Human Lung Cells (W138); Human Hepatocytes (Hep G2); mouse breast tumor cells (MMT 060562); TRI cells (as described in, for example, Mather et al., Annals NY Acad Sci 383, 44-68 (1982)); MRC 5 cells and FS4 cells. Other applicable mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including dhfr ^- CHO cells (Urlaub et al., Proc Natl Acad Sci USA 77, 4216 (1980)); and myeloma cell lines, such as YO, NS0 , P3X63 and Sp2/0. For a review of certain mammalian host cell lines suitable for protein production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Volume 248 (Edited by BKC Lo, Humana Press, Totowa, NJ), pages 255-268 (2003 ). Host cells include cultured cells, such as cultured mammalian cells, yeast cells, insect cells, bacterial cells, and plant cells (to name a few), and also include transgenic animals, transgenic plants, or cultured plants Or cells contained in animal tissues. In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell, such as Chinese hamster ovary (CHO) cells, human fetal kidney (HEK) cells or lymphocytes (e.g., Y0, NS0, Sp20 cells).

Standard techniques for expressing foreign genes in these systems are known in the art. Cells expressing mutant IL-2 polypeptides fused to the heavy or light chain of an antibody can be engineered to also express other antibody chains, so that the expressed mutant IL-2 fusion product has a heavy chain and a light chain Antibody.

In one embodiment, there is provided a method of preparing an immunoconjugate according to the present invention, wherein the method comprises culturing the polynucleus comprising one or more encoding immunoconjugates as provided herein under conditions suitable for expressing the immunoconjugate Utilize the host cell, and optionally recover the immunoconjugate from the host cell (or host cell culture medium).

In the immunoconjugate of the present invention, the mutant IL-2 polypeptide may be genetically fused with the antibody, or may be chemically bound to the antibody. The gene fusion of IL-2 polypeptide and antibody can be designed so that the IL-2 sequence is directly fused with the polypeptide or indirectly fused via a linker sequence. The composition and length of the linker can be determined according to methods well known in the art and its efficacy can be tested. Specific connecting peptides are described herein. Additional sequences may also be included to incorporate the cleavage site to separate the individual components of the fusion if necessary, such as the endopeptidase recognition sequence. In addition, it is also possible to chemically synthesize IL-2 fusion proteins using peptide synthesis methods well known in the art (for example, Merrifield solid phase synthesis). A well-known chemical binding method can be used to chemically bind the mutant IL-2 polypeptide to other molecules (eg, antibodies). For this purpose, bifunctional crosslinkers can be used, such as homofunctional and heterofunctional crosslinkers well known in the art. The type of cross-linking agent used depends on the nature of the molecule coupled to IL-2 and can be easily identified by those skilled in the art. Alternatively or in addition, the mutant IL-2 and/or molecule to which it is desired to bind can be chemically derivatized so that the two can be combined in separate reactions that are also well known in the art.

The immunoconjugate of the present invention includes an antibody. The method of producing antibodies is well known in the art (see, for example, Harlow and Lane, "Antibodies, a laboratory manual", Cold Spring Harbor Laboratory, 1988). Non-naturally occurring antibodies can be constructed using solid-phase peptide synthesis, can be produced recombinantly (for example, as described in US Patent No. 4,186,567) or can be screened for combinations comprising variable heavy chains and variable light chains, for example Library to obtain (see, for example, McCafferty, U.S. Patent No. 5,969,108). Immunoconjugates, antibodies and methods for their production are also described in detail in, for example, PCT Publication Nos. WO 2011/020783, WO 2012/107417, and WO 2012/146628, each of which is cited in full The method is incorporated into this article.

Antibodies of any animal species can be used in the immunoconjugate of the present invention. Non-limiting antibodies suitable for use in the present invention may be of murine, primate or human origin. If the immunoconjugate is intended for human use, a chimeric form of the antibody can be used, in which the constant region of the antibody is derived from a human. Antibodies in humanized or fully human form can also be prepared according to methods well known in the art (see, for example, US Patent No. 5,565,332 to Winter). Humanization can be achieved by a variety of methods, including (but not limited to): (a) grafting non-human (such as donor antibody) CDRs to human (such as acceptor antibody) framework regions and constant regions, with or without retention Key framework residues (such as those residues that have an important role in retaining good antigen binding affinity or antibody function); (b) Only non-human specificity determining regions (SDR or a-CDR; for antibody-antigen interactions) Residues that have a key role in terms of function) are transplanted to human framework regions and constant regions; or (c) the entire non-human variable domain is transplanted, but the surface residues are replaced with human-like segments to "mask it"". Humanized antibodies and their preparation methods are reviewed in, for example, Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are further described in, for example, Riechmann et al., Nature 332:323-329 (1988); Queen et al. , Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321 and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005 ) (Describe specificity determining region (SDR) transplantation); Padlan, Mol. Immunol. 28:489-498 (1991) (describe "surface remodeling");Dall'Acqua et al., Methods 36:43-60 (2005 ) (Description "FR reorganization"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer , 83:252-260 (2000) (description of FR reorganization "guide Method of choosing). Human framework regions that can be used for humanization include (but are not limited to): framework regions selected using the "best-fit" method (see, for example, Sims et al., J. Immunol. 151:2296 (1993)) ; Framework regions derived from the common sequence of human antibodies with a specific subgroup of light chain or heavy chain variable regions (see, for example, Carter et al., Proc. Natl. Acad. Sci. USA , 89:4285 (1992); and Presta et al., J. Immunol. , 151:2623 (1993)); human mature (somatic mutation) framework regions or human germline framework regions (see, for example, Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008) )); and the framework regions derived from the screening FR library (see, for example, Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 ( 1996)).

Various techniques known in the art can be used to produce human antibodies. Human antibodies are generally described in van Dijk and van de Winkel, Curr Opin Pharmacol 5, 368-74 (2001) and Lonberg, Curr Opin Immunol 20, 450-459 (2008). Human antibodies can be prepared by administering immunogens to transgenic animals that have been engineered to produce complete human antibodies or complete antibodies with human variable regions in response to an antigen challenge. Such animals usually contain all or part of the human immunoglobulin locus, which replaces the endogenous immunoglobulin locus, or exists outside the chromosomes or is randomly integrated into the animal chromosomes. In such transgenic mice, the endogenous immunoglobulin locus is generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, for example ^{, U.S. Patent Nos. 6,075,181 and 6,150,584 describing XENOMOUSE TM} technology; U.S. Patent No. 5,770,429 describing HUMAB® technology; National Patent No. 7,041,870 describing U.S. KM MOUSE® technology; and U.S. Patent describing VELOCIMOUSE® technology Application Publication No. US 2007/0061900. The human variable regions of intact antibodies produced by such animals can be further modified, for example, by combining with different human constant regions.

Human antibodies can also be prepared by methods based on fusion tumors. Human myeloma and mouse-human hybrid myeloma cell lines for the production of human monoclonal antibodies have been described. (See, for example, Kozbor J. Immunol. , 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications , pages 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol. , 147: 86 (1991)). Human antibodies produced by human B-cell fusion tumor technology are also described in Li et al., Proc. Natl. Acad. Sci. USA , 103:3557-3562 (2006). Other methods include, for example, U.S. Patent No. 7,189,826 (describes the production of single human IgM antibodies from fusion tumor cell lines) and described in Ni, Xiandai Mianyixue 26(4):265-268 (2006) (description of human-human fusion tumors) Of their methods. Human fusion tumor technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology , 20(3):927-937 (2005), and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology , 27(3): 185-91 (2005).

Human antibodies can also be produced by isolation from human antibody repertoires, as described herein.

Antibodies suitable for use in the present invention can be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. The method for screening combinatorial libraries is reviewed in, for example, Nature Reviews 16:498-508 (2016) by Lerner et al. For example, various methods for generating phage display libraries and screening such libraries for antibodies with desired binding characteristics are known in the art. Such methods are reviewed in, for example, Frenzel et al. mAbs 8:1177-1194 (2016); Bazan et al. Human Vaccines and Immunotherapeutics 8:1817-1828 (2012) and Zhao et al. Critical Reviews in Biotechnology 36:276-289 (2016) and Hoogenboom et al. Methods in Molecular Biology 178:1-37 (O'Brien et al., Human Press, Totowa, NJ, 2001) and Marks and Bradbury’s Methods in Molecular Biology 248:161-175 (Lo Editor, Human Press, Totowa, NJ, 2003).

In some phage display methods, the lineages of the VH and VL genes are respectively selected by polymerase chain reaction (PCR) and randomly recombined in a phage library, and then can be used as described in Annual Review of Immunology 12: 433- by Winter et al. As described in 455 (1994), screening for antigen-binding phages. Phages typically present antibody fragments in the form of single chain Fv (scFv) fragments or Fab fragments. The library from immunized sources provides high-affinity antibodies to the immunogen without the need to construct fusion tumors. Alternatively, the native lineage can be cloned (e.g. from humans) to provide a single source of antibodies against a wide range of non-self antigens and self-antigens without any immunization, such as Griffiths et al. EMBO Journal 12: 725-734 ( 1993). Finally, the native library can also be prepared synthetically by cloning unrearranged V gene segments from stem cells, and using PCR primers that contain random sequences to encode highly variable CDR3 regions and achieve in vitro rearrangement, such as Described in Hoogenboom and Winter Journal of Molecular Biology 227: 381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Patent Nos. 5,750,373, 7,985,840, 7,785,903, and 8,679,490, and U.S. Patent Publications No. 2005/0079574, No. 2007/0117126, No. 2007/0237764 No. and No. 2007/0292936. Other examples of methods known in the art for screening combinatorial libraries for antibodies with one or more desired activities include ribosomal and mRNA presentation, and antibodies used on bacteria, mammalian cells, insect cells, or yeast cells Methods of presentation and selection. A method for rendering the surface of yeast reviewed in e.g. the Scholler et al Methods in Molecular Biology 503: 135-56 ( 2012) and the Cherf et al. Methods in Molecular biolo gy 1319: 155-175 (2015) and Zhao et al. Methods of in Molecular Biology 889:73-84 (2012). Methods for ribosome presentation are described in, for example, He et al., Nucleic Acids Research 25:5132-5134 (1997) and Hanes et al., PNAS 94:4937-4942 (1997).

It may be necessary to further chemically modify the immunoconjugate of the present invention. For example, the problem of immunogenicity and short half-life can be improved by combining with substantially linear polymers such as polyethylene glycol (PEG) or polypropylene glycol (PPG) (see, for example, WO 87/00056).

The immunoconjugate prepared as described herein can be prepared by techniques known in the art (such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography and similar techniques). )purification. The actual conditions used to purify a particular protein depend in part on factors such as net charge, hydrophobicity, hydrophilicity, etc., and are obvious to those skilled in the art. For affinity chromatography purification, antibodies, ligands, receptors, or antigens bound by immunoconjugates can be used. For example, antibodies that specifically bind to mutant IL-2 polypeptides can be used. For affinity chromatography, a matrix with protein A or protein G can be used to purify the immunoconjugate of the present invention. For example, the immunoconjugates can be separated using protein A or G affinity chromatography and size exclusion chromatography performed in sequence, substantially as described in the examples. The purity of the immunoconjugate can be determined by any of a variety of well-known analytical methods, including gel electrophoresis, high pressure liquid chromatography and the like.

Compositions, formulations and routes of administration In another aspect, the present invention provides pharmaceutical compositions comprising an immunoconjugate as described herein, for example, for use in any of the following treatment methods. In one embodiment, the pharmaceutical composition includes any of the immunoconjugates provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition comprises any one of the immunoconjugates provided herein and at least one additional therapeutic agent, for example as described below.

In addition, there is provided a method for producing the immunoconjugate of the present invention in a form suitable for in vivo administration, the method comprising (a) obtaining the immunoconjugate according to the present invention, and (b) using at least one pharmaceutically acceptable The carrier formulates the immunoconjugate, wherein the immunoconjugate preparation is formulated for in vivo administration.

The pharmaceutical composition of the present invention comprises a therapeutically effective amount of the immunoconjugate dissolved or dispersed in a pharmaceutically acceptable carrier. The phrase "pharmaceutically or pharmacologically acceptable" means that molecular entities and compositions are generally non-toxic to the recipient at the dose and concentration used, that is, when appropriate, when administered to animals (such as humans) At times, no harmful allergic reactions or other adverse reactions will occur. Those skilled in the art will know the preparation of pharmaceutical compositions containing immunoconjugates and optionally additional active ingredients according to the present invention, as exemplified in Remington's Pharmaceutical Sciences, 18th edition, Mack Printing Company, 1990. The way of citation is incorporated into this article. In addition, with regard to the administration of drugs to animals (such as humans), it should be understood that the preparations should meet the sterility, fever, overall safety and purity standards required by the FDA Office of Biological Standards or corresponding agencies in other countries. The preferred composition is a lyophilized formulation or an aqueous solution. As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives ( Such as antibacterial agents, antifungal agents), isotonic agents, absorption delay agents, salts, antioxidants, proteins, drugs, drug stabilizers, polymers, gels, binders, excipients, disintegrants, lubricants , Sweeteners, flavors, dyes, and similar materials and combinations thereof (see, for example, Remington's Pharmaceutical Sciences, 18th edition, Mack Printing Company, 1990, pages 1289-1329, which are incorporated herein by reference). Unless any conventional carrier is incompatible with the active ingredient, its use in therapeutic or pharmaceutical compositions is considered.

The immunoconjugate (and any other therapeutic agent) of the present invention can be administered by any suitable method, including parenteral, intrapulmonary, and intranasal, and if necessary, for local treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Depending in part on the short-term or long-term nature of the administration, the administration can be carried out by any suitable route (for example, by injection, such as intravenous or subcutaneous injection).

Parenteral compositions include those designed for administration by injection (e.g., subcutaneous, intradermal, intralesional, intravenous, intraarterial, intramuscular, intrathecal, or intraperitoneal injection). For injection, the immunoconjugate of the present invention may be in an aqueous solution, preferably a physiologically compatible buffer (such as Hank's solution, Ringer's solution or physiological saline buffer). Liquid) in the deployment. The solution may contain formulation agents such as suspending agents, stabilizers and/or dispersants. Alternatively, the immunoconjugate may be in powder form, which is reconstituted with a suitable vehicle (e.g., sterile pyrogen-free water) before use. When necessary, a sterile injectable solution is prepared by incorporating the required amount of the immunoconjugate of the present invention together with various other ingredients listed below in an appropriate solvent. Sterility can be easily achieved by, for example, sterile filtration membrane filtration. Generally speaking, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and/or other ingredients. When sterile powders are used to prepare sterile injectable solutions, suspensions or emulsions, the preferred preparation methods are vacuum drying and freeze-drying techniques, which use pre-sterile filtered liquid media to produce powders of the active ingredient and any other required ingredients . If necessary, the liquid medium should be buffered, and the liquid diluent should first be used to produce isotonicity before being injected with enough saline or glucose. The composition must be stable under the conditions of manufacture and storage, and must avoid contamination by microorganisms such as bacteria and fungi. It should be understood that endotoxin contamination should be kept at a minimum safety level, such as less than 0.5 ng/mg protein. Suitable pharmaceutically acceptable carriers include (but are not limited to): buffers, such as phosphate, citrate and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as decachloride Octaalkyl dimethyl benzyl ammonium; hexahydroxy quaternary ammonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl paraben Esters, such as methyl or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) ) Polypeptides; proteins, such as serum albumin, gelatin or immunoglobulin; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, asparagine, histidine , Arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; Salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants, such as polyethylene glycol (PEG). Aqueous injection suspensions may contain compounds that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, polydextrose or the like. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compound to allow the preparation of highly concentrated solutions. In addition, the suspension of the active compound can be prepared as an oily injection suspension if necessary. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil; or synthetic fatty acid esters, such as ethyl oleate or triglycerides; or liposomes.

The active ingredient can be encapsulated in microcapsules, such as microcapsules prepared by coacervation technology or by interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively ; Coated in colloidal drug delivery systems (such as liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (18th edition, Mack Printing Company, 1990). Sustained release formulations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing polypeptides, which matrices are in the form of shaped articles, such as films or microcapsules. In certain embodiments, the absorption of the injectable composition can be prolonged by using an agent that delays absorption in the composition, such as aluminum monostearate, gelatin, or a combination thereof.

In addition to the previously described composition, the immunoconjugate can also be formulated into a depot preparation. Such long acting formulations can be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Therefore, for example, the immunoconjugate can be formulated with suitable polymers or hydrophobic materials (for example, as an emulsion in an acceptable oil) or with ion exchange resins, or as a sparingly soluble derivative, such as a sparingly soluble salt. .

The pharmaceutical composition containing the immunoconjugate of the present invention can be manufactured by means of a conventional mixing, dissolving, emulsifying, encapsulating, coating or freeze-drying process. The pharmaceutical composition can be formulated in a conventional manner using one or more carriers, diluents, excipients, or adjuvants that are physiologically acceptable for processing the protein into a preparation that can be used in medicine. The appropriate formulation depends on the chosen route of administration.

The immunoconjugate can be formulated into a composition in free acid or base, neutral or salt form. A pharmaceutically acceptable salt is a salt that substantially retains the biological activity of the free acid or base. Such salts include acid addition salts, such as salts formed with free amine groups of protein compositions, or salts formed with inorganic acids (such as hydrochloric acid or phosphoric acid) or organic acids (such as acetic acid, oxalic acid, tartaric acid, or mandelic acid). Salts with free carboxyl groups can also be derived from inorganic bases, such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or iron hydroxide; or organic bases, such as isopropylamine, trimethylamine, histidine or general Procaine. Compared to the corresponding free base form, pharmaceutical salts tend to be more soluble in aqueous and other protic solvents.

Treatment methods and compositions Any of the immunoconjugates provided herein can be used in treatment methods. The immunoconjugate of the present invention can be used as an immunotherapeutic agent, such as an immunotherapeutic agent for treating cancer.

In order to be used in a therapeutic method, the immunoconjugate of the present invention will be formulated, administered and administered in a manner consistent with good medical practice. In this case, the consideration factors include the specific disease being treated, the specific mammal being treated, the clinical condition of individual patients, the etiology of the disease, the site of drug delivery, the method of administration, the time course of administration, and others known to the medical practitioner factor.

The immunoconjugates of the present invention may be particularly suitable for the treatment of disease states in which stimulation of the host immune system is beneficial, in particular, diseases in which an enhanced cellular immune response is required. Such disease states may include disease states in which the host immune response is insufficient or lacking. Disease states in which the immunoconjugates of the present invention can be administered include, for example, tumors or infections in which the cellular immune response will be a key mechanism of specific immunity. The immunoconjugate of the present invention can be administered by itself or in any suitable pharmaceutical composition.

In one aspect, the immunoconjugate of the present invention for use as a medicament is provided. In other aspects, the immunoconjugate of the present invention for use in the treatment of diseases is provided. In certain embodiments, the immunoconjugates of the present invention for use in therapeutic methods are provided. In one embodiment, the present invention provides an immunoconjugate as described herein for use in the treatment of a disease in an individual in need. In certain embodiments, the present invention provides an immunoconjugate for use in a method of treating an individual suffering from a disease, which comprises administering to the individual a therapeutically effective amount of the immunoconjugate. In certain embodiments, the disease to be treated is a proliferative disorder. In a specific embodiment, the disease is cancer. In certain embodiments, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, for example, if the disease to be treated is cancer, administering an anticancer agent. In other embodiments, the present invention provides immunoconjugates for stimulating the immune system. In certain embodiments, the present invention provides an immune conjugate used in a method of stimulating the immune system of an individual, the method comprising administering to the individual an effective amount of an immune conjugate that stimulates the immune system. The "individual" according to any of the above embodiments is a mammal, preferably a human. The "stimulation of the immune system" according to any of the above embodiments may include any one or more of the following: overall improvement of immune function, improvement of T cell function, improvement of B cell function, recovery of lymphocyte function, IL -2 receptor expression enhancement, T cell response enhancement, natural killer cell activity or lymphoid mediator activated killer (LAK) cell activity enhancement, and the like.

In another aspect, the present invention provides a use of the immunoconjugate of the present invention for the manufacture or preparation of a medicament. In one embodiment, the medicament is used to treat a disease in an individual in need. In one embodiment, the drug is used in a method of treating a disease, the method comprising administering a therapeutically effective amount of the drug to an individual suffering from the disease. In certain embodiments, the disease to be treated is a proliferative disorder. In a specific embodiment, the disease is cancer. In one embodiment, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, for example, if the disease to be treated is cancer, administering an anticancer agent. In another embodiment, the agent is used to stimulate the immune system. In another embodiment, the agent is used in a method of stimulating the immune system of an individual, and the method comprises administering to the individual an effective amount of an agent that stimulates the immune system. The "individual" according to any of the above embodiments may be a mammal, preferably a human. The "stimulation of the immune system" according to any of the above embodiments may include any one or more of the following: overall improvement of immune function, improvement of T cell function, improvement of B cell function, recovery of lymphocyte function, IL -2 receptor expression enhancement, T cell response enhancement, natural killer cell activity or lymphoid mediator activated killer (LAK) cell activity enhancement, and the like.

In another aspect, the present invention provides a method of treating an individual's disease. In one embodiment, the method comprises administering a therapeutically effective amount of the immunoconjugate of the invention to an individual suffering from such diseases. In one embodiment, a composition comprising the immunoconjugate of the present invention is administered to the individual, the composition is in a pharmaceutically acceptable form. In certain embodiments, the disease to be treated is a proliferative disorder. In a specific embodiment, the disease is cancer. In certain embodiments, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, for example, if the disease to be treated is cancer, administering an anticancer agent. In another aspect, the present invention provides a method for stimulating the immune system of an individual, which comprises administering to the individual an effective amount of an immune conjugate that stimulates the immune system. The "individual" according to any of the above embodiments may be a mammal, preferably a human. The "stimulation of the immune system" according to any of the above embodiments may include any one or more of the following: overall improvement of immune function, improvement of T cell function, improvement of B cell function, recovery of lymphocyte function, IL -2 receptor expression enhancement, T cell response enhancement, natural killer cell activity or lymphoid mediator activated killer (LAK) cell activity enhancement, and the like.

In some embodiments, the disease to be treated is a proliferative disorder, specifically cancer. Non-limiting examples of cancers include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectum Cancer, stomach cancer, prostate cancer, blood cancer, skin cancer, squamous cell cancer, bone cancer and kidney cancer. Other cell proliferative disorders that can be treated with the immunoconjugates of the present invention include (but are not limited to) neoplasms located in the following: abdomen, bones, breasts, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal glands, Parathyroid, pituitary, testis, ovary, thymus, thyroid), eyes, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, chest area and genitourinary system. It also includes precancerous conditions or lesions and cancer metastasis. In certain embodiments, the cancer is selected from the group consisting of kidney cancer, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer, head and neck cancer, prostate cancer, and bladder cancer. Those familiar with this technology can easily realize that in many cases, immunoconjugates may not provide a cure, but may only provide partial benefits. In some embodiments, physiological changes with some benefits are also considered therapeutically beneficial. Therefore, in some embodiments, the amount of the immunoconjugate that provides a physiological change is regarded as an "effective amount" or a "therapeutically effective amount". The subject, patient or individual in need of treatment is usually a mammal, more specifically a human.

In some embodiments, an effective amount of the immunoconjugate of the invention is administered to the cell. In other embodiments, a therapeutically effective amount of the immunoconjugate of the present invention is administered to an individual for the treatment of diseases.

In order to prevent or treat diseases, the appropriate dosage of the immunoconjugate of the present invention (when used alone or in combination with one or more other additional therapeutic agents) depends on the following: type of disease to be treated, route of administration, patient weight, molecular type (For example, including or not including the Fc domain), severity and course of the disease, whether the immune conjugate was administered for preventive or therapeutic purposes, pre- or concurrent therapeutic intervention, patient clinical history and response to the immune conjugate, and indications The judgment of the physician. The practitioner responsible for the administration will determine the concentration of the active ingredient in the composition and the dosage applicable to an individual in any case. Various administration schedules are considered herein, including (but not limited to) single administration or multiple administrations at different time points, rapid administration and pulse infusion.

The immunoconjugate should be administered to the patient at one time or after a series of treatments. Depending on the type and severity of the disease, an immunoconjugate of about 1 µg/kg to 15 mg/kg (for example, 0.1 mg/kg-10 mg/kg) can be the initial candidate dose for administration to the patient, regardless of whether by, for example, One or more divided doses or by continuous infusion. Depending on the factors mentioned above, a typical daily dose may range from about 1 μg/kg to 100 mg/kg or higher. For repeated administrations over several days or longer, depending on the condition, the treatment will usually continue until the desired suppression of disease symptoms occurs. An exemplary dose of the immunoconjugate will be in the range of about 0.005 mg/kg to about 10 mg/kg. In other non-limiting examples, the dosage may also include about 1 microgram/kg/body weight, about 5 micrograms/kg/body weight, about 10 micrograms/kg/body weight, about 50 micrograms/kg/body weight, about 100 micrograms/kg/body weight per administration. Μg/kg/body weight, about 200 μg/kg/body weight, about 350 μg/kg/body weight, about 500 μg/kg/body weight, about 1 mg/kg/body weight, about 5 mg/kg/body weight, about 10 mg/ Kg/body weight, about 50 mg/kg/body weight, about 100 mg/kg/body weight, about 200 mg/kg/body weight, about 350 mg/kg/body weight, about 500 mg/kg/body weight to about 1000 mg/kg/ Body weight or greater, and any range that can be derived from it. In the non-limiting example of the derivable range of the values listed herein, based on the above values, the range that can be administered is from about 5 mg/kg/body weight to about 100 mg/kg/body weight, and about 5 mg/kg/body weight to about 5 mg/kg/body weight. About 500 mg/kg/body weight, etc. Therefore, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 5.0 mg/kg, or 10 mg/kg (or any combination thereof) can be administered to the patient. Such doses may be administered intermittently, for example every week or every three weeks (e.g., allowing the patient to receive about two to about twenty or for example about six doses of the immunoconjugate). An initial higher starting dose can be administered, followed by one or more lower doses. However, other dosing regimens may be applicable. The progress of this therapy is easily monitored by conventional techniques and analysis.

The immunoconjugate of the present invention will usually be used in an amount effective to achieve the intended purpose. In order to be used for the treatment or prevention of disease conditions, the immunoconjugate of the present invention or the pharmaceutical composition thereof is administered or administered in a therapeutically effective amount. The determination of the therapeutically effective dose is completely within the ability of those who are familiar with the technology, especially according to the detailed disclosure provided in this article.

Regarding systemic administration, the therapeutically effective dose can be estimated first based on in vitro analysis (such as cell culture analysis). It may then be formulated in animal models to achieve a circulating concentration range of doses, including the IC ₅₀ as determined in cell culture, such as. Such information can be used to more accurately determine the dose applicable to humans.

Techniques well known in the art can also be used to estimate the initial dose based on in vivo data (such as animal models). Generally, those who are familiar with this technology can easily optimize the administration of drugs to humans based on animal data.

The dosage and time interval can be adjusted individually to provide sufficient plasma levels of the immune conjugate to maintain the therapeutic effect. The usual injection dosage for patients is in the range of about 0.1 to 50 mg/kg/day, usually about 0.5 to 1 mg/kg/day. The therapeutically effective plasma content can be achieved by administering multiple doses daily. The plasma content can be measured by, for example, HPLC.

In the case of local administration or selective absorption, the local effective concentration of the immunoconjugate may not be related to plasma concentration. Those familiar with this technique can optimize the therapeutically effective local dose without undue experimentation.

The therapeutically effective dose of the immunoconjugates described herein will generally provide therapeutic benefit without causing substantial toxicity. The toxicity and therapeutic efficacy of the immunoconjugate in cell culture or laboratory animals can be determined by standard medical procedures. Cell culture analysis and animal studies can be used to determine LD ₅₀ (50% population lethal dose) and ED ₅₀ (50% population therapeutically effective dose). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD ₅₀ / ED _50. Immunoconjugates exhibiting a large therapeutic index are preferred. In one embodiment, the immunoconjugate according to the present invention exhibits a high therapeutic index. The data obtained from cell culture analysis and animal studies can be used to formulate a range of dosages suitable for humans. The dosage is preferably within a range of circulating concentrations, which includes the ED _{50 with} little or no toxicity. The dosage may vary within this range depending on a variety of factors, such as the dosage form used, the route of administration used, the individual's condition and the like. The exact formula, route of administration and dosage can be selected by individual physicians in consideration of the patient's condition. (See, for example, Fingl et al., The Pharmacological Basis of Therapeutics, 1975, Chapter 1, page 1, which is incorporated herein by reference in its entirety).

The attending physician of a patient treated with the immunoconjugate of the present invention knows how and when to terminate, interrupt or adjust the administration due to toxicity, organ dysfunction and the like. Conversely, the attending physician is also known to adjust the treatment to a higher level when the clinical response is insufficient (to rule out toxicity). The amount of the dose administered in the management of the condition of concern will vary with the severity of the condition to be treated, the route of administration and similar factors. The severity of the condition can be evaluated, for example, in part by standard prognostic evaluation methods. In addition, the dosage and possible frequency of administration will also vary according to the age, weight and response of individual patients.

The maximum therapeutic dose of the immunoconjugate containing the mutant IL-2 polypeptide as described herein can be increased relative to the maximum therapeutic dose used for the immunoconjugate containing wild-type IL-2.

Other agents and therapies The immunoconjugates according to the present invention can be administered in combination with one or more other therapeutic agents. For example, the immunoconjugates of the invention can be co-administered with at least one additional therapeutic agent. The term "therapeutic agent" encompasses any agent administered to treat a symptom or disease in an individual in need of such treatment. Such additional therapeutic agents may contain any active ingredients suitable for the treatment of specific indications, preferably those active ingredients with complementary activities that do not adversely affect each other. In certain embodiments, other therapeutic agents are immunomodulators, cytostatic agents, cell adhesion inhibitors, cytotoxic agents, apoptosis activators, or agents that enhance the sensitivity of cells to apoptosis-inducing agents. In a specific embodiment, the additional therapeutic agent is an anticancer agent, such as a microtubule disrupting agent, antimetabolites, topoisomerase inhibitors, DNA intercalators, alkylating agents, hormone therapy, kinase inhibitors, receptors Antagonist, tumor cell apoptosis activator or anti-angiogenesis agent.

Such other agents are preferably present in combination in an amount effective to achieve the intended purpose. The effective amount of such other agents depends on the amount of the immunoconjugate used, the type of disorder or treatment, and other factors as described above. The immunoconjugate is generally used at the same dosage and route of administration as described herein, or at 1 to 99% of the dosage described herein, or at any dosage and any route judged empirically/clinically as appropriate .

Such combination therapies mentioned above encompass combined administration (wherein two or more therapeutic agents are included in the same or separate composition), and separate administration (in this case, administration of the immunoconjugate of the present invention ) Can occur before, at the same time and/or after the administration of the additional therapeutic agent and/or adjuvant. The immunoconjugate of the present invention can also be used in combination with radiation therapy.

Articles In another aspect of the present invention, there is provided an article containing materials suitable for treating, preventing and/or diagnosing the conditions described above. The product includes the container and the markings or drug inserts on the container or accompanying the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container can be formed of a variety of materials, such as glass or plastic. The container contains a composition alone or in combination with another composition effective in treating, preventing and/or diagnosing the condition, and may have a sterile access port (for example, the container may be an intravenous solution having a stopper pierced by a hypodermic injection needle Bag or vial). At least one active agent in the composition is the immunoconjugate of the present invention. The label or package insert indicates that the composition is used to treat the selected condition. In addition, the product may comprise: (a) a first container containing the composition, wherein the composition contains the immunoconjugate of the present invention; and (b) a second container containing the composition, wherein the composition contains another Cytotoxic agents or other therapeutic agents. The article of this embodiment of the present invention may further include a package insert indicating that the composition can be used to treat a specific condition. Alternatively or in addition, the product may further comprise a second (or third) container, which contains a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution (Ringer's solution) and dextrose solution. It may further include other materials desired from a commercial and user point of view, including other buffers, diluents, filters, needles and syringes.

Amino acid sequence Amino acid sequence SEQ ID NO HCDR1_anti-CD8 DTYIH 1 HCDR2_anti-CD8 RIDPANDNTLYASKFQG 2 HCDR3_anti-CD8 GYGYYVFDH 3 LCDR1_anti-CD8 RTSRSISQYLA 4 LCDR2_anti-CD8 SGSTLQS 5 LCDR3_anti-CD8 QQHNENPLT 6 VH_anti-CD8 EVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYIHWVRQAPGQGLEWIGRIDPANDNTLYASKFQGRATITADTSTSTAYLELSSLRSEDTAVYYCGRGYGYYVFDHWGQGTLVTVSS 7 VL_anti-CD8 DVQITQSPSSLSASVGDRVTITCRTSRSISQYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHNENPLTFGQGTKVEIK 8 Chain A: LC (CD8-IL2v OA/TA) DVQITQSPSSLSASVGDRVTITCRTSRSISQYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHNENPLTFGQGTKVEIKRTTKTLTKVAAPSVFIFPPSDEQLKSGTASVVCLLKANNFYPREAKTLSVQWKESVVCLLKANNFYPREAKTLSVQGEDYSVVCLLKANNFYPREAKTLSVQWKESVVCLLKANNFYPREAKTLSVQWKESVSSVSLQSGV 9 Chain H: socket (CD8-IL2v OA/TA) EVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYIHWVRQAPGQGLEWIGRIDPANDNTLYASKFQGRATITADTSTSTAYLELSSLRSEDTAVYYCGRGYGYYVFDHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 10 Chain K: Pestle (CD8-IL2v TA) EVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYIHWVRQAPGQGLEWIGRIDPANDNTLYASKFQGRATITADTSTSTAYLELSSLRSEDTAVYYCGRGYGYYVFDHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSAPASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFAMPKKATELKHLQCLEEELKPLEEVLNGAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT 11 Chain K: Pestle (CD8-IL2v OA) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSAPASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFAMPKKATELKHLQCLEEELKPLEEVLNGAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT 12 Human IL-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 13 Human IL-2 (T3A, F42A, Y45Y, L72G, C125A) APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFAMPKKATELKHLQCLEEELKPLEEVLNGAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT 14 Linker GGGGSGGGGSGGGGS 15 Human IL-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 16 Human IL-2 (T3A, F42A, Y45Y, L72G, C125A) APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFAMPKKATELKHLQCLEEELKPLEEVLNGAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT 17 Linker GGGGSGGGGSGGGGS 18 hIL-2 signal peptide MYRMQLLSCIALSLALVTNS 19 Human IL-2 (C125A) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT 20 Human IgG1 Fc domain DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPVSNKALPAPIEKTISKAKGQPREPVSKVYTLPPSRTKNQVSLTCLVKGFYPSNYCSHYGLSHYPSRTKNQVSLTCLVKGFYPSNYKSLYKNQVSLTCLVKGFYPSNYSKAKGQPREPVSKGVHSGNSH twenty one Human κ CL domain RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC twenty two Human lambda CL domain QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS twenty three Human IgG1 heavy chain constant region (CH1-CH2-CH3) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP twenty four muFAP HC-Fc (DD)-muIL2v EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVAISKDDPEVQFSWFVDDVEVHTAQTKPREEQINSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFGAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITNFFPEDITVEWQWNGQPAENYDNTQPIMDTDGSYFVYSDLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGGGGGSGGGGSGGGGSAPASSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNLKLPRMLTAKFALPKQATELKDLQCLEDELGPLRHVLDGTQSKSFQLEDAENFISNIRVTVVKLKGSDNTFECQFDDESATVVDFLRRWIAFAQSIISTSPQ 25 muFAP HC-Fc (KK) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVAISKDDPEVQFSWFVDDVEVHTAQTKPREEQINSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFGAPIEKTISKTKGRPKAPQVYTIPPPKKQMAKDKVSLTCMITNFFPEDITVEWQWNGQPAENYKNTQPIMKTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK 26 muFAP LC EIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGIMLPPTFGQGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSTLTKDEPNVKWKIDGSERQNGVLNSWTSTLTKDERNVKWKIDGSERQNGVLNSWTDNSERTCRNVKFNYSMHTSKD 27 LCDR3 anti-CD8-Okt8.v11 QQVNEFPPT 28 VL_anti-CD8-Okt8.v11 DVQITQSPSSLSASVGDRVTITCRTSRSISQYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVNEFPPTFGQGTKVEIKRTTKTVAAPSVFIFPPSDEQLKSGTASVVCLLKANNFYPREAKTLSVQGEDYSVVCLLKANNFYPREAKTLSVQWKESVVCLLKANNFYPREAKTLSVQWKESVVCLLKANNFYPREAKTLSVQGEDYKESVVCLLKANNFYPEDFATYYCQQVNEFPPTFGQGTKVEIKRTTKVAAPSVFIFPPSD 29 Chain A: LC (CD8-(Okt8.v11)IL2v OA/TA) DVQITQSPSSLSASVGDRVTITCRTSRSISQYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVNEFPPTFGQGTKVEIKRTTKTVAAPSVFIFPPSDEQLKSGTASVVCLLKANNFYPREAKTLSVQGEDYSVVCLLKANNFYPREAKTLSVQWKESVVCLLKANNFYPREAKTLSVQWKESVVCLLKANNFYPREAKTLSVQGEDYKESVVCLLKANNFYPEDFATYYCQQVNEFPPTFGQGTKVEIKRTTKVAAPSVFIFPPSD 30

Examples The following are examples of the methods and compositions of the present invention. It should be understood that various other embodiments may be practiced in view of the general description provided above.

Instance 1

Example 1.1 Production of anti-CD8-IL2v TA conjugates and anti-CD8-IL2v OA conjugates targeting human CD8 The abbreviation "TA" means "two arms" in this article. The abbreviation "OA" means "single arm" in this article. The terms "anti-CD8-IL2v TA" and "CD8-IL2v TA" are used interchangeably herein. The terms "anti-CD8-IL2v OA" and "CD8-IL2v OA" are used interchangeably herein.

The performance of all genes is under the control of the human CMV promoter-intron A-5'UTR cassette. The BGH polyadenylation signal is located downstream of the gene. In order to be used in HEK293 EBNA cells, the vector contains oriP elements for stabilizing the free form and maintaining plastids.

1.2 Examples of production of IgG-like protein in HEK293 EBNA cells by transient transfection of HEK293 EBNA cells to produce IgG-IL2 molecule. For anti-CD8-IL2v TA, these cells use the corresponding expression vector at a ratio of 1:1:2 ("vector heavy chain (VH-CH1-CH2-CH3)": "vector heavy chain (VH-CH1-CH2- CH3-IL2v)": "Vector light chain (VL-CL)") transfection. For anti-CD8-IL2v OA, these cells use the corresponding expression vector at a ratio of 1:1:1 ("vector heavy chain (VH-CH1-CH2-CH3)": "vector heavy chain (CH2-CH3-IL2v) ": "Vector light chain (VL-CL)") transfection. The cells were centrifuged, and the medium was replaced with pre-warmed CD CHO medium (Thermo Fisher, catalog number 10743029). The expression vector was mixed in CD CHO medium, PEI (Polyethylenimine, Polysciences, Inc, catalog number 23966-1) was added, the solution was vortexed and incubated at room temperature for 10 minutes. Subsequently, the cells (2 million cells/ml) were mixed with the carrier/PEI solution, transferred to a flask and incubated at 37°C for 3 hours in a shaking incubator _{with a 5% CO 2 atmosphere.} After incubation, add Excell medium (W. Zhou and A. Kantardjieff, Mammalian Cell Cultures for Biologics Manufacturing, digital object identification code: 10.1007/978-3-642-54050-9) with supplements (80% of the total volume) ; 2014). On the first day after transfection, add supplements (feeding material, 12% of the total volume). After 7 days, the cell supernatant was harvested by centrifugation and subsequent filtration (0.2 μm filter), and the protein was purified from the harvested supernatant by standard methods as indicated below.

Example 1.3 Purification of IgG-like proteins Refer to the standard protocol to purify proteins from filtered cell culture supernatants. In short, by protein A affinity chromatography (equilibration buffer: 20 mM sodium citrate, 20 mM sodium phosphate, pH 7.5; dissociation buffer: 20 mM sodium citrate, 100 mM NaCl, 100 mM glycine, pH 3.0) Purify Fc-containing protein from cell culture supernatant. Dissolution is achieved at pH 3.0, and the sample is immediately pH neutralized. The protein was concentrated by centrifugation (Millipore Amicon® ULTRA-15 (Technical Code: UFC903096)), and the aggregated protein was combined with size exclusion chromatography in 20 mM histidine and 140 mM sodium chloride (pH 6.0). The monomeric proteins are separated.

Example 1.4 Analysis of IgG-like protein. The purified protein was measured by measuring the absorption at 280 nm using the mass extinction coefficient calculated based on the amino acid sequence according to Pace, et al., Protein Science, 1995, 4, 2411-1423. concentration. The purity and molecular weight of the protein were analyzed by CE-SDS using LabChipGXII (Perkin Elmer) in the presence and absence of reducing agents. The aggregate content was determined using analytical size exclusion column (TSKgel G3000 SW XL or UP-SW3000) at 25°C in operating buffer (25 mM K ₂ HPO ₄ , 125 mM NaCl, 200 mM L- Arginine monohydrochloride, pH 6.7 or 200 mM KH ₂ PO ₄ , 250 mM KCl pH 6.2) equilibrate in HPLC chromatography.

The two IgG-IL2v constructs were purified with comparable and good quality, and the monomer content determined by size exclusion chromatography was higher than 98%. The percentage of the main peak (determined by CE-SDS) is higher than 92%. The yield after purification (the purified amount in mg divided by the volume of the supernatant produced in L) was 2.1 mg/L (anti-CD8-IL2v TA) and 7.8 mg/L (anti-CD8- IL2v OA).

The anti-CD8-IL2v TA consists of the following (format: A2HK): two polypeptides with the amino acid sequence of SEQ ID NO: 9, one polypeptide with the amino acid sequence of SEQ ID NO: 10, and a polypeptide with SEQ ID NO: A polypeptide of 11 amino acid sequence. Anti-CD8-IL2v OA consists of the following (format: AHK): a polypeptide with the amino acid sequence of SEQ ID NO: 9, a polypeptide with the amino acid sequence of SEQ ID NO: 10, and a polypeptide with SEQ ID NO: 12 A polypeptide of the amino acid sequence.

Anti-CD8 (OKT8.v11)-IL2v TA consists of the following (format: A2HK): two polypeptides with the amino acid sequence of SEQ ID NO: 30, one polypeptide with the amino acid sequence of SEQ ID NO: 10, and A polypeptide having the amino acid sequence of SEQ ID NO: 11. The terms "anti-CD8(OKT8.v11)-IL2v TA", "anti-CD8-IL2v OKT8.v11 TA" and "CD8-IL2v OKT8.v11 TA" are used interchangeably in this article. Anti-CD8 (OKT8.v11)-IL2v OA consists of the following (format: AHK): a polypeptide with the amino acid sequence of SEQ ID NO: 30, a polypeptide with the amino acid sequence of SEQ ID NO: 10, and A polypeptide of the amino acid sequence of SEQ ID NO: 12. The terms "anti-CD8(OKT8.v11)-IL2v OA", "anti-CD8-IL2v OKT8.v11 OA" and "CD8-IL2v OKT8.v11 OA" are used interchangeably in this article.

The conjugate prepared in this example is further used in the following examples.

Instance 2

And anti-CD8-IL2v TA binding anti-CD8-IL2v OA and CD8 T cells of the counts were isolated from PBMC of a healthy donor fresh, and transferred to a round bottom 96 well plates (100'000 cells per well). The cells were washed with FACS buffer (PBS, 2% FBS, 5 mM EDTA, 0.025% NaN ₃ ) and stained with 30 μl of FACS buffer containing indicator molecules at 4° C. for 30 min. The cells were washed twice with FACS buffer to remove unbound molecules. Then, 20 µl of the diluted FITC anti-human Fc specific secondary antibody (diluted 1:50, 109-096-098, Jackson ImmunoResearch) and CD3 PE antibody (344806, BioLegend) and used to detect CD8 T cells CD8 APC-Cy7 antibody (557834, BD Bioscience) was added to the cells together. After incubation for 30 min at 4°C, unbound antibody was removed by washing twice with FACS buffer. Finally, the cells were resuspended in FACS buffer and measured using BD Fortessa, which gated ^{CD3 +} CD8 ^{+ cells (CD8 T cells).}

Evaluation of anti-CD8-IL2v TA and anti-CD8-IL2v OA molecules with FAP-IL2v (as disclosed, for example, in WO2012107417A1, which is incorporated by reference; FAP-IL2v includes polypeptides according to SEQ ID NOs: 25, 26, and 27 ) Compared to the ability to bind to CD8 T cells in PBMC. As shown in Figure 2, anti-CD8-IL2v TA showed extremely strong binding to CD8 T cells, and anti-CD8-IL2v OA bound to CD8 T cells weakly but still much stronger than FAP-IL2v. This is consistent with the fact that anti-CD8-IL2v OA can only bind to CD8 monovalently, while anti-CD8-IL2v TA can bind to CD8 divalently, making the latter have a higher affinity for CD8. As seen by FAP-IL2v, the contribution of IL2v to the binding of CD8 T cells is only minimal, because the IL2Rβ/γ performance of resting CD8 T cells is lower than that of CD8 (Figure 2).

Instance 3

Phosphorylation of STAT5 in immune cells after treatment with anti- CD8-IL2v TA and anti- CD8-IL2v OA . Freshly isolated PBMCs from healthy donors were inoculated into warm medium (RPMI1640, 10% FCS, 2 mM bran) in round bottom 96-well plates. Amino acid) (200'000 cells/well). The culture plate was centrifuged at 300 g for 10 min and the supernatant was removed. The cells were resuspended in 100 µl of medium containing IL2v conjugate and stimulated at 37°C for 20 min. In order to maintain the phosphorylation state, the cells were fixed immediately after stimulation with an equal amount of pre-warmed Cytofix buffer (554655, BD Bioscience) at 37°C for 10 min. Subsequently, the culture plate was centrifuged at 300 g for 10 min and the supernatant was removed. In order to achieve intracellular staining, cells were permeated in 200 µl Phosflow Perm Buffer III (558050, BD Bioscience) at 4°C for 30 min. Then, the cells were washed twice with 150 µl of cold FACS buffer and split into two round-bottom 96-well plates and stained with 20 µl of antibody mixture I or II each for 60 min in the refrigerator. Antibody mixture I was used to stain pSTAT5 in CD4 T cells and regulatory T cells, and antibody mixture II was used to stain pSTAT5 in CD8 T cells and NK cells. Subsequently, the cells were washed twice with FACS buffer and resuspended in 200 µl of FACS buffer containing 2% PFA per well. Analyze using the BD Fortessa flow cytometer that gates CD8 T cells (CD3 ⁺ CD8 ⁺ ), NK cells (CD3 ^- CD56 ⁺ , CD4 T cells (CD4 ⁺ ) and Treg (CD4 ⁺ CD25 ⁺ FoxP3 ⁺⁾ table 1. FACS antibody mixture I (CD4 T cells and regulatory T cells) Antibody Volume/sample CD4 PE/Cy7, pure SK3, mouse IgG1, κ (557852, BD Bioscience) 0.5 μl/hole CD25 APC, pure line M-A251, mouse IgG1, κ (356110, BioLegend) 0.5 μl/hole PE mouse anti-human FoxP3 pure line 259D/C7 (560046, BD Bioscience) 1 μl/well A488 pSTAT5 (pY694), pure line 47, mouse IgG1 (562075, BD Bioscience) 0.5 μl/hole Table 2. FACS Antibody Mix II (CD8 T cells and NK cells) Antibody Volume/sample CD3 PE/Cy7, pure UCHT1, mouse IgG1, κ (300420, BioLegend) 0.5 μl/hole CD56 APC, pure line HCD56, mouse IgG1, κ (318310, BioLegend) 0.5 μl/hole CD8 PE, pure HIT8a, mouse IgG1 (555635, BD Bioscience) 0.5 μl/hole A488 pSTAT5 (pY694), pure line 47, mouse IgG1 (BD Bioscience) 0.5 μl/hole

The functional activity of anti-CD8-IL2v TA and anti-CD8-IL2v OA in inducing STAT5 phosphorylation was compared with the functional activity of FAP-IL2v on different immune cell subsets in PBMC. STAT5 phosphorylation is used as a marker for IL2 receptor (IL2R) activation. As shown in Figure 3, on CD4 T cells and regulatory T cells (Treg), all three tested molecules have the same activity in inducing phosphorylation of STAT5. On CD8 T cells, anti-CD8-IL2v TA and anti-CD8-IL2v OA have much higher potency in inducing phosphorylation of STAT5, among which anti-CD8-IL2v TA is slightly more potent than anti-CD8-IL2v OA. On NK cells, anti-CD8-IL2v TA and anti-CD8-IL2v OA are stronger than FAP-IL2v, which may be due to some of the NK cells that are CD8 positive. These data indicate that targeting IL2v to CD8 T cells can strongly enhance IL2R activation via IL2v, and this effect is only cis-mediated, because the activation of IL2R on CD8-negative T cells does not increase (Figure 3).

Instance 4

Proliferation of immune cells after treatment with CD8-IL2v and CD8-IL2v OA . PBMC freshly isolated from healthy donors used CFSE (5(6)-carboxyfluorescein diacetate N-succinimidyl ester, 21888, Sigma-Aldrich) label. 30 million PBMCs were briefly washed once with PBS. At the same time, CSFE stock solution (2 mM in DMSO) was diluted 1:20 in PBS. Resuspend the PBMC in 30 ml of pre-warmed PBS, add 30 μl of CFSE solution and mix the cells immediately. For optimal labeling, incubate cells at 37°C for 15 min. Next, add 10 ml of pre-warmed medium (RPMI1640, 10% FCS, 1% glutamic acid) to stop the labeling reaction. The cells were briefly centrifuged at 400 g for 10 min and resuspended in 20 ml of fresh medium and incubated for another 30 min at 37°C. Finally, the cells were washed once with medium and resuspended in fresh medium at 1 million cells per milliliter. The labeled PBMCs were seeded in round bottom 96-well plates (100'000 cells per well) and treated with indicator molecules for 5 days. After incubation, the cells were washed once with FACS buffer, and CD3 APC-Cy7 (557834, BD Bioscience), CD8 APC (pure SK1, 344722, BioLegend), CD4 PE (300508, BioLegend) and CD56 BV421 were used at 4°C. (318328, BioLegend) 20 μl mixture in FACS buffer was stained for 30 min. Subsequently, before fixing PBMC with FACS buffer containing 1% PFA and measuring fluorescence with BD Fortessa, PBMC was washed twice with FACS buffer. The proliferation was measured by measuring CD8 T cells (CD3 ⁺ CD8 ⁺ ), CD4 T cells (CD3 ⁺ CD4 ⁺ ) and NK cells (CD3 ^- CD56 ⁺ ) in CFSE dilution.

The ability of CD8-IL2v and CD8-IL2v OA to induce the proliferation of CD8 T cells, CD4 T cells and NK cells compared with FAP-IL2v was tested. As shown in Figure 4, CD8-IL2v TA and CD8-IL2v OA have similar activities to induce the proliferation of CD4 T cells and NK cells as FAP-IL2v. In contrast, on CD8 T cells, the two molecules are much more potent than FAP-IL2v in inducing proliferation. This is due to the direct targeting of the molecules to these cells via CD8. CD8-IL2v TA is about 1300 times more potent, and CD8-IL2v OA is about 200 times more potent. This is consistent with the observed differences in binding on CD8 T cells and phosphorylation of STAT5.

Instance 5

Phosphorylation of STAT5 in immune cells after treatment with CD8-IL2v TA and CD8-IL2v OKT8.v11 TA . Freshly isolated PBMCs from healthy donors were inoculated in warm medium (RPMI1640, 10% FCS, 2 mM) in round bottom 96-well plates. Glutamic acid) (200'000 cells/well). The culture plate was centrifuged at 300 g for 10 min and the supernatant was removed. The cells were resuspended in 100 µl of medium containing IL2v molecules and stimulated at 37°C for 20 min. In order to maintain the phosphorylation state, the cells were fixed immediately after stimulation with an equal amount of pre-warmed Cytofix buffer (554655, BD Bioscience) at 37°C for 10 min. Subsequently, the culture plate was centrifuged at 300 g for 10 min and the supernatant was removed. In order to achieve intracellular staining, cells were permeated in 200 µl Phosflow Perm Buffer III (558050, BD Bioscience) at 4°C for 30 min. Then, the cells were washed twice with 150 µl of cold FACS buffer and split into two round-bottom 96-well plates and stained with 20 µl of antibody mixture I or II each for 60 min in the refrigerator. Antibody mixture I was used to stain pSTAT5 in CD4 T cells and regulatory T cells, and antibody mixture II was used to stain pSTAT5 in CD8 T cells and NK cells. Subsequently, the cells were washed twice with FACS buffer and resuspended in 200 µl of FACS buffer containing 2% PFA per well. Analyze using the BD Fortessa flow cytometer that gates CD8 T cells (CD3 ⁺ CD8 ⁺ ), NK cells (CD3 ^- CD56 ⁺ , CD4 T cells (CD4 ⁺ ) and Treg (CD4 ⁺ CD25 ⁺ FoxP3 ⁺⁾ Table 3. FACS antibody mixture I (CD4 T cells and regulatory T cells) Antibody Volume/sample CD4 PE/Cy7, pure SK3, mouse IgG1, κ (557852, BD Bioscience) 0.5 μl/hole CD25 APC, pure line M-A251, mouse IgG1, κ (356110, BioLegend) 0.5 μl/hole PE mouse anti-human FoxP3 pure line 259D/C7 (560046, BD Bioscience) 1 μl/well A488 pSTAT5 (pY694), pure line 47, mouse IgG1 (562075, BD Bioscience) 0.5 μl/hole Table 4. FACS Antibody Mix II (CD8 T cells and NK cells) Antibody Volume/sample CD3 PE/Cy7, pure UCHT1, mouse IgG1, κ (300420, BioLegend) 0.5 μl/hole CD56 APC, pure line HCD56, mouse IgG1, κ (318310, BioLegend) 0.5 μl/hole CD8 PE, pure HIT8a, mouse IgG1 (555635, BD Bioscience) 0.5 μl/hole A488 pSTAT5 (pY694), pure line 47, mouse IgG1 (BD Bioscience) 0.5 μl/hole

The functional activity of CD8-IL2v TA and CD8-IL2v OKT8.v11 TA in inducing STAT5 phosphorylation was compared with the functional activity of FAP-IL2v on different immune cell subsets in PBMC. STAT5 phosphorylation is used as a marker for IL2 receptor (IL2R) activation. On CD4 T cells and regulatory T cells (Treg), all three tested molecules showed the same activity in inducing phosphorylation of STAT5. On CD8 T cells, CD8-IL2v TA and CD8-IL2v OKT8.v11 TA showed much higher potency in inducing phosphorylation of STAT5, but CD8-IL2v TA and CD8-IL2v OKT8.v11 TA showed much higher potency in activation. There is no difference. On NK cells, CD8-IL2v TA and CD8-IL2v OKT8.v11 TA are stronger than FAP-IL2v, possibly because some NK cells are CD8 positive. These data indicate that targeting IL2v to CD8 T cells can strongly enhance the activation of IL2R via IL2v, and this effect is only cis-mediated, because the activation of IL2R on CD8-negative T cells does not increase (Figure 5). * * *

Although for the purpose of clear understanding, the foregoing invention has been described in considerable detail with the help of illustrations and examples, the description and examples should not be construed as limiting the scope of the invention. The disclosures of all patents and scientific documents cited in this article are expressly incorporated into this article by reference in their entirety.

Figure 1A and Figure 1B. Schematic representation of the type of IgG-IL-2 immunoconjugate containing mutant IL-2 polypeptide. (Figure 1A) Two-arm anti-CD8; (Figure 2A) One-arm anti-CD8.

Figure 2. Two-arm anti-CD8-IL2v (CD8-IL2v TA) and single-arm anti-CD8-IL2v (CD8-IL2v TA) compared with the binding of anti-FAP-IL2v (FAP-IL2v) to CD8 T cells in resting PBMC by flow cytometry Arm anti-CD8-IL2v (CD8-IL2v OA) binding to CD8 T cells. A fluorescently labeled anti-human Fc-specific secondary antibody is used to detect the molecule. CD3 and CD8 staining of PBMC was used to identify CD8 T cells.

Figure 3A , Figure 3B , Figure 3C, and Figure 3D. Flow cytometry was used to determine the CD8 T cells (Figure 3A), after the resting PBMC were treated with CD8-IL2v TA, CD8-IL2v OA, and FAP-IL2v. STAT5 phosphorylation in CD4 T cells (Figure 3C), regulatory T cells (Figure 3D) and NK cells (Figure 3D).

Figure 4A , Figure 4B and Figure 4C. Determination of NK cells (Figure 4C) and CD4 T cells (Figure 4B) in PBMC with CD8-IL2v TA, CD8-IL2v OA and FAP-IL2v by flow cytometry And the proliferation of CD8 T cells (Figure 4C).

Figure 5A , Figure 5B , Figure 5C and Figure 5D. Flow cytometry was used to determine the CD8 T cells after the resting PBMC were treated with CD8-IL2v TA, CD8-IL2v OKT8.v11 TA and FAP-IL2v (Figure 5A), STAT5 phosphorylation in NK cells (Figure 5B), CD4 T cells (Figure 5C) and regulatory T cells (Figure 5D).

Claims (35)

An immunoconjugate comprising a mutant interleukin-2 (IL-2) polypeptide and an antibody that binds to CD8, wherein the IL-2 polypeptide contains amino acid substitutions F42A, Y45A and L72G (relative to human IL- 2 Sequence numbered in SEQ ID NO: 13) mutant IL-2 polypeptide. Such as the immunoconjugate of claim 1, Wherein the mutant IL-2 polypeptide is a human IL-2 molecule, which contains amino acid substitutions F42A, Y45A and L72G (numbering relative to the human IL-2 sequence SEQ ID NO: 13); and Wherein the antibody comprises: (a) a heavy chain variable region (VH), which comprises the heavy chain complementarity determining region (HCDR) containing the amino acid sequence of SEQ ID NO: 1, 1. containing the amino group of SEQ ID NO: 2 The HCDR of the acid sequence 2, the HCDR 3 containing the amino acid sequence of SEQ ID NO: 3; and (b) the light chain variable region (VL), which includes the light chain containing the amino acid sequence of SEQ ID NO: 4 Complementarity determining region (LCDR) 1. LCDR 2 containing the amino acid sequence of SEQ ID NO: 5 and LCDR 3 containing the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 28. The immunoconjugate of claim 1 or 2, wherein the IL-2 polypeptide is a mutant IL-2 polypeptide, Wherein the mutant IL-2 polypeptide is a human IL-2 molecule, which contains amino acid substitutions F42A, Y45A and L72G (numbering relative to the human IL-2 sequence SEQ ID NO: 13); and Wherein the antibody comprises: (a) heavy chain variable region (VH), which comprises at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 7 And (b) the light chain variable region (VL), which comprises at least about 95%, 96%, 97%, 98% of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 29 %, 99% or 100% identical amino acid sequence. The immunoconjugate of claim 1 or 2, wherein the mutant IL-2 polypeptide further comprises an amino acid substituted for T3A and/or an amino acid substituted for C125A. The immunoconjugate of claim 1 or 2, wherein the mutant IL-2 polypeptide comprises the sequence of SEQ ID NO: 14. The immunoconjugate of claim 1 or 2, wherein the immunoconjugate comprises not more than one mutant IL-2 polypeptide. The immunoconjugate of claim 1 or 2, wherein the antibody comprises an Fc domain composed of a first subunit and a second subunit. The immunoconjugate according to claim 7, wherein the Fc domain is of the IgG class, especially the IgG ₁ subclass Fc domain. The immunoconjugate of claim 7, wherein the Fc domain is a human Fc domain. The immunoconjugate of claim 1 or 2, wherein the antibody is of the IgG class, especially the IgG ₁ subclass immunoglobulin. The immunoconjugate of claim 7, wherein the Fc domain comprises a modification that promotes the binding of the first unit and the second unit of the Fc domain. The immunoconjugate according to claim 7, wherein in the CH3 domain of the first unit of the Fc domain, an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby in the first The CH3 domain of the subunit produces a protuberance that can be located in the cavity in the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain, the amino acid residues have relatively Amino acid residues with a small side chain volume are replaced, thereby creating a cavity in the CH3 domain of the second subunit in which the protuberance in the CH3 domain of the first subunit can be positioned. The immunoconjugate of claim 9, wherein in the first unit of the Fc domain, the threonine residue at position 366 is replaced by a tryptophan residue (T366W), and in the second unit of the Fc domain In the unit, the tyrosine residue at position 407 is replaced by a valine residue (Y407V), and optionally the threonine residue at position 366 is replaced by a serine residue (T366S) and the position at 368 Leucine residues were replaced by alanine residues (L368A) (numbering according to Kabat EU index). The immunoconjugate of claim 13, wherein in the first unit of the Fc domain, in addition, the serine residue at position 354 is replaced with a cysteine residue (S354C) or the glutamine at position 356 The acid residue is replaced by a cysteine residue (E356C), and in the second unit of the Fc domain, in addition, the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C) ( According to the Kabat EU index number). The immunoconjugate of claim 9, wherein the mutant IL-2 polypeptide is at its amino terminal amino acid and a subunit of the Fc domain, specifically, the carboxy terminal amine of the first unit of the Fc domain Base acid fusion, optionally via connecting peptide fusion. The immunoconjugate of claim 15, wherein the connecting peptide has the amino acid sequence of SEQ ID NO: 15. The immunoconjugate of claim 9, wherein the Fc domain comprises one or more amino acid substitutions, the substitutions reducing binding to Fc receptors (specifically Fcγ receptors), and/or effector functions (specifically Antibody-dependent cell-mediated cytotoxicity (ADCC). The immunoconjugate of claim 17, wherein the one or more amino acid substitutions are at one or more positions selected from the group consisting of L234, L235 and P329 (Kabat EU index number). The immunoconjugate of claim 9, wherein each subunit in the Fc domain includes amino acid substitutions L234A, L235A and P329G (Kabat EU index number). Such as the immunoconjugate of claim 1 or 2, which comprises: comprising at least about 80%, 85%, 90%, 95%, 96%, 97%, 98 of the sequence of SEQ ID NO: 9 or SEQ ID NO: 30 A polypeptide with an amino acid sequence that is %, 99%, or 100% identical; comprising at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% with the sequence of SEQ ID NO: 10 Or a polypeptide with a 100% identical amino acid sequence; and comprising at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% with the sequence of SEQ ID NO: 11 or SEQ ID NO: 12 , 99% or 100% identical peptides with amino acid sequence. The immunoconjugate of claim 1 or 2, which comprises: a polypeptide comprising the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 30; a polypeptide comprising the amino acid sequence of SEQ ID NO: 10; and A polypeptide of the amino acid sequence of SEQ ID NO: 11. The immunoconjugate of claim 1 or 2, which comprises: a polypeptide comprising the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 30; a polypeptide comprising the amino acid sequence of SEQ ID NO: 10; and A polypeptide of the amino acid sequence of SEQ ID NO: 12. Such as the immunoconjugate of claim 1 or 2, which basically consists of a mutant IL-2 polypeptide and an IgG ₁ immunoglobulin molecule joined by a linker sequence. One or more isolated polynucleotides, which encode the immunoconjugate according to any one of claims 1 to 23. One or more vectors, in particular expression vectors, which comprise polynucleotides as in claim 24. A host cell comprising the polynucleotide of claim 24 or the vector of claim 25. A method for producing an immunoconjugate, the immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to CD8, the method comprising (a) culturing a host as in claim 26 under conditions suitable for expressing the immunoconjugate Cells, and optionally (b) recover the immunoconjugate. An immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to CD8, the immunoconjugate being produced by the method of claim 27. A pharmaceutical composition comprising the immunoconjugate according to any one of claims 1 to 23 or 28 and a pharmaceutically acceptable carrier. Such as the immunoconjugate of claim 1 or 2, which is used as a medicament. Such as the immunoconjugate of claim 1 or 2, which is used for the treatment of diseases. The immunoconjugate of claim 31, wherein the disease is cancer. An immunoconjugate according to any one of claims 1 to 23 or 28, which is used for the manufacture of a medicament for the treatment of diseases. Such as the use of claim 33, wherein the disease is cancer. A use of the immunoconjugate according to any one of claims 1 to 23 or 28, which is used to manufacture a medicament for stimulating the immune system of an individual.

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