Conjugates of checkpoint inhibitors and IL-2, and uses thereof

Abstract

The present disclosure relates to modified anti-PD-1 polypeptides, pharmaceutical compositions comprising the modified anti-PD-1 polypeptides, methods of making the anti-PD-1 polypeptides, and methods of using the modified anti-PD-1 polypeptides for the treatment of disease. In one aspect, the disclosure relates to a method of treating cancer in a subject using the modified anti-PD-1 polypeptides.

Description

[Background technology]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 219,981, filed July 9, 2021, and U.S. Provisional Patent Application No. 63 / 219,989, filed July 9, 2021, which are incorporated by reference in their entireties.

[0002] In 2021, an estimated 1.8 million new cases of cancer will be diagnosed in the United States, and more than 600,000 people will die from the disease. Immunotherapies harness the subject's immune system to aid in the treatment of disease. Immunotherapies can be designed to activate or suppress the immune system depending on the nature of the disease being treated. The goal of various immunotherapies for the treatment of cancer is to stimulate the immune system so that it recognizes and destroys tumors or other cancerous tissues.

[0003] Programmed cell death protein 1 (PD-1) is a cell surface protein that controls the immune system's response to cells in the human body by downregulating the immune system and promoting self-tolerance by suppressing T-cell inflammatory activity. Programmed cell death ligand 1 (PD-L1) is a type 1 transmembrane protein that suppresses the adaptive arm of the immune system. PD-1 and PD-L1 pathways represent adaptive immune system resistance mechanisms exerted by tumor cells in response to intrinsic immune antitumor activity. PD-1 inhibitors, such as anti-PD-1 polypeptides and anti-PD-1 antigen-binding fragments, are checkpoint inhibitor anticancer drugs that block the activity of the PD-1 immune checkpoint protein. However, monotherapy alone is often insufficient to obtain sustained responses in cancer patients. Thus, improved therapies for treating cancer are needed. Summary of the Invention

[0004] Described herein are anti-programmed cell death protein 1 (PD-1)-interleukin 2 (IL2) immunoconjugates and uses thereof.

[0005] In one aspect, described herein is a composition comprising a polypeptide that selectively binds programmed cell death protein 1 (PD-1), a modified IL-2 polypeptide, and a linker, the linker comprising a first attachment point covalently attached to a non-terminal residue of the modified IL-2 polypeptide and a second attachment point covalently attached to the polypeptide that selectively binds PD-1.

[0006] In one embodiment, described herein is a composition comprising a polypeptide that selectively binds to PD-1, a modified IL-2 polypeptide, and a linker, the linker comprising a first attachment point covalently attached to the modified IL-2 polypeptide and a second attachment point covalently attached to a non-terminal residue of the polypeptide that selectively binds to PD-1.

[0007] In another embodiment, described herein is a composition comprising a polypeptide that selectively binds to PD-1, a modified IL-2 polypeptide, and a chemical linker, the chemical linker comprising a first attachment point covalently attached to the modified IL-2 polypeptide and a second attachment point covalently attached to the polypeptide that selectively binds to PD-1.

[0008] In another embodiment, described herein is a composition comprising a polypeptide that selectively binds to PD-1, a modified IL-2 polypeptide, and a chemical linker, the chemical linker comprising a first attachment point covalently attached to the modified IL-2 polypeptide and a second attachment point covalently attached to the polypeptide that selectively binds to PD-1, wherein the modified IL-2 polypeptide is targeted to the IL-2 receptor β subunit.

[0009] In another aspect, described herein is a composition comprising an IL-2 polypeptide, the IL-2 polypeptide comprising a first polymer linked to amino acid residue 42, where the amino acid residue position numbering of the modified IL-2 polypeptide is based on SEQ ID NO:1 as a reference sequence, and a polypeptide that selectively binds to programmed cell death protein 1 (PD-1).

[0010] In another aspect, described herein is a composition comprising: (a) an antibody or antigen-binding fragment that selectively binds programmed cell death protein 1 (PD-1) and comprises an Fc region, wherein the Fc region comprises an amino acid sequence having 90% or greater identity to SEQ ID NO:105; (b) one or more linkers covalently attached to the Fc region at an amino acid residue selected from the group consisting of: (i) positions 25-35 of SEQ ID NO:105; (ii) positions 70-80 of SEQ ID NO:105; and (iii) positions 95-105 of SEQ ID NO:105; and (c) one or more cytokines covalently attached to the linker.

[0011] In another aspect, described herein is a composition comprising: (a) an antibody or antigen-binding fragment thereof that selectively binds to PD-1 and comprises an Fc region; (b) one or more linkers covalently attached to the Fc region at amino acid residues selected from the group consisting of K246, K248, K288, K290, and K317 (Eu numbering); and (c) one or more cytokines covalently attached to the one or more linkers.

[0012] A polypeptide that selectively binds to PD-1 can be, for example, a recombinant protein, such as an antibody, or a synthetic protein.

[0013] In another aspect, described herein are pharmaceutical compositions that include a) a composition described herein and b) one or more pharma- ceutically acceptable carriers or excipients.

[0014] In another aspect, described herein is a method of treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of any of the compositions described herein or the pharmaceutical compositions described herein.

[0015] In another aspect, described herein is a method of making a composition described herein, the method comprising the steps of a) covalently attaching a reactive group to a specific residue of a polypeptide that selectively binds to PD-1, b) contacting the reactive group with a complementary reactive group attached to a cytokine, and c) forming the composition.

[0016] In another aspect, described herein is a method of making a composition comprising a polypeptide that selectively binds programmed cell death protein 1 (PD-1), a modified IL-2 polypeptide, and a linker, the linker comprising a first point of attachment covalently attached to a non-terminal residue of the modified IL-2 polypeptide and a second point of attachment covalently attached to the polypeptide that selectively binds PD-1, the method comprising the steps of: a) providing an anti-PD-1 antibody or antigen-binding fragment having at least one receptor amino acid residue that is reactive with the linker in the presence of a coupling enzyme; and b) reacting the antibody or antigen-binding fragment with a linker that comprises a primary amine, the linker comprising a reactive group (R), in the presence of an enzyme that can cause formation of a covalent bond between at least one receptor amino acid residue and the linker, wherein the covalent bond is not present in the R moiety, the method being performed under conditions sufficient for the at least one receptor amino acid residue to form a covalent bond via the linker to the reactive group, the covalent bond comprising the second point of attachment of the linker.

[0017] In another embodiment, a method of making a composition comprising a polypeptide that selectively binds to PD-1, a modified IL-2 polypeptide, and a linker, the linker comprising a first attachment point covalently attached to a residue of the modified IL-2 polypeptide and a second attachment point covalently attached to the polypeptide that selectively binds to PD-1, the method comprising the steps of a) providing a polypeptide that selectively binds to PD-1 having at least one receptor amino acid residue that is reactive with the linker in the presence of a functionalized Fc-binding affinity peptide; and b) reacting said polypeptide that selectively binds to PD-1 with a linker precursor that comprises a reactive group (R) capable of binding to a receptor amino acid residue, the method being performed under conditions sufficient for the at least one receptor amino acid residue to form a covalent bond to the reactive group via the linker, the covalent bond comprising the second attachment point of the linker.

[0018] Further aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, in which only illustrative embodiments of the present disclosure are shown and described. As will be understood, the present disclosure is capable of other and different embodiments, and its several details are capable of modification in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive. Incorporation by Reference

[0019] All publications, patents, and patent applications mentioned in this specification are incorporated by reference herein to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, the present specification is intended to supersede and / or take precedence over such conflicting material. [Brief description of the drawings]

[0020] [Figure 1A]FIG. 1 shows anti-PD1-IL2 immunocytokines of the present disclosure and their interaction with activated T cells via IL2Rβ / γ upregulation and PD-1 inhibition. [Figure 1B] FIG. 1 shows the structure of modified conjugable cytokine composition AB. [Figure 2A] FIG. 1 shows site-selective modification of anti-PD1 antibodies by chemical modification techniques to introduce one or two conjugation handles. [Figure 2B] FIG. 1 shows Q-TOF mass spectra of pembrolizumab and unmodified pembrolizumab with a DBCO conjugation handle. [Figure 2C] FIG. 1 shows site-selective conjugation of modified IL2 cytokine to generate PD1-IL2 with DAR1, DAR2 or mixed DARs of 1-2. [Figure 2D] FIG. 1 shows the TIC chromatogram (top) and intact RP-HPLC (bottom) profile of crude pembrolizumab and Composition AB conjugation reaction. [Figure 2E] FIG. 1 shows the Q-TOF mass spectral profile of crude pembrolizumab conjugated in Composition AB conjugation reaction showing the formation of drug-antibody ratio 1 (DAR1) and drug-antibody ratio 2 (DAR2) species. [Figure 2F] FIG. 1 shows the intact RP-HPLC (top left) profile of purified pembrolizumab conjugated to Composition AB. [Figure 2G] FIG. 1 shows the Q-TOF mass spectral profile of purified pembrolizumab conjugated to Composition AB with mixed drug-to-antibody ratios (DAR). [Figure 2H] FIG. 1 shows SEC-HPLC of purified immunocytokines including pembrolizumab conjugated to Composition AB. [Diagram 3]Figure 1 shows a plot measuring the ability of unmodified and conjugated anti-PD1 antibodies to bind to PD1 / CD279 ligand, with ELISA signal on the y-axis and dosage of biotinylated PD-1 protein on the x-axis. The unconjugated reference antibodies are pembrolizumab, nivolumab and LZM-009. The conjugated antibodies tested in this figure are compositions A, C, D, E, F, G, and H. [Figure 4] Figure 1 shows a plot measuring the ability of unmodified and conjugated anti-PD1 antibodies to disrupt the PD1 / PDL1 pathway, with the mean luminescence intensity of the effector cell NFAT-RE reporter shown on the y-axis and the dosage of unmodified and conjugated anti-PD1 antibodies shown on the x-axis. The unconjugated reference antibody is pembrolizumab, and the conjugated antibody tested in this figure is Composition B. The IL-2 polypeptides tested in this figure are Proleukin and Composition AA. [Diagram 5] Figure 1 shows a plot measuring the ability of unmodified and conjugated anti-PD1 antibodies to bind to human neonatal Fc receptor (FcRn) at pH 6, with the average AlphaLISA® FcRn-IgG signal shown on the y-axis and the dosage of unmodified and conjugated anti-PD1 antibodies shown on the x-axis. The unconjugated reference antibodies are pembrolizumab and LZM-009. The conjugated antibodies tested are compositions A, D, E, H, J, and K. [Figure 6A]FIG. 1 shows a plot measuring the ability of unmodified and conjugated anti-PD1 antibodies to bind to human Fc gamma receptor I (CD64), human Fc gamma receptor IIa (CD32a), human Fc gamma receptor IIb (CD32b), and human Fc gamma receptor IIIa (CD16), with the average ELISA signal shown on the y-axis and the dosage of unmodified and conjugated anti-PD1 antibodies shown on the x-axis. The unconjugated reference antibodies are pembrolizumab and LZM-009. The conjugated antibodies tested are compositions A, C, D, and H. [Figure 6B] Figure 1 shows a plot measuring the ability of conjugated anti-PD1 antibodies to bind to human Fc gamma receptor I (CD64), human Fc gamma receptor IIa (CD32a), human Fc gamma receptor IIb (CD32b), and human Fc gamma receptor IIIa (CD16), with the average ELISA signal shown on the y-axis and the dosage of conjugated anti-PD1 antibody shown on the x-axis. The conjugated antibody compositions tested from top to bottom are compositions E, J, and K, respectively. [Figure 7A] FIG. 1 shows plots measuring surface expression levels of PD-1 / CD279 on parental non-transduced Mo7e (PD1−) and stably transduced (PD1+) Mo7e cells. [Figure 7B]Figure 1 shows EC50 values ​​of phosphorylated signal transduction and activator of transcription 5 (pSTAT5) on the y-axis in parental PD1-Mo7e cells as "PD1-" or stable PD1+Mo7e cells as "PD1+" by treatment of modified IL-2 polypeptides or immunocytokines as listed on the x-axis. Measurements shown for PD1-negative cells are shown in solid symbols, and PD1-positive cells are shown in grey open symbols. The unconjugated modified IL-2 polypeptides tested in this figure are Proleukin, and composition AB. The modified IL-2 anti-PD-1 immunocytokines are compositions A, C, and H. Composition O, a Her2-targeted IL-2 immunocytokine, is shown as a negative control. [Figure 8] Figure 1 shows plots measuring the effect of modified IL-2 polypeptides unconjugated and conjugated to anti-PD1 antibodies on the induction of Teff and Treg cells in human T cells in vitro. Showing the mean fluorescence intensity of phosphorylated signal transducer and activator of transcription 5 (pSTAT5) on the y-axis as a dose response to modified IL-2 polypeptide or immunocytokine on the x-axis. The modified IL-2 polypeptide tested is composition AA. The IL-2 anti-PD-1 immunocytokine tested is composition A, B, and C. [Figure 9A] FIG. 1 shows plots measuring surface expression levels of PD-1 / CD279 on resting memory (CD45RA-) and naive (CD45RA+) CD8+ Teff cells freshly isolated from peripheral blood of healthy donors, with illustrations of the indicated cell types. [Figure 9B]Figure 1 shows the dose-response effect of conjugating IL-2 to PD-1 on CD8+ Teff cells. Figure 2 shows plots measuring the effect of modified IL-2 polypeptides not conjugated to anti-PD1 antibodies and conjugated to anti-PD1 antibodies on the induction of resting memory (CD45RA-) and naive (CD45RA+) CD8+ Teff cells in in vitro samples of human T cells, with the mean fluorescence intensity of phosphorylated signal transduction and activator of transcription 5 (pSTAT5) shown on the y-axis and the dosage of modified IL-2 polypeptide and immunocytokine shown on the x-axis. The modified IL-2 polypeptide tested in this figure is composition AA. The IL-2 anti-PD-1 immunocytokine tested in this figure is composition B. Her2-targeting immunocytokine composition N (trastuzumab antibody conjugated to IL-2 polypeptide) is shown as a negative control. [Figure 10A] FIG. 1 shows plots measuring the effect of modified IL-2 polypeptides unconjugated to anti-PD1 antibodies and conjugated modified IL-2 polypeptides on the induction of resting naive (CD45RA+) CD8+ Teff cells in in vitro samples of human T cells in the presence or absence of excess unconjugated anti-PD1 antibody pembrolizumab, where the mean fluorescence intensity of phosphorylated signal transduction and activator of transcription 5 (pSTAT5) is shown on the y-axis and dosages of modified IL-2 polypeptides and immunocytokines are shown on the x-axis. The modified IL-2 polypeptide tested in this figure is composition AA, and the immunocytokines tested in this figure are composition B and the Her2-targeted immunocytokine composition N (trastuzumab antibody conjugated to an IL-2 polypeptide) as a control. [Figure 10B]FIG. 1 shows plots measuring the effect of modified IL-2 polypeptides unconjugated to anti-PD1 antibodies and conjugated modified IL-2 polypeptides on the induction of resting memory (CD45RA-) CD8+ Teff cells in in vitro samples of human T cells in the presence or absence of excess of unconjugated anti-PD1 antibody pembrolizumab, where the mean fluorescence intensity of phosphorylated signal transduction and activator of transcription 5 (pSTAT5) is shown on the y-axis and dosages of modified IL-2 polypeptide and immunocytokine are shown on the x-axis. The modified IL-2 polypeptide tested in this figure is composition AA, and the immunocytokine tested in this figure is composition B and the Her2-targeted immunocytokine composition N (trastuzumab antibody conjugated to an IL-2 polypeptide) as a control. [Figure 11A] Figure 1 shows plots illustrating the effect of PD-1 targeted and non-targeted immunocytokines on the growth of CT26 syngeneic colon cancer tumors in hPD1-humanized BALB / c mice. The immunocytokine tested in this figure is Composition A, which was tested as a single agent at 1 mg / kg and 2.5 mg / kg following a single injection schedule. A control Her2 targeted immunocytokine Composition O (trastuzumab antibody conjugated to an IL-2 polypeptide) was also tested at 2.5 mg / kg (mean ± SEM). [Figure 11B] Figure 1 shows a bar graph illustrating the effect of PD-1 targeted and non-targeted immunocytokines on the growth of CT26 syngeneic colon cancer tumors in hPD1 humanized BALB / c mice after 7 days of treatment. The immunocytokine tested in this figure is Composition A, which was tested as a single agent at 1 mg / kg and 2.5 mg / kg following a single injection schedule. A control Her2 targeted immunocytokine Composition O (trastuzumab antibody conjugated to an IL-2 polypeptide) was also tested at 2.5 mg / kg (mean ± SEM; **One-way ANOVA P-value < 0.001). [Figure 12A]Figure 1 shows plots illustrating the effect of PD-1 targeted and non-targeted immunocytokines on the expansion of naive (CD62Lhigh CD44low) CD8+ T cells in the blood and tumors of hPD1-humanized BALB / c mice bearing CT26 tumors 7 days after treatment. The immunocytokine tested in this figure is Composition A, which was tested as a single agent at 1 mg / kg and 2.5 mg / kg following a single injection schedule. A control Her2 targeted immunocytokine Composition O (trastuzumab antibody conjugated to an IL-2 polypeptide) was also tested at 2.5 mg / kg (n=3; mean±SEM). [Figure 12B] Figure 1 shows plots illustrating the effect of PD-1 targeted and non-targeted immunocytokines on the expansion of effector memory (CD62L negative CD44 high) CD8+ T cells in the blood and tumors of hPD1 humanized BALB / c mice bearing CT26 tumors 7 days after treatment. The immunocytokine tested in this figure is Composition A, which was tested as a single agent at 1 mg / kg and 2.5 mg / kg following a single injection schedule. A control Her2 targeted immunocytokine Composition O (trastuzumab antibody conjugated to an IL-2 polypeptide) was also tested at 2.5 mg / kg (n=3; mean ± SEM). [Figure 13A] FIG. 1 shows plots illustrating the effect of PD-1 targeting and non-targeting of immunocytokines on their persistence in the blood and tumors of hPD1-humanized BALB / c mice bearing CT26 tumors, with plasma or tumor concentrations of PD-1-targeting and control immunocytokines shown on the y-axis and time shown on the x-axis. The immunocytokine tested in this figure is Composition A, which was tested as a single agent at 1 mg / kg and 2.5 mg / kg after a single injection schedule. A control Her2-targeting immunocytokine Composition O (trastuzumab antibody conjugated to an IL-2 polypeptide) was also tested at 2.5 mg / kg (n=3; mean±SD). [Figure 13B]FIG. 1 shows how PD-1 targeting leads to the gradual accumulation of PD1-IL2 immunocytokine in tumors over a 7-day period. In contrast, non-targeted control immunocytokine (Her2-IL2) shows no intratumoral accumulation and disappears within 4 days. Analysis was performed in hPD1-humanized BALB / c mice bearing CT26 tumors, with the tumor / plasma concentration ratios of PD-1-targeted and control immunocytokine shown on the y-axis and time shown on the x-axis. The immunocytokine analyzed is composition A, which was tested as a single agent at 1 mg / kg and 2.5 mg / kg after a single application. The control Her2-targeted immunocytokine composition O (trastuzumab antibody conjugated to an IL-2 polypeptide) was tested at 2.5 mg / kg (n=3; mean±SEM). [Figure 14A] Figure 1 shows a plot illustrating the effect of a single injection of conjugated anti-PD1 antibody on the growth of MC38 syngeneic colon carcinoma tumors in hPD1 C57BL / / 6 mice. The immunocytokine tested in this figure is Composition H, tested as a single agent at 1 mg / kg as a single injection (n=8; mean±SEM). [Figure 14B] Figure 1 shows a bar graph illustrating the effect of a single injection of conjugated anti-PD1 antibody on the growth of MC38 syngeneic colon carcinoma tumors in hPD1 C57BL / / 6 mice 7 days after treatment. The immunocytokine tested in this figure is Composition H, tested as a single agent at a single injection of 1 mg / kg (n=8 animals; mean±SEM; **One-way ANOVA P-value<0.005). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Anti-PD-1 polypeptides are disclosed herein. In some embodiments, the anti-PD-1 polypeptides are conjugated to cell signaling molecules such as cytokines. In some embodiments, the cytokine is IL-2. FIG. 1A shows an exemplary immunocytokine comprising an anti-PD-1 polypeptide conjugated to an IL-2 cytokine. The anti-PD-1 antibody / IL-2 immunocytokine (herein referred to as PD1-IL2) of the present disclosure may have superior efficacy and potentially improved tolerability by subjects. In some embodiments, the anti-PD-1-IL-2 immunocytokine of the present disclosure may directly target tumor infiltrating lymphocytes (TILs). In some embodiments, the anti-PD-1-IL-2 immunocytokine may significantly reduce the therapeutic dose of anti-PD-1 polypeptide or IL-2 for a subject with a disease such as cancer.

[0022] Anti-PD-1-IL-2 immunocytokines may act by one or more mechanisms of action. In some embodiments, anti-PD-1-IL-2 immunocytokines can inhibit PD-1 by targeting PD-1 and CD8+ T cells within tumors. In some embodiments, anti-PD-1-IL-2 immunocytokines can activate T cells and NK cells via IL-2Rβγ. The following description and examples illustrate the embodiments of the present disclosure in detail. It should be understood that the present disclosure is not limited to the specific embodiments described herein and may therefore vary. Those skilled in the art will recognize that there are many variations and modifications of the present disclosure that fall within the scope of the present disclosure.

[0023] Although various features of the present disclosure may be described in the context of a single embodiment, these features may also be provided separately or in any suitable combination. Conversely, although the present disclosure may be described herein for clarity in the context of separate embodiments, the present disclosure may also be implemented in a single embodiment. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. definition

[0024] All terms are intended to be understood as those terms are understood by one of ordinary skill in the art. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0025] The following definitions supplement the definitions in the art, and are directed to the current application, and should not be attributed to related or unrelated cases, such as co-owned patents or applications.Any method and material similar or equivalent to those described herein can be used to carry out the test of the present disclosure, but preferred materials and methods are described herein.Therefore, the terms used herein are only intended to describe specific embodiments, and are not intended to be limiting.

[0026] Thus, the terminology used herein is for the purpose of describing particular instances only and is not intended to be limiting. In this application, the use of the singular includes the use of the plural unless specifically stated otherwise. As used herein, the singular forms "a," "an," and "the" are intended to include the plural unless the context clearly dictates otherwise. In this application, the use of "or" means "and / or" unless otherwise specified. As used herein, the terms "and / or" and "any combination thereof" and their grammatical equivalents can be used interchangeably. These terms can convey that any combination is specifically contemplated. For illustrative purposes only, the following phrases "A, B, and / or C" or "A, B, C, or any combination thereof" can mean "A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C." The term "or" can be used conjunctively or disjunctively unless the context specifically dictates disjunctive use.

[0027] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within one standard deviation or more than one standard deviation, according to the conventions of the art. Alternatively, "about" can mean within a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, within 5-fold, or within 2-fold of a value. When a particular value is described in this application and claims, unless otherwise specified, the term "about" means within an acceptable error range for the particular value.

[0028] As used in the specification and claims, "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the disclosure, and vice versa. Additionally, the compositions of the disclosure can be used to achieve the methods of the disclosure.

[0029] Reference herein to "some embodiments," "an embodiment," "one embodiment," or "other embodiments" means that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least some embodiments of the present disclosure, but not necessarily in all embodiments. To facilitate understanding of this disclosure, certain terms and phrases are defined below.

[0030] Groups that are "attached" or "covalently attached" to a residue of an IL-2 polypeptide are referred to herein. As used herein, "attached" or "covalently attached" means that the group is tethered to the indicated residue, and such tethering may include a linking group (i.e., a linker). Thus, when referring to a group that is "attached" or "covalently attached" to a residue, it is expressly intended that such linking groups are also encompassed.

[0031] Binding affinity refers to the strength of the binding interaction between a single molecule and its ligand / binding partner. A higher binding affinity refers to a stronger binding than a lower binding affinity. In some cases, binding affinity is measured by the dissociation constant (K D ) is measured by K D When comparing values, a binding interaction with a lower value has a higher binding affinity than a binding interaction with a higher value. For protein-ligand interactions, K D is calculated according to the following formula:

[0032]

number

[0033] As used herein, it refers to a particular amino acid sequence (e.g., a polypeptide sequence) that has a certain percent sequence identity to a reference sequence or refers to a residue at a position corresponding to a position of a reference sequence. Sequence identity is measured by the protein-protein BLAST algorithm using the parameters of Matrix BLOSUM62, Gap Costs Existence: 11, Extension: 1, and Compositional Adjustments Conditional Compositional Score Matrix Adjustment. This alignment algorithm is also used to evaluate whether residues are at "corresponding" positions through the analysis of the alignment of the two sequences being compared.

[0034] The term "pharmaceutical acceptable" refers to that approved or approved by a regulatory agency of the Federal or state government, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias, for use in animals, including humans.

[0035] A "pharmaceutical acceptable excipient, carrier, or diluent" refers to an excipient, carrier, or diluent that can be administered to a subject together with a drug, does not destroy its pharmacological activity, and is non-toxic when administered in a dosage sufficient to deliver a therapeutic amount of the drug.

[0036] A "pharmaceutically acceptable salt" suitable for this disclosure may be any acid or base salt generally considered in the art to be suitable for use in contact with human or animal tissues without undue toxicity, irritation, allergic response, or other problem or complication. Such salts include inorganic and organic acid salts of basic residues such as amines, and alkali or organic salts of acidic residues such as carboxylic acids. Particular pharmaceutical salts include, but are not limited to, salts of acids such as hydrochloric acid, phosphoric acid, hydrobromic acid, malic acid, glycolic acid, fumaric acid, sulfuric acid, sulfamic acid, sulfanilic acid, formic acid, toluenesulfonic acid, methanesulfonic acid, benzenesulfonic acid, ethanedisulfonic acid, 2-hydroxyethylsulfonic acid, nitric acid, benzoic acid, 2-acetoxybenzoic acid, citric acid, tartaric acid, lactic acid, stearic acid, salicylic acid, glutamic acid, ascorbic acid, pamoic acid, succinic acid, fumaric acid, maleic acid, propionic acid, hydroxymaleic acid, hydroiodic acid, phenylacetic acid, alkanoic acids such as acetic acid, HOOC-(CH2)n-COOH, where n is 0 to 4. Similarly, pharma-ceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium. Those skilled in the art will recognize from this disclosure and knowledge of the art that additional pharma-ceutically acceptable salts include those listed in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, p.1418 (1985). In general, pharma-ceutically acceptable acid or base salts can be synthesized from parent compounds that contain a basic or acidic moiety by any conventional chemical method. Briefly, such salts can be prepared by reacting the free acid or free base forms of these compounds with a stoichiometric amount of the appropriate base or acid in a suitable solvent.

[0037] Ranges provided herein are understood to be shorthand for all values ​​within the range. For example, the range of 1 to 50 is understood to include any number, combination of numbers, or subranges from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, as well as all intervening decimal values ​​between the aforementioned integers, such as 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to subranges, "nested subranges" extending from either end of the range are specifically contemplated. For example, nested subranges of the exemplary range of 1 to 50 could include 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

[0038] Certain formulas and other examples provided herein depict the triazole reaction products resulting from the azide-alkyne cycloaddition reaction. Although such formulas generally depict only a single positional isomer of the resulting triazole formed in the reaction, the formulas are intended to encompass both resulting positional isomers. Thus, the formulas may depict only a single positional isomer (e.g.,

[0039] [ka] ), other positional isomers (e.g.,

[0040] [ka] ) are also intended to be included.

[0041] The term "subject" refers to an animal that is the object of treatment, observation, or experiment. By way of example only, a subject includes, but is not limited to, a human or non-human mammal, such as a mammal, including but not limited to a non-human primate, bovine, equine, canine, ovine, or feline.

[0042] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and indicates that the description includes examples where the event or circumstance occurs or examples where the event or circumstance does not occur.

[0043] The term "moiety" refers to a specific segment or functional group of a molecule. A chemical moiety is often recognized as a chemical entity that is embedded in or appended to a molecule.

[0044] As used herein, the term “number average molecular weight” (Mn) refers to the statistical average molecular weight of all individual units in a sample, and is represented by the formula (1):

[0045]

number

[0046] As used herein, the term “weight average molecular weight” (Mw) refers to a molecular weight of a compound represented by the formula (2):

[0047]

number

[0048] As used herein, "peak molecular weight" (Mp) means the molecular weight of the highest peak in a given analytical method (e.g., mass spectrometry, size exclusion chromatography, dynamic light scattering, analytical centrifugation, etc.).

[0049] As used herein, a "non-standard" amino acid can refer to an amino acid residue in either D- or L-form that is not among the 20 standard amino acids commonly incorporated into naturally occurring proteins.

[0050] As used herein, "conjugation handle" refers to a reactive group that can form a bond when contacted with a complementary reactive group. In some cases, the conjugation handle preferably does not have substantial reactivity with other molecules that do not contain the intended complementary reactive group. Non-limiting examples of conjugation handles, their respective complementary conjugation handles, and corresponding reaction products can be found in the table below. Although the table headings list specific reactive groups under the headings "conjugation handle" or "complementary conjugation handle," it is intended that any reference to a conjugation handle may instead include the complementary conjugation handle listed in the table (e.g., trans-cyclooctene may be a conjugation handle, in which case tetrazine is the complementary conjugation handle). In some cases, amine conjugation handles and amine-complementary conjugation handles are less preferred for use in biological systems due to the ubiquitous presence of amines in biological systems and the increased likelihood of off-target conjugation.

[0051] [Table 1]

[0052] Throughout this application, a prefix is ​​used before the term "conjugation" to indicate the function to which the conjugation handle is linked. For example, a "protein conjugation handle" is a conjugation handle attached (directly or via a linker) to a protein, an "antibody conjugation handle" is a conjugation handle attached (directly or via a linker) to an antibody, and a "linker conjugation handle" is a conjugation handle attached to a linker group (e.g., a bifunctional linker used to link a synthetic protein to an antibody).

[0053] The term "alkyl" refers to a straight or branched hydrocarbon chain radical having from 1 to 20 carbon atoms and attached to the rest of the molecule by a single bond. Alkyl containing up to 10 carbon atoms includes C1-C 10 Similarly, for example, an alkyl containing up to 6 carbon atoms is referred to as a C1-C6 alkyl. Alkyl containing other numbers of carbon atoms (and other moieties defined herein) are similarly represented. Alkyl groups include C1-C 10Examples of alkyl include, but are not limited to, alkyl, C1-C9 alkyl, C1-C8 alkyl, C1-C7 alkyl, C1-C6 alkyl, C1-C5 alkyl, C1-C4 alkyl, C1-C3 alkyl, C1-C2 alkyl, C2-C8 alkyl, C3-C8 alkyl, and C4-C8 alkyl. Representative alkyl groups include, but are not limited to, methyl, ethyl, -propyl, 1-methylethyl, -butyl, -pentyl, 1,1-dimethylethyl, 3-methylhexyl, 2-methylhexyl, 1-ethyl-propyl, and the like. In some embodiments, alkyl is methyl or ethyl. In some embodiments, alkyl is -CH(CH3)2 or -C(CH3)3. Unless specifically stated otherwise in the specification, haloalkyl groups may be optionally substituted. "Alkylene" or "alkylene chain" refers to a straight or branched divalent hydrocarbon chain connecting the remainder of the molecule to a radical group. In some embodiments, alkylene is -CH-, -CHCH-, or -CHCHCH-. In some embodiments, alkylene is -CH-. In some embodiments, alkylene is -CHCH-. In some embodiments, alkylene is -CHCHCH-. In some embodiments, alkylene is -CHCHCH-. Unless specifically stated otherwise in the specification, a haloalkyl group may be optionally substituted.

[0054] The term "alkenylene" or "alkenylene chain" refers to a straight or branched divalent hydrocarbon chain in which there is at least one carbon-carbon double bond connecting the remainder of the molecule to a radical group. In some embodiments, the alkenylene is -CH=CH-, -CHCH=CH-, or -CH=CHCH-. In some embodiments, the alkenylene is -CH=CH-. In some embodiments, the alkenylene is -CHCH=CH-. In some embodiments, the alkenylene is -CH=CHCH-. In some embodiments, the alkenylene is -CH=CHCH-.

[0055] The term "alkynyl" refers to a type of alkyl group in which at least one carbon-carbon triple bond is present. In one embodiment, an alkenyl group has the formula -C≡CRX wherein R x refers to the remainder of the alkynyl group. In some embodiments, R x is H or alkyl. In some embodiments, alkynyl is selected from ethynyl, propynyl, butynyl, pentynyl, hexynyl, etc. Non-limiting examples of alkynyl groups include -C≡CH, -C≡CCH3, -C≡CCH2CH, and -CH2C o Examples include CH.

[0056] The term "aryl" refers to a radical that contains at least one aromatic ring in which each of the atoms that form the ring is a carbon atom. The aryl group may be optionally substituted. Examples of aryl groups include, but are not limited to, phenyl and naphthyl. In some embodiments, the aryl is phenyl. Depending on the structure, the aryl group may be a monoradical or a diradical (i.e., an arylene group). Unless specifically stated otherwise in the specification, the term "aryl" or the prefix "ar-" (such as "aralkyl") is meant to include aryl radicals that are optionally substituted. In some embodiments, the aryl group contains a partially reduced cycloalkyl group as defined herein (e.g., 1,2-dihydronaphthalene). In some embodiments, the aryl group contains a fully reduced cycloalkyl group as defined herein (e.g., 1,2,3,4-tetrahydronaphthalene). When the aryl contains a cycloalkyl group, the aryl is bonded to the remainder of the molecule through an aromatic ring carbon atom. The aryl radical may be a monocyclic or polycyclic (e.g., bicyclic, tricyclic, or tetracyclic) ring system that may include fused, spiro, or bridged ring systems.

[0057] "Cycloalkyl" refers to a monocyclic or polycyclic non-aromatic radical in which each of the atoms forming the ring (i.e., skeletal atoms) is a carbon atom. In some embodiments, the cycloalkyl is saturated or partially unsaturated. In some embodiments, the cycloalkyl is a spirocyclic or bridged compound. In some embodiments, the cycloalkyl may be fused to an aromatic ring (in which case the cycloalkyl is attached through a non-aromatic ring carbon atom). Cycloalkyl groups include groups having 3-10 ring atoms. Representative cycloalkyls include, but are not limited to, cycloalkyls having 3-10 carbon atoms, 3-8 carbon atoms, 3-6 carbon atoms, or 3-5 carbon atoms. Monocyclic cycloalkyls include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. In some embodiments, the monocyclic cycloalkyl is cyclopentyl. In some embodiments, the monocyclic cycloalkyl is cyclopentenyl or cyclohexenyl. In some embodiments, the monocyclic cycloalkyl is cyclopentenyl. Polycyclic radicals include, for example, adamantyl, 1,2-dihydronaphthalenyl, 1,4-dihydronaphthalenyl, tetranyl, decalinyl, 3,4-dihydronaphthalenyl-1(2H)-one, spiro[2.2]pentyl, norbornyl, and bicyclo[1.1.1]pentyl. Unless otherwise specifically stated in the specification, cycloalkyl groups may be optionally substituted.

[0058] The term "heteroalkylene" or "heteroalkylene chain" refers to a straight or branched divalent heteroalkyl chain that connects the remainder of the molecule to a radical group. Unless otherwise specifically stated in the specification, a heteroalkyl or heteroalkylene group may be optionally substituted as described below. Representative heteroalkylene groups include, but are not limited to, -CH2-O-CH2-, -CH2-N(alkyl)-CH2-, -CH2-N(aryl)-CH2-, -OCH2CHO-, -OCH2CHOCH2CHO-, or -OCH2CHOCH2CHOCH2CHO-.

[0059] The term "hetercycloalkyl" refers to a cycloalkyl group containing at least one heteroatom selected from nitrogen, oxygen, and sulfur. Unless otherwise specifically stated in the specification, a heterocycloalkyl radical can be a monocyclic or bicyclic ring system, which can include fused (when fused to an aryl or heteroaryl ring, the heterocycloalkyl is attached via a non-aromatic ring atom) or bridged ring systems. The nitrogen, carbon, or sulfur atoms in the heterocyclyl radical can be optionally oxidized. The nitrogen atom can be optionally quaternized. The heterocyclyl radical can be partially or fully saturated. Examples of such heterocyclyl radicals include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, tetrahydroquinolyl, tetrahydroisoquinolyl, decahydroquinolyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. The term heterocycloalkyl also includes all ring forms of carbohydrates, including but not limited to monosaccharides, disaccharides, and oligosaccharides. Unless otherwise specified, heterocycloalkyls have 2-12 carbons in the ring. In some embodiments, heterocycloalkyls have 2-10 carbons in the ring. In some embodiments, heterocycloalkyls have 2-10 carbons and 1 or 2 N atoms in the ring. In some embodiments, heterocycloalkyls have 2-10 carbons and 3 or 4 N atoms in the ring. In some embodiments, heterocycloalkyls have 2-12 carbons, 0-2 N atoms, 0-2 O atoms, 0-2 P atoms, and 0-1 S atoms in the ring.In some embodiments, a heterocycloalkyl has 2-12 carbons, 1-3 N atoms, 0-1 O atoms, and 0-1 S atoms in the ring. When referring to the number of carbon atoms in a heterocycloalkyl, it is understood that the number of carbon atoms in the heterocycloalkyl is not the same as the total number of atoms (including heteroatoms) that make up the heterocycloalkyl (i.e., skeletal atoms of the heterocycloalkyl ring). Unless otherwise specifically indicated in the specification, a heterocycloalkyl group may be optionally substituted.

[0060] The term "heteroaryl" refers to an aryl group containing one or more ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the heteroaryl is monocyclic or bicyclic. Specific examples of monocyclic heteroaryl include pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, pyridazinyl, triazinyl, oxadiazolyl, thiadiazolyl, furazanyl, indolizine, indole, benzofuran, benzothiophene, indazole, benzimidazole, purine, quinolizine, quinoline, isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline, 1,8-naphthyridine, and pteridine. Specific examples of monocyclic heteroaryls include pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, pyridazinyl, triazinyl, oxadiazolyl, thiadiazolyl, and furazanyl. Specific examples of bicyclic heteroaryls include indolizine, indole, benzofuran, benzothiophene, indazole, benzimidazole, purine, quinolizine, quinoline, isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline, 1,8-naphthyridine, and pteridine. In some embodiments, the heteroaryl is pyridinyl, pyrazinyl, pyrimidinyl, thiazolyl, thienyl, thiadiazolyl, or furyl. In some embodiments, the heteroaryl contains 0-6 N atoms in the ring. In some embodiments, the heteroaryl contains 1-4 N atoms in the ring. In some embodiments, the heteroaryl contains 4-6 N atoms in the ring. In some embodiments, the heteroaryl contains 0-4 N atoms, 0-1 O atoms, 0-1 P atoms, and 0-1 S atoms in the ring. In some embodiments, the heteroaryl contains 1-4 N atoms, 0-1 O atoms, and 0-1 S atoms in the ring. In some embodiments, the heteroaryl is a C1-C9 heteroaryl.In some embodiments, the monocyclic heteroaryl is a C1-C5 heteroaryl. In some embodiments, the monocyclic heteroaryl is a 5- or 6-membered heteroaryl. In some embodiments, the bicyclic heteroaryl is a C6-C9 heteroaryl. In some embodiments, the heteroaryl group comprises a partially reduced cycloalkyl or heterocycloalkyl group as defined herein (e.g., 7,8-dihydroquinoline). In some embodiments, the heteroaryl group comprises a fully reduced cycloalkyl or heterocycloalkyl group as defined herein (e.g., 5,6,7,8-tetrahydroquinoline). When the heteroaryl comprises a cycloalkyl or heterocycloalkyl group, the heteroaryl is bonded to the remainder of the molecule through a heteroaromatic ring carbon or heteroatom. The heteroaryl radical may be a monocyclic or polycyclic (e.g., bicyclic, tricyclic, or tetracyclic) ring system, which may include fused, spiro, or bridged ring systems.

[0061] The term "optionally substituted" or "substituted" means that the referenced group is optionally substituted with one or more additional groups individually and independently selected from D, halogen, -CN, -NH, -NH(alkyl), -N(alkyl), -OH, -COH, -COalkyl, -C(=O)NH, -C(=O)NH(alkyl), -C(=O)N(alkyl), -S(=O)NH, -S(=O)NH(alkyl), -S(=O)N(alkyl), alkyl, cycloalkyl, fluoroalkyl, heteroalkyl, alkoxy, fluoroalkoxy, heterocycloalkyl, aryl, heteroaryl, aryloxy, alkylthio, arylthio, alkylsulfoxide, arylsulfoxide, alkylsulfone, and arylsulfone. In some other embodiments, the optional substituents are independently selected from D, halogen, -CN, -NH, -NH(CH), -N(CH), -OH, -COH, -CO(C-C alkyl), -C(=O)NH, -C(=O)NH(C-C alkyl), -C(=O)N(C-C alkyl), -S(=O)NH, -S(=O)NH(C-C alkyl), -S(=O)N(C-C alkyl), C-C alkyl, C-C cycloalkyl, C-C fluoroalkyl, C-C heteroalkyl, C-C alkoxy, C-C fluoroalkoxy, -SC-C alkyl, -S(=O)C-C alkyl, and -S(=O)C-C alkyl. In some embodiments, optional substituents are independently selected from D, halogen, -CN, -NH2, -OH, -NH(CH3), -N(CH3)2, -NH(cyclopropyl), -CH3, -CH2CH3, -CF3, -OCH3, and -OCF3. In some embodiments, a substituted group is substituted with one or two of the preceding groups. In some embodiments, optional substituents on an aliphatic carbon atom (acyclic or cyclic) include oxo (=O).

[0062] As used herein, "AJICAP™ Technology," "AJICAP™ Method," and similar terms refer to a system and method (currently manufactured by Ajinomoto Bio-Pharma Services, Inc. ("Ajinomoto")) for site-specific functionalization of antibodies and related molecules using affinity peptides to deliver the desired functionalization to a desired site. General protocols for the AJICAP™ methodology can be found at least in WO 2018199337(A1), WO 2019240288(A1), WO 2019240287(A1), WO 2020090979(A1), Matsuda et al., Mol.Pharmaceutics 2021, 18, 4058-4066, and Yamada et al., Affinity Peptide Mediated Regiodivergent Functionalization of Native Antibodies. Angew.Chem., Int. Ed. 2019, 58, 5592-5597, and in particular in Examples 2-4 of US Patent Application Publication No. 20200190165(A1). In some embodiments, such methodologies specifically incorporate a desired functionalization at a lysine residue at a position selected from positions 246, 248, 288, 290, and 317 (EU numbering) of an antibody Fc region (e.g., an IgG1 Fc region). In some embodiments, the desired functionalization is incorporated at residue position 248 (EU numbering) of the antibody Fc region. In some embodiments, position 248 corresponds to the 18th residue of the human IgG CH2 region (EU numbering).

[0063] Composition AA refers to a modified IL-2 polypeptide having the sequence shown in SEQ ID NO:3, containing an approximately 0.5 kDa PEG group attached to residue Y45 and a second approximately 0.5 kDa PEG group attached to residue F42Y.

[0064] Composition AB refers to a modified IL-2 polypeptide having the sequence shown in SEQ ID NO:3, containing an approximately 0.5 kDa PEG group attached to residue Y45 and a 0.5 kDa PEG group capped with an azide functionality that facilitates conjugation at residue F42Y. An illustrative image of Composition AB is shown in FIG. 1B. Composition AB and related modified IL-2 polypeptides are described in WO2021140416(A2), which is incorporated by reference in its entirety. The polymer attached to Composition AB acts to disrupt the interaction of Composition AB with the IL-2 receptor α subunit and bias the molecule in favor of IL-2 receptor β subunit signaling, thus enhancing the IL-2 polypeptide's T signaling in vivo compared to WT IL-2. eff Enhances the ability of cells to expand and / or stimulate.

[0065] Composition AC refers to a modified IL-2 polypeptide having the sequence shown in SEQ ID NO: 3, containing an approximately 0.5 kDa PEG group attached to residues F42Y and Y45. Composition AC contains an azide conjugation handle attached to the N-terminal A residue via an approximately 0.5 kDa PEG (see Structure 7 provided herein) coupled via a glutaric acid linker functionality.

[0066] Composition A refers to an anti-PD-1 antibody / IL-2 conjugate prepared from the reaction of composition AB with the anti-PD-1 antibody pembrolizumab or LZM-009. Composition A is formed from the reaction of an azide functional group of composition AB with a DBCO functional group attached to residue K248 of the Fc region of pembrolizumab (EU numbering). The DBCO functional group is attached to pembrolizumab using the affinity peptide system with AJICAP technology from Ajinomoto Co. Composition A has a drug-to-antibody ratio of 1.

[0067] Composition B is formed from the reaction of the azide functional group of Composition AB with a DBCO functional group attached to residue K248 of the Fc region of pembrolizumab (EU numbering). The DBCO functional group is attached to pembrolizumab using the affinity peptide system from AJICAP technology from Ajinomoto Co. Composition B has a drug-to-antibody ratio of 1.5.

[0068] Composition C is formed from the reaction of the azide functional group of composition AB with a DBCO functional group attached to residue K248 of the Fc region of pembrolizumab (EU numbering). The DBCO functional group is attached to pembrolizumab using the affinity peptide system from AJICAP technology from Ajinomoto Co. Composition C has a drug-to-antibody ratio of 2.

[0069] Composition D is formed from the reaction of the azide functional group of composition AB with a DBCO functional group attached to residue K288 of the Fc region of pembrolizumab (EU numbering). The DBCO functional group is attached to pembrolizumab using the affinity peptide system from AJICAP technology from Ajinomoto Co. Composition D has a drug-to-antibody ratio of 1.

[0070] Composition E is formed from the reaction of the azide functional group of composition AB with a DBCO functional group attached to residue K288 of the Fc region of pembrolizumab (EU numbering). The DBCO functional group is attached to pembrolizumab using the affinity peptide system from AJICAP technology from Ajinomoto Co. Composition E has a drug-to-antibody ratio of 2.

[0071] Composition F is formed from the reaction of the azide functional group of composition AC with a DBCO functional group attached to residue K248 of the Fc region of pembrolizumab (EU numbering). The DBCO functional group is attached to pembrolizumab using the affinity peptide system from AJICAP technology from Ajinomoto Co. Composition F has a drug-to-antibody ratio of 1.

[0072] Composition G is formed from the reaction of the azide functional group of composition AC with a DBCO functional group attached to residue K248 of the Fc region of pembrolizumab (EU numbering). The DBCO functional group is attached to pembrolizumab using the affinity peptide system from AJICAP technology from Ajinomoto Co. Composition G has a drug-to-antibody ratio of 2.

[0073] Composition H is formed from the reaction of the azide functional group of composition AB with a DBCO functional group attached to residue K248 of the Fc region of LZM-009 (EU numbering). The DBCO functional group is added to LZM-009 using the affinity peptide system from AJICAP technology from Ajinomoto Co. Composition H has a drug-to-antibody ratio of 1.

[0074] Composition I is formed from the reaction of the azide functional group of Composition AB with a DBCO functional group attached to residue K248 of the Fc region of LZM-009 (EU numbering). The DBCO functional group is added to LZM-009 using the affinity peptide system from AJICAP technology from Ajinomoto Co. Composition I has a drug-to-antibody ratio of 2.

[0075] Composition J is formed from the reaction of the azide functional group of composition AB with a DBCO functional group attached to residue K288 of the Fc region of LZM-009 (EU numbering). The DBCO functional group is added to LZM-009 using the affinity peptide system from AJICAP technology from Ajinomoto Co. Composition J has a drug-to-antibody ratio of 1.

[0076] Composition K is formed from the reaction of the azide functional group of composition AB with a DBCO functional group attached to residue K288 of the Fc region of LZM-009 (EU numbering). The DBCO functional group is added to LZM-009 using the affinity peptide system from AJICAP technology from Ajinomoto Co. Composition K has a drug-to-antibody ratio of 2.

[0077] Composition N is formed from the reaction of the azide functional group of composition AB with a DBCO functional group attached to residue K248 of the Fc region of trastuzumab (EU numbering). The DBCO functional group is attached to trastuzumab using the affinity peptide system from AJICAP technology from Ajinomoto Co. Composition N has a 1 drug-to-antibody ratio of 1.6.

[0078] Composition O is formed from the reaction of the azide functional group of composition AB with a DBCO functional group attached to residue K248 of the Fc region of trastuzumab (EU numbering). The DBCO functional group is attached to trastuzumab using the affinity peptide system from AJICAP technology from Ajinomoto Co. Composition O has a drug-antibody ratio of 1. An overview of all immunocytokine compositions is given in the table below.

[0079] [Table 2]

[0080] Anti-PD-1 Polypeptides Conjugated to Cytokines Programmed cell death protein 1 (also known as PD-1 and CD279) is a cell surface receptor that plays a role in downregulating the immune system and promoting self-tolerance by suppressing T-cell inflammatory activity. PD-1 is an immune cell inhibitory molecule expressed on activated B cells, T cells, and myeloid cells. PD-1 represents an immune checkpoint and protects against autoimmunity through a dual mechanism of promoting apoptosis (programmed cell death) in antigen-specific T cells in lymph nodes while simultaneously reducing apoptosis in regulatory T cells. PD-1 is a member of the CD28 / CTLA-4 / ICOS costimulatory receptor family that delivers negative signals that affect T-cell and B-cell immunity. PD-1 is a monomer both in solution and on the cell surface, in contrast to CTLA-4 and other family members, which are all disulfide-linked homodimers. Signaling through the PD-1 inhibitory receptor when bound to its ligand, PD-L1, suppresses immune responses to self-antigens and tumors and plays a role in maintaining peripheral immune tolerance. The interaction between PD-1 and PD-L1 results in a reduction in tumor-infiltrating lymphocytes, a reduction in T cell receptor-mediated proliferation, and immune evasion by cancerous cells. A non-limiting exemplary human PD-1 amino acid sequence is MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL (SEQ ID NO: 120).

[0081] Provided herein are polypeptides, e.g., antibodies and anti-PD-1 antigen-binding fragments, that bind to programmed cell death protein 1 (PD-1) conjugated to one or more cytokine molecules or derivatives thereof. The conjugates provided herein deliver cytokines and polypeptides that selectively bind to PD-1 to target cells, e.g., CD8+ T effector (T eff ) cells. This simultaneous delivery of both agents to the same cells has many advantages, including improved IL-2 polypeptide selectivity, increased therapeutic potential of IL-2, and potentially reduced risk of side effects from administration of IL-2 therapy.

[0082] The conjugate compositions provided herein utilize linkers to link a polypeptide that binds PD-1 to a cytokine, such as an IL-2 polypeptide and its derivatives. In some embodiments, the linker is attached to each moiety, i.e., a polypeptide that selectively binds to PD-1 and a cytokine, at a specific residue or a specific subset of residues. In some embodiments, the linker is attached to each moiety site-selectively, such that the population of conjugates is substantially homogenous. This can be accomplished in a variety of ways, as provided herein, including site-selective addition of reagents for the conjugation reaction to the moiety to be conjugated, synthesis or other preparation of the moiety to be conjugated with the desired reagents for the conjugation reaction, or a combination of these two approaches. Using these approaches, the attachment site (e.g., a specific amino acid residue) of the linker for each moiety can be precisely selected. Furthermore, these approaches allow for a variety of linkers to be used in the compositions that are not limited to amino acid residues as required for fusion proteins. This combination of linker selection and precise attachment to the moieties, in some embodiments, allows the linker to also function to modulate the activity of one of the moieties, for example, when the linker is attached to a cytokine at a position that interacts with the cytokine's receptor.

[0083] Anti-PD-1 Polypeptides In some embodiments, an anti-PD-1 polypeptide of the present disclosure specifically binds to PD-1. An anti-PD-1 polypeptide selectively binds or preferentially binds to a target if it binds with higher affinity, avidity, more readily, and / or with a longer duration than it binds to other substances. Thus, "specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding. Generally, but not necessarily, reference to specific binding refers to preferential binding in which the affinity of the antibody or antigen-binding fragment thereof is at least 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 20-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, at least 100-fold greater, or at least 1000-fold greater than the affinity of the antibody for an unrelated amino acid sequence. The anti-PD-1 polypeptides or anti-PD-1 antigen-binding fragments of the disclosure can block the interaction of PD-1 with a ligand (e.g., PD-L1).

[0084] As used herein, the term "antibody" refers to an immunoglobulin (Ig), polypeptide, or protein having a binding domain that is or is homologous to an antigen-binding domain. The term further includes "antigen-binding fragment" and other interchangeable terms for similar binding fragments described below. Native antibodies and native immunoglobulins (Ig) are generally heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains and two identical heavy chains. Each light chain is typically linked to a heavy chain by one covalent disulfide bond, although the number of disulfide bonds varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain ("V") at one end and a variable domain ("V") at the other end. H "), followed by several constant domains ("C H Each light chain has at one end a variable domain ("V") followed by a L ") at the other end and a constant domain ("C L "). The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain and the heavy chain variable domains.

[0085] In some cases, the antibody or antigen-binding fragment comprises an isolated antibody or antigen-binding fragment, a purified antibody or antigen-binding fragment, a recombinant antibody or antigen-binding fragment, a modified antibody or antigen-binding fragment, or a synthetic antibody or antigen-binding fragment.

[0086] The antibody and antigen-binding fragment herein can be partially or entirely synthetically produced.Antibody or antigen-binding fragment can be a polypeptide or protein having a binding domain that can be an antigen-binding domain or can be homologous to an antigen-binding domain.In one example, antibody or antigen-binding fragment can be produced in a suitable in vivo animal model and then isolated and / or purified.

[0087] Depending on the amino acid sequence of the constant domain of its heavy chain, immunoglobulins (Ig) can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Ig or a portion thereof can be human Ig in some cases. In some cases, the CH3 domain can be derived from an immunoglobulin. In some cases, a chain or portion of an antibody or antigen-binding fragment, modified antibody or antigen-binding fragment, or binder can be derived from Ig. In such cases, the Ig can be or is derived from IgG, IgA, IgD, IgE, or IgM. If the Ig is an IgG, it can be a subtype of IgG, which can include IgG1, IgG2a, IgG2b, IgG3, or IgG4. In some cases, the CH3 domain may be derived from or may be derived from an immunoglobulin selected from the group consisting of IgG, IgA, IgD, IgE, and IgM. In some embodiments, the antibodies or antigen-binding fragments described herein comprise or are derived from IgG. In some cases, the antibodies or antigen-binding fragments comprise or are derived from IgG1. In some cases, the antibodies or antigen-binding fragments comprise or are derived from IgG4. In some embodiments, the antibodies or antigen-binding fragments described herein comprise or are derived from IgM or are monomeric forms of IgM. In some embodiments, the antibodies or antigen-binding fragments described herein comprise or are derived from IgE. In some embodiments, the antibodies or antigen-binding fragments described herein comprise or are derived from IgD. In some embodiments, the antibodies or antigen-binding fragments described herein comprise or are derived from IgA.

[0088] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa ("κ" or "K") or lambda ("λ"), based on the amino acid sequences of their constant domains.

[0089] The "variable region" of an antibody refers to the variable region of an antibody light chain or the variable region of an antibody heavy chain, either alone or in combination. The variable regions of the heavy and light chains each consist of four framework regions (FRs) linked by three complementarity determining regions (CDRs), also known as hypervariable regions. The CDRs of each chain are held together in close proximity by the FRs and, together with the CDRs of the other chain, contribute to the formation of the antigen-binding site of the antibody. There are at least two techniques for determining CDRs: (1) interspecies sequence variability (e.g., Kabat et al., Sequences of Proteins of Immunological Interest, (5th Ed., 1991, National Institutes of Health, Bethesda Md. (1991), pages 647-669; hereafter referred to as "Kabat"); (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-Iazikani et al., ((1997) J. Molec. Biol. 273:927-948)). As used herein, CDRs may refer to CDRs defined by either approach or a combination of both approaches.

[0090] With respect to antibodies, the term "variable domain" refers to the variable domains of antibodies that are used in the binding and specificity of each particular antibody for its particular antigen. However, variability is not uniformly distributed throughout the variable domains of antibodies. Rather, it is concentrated in three segments called hypervariable regions (also known as CDRs) of both the light and heavy chain variable domains. The more highly conserved parts of the variable domains are called "framework regions" or "FRs". Unmodified heavy and light chain variable domains each contain four FRs (FR1, FR2, FR3 and FR4) and mainly adopt a beta-sheet configuration with the three CDRs interspersed, which form loops that connect and in some cases form part of the beta-sheet structure. The CDRs of each chain are held together in close proximity by the FRs and, together with the CDRs of the other chain, contribute to the formation of the antigen-binding site of the antibody (see Kabat).

[0091] The terms "hypervariable region" and "CDR" as used herein refer to the amino acid residues of an antibody that are involved in antigen binding. CDRs bind complementary to an antigen and H Chain and V LEach of the chains contains amino acid residues from three sequence regions known as CDR1, CDR2 and CDR3. In the light chain variable domain, the CDRs typically correspond to approximately residues 24-34 (CDRL1), 50-56 (CDRL2) and 89-97 (CDRL3), and in the heavy chain variable domain, the CDRs typically correspond to approximately residues 31-35 (CDRH1), 50-65 (CDRH2) and 95-102 (CDRH3) according to Kabat. It is understood that the amino acid numbering may differ since the CDRs of different antibodies may contain insertions. The Kabat numbering system accounts for such insertions using a numbering scheme that utilizes letters added to specific residues (e.g., 27A, 27B, 27C, 27D, 27E and 27F of CDRL1 in the light chain) to reflect any insertions in the numbering between different antibodies. Alternatively, in a light chain variable domain, the CDRs typically correspond to about residues 26-32 (CDRL1), 50-52 (CDRL2), and 91-96 (CDRL3), and in a heavy chain variable domain, the CDRs typically correspond to about residues 26-32 (CDRH1), 53-55 (CDRH2), and 96-101 (CDRH3) according to Chothia and Lesk (J. Mol. Biol., 196:901-917 (1987)).

[0092] As used herein, "framework region," "FW," or "FR" refers to framework amino acid residues that form part of an antigen binding pocket or groove. In some embodiments, the framework residues form loops that are part of the antigen binding pocket or groove, and the amino acid residues within the loops may or may not contact the antigen. Framework regions generally include the regions between the CDRs. In the light chain variable domain, the FRs typically correspond to about residues 0-23 (FRL1), 35-49 (FRL2), 57-88 (FRL3), and 98-109, and in the heavy chain variable domain, the FRs typically correspond to about residues 0-30 (FRH1), 36-49 (FRH2), 66-94 (FRH3), and 103-133 according to Kabat. As noted above in the Kabat numbering for the light chain, the heavy chain accounts for insertions in a similar manner (e.g., 35A, 35B of CDRH1 in the heavy chain). Alternatively, in light chain variable domains, FRs typically correspond to approximately residues 0-25 (FRL1), 33-49 (FRL2), 53-90 (FRL3), and 97-109 (FRL4), and in heavy chain variable domains, FRs typically correspond to approximately residues 0-25 (FRH1), 33-52 (FRH2), 56-95 (FRH3), and 102-113 (FRH4) according to Chothia and Lesk, Id. The loop amino acids of the FRs can be assessed and determined by examination of the three-dimensional structure of the antibody heavy chain and / or antibody light chain. The three-dimensional structure can be analyzed for amino acid positions that are solvent accessible, as such positions are more likely to form loops and / or provide antigen contacts within the antibody variable domain. Some of the solvent accessible positions can tolerate amino acid sequence diversity, while other positions (e.g., structural positions) generally have less diversity. The three-dimensional structure of the antibody variable domain can be derived from a crystal structure or protein modeling.

[0093] In this disclosure, where appropriate, the following abbreviations (in parentheses) are used according to convention: heavy chain (H chain), light chain (L chain), heavy chain variable region (VH), light chain variable region (VL), complementarity determining region (CDR), first complementarity determining region (CDR1), second complementarity determining region (CDR2), third complementarity determining region (CDR3), heavy chain first complementarity determining region (VH CDR1), heavy chain second complementarity determining region (VH CDR2), heavy chain third complementarity determining region (VH CDR3), light chain first complementarity determining region (VL CDR1), light chain second complementarity determining region (VL CDR2), and light chain third complementarity determining region (VL CDR3).

[0094] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. An "Fc region" may be a native sequence Fc region or a variant Fc region. Although the boundaries of an immunoglobulin heavy chain Fc region may vary, the human IgG heavy chain Fc region is generally defined to extend from an amino acid residue at position Cys226, or from an amino acid residue at Pro230, to the carboxyl terminus thereof. The numbering of residues in the Fc region is that of the EU index, as in Kabat. The Fc region of an immunoglobulin generally comprises two constant domains, CH2 and CH3.

[0095] An "antibody" useful in the present disclosure includes, but is not limited to, a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a bispecific antibody, a multispecific antibody, a heteroconjugated antibody, a humanized antibody, a human antibody, a grafted antibody, a deimmunized antibody, a mutant thereof, a fusion thereof, an immunoconjugate thereof, an antigen-binding fragment thereof, and / or any other modified configuration of an immunoglobulin molecule that contains an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. In certain embodiments of the methods and conjugates provided herein, the antibody requires an Fc region to allow attachment of a linker between the antibody and the protein (e.g., attachment of a linker using an affinity peptide, such as in AJICAP™ technology).

[0096] In some cases, the antibody is a monoclonal antibody. As used herein, "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Typically, in contrast to polyclonal antibody preparations that contain different antibodies against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen (epitope). The modifier "monoclonal" refers to the character of the antibody obtained from a population of substantially homogeneous antibodies and should not be construed as requiring production of the antibody by a particular method.

[0097] In some cases, the antibody is a humanized antibody. As used herein, "humanized" antibody refers to a form of a non-human (e.g., murine) antibody that is a specific chimeric immunoglobulin, immunoglobulin chain, or fragment thereof that contains minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit that has the desired specificity, affinity, and biological activity. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance. Generally, a humanized antibody comprises substantially all of at least one, typically two, variable domains, with all or substantially all of the CDR regions corresponding to those of a non-human immunoglobulin, and all or substantially all of the FR regions being those of a human immunoglobulin consensus sequence. The humanized antibody will also optimally comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically a human immunoglobulin constant region or domain (Fc). The antibody may have an Fc region modified, for example, as described in WO 99 / 58572. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, or six) that have been altered relative to the original antibody, also referred to as one or more CDRs "derived from" one or more CDRs from the original antibody.

[0098] Optionally, the antibodies or antigen-binding fragments described herein can be evaluated for immunogenicity and can be deimmunized (i.e., the antibody is rendered less immunoreactive by altering one or more T cell epitopes). As used herein, a "deimmunized antibody" means that one or more T cell epitopes in the antibody sequence have been modified such that the T cell response following administration of the antibody to a subject is reduced compared to an antibody that has not been deimmunized. Analysis of the immunogenicity and T cell epitopes present in the antibodies and antigen-binding fragments described herein can be performed through the use of software and specific databases. Exemplary software and databases include iTope™, developed by Antitope Ltd., Cambridge, UK. iTope™ is an in silico technology for the analysis of peptide binding to human MHC class II alleles. The iTope™ software predicts peptide binding to human MHC class II alleles, thereby providing an initial screening for the location of such "potential T cell epitopes." The iTope™ software predicts favorable interactions between amino acid side chains of peptides and specific binding pockets within the binding grooves of the 34 human MHC class II alleles. The locations of key binding residues are obtained by in silico generation of 9-mer peptides that overlap by one amino acid spanning the test antibody variable region sequence. Each 9-mer peptide can be tested against each of the 34 MHC class II alleles and scored based on their potential "fit" and interaction with the MHC class II binding groove. Peptides that yield high average binding scores (>0.55 on the iTope™ scoring function) for >50% of the MHC class II alleles are considered potential T-cell epitopes. In such regions, the core 9 amino acid sequence for peptide binding within the MHC class II groove is analyzed to determine the MHC class II pocket residues (P1, P4, P6, P7, P9) and possible T-cell receptor (TCR) contact residues (P1, P2, P3, P5, P8).After identifying any T cell epitope, amino acid residue changes, substitutions, additions, and / or deletions can be introduced to eliminate the identified T cell epitope. Such changes can be made to preserve the structure and function of the antibody while still eliminating the identified epitope. Exemplary changes can include, but are not limited to, conservative amino acid changes.

[0099] The antibody may be a human antibody. As used herein, "human antibody" refers to an antibody having an amino acid sequence that corresponds to that of an antibody produced by a human, and / or an antibody made using any suitable technique for making a human antibody. This definition of a human antibody includes an antibody that comprises at least one human heavy chain polypeptide or at least one human light chain polypeptide. One such example is an antibody that comprises a mouse light chain and a human heavy chain polypeptide. In one embodiment, the human antibody is selected from a phage library, which expresses a human antibody. Human antibodies can also be made by introducing human immunoglobulin loci into a transgenic animal, such as a mouse in which the endogenous immunoglobulin genes have been partially or completely inactivated. Alternatively, human antibodies can be prepared by immortalizing human B lymphocytes that produce antibodies against a target antigen (such B lymphocytes can be collected from an individual or immunized in vitro).

[0100] Any of the antibodies herein may be bispecific. A bispecific antibody is an antibody that has binding specificities for at least two different antigens and can be prepared using the antibodies disclosed herein. Traditionally, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, where the two heavy chains have different specificities. A bispecific antibody may be composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm and a hybrid immunoglobulin heavy-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure with an immunoglobulin light chain in only half of the bispecific molecule facilitates the separation of the desired bispecific compound from undesired immunoglobulin chain combinations.

[0101] According to one approach to making bispecific antibodies, antibody variable domains (antibody-antigen binding sites) with the desired binding specificity are fused to immunoglobulin constant domain sequences. The fusion can be with an immunoglobulin heavy chain constant domain, including at least a portion of the hinge, CH2 and CH3 regions. The first heavy chain constant region (CH1), containing the site necessary for light chain binding, can be present in at least one of the fusions. DNA encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors and co-transfected into a suitable host organism. This provides great flexibility in adjusting the mutual ratios of the three polypeptide fragments, in embodiments where optimal yields are obtained when the ratios of the three polypeptide chains used in the construction are not equal. However, it is possible to insert the coding sequences for two or all three polypeptide chains into one expression vector, when expression of at least two polypeptide chains in equal ratios results in high yields, or when the ratio is not particularly important.

[0102] In some cases, the antibody herein is a chimeric antibody. "Chimeric" forms of non-human (e.g., mouse) antibodies include chimeric antibodies that contain minimal sequences derived from non-human Ig. In most cases, chimeric antibodies are mouse antibodies in which at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin, is inserted in place of the mouse Fc. Chimeric or hybrid antibodies can also be prepared in vitro using suitable methods of synthetic protein chemistry involving cross-linking agents. For example, immunotoxins can be constructed using disulfide exchange reactions or by forming thioether bonds. Examples of reagents suitable for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.

[0103] Provided herein are antibodies and antigen-binding fragments thereof, modified antibodies and antigen-binding fragments thereof, and binding agents that specifically bind to one or more epitopes on one or more target antigens. In one example, the binding agent selectively binds to an epitope on a single antigen. In another example, the binding agent is bivalent and selectively binds to two different epitopes on a single antigen, or binds to two different epitopes on two different antigens. In another example, the binding agent is multivalent (i.e., trivalent, tetravalent, etc.), and the binding agent binds to three or more different epitopes on a single antigen, or binds to three or more different epitopes on two or more (multiple) antigens.

[0104] Any antigen-binding fragment of the antibodies herein is also contemplated. The terms "antigen-binding portion of an antibody," "antigen-binding fragment," "antigen-binding domain," "antibody fragment," or "functional fragment of an antibody" are used interchangeably herein to refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Exemplary antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, bispecific F(ab')2, trispecific F(ab')2, variable fragment (Fv), single chain variable fragment (scFv), dsFv, bispecific scFv, variable heavy domain, variable light domain, variable NAR domain, bispecific scFv, AVIMER®, minibodies, diabodies, bispecific diabodies, triabodies, tetrabodies, minibodies, maxibodies, camelids, VHH, minibodies, intrabodies, fusion proteins comprising an antibody portion (e.g., a domain antibody), single chain binding polypeptides, scFv-Fc, Fab-Fc, bispecific T cell engagers (BiTEs; two scFvs produced as a single polypeptide chain, each scFv comprising a CDR combination or a VL / VL combination as described herein. Examples of suitable antibodies include tetravalent tandem diabodies (TandAbs; antibody fragments produced as non-covalent homodimeric folds in a head-to-tail arrangement, e.g., TandAbs comprising scFvs, where the scFvs comprise amino acid sequences comprising a CDR combination or a VL / VL combination as described herein), dual affinity retargeting antibodies (DARTs; different scFvs connected by a stabilized interchain disulfide bond), bispecific antibodies (bscAbs; two single chain Fv fragments linked via a glycine-serine linker), single domain antibodies (sdAbs), fusion proteins, or bispecific disulfide stabilized Fv antibody fragments (dsFv-dsFv'; two different disulfide stabilized Fv antibody fragments linked by a flexible linker peptide). In certain embodiments of the invention, full-length antibodies (e.g., antigen binding fragment and Fc region) are preferred.

[0105] Heteroconjugate polypeptides comprising two covalently linked antibodies or antibody antigen-binding fragments are also within the scope of the present disclosure. Appropriate linkers may be used to multimerize the binding agents. Non-limiting examples of linking peptides include, but are not limited to, (GS) n (SEQ ID NO: 24), (GGS) n (SEQ ID NO: 25), (GGGS) n (SEQ ID NO:26), (GGSG) n (SEQ ID NO: 27), or (GGSGG) n (SEQ ID NO:28), (GGGGS) n (SEQ ID NO:29), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. For example, the linking peptide can be (GGGGS)3 (SEQ ID NO:30) or (GGGGS)4 (SEQ ID NO:31). In some embodiments, the linking peptide bridges about 3.5 nm between the carboxy terminus of one variable region and the amino terminus of the other variable region. Linkers of other sequences have been designed and used. The linker can then be modified for additional functions, such as drug attachment or attachment to a solid support.

[0106] As used herein, the term "avidity" refers to the resistance of a complex of two or more agents to dissociation after dilution. Apparent affinity can be determined by methods such as enzyme-linked immunosorbent assay (ELISA) or any other suitable technique. Avidity can be determined by methods such as Scatchard analysis or any other suitable technique.

[0107] As used herein, the term "affinity" refers to the equilibrium constant for the reversible binding of two agents, K D The binding affinity (K D) is 500nM, 475nM, 450nM, 425nM, 400nM, 375nM, 350nM, 325nM, 300nM, 275nM, 250nM, 225nM, 200nM, 175nM, 150nM, 125nM, 100nM, 90nM, 80nM, 70nM, 50nM, 50nM, 49nM, 48nM, 47nM, 46nM, 45nM, 44nM, 43nM, 42nM, 41nM, 40nM, 39nM, 38nM, 37nM, 36nM, 35nM, 34nM, 33nM, 32nM, 3 1nM, 30nM, 29nM, 28nM, 27nM, 26nM, 25nM, 24nM, 23nM, 22nM, 21nM, 20nM, 19nM, 18nM, 17nM, 16nM, 15nM, 14nM, 13nM, 12nM, 11nM, 10nM, 9n M, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, 1nM, 990pM, 980pM, 970pM, 960pM, 950pM, 940pM, 930pM, 920pM, 910pM, 900pM, 890pM, 880pM, 870pM , 860pM, 850pM, 840pM, 830pM, 820pM, 810pM, 800pM, 790pM, 780pM, 770pM, 760pM, 750pM, 740pM, 730pM, 720pM, 710pM, 700pM, 690pM, 6 80pM, 670pM, 660pM, 650pM, 640pM, 630pM, 620pM, 610pM, 600pM, 590pM, 580pM, 570pM, 560pM, 550pM, 540pM, 530pM, 520pM, 510pM, 500p M, 490pM, 480pM, 470pM, 460pM, 450pM, 440pM, 430pM, 420pM, 410pM, 400pM, 390pM, 380pM, 370pM, 360pM, 350pM, 340pM, 330pM, 320pM, 310pM, 300pM, 290pM, 280pM, 270pM, 260pM, 250pM, 240pM, 230pM, 220pM, 210pM, 200pM, 190pM, 180pM, 170pM, or any integer therebetween.Binding affinity can be determined using surface plasmon resonance (SPR), KINEXA® biosensor, scintillation proximity assay, enzyme-linked immunosorbent assay (ELISA), ORIGEN immunoassay (IGEN), fluorescence quenching, fluorescence transfer, yeast display, or any combination thereof. Binding affinity can also be screened using a suitable bioassay.

[0108] As used herein, the term "avidity" refers to the resistance of a complex of two or more agents to dissociation after dilution. Apparent affinity can be determined by methods such as enzyme-linked immunosorbent assay (ELISA) or any other technique familiar to those skilled in the art. Avidity can be determined by methods such as Scatchard analysis or any other technique familiar to those skilled in the art.

[0109] Affinity matured antibodies are also provided herein. The following methods can be used to adjust the affinity of an antibody and to characterize the CDRs. One method to characterize the CDRs of an antibody and / or to change (e.g., improve) the binding affinity of a polypeptide such as an antibody is called "library scanning mutagenesis". In general, library scanning mutagenesis works as follows: One or more amino acid positions in the CDRs are replaced with two or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) amino acids. This generates a small library of clones (in some embodiments, one for each amino acid position analyzed), each with a complexity of two or more members (when two or more amino acids are replaced at every position). In general, the library also includes clones that contain the native (unsubstituted) amino acid. A small number of clones from each library, for example about 20-80 clones (depending on the complexity of the library), can be screened for binding specificity or binding affinity to the target polypeptide (or other binding target) and candidates with increased, the same, decreased, or no binding are identified. Binding affinity can be determined using Biacore surface plasmon resonance analysis, which detects differences in binding affinity of about 2-fold or greater.

[0110] In some cases, the antibody or antigen-binding fragment is bispecific or multispecific and can specifically bind to two or more antigens. In some cases, such bispecific or multispecific antibodies or antigen-binding fragments can specifically bind to two or more different antigens. In some cases, the bispecific antibody or antigen-binding fragment can be a bivalent antibody or antigen-binding fragment. In some cases, the multispecific antibody or antigen-binding fragment can be a bivalent antibody or antigen-binding fragment, a trivalent antibody or antigen-binding fragment, or a tetravalent antibody or antigen-binding fragment.

[0111] The antibodies or antigen-binding fragments described herein can be isolated, purified, recombinant, or synthetic.

[0112] The antibodies described herein can be made by any suitable method. Antibodies can often be produced in large quantities, especially when high-level expression vectors are utilized.

[0113] In one embodiment, an anti-PD1 antibody or antigen-binding fragment of the disclosure comprises a combination of a heavy chain variable region (VH) and a light chain variable region (VL) described herein. In another embodiment, an anti-PD1 antibody or antigen-binding fragment of the disclosure comprises a combination of complementarity determining regions (VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3) described herein. In one embodiment, the anti-PD-1 antibody or anti-PD-1 antigen-binding fragment of the disclosure is a modified tislelizumab, Baizean, 0KVO411B3N, BGB-A317, hu317-1 / IgG4mt2, sintilimab, Tyvyt, IBI-308, toripalimab, TeRuiPuLi, Terepril, Tuoyi, JS-001, TAB-001, camrelizumab, HR-301210, INCSHR-01210, SHR-1210 , cemiplimab, cemiplimab-rwlc, LIBTAYO®, 6QVL057INT, H4H7798N, REGN-2810, SAR-439684, lambrolizumab, pembrolizumab, KEYTRUDA®, MK-3475, SCH-900475, h409A11, nivolumab, nivolumab BMS, OPDIVO®, BMS-936558, MDX-1106, ONO-4538, prorugolimab (P rolgolimab), Forteca, BCD-100, Penprimab, AK-105, Zimberelimab, AB-122, GLS-010, WBP-3055, Balstilimab, 1Q2QT5M7EO, AGEN-2034, AGEN-2034w, Genolimuzumab, Geptanolimab, APL-501, CBT-501, GB-226, Dostallimab, ANB-011, GSK-4057190A, P0GVQ 9A4S5, TSR-042, WBP-285, selpullimab, HLX-10, CS-1003, retifanlimab, 2Y3T5IF01Z, INCMGA-00012, INCMGA-0012, MGA-012, sasanlimab, LZZ0IC2EWP, PF-06801591, RN-888, spartalizumab, NVP-LZV-184, PDR-001, QOG25L6Z8Z, leratolimab / nivolumab,BMS-986213, cetrelimab, JNJ-3283, JNJ-63723283, LYK98WP91F, tevotelimab, MGD-013, BCD-217, BAT-1306, HX-008, MEDI-5752, JTX-4014, kadonilimab, AK-104, BI-754091, pidilizumab, CT-011, MDV-930 0, YBL-006, AMG-256, RG-6279, RO-7284755, BH-2950, ​​IBI-315, RG-6139, RO-7247669, ONO-4 685, AK-112, 609-A, LY-3434172, T-3011, MAX-10181, AMG-404, IBI-318, MGD-019, INCB-086 550, ONCR-177, LY-3462817, RG-7769, RO-7121661, F-520, XmAb-23104, Pd-1-pik, SG-001, S -95016, Sym-021, LZM-009, Budigalimab, 6VDO4TY3OO, ABBV-181, PR-1648817, CC- 90006, XmAb-20717, 2661380, AMP-224, B7-DCIg, EMB-02, ANB-030, PRS-332, [89Zr]deferoxamide-pembrolizumab, 89Zr-Df-pembrolizumab, [89Zr]Df-pembrolizumab, STI-1110, STI-A1110, CX-188, mPD-1 Pb-Tx, MCLA-134, 244C8, ENUM 224C8, ENUM C8, 388D4, ENUM 388D4, ENUM D4, MEDI0680, or AMP-514.

[0114] In one embodiment, the anti-PD-1 antibody or anti-PD-1 antigen-binding fragment of the disclosure comprises a modified Tislelizumab, Sintilimab, Toripalimab, Terepril, Camrelizumab, Cemiplimab, Pembrolizumab, Nivolumab, Prorugolimab, Penprimab, Zimverelimab, Balstilimab, Genolimuzumab, Geptanolimab, Dostallimab, Serpulimumab, Retifanlimab, Sasanlimab, Spartalizumab, Cetrelimab, Tebotelimab, Kadonilimab, A Pidilizumab, LZM-009, or Budigalimab.

[0115] In some embodiments, the anti-PD-1 polypeptide is nivolumab, pembrolizumab, LZM-009, dostallimab, sintilimab, spartalizumab, tislelizumab, or cemiplimab. In some embodiments, the anti-PD-1 polypeptide is dostallimab, sintilimab, spartalizumab, or tislelizumab. In some embodiments, the anti-PD-1 polypeptide is nivolumab, pembrolizumab, LZM-009, or cemiplimab.

[0116] In some embodiments, the anti-PD-1 polypeptide is modified pembrolizumab. In some embodiments, the anti-PD-1 polypeptide is modified with mAB3. In some embodiments, the anti-PD-1 polypeptide is modified with mAB4.

[0117] Generic or biosimilar versions of the antibodies named herein that share the same amino acid sequence as the indicated antibody are also intended to be encompassed when the antibody name is used. In some embodiments, the anti-PD-1 antibody is a biosimilar of Tislelizumab, Sintilimab, Toripalimab, Terepril, Camrelizumab, Cemiplimab, Pembrolizumab, Nivolumab, Prorugolimab, Penprimab, Zimverelimab, Balstilimab, Genolimuzumab, Geptanolimab, Dostallimab, Serpulimumab, Retifanlimab, Sasanlimab, Spartalizumab, Cetrelimab, Tebotelimab, Kadonilimab, A Pidilizumab, LZM-009, or Budigalimab. In some embodiments, the anti-PD-1 antibody is a biosimilar of any one of the antibodies provided herein.

[0118] Table 1 provides sequences of exemplary anti-PD-1 polypeptides (e.g., anti-PD-1 antibodies) and anti-PD-1 antigen-binding fragments that can be modified to prepare anti-PD-1 immunoconjugates. Table 1 also provides combinations of CDRs that can be utilized in modified anti-PD-1 immunoconjugates. References herein to anti-PD-1 polypeptides may alternatively refer to anti-PD-1 antigen-binding fragments.

[0119] [Table 3-1]

[0120] [Table 3-2]

[0121] [Table 3-3]

[0122] [Table 3-4]

[0123] [Table 3-5]

[0124] [Table 3-6]

[0125] [Table 3-7]

[0126] [Table 3-8]

[0127] [Table 3-9]

[0128] [Table 3-10]

[0129] [Table 3-11]

[0130] [Table 3-12]

[0131] An anti-PD-1 polypeptide or antigen-binding fragment may comprise a VH having the amino acid sequence of any one of SEQ ID NOs: 32, 34, 36, 38, 40, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, and 78. An anti-PD-1 polypeptide or antigen-binding fragment may comprise a VH having the amino acid sequence of any one of SEQ ID NOs: 33, 35, 37, 39, 41, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, and 79.

[0132] In one example, an anti-PD-1 polypeptide or antigen-binding fragment comprises a VH having the amino acid sequence of SEQ ID NO:32 and a VL having the amino acid sequence of SEQ ID NO:33. In another example, an anti-PD-1 polypeptide or antigen-binding fragment comprises a VH having the amino acid sequence of SEQ ID NO:34 and a VL having the amino acid sequence of SEQ ID NO:35. In another example, an anti-PD-1 polypeptide or antigen-binding fragment comprises a VH having the amino acid sequence of SEQ ID NO:36 and a VL having the amino acid sequence of SEQ ID NO:37. In another example, an anti-PD-1 polypeptide or antigen-binding fragment comprises a VH having the amino acid sequence of SEQ ID NO:38 and a VL having the amino acid sequence of SEQ ID NO:39. In another example, an anti-PD-1 polypeptide or antigen-binding fragment comprises a VH having the amino acid sequence of SEQ ID NO:40 and a VL having the amino acid sequence of SEQ ID NO:41. In another example, an anti-PD-1 polypeptide or antigen-binding fragment comprises a VH having the amino acid sequence of SEQ ID NO:46 and a VL having the amino acid sequence of SEQ ID NO:47. In another example, the anti-PD-1 polypeptide or antigen-binding fragment comprises a VH having the amino acid sequence of SEQ ID NO:48 and a VL having the amino acid sequence of SEQ ID NO:49. In another example, the anti-PD-1 polypeptide or antigen-binding fragment comprises a VH having the amino acid sequence of SEQ ID NO:50 and a VL having the amino acid sequence of SEQ ID NO:51. In another example, the anti-PD-1 polypeptide or antigen-binding fragment comprises a VH having the amino acid sequence of SEQ ID NO:52 and a VL having the amino acid sequence of SEQ ID NO:53. In another example, the anti-PD-1 polypeptide or antigen-binding fragment comprises a VH having the amino acid sequence of SEQ ID NO:54 and a VL having the amino acid sequence of SEQ ID NO:55. In another example, the anti-PD-1 polypeptide or antigen-binding fragment comprises a VH having the amino acid sequence of SEQ ID NO:56 and a VL having the amino acid sequence of SEQ ID NO:57. In another example, the anti-PD-1 polypeptide or antigen-binding fragment comprises a VH having the amino acid sequence of SEQ ID NO:58 and a VL having the amino acid sequence of SEQ ID NO:59.In another example, the anti-PD-1 polypeptide or antigen-binding fragment comprises a VH having the amino acid sequence of SEQ ID NO:60 and a VL having the amino acid sequence of SEQ ID NO:61. In another example, the anti-PD-1 polypeptide or antigen-binding fragment comprises a VH having the amino acid sequence of SEQ ID NO:62 and a VL having the amino acid sequence of SEQ ID NO:63. In another example, the anti-PD-1 polypeptide or antigen-binding fragment comprises a VH having the amino acid sequence of SEQ ID NO:64 and a VL having the amino acid sequence of SEQ ID NO:65. In another example, the anti-PD-1 polypeptide or antigen-binding fragment comprises a VH having the amino acid sequence of SEQ ID NO:66 and a VL having the amino acid sequence of SEQ ID NO:67. In another example, the anti-PD-1 polypeptide or antigen-binding fragment comprises a VH having the amino acid sequence of SEQ ID NO:68 and a VL having the amino acid sequence of SEQ ID NO:69. In another example, the anti-PD-1 polypeptide or antigen-binding fragment comprises a VH having the amino acid sequence of SEQ ID NO:70 and a VL having the amino acid sequence of SEQ ID NO:71. In another example, the anti-PD-1 polypeptide or antigen-binding fragment comprises a VH having the amino acid sequence of SEQ ID NO: 72 and a VL having the amino acid sequence of SEQ ID NO: 73. In another example, the anti-PD-1 polypeptide or antigen-binding fragment comprises a VH having the amino acid sequence of SEQ ID NO: 74 and a VL having the amino acid sequence of SEQ ID NO: 75. In another example, the anti-PD-1 polypeptide or antigen-binding fragment comprises a VH having the amino acid sequence of SEQ ID NO: 76 and a VL having the amino acid sequence of SEQ ID NO: 77. In another example, the anti-PD-1 polypeptide or antigen-binding fragment comprises a VH having the amino acid sequence of SEQ ID NO: 78 and a VL having the amino acid sequence of SEQ ID NO: 79.

[0133] In one example, an anti-PD-1 polypeptide or antigen-binding fragment comprises a VH CHR1 having the amino acid sequence of SEQ ID NO: 80, a VH CHR2 having the amino acid sequence of SEQ ID NO: 81, a VH CHR3 having the amino acid sequence of SEQ ID NO: 82, a VL CHR1 having the amino acid sequence of SEQ ID NO: 83, a VL CHR2 having the amino acid sequence of SEQ ID NO: 84, and a VL CHR3 having the amino acid sequence of SEQ ID NO: 85. In one example, an anti-PD-1 polypeptide or antigen-binding fragment comprises a VH CHR1 having the amino acid sequence of SEQ ID NO: 86, a VH CHR2 having the amino acid sequence of SEQ ID NO: 87, a VH CHR3 having the amino acid sequence of SEQ ID NO: 88, a VL CHR1 having the amino acid sequence of SEQ ID NO: 89, a VL CHR2 having the amino acid sequence of SEQ ID NO: 90, and a VL CHR3 having the amino acid sequence of SEQ ID NO: 91. In one example, an anti-PD-1 polypeptide or anti-PD-1 antigen-binding fragment comprises a VH CHR1 having the amino acid sequence of SEQ ID NO: 92, a VH CHR2 having the amino acid sequence of SEQ ID NO: 93, a VH CHR3 having the amino acid sequence of SEQ ID NO: 94, a VL CHR1 having the amino acid sequence of SEQ ID NO: 95, a VL CHR2 having the amino acid sequence of SEQ ID NO: 96, and a VL CHR3 having the amino acid sequence of SEQ ID NO: 97. In one example, an anti-PD-1 polypeptide or anti-PD-1 antigen-binding fragment comprises a VH CHR1 having the amino acid sequence of SEQ ID NO: 98, a VH CHR2 having the amino acid sequence of SEQ ID NO: 99, a VH CHR3 having the amino acid sequence of SEQ ID NO: 100, a VL CHR1 having the amino acid sequence of SEQ ID NO: 101, a VL CHR2 having the amino acid sequence of SEQ ID NO: 102, and a VL CHR3 having the amino acid sequence of SEQ ID NO: 103.

[0134] In one example, the anti-PD-1 polypeptide comprises a fusion protein. Such a fusion protein can be, for example, a two-sided Fc fusion protein comprising the extracellular domain (ECD) of programmed cell death 1 (PD-1) expressed in CHO-K1 cells and the ECD of tumor necrosis factor (ligand) superfamily member 4 (TNFSF4 or OX40L) fused via the hinge-CH2-CH3 Fc domain of human IgG4, where the fusion protein has the exemplary amino acid sequence of SEQ ID NO: 104.

[0135] Anti-Her2 antibodies are also provided herein. Anti-Her2 antibodies can be conjugated to IL-2 polypeptides provided herein. In some embodiments, the anti-Her2 antibody is trastuzumab (Herceptin, Roche Herclon, RG597, RO452317). Trastuzumab is EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP The VH sequence consists of CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (sequence number 121).

[0136] The VL sequence of trastuzumab is DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (sequence number 122).

[0137] Modifications to the Fc region Disclosed herein are anti-PD-1 polypeptides, wherein the anti-PD-1 polypeptide comprises an Fc region, and the Fc region comprises at least one covalently attached chemical linker. In some embodiments, the chemical linker is covalently attached to an asparagine, glutamine, cysteine, or lysine residue. In some embodiments, the chemical linker is covalently attached to a lysine or cysteine ​​residue. In some embodiments, the chemical linker is covalently attached to a lysine residue. In some embodiments, the chemical linker is covalently attached to a constant region of the anti-PD-1 polypeptide. In some embodiments, the chemical linker is covalently attached to a constant region of the anti-PD-1 polypeptide.

[0138] In some embodiments, the anti-PD-1 polypeptide comprises an Fc region. In some embodiments, the Fc region is an IgG Fc region, an IgA Fc region, an IgD Fc region, an IgM Fc region, or an IgE Fc region. In some embodiments, the Fc region is an IgG Fc region, an IgA Fc region, or an IgD Fc region. In some embodiments, the Fc region is a human Fc region. In some embodiments, the Fc region is humanized. Fc region. In some embodiments, the Fc region is an IgG Fc region. In some embodiments, the IgG Fc region is an IgG1 Fc region, an IgG2a Fc region, or an IgG4 Fc region. In some embodiments, the IgG Fc region is an IgG1 Fc region, an IgG2a Fc region, or an IgG4 Fc region.

[0139] One or more mutations may be introduced into the Fc region to reduce the Fc-mediated effector functions of the antibody or antigen-binding fragment, such as antibody-dependent cellular cytotoxicity (ADCC) and / or complement function. In some cases, the modified Fc comprises a humanized IgG4 kappa isotype containing an S228P Fc mutation. In some cases, the modified Fc comprises a human IgG1 in which the heavy chain CH2 domain has been engineered with triple mutations, such as (a) L238P, L239E, and P335S; or (2) K248; K288, and K317.

[0140] In some embodiments, Fc region sequence is SEQ ID NO:105(Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro1 Glu Xaa Xaa Gly Xaa Pro Ser Val Phe Leu Phe Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asp Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Xaa Glu Xaa Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Xaa Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser ProGly, where Xaa can be any naturally occurring amino acid).

[0141] In some embodiments, the Fc region comprises one or more mutations that render the Fc region amenable to modification or conjugation with specific residues, such as by incorporating a cysteine ​​residue at a position in SEQ ID NO: 105 that does not contain a cysteine. Alternatively, the Fc region may be modified to incorporate a modified natural or non-natural amino acid that comprises a binding handle, such as one linked to a modified natural or non-natural amino acid via a linker. In some embodiments, the Fc region does not comprise any mutations that facilitate attachment of the linker to an additional cytokine (e.g., an IL-2, IL-7, or IL-18 polypeptide). In some embodiments, the chemical linker is attached to the native residue shown in SEQ ID NO: 105. In some embodiments, the chemical linker is attached to the native lysine residue of SEQ ID NO: 105.

[0142] In some embodiments, the chemical linker may be covalently attached to an amino acid residue in the Fc region of the anti-PD-1 polypeptide. In some embodiments, the chemical linker is covalently attached to a non-terminal residue in the Fc region. In some embodiments, the non-terminal residue is in the CH1, CH2, or CH3 region of the anti-PD-1 polypeptide. In some embodiments, the non-terminal residue is in the CH2 region of the anti-PD-1 polypeptide.

[0143] In some embodiments, the chemical linker is attached to the Fc region at any one of amino acid residues at positions 10 to 90 of SEQ ID NO: 105. In some embodiments, the chemical linker is attached to the Fc region at any one of amino acid residues at positions 10 to 20, 10 to 30, 10 to 40, 10 to 50, 10 to 60, 10 to 70, 1 to 80, 10 to 90, 10 to 100, 10 to 110, 10 to 120, 10 to 130, 10 to 140, 10 to 150, 10 to 160, 10 to 170, 10 to 180, 10 to 190, or 10 to 200 of SEQ ID NO: 105. In some embodiments, the chemical linker is attached to the Fc region at any one of amino acid residues at positions 10 to 30, 50 to 70, or 80 to 100 of SEQ ID NO: 105. In some embodiments, the chemical linker is attached to the Fc region at any one of amino acid residues 20 to 40, 65 to 85, or 90 to 110 of SEQ ID NO: 105. In some embodiments, the chemical linker is attached to the Fc region at any one of amino acid residues 15 to 26, 55 to 65, or 85 to 90 of SEQ ID NO: 105. In some embodiments, the chemical linker is attached to the Fc region at any one of amino acid residues 25 to 35, 70 to 80, or 95 to 105 of SEQ ID NO: 105. In some embodiments, the chemical linker is attached to the Fc region at any one of amino acid residues 30, 32, 72, 74, 79, or 101 of SEQ ID NO: 105. In some embodiments, the chemical linker is attached to the Fc region at any one of amino acid residues K30, K32, K72, K74, Q79, or K101 of SEQ ID NO: 105. In some embodiments, the chemical linker is attached to the Fc region at amino acid residue 30 of SEQ ID NO:105. In some embodiments, the chemical linker is attached to the Fc region at amino acid residue 32 of SEQ ID NO:105. In some embodiments, the chemical linker is attached to the Fc region at amino acid residue 72 of SEQ ID NO:105. In some embodiments, the chemical linker is attached to the Fc region at amino acid residue 74 of SEQ ID NO:105. In some embodiments, the chemical linker is attached to the Fc region at amino acid residue 79 of SEQ ID NO:105.In some embodiments, the chemical linker is attached to the Fc region at amino acid residue 101 of SEQ ID NO:105.

[0144] In some embodiments, the chemical linker is covalently attached to an amino acid residue of the polypeptide that selectively binds to a cancer or inflammation-associated antigen (e.g., an anti-PD-1 antibody) such that the function of the polypeptide is maintained (e.g., without denaturing the polypeptide). For example, when the polypeptide is an antibody such as human IgG (e.g., human IgG1), exposed lysine residues, exposed glutamine residues, and exposed tyrosine residues are present at the following positions (see website imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html according to EU numbering). Exemplary exposed lysine residues: CH2 domain (positions 246, 248, 274, 288, 290, 317, 320, 322, and position 338) CH3 domain (positions 360, 414, and 439). Exemplary exposed glutamine residue: CH2 domain (position 295). Exemplary exposed tyrosine residues: CH2 domain (positions 278, 296, and 300) CH3 domain (position 436).

[0145] Human IgG, such as human IgG1, may also be modified with lysine, glutamine or tyrosine residues at any one of the positions listed above to provide ideally surface-exposed residues for subsequent modification.

[0146] In some embodiments, the chemical linker is covalently attached to an amino acid residue in the constant region of the anti-PD-1 antibody. In some embodiments, the chemical linker is covalently attached to an amino acid residue in the CH1, CH2, or CH3 region. In some embodiments, the chemical linker is covalently attached to an amino acid residue in the CH2 region. In some embodiments, the chemical linker may be covalently attached to one residue selected from the following groups of residues according to EU numbering in human IgG Fc: amino acid residues 1-478, amino acid residues 2-478, amino acid residues 1-477, amino acid residues 2-477, amino acid residues 10-467, amino acid residues 30-447, amino acid residues 50-427, amino acid residues 100-377, amino acid residues 150-327, amino acid residues 200-327, amino acid residues 240-327, and amino acid residues 240-320.

[0147] In some embodiments, the chemical linker is covalently attached to a lysine or glutamine residue of a human IgG Fc region. In some embodiments, the chemical linker is covalently attached to Lys246 of the Fc region of the anti-PD-1 polypeptide, where the amino acid residue position numbering is based on Eu numbering. In some embodiments, the chemical linker is covalently attached to Lys248 of the Fc region of the anti-PD-1 polypeptide, where the amino acid residue position numbering is based on Eu numbering. In some embodiments, the chemical linker is covalently attached to Lys288 of the Fc region of the anti-PD-1 polypeptide, where the amino acid residue position numbering is based on Eu numbering. In some embodiments, the chemical linker is covalently attached to Lys290 of the Fc region of the anti-PD-1 polypeptide, where the amino acid residue position numbering is based on Eu numbering. In some embodiments, the chemical linker is covalently attached to Gln295 of the Fc region of the antibody polypeptide, where the amino acid residue position numbering is based on Eu numbering. In some embodiments, the chemical linker is covalently attached to Lys317 of the anti-PD-1 polypeptide, where the amino acid residue position numbers are based on Eu numbering.

[0148] In some embodiments, the chemical linker may be covalently attached to an amino acid residue selected from a subset of amino acid residues. In some embodiments, the subset includes 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues of the Fc region of the anti-PD-1 polypeptide. In some embodiments, the chemical linker may be covalently attached to one of two lysine residues of the Fc region of the anti-PD-1 polypeptide.

[0149] In some embodiments, the anti-PD-1 polypeptide will comprise two linkers covalently attached to the Fc region of the anti-PD-1 polypeptide. In some embodiments, each of the two linkers is covalently attached to a different heavy chain of the anti-PD-1 polypeptide. In some embodiments, each of the two linkers is covalently attached to a different heavy chain of the anti-PD-1 polypeptide at the same residue position. In some embodiments, each of the two linkers is covalently attached to a different heavy chain of the anti-PD-1 polypeptide at a different residue position. When the two linkers are covalently attached to different residue positions, any combination of residue positions provided herein can be used in combination.

[0150] In some embodiments, the first chemical linker is covalently attached to Lys248 of the first Fc region of the anti-PD-1 polypeptide and the second chemical linker is covalently attached to Lys288 of the second Fc region of the anti-PD-1 polypeptide, where the residue position numbers are based on Eu numbering. In some embodiments, the first chemical linker is covalently attached to Lys246 of the first Fc region of the anti-PD-1 polypeptide and the second chemical linker is covalently attached to Lys288 of the second Fc region of the anti-PD-1 polypeptide, where the residue position numbers are based on Eu numbering. In some embodiments, the first chemical linker is covalently attached to Lys248 of the first Fc region of the anti-PD-1 polypeptide and the second chemical linker is covalently attached to Lys317 of the second Fc region of the anti-PD-1 polypeptide, where the residue position numbers are based on Eu numbering. In some embodiments, the first chemical linker is covalently attached to Lys246 of the first Fc region of the anti-PD-1 polypeptide and the second chemical linker is covalently attached to Lys317 of the second Fc region of the anti-PD-1 polypeptide, where the residue position numbers are based on Eu numbering. In some embodiments, the first chemical linker is covalently attached to Lys288 of the first Fc region of the anti-PD-1 polypeptide and the second chemical linker is covalently attached to Lys317 of the second Fc region of the anti-PD-1 polypeptide, where the residue position numbers are based on Eu numbering.

[0151] Methods for modifying the Fc region Also provided herein are methods for preparing modified Fc regions of polypeptides that selectively bind programmed cell death protein 1 (PD-1), e.g., methods for attaching a linker, conjugation handle, or cytokine to a polypeptide that selectively binds PD-1. A variety of methods are known in the art for site-specific modification of the Fc region of an antibody or other polypeptide that binds PD-1. Modification with an affinity peptide designed to site-specifically attach the linker to the antibody

[0152] In some embodiments, the Fc region is modified to incorporate a linker, a conjugation handle, or a combination thereof. In some embodiments, the modification is performed by contacting the Fc region with an affinity peptide having a payload configured to attach a linker or other group to the Fc region, for example, to a specific residue of the Fc region. In some embodiments, the linker is attached using a reactive group (e.g., an N-hydroxysuccinimide ester) that forms a bond with a residue of the Fc region. In some embodiments, the affinity peptide comprises a cleavable linker. The cleavable linker is configured on the affinity peptide such that after the linker or other group is attached to the Fc region, the affinity peptide can be removed, leaving only the desired linker or other group attached to the Fc region. The linker or other group can then be used to further add additional groups to the Fc region, such as a cytokine or a linker attached to the cytokine.

[0153] Non-limiting examples of such affinity peptides can be found at least in WO2018199337(A1), WO2019240288(A1), WO2019240287(A1), and WO2020090979(A1), each of which is incorporated by reference as if set forth in its entirety herein. In some embodiments, the affinity peptide is a peptide modified to deliver one or more specific residues of the Fc region of an antibody to the linker / conjugation handle payload. In some embodiments, the affinity peptide is: (1) QETNPTENLYFQQKNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDDC (SEQ ID NO: 106); (2) QTADNQKNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDCSQSANLLAEAQQLNDAQAPQA (SEQ ID NO: 107); (3) QETKNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDDC (SEQ ID NO: 108); (4) QETFNKQCQRRFYEALHDPNLNEEQRNARIRSIRDDDC (SEQ ID NO: 109); (5) QETFNMQCQRRFYEALHDPNLNKEQRNARIRSIRDDDC (SEQ ID NO: 110); (6) QETFNMQCQRRFYEALHDPNLNEEQRNARIRS (7) QETMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO:111); (8) QETQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO:113); (9) QETCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO:114); (10) QETRGNCAYHKGQLVWCTYH (SEQ ID NO:115); and (11) QETRGNCAYHKGQIIWCTYH (SEQ ID NO:116), or the corresponding peptide truncated by 1, 2, 3, 4, or 5 residues at the N-terminus.As provided herein, an exemplary affinity peptide having a cleavable linker and a conjugation handle payload capable of attaching the payload to residue K248 of an antibody is shown below (reported in Matsuda et al., Chemical Site-Specific Conjugation Platform to Improve the Pharmacokinetics and Therapeutic Index of Antibody-Drug Conjugates, Mol. Pharmaceutics 2021, 18, 11, 4058-4066).

[0154] [ka]

[0155] Alternative affinity peptides targeting alternative residues in the Fc region are described in the references cited above for the AJICAP™ technology, and such affinity peptides can be used to attach desired functional groups to alternative residues in the Fc region (e.g., K246, K288, etc.). For example, the disulfide groups of the affinity peptides described above can be replaced with thioesters instead to provide sulfhydryl protecting groups as the cleavable portion of the linking group (e.g., the relevant portion of the affinity peptide is

[0156] [ka] or another of the cleavable linkers discussed below).

[0157] The affinity peptide of the present disclosure may include a cleavable linker. In some embodiments, the cleavable linker of the affinity peptide connects the affinity peptide to a group that will bind to the Fc region and is configured so that the peptide can be cleaved after the group containing the linker or conjugation handle is attached. In some embodiments, the cleavable linker is a divalent group. In some embodiments, the cleavable linker is a thioester group, an ester group, a sulfane group; a methanimine group; an oxyvinyl group; a thiopropanoate group; an ethane-1,2-diol group; a (imidazol-1-yl)methan-1-one group; a selenoether group; a silyl ether group; a dioxysilane group; an ether group; a dioxymethane group; a tetraoxospiro[5.5]undecane group; an acetamidoethyl phosphoramidite group; a bis(methylthio)-pyrazolopylidene ...bis(methylthio)-pyrazolopylidene group; a bis(methylthio)-pyrazolopylidene group; a bis(methylthio)-pyrazolopylidene group; a bis(methylthio)-pyrazolopylidene group; a bis(methylthio)-pyrazolopylidene group; a bis(methylthio)-pyrazolopylidene group; a bis(methylthio)-pyrazolopylidene The linker may comprise a benzoyl-dione group; a 2-oxo-2-phenylethyl formate group; a 4-oxybenzyl carbamate group; a 2-(4-hydroxy-oxyphenyl) diazinyl) benzoic acid group; a 4-amino-2-(2-amino-2-oxoethyl)-4-oxobut-2-enoic acid group; a 2-(2-methylenehydrazinyl) pyridine group; an N'-methyleneformohydrazide group; or an isopropyl carbamate group, all of which are unsubstituted or substituted. The composition and attachment points of the cleavable linker to the affinity peptide, as well as related methods of use, are described at least in WO2018199337(A1), WO2019240288(A1), WO2019240287(A1), and WO2020090979(A1).

[0158] In some embodiments, the cleavable linker is

[0159] [ka] and During the ceremony, - one of A or B is a point of attachment for a linker, and the other of A or B is a point of attachment to an affinity peptide; -Each R2a are independently H or optionally substituted alkyl; -Each R 2b are independently H or optionally substituted alkyl; -R 2c is H or optionally substituted alkyl; -J is a methylene, N, S, Si or O atom; -r is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0160] The affinity peptide comprises a reactive group configured to allow covalent attachment of a linker / conjugation handle to the Fc region. In some embodiments, the reactive group is selective for a specific amino acid residue functional group, such as a lysine residue, a tyrosine residue, a serine residue, a cysteine ​​residue, or a non-natural amino acid residue, of the Fc region that has been incorporated to facilitate attachment of the linker. The reactive group can be any suitable functional group, such as an activated ester (e.g., an N-hydroxysuccinimide ester or derivative thereof, a pentafluorophenyl ester, etc.) for reaction with lysine or a sulfhydryl reactive group (e.g., an α-β unsaturated carbonyl or a Michael acceptor such as a maleimide) for reaction with cysteine. In some embodiments, the reactive group is:

[0161] [ka] and During the ceremony, -Each R 5a , R 5b , and R 5c are independently H, halogen, or optionally substituted alkyl; - each j is 1, 2, 3, 4, or 5, - each k is 1, 2, 3, 4, or 5.

[0162] In some embodiments, an affinity peptide is used to deliver a reactive moiety to a desired amino acid residue such that the reactive moiety is exposed upon cleavage of the cleavable linker. As a non-limiting example, the reactive group forms a covalent bond with a desired residue of the Fc region of a polypeptide that selectively binds to anti-PD-1 due to the interaction between the affinity peptide and the Fc region. Following this covalent bond formation, the cleavable linker is cleaved under appropriate conditions to reveal the reactive moiety (e.g., if the cleavable linker comprises a thioester, a free sulfhydryl group will be attached to the Fc region after cleavage of the cleavable linker). This new reactive moiety can then be used to subsequently add additional moieties, such as conjugation handles, by a reagent that comprises a conjugation handle tethered to the sulfhydryl reactive group (e.g., an α-halogenated carbonyl group, an α-β unsaturated carbonyl group, a maleimide group, etc.).

[0163] In some embodiments, an affinity peptide is used to deliver a free sulfhydryl group to the lysine of the Fc region. In some embodiments, the free sulfhydryl group is then reacted with a bifunctional linking reagent to attach a new conjugation handle to the Fc region. In some embodiments, the new conjugation handle is then used to form a linker to the bound cytokine. In some embodiments, the new conjugation handle is an alkyne functional group. In some embodiments, the new conjugation handle is a DBCO functional group.

[0164] Exemplary bifunctional linking reagents useful for this purpose are of the formula ABC, where A is a sulfhydryl-reactive conjugation handle (e.g., maleimide, α,β-unsaturated carbonyl, a-carbonyl halide), B is a lining group, and C is a new conjugation handle (e.g., an alkyne such as DBCO). Specific non-limiting examples of bifunctional linking reagents include:

[0165] [ka] and related molecules (e.g., isomers), In the formula, each n is independently an integer from 1 to 6, and each m is independently an integer from 1 to 30.

[0166] Alternatively, the affinity peptide can be constructed such that a conjugation handle is added to the Fc region (such as by a linker group) immediately following covalent bond formation between the reactive group and a residue of the Fc region. In such a case, the affinity peptide is cleaved and the conjugation handle is immediately ready for subsequent conjugation to an IL-2 polypeptide (or other cytokine).

[0167] Alternative Binding Method – Enzyme Mediated Although affinity peptide-mediated modification of an antibody Fc region has many advantages over other methods that can be used to site-specifically modify an Fc region (e.g., ease of use, ability to rapidly generate many different antibody conjugates, ability to use many "off the shelf" commercially available antibodies without the need to undergo time-consuming protein engineering, etc.), other methods of making the modifications are contemplated as being within the scope of this disclosure.

[0168] In some embodiments, the disclosure generally relates to transglutaminase-mediated site-specific antibody-drug conjugates (ADCs) comprising: 1) a glutamine-containing tag, an endogenous glutamine (e.g., a native glutamine without engineering, e.g., a glutamine in a variable domain, CDR, etc.), and / or an endogenous glutamine made reactive by antibody engineering or an engineered transglutaminase; and 2) an amine donor agent comprising an amine donor unit, a linker, and a drug moiety. Non-limiting examples of such transglutaminase-mediated site-specific modifications can be found in at least the following publications: WO2020188061, US2022133904, US2019194641, US2021128743, U.S. Pat. No. 9,764,038, U.S.10,675,359, U.S. Pat. No. 9,717,803, U.S.10,434,180, U.S. Pat. No. 9,427,478, which are incorporated by reference as if set forth in their entireties herein.

[0169] In another aspect, the disclosure provides an engineered Fc-containing polypeptide conjugate comprising the formula: (Fc-containing polypeptide-TA), where T is an engineered acyl donor glutamine-containing tag at a specific site, A is an amine donor agent that is site-specifically conjugated to the acyl donor glutamine-containing tag at the carboxyl terminus, amino terminus, or another site of the Fc-containing polypeptide, where the acyl donor glutamine-containing tag comprises the amino acid sequence XXQX, where X is any amino acid (e.g., X can be the same or different amino acid), and where the engineered Fc-containing polypeptide conjugate comprises an amino acid substitution at position 295 for glutamine to asparagine (Q295N; EU numbering scheme).

[0170] In some embodiments, the acyl donor glutamine-containing tag is not spatially adjacent to a reactive Lys (e.g., the ability to form a covalent bond as an amine donor in the presence of an acyl donor and transglutaminase) in the polypeptide or Fc-containing polypeptide. In some embodiments, the polypeptide or Fc-containing polypeptide comprises an amino acid modification at the last amino acid position of the carboxyl terminus compared to the wild-type polypeptide at the same position. The amino acid modification can be an amino acid deletion, insertion, substitution, mutation, or any combination thereof.

[0171] In some embodiments, the polypeptide conjugate comprises a full-length antibody heavy chain and an antibody light chain, and the acyl donor glutamine-containing tag is located at the carboxyl-terminus of the heavy chain, the light chain, or both the heavy and light chains.

[0172] In some embodiments, the polypeptide conjugate comprises an antibody, which is a monoclonal antibody, a polyclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, a bispecific antibody, a minibody, a diabody, or an antibody fragment. In some embodiments, the antibody is an IgG.

[0173] In another aspect, described herein is a method of preparing an engineered Fc-containing polypeptide conjugate comprising the formula: (Fc-containing polypeptide-TA), where T is an engineered acyl donor glutamine-containing tag at a specific site, and A is an amine donor agent that is site-specifically conjugated to the acyl donor glutamine-containing tag at the carboxyl terminus, amino terminus, or another site of the Fc-containing polypeptide, and the acyl donor glutamine-containing tag comprises the amino acid sequence XXQX, where X is any amino acid (e.g., X can be the same or different amino acids), and The engineered Fc-containing polypeptide conjugate comprises an amino acid substitution of glutamine to asparagine at position 295 (Q295N; EU numbering scheme), and the method includes the steps of a) providing an engineered (Fc-containing polypeptide)-T molecule comprising an Fc-containing polypeptide and an acyl donor glutamine-containing tag; b) contacting an amine donor agent with the engineered (Fc-containing polypeptide)-T molecule in the presence of transglutaminase; and c) covalently linking the engineered (Fc-containing polypeptide)-T to the amine donor agent to form the engineered Fc-containing polypeptide conjugate.

[0174] In another aspect, described herein is a method of preparing an engineered polypeptide conjugate comprising the formula: polypeptide-TA, where T is an engineered acyl donor glutamine-containing tag at a specific site and A is an amine donor agent, the amine donor agent being site-specifically conjugated to the acyl donor glutamine-containing tag at the carboxyl terminus, amino terminus, or another site of the polypeptide, the acyl donor glutamine-containing tag comprising the amino acid sequence LLQGPX (SEQ ID NO: 153) (wherein X is A or P), or GGLLQGPP (SEQ ID NO: 154), the method comprising: a) providing an engineered polypeptide-T molecule comprising a polypeptide and the acyl donor glutamine-containing tag; b) contacting the amine donor agent with the engineered polypeptide-T molecule in the presence of a transglutaminase; and c) covalently attaching the engineered polypeptide-T to the amine donor agent to form an engineered Fc-containing polypeptide conjugate.

[0175] In some embodiments, an engineered polypeptide conjugate described herein (e.g., an engineered Fc-containing polypeptide conjugate, an engineered Fab-containing polypeptide conjugate, or an engineered antibody conjugate) has a conjugation efficiency of at least about 51%. In another aspect, the invention provides a pharmaceutical composition comprising an engineered polypeptide conjugate described herein (e.g., an engineered Fc-containing polypeptide conjugate, an engineered Fab-containing polypeptide conjugate, or an engineered antibody conjugate) and a pharma- ceutically acceptable excipient.

[0176] In some embodiments, methods of conjugating a moiety of interest (Z) to an antibody are described herein, comprising: (a) providing an antibody having at least one acceptor amino acid residue (e.g., a naturally occurring amino acid) (e.g., in the primary sequence of the constant region) that is reactive with a linking reagent (linker) in the presence of a coupling enzyme, such as a transamidase; and (b) reacting said antibody with a linking reagent (e.g., a linker comprising a primary amine) that comprises a reactive group (R), optionally protected or optionally unprotected, in the presence of an enzyme capable of forming a covalent bond between the acceptor amino acid residue and the linking reagent (other than the R moiety), under conditions sufficient to obtain an antibody comprising an acceptor amino acid residue (covalently) linked to the reactive group (R) via the linking reagent. Optionally, said acceptor residue of the antibody or antibody fragment is adjacent to a non-aspartic acid residue at the +2 position. Optionally, the residue at the +2 position is a non-aspartic acid residue. In one embodiment, the residue at the +2 position is a non-aspartic acid, non-glutamine residue. In one embodiment, the residue at position +2 is a non-aspartic acid, non-asparagine residue. In one embodiment, the residue at position +2 is a non-negatively charged amino acid (an amino acid other than aspartic acid or glutamic acid). Optionally, the acceptor glutamine is within the Fc domain of the antibody heavy chain, and optionally further within the CH2 domain. Optionally, the antibody does not contain heavy chain N297-linked glycosylation. Optionally, the acceptor glutamine is at position 295 and the residue at position +2 is the residue at position 297 (EU index numbering) of the antibody heavy chain.

[0177] In one aspect, a method of conjugating a moiety of interest (Z) to an antibody is described herein, comprising: (a) providing an antibody having at least one acceptor glutamine residue; and (b) reacting said antibody with a linker comprising a reactive group (R), preferably a primary amine comprising a protected reactive group (lysine-based linker), in the presence of transglutaminase (TGase) under conditions sufficient to obtain an antibody comprising an acceptor glutamine (covalently) linked to the reactive group (R) via said linker. Optionally, said acceptor glutamine residue of the antibody or antibody fragment is adjacent to a non-aspartic acid residue at the +2 position. Optionally, the residue at the +2 position is a non-aspartic acid residue. In one embodiment, the residue at the +2 position is a non-aspartic acid, non-glutamine residue. In one embodiment, the residue at the +2 position is a non-aspartic acid, non-asparagine residue. In one embodiment, the residue at the +2 position is a non-negatively charged amino acid (an amino acid other than aspartic acid or glutamic acid). Optionally, the acceptor glutamine is within the Fc domain of the antibody heavy chain, and optionally further within the CH2 domain. Optionally, the antibody does not contain heavy chain N297-linked glycosylation. Optionally, the acceptor glutamine is at position 295, and the residue at position +2 is the residue at position 297 (EU index numbering) of the antibody heavy chain.

[0178] The antibody comprising the receptor residue or acceptor glutamine residue linked to the reactive group (R) via a linker comprising a primary amine (lysine-based linker) can then be reacted with a reaction partner comprising a moiety of interest (Z) to generate an antibody comprising the receptor residue or acceptor glutamine residue linked to the moiety of interest (Z) via a linker. Thus, in one embodiment, the method further comprises step (c): (i) reacting the antibody of step b) comprising the acceptor glutamine linked to the reactive group (R) via a linker comprising a primary amine (lysine-based linker), optionally immobilized on a solid support, with (ii) a compound comprising the moiety of interest (Z) and a reactive group (R') capable of reacting with the reactive group R, under conditions sufficient to obtain an antibody comprising the acceptor glutamine linked to the moiety of interest (Z) via a linker comprising a primary amine (lysine-based linker). Preferably, the aforementioned compound comprising the moiety of interest (Z) and the reactive group (R') capable of reacting with the reactive group R is provided in less than 80-fold, 40-fold, 20-fold, 10-fold, 5-fold or 4 molar equivalents relative to the antibody. In one embodiment, the antibody comprises two acceptor glutamines and the compound comprising the moiety of interest (Z) and the reactive group (R') is provided in less than 10 molar equivalents relative to the antibody. In one embodiment, the antibody comprises two acceptor glutamines and the compound comprising the moiety of interest (Z) and the reactive group (R') is provided in less than 5 molar equivalents relative to the antibody. In one embodiment, the antibody comprises four acceptor glutamines and the compound comprising the moiety of interest (Z) and the reactive group (R') is provided in less than 20 molar equivalents relative to the antibody. In one embodiment, the antibody comprises four acceptor glutamines and the compound comprising the moiety of interest (Z) and the reactive group (R') is provided in less than 10 molar equivalents relative to the antibody. In one embodiment, steps (b) and / or (c) are performed under aqueous conditions.Optionally, step (c) comprises immobilizing a sample of the antibody comprising the functionalized acceptor glutamine residue on a solid support to provide a sample comprising immobilized antibody, reacting the sample comprising the immobilized antibody with a compound, optionally recovering any unreacted compound and reintroducing such recovered compound to the solid support for reaction with the immobilized antibody, and eluting the antibody conjugate to provide a composition comprising the Z moiety.

[0179] Conjugation Handle Chemistry In some embodiments, a suitably modified Fc region of a polypeptide that selectively binds PD-1 comprises a conjugation handle that is used to conjugate the polypeptide that selectively binds PD-1 to an IL-2 polypeptide.

[0180] Any suitable reactive group capable of reacting with a complementary reactive group attached to an IL-2 polypeptide can be used as a conjugation handle. In some embodiments, the conjugation handle comprises reagents for metal-mediated processes such as Cu(I)-catalyzed or "copper-free" alkyne-azide triazole forming reactions (e.g., strain-promoted cycloaddition), Staudinger ligation, inverse electro-demanded Diels-Alder (IEDDA) reactions, "photoclick" chemistry, tetrazine cycloaddition with trans-cycloctene, or olefin metathesis and Suzuki-Miyaura or Sonogashira cross-coupling.

[0181] In some embodiments, the conjugation handle comprises a reagent for a "copper-free" alkyne azide triazole forming reaction. Non-limiting examples of alkynes for the aforementioned alkyne azide triazole forming reaction include cyclooctyne reagents (e.g., (1R,8S,9s)bicyclo[6.1.0]non-4-yn-9-ylmethanol-containing reagents, dibenzocyclooctyne-amine reagents, difluorocyclooctyne, or derivatives thereof). In some embodiments, the alkyne functional group is attached to the Fc region. In some embodiments, the azide functional group is attached to the Fc region.

[0182] In some embodiments, the conjugation handle comprises a reactive group selected from azide, alkyne, tetrazine, halide, sulfhydryl, disulfide, maleimide, activated ester, alkene, aldehyde, ketone, imine, hydrazine, and hydrazide. In some embodiments, the IL-2 polypeptide comprises a reactive group complementary to the conjugation handle of the Fc region. In some embodiments, the conjugation handle and the complementary conjugation handle comprise "click" chemistry reagents. Exemplary groups of click chemistry residues are provided in Hein et al., "Click Chemistry, A Powerful Tool for Pharmaceutical Sciences," Pharmaceutical Research volume 25, pages 2216-2230 (2008); Thirumurugan et al., "Click Chemistry for Drug Development and Diverse Chemical-Biology Applications," Chem. Rev. 2013, 113, 7, 4905-4979; U.S. Patent Application Publication No. 20160107999(A1); U.S. Patent No. 10266502; U.S. Patent Application Publication No. 20190204330(A1), each of which is incorporated by reference in its entirety. Linker Structure

[0183] In some embodiments, the linker used to link a polypeptide that selectively binds PD-1 and a cytokine (e.g., an IL-2 polypeptide) includes a point of attachment on both moieties. The point of attachment can be any of the residues provided herein to facilitate attachment. The linker structure can be any suitable structure for creating a spatial linkage between the two moieties. In some embodiments, the linker provides a covalent bond between both moieties. In some embodiments, the linker is a chemical linker (e.g., not a polypeptide expressed as a fusion protein).

[0184] In some embodiments, the linker comprises a polymer. In some embodiments, the linker comprises a water-soluble polymer. In some embodiments, the linker comprises a poly(alkylene oxide), a polysaccharide, a poly(vinylpyrrolidone), a poly(vinyl alcohol), a polyoxazoline, a poly(acryloylmorpholine), or a combination thereof. In some embodiments, the linker comprises a poly(alkylene oxide). In some embodiments, the poly(alkylene oxide) is polyethylene glycol or polypropylene glycol, or a combination thereof. In some embodiments, the poly(alkylene oxide) is polyethylene glycol.

[0185] In some embodiments, the linker is a bifunctional linker. In some embodiments, the bifunctional linker comprises an amide group, an ester group, an ether group, a thioether group, or a carbonyl group. In some embodiments, the linker comprises a non-polymeric linker. In some embodiments, the linker comprises a non-polymeric bifunctional linker. In some embodiments, the non-polymeric bifunctional linker comprises succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate; maleimidocaproyl; valine-citrulline; allyl(4-methoxyphenyl)dimethylsilane; 6-(allyloxycarbonylamino)-1-hexanol; 4-aminobutyraldehyde diethyl acetal; or (E)-N-(2-aminoethyl)-4-{2-[4-(3-azidopropoxy)phenyl]diazenyl}benzamide hydrochloride.

[0186] The linker may be branched or linear. In some embodiments, the linker is linear. In some embodiments, the linker is branched. In some embodiments, the linker comprises a linear portion of the chain of at least 10, 20, 50, 100, 500, 1000, 2000, 3000, or 5000 atoms (e.g., between the first and second attachment points). In some embodiments, the linker comprises a linear portion of the chain of at least 10, 20, 30, 40, or 50 atoms. In some embodiments, the linker comprises a linear portion of at least 10 atoms. In some embodiments, the linker is branched and comprises a linear portion of the chain of at least 10, 20, 50, 100, 500, 1000, 2000, 3000, or 5000 atoms.

[0187] In some embodiments, the linker has a molecular weight of about 200 Daltons to about 2000 Daltons. In some embodiments, the linker has a molecular weight of 200 Daltons to 100,000 Daltons. In some embodiments, the linker has a molecular weight of 200 Daltons to 500 Daltons, 200 Daltons to 750 Daltons, 200 Daltons to 1,000 Daltons, 200 Daltons to 5,000 Daltons, 200 Daltons to 10,000 Daltons, 200 Daltons to 20,000 Daltons, 200 Daltons to 50,000 Daltons, 200 Daltons to 100,000 Daltons, 500 Daltons to 750 Daltons, 500 Daltons to 500 Daltons. 1,000 Daltons, 500 Daltons to 5,000 Daltons, 500 Daltons to 10,000 Daltons, 500 Daltons to 20,000 Daltons, 500 Daltons to 50,000 Daltons, 500 Daltons to 100,000 Daltons, 750 Daltons to 1,000 Daltons, 750 Daltons to 5,000 Daltons, 750 Daltons to 10,000 Daltons, 750 Daltons to 20,000 Daltons, 750 Daltons to 100,000 Daltons 1000 Daltons to 50,000 Daltons, 750 Daltons to 100,000 Daltons, 1,000 Daltons to 5,000 Daltons, 1,000 Daltons to 10,000 Daltons, 1,000 Daltons to 20,000 Daltons, 1,000 Daltons to 50,000 Daltons, 1,000 Daltons to 100,000 Daltons, 5,000 Daltons to 10,000 Daltons, 5,000 Daltons to 20,000 Daltons, 5,0 In some embodiments, the linker has a molecular weight of 200 daltons, 500 daltons, 750 daltons, 1,000 daltons, 5,000 daltons, 10,000 daltons, 20,000 daltons, 50,000 daltons, or 100,000 daltons.In some embodiments, the linker has a molecular weight of at least 200 daltons, 500 daltons, 750 daltons, 1,000 daltons, 5,000 daltons, 10,000 daltons, 20,000 daltons, or 50,000 daltons. In some embodiments, the linker has a molecular weight of at most 500 daltons, 750 daltons, 1,000 daltons, 5,000 daltons, 10,000 daltons, 20,000 daltons, 50,000 daltons, or 100,000 daltons. In preferred embodiments, the linker has a molecular weight of less than 5000 daltons, less than 4000 daltons, less than 3000 daltons, or less than 2000 daltons, and the linker is monodisperse (e.g., there is a high degree of uniformity of linker structures between the polypeptide that specifically binds to PD-L1 and the IL-2 polypeptide (or other cytokine) in the population of conjugate compositions herein).

[0188] In some embodiments, the linker comprises one or more pairs of conjugation handles and their complementary conjugation handles that are reaction products. In some embodiments, the reaction products comprise triazoles, hydrazones, pyridazines, sulfides, disulfides, amides, esters, ethers, oximes, alkenes, or any combination thereof. In some embodiments, the reaction products comprise triazoles. The reaction products can be separated from the first and second attachment points by any portion of the linker. In some embodiments, the reaction products are substantially at the center of the linker. In some embodiments, the reaction products are substantially closer to one attachment point than the other attachment point.

[0189] In some embodiments, the linker comprises a structure of formula (X):

[0190] [ka] In the formula, L 1 , L 2 , L 3 , L 4 , L 5 , L6 , L 8 , and L 9 each independently represents -O-, -NR L -, -N(R L )2 + -,-OP(=O)(OR L )O-, -S-, -S(=O)-, -S(=O)2-, -C(=O)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -C(=O)NR L -, -NR L C(=O)-, -OC(=O)NR L -, -NR L C(=O)O-, -NR L C(=O)NR L -, -NR L C(=S)NR L -, -CR L =N-, -N=CR L , -NR L S(=O)2-, -S(=O)2NR L -, -C(=O)NR L S(=O)2-, -S(=O)2NR L C(=O)-, substituted or unsubstituted C1-C6 alkylene, substituted or unsubstituted C1-C6 heteroalkylene, substituted or unsubstituted C2-C6 alkenylene, substituted or unsubstituted C2-C6 alkynylene, substituted or unsubstituted C6-C 20 Arylene, substituted or unsubstituted C2-C 20 Heteroarylene, -(CH2-CH2-O) qa -, -(O-CH2-CH2) qb -, -(CH2-CH(CH3)-O) qc -, -(O-CH(CH3)-CH2) qd -, a reaction product of a conjugation handle and a complementary conjugation handle, or absent, Each R Lare independently hydrogen, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C5 alkynyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C2-C7 heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; Each of qa, qb, qc, and qd independently represents an integer of 1 to 100; During the ceremony, each

[0191] [ka] is a point of attachment to a polypeptide that selectively binds to PD-1 or a cytokine (eg, an IL-2 polypeptide).

[0192] In some embodiments, the linker has the formula (X a ) structure,

[0193] [ka] In the formula, L 1 , L 2 , L 3 , L 4 , L 5 , L 6 , L 7 , L 8 , and L 9 each independently represents -O-, -NR L -, -(C1-C6 alkylene)NR L -, -NR L (C1-C6 alkylene)-, -N(R L )2 + -, -(C1-C6 alkylene)N(R L )2 + -, -N(R L )2 + -(C1-C6 alkylene)-, -OP(=O)(OR L)O-, -S-, -(C1-C6 alkylene)S-, -S(C1-C6 alkylene)-, -S(=O)-, -S(=O)2-, -C(=O)-, -(C1-C6 alkylene)C(=O)-, -C(=O)(C1-C6 alkylene)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -C(=O)NR L -, -C(=O)NR L (C1-C6 alkylene)-, -(C1-C6 alkylene)C(=O)NR L -, -NR L C(=O)-, -(C1-C6 alkylene)NR L C(=O)-, -NR L C(=O)(C1-C6 alkylene)-, -OC(=O)NR L -, -NR L C(=O)O-, -NR L C(=O)NR L -, -NR L C(=S)NR L -, -CR L =N-, -N=CR L , -NR L S(=O)2-, -S(=O)2NR L -, -C(=O)NR L S(=O)2-, -S(=O)2NR L C(=O)-, substituted or unsubstituted C1-C6 alkylene, substituted or unsubstituted C1-C6 heteroalkylene, substituted or unsubstituted C2-C6 alkenylene, substituted or unsubstituted C2-C6 alkynylene, substituted or unsubstituted C6-C 20 Arylene, substituted or unsubstituted C2-C 20 Heteroarylene, -(CH2-CH2-O) qa -, -(O-CH2-CH2) qb -, -(CH2-CH(CH3)-O) qc -, -(O-CH(CH3)-CH2) qd -, the reaction product of a conjugation handle and a complementary conjugation handle, or absent; (C1-C6 alkylene); Each R Lare independently hydrogen, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C5 alkynyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C2-C7 heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; Each of qa, qb, qc, and qd independently represents an integer of 1 to 100; During the ceremony, each

[0194] [ka] is a point of attachment to a polypeptide that selectively binds to PD-1 or a cytokine (eg, an IL-2 polypeptide).

[0195] In some embodiments, the linker comprises a structure of formula (X'):

[0196] [ka] In the formula, each L' is independently -O-, -NR L -, -(C1-C6 alkylene)NR L -, -NR L (C1-C6 alkylene)-, -N(R L )2 + -, -(C1-C6 alkylene)N(R L )2 + -, -N(R L )2 + -(C1-C6 alkylene)-, -OP(=O)(OR L )O-, -S-, -(C1-C6 alkylene)S-, -S(C1-C6 alkylene)-, -S(=O)-, -S(=O)2-, -C(=O)-, -(C1-C6 alkylene)C(=O)-, -C(=O)(C1-C6 alkylene)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -C(=O)NR L -, -C(=O)NR L(C1-C6 alkylene)-, -(C1-C6 alkylene)C(=O)NR L -, -NR L C(=O)-, -(C1-C6 alkylene)NR L C(=O)-, -NR L C(=O)(C1-C6 alkylene)-, -OC(=O)NR L -, -NR L C(=O)O-, -NR L C(=O)NR L -, -NR L C(=S)NR L -, -CR L =N-, -N=CR L , -NR L S(=O)2-, -S(=O)2NR L -, -C(=O)NR L S(=O)2-, -S(=O)2NR L C(=O)-, substituted or unsubstituted C1-C6 alkylene, substituted or unsubstituted C1-C6 heteroalkylene, substituted or unsubstituted C2-C6 alkenylene, substituted or unsubstituted C2-C6 alkynylene, substituted or unsubstituted C6-C 20 Arylene, substituted or unsubstituted C2-C 20 Heteroarylene, -(CH2-CH2-O) qa -, -(O-CH2-CH2) qb -, -(CH2-CH(CH3)-O) qc -, -(O-CH(CH3)-CH2) qd -, the reaction product of a conjugation handle and a complementary conjugation handle, or absent; (C1-C6 alkylene); Each R L are independently hydrogen, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C5 alkynyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C2-C7 heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; Each of qa, qb, qc, and qd independently represents an integer of 1 to 100; g is an integer from 1 to 100; During the ceremony, each

[0197] [ka] is the point of attachment to the modified IL-2 polypeptide or antibody or antigen-binding fragment.

[0198] In some embodiments, the linker of Formula (X) or Formula (Xa) or Formula (X′) comprises the following structure or a positional isomer thereof:

[0199] [ka] During the ceremony,

[0200] [ka] is a first point of attachment to a lysine residue of a polypeptide that selectively binds PD-1, L is a linking group;

[0201] [ka] is the point of attachment to the linking group that is attached to the first point of attachment.

[0202] In some embodiments, L is the structure

[0203] [ka] having wherein each n is independently an integer from 1 to 6, and each m is an integer from 1 to 30. In some embodiments, each m is independently 2 or 3. In some embodiments, each m is an integer from 1 to 24, 1 to 18, 1 to 12, or 1 to 6.

[0204] In some embodiments, the linker of Formula (X) or Formula (Xa) or Formula (X′) comprises the following structure or a positional isomer thereof:

[0205] [ka] During the ceremony,

[0206] [ka] is a first point of attachment to a lysine residue of a polypeptide that selectively binds PD-1, L is a linking group;

[0207] [ka] is the point of attachment to the linking group that is attached to the first point of attachment.

[0208] In some embodiments, L″ is

[0209] [ka] The structure is wherein each n is independently an integer from 1 to 6, and each m is independently an integer from 1 to 30. In some embodiments, each m is independently 2 or 3. In some embodiments, each m is an integer from 1 to 24, 1 to 18, 1 to 12, or 1 to 6.

[0210] In some embodiments, L or L″ are each independently

[0211] [ka] 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more subunits selected from wherein each n is independently an integer from 1 to 30. In some embodiments, each n is independently an integer from 1 to 6. In some embodiments, L or L″ comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 subunits.

[0212] In some embodiments, L or L″ has the structure of formula (X″):

[0213] [ka] In the formula, L 1a , L 2a , L 3a , L 4a , L 5a each independently represents -O-, -NR La -, -(C1-C6 alkylene)NR La -, -NR La (C1-C6 alkylene)-, -N(R L )2 + -, -(C1-C6 alkylene)N(R La )2 + (C1-C6 alkylene)-, -N(R L )2 + -,-OP(=O)(OR La )O-, -S-, -(C1-C6 alkylene)S-, -S(C1-C6 alkylene)-, -S(=O)-, -S(=O)2-, -C(=O)-, -(C1-C6 alkylene)C(=O)-, -C(=O)(C1-C6 alkylene)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -C(=O)NR La -, -C(=O)NR La (C1-C6 alkylene)-, -(C1-C6 alkylene)C(=O)NR La -, -NR La C(=O)-, -(C1-C6 alkylene)NR La C(=O)-, -NR La C(=O)(C1-C6 alkylene)-, -OC(=O)NR La -, -NR La C(=O)O-, -NR La C(=O)NR La -, -NRLa C(=S)NR La -, -CR La =N-, -N=CR La , -NR La S(=O)2-, -S(=O)2NR La -, -C(=O)NR La S(=O)2-, -S(=O)2NR La C(=O)-, substituted or unsubstituted C1-C6 alkylene, substituted or unsubstituted C1-C6 heteroalkylene, substituted or unsubstituted C2-C6 alkenylene, substituted or unsubstituted C2-C6 alkynylene, substituted or unsubstituted C6-C 20 Arylene, substituted or unsubstituted C2-C 20 Heteroarylene, -(CH2-CH2-O) qe -, -(O-CH2-CH2) qf -, -(CH2-CH(CH3)-O) qg -, -(O-CH(CH3)-CH2) qh -, a reaction product of a conjugation handle and a complementary conjugation handle, or absent, Each R La are independently hydrogen, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C5 alkynyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C2-C7 heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; Each of qe, qf, qg and qh independently represents an integer of 1 to 100.

[0214] In some embodiments, L or L″ comprises a linear chain of 2 to 10, 2 to 15, 2 to 20, 2 to 25, or 2 to 30 atoms. In some embodiments, the linear chain comprises one or more alkyl groups (e.g., lower alkyl (C1-C4)), one or more aromatic groups (e.g., phenyl), one or more amide groups, one or more ether groups, one or more ester groups, or any combination thereof.

[0215] In some embodiments, the linking group attached to the first point of attachment (e.g., the point of attachment to a cytokine) comprises poly(ethylene glycol). In some embodiments, the linking group comprises from about 2 to about 30 poly(ethylene glycol) units. In some embodiments, the linking group attached to the first point of attachment (e.g., the point of attachment to a cytokine) is a functional group attached to a cytokine provided herein that comprises an azide (e.g., a triazole is the reaction product of an azide).

[0216] In some embodiments, L is -O-, -NR L -, -N(R L )2 + -,-OP(=O)(OR L )O-, -S-, -S(=O)-, -S(=O)2-, -C(=O)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -C(=O)NR L -, -NR L C(=O)-, -OC(=O)NR L -, -NR L C(=O)O-, -NR L C(=O)NR L -, -NR L C(=S)NR L -, -CR L =N-, -N=CR L , -NR L S(=O)2-, -S(=O)2NR L -, -C(=O)NR L S(=O)2-, -S(=O)2NR L C(=O)-, substituted or unsubstituted C1-C6 alkylene, substituted or unsubstituted C1-C6 heteroalkylene, substituted or unsubstituted C2-C6 alkenylene, substituted or unsubstituted C2-C6 alkynylene, substituted or unsubstituted C6-C 20 Arylene, substituted or unsubstituted C2-C 20 Heteroarylene, -(CH2-CH2-O) qa -, -(O-CH2-CH2) qb -, -(CH2-CH(CH3)-O) qc -, -(O-CH(CH3)-CH2) qd-, where each R L are independently hydrogen, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C5 alkynyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C2-C7 heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, and each of qa, qb, qc, and qd is independently an integer of 1 to 100.

[0217] In some embodiments, the reaction product of the conjugation handle and the complementary conjugation handle independently comprises a triazole, a hydrazone, a pyridazine, a sulfide, a disulfide, an amide, an ester, an ether, an oxime, or an alkene. In some embodiments, the reaction product of the conjugation handle and the complementary conjugation handle comprises a triazole. In some embodiments, the reaction product of the conjugation handle and the complementary conjugation handle comprises

[0218] [ka] or a positional isomer or derivative thereof.

[0219] In some embodiments, the linker is a cleavable linker. In some embodiments, the cleavable linker is cleaved near or in the tumor microenvironment. In some embodiments, the tumor is mechanically or physically cleaved near or in the tumor microenvironment. In some embodiments, the tumor is chemically cleaved near or in the tumor microenvironment. In some embodiments, the cleavable linker is a reduction-sensitive linker. In some embodiments, the cleavable linker is an oxidation-sensitive linker. In some embodiments, the cleavable linker is cleaved near or in the tumor microenvironment as a result of pH. In some embodiments, the linker is by tumor metabolism near or in the tumor microenvironment. In some embodiments, the cleavable linker is cleaved by proteases near or in the tumor microenvironment.

[0220] IL-2 cytokine Cytokines are proteins produced in the body that are important in cell signaling. Cytokines can regulate the immune system, and cytokine therapy utilizes the immunomodulatory properties of molecules to enhance the immune system of subjects and kill cancer cells. Disclosed herein are anti-PD-1 polypeptides conjugated to cytokines that can exhibit enhanced biological activity.

[0221] Interleukin-2 (IL-2) is a cytokine signaling molecule important in regulating the immune system. IL-2 is involved in helping the immune system differentiate between foreign and endogenous cell types, thereby preventing the immune system from attacking the subject's own cells. IL-2 achieves its activity through interactions with the IL-2 receptor (IL-2R) expressed by lymphocytes. Through these binding interactions, IL-2 mediates the expression of T effector (T eff ) cells, natural killer (NK) cells, and regulatory T cells (T reg ) the population of subjects can be adjusted.

[0222] IL-2 has been used to treat cancer, both alone and in combination with other therapies.However, the use of IL-2 as treatment has been limited by the toxicity of IL-2, undesirable side effects such as vascular leakage syndrome, and the short half-life of IL-2.Conjugation of IL-2 to the anti-PD-1 polypeptide of the present disclosure can improve the selectivity of IL-2 polypeptide, enhance the therapeutic potential of IL-2, and potentially reduce the risk of side effects from the administration of IL-2 therapy.

[0223] The present disclosure describes anti-PD-1 polypeptides conjugated to modified interleukin-2 (IL-2) polypeptides and their use as therapeutic agents. The modified IL-2 polypeptides provided herein can be used as immunotherapies or as part of other immunotherapeutic regimens. Such modified IL-2 polypeptides may exhibit binding properties for the IL-2 receptor (IL-2R) that differ from wild-type IL-2. In one aspect, the modified IL-2 polypeptides described herein have a decreased affinity for the IL-2Rαβγ complex (IL-2Rα). In some embodiments, the modified IL-2 polypeptides have an increased affinity for the IL-2Rβγ complex (IL-2Rβ). In some embodiments, the binding affinity between the modified IL-2 polypeptide and IL-2Rβ is the same or higher than the binding affinity between wild-type IL-2 and IL-2Rβ. Non-limiting examples of IL-2 amino acid sequences utilized in the embodiments described herein are provided in Table 8 below.

[0224] In some embodiments of the present disclosure, the IL-2 polypeptide is preferably biased in favor of signaling through the IL-2 receptor beta subunit compared to wild-type IL-2. In some embodiments, this is accomplished by one or both of a) inhibiting or decreasing binding of the IL-2 polypeptide to the IL-2 receptor alpha subunit (e.g., by mutations in residues that contact the alpha subunit, addition of a polymer to residues that contact the alpha subunit, or attachment of a linker to a polypeptide that binds PD-1 to residues that contact the alpha subunit) and / or b) enhancing binding of the IL-2 polypeptide to the beta subunit of the IL-2 receptor (e.g., by mutations in residues that contact the beta subunit that enhance binding). In some embodiments, the IL-2 polypeptide of the immunocytokine compositions provided herein is biased toward the IL-2 receptor beta subunit compared to wild-type IL-2. Non-limiting examples of IL-2 polypeptides having modifications biased towards IL-2 receptor beta signaling are described, for example, in WO2021140416(A2), WO2012065086(A1), WO2019028419(A1), WO2012107417(A1), WO2018119114(A1), WO2012062228(A2), WO2019104092(A1), WO2012088446(A1), and WO2015164815(A1), each of which is incorporated by reference as if set forth in its entirety herein.

[0225] Attachment points of chemical linkers to the IL-2 polypeptide Provided herein are compositions comprising a polypeptide, e.g., an antibody, that binds to PD-1, connected to an IL-2 polypeptide by a chemical linker. As discussed above, the chemical linker can be attached to the anti-PD-1 polypeptide at any of the positions provided herein. The second attachment point of the linker is attached to the IL-2 polypeptide provided herein.

[0226] In some embodiments, the chemical linker is attached to the IL-2 polypeptide at an amino acid residue. In some embodiments, the chemical linker is attached to an amino acid residue corresponding to any one of amino acid residues 1-133 of SEQ ID NO:1. In some embodiments, the chemical linker is attached to a non-terminal amino acid residue (e.g., any one of amino acid residues 2-132 of SEQ ID NO:1, or any one of amino acid residues 1-133 of SEQ ID NO:1, extended at either the N-terminus or C-terminus by one or more amino acid residues). In some embodiments, the chemical linker is attached to a non-terminal amino acid residue of the IL-2 polypeptide, and the IL-2 polypeptide comprises either an N-terminal truncation or a C-terminal truncation relative to SEQ ID NO:1.

[0227] In some embodiments, the chemical linker is attached to the IL-2 polypeptide at an amino acid residue that interacts with an IL-2 receptor (IL-2R) protein or subunit. In some embodiments, the chemical linker is attached to an amino acid residue that interacts with the IL-2R alpha subunit (IL-2R alpha), the IL-2R beta subunit (IL-2R beta), or the IL-2R gamma subunit (IL-2R gamma). In some embodiments, the chemical linker is attached to an amino acid residue that interacts with the IL-2R alpha subunit (IL-2R alpha). In some embodiments, the chemical linker is attached to an amino acid residue that interacts with the IL-2R beta subunit (IL-2R beta). In some embodiments, the chemical linker is attached to an amino acid residue that interacts with the IL-2R gamma subunit (IL-2R gamma).

[0228] In some embodiments, the attachment point to the IL-2 polypeptide is selected such that the interaction of the IL-2 polypeptide with at least one IL-2 receptor subunit is reduced or blocked. In some embodiments, the attachment point is selected such that the interaction of the IL-2 polypeptide with IL-2 Rα is reduced or blocked. In some embodiments, the attachment point is selected such that the interaction of the IL-2 polypeptide with IL-2Rβ is reduced.

[0229] In some embodiments, the linker is attached to the IL-2 polypeptide at a residue that disrupts binding between the IL-2 polypeptide and the IL-2 receptor alpha subunit (IL-2Rα). Examples of these residues include residues 3, 5, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 60, 61, 62, 63, 64, 65, 67, 68, 69, 71, 72, 103, 104, 105, and 107, as described, for example, in WO2019028419(A1), WO2020056066(A1), WO2021140416(A2), and WO2021216478(A1), each of which is incorporated herein by reference as if set forth in its entirety.

[0230] In some embodiments, the linker is attached to the IL-2 polypeptide at any one of amino acid residues at positions 1 to 110, where the numbering of the residue positions of the modified IL-2 polypeptide is based on SEQ ID NO: 1 as the reference sequence. In some embodiments, the linker is attached to the IL-2 polypeptide at any one of amino acid residues at positions 1 to 10, 1 to 20, 1 to 30, 30 to 50, 30 to 70, 30 to 100, 40 to 50, 40 to 70, 40 to 100, or 40 to 110. In some embodiments, the linker is attached to the IL-2 polypeptide at any one of amino acid residues at positions 1, 35, 37, 38, 41, 42, 43, 44, 45, 60, 61, 62, 64, 65, 68, 69, 71, 72, 104, and 105, and 107, where the numbering of the amino acid residue positions of the modified IL-2 polypeptide is based on SEQ ID NO: 1 as the reference sequence. In some embodiments, the linker is attached to the IL-2 polypeptide at any one of amino acid residues at positions 1, 35, 37, 38, 41, 42, 43, 44, 45, 60, 61, 62, 64, 65, 68, 69, 71, 72, 104, and 105, and 107, where the amino acid residue position numbering of the modified IL-2 polypeptide is based on SEQ ID NO: 1 as a reference sequence. In some embodiments, the linker is attached to the IL-2 polypeptide at any one of amino acid residues at positions 1, 35, 37, 38, 41, 42, 43, 44, 60, 61, 62, 64, 65, 68, 69, 71, 72, 104, and 105, and 107, where the amino acid residue position numbering of the modified IL-2 polypeptide is based on SEQ ID NO: 1 as a reference sequence. In some embodiments, the linker is attached to the IL-2 polypeptide at any one of amino acid residues at positions 1, 35, 37, 38, 41, 43, 44, 60, 61, 62, 64, 65, 68, 69, 71, 72, 104, and 105, and 107, where the amino acid residue position numbering of the modified IL-2 polypeptide is based on SEQ ID NO: 1 as the reference sequence.In some embodiments, the linker is attached to the IL-2 polypeptide at any one of amino acid residues at positions 1, 35, 37, 38, 39, 40, 41, 42, 43, 44, 45, or 46. In some embodiments, the linker is attached to the IL-2 polypeptide at any one of amino acid residues at positions 1, 41, 42, 43, 44, and 45, where the amino acid residue position numbering of the modified IL-2 polypeptide is based on SEQ ID NO: 1 as the reference sequence. In some embodiments, the linker is attached at amino acid residue 1, 42, or 45. In some embodiments, the linker is attached at amino acid residue 1. In some embodiments, the linker is attached at amino acid residue 42. In some embodiments, the linker is attached at amino acid residue 45.

[0231] In some embodiments, the linker is attached to an amino acid residue that is a natural amino acid residue of the IL-2 polypeptide shown in SEQ ID NO:1. In some embodiments, the linker is attached to an amino acid residue that is a modified version of the natural amino acid residue of the IL-2 polypeptide shown in SEQ ID NO:1. Non-limiting examples of such modifications include the incorporation or attachment of a conjugation handle (including via a linker) to the natural amino acid residue, or the attachment of a chemical linker to the natural amino acid using any compatible method. In some embodiments, the linker is attached to an amino acid residue that is a substituted amino acid residue compared to the IL-2 polypeptide of SEQ ID NO:1. The substitution can be to a natural amino acid that is more suitable for attachment of additional functional groups (e.g., aspartic acid, cysteine, glutamic acid, lysine, serine, threonine or tyrosine), derivatives of modified versions of any naturally occurring amino acid, or any non-natural amino acid (e.g., an amino acid containing a desired reactive group, e.g., click chemistry reagents such as azides, alkynes, etc.). Non-limiting examples of amino acids that may be substituted include, but are not limited to, -α-(9-fluorenylmethyloxycarbonyl)-L-biphenylalanine (Fmoc-L-Bip-OH) and N-α-(9-fluorenylmethyloxycarbonyl)-O-benzyl-L-tyrosine (Fmoc-L-Tyr(Bzl)-OH.Exemplary non-standard amino acids include p-acetyl-L-phenylalanine, p-iodo-L-phenylalanine, p-methoxyphenylalanine, O-methyl-L-tyrosine, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcp-serine, L-Dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-boronophenylalanine, O-propargyltyrosine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-bromophenylalanine, selenoside, and the like. The non-standard amino acids include steine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, azido-lysine (AzK), analogs of tyrosine amino acids; analogs of glutamine amino acids; analogs of phenylalanine amino acids; analogs of serine amino acids; analogs of threonine amino acids; alkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynyl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or amino substituted amino acids, β-amino acids; cyclic amino acids other than proline or histidine; aromatic amino acids other than phenylalanine, tyrosine, or tryptophan; or combinations thereof. In some embodiments, the non-standard amino acids are selected from β-amino acids, homoamino acids, cyclic amino acids, and amino acids with derivatized side chains. In some embodiments, the non-standard amino acid is β-alanine, β-aminopropionic acid, piperidinic acid, aminocaproic acid, aminoheptanoic acid, aminopimelic acid, desmosine, diaminopimelic acid, N. α -Ethylglycine, N α -ethylasparagine, hydroxylysine, allo-hydroxylysine, isodesmosine, allo-isoleucine, ω-methylarginine, N α -Methylglycine, Nα -Methylisoleucine, N α -Methylvaline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N α -Acetylserine, N α -Formylmethionine, 3-methylhistidine, 5-hydroxylysine, and / or other similar amino acids.

[0232] In some embodiments, the linker is attached at the non-natural amino acid residue. In some embodiments, the non-natural amino acid residue comprises a conjugation handle. In some embodiments, the conjugation handle facilitates the addition of the linker to the modified IL-2 polypeptide. The conjugation handle can be any of the conjugation handles provided herein. In some embodiments, the linker is site-specifically covalently attached to the non-natural amino acid. Non-limiting examples of amino acid residues that comprise a conjugation handle can be found, for example, at least in WO2015054658(A1), WO2014036492(A1), and WO2021133839(A1), WO2006069246(A2), and WO2007079130(A2), each of which is incorporated by reference as if set forth herein in its entirety.

[0233] In some embodiments, the linker is attached to an amino acid residue substituted with a natural amino acid. In some embodiments, the linker is attached to an amino acid residue substituted with a cysteine, lysine, or tyrosine residue. In some embodiments, the linker is attached to a residue substituted with a cysteine ​​residue. In some embodiments, the linker is attached to an amino acid residue substituted with a lysine residue. In some embodiments, the linker is attached to an amino acid residue substituted with a tyrosine residue.

[0234] In some embodiments, the linker is attached to the N-terminus, A1, K35, F42Y, K43, F44Y, or Y45 amino acid residues. In some embodiments, the linker is attached to the N-terminus, A1, F42Y, or Y45 amino acid residues. In some embodiments, the linker is attached to the amino terminal residue. In some embodiments, the linker is attached to amino acid residue A1. In some embodiments, the linker is attached to amino acid residue F42Y. In some embodiments, the linker is attached to amino acid residue Y45. Modifications to IL-2 Polypeptides

[0235] In some embodiments, the modified IL-2 polypeptides described herein contain one or more modified amino acid residues. Such modifications can take the form of mutations of the wild-type IL-2 polypeptide, such as the amino acid sequence of SEQ ID NO:1, additions and / or deletions of amino acids from the sequence of SEQ ID NO:1, or additions of moieties to amino acid residues. In some embodiments, the modified IL-2 polypeptides described herein include a deletion of a first amino acid from the sequence of SEQ ID NO:1. In some embodiments, the modified IL-2 polypeptides described herein include a C125S mutation, using the sequence of SEQ ID NO:1 as a reference sequence. Moieties that may be added to amino acid residues include, but are not limited to, polymers, linkers, spacers, and combinations thereof. When added to specific amino acid residues, these moieties can modulate the activity or other properties of the modified IL-2 polypeptide compared to wild-type IL-2. In some embodiments, the modified IL-2 polypeptides include two modifications within the range of amino acid residues 35-46. In some embodiments, one modification is within the range of amino acid residues 40-43. In some embodiments, one modification is at amino acid residue 42. In some embodiments, one modification is within amino acid residues 44 to 46. In some embodiments, one modification is at amino acid residue 45.

[0236] In some embodiments, the modified IL-2 polypeptides described herein contain one or more modified amino acid residues. For example, the addition of a polymer to a particular amino acid residue may have the effect of disrupting the binding interaction of the modified IL-2 polypeptide with IL-2R, particularly the αβγ complex. In some embodiments, the residues to which a polymer is added to disrupt this interaction include F42 and Y45. In some embodiments, the polymer added to residue 42 or 45 also acts as a linker between the IL-2 polypeptide and the polypeptide that binds to PD-1.

[0237] In some embodiments, the polymer is a water-soluble polymer, e.g., a polyethylene glycol (PEG) polymer. The F42 residue can be mutated to another residue to facilitate the addition of a PEG polymer (or linker), e.g., to a tyrosine residue. Polymers may be added to one or both of residues F42 and Y45, or variants thereof. These polymers may be in the form of a linker between the IL-2 polypeptide and the polypeptide that selectively binds to PD-1, or may be additional polymers in addition to the linker. In some embodiments, the modified IL-2 polypeptide comprises one or more amino acid mutations selected from Table 2.

[0238] [Table 4]

[0239] In some embodiments, the modified IL-2 polypeptides provided herein contain one or more amino acid mutations selected from Table 3.

[0240] [Table 5]

[0241] In some embodiments, the modified IL-2 polypeptides provided herein comprise one or more polymers selected from Table 4.

[0242] [Table 6]

[0243] In some embodiments, modified IL-2 polypeptides provided herein include mutations and polymers provided in Table 5. In some embodiments, one or more of the polymers in the table is replaced with or includes a portion of the linker attached to the polypeptide that binds PD-1.

[0244] [Table 7]

[0245] In some cases, the modified IL-2 polypeptides described herein may be recombinant. The modified IL-2 polypeptides described herein may also be chemically synthesized rather than expressed as recombinant polypeptides. Synthetic IL-2 polypeptides are described at least in WO2021140416(A2), US20190023760(A1), and Asahina et al., Angew.Chem.Int.Ed.2015,54,8226-8230, each of which is incorporated by reference as if set forth herein in its entirety. The modified IL-2 polypeptides can be produced by synthesizing one or more fragments of a full-length modified IL-2 polypeptide, ligating the fragments together, and folding the ligated full-length polypeptide. In some embodiments, the modified IL-2 polypeptide comprises an F42Y mutation in the amino acid sequence, a first PEG polymer of about 500 Da covalently attached to amino acid residue F42Y, a second PEG polymer of about 500 Da covalently attached to amino acid residue Y45, and an optional third PEG polymer of about 6 kDa covalently attached to the N-terminus of the modified IL-2 polypeptide. In some embodiments, the PEG polymer comprises part of a linker connecting the IL-2 polypeptide to a polypeptide that binds PD-1.

[0246] In some embodiments, the chemically synthesized IL-2 polypeptide comprises a conjugation handle attached to one or more residues to facilitate attachment of a linker to a polypeptide that selectively binds to PD-1. The conjugation handle may be any such conjugation handle provided herein and may be attached to any residue to which a linker may be attached. In some embodiments, the conjugation handle is attached to residue 42 or 45 of the IL-2 polypeptide. In some embodiments, the conjugation handle comprises an azide or an alkyne. Alternatively, in some embodiments, the conjugation handle is incorporated into a non-natural amino acid or a modified natural amino acid of the recombinant IL-2 polypeptide. Recombinant IL-2 polypeptides with non-natural amino acids can be made using methods described, for example, in Patent Cooperation Treaty Publications WO2016115168, WO2002085923, WO2005019415, and WO2005003294.

[0247] In some embodiments, the modified IL-2 polypeptide, when administered to a subject, inhibits T effectors (T eff In some embodiments, the modified IL-2 polypeptide, when administered to a subject, enhances regulatory T cells (T reg ) while preventing preferential activation of T eff or enhances NK cell proliferation. In some embodiments, the modified IL-2 polypeptide increases CD8+ T and NK cells. In some embodiments, the modified IL-2 polypeptide increases T close to 1 when administered to a subject. eff / T reg This gives rise to the ratio:

[0248] In one aspect, a modified polypeptide is described herein, comprising a modified interleukin-2 (IL-2) polypeptide, the modified IL-2 polypeptide comprising a first polymer covalently attached thereto. A modified polypeptide is described herein, comprising a modified interleukin-2 (IL-2) polypeptide, the modified IL-2 polypeptide comprising a first polymer covalently attached to residue F42Y, the residue position numbering of the modified IL-2 polypeptide being based on SEQ ID NO: 1 as a reference sequence. In some embodiments, the first polymer is the same as the linker that connects the IL-2 polypeptide and the polypeptide that selectively binds to PD-1. In some embodiments, the first polymer is an additional polymer that is different from the linker. In another aspect, described herein is a modified polypeptide comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide exhibits reduced functional activity against cells expressing a high affinity heterotrimeric IL-2 receptor (IL-2Rα / β / γ) and greater functional activity against cells expressing an intermediate affinity heterodimeric IL-2 receptor (IL-2Rβ / γ), as measured by median effective concentration (EC50) in an agonist assay against primary Tregs (expressing IL-2Rα / β / γ receptors) and resting CD8+ Teffs (expressing IL-2Rβ / γ receptors), wherein the ratio of the EC50 value of the modified IL-2 polypeptide for IL-2Rα to the EC50 value of the modified IL-2 polypeptide for IL-2Rβ is less than 3:1. In some cases, the modified IL-2 polypeptide is a modified IL-2 polypeptide described herein, a modified IL-2 polypeptide provided in Table 8 or Table 5, a modified IL-2 polypeptide having a mutation provided in Table 2 or Table 3, and / or a modified IL-2 polypeptide having a polymer provided in Table 4.

[0249] biological activity In some embodiments, immunoconjugates comprising a modified IL-2 polypeptide conjugated to an anti-PD-1 polypeptide provided herein exhibit enhanced affinity for immune cells expressing high levels of PD-1 (CD279) located within tumors (e.g., tumor-infiltrating lymphocytes (TILs)) or tumor-perfused lymph nodes, but exhibit reduced affinity for peripheral immune cells expressing low or intermediate levels of surface PD-1.

[0250] In some embodiments, an immunoconjugate comprising a modified IL-2 polypeptide conjugated to an anti-PD-1 polypeptide exhibits enhanced exposure in a tumor or tumor-perfused lymph nodes compared to exposure in plasma compared to a non-targeted IL-2 polypeptide or a non-targeted IL-2 immunoconjugate. In some embodiments, the ratio of exposure in a tumor or tumor-perfused lymph nodes to exposure in plasma or serum of a PD1-IL2 immunoconjugate is at least 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold or more compared to a non-targeted IL-2 immunoconjugate or an unconjugated IL-2 polypeptide. In some embodiments, the half-life of a PD1-IL2 immunoconjugate in a tumor or tumor-perfused lymph nodes is 10-fold to 100-fold higher compared to its half-life in plasma or serum. In some embodiments, the ratio of exposure in the tumor or tumor-perfused lymph nodes to exposure in plasma or serum of a PD1-IL2 immunoconjugate is 10-fold to 100-fold, 20-fold to 100-fold, 30-fold to 100-fold, 40-fold to 100-fold, 20-fold to 75-fold, 30-fold to 75-fold, 40-fold to 100-fold, or 40-fold to 75-fold higher compared to a non-targeted IL-2 immunoconjugate or unconjugated IL-2 polypeptide.

[0251] In some embodiments, the PD1-IL2 immunoconjugates exhibit an increased ratio of tumor or tumor-perfused lymph node exposure to plasma or serum exposure compared to an IL-2 immunoconjugate or IL-2 polypeptide that does not target PD-1. In some embodiments, the ratio of tumor or tumor-perfused lymph node exposure to plasma or serum exposure of the anti-PD1-IL-2 immunoconjugate is at least 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold higher compared to an IL-2 immunoconjugate or IL-2 polypeptide that does not target PD-1. In some embodiments, the ratio of tumor or tumor-perfused lymph node exposure to plasma or serum exposure of the anti-PD1-IL-2 immunoconjugate is 10-fold to 100-fold higher compared to an IL-2 immunoconjugate or IL-2 polypeptide that does not target PD-1. In some embodiments, the ratio of tumor or tumor-perfused lymph node exposure to plasma or serum exposure of a PD1-IL2 immunoconjugate is 10-fold to 100-fold, 20-fold to 100-fold, 30-fold to 100-fold, 40-fold to 100-fold, 20-fold to 75-fold, 30-fold to 75-fold, 40-fold to 100-fold, or 40-fold to 75-fold higher than a non-targeted IL-2 immunoconjugate or IL-2 polypeptide.

[0252] In some embodiments, the ratio of expansion of immune cell populations in tumors (e.g., tumor infiltrating lymphocytes (TILs)) and tumor-perfused lymph nodes to expansion of immune cell populations in other tissues (e.g., the same type of immune cells in other tissues) induced by the PD1-IL2 immunoconjugate is at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 or more. In some embodiments, the ratio of expansion of immune cell populations in tumors and tumor-perfused lymph nodes to expansion of immune cell populations in other tissues induced by the PD1-IL2 immunoconjugate is about 1.5-10, about 2-10, about 2.5-10, about 3-10, about 1.5-8, about 2-8, about 2.5-8, about 3-8, about 1.5-6, about 2-6, about 2.5-6, or about 3-6. In some embodiments, the immune cell population is at least one selected from naive CD8+ cells, CD4+ helper cells, CD8+ central memory cells, CD8+ effector memory cells, NK cells, NKT cells, or any combination thereof. In some embodiments, the ratio is measured at a specified time (e.g., 6 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days) after administration.

[0253] In some embodiments, a PD1-IL2 immunoconjugate comprising a modified IL-2 polypeptide conjugated to an anti-PD-1 polypeptide exhibits enhanced potency due to cis-signaling of the modified IL-2 polypeptide to cells expressing high levels of PD-1 compared to cells that do not express PD-1 or express only moderate levels. In some embodiments, for the PD1-IL2 immunoconjugate, the ratio of the EC50 value of IL-2 pathway engagement in cells that do not express PD-1 or express only moderate levels (pSTAT5 assay) to the EC50 value of IL-2 pathway engagement in cells that express high levels of PD-1 (pSTAT5 assay) is at least 10, at least 50, at least 100, at least 250, at least 500, at least 750, at least 1000, at least 1500, at least 2000, at least 2500, or at least 3000. In some embodiments, the ratio of the EC50 value for IL-2 pathway engagement (pSTAT5 assay) in cells expressing high levels of PD-1 to the EC50 value for IL-2 pathway engagement (pSTAT5 assay) in cells that do not express PD-1 or express only moderate levels of PD-1 for a PD1-IL2 immunoconjugate is at least 10-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, or at least 1000-fold greater than an IL-2 immunoconjugate or IL-2 polypeptide that does not target PD-1.

[0254] In some embodiments, the modified IL-2 polypeptide exhibits an activity that differs from wild-type IL-2. These modified biological activities provided herein below, in some embodiments, apply not only to the IL-2 polypeptide alone (e.g., not conjugated or otherwise linked to a polypeptide that binds PD-1), but also when the IL-2 polypeptide is conjugated or otherwise linked to a polypeptide that binds PD-1 (e.g., the modified biological activity is retained when conjugated or linked). Thus, when a modified IL-2 polypeptide is described herein as having an indicated activity, it is also contemplated that the immunocytokine compositions provided herein (e.g., an IL-2 polypeptide linked to a polypeptide that binds PD-1) have the same activity.

[0255] In some embodiments, the modified IL-2 polypeptides described herein can expand CD4+ helper cells, CD8+ central memory cells, CD8+ effector memory cells, naive CD8+ cells, natural killer (NK) cells, natural killer T (NKT) cell populations, or combinations thereof. In some cases, the modified IL-2 polypeptide is a modified IL-2 polypeptide described herein, a modified IL-2 polypeptide provided in Table 8 or Table 5, a modified IL-2 polypeptide having a mutation provided in Table 2 or Table 3, and / or a modified IL-2 polypeptide having a polymer provided in Table 4.

[0256] In some embodiments, the modified IL-2 polypeptides described herein are capable of inhibiting effector T cells (T eff In some embodiments, the modified IL-2 polypeptide is used to expand a cell population of T cells when the modified IL-2 polypeptide is contacted with the population. effThe cell population of cells is expanded by at least 1%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, or at least 200%. In some embodiments, the modified IL-2 polypeptide is a T cell that is expressed by a T cell population when the modified IL-2 polypeptide is contacted with the population. eff The cell population of cells is expanded by at least 20%. In some embodiments, the modified IL-2 polypeptide enhances T cell proliferation and proliferation when the modified IL-2 polypeptide is contacted with the population. eff The cell population of cells is expanded by at least 30%. In some embodiments, the modified IL-2 polypeptide enhances T cell proliferation and proliferation when the modified IL-2 polypeptide is contacted with the population. eff The cell population of cells is expanded by at least 40%. In some embodiments, the modified IL-2 polypeptide enhances T cell proliferation and proliferation when the modified IL-2 polypeptide is contacted with the population. eff The cell population of cells is expanded by at least 50%. In some embodiments, the modified IL-2 polypeptide enhances T cell proliferation and proliferation when the modified IL-2 polypeptide is contacted with the population. eff The cell population of cells is expanded by at least 100%. In some embodiments, the modified IL-2 polypeptide enhances T cell proliferation and proliferation when the modified IL-2 polypeptide is contacted with the population. eff Expand the cell population by at least 200%.

[0257] In some embodiments, the modified IL-2 polypeptides described herein are capable of inhibiting effector T cells (T eff In some embodiments, the modified IL-2 polypeptide is used to expand a cell population of T cells when the modified IL-2 polypeptide is contacted with the population. eff The cell population of cells is expanded by at most 5%, at most 10%, at most 20%, at most 30%, at most 40%, at most 50%, at most 75%, at most 100%, or at most 500%. In some embodiments, the modified IL-2 polypeptide expands the T cell population when the modified IL-2 polypeptide is contacted with the population.eff In some embodiments, the modified IL-2 polypeptide is administered to a cell population of cells by at most 5%. eff In some embodiments, the modified IL-2 polypeptide enhances T cell proliferation and / or proliferation by at least 20% when the modified IL-2 polypeptide is contacted with the population. eff In some embodiments, the modified IL-2 polypeptide expands the cell population of cells by at most 50% when the modified IL-2 polypeptide is contacted with the population. eff In some embodiments, the modified IL-2 polypeptide enhances T cell proliferation and / or proliferation by up to 100% when the modified IL-2 polypeptide is contacted with the population. eff Expand the cell population by up to 500%.

[0258] In some embodiments, the T cells expanded by the modified IL-2 polypeptides described herein reg T for cell population expansion eff The cell population expansion ratio of cells is about 0.1 to about 15, about 0.5 to about 10, about 0.75 to about 5, or about 1 to about 2. In some embodiments, the T cells expanded by the modified IL-2 polypeptide are reg T for cell population expansion eff The cell population expansion ratio of cells is 0.1 to 15. In some embodiments, the T cells expanded with the modified IL-2 polypeptide are reg T for cell population expansion eff The cell population expansion ratio of cells is 0.1-0.5, 0.1-0.75, 0.1-1, 0.1-2, 0.1-5, 0.1-10, 0.1-15, 0.5-0.75, 0.5-1, 0.5-2, 0.5-5, 0.5-10, 0.5-15, 0.75-1, 0.75-2, 0.75-5, 0.75-10, 0.75-15, 1-2, 1-5, 1-10, 1-15, 2-5, 2-10, 2-15, 5-10, 5-15, 10-15, or any number or range therebetween.reg T for cell population expansion eff The cell population expansion ratio of cells is 0.1, 0.5, 0.75, 1, 2, 5, 10, or 15. In some embodiments, the T cells expanded by the modified IL-2 polypeptide are reg T for cell population expansion eff The cell population expansion ratio of cells is 0.1, 0.5, 0.75, 1, 2, 5, or 10. In some embodiments, the T cells expanded by the modified IL-2 polypeptide are reg T for cell population expansion eff The cell population expansion ratios of cells are 0.5, 0.75, 1, 2, 5, 10, or 15.

[0259] In some embodiments, the cell population expanded by the modified IL-2 polypeptides provided herein is an in vitro cell population, an in vivo cell population, or an ex vivo cell population. In some embodiments, the cell population is an in vitro cell population. In some embodiments, the cell population is an in vivo cell population. In some embodiments, the cell population is an ex vivo cell population. The cell population can be a population of CD4+ helper cells, CD8+ central memory cells, CD8+ effector memory cells, naive CD8+ cells, natural killer (NK) cells, natural killer T (NKT) cells, or a combination thereof.

[0260] In some embodiments, the level of cells is measured 1 hour after injection of the modified IL-2 polypeptide. In some embodiments, the level of cells is measured 2 hours after injection of the modified IL-2 polypeptide. In some embodiments, the level of cells is measured 4 hours after injection of the modified IL-2 polypeptide. In some embodiments, the level of cells is measured 30 minutes after injection of the modified IL-2 polypeptide (e.g., for in vitro experiments). In some embodiments, the level of cells is measured at extended time points (e.g., 6 hours, 12 hours, 24 hours, 72 hours, 96 hours, 120 hours, 144 hours, 168 hours, etc.), particularly for in vivo experiments.

[0261] In some embodiments, the immunoconjugate compositions provided herein (e.g., a polypeptide that binds PD-1 (e.g., an anti-PD-1 antibody such as, for example, pembrolizumab or LZM-009) linked to an IL-2 polypeptide via a linker) maintain the binding affinity associated with at least one of the components after formation of the bond between the two groups. For example, in an immunoconjugate composition comprising an anti-PD-1 antibody or antigen-binding fragment linked to an IL-2 polypeptide, in some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof retains binding to one or more Fc receptors. In some embodiments, the compositions exhibit binding to one or more Fc receptors that is reduced by about 5-fold or less, about 10-fold or less, about 15-fold or less, or about 20-fold or less compared to the unconjugated antibody. In some embodiments, the one or more Fc receptors are FcRn receptors, FcγRI receptors (CD64), FcγRIIa receptors (CD32α), FcγRIIβ receptors (CD32β), FcγRIII receptors (CD16a), or any combination thereof. In some embodiments, binding of the composition to each of the FcRn receptors, FcγRI receptors (CD64), FcγRIIa receptors (CD32α), and FcγRIIβ receptors (CD32β), FcγRIII receptors (CD16a) is reduced by about 10-fold or less compared to the unconjugated antibody.

[0262] In some embodiments, binding of a polypeptide that binds to PD-1 (e.g., an antibody) to PD-1 is not substantially affected by conjugation to an IL-2 polypeptide, hi some embodiments, binding of the polypeptide to PD-1 is reduced by about 5% or less compared to the unconjugated antibody.

[0263] site-specific modification In some embodiments, the modified IL-2 polypeptide described herein comprises one or more modifications at one or more amino acid residues. In some embodiments, the residue position numbering of the modified IL-2 polypeptide is based on SEQ ID NO: 1 as a reference sequence. In some embodiments, the amino acid residue position numbering of the modified IL-2 polypeptide is based on a wild-type human IL-2 polypeptide as a reference sequence. In some embodiments, the modified IL-2 polypeptide is a modified IL-2 polypeptide described herein, a modified IL-2 polypeptide provided in Table 8 or Table 5, a modified IL-2 polypeptide having a mutation provided in Table 2 or Table 3, and / or a modified IL-2 polypeptide having a polymer provided in Table 4.

[0264] Modifications to the polypeptides described herein include mutations of the wild-type version of the protein or protein fragment, addition of various functionalities, deletion of amino acids, addition of amino acids, or any other alteration. Functional groups that can be added to the polypeptide include polymers, linkers, alkyl groups, detectable molecules such as chromophores or fluorophores, reactive functional groups, or any combination thereof. In some embodiments, functional groups are added to individual amino acids of the polypeptide. In some embodiments, functional groups are added site-specifically to the polypeptide. In some embodiments, the functional group comprises at least a portion of a linker used to link the IL-2 polypeptide to a polypeptide that selectively binds to PD-1.

[0265] In some embodiments, the modified IL-2 polypeptide described herein comprises a modification at an amino acid residue from the region of residues 35-46, where the numbering of the residues is based on SEQ ID NO:1. In some embodiments, the modification is at K35, L36, T37, R38, M39, L40, T41, F42, K43, F44, Y45, or M46. In some embodiments, the modification is at F42. In some embodiments, the modification is Y45. In some embodiments, the modified IL-2 polypeptide comprises a modification at the N-terminal residue. In some embodiments, the modified IL-2 polypeptide comprises a C125S mutation. In some embodiments, the modified IL-2 polypeptide comprises an A1 deletion. In some embodiments, the modification comprises the attachment of a linker that is used to attach the IL-2 polypeptide to a polypeptide that selectively binds to PD-1.

[0266] In some embodiments, the modified IL-2 polypeptide described herein comprises a first polymer covalently attached to any amino acid residue between 35-46, where the amino acid residue position numbering of the modified IL-2 polypeptide is based on SEQ ID NO: 1 as the reference sequence. In some embodiments, the modified IL-2 polypeptide comprises a first polymer covalently attached to any amino acid residue between 39-43. In some embodiments, the modified IL-2 polypeptide comprises a first polymer covalently attached to amino acid residue F42. In some embodiments, the modified IL-2 polypeptide comprises a first polymer covalently attached to amino acid residue F42Y. In some embodiments, the modified IL-2 polypeptide comprises a first polymer covalently attached to any amino acid residue between 44-46. In some embodiments, the modified IL-2 polypeptide comprises a first polymer covalently attached to amino acid residue Y45. In some embodiments, the first polymer is part of a linker that connects the IL-2 polypeptide to a polypeptide that selectively binds to PD-1. In some embodiments, the first polymer is a separate modification from the linker that attaches the IL-2 polypeptide to the polypeptide that selectively binds to PD-1.

[0267] In some embodiments, the modified IL-2 polypeptides described herein comprise one or more PEGylated tyrosines located at amino acid residues in the region from amino acid residue 35 to amino acid residue 45. In some embodiments, the one or more PEGylated tyrosines are located at amino acid residue 42, amino acid residue 45, or both. In some embodiments, one or more PEGylated tyrosines are located at amino acid residue 42. In some embodiments, one or more PEGylated tyrosines are located at amino acid residue 45. In some embodiments, one or more PEGylated tyrosines are located at both amino acid residue 42 and amino acid residue 45. In some embodiments, the modified IL-2 polypeptides comprise two PEGylated tyrosines, each independently having the structure of formula (I). A non-limiting set of modified IL-2 polypeptides provided herein with various linker attachment points and polymers provided herein is shown in Table 7 below.

[0268] [Table 8]

[0269] In some embodiments, the modified IL-2 polypeptide is synthetic. In one aspect, modified IL-2 polypeptides comprising one or more amino acid substitutions are disclosed herein. In some embodiments, the modified IL-2 polypeptide comprises F42Y and Y45. In some embodiments, the modified IL-2 polypeptide comprises a homoserine (Hse) residue located at any one of amino acid residues 35-45. In some embodiments, the modified IL-2 polypeptide comprises an Hse residue located at any one of amino acid residues 61-81. In some embodiments, the modified IL-2 polypeptide comprises an Hse residue located at any one of amino acid residues 94-114. In some embodiments, the modified IL-2 polypeptide comprises one, two, three, or more Hse residues. In some embodiments, the modified IL-2 polypeptide comprises Hse41, Hse71, Hse104, or a combination thereof. In some embodiments, the modified IL-2 polypeptide comprises Hse41, Hse71, and Hse104. In some embodiments, the modified IL-2 polypeptide comprises at least two amino acid substitutions, the at least two amino acid substitutions being selected from (a) a homoserine (Hse) residue located at any one of amino acid residues 35-45, (b) a homoserine residue located at any one of amino acid residues 61-81, and (c) a homoserine residue located at any one of amino acid residues 94-114. In some embodiments, the modified IL-2 polypeptide comprises Hse41 and Hse71. In some embodiments, the modified IL-2 polypeptide comprises Hse41 and Hse104. In some embodiments, the modified IL-2 polypeptide comprises Hse71 and Hse104. In some embodiments, the modified IL-2 polypeptide comprises Hse41. In some embodiments, the modified IL-2 polypeptide comprises Hse71. In some embodiments, the modified IL-2 polypeptide comprises Hse104. In some embodiments, the modified IL-2 polypeptide comprises one, two, three or more norleucine (Nle) residues.In some embodiments, the modified IL-2 polypeptide comprises an Nle residue located at any one of residues 18-28. In some embodiments, the modified IL-2 polypeptide comprises one or more Nle residues located at any one of amino acid residues 34-50. In some embodiments, the modified IL-2 polypeptide comprises an Nle residue located at any one of amino acid residues 20-60. In some embodiments, the modified IL-2 polypeptide comprises three Nle substitutions. In some embodiments, the modified IL-2 polypeptide comprises Nle23, Nle39 and Nle46. In some embodiments, the modified IL-2 polypeptide comprises SEQ ID NO:3. In some embodiments, the modified IL-2 polypeptide comprises SEQ ID NO:3 with an A1 deletion. In some embodiments, the modified IL-2 polypeptide comprises SEQ ID NO:4. In some embodiments, the modified IL-2 polypeptide comprises an A1 deletion. In some embodiments, the modified IL-2 polypeptide comprises SEQ ID NO:4 with an A1 deletion.

[0270] In some embodiments, the modified IL-2 polypeptide provided herein comprises the amino acid sequence of any one of SEQ ID NOs: 3-23 provided in Table 8. In some embodiments, the modified IL-2 polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 3-23. In some embodiments, the modified IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the modified IL-2 polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to the sequence of SEQ ID NO: 3. In some embodiments, the modified IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the modified IL-2 polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to the sequence of SEQ ID NO: 4. In some embodiments, the modified IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, the modified IL-2 polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to the sequence of SEQ ID NO:9. In some embodiments, the modified IL-2 polypeptide comprises an amino acid sequence of SEQ ID NO:10. In some embodiments, the modified IL-2 polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to the sequence of SEQ ID NO:10. In some embodiments, the modified IL-2 polypeptide comprises an amino acid sequence of SEQ ID NO:11. In some embodiments, the modified IL-2 polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to the sequence of SEQ ID NO:11. In some embodiments, the modified IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO:12. In some embodiments, the modified IL-2 polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the sequence of SEQ ID NO: 12. In some embodiments, the modified IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 13.In some embodiments, the modified IL-2 polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to the sequence of SEQ ID NO: 13. In some embodiments, the modified IL-2 polypeptide comprises an amino acid sequence of SEQ ID NO: 14. In some embodiments, the modified IL-2 polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to the sequence of SEQ ID NO: 14. In some embodiments, the modified IL-2 polypeptide comprises an amino acid sequence of SEQ ID NO: 15. In some embodiments, the modified IL-2 polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to the sequence of SEQ ID NO: 15. In some embodiments, the modified IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 17. In some embodiments, the modified IL-2 polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to the sequence of SEQ ID NO: 17. In some embodiments, the modified IL-2 polypeptide comprises an amino acid sequence of SEQ ID NO: 18. In some embodiments, the modified IL-2 polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to the sequence of SEQ ID NO: 18. In some embodiments, the modified IL-2 polypeptide comprises an amino acid sequence of SEQ ID NO: 19. In some embodiments, the modified IL-2 polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical to the sequence of SEQ ID NO: 19. In some embodiments, the modified IL-2 polypeptide comprises an amino acid sequence of SEQ ID NO: 20. In some embodiments, the modified IL-2 polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the sequence of SEQ ID NO: 20. In some embodiments, the modified IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 21. In some embodiments, the modified IL-2 polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the sequence of SEQ ID NO: 21.In some embodiments, the modified IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 22. In some embodiments, the modified IL-2 polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the sequence of SEQ ID NO: 22. In some embodiments, the modified IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 23. In some embodiments, the modified IL-2 polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the sequence of SEQ ID NO: 23.

[0271] In some embodiments, the modified IL-2 polypeptides described herein comprise at least 3, at least 4, at least 5, at least 6, at least 7, or at least 9 amino acid substitutions. In some embodiments, the modified IL-2 polypeptides comprise 3-9 amino acid substitutions. In some embodiments, the modified IL-2 polypeptides comprise 3 or 4 amino acid substitutions, 3-5 amino acid substitutions, 3-6 amino acid substitutions, 3-7 amino acid substitutions, 3-9 amino acid substitutions, 4 or 5 amino acid substitutions, 4-6 amino acid substitutions, 4-7 amino acid substitutions, 4-9 amino acid substitutions, 5 or 6 amino acid substitutions, 5-7 amino acid substitutions, 5-9 amino acid substitutions, 6 or 7 amino acid substitutions, 6-9 amino acid substitutions, or 7-9 amino acid substitutions. In some embodiments, the modified IL-2 polypeptides comprise 3 amino acid substitutions, 4 amino acid substitutions, 5 amino acid substitutions, 6 amino acid substitutions, 7 amino acid substitutions, or 9 amino acid substitutions. In some embodiments, the modified IL-2 polypeptide contains at most 4 amino acid substitutions, 5 amino acid substitutions, 6 amino acid substitutions, 7 amino acid substitutions, or 9 amino acid substitutions. In some embodiments, the one or more amino acid substitutions are selected from Table 2. In some embodiments, the one or more amino acid substitutions are selected from Table 3. In some embodiments, the modified IL-2 polypeptide is a modified IL-2 polypeptide described herein, a modified IL-2 polypeptide provided in Table 8 or Table 5, a modified IL-2 polypeptide having a mutation provided in Table 2 or Table 3, and / or a modified IL-2 polypeptide having a polymer provided in Table 4.

[0272] In some embodiments, the modified IL-2 polypeptides described herein comprise a second modification. In some embodiments, the modified IL-2 polypeptides comprise a third modification. In some embodiments, the modified IL-2 polypeptides comprise a second and a third modification.

[0273] In some embodiments, a modified IL-2 polypeptide described herein comprises at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 3. In some embodiments, sequence identity is measured by the protein-protein BLAST algorithm using parameters of Matrix BLOSUM62, Gap Costs Existence:11, Extension:1, and Compositional Adjustments Conditional Compositional Score Matrix Adjustment.

[0274] Modified IL-2 polypeptides as described herein may include one or more non-standard amino acids. In some embodiments, in some cases, Tyr45 and / or Phe42 are substituted with a non-standard amino acid. In some embodiments, one or more amino acids located at the positions provided in Table 2 and / or Table 3 are substituted with one or more non-standard amino acids. Non-standard amino acids include, but are not limited to, N-α-(9-fluorenylmethyloxycarbonyl)-L-biphenylalanine (Fmoc-L-Bip-OH) and N-α-(9-fluorenylmethyloxycarbonyl)-O-benzyl-L-tyrosine (Fmoc-L-Tyr(Bzl)-OH).Exemplary non-standard amino acids include p-acetyl-L-phenylalanine, p-iodo-L-phenylalanine, p-methoxyphenylalanine, O-methyl-L-tyrosine, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcp-serine, L-Dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-boronophenylalanine, O-propargyltyrosine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-bromophenylalanine, selenoside, and the like. The non-standard amino acids include steine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, azido-lysine (AzK), analogs of tyrosine amino acids; analogs of glutamine amino acids; analogs of phenylalanine amino acids; analogs of serine amino acids; analogs of threonine amino acids; alkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynyl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or amino substituted amino acids, β-amino acids; cyclic amino acids other than proline or histidine; aromatic amino acids other than phenylalanine, tyrosine, or tryptophan; or combinations thereof. In some embodiments, the non-standard amino acids are selected from β-amino acids, homoamino acids, cyclic amino acids, and amino acids with derivatized side chains. In some embodiments, the non-standard amino acid is β-alanine, β-aminopropionic acid, piperidinic acid, aminocaproic acid, aminoheptanoic acid, aminopimelic acid, desmosine, diaminopimelic acid, N. α -Ethylglycine, N α -ethylasparagine, hydroxylysine, allo-hydroxylysine, isodesmosine, allo-isoleucine, ω-methylarginine, N α -Methylglycine, Nα -Methylisoleucine, N α -Methylvaline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N α -Acetylserine, N α -formylmethionine, 3-methylhistidine, 5-hydroxylysine, and / or other similar amino acids. In some embodiments, Tyr45 and / or Phe42 are replaced with modified tyrosine residues. In some embodiments, the modified tyrosine residues include amino, azido, allyl, ester and / or amide functional groups. In some embodiments, the modified tyrosine residues at positions 42 or 45 are used as attachment points for linkers that connect the IL-2 polypeptide to a polypeptide that selectively binds PD-1. In some embodiments, the modified tyrosine residues at positions 42 and / or 45 have a structure constructed from a precursor structure 1, structure 2, structure 3, structure 4, or structure 5, where structure 1 is

[0275] [ka] and Structure 2 is

[0276] [ka] and Structure 3 is

[0277] [ka] and Structure 4 is

[0278] [ka] and Structure 5 is

[0279] [ka] It is.

[0280] polymer In some embodiments, the modified IL-2 polypeptides described herein comprise one or more polymers covalently attached thereto. In some embodiments, the modified IL-2 polypeptides described comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polymers covalently attached to the modified IL-2 polypeptide. In some embodiments, the modified IL-2 polypeptides described comprise a first polymer. In some embodiments, the first polymer comprises at least a portion of a linker that connects the IL-2 polypeptide to a polypeptide that selectively binds to PD-1. In some cases, the modified IL-2 polypeptide is a modified IL-2 polypeptide described herein, a modified IL-2 polypeptide provided in Table 4, a modified IL-2 polypeptide having a mutation provided in Table 2 or Table 3, and / or a modified IL-2 polypeptide having a polymer provided in Table 3.

[0281] In some embodiments, the first polymer comprises a water-soluble polymer. In some embodiments, the water-soluble polymer comprises a poly(alkylene oxide), a polysaccharide, a poly(vinylpyrrolidone), a poly(vinyl alcohol), a polyoxazoline, a poly(acryloylmorpholine), or a combination thereof. In some embodiments, the water-soluble polymer is a poly(alkylene oxide). In some embodiments, the water-soluble polymer is a polysaccharide. In some embodiments, the water-soluble polymer is a poly(ethylene oxide).

[0282] In some embodiments, the modified IL-2 polypeptide described herein comprises a first polymer covalently attached to the N-terminus of the IL-2 polypeptide. In some embodiments, the modified IL-2 polypeptide comprises a second polymer covalently attached thereto. In some embodiments, the modified IL-2 polypeptide comprises a second and a third polymer covalently attached thereto. In some embodiments, the second polymer is covalently attached to amino acid residue 42 or 45, where the amino acid residue position numbering of the modified IL-2 polypeptide is based on SEQ ID NO: 1 as the reference sequence. In some embodiments, the second polymer is covalently attached to amino acid residue F42Y or Y45, where the amino acid residue position numbering of the modified IL-2 polypeptide is based on SEQ ID NO: 1 as the reference sequence. In some embodiments, the second and the third polymer are covalently attached to amino acid residues 42 and 45, where the amino acid residue position numbering of the modified IL-2 polypeptide is based on SEQ ID NO: 1 as the reference sequence. In some embodiments, the second and third polymers are covalently attached to amino acid residues F42Y and Y45, where the amino acid residue position numbering of the modified IL-2 polypeptide is based on SEQ ID NO: 1 as the reference sequence. In some embodiments, at least one of the first, second or third polymers comprises at least a portion of a linker used to link the IL-2 polypeptide to a polypeptide that selectively binds to PD-1.

[0283] In some embodiments, the attached polymer, such as the first polymer, has a weight average molecular weight of about 120 Daltons to about 1,000 Daltons. In some embodiments, the polymer has a weight average molecular weight of about 120 daltons to about 250 daltons, about 120 daltons to about 300 daltons, about 120 daltons to about 400 daltons, about 120 daltons to about 500 daltons, about 120 daltons to about 1,000 daltons, about 250 daltons to about 300 daltons, about 250 daltons to about 400 daltons, about 250 daltons to about 500 daltons, about 250 daltons to about 1,000 daltons, about 300 daltons to about 400 daltons, about 300 daltons to about 500 daltons, about 300 daltons to about 1,000 daltons, about 400 daltons to about 500 daltons, about 400 daltons to about 1,000 daltons, or about 500 daltons to about 1,000 daltons. In some embodiments, the polymer has a weight average molecular weight of about 120 daltons, about 250 daltons, about 300 daltons, about 400 daltons, about 500 daltons, or about 1,000 daltons. In some embodiments, the polymer has a weight average molecular weight of at least about 120 daltons, about 250 daltons, about 300 daltons, about 400 daltons, or about 500 daltons. In some embodiments, the polymer has a weight average molecular weight of at most about 250 daltons, about 300 daltons, about 400 daltons, about 500 daltons, or about 1,000 daltons.

[0284] In some embodiments, the first polymer comprises a water-soluble polymer. In some embodiments, the water-soluble polymer comprises a poly(alkylene oxide), a polysaccharide, a poly(vinylpyrrolidone), a poly(vinyl alcohol), a polyoxazoline, a poly(acryloylmorpholine), or a combination thereof. In some embodiments, the water-soluble polymer is a poly(alkylene oxide), such as a polyethylene glycol (e.g., polyethylene oxide). In some embodiments, each water-soluble polymer is a polyethylene glycol. In some embodiments, the water-soluble polymer comprises a modified poly(alkylene oxide). In some embodiments, the modified poly(alkylene oxide) comprises one or more linker groups. In some embodiments, the one or more linker groups comprise a bifunctional linker, such as an amide group, an ester group, an ether group, a thioether group, a carbonyl group. In some embodiments, the one or more linker groups comprise an amide linker group. In some embodiments, the modified poly(alkylene oxide) comprises one or more spacer groups. In some embodiments, the spacer group comprises a substituted or unsubstituted C1-C6 alkylene group. In some embodiments, the spacer group comprises --CH2-, -CH2CH2-, or -CH2CH2CH2-. In some embodiments, the linker group is the product of a bioorthogonal reaction (e.g., a biocompatible and selective reaction). In some embodiments, the bioorthogonal reaction is a metal-mediated process such as a Cu(I)-catalyzed or "copper-free" alkyne-azide triazole-forming reaction, Staudinger ligation, an inverse electro-demanded Diels-Alder (IEDDA) reaction, "photoclick" chemistry, or olefin metathesis and Suzuki-Miyaura or Sonogashira cross-coupling. In some embodiments, the first polymer is attached to the IL-2 polypeptide via click chemistry. In some embodiments, the first polymer comprises at least a portion of a linker that attaches the IL-2 polypeptide to a polypeptide that selectively binds PD-1.

[0285] In some embodiments, the modified IL-2 polypeptides provided herein comprise reactive groups that facilitate conjugation of the modified IL-2 polypeptides to derivatized molecules or moieties, such as antibodies and polymers. In some embodiments, the reactive group comprises one or more of an active ester derived from a carboxylic acid, a mixed anhydride, an acyl halide, an acyl azide, an alkyl halide, an N-maleimide, an imino ester, an isocyanate, and an isothiocyanate. In some embodiments, the reactive group comprises an azide. In some embodiments, the reactive group forms part of a linker that links the IL-2 polypeptide to a polypeptide that selectively binds to PD-1.

[0286] In some embodiments, the modified IL-2 polypeptides provided herein comprise a chemical reagent covalently attached to an amino acid residue. In some embodiments, the chemical reagent comprises a bioorthogonal reagent. In some embodiments, the chemical reagent comprises an azide. In some embodiments, the chemical reagent comprises an alkyne. In some embodiments, the chemical reagent is attached to amino acid residues 35-46, where the position numbering of the amino acid residues is based on SEQ ID NO:1 as a reference sequence. In some embodiments, the chemical reagent is attached to amino acid residues 39-43, where the position numbering of the amino acid residues is based on SEQ ID NO:1 as a reference sequence. In some embodiments, the chemical reagent is attached to amino acid residue 42, where the position numbering of the amino acid residues is based on SEQ ID NO:1 as a reference sequence. In some embodiments, the chemical reagent is attached to amino acid residue F42Y, where the position numbering of the amino acid residues is based on SEQ ID NO:1 as a reference sequence. In some embodiments, the chemical reagent is attached to amino acid residues 44-46, where the position numbering of the amino acid residues is based on SEQ ID NO:1 as a reference sequence. In some embodiments, the chemical reagent is attached to amino acid residue 45, where the amino acid residue position numbering is based on SEQ ID NO: 1 as the reference sequence. In some embodiments, the chemical reagent is attached to any of the amino acid residues shown in Table 2 or Table 3. In some embodiments, the chemical reagent forms part of a linker that connects the IL-2 polypeptide to a polypeptide that selectively binds to PD-1.

[0287] In some embodiments, the water soluble polymer comprises 1-10 polyethylene glycol chains.

[0288] In some embodiments, the modified IL-2 polypeptide described herein further comprises a second polymer covalently attached to the modified IL-2 polypeptide. In some embodiments, the second polymer is covalently attached to the amino acid residue region from residue 40 to residue 50. In some embodiments, the second polymer is covalently attached to amino acid residue Y45. In some embodiments, the second polymer is covalently attached to the N-terminus of the modified IL-2 polypeptide. In some embodiments, the second polymer comprises at least a portion of a linker that links the IL-2 polypeptide to a polypeptide that selectively binds to PD-1.

[0289] In some embodiments, the second polymer has a weight average molecular weight of about 120 daltons to about 1,000 daltons. In some embodiments, the second polymer has a weight average molecular weight of about 120 daltons to about 250 daltons, about 120 daltons to about 300 daltons, about 120 daltons to about 400 daltons, about 120 daltons to about 500 daltons, about 120 daltons to about 1,000 daltons, about 250 daltons to about 300 daltons, about 250 daltons to about 400 daltons, about 250 daltons to about 500 daltons, about 250 daltons to about 1,000 daltons, about 300 daltons to about 400 daltons, about 300 daltons to about 500 daltons, about 300 daltons to about 1,000 daltons, about 400 daltons to about 500 daltons, about 400 daltons to about 1,000 daltons, or about 500 daltons to about 1,000 daltons. In some embodiments, the second polymer has a weight average molecular weight of about 120 daltons, about 250 daltons, about 300 daltons, about 400 daltons, about 500 daltons, or about 1,000 daltons. In some embodiments, the second polymer has a weight average molecular weight of at least about 120 daltons, about 250 daltons, about 300 daltons, about 400 daltons, or about 500 daltons. In some embodiments, the second polymer has a weight average molecular weight of at most about 250 daltons, about 300 daltons, about 400 daltons, about 500 daltons, or about 1,000 daltons.

[0290] In some embodiments, the second polymer comprises a water soluble polymer. In some embodiments, the water soluble polymer comprises a poly(alkylene oxide), a polysaccharide, a poly(vinylpyrrolidone), a poly(vinyl alcohol), a polyoxazoline, a poly(acryloylmorpholine), or a combination thereof. In some embodiments, the water soluble polymer is a poly(alkylene oxide). In some embodiments, the water soluble polymer is a poly(ethylene oxide). In some embodiments, the second polymer is attached to the IL-2 polypeptide via click chemistry. In some embodiments, the second polymer comprises at least a portion of a linker that attaches the IL-2 polypeptide to a polypeptide that selectively binds PD-1.

[0291] In some embodiments, the second water-soluble polymer comprises 1 to 10 polyethylene glycol chains.

[0292] In some embodiments, the modified IL-2 polypeptide described herein further comprises a third polymer covalently attached to the modified IL-2 polypeptide. In some embodiments, the third polymer is covalently attached to the amino acid residue region from amino acid residue 40 to amino acid residue 50. In some embodiments, the third polymer is covalently attached to amino acid residue Y45. In some embodiments, the third polymer is covalently attached to the N-terminus of the modified IL-2 polypeptide.

[0293] In some embodiments, each polymer comprises a water-soluble polymer. In some embodiments, the water-soluble polymer comprises a poly(alkylene oxide), a polysaccharide, a poly(vinylpyrrolidone), a poly(vinyl alcohol), a polyoxazoline, a poly(acryloylmorpholine), or a combination thereof. In some embodiments, each water-soluble polymer is a poly(alkylene oxide). In some embodiments, each water-soluble polymer is a polyethylene glycol.

[0294] In some embodiments, each of the first polymer and the second polymer independently comprises 1 to 5 polyethylene glycol chains, hi some embodiments, each of the first polymer and the second polymer independently comprises a single polyethylene glycol chain.

[0295] In some embodiments, each of the polyethylene glycol chains is independently linear or branched. In some embodiments, each of the polyethylene glycol chains is linear polyethylene glycol. In some embodiments, each of the polyethylene glycol chains is branched polyethylene glycol. For example, in some embodiments, each of the first and second polymers comprises a linear polyethylene glycol chain.

[0296] In some embodiments, each of the polyethylene glycol chains is independently end-capped with a hydroxy, alkyl, alkoxy, amido or amino group. In some embodiments, each of the polyethylene glycol chains is independently end-capped with an amino group. In some embodiments, each of the polyethylene glycol chains is independently end-capped with an amide group. In some embodiments, each of the polyethylene glycol chains is independently end-capped with an alkoxy group. In some embodiments, each of the polyethylene glycol chains is independently end-capped with an alkyl group. In some embodiments, each of the polyethylene glycol chains is independently end-capped with a hydroxy group.

[0297] In some embodiments, the modified IL-2 polypeptide comprises one or more PEGylated tyrosines having the structure of formula (I):

[0298] [ka] In the formula, n is an integer selected from 4 to 30. In some embodiments, n is 4 to 6, 4 to 8, 4 to 10, 4 to 15, 4 to 20, 4 to 25, 4 to 30, 6 to 8, 6 to 10, 6 to 15, 6 to 20, 6 to 25, 6 to 30, 8 to 10, 8 to 15, 8 to 20, 8 to 25, 8 to 30, 10 to 15, 10 to 20, 10 to 25, 10 to 30, 15 to 20, 15 to 25, 15 to 30, 20 to 25, 20 to 30, or 25 to 30. In some embodiments, n is 4, 6, 8, 10, 15, 20, 25, or 30. In some embodiments, n is at least 4, 6, 8, 10, 15, 20, or 25. In some embodiments, n is at most 6, 8, 10, 15, 20, 25, or 30. In one aspect, the modified IL-2 polypeptides described herein comprise one or two water soluble polymers covalently attached to one or two amino acid residues. For example, in some embodiments, the modified IL-2 polypeptides comprise one or two water soluble polymers having the properties and binding sites shown in Table 6.

[0299] [Table 9] In some embodiments, the water soluble polymer that can be attached to the modified IL-2 polypeptide has the structure of formula (D):

[0300] [ka] Includes.

[0301] In some embodiments, the polymer is synthesized from a suitable precursor material. In some embodiments, the polymer is synthesized from a precursor material of Structure 6, Structure 7, Structure 8, or Structure 9, where Structure 6 is:

[0302] [ka] and Structure 7 is

[0303] [ka] and Structure 8 is

[0304] [ka] and Structure 9 is

[0305] [ka] It is.

[0306] Orthogonal Payloads The anti-PD-1-IL-2 immunoconjugates of the present disclosure may include a bi-orthogonal payload. In one non-limiting example, the anti-PD-1-IL-2 immunoconjugate may include an anti-PD-1 polypeptide, one modified IL-2 polypeptide, and one payload linked to the anti-PD-1 polypeptide by a chemical orthogonal linking group. The orthogonal payload may be an amino acid, an amino acid derivative, a peptide, a protein, a cytokine, an alkyl group, an aryl or heteroaryl group, a therapeutic small molecule drug, a polyethylene glycol (PEG) moiety, a lipid, a sugar, biotin, a biotin derivative, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or a peptide nucleic acid (PNA), any of which may be substituted, unsubstituted, modified, or unmodified. In some embodiments, the orthogonal payload is a therapeutic small molecule. In some embodiments, the orthogonal payload is a PEG moiety. In some embodiments, the orthogonal payload is an additional cytokine, for example, IL-7 or IL-18. In one illustrative example, human IL-7 has the amino acid sequence DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH (SEQ ID NO: 117), or is a modified IL-7. In one illustrative example, human IL-18 has the amino acid sequence YFIAEDDENLESDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 118), or is a modified IL-18. In some cases, a conjugation handle can be added to one or more of Cys68, Glu69, Lys70 of IL-18.

[0307] Pharmaceutical Compositions In one aspect, described herein is a pharmaceutical composition comprising a polypeptide that selectively binds PD-1 linked to a modified IL-2 polypeptide described herein and a pharma- ceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical composition further comprises one or more excipients, including but not limited to, carbohydrates, inorganic salts, antioxidants, surfactants, buffering agents, or any combination thereof. In some embodiments, the pharmaceutical composition further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more excipients, including but not limited to, carbohydrates, inorganic salts, antioxidants, surfactants, buffering agents, or any combination thereof.

[0308] In some embodiments, the pharmaceutical composition further comprises a carbohydrate. In certain embodiments, the carbohydrate is selected from the group consisting of fructose, maltose, galactose, glucose, D-mannose, sorbose, lactose, sucrose, trehalose, cellobiose, raffinose, melezitose, maltodextrin, dextran, starch, mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myo-inositol, cyclodextrin, and combinations thereof.

[0309] Alternatively, or in addition, the pharmaceutical composition further comprises an inorganic salt, hi certain embodiments, the inorganic salt is selected from the group consisting of sodium chloride, potassium chloride, magnesium chloride, calcium chloride, sodium phosphate, potassium phosphate, sodium sulfate, or a combination thereof.

[0310] Alternatively or additionally, the pharmaceutical composition comprises an antioxidant. In certain embodiments, the antioxidant is selected from the group consisting of ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, potassium metabisulfite, propyl gallate, sodium metabisulfite, sodium thiosulfate, vitamin E, 3,4-dihydroxybenzoic acid, and combinations thereof.

[0311] Alternatively or additionally, the pharmaceutical composition further comprises a surfactant, hi certain embodiments, the surfactant is selected from the group consisting of polysorbates, sorbitan esters, lipids, phospholipids, phosphatidylethanolamines, fatty acids, fatty acid esters, steroids, EDTA, zinc, and combinations thereof.

[0312] Alternatively, or in addition, the pharmaceutical composition further comprises a buffer. In certain embodiments, the buffering agent is selected from the group consisting of citric acid, sodium phosphate, potassium phosphate, acetate, ethanolamine, histidine, an amino acid, tartaric acid, succinic acid, fumaric acid, lactic acid, Tris, HEPES, or a combination thereof.

[0313] In some embodiments, the pharmaceutical composition is formulated for parenteral or enteral administration. In some embodiments, the pharmaceutical composition is formulated for intravenous (IV) or subcutaneous (SQ) administration. In some embodiments, the pharmaceutical composition is in lyophilized form.

[0314] In one aspect, described herein is a liquid or lyophilized composition comprising a described polypeptide that selectively binds PD-1 linked to a modified IL-2 polypeptide. In some embodiments, the modified IL-2 polypeptide is a lyophilized powder. In some embodiments, the lyophilized powder is resuspended in a buffer. In some embodiments, the buffer solution comprises a buffer, a sugar, a salt, a surfactant, or any combination thereof. In some embodiments, the buffer solution comprises a phosphate. In some embodiments, the phosphate is sodium Na2HPO4. In some embodiments, the salt is sodium chloride. In some embodiments, the buffer comprises phosphate buffered saline. In some embodiments, the buffer solution comprises mannitol. In some embodiments, the lyophilized powder is suspended in a solution comprising about 10 mM Na2HPO4 buffer, about 0.022% SDS, and about 50 mg / mL mannitol, and having a pH of about 7.5.

[0315] Dosage form The polypeptide that selectively binds PD-1 linked to a modified IL-2 polypeptide described herein may be in a variety of dosage forms. In some embodiments, the polypeptide that selectively binds PD-1 linked to a modified IL-2 polypeptide is administered as a reconstituted lyophilized powder. In some embodiments, the polypeptide that selectively binds PD-1 linked to a modified IL-2 polypeptide is administered as a suspension. In some embodiments, the polypeptide that selectively binds PD-1 linked to a modified IL-2 polypeptide is administered as a solution. In some embodiments, the polypeptide that selectively binds PD-1 linked to a modified IL-2 polypeptide is administered as an injectable solution. In some embodiments, the polypeptide that selectively binds PD-1 linked to a modified IL-2 polypeptide is administered as an IV solution. In some embodiments, the polypeptide that selectively binds PD-1 linked to a modified IL-2 polypeptide is administered by subcutaneous or intramuscular administration.

[0316] Treatment Method In one aspect, described herein is a method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a polypeptide that selectively binds to PD-1 linked to a modified IL-2 polypeptide or pharmaceutical composition as described herein. In some embodiments, the cancer is a solid cancer. The cancer or tumor can be, for example, a primary cancer or tumor or a metastatic cancer or tumor. Treated cancers and tumors include melanoma, lung cancer (e.g., non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), etc.), carcinoma (e.g., cutaneous squamous cell carcinoma (CSCC), urothelial carcinoma (UC), renal cell carcinoma (RCC), hepatocellular carcinoma (HCC), head and neck squamous cell carcinoma (HNSCC), esophageal squamous cell carcinoma (ESCC), gastroesophageal junction (GEJ) cancer, endometrial carcinoma (EC), Merkel cell carcinoma (MCC), etc.), bladder cancer (BC), These include, but are not limited to, microsatellite instability-high (MSI-H) / mismatch repair deficient (dMMR) solid tumors (e.g., colorectal cancer (CRC)), tumor mutation burden-high (TMB-H) solid tumors, triple-negative breast cancer (TNBC), gastric cancer (GC), cervical cancer (CC), pleural mesothelioma (PM), classical Hodgkin lymphoma (cHL), or primary mediastinal large B-cell lymphoma (PMBCL).

[0317] Combination therapy with one or more additional active agents is contemplated herein. In some embodiments, the second therapeutic agent is selected based on tumor type, tumor-derived tissue, tumor stage, or mutation of genes expressed by tumor. For example, anti-PD-1 antibody can be administered in combination with one or more of chemotherapeutic agents, immune checkpoint inhibitors, immune agonists, biological cancer agents, low molecular weight anticancer agents, synthetic peptide anticancer agents, anticancer protein degraders, cancer-specific agents, cytokine therapy, antiangiogenic agents, drugs that target cancer metabolism, antibodies that mark cancer cell surface for destruction, antibody-drug conjugates, cell therapy, commonly used anti-neoplastic agents, CAR-T therapy, oncolytic viruses, non-pharmaceutical therapies, neurotransmission blocking agents, or nerve growth factor blocking agents.

[0318] In some embodiments, the cancer is a solid cancer. In some embodiments, the solid cancer is adrenal gland cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, carcinoid cancer, cervical cancer, colorectal cancer, esophageal cancer, eye cancer, gallbladder cancer, gastrointestinal stromal tumor, germ cell cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrine cancer, oral cancer, oropharyngeal cancer, ovarian cancer, pancreatic cancer, childhood cancer, penile cancer, pituitary cancer, prostate cancer, skin cancer, soft tissue cancer, spinal cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, ureter cancer, uterine cancer, vaginal cancer, or vulvar cancer.

[0319] In some embodiments, the cancer is a blood cancer. In some embodiments, the blood cancer is leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, AIDS-related lymphoma, multiple myeloma, plasmacytoma, post-transplant lymphoproliferative disorder, or Waldenstrom's macroglobulinemia.

[0320] An effective response occurs when a subject experiences partial or total alleviation or reduction of signs or symptoms of the disease, a decrease in tumor burden, or an increase in time to tumor burden (tumor progression), including, but not limited to, an increase in survival time. Predicted progression-free survival can be measured in months to years, depending on prognostic factors, including the number of disease recurrences, stage, and other factors. Prolonged survival can include, but is not limited to, at least 1 month (month), about at least 2 months, about at least 3 months, about at least 4 months. Prolonged survival can include, but is not limited to, at least 6 months, about at least 1 year, about at least 2 years, about at least 3 years, about at least 4 years, about at least 5 years, and the like. Overall survival or progression-free survival can also be measured in months to years. Alternatively, an effective response can be when the subject's symptoms or cancer burden remain quiescent and do not worsen. Further indications for treatment of indications are described in more detail below. In some examples, the cancer or tumor is reduced by at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0321] In some embodiments, the polypeptide that selectively binds PD-1 linked to a modified IL-2 polypeptide is administered in a single dose of an effective amount of the modified IL-2 polypeptide, and in further embodiments, (i) the polypeptide that selectively binds PD-1 linked to a modified IL-2 polypeptide is administered once daily, or (ii) the polypeptide that selectively binds PD-1 linked to a modified IL-2 polypeptide is administered multiple times over the course of a day to the subject. In some embodiments, the polypeptide that selectively binds to PD-1 linked to a modified IL-2 polypeptide is administered daily, every other day, three times a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every three days, once every four days, once every five days, once every six days, every other week, three times a week, four times a week, five times a week, six times a week, once a month, twice a month, three times a month, once every two months, once every three months, once every four months, once every five months, or once every six months. Administration includes, but is not limited to, injection by any suitable route (e.g., parenteral, enteral, intravenous, subcutaneous, etc.). In a preferred embodiment, the composition is administered weekly, once every two weeks, once every three weeks, or once every four weeks.

[0322] Manufacturing method In one aspect, described herein is a method of making a composition, the method comprising providing a polypeptide that selectively binds to PD-1, where the polypeptide that selectively binds to PD-1 comprises a reactive group (e.g., a conjugation handle), contacting the reactive group with a complementary reactive group attached to a cytokine, and forming a composition. The resulting composition is any of the compositions provided herein.

[0323] In some embodiments, the polypeptide that selectively binds to PD-1 is an antibody or an antigen-binding fragment thereof. In some embodiments, providing an antibody comprising a reactive group comprises attaching the reactive group to the antibody. In some embodiments, the reactive group is site-specifically added. In some embodiments, attaching the reactive group to the antibody comprises contacting the antibody with an affinity group comprising a reactive functional group that forms a bond with a specific residue of the antibody. In some embodiments, attaching the reactive group to the antibody comprises contacting the antibody with an enzyme. In some embodiments, the enzyme is configured to site-specifically attach the reactive group to a specific residue of the antibody. In some embodiments, the enzyme is a glycosylation enzyme or a transglutaminase enzyme.

[0324] In some embodiments, the method further comprises conjugating the cytokine with a complementary reactive group. In some embodiments, conjugating the cytokine with a complementary reactive group comprises chemically synthesizing the cytokine.

[0325] In some embodiments, the method comprises producing a modified IL-2 polypeptide. In some embodiments, the method comprises synthesizing two or more fragments of a modified IL-2 polypeptide and ligating the fragments. In some embodiments, the method comprises a. synthesizing two or more fragments of a modified IL-2 polypeptide, b. ligating the fragments, and c. folding the ligated fragments.

[0326] In some embodiments, the two or more fragments of the modified IL-2 polypeptide are chemically synthesized. In some embodiments, the two or more fragments of the modified IL-2 polypeptide are synthesized by solid phase peptide synthesis. In some embodiments, the two or more fragments of the modified IL-2 polypeptide are synthesized on an automated peptide synthesizer.

[0327] In some embodiments, the modified IL-2 polypeptide is ligated from 2, 3, 4, 5, 6, 7, 8, 9, 10 or more peptide fragments. In some embodiments, the modified peptide is ligated from two peptide fragments. In some embodiments, the modified IL-2 polypeptide is ligated from three peptide fragments. In some embodiments, the modified IL-2 polypeptide is ligated from four peptide fragments. In some embodiments, the modified IL-2 polypeptide is ligated from 2-10 peptide fragments.

[0328] In some embodiments, two or more fragments of the modified IL-2 polypeptide are ligated together. In some embodiments, three or more fragments of the modified IL-2 polypeptide are ligated in a sequential manner. In some embodiments, three or more fragments of the modified IL-2 polypeptide are ligated in a one-pot reaction.

[0329] In some embodiments, the ligated fragment is folded. In some embodiments, the folding comprises forming one or more disulfide bonds in the modified IL-2 polypeptide. In some embodiments, the ligated fragment is subjected to a folding step. In some embodiments, the ligated fragment is folded using methods known in the art. In some embodiments, the ligated or folded polypeptide is further modified by attaching one or more polymers thereto. In some embodiments, the ligated or folded polypeptide is further modified by PEGylation. In some embodiments, the modified IL-2 polypeptide is synthetic. IL-2 Polypeptide Sequence (SEQ ID NO:

[0330] [Table 10-1]

[0331] [Table 10-2]

[0332] [Table 10-3] In Table 8 above, Nle is a norleucine residue and Hse is a homoserine residue.

[0333] Although the present disclosure and its advantages have been described in detail, it should be understood that various changes, substitutions, and alterations can be made therein without departing from the spirit and scope of the present disclosure as defined in the appended claims.

[0334] The present disclosure is further described in the following examples, which are provided for illustrative purposes only and are not intended to limit the disclosure in any way. EXAMPLES

[0335] Example 1: Preparation of Pembrolizumab-(IL-2 Polypeptide) Immunoconjugate Modified pembrolizumab polypeptides are prepared utilizing the methods described in Examples 2-4 of US Patent Publication No. 20200190165(A1). Briefly, azicapping with DBCO conjugation groups (AJICAP™ by Ajinomoto Bio-Pharma Services, Inc., which is described at least in WO 2018199337(A1), WO 2019240288(A1), WO 2019240287(A1), WO 2020090979(A1), Matsuda et al., Mol. Pharmaceutics 2021, 18, 4058-4066, and Yamada et al., Affinity Peptide Mediated Regiodivergent Functionalization of Native Pembrolizumab polypeptide (as found in Antibodies. Angew. Chem., Int. Ed. 2019, 58, 5592-5597, and in particular in Examples 2-4 of US Patent Publication No. 20200190165(A1)) was reacted with (IL-2 polypeptide) azide polypeptide for 48 hours at room temperature. Crude pembrolizumab-(IL-2 polypeptide) immunoconjugates were separated using hydrophobic interaction chromatography, ion exchange chromatography, size exclusion chromatography, capture with DBCO-PEG, Protein A chromatography or DBCO resin column purification.

[0336] An exemplary process for the AJICAP™ methodology is as follows: A modified antibody (e.g., an anti-PD-1 antibody such as pembrolizumab, LZM-009) containing a DBCO conjugation handle is prepared using a protocol modified from Examples 2-4 of US Patent Publication No. 20200190165(A1). Briefly, an anti-PD1 antibody having a free sulfhydryl group attached to a lysine residue side chain in the Fc region is prepared by contacting the antibody with an affinity peptide configured to deliver a protected version of the sulfhydryl group (e.g., a thioester or reducible disulfide) to the lysine residue. An exemplary peptide capable of carrying out this reaction is shown below, as reported in Matsuda et al., Pharmaceutics 2021, 18, 4058-4066, which selectively attached a sulfhydryl group via an NHS ester at residue K248 in the Fc region of the following antibody:

[0337] [ka] Alternative affinity peptides targeting alternative residues in the Fc region are described in the references cited above for the AJICAP™ technology, and such affinity peptides can be used to attach desired functional groups to alternative residues in the Fc region (e.g., K246, K288, etc.). For example, the disulfide groups of the affinity peptides described above can instead be replaced with thioesters to provide sulfhydryl protecting groups (e.g., the relevant portion of the affinity peptide is

[0338] [ka] (It will have the structure:

[0339] The protecting group is then removed to reveal the free sulfhydryl (e.g., by hydrolysis of the thioester or reduction of the disulfide with TCEP). The free sulfhydryl is then reacted with a bifunctional reagent containing a bromoacetamide or bromoketone group linked to a DBCO conjugation handle via a linking group (e.g., bromoacetamide-dPEG®4-amide-DBCO). This method can be used to produce antibodies with one DBCO group present (DAR1) and / or two DBCO groups attached to the antibody (DAR2, one DBCO group linked to each Fc of the antibody). The desired azide-modified IL-2 polypeptide (e.g., composition AB) is then reacted with the DBCO-modified antibody to produce the immunocytokine.

[0340] In another embodiment, an antibody containing a single DBCO conjugation handle is prepared by first reacting an excess of an anti-PD-1 antibody with an appropriately loaded affinity peptide to introduce a single sulfhydryl, after appropriate removal of the protecting group (e.g., disulfide reduction or thioester cleavage). A bifunctional linking group with a sulfhydryl-reactive conjugation handle and a DBCO conjugation handle (e.g., bromoacetamido-dPEG®4-amide-DBCO) is then reacted with the single sulfhydryl to produce a single DBCO-containing antibody. The single DBCO-containing antibody is then conjugated with an appropriate azide-containing IL-2 (e.g., composition AB) to obtain an anti-PD-1-IL-2 immunoconjugate with a DAR of 1.

[0341] Figure 2A shows the site-selective modification of anti-PD1 antibodies by chemical modification techniques to introduce one or two conjugation handles. Figure 2B shows the Q-TOF mass spectra of unmodified pembrolizumab and pembrolizumab with conjugation to the DBCO conjugation handle using AJICAP technology. Figure 2C shows the site-selective conjugation of IL2 cytokine to generate PD1-IL2 with DAR1 or DAR2. A population of PD1-IL2 with mixed DARs of 1 to 2 can also be prepared. Figure 2D shows the TIC chromatogram (top) and intact RP-HPLC (bottom) profiles of crude pembrolizumab and composition AB conjugation reaction mixtures. DAR0 represents pembrolizumab conjugated with no IL-2, DAR1 represents pembrolizumab conjugated with one IL-2, and DAR2 represents pembrolizumab conjugated with two IL-2. Figure 2E shows the Q-TOF mass spectrum profile of crude pembrolizumab and (composition AB) conjugation reaction mixture showing the formation of DAR1 and DAR2 species. Figure 2F shows a representative RP-HPLC chromatogram of purified PD1-IL2, Figure 2G shows a representative Q-TOF mass spectrum of purified PD1-IL2, and Figure 2H shows a representative analytical SEC of purified PD1-IL2 (composition B).

[0342] Table 9 below summarizes various immunocytokines that may be prepared according to the methods provided herein or similar methods described elsewhere.

[0343] [Table 11]

[0344] Example 2: PD-1 Binding ELISA Assay (Figure 3) The interaction of unmodified and conjugated anti-PD1 antibodies with PD-1 (CD279) was measured by ELISA assay. For these studies, Corning high-binding half-area plates (Fisher Scientific, Reinach, Switzerland) were coated with 25 μl of unmodified or conjugated anti-PD1 antibodies at 2.5 μg / ml in PBS overnight at 4° C. The plates were then washed four times with 100 μl of PBS-0.02% Tween 20. The plate surface was blocked with 25 μl of PBS-0.02% Tween 20-1% BSA for 1 h at 37° C. The plates were then washed four times with 100 μl of PBS-0.02% Tween 20. Twenty-five microliters of recombinant biotinylated PD1 / CD279 protein from Biolegend (789406, London, UK) was added in 5-fold serial dilutions starting from 23 nM to 0.0003 nM in PBS-0.02% Tween20-0.1% BSA and incubated for 2 hours at 37°C. The plate was then washed four times with 100 μl of PBS-0.02% Tween20. Twenty-five microliters of streptavidin-horseradish peroxidase (#RABHRP 3, Merck, Buchs, Switzerland) diluted 1:500 in PBS-0.02% Tween20-0.1% BSA was added to each well and incubated for 30 minutes at room temperature. The plate was then washed four times with 100 μl of PBS-0.02% Tween20. Fifty microliters of TMB substrate reagent (catalog no. 07, Merck, Buchs, Switzerland) was added to each well and incubated for 5 min at 37° C. After 5 min at 37° C., the horseradish peroxidase reaction was stopped by adding 50 μl / well of 0.5 M H2SO4 stop solution. The ELISA signal was then measured at 450 nm on an EnSpire plate reader from Perkin Elmer (Schwerzenbach, Switzerland).

[0345] [Table 12]

[0346] Figure 3 shows a plot illustrating the ability of unmodified and conjugated anti-PD1 antibodies to bind to PD1 / CD279 ligands, with ELISA signal shown on the y-axis and dosage of biotinylated PD-L1 protein shown on the x-axis. The unconjugated reference and conjugated antibodies tested in this figure are compositions pembrolizumab, nivolumab, LZM-009, and compositions A, C, D, E, F, G, and H, respectively.

[0347] PD-1 / PD-L1 Blockade Bioassay: A PD-1 / PD-L1 blockade bioassay was used to determine the ability of pembrolizumab-(IL-2 polypeptide) immunoconjugates to block PD-1 / PD-L1 interaction.

[0348] The ability of unmodified and conjugated anti-PD1 antibodies to disrupt the PD1 / PDL1 pathway was measured using the PD-1 / PD-L1 Blockade Bioassay from Promega (catalog no. J1250, Madison, WI, USA). The PD-1 / PD-L1 Blockade Bioassay is a bioluminescent cell-based assay based on co-culture of effector cells with target cells mimicking the immune synapse. Jurkat T cells expressing human PD-1 and a luciferase reporter driven by an NFAT response element (NFAT-RE) are activated by CHO-K1 cells expressing human PD-L1 and an engineered cell surface protein designed to activate the cognate TCR of Jurkat. The simultaneous interaction PD-1 / PD-L1 inhibits TCR signaling and suppresses NFAT-RE-mediated luminescence. Addition of either anti-PD-1 or anti-PD-L1 antibodies, which block the PD-1 / PD-L1 interaction, releases an inhibitory signal, restoring TCR activation and resulting in an increase in the signal of the NFAT-RE luminescent reporter.

[0349] Briefly, PD-L1 aAPC / CHO-K1 target cells were seeded into white tissue culture-96 well plates and cultured overnight at 37°C / 5% CO2. Test molecules were measured in 4-fold serial dilutions starting at 1 μM down to 0.002 nM and pre-incubated on the target cells for 10 min before addition of freshly thawed PD-1 Jurkat effector cells. After 6 h at 37°C / 5% CO2, active NFAT-RE luminescent reporter was assessed by addition of Bio-Glo reagent and measured on an EnSpire plate reader (1 sec / well) from Perkin Elmer (Schwerzenbach, Switzerland).

[0350] Figure 4 shows plots illustrating the ability of anti-PD1 and conjugated anti-PD1 antibodies to block the PD1 / PDL1 pathway, with the mean luminescence intensity of the effector cell NFAT-RE reporter shown on the y-axis and the dosage of unmodified and conjugated anti-PD1 antibodies shown on the x-axis. The unconjugated reference and conjugated antibodies tested in this figure are pembrolizumab and composition B, respectively. The modified IL-2 polypeptides tested in this figure are Proleukin and composition AA.

[0351] The interaction of unmodified and conjugated anti-PD1 antibodies with human neonatal Fc receptor (FcRn) at pH 6 was measured using the AlphaLISA® Human FcRn Binding Kit (AL3095C) from Perkin Elmer (Schwerzenbach, Switzerland). AlphaLISA® detection of FcRn and IgG binding uses IgG-coated AlphaLISA® acceptor beads to interact with biotinylated human FcRn captured on streptavidin-coated donor beads. Binding of the reference IgG to FcRn brings the donor and acceptor beads into close proximity, allowing the transfer of singlet oxygen that triggers a cascade of energy transfer reactions within the acceptor beads, resulting in a sharp peak of emission at 615 nm. Addition of free IgG antibody to the AlphaLISA® mixture creates competition for binding of FcRn to the reference antibody, resulting in loss of signal.

[0352] Briefly, test molecules were measured in serial dilutions starting from 5 μM down to 64 pM and incubated with an AlphaLISA® reaction mixture consisting of 800 nM recombinant biotinylated human FcRn, 40 μg / ml human IgG-conjugated receptor beads and 40 μg / ml streptavidin-coated donor beads in MES buffer pH 6. After 90 min at 23° C. in the dark, the AlphaLISA® signal was measured on an EnSpire plate reader from Perkin Elmer (excitation at 680 nm, emission at 615 nm) (Schwerzenbach, Switzerland).

[0353] Figure 5 shows a plot illustrating the ability of unmodified and conjugated anti-PD1 antibodies to bind to human neonatal Fc receptor (FcRn) at pH 6, with the average AlphaLISA® FcRn-IgG signal shown on the y-axis and the dosage of unmodified and conjugated anti-PD1 antibodies shown on the x-axis. The unconjugated reference and conjugated antibodies tested in this figure are pembrolizumab, LZM-009, and compositions A, D, E, H, J, K, respectively.

[0354] [Table 13]

[0355] Example 3: Human FcγR binding assay (Figure 6) The interaction of unmodified and conjugated anti-PD1 antibodies with human Fc gamma receptor I (FcγRI / CD64), human Fc gamma receptor IIa (FcγRIIa / CD32a), inhibitory human Fc gamma receptor IIb (FcγRIIb / CD32b) and human Fc gamma receptor III FcγR3a / CD16 was measured by ELISA. Briefly, Corning high-binding half-area plates (Fisher Scientific, Reinach, Switzerland) were coated with 25 μl of unmodified and conjugated anti-PD1 antibodies at 2.5 μg / ml in PBS overnight at 4° C. The plates were then washed four times with 100 μl of PBS-0.02% Tween 20. The plate surface was blocked with 25 μl of PBS-0.02% Tween 20-1% BSA at 37° C. for 1 h. Plates were then washed 4 times with 100 μl of PBS-0.02% Tween 20. Then 25 microliters of either recombinant human Fc gamma RI / CD64 protein (R&D systems, 1257-FC-050, CF), recombinant human Fc gamma RIIA / CD32a (H167) protein (R&D systems, 9595-CD-050, CF), recombinant human Fc gamma RIIB / CD32b Avi tag protein (R&D systems, AVI1875-050, CF), or recombinant human Fc gamma RIIIA / CD16a protein (R&D systems, 4325-FC-050; CF) were added in 5-fold serial dilutions ranging from 1000 nM to 0.001 nM in PBS-0.02% Tween 20-0.1% BSA and incubated at 37° C. for 2 hours. The plates were then washed four times with 100 μl of PBS-0.02% Tween 20. Twenty-five microliters of streptavidin-horseradish peroxidase (#RABHRP3, Merck, Buchs, Switzerland) diluted 1:500 in PBS-0.02% Tween 20-0.1% BSA was added to each well and incubated for 30 minutes at room temperature. The plates were then washed four times with 100 μl of PBS-0.02% Tween 20.Fifty microliters of TMB substrate reagent (cat. no. 07, Merck, Buchs, Switzerland) was added to each well and incubated for 5 min at 37° C. After 5 min at 37° C., the horseradish peroxidase reaction was stopped by adding 50 μl / well of 0.5 M H2SO4 stop solution. The ELISA signal was then measured at 450 nm in an EnSpire plate reader from Perkin Elmer (Schwerzenbach, Switzerland). After 90 min at 23° C. in the dark.

[0356] [Table 14] 6A shows plots illustrating the ability of unmodified and conjugated anti-PD1 antibodies to bind to human Fc gamma receptor I (CD64), human Fc gamma receptor IIa (CD32a), human Fc gamma receptor IIb (CD32b), and human Fc gamma receptor IIIa (CD16), with the average ELISA signal shown on the y-axis and the dosage of unmodified and conjugated anti-PD1 antibodies shown on the x-axis. The unconjugated reference antibodies are pembrolizumab, LZM-009, and composition A, and the conjugated antibodies tested in this figure are compositions C, D, and H. 6B shows plots illustrating the ability of unmodified and conjugated anti-PD1 antibodies to bind to human Fc gamma receptor I (CD64), human Fc gamma receptor IIa (CD32a), human Fc gamma receptor IIb (CD32b), and human Fc gamma receptor IIIa (CD16), with the average ELISA signal shown on the y-axis and the dosage of unmodified and conjugated anti-PD1 antibodies shown on the x-axis. The conjugated antibodies tested in this figure are compositions E, J, and K.

[0357] Example 4: PD1 陽性 Mo7e cells vs. PD1 陰性IL2-induced pSTaT5 activation in Mo7e cells (Figures 7A-7B) We established a human Mo7e cell line that stably expresses human PD-1. 5 Mo7e cells were transduced with lentiviral particles carrying the human PD1 gene (PDCD1 NM_005018; Origene, Cat. No. RC210364L3V) at an MOI (multiplicity of infection) of 4. Spin infection was performed at 1260 g for 90 min at 37°C in the presence of 5 μg / ml polybrene and 10 mM HEPES in complete culture medium (RPMI, 20% FBS, 10 ng / ml GM-CSF). Five days after transduction, PD-1 positive cells were selected by adding 0.75 μg / ml puromycin. Stable and homogenous expression of PD-1 was confirmed by surface staining.

[0358] Experiments were performed to determine the effect of various IL-2 polypeptides on PD-1 negative (parental untransduced line) and PD-1 expressing (transduced) Mo7e cells. Cells were dispensed at 100,000 cells / well and stimulated with 10-fold serial dilutions of modified IL-2 polypeptides not conjugated to anti-PD1 antibodies and unconjugated modified IL-2 polypeptides at starting concentrations ranging from 949 nM to 10 pM for 40 min at 37°C / 5% CO2. After incubation, cells were fixed and permeabilized using a transcription factor phosphobuffer kit (BD Biosciences), followed by surface and intracellular immunostaining of PD-1 and pSTaT5 to allow cell identification and measurement of the level of Stat5 (signal transducer and activator of transcription 5) phosphorylation. FACS (fluorescence-activated cell sorting) measurements were performed using a Quanteon flow cytometer from Acea.

[0359] Figure 7A shows parental non-transduced Mo7e (PD1 - ) and stably transduced (PD1 + 3A-3C show plots illustrating surface expression levels of PD-1 / CD279 on Mo7e cells.

[0360] FIG. 7B shows PD1 in vitro. 陰性 (solid symbols) and PD1 陽性 (Grey open symbols) Plots illustrating the effect of modified IL-2 polypeptides unconjugated and conjugated to anti-PD1 antibodies on induction of the IL-2 signaling pathway in Mo7e cells, showing the mean EC 50 Values ​​are shown on the y-axis, and dosages of modified IL-2 polypeptides and immunocytokines are shown on the x-axis. The modified IL-2 polypeptides tested in this figure are Proleukin and Composition AB. The immunocytokines tested in this figure are Compositions A, C, H, L and Her2-targeting immunocytokine Composition O (Trastuzumab antibody conjugated to an IL-2 polypeptide) as a control.

[0361] [Table 15]

[0362] Example 5: IL2-induced pStat5 activation in primary T cells (Figures 8-10) Experiments were performed to determine the effect of various IL-2 polypeptides on human T cell populations. Primary pan T cells (CD4+T cells, CD8+T cells, and Tregs) were obtained from buffy coats of healthy donors by peripheral blood mononuclear cell (PBMC) purification using ficoll gradient centrifugation, followed by negative isolation with magnetic beads, and then cryopreservation until use. Pan T cells were thawed and allowed to recover overnight in T cell medium (RPMI 10% FCS, 1% glutamine, 1% NEAA, 25 μM βMeoH, 1% NaPyrovate), and after two washing steps with PBS, the cells were resuspended in PBS. If necessary, cells were preincubated with 100 nM of the unconjugated anti-PD1 antibody pembrolizumab for 20 min at 37 °C. Cells were then dispensed at 200,000 cells / well and stimulated with modified IL-2 polypeptides not conjugated to anti-PD1 antibodies and 3.16-fold serial dilutions of unconjugated modified IL-2 polypeptides at starting concentrations ranging from 316 nM to 3 pM for 40 min at 37°C / 5% CO2. After incubation, cells were fixed and permeabilized using a transcription factor phosphobuffer kit, followed by surface and intracellular immunostaining for CD4, CD8, CD25, FoxP3, CD45RA and pSTaT5 to allow identification of cell subsets and measurement of the level of Stat5 (signal transducer and activator of transcription 5) phosphorylation. FACS (fluorescence-activated cell sorting) measurements were performed using either NovoCyte or Quanteon flow cytometers from Acea.

[0363] The pStat5 MFI (medium fluorescence intensity) signals of the following T cell subsets were plotted against the concentration of wild-type or modified IL-2 polypeptides: 50% effective concentration (EC 50 ) was calculated based on a variable slope, four-parameter analysis using GraphPad PRISM software.

[0364] Gating strategies for identifying T cell subsets

[0365] [Table 16]

[0366] [Table 17]

[0367] FIG. 8 shows T cell proliferation in an in vitro sample of human T cells. eff and T reg 1 shows a plot illustrating the effect of modified IL-2 polypeptides not conjugated to anti-PD1 antibodies and modified IL-2 polypeptides conjugated to anti-PD1 antibodies on cell induction, with the mean fluorescence intensity of phosphorylated signal transduction and activator of transcription 5 (pSTAT5) shown on the y-axis and the dosage of modified IL-2 polypeptides and immunocytokines shown on the x-axis. The modified IL-2 polypeptide tested in this figure is composition AA. The immunocytokines tested in this figure are compositions A, B, and C.

[0368] FIG. 9A shows resting memory (CD45RA-) and naive (CD45RA+) CD8+ T cells freshly isolated from peripheral blood of healthy donors. eff 1 shows plots illustrating surface expression levels of PD-1 / CD279 on cells.

[0369] FIG. 9B shows the expression of resting memory (CD45RA−) and naive (CD45RA+) CD8+ T cells in an in vitro sample of human T cells. eff1 shows plots illustrating the effect of modified IL-2 polypeptides unconjugated and conjugated to anti-PD1 antibodies on cell induction, with the mean fluorescence intensity of phosphorylated signal transduction and activator of transcription 5 (pSTAT5) shown on the y-axis and the dosage of modified IL-2 polypeptide and immunocytokine shown on the x-axis. The modified IL-2 polypeptide tested in this figure is composition AA, and the immunocytokine tested in this figure is composition B and immunocytokine composition N (trastuzumab antibody conjugated to an IL-2 polypeptide) as a control.

[0370] FIG. 10A shows the expression of resting naive (CD45RA+) CD8+ T cells in in vitro samples of human T cells in the presence or absence of excess unconjugated anti-PD1 antibody pembrolizumab. eff Figure 1 shows plots measuring the effect of modified IL-2 polypeptides unconjugated and conjugated to anti-PD1 antibodies on cell induction, where the mean fluorescence intensity of phosphorylated signal transduction and activator of transcription 5 (pSTAT5) is shown on the y-axis and the dosage of modified IL-2 polypeptide and immunocytokine is shown on the x-axis. The modified IL-2 polypeptide tested in this figure is composition AA, and the immunocytokine tested in this figure is composition B and the Her2-targeted immunocytokine composition N (trastuzumab antibody conjugated to an IL-2 polypeptide) as a control.

[0371] FIG. 10B shows the expression of resting memory (CD45RA−) CD8+ T cells in in vitro samples of human T cells in the presence or absence of excess unconjugated anti-PD1 antibody pembrolizumab. effFigure 1 shows plots measuring the effect of modified IL-2 polypeptides unconjugated and conjugated to anti-PD1 antibodies on cell induction, where the mean fluorescence intensity of phosphorylated signal transduction and activator of transcription 5 (pSTAT5) is shown on the y-axis and the dosage of modified IL-2 polypeptide and immunocytokine is shown on the x-axis. The modified IL-2 polypeptide tested in this figure is composition AA, and the immunocytokine tested in this figure is composition B and the Her2-targeted immunocytokine composition N (trastuzumab antibody conjugated to an IL-2 polypeptide) as a control.

[0372] Example 6: PK / PD studies in tumor-bearing mice (Figures 11-13) In vivo PK / PD studies were performed in mice. Naive 6- to 8-week-old BALB / c-hPD1 female mice (GemPharmatech Co. Ltd, Nanjing, China) were inoculated with wild-type CT26 tumor cells (3 × 10 5 ) was inoculated subcutaneously into the left flank. Animals were randomized (using Excel-based randomization software with stratified randomization based on tumor volume) until the mean tumor volume reached approximately 186 mm 3Treatment was initiated when tumor volume reached 100 μg / kg. Animals treated with Composition A received a single 10 mL / kg bolus intravenous (iv) injection of 1, and 2.5 mg / kg of PD-1 antibody conjugated to a modified IL-2 polypeptide. Animals treated with control Her2-targeting immunocytokine Composition O (trastuzumab antibody conjugated to an IL-2 polypeptide) received a single 10 mL / kg bolus intravenous (iv) injection of 2.5 mg / kg of anti-Her2 antibody conjugated to a modified IL-2 polypeptide. After inoculation, animals were checked daily for morbidity and mortality. At that time, animals were checked for tumor growth and effects on normal behavior, including motility, food and water consumption, weight gain / loss (body weight was measured twice a week), eye / hair matting, and any other abnormal effects. Tumor size was measured bidimensionally three times a week using calipers, and volume was calculated according to the formula V=0.5a×b 2 Using mm 3 The tumor size was expressed as a function of time (a) and (b) where a and b are the long and short diameters of the tumor, respectively. Mortality and observed clinical signs were recorded based on the number of animals in each subset.

[0373] The pharmacokinetic study included nine time points (5 min, 1 h, 6 h, 12 h, 24 h, 72 h, 96 h, 120 h, and 168 h) with three mice sampled per time point. At the indicated time points, blood samples were collected in the presence of EDTA via either tail vein sampling or cardiac puncture (endpoint). Additionally, 72 h, 96 h, 120 h, and 168 h after injection, three mice from each group were sacrificed and tumor samples were collected.

[0374] Fresh tumor samples from each mouse were individually minced and digested with the enzyme mix in a C-tube. The C-tube was mounted on the sleeve of a Gentle MACS Dissociator before one round of program "m_imptumor_01_01". The C-tube was then incubated at 37°C for 30 min, after which another round of program "m_imptumor_01_01" was performed. The digested tissue was filtered through a 70 μm cell strainer. The cells were washed twice with DPBS before staining.

[0375] Mouse blood collected in the presence of EDTA was immediately centrifuged at 4,000 rpm for 5 minutes at 4°C. The collected plasma (supernatant) was stored at -80°C until bioanalysis. One volume of cell pellet was then mixed with 20 volumes of 1x red blood cell lysis solution. It was then incubated for 3 minutes and centrifuged. If the blood was not sufficiently lysed, it was suspended again in 2 mL of 1x red blood cell lysis solution and incubated for 3 minutes.

[0376] The cells were washed twice with DPBS. 100 μL of resuspended cells (at a concentration of 10 million / mL) were seeded in a 96V-well plate per test. After centrifugation, the cells were suspended in 100 μL of DPBS. BV510 live / dead was added and incubated for 30 min at 4°C in the dark. The cells were washed twice with DPBS. After extracellular antibody incubation, the cells were washed twice with staining buffer, fixed and permeabilized for 30 min. Purified rat anti-mouse CD16 / CD32 was added for 5 min incubation and the cells were stained according to the procedure indicated in the specific intracellular antibody specifications. The cells were then washed twice and suspended in 200 μL of staining buffer. The stained cells were analyzed by a BD Fortessa X20 flow cytometer.

[0377] [Table 18]

[0378] Figure 11A shows plots illustrating the effect of PD-1 targeted and non-targeted immunocytokines on the growth of CT26 syngeneic colon cancer tumors in hPD1 humanized BALB / c mice. The immunocytokine tested in this figure is Composition A, which was tested as a single agent at 1 mg / kg and 2.5 mg / kg following a single injection schedule. A control Her2 targeted immunocytokine Composition O (trastuzumab antibody conjugated to an IL-2 polypeptide) was also tested at 2.5 mg / kg (mean ± SEM).

[0379] 11B is a bar graph illustrating the effect of PD-1-targeted and non-targeted immunocytokines on the growth of CT26 syngeneic colon cancer tumors in hPD1-humanized BALB / c mice after 7 days of treatment. The immunocytokine tested in this figure is Composition A, which was tested as a single agent at 1 mg / kg and 2.5 mg / kg following a single injection schedule. A control Her2-targeted immunocytokine Composition O (trastuzumab antibody conjugated to an IL-2 polypeptide) was also tested at 2.5 mg / kg (mean ± SEM; **One-way ANOVA P-value <0.001).

[0380] FIG. 12A shows the expression of naive (CD62L)-Humanized BALB / c mice in the blood and tumors of CT26 tumor-bearing hPD1-humanized BALB / c mice 7 days after treatment. high CD44 low 1 shows plots illustrating the effect of PD-1 targeted and non-targeted immunocytokines on CD8+ T cell expansion. The immunocytokine tested in this figure is Composition A, which was tested as a single agent at 1 mg / kg and 2.5 mg / kg following a single injection schedule. A control Her2 targeted immunocytokine Composition O (trastuzumab antibody conjugated to an IL-2 polypeptide) was also tested at 2.5 mg / kg (n=3; mean±SEM).

[0381] FIG. 12B shows effector memory (CD62L) in the blood and tumors of hPD1-humanized BALB / c mice bearing CT26 tumors 7 days after treatment. 陰性 CD44high 1 shows plots illustrating the effect of PD-1 targeted and non-targeted immunocytokines on CD8+ T cell expansion. The immunocytokine tested in this figure is Composition A, which was tested as a single agent at 1 mg / kg and 2.5 mg / kg following a single injection schedule. A control Her2 targeted immunocytokine Composition O (trastuzumab antibody conjugated to an IL-2 polypeptide) was also tested at 2.5 mg / kg (n=3; mean±SEM).

[0382] Figure 13A shows plots illustrating the effect of PD-1 targeting and non-targeting of immunocytokines on their persistence in the blood and tumors of hPD1-humanized BALB / c mice bearing CT26 tumors, with plasma or tumor concentrations of PD-1-targeting and control immunocytokines shown on the y-axis and time shown on the x-axis. The immunocytokine tested in this figure is Composition A, which was tested as a single agent at 1 mg / kg and 2.5 mg / kg after a single injection schedule. A control Her2-targeting immunocytokine Composition O (trastuzumab antibody conjugated to an IL-2 polypeptide) was also tested at 2.5 mg / kg (n=3; mean ± SD).

[0383] FIG. 13B shows plots illustrating the effect of PD-1 targeting and non-targeting of immunocytokines on their persistence in tumors compared to blood of hPD1-humanized BALB / c mice bearing CT26 tumors, with the ratio of tumor / plasma concentrations of PD-1-targeting and control immunocytokines shown on the y-axis and time shown on the x-axis. The immunocytokine tested in this figure is Composition A, which was tested as a single agent at 1 mg / kg and 2.5 mg / kg after a single injection schedule. A control Her2-targeting immunocytokine Composition O (trastuzumab antibody conjugated to an IL-2 polypeptide) was also tested at 2.5 mg / kg (n=3; mean±SEM).

[0384] Example 7: Efficacy Study (Figures 14A-B) In vivo efficacy studies were performed in mice. Naive 6-8 week-old C57BL / 6-hPD1 female mice (GemPharmatech Co. Ltd, Nanjing, China) were inoculated with MC38 tumor cells (3 × 10 5 ) was inoculated subcutaneously into the upper right flank. Animals were randomized (using Excel-based randomization software with stratified randomization based on tumor volume) until the mean tumor volume was approximately 90 mm 3 Treatment was initiated when the tumor volume reached 100 μg / kg. Animals treated with Composition H were injected intravenously (iv) with 1 mg / kg PD-1 antibody conjugated to a modified IL-2 polypeptide in a single bolus at 10 mL / kg. After inoculation, animals were checked daily for morbidity and mortality. At that time, animals were checked for tumor growth and effects on normal behavior, including motility, food and water consumption, weight gain / loss (body weight was measured twice weekly), eye / fur matting, and any other abnormal effects. The primary endpoint was tumor growth delay or complete tumor regression. Tumor size was measured bidimensionally three times weekly using calipers, and volume was calculated according to the formula V=0.5a×b 2 Using mm 3 The tumor size was expressed as a function of time (a) and (b) where a and b are the long and short diameters of the tumor, respectively. Mortality and observed clinical signs were recorded based on the number of animals in each subset.

[0385] Figure 14A shows plots illustrating the effect of a single injection of conjugated anti-PD1 antibodies on the growth of MC38 syngeneic colon carcinoma tumors in hPD1 C57BL / / 6 mice. The immunocytokine tested in this figure is Composition H, tested as a single agent at 1 mg / kg as a single iv injection (n=8; mean±SEM).

[0386] Figure 14B is a bar graph illustrating the effect of a single injection of conjugated anti-PD1 antibodies on the growth of MC38 syngeneic colon carcinoma tumors in hPD1 C57BL / / 6 mice 7 days after treatment. The immunocytokine tested in this figure is Composition H, tested as a single agent at a single iv injection of 1 mg / kg (n=8 animals; mean±SEM; **One-way ANOVA P-value<0.005).

[0387] Example 8: Synthesis of Composition AB Modified IL-2 polypeptide composition AB containing azido-PEG attached to residues F42Y, a PEG group at Y45, and having the amino acid sequence of SEQ ID NO:3 was synthesized by ligating individual peptides synthesized using solid phase peptide synthesis (SPPS). Individual peptides were synthesized on an automated peptide synthesizer using the methods described below. Related modified IL-2s provided herein were synthesized using similar protocols.

[0388] Commercially available reagents were purchased from Sigma-Aldrich, Acros, Merck or TCI Europe and used without further purification. Fmoc amino acids with side chain protecting groups suitable for solid phase peptide synthesis were purchased from Novabiochem, Christof Senn Laboratories AG or PeptART and were used as supplied. Polyethylene glycol derivatives used in peptide synthesis were purchased from Polypure. HPLC grade CH3CN from Sigma Aldrich was used for analytical and preparative HPLC purification.

[0389] High-resolution mass spectra (FTMS) of peptides and proteins were measured on a Bruker solariX (9.4T magnet) equipped with a dual ESI / MALDI-FTICR source using 4-hydroxy-α-cyanocinnamic acid (HCCA) as the matrix. CD spectra were recorded on a Jasco J-715 spectrometer equipped with a 1.0 mm path length cell. Spectra were collected at 25°C in continuous scan mode with standard sensitivity (100 mdeg), 0.5 nm data pitch, 50 nm / min scan speed, 1 nm bandwidth and 5 accumulations.

[0390] Peptides and protein fragments were analyzed and purified by reversed-phase high-performance liquid chromatography (RP-HPLC). Peptide analysis and reaction monitoring were performed on an analytical Jasco instrument equipped with a dual pump, mixer and in-line degasser, autosampler, variable wavelength UV detector (simultaneous monitoring of eluents at 220 nm and 254 nm) and an injector with a 100 μl injection loop. Purification of peptide fragments was performed on a Gilson preparative instrument equipped with a 20 mL injection loop. In both cases, the mobile phase was MilliQ-H2O with 0.1% TFA (buffer A) and HPLC grade CH3CN with 0.1% TFA (buffer B). Analytical HPLC was performed with a bioZen™ intact C4 column (3.6 μm, 150 × 4.6 mm) or a Shiseido Capcell Pak MG III (5 μm, 150 × 4.6 mm) column at a flow rate of 1 mL / min. Preparative HPLC was performed using a Shiseido Capcell Pak UG80 C18 column (5 μm, 50 mm i.d.×250 mm) at a flow rate of 40 mL / min.

[0391] Peptide segments were synthesized using Fmoc SPPS chemistry on a Syro I or CS Bio 136X peptide synthesizer. The following Fmoc amino acids with side chain protecting groups were used: Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Cys(Acm), Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-His(1-Trt)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Nle-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH. Fmoc-pseudoproline dipeptides were incorporated into the synthesis when necessary. Fmoc deprotection was performed with 20% piperidine in DMF (2 × 8 min) and monitored with UV at 304 nm using a feedback loop to ensure complete Fmoc removal. Coupling was performed at room temperature or 50 °C using Fmoc-amino acids (3.0–5.0 equiv. relative to resin substitution), HCTU or HATU (2.9–4.9 equiv.) as coupling reagents, and DIPEA or NMM (6–10 equiv.) in DMF. After preactivation for 3 min, the solution was transferred and reacted with the peptide on the resin for 30 min or 2 h, depending on the amino acid. In some cases, double coupling was required. After coupling, the resin was treated with 20% acetic anhydride in DMF to cap unreacted free amines. LiCl washes were performed as necessary. Deprotection of the allyl ester was accomplished using phenylsilane (24 equiv.) and palladium(0) tetrakis(triphenylphosphine) (0.5 equiv.) in anhydrous dichloromethane.

[0392] The synthesis of peptide segments by SPPS was monitored by microcleavage and analysis of the corresponding resin. The peptides were cleaved from the resin using a mixture of 95:2.5:2.5 TFA:DODT:H2O (α-keto acid segments synthesized on α-keto acid resin) or 95:2.5:2.5 TFA:TIPS:H2O (peptides synthesized on 2-chlorotrityl polystyrene resin) for 2 h. The resin was filtered off and the filtrate was evaporated, treated with cold diethyl ether, triturated and centrifuged. The ether layer was carefully decanted and the residue was resuspended in diethyl ether, triturated and centrifuged. The ether wash was repeated twice.

[0393] 1.1 Synthesis of the composition AB variant of IL-2 Synthesis of IL-2(1-39)-Leu-α-keto acid

[0394] [ka] IL2(1-39)-Leu-α-keto acid (see SEQ ID NO: 3) was synthesized on Rink-amide resin preloaded with protected Fmoc-α-Leu-keto acid with a substitution capacity of 0.25 mmol / g. To do so, Fmoc-Rink amide MBHA resin (4 g) was preswelled in DMF for 15 min and Fmoc deprotection was performed. Fmoc-leucine-protected α-keto acid (795 mg, 1 mmol, 1.00 equiv.) was dissolved in 40 mL of DMF and preactivated with HATU (361 mg, 0.95 mmol, 0.95 equiv.) and DIPEA (348 μL, 2 mmol, 2.00 equiv.). Coupling was allowed to proceed for 6 h at room temperature. The resin was then capped before Fmoc deprotection. The synthesis of the segments was carried out by automated Fmoc SPPS using the procedure described in the General Methods section with a 0.250 mmol scale-up to Ala1. The progress of the peptide synthesis was monitored by microcleavage and analysis using a mixture of (95:2.5:2.5) TFA:DODT:HO for 1.5 h. HPLC analysis was performed using a C18 column at 60 °C. The peptide was cleaved from the resin using a mixture of 95:2.5:2.5 TFA:DODT:HO (15 mL / g resin) for 2 h according to the procedure described in General Methods. Purification of crude IL2(1-39) was performed by preparative HPLC using a Shiseido capcell pak C18 column (50 × 250 mm) with a gradient of 30-80% CH3CN containing 0.1% TFA in 30 min. Pure product fractions were pooled and lyophilized to give 650 mg of pure IL2(1-39)-Leu-α-ketoacid (69% yield for peptide synthesis, resin cleavage and purification steps). Analytical HPLC and ESI-HRMS were used to confirm the purity and accurate mass of the product. m / z calculated for C 204 H 346 N 56 O 61 [M]:4556.5694;measured:4556.5783.

[0395] Synthesis of Opr-IL2(42-69) photoprotected Leu-α-keto acids of composition AB

[0396]

change

[0397] Synthesis of Fmoc-Opr IL2(72-102)-Phe-α-keto acid of composition AB

[0398] [ka] Fmoc-Opr IL2(72-102)-phenylalanine-α-keto acid was synthesized on Rink amide ChemMatrix resin preloaded with Fmoc-Phe-protonated α-keto acid with a substitution capacity of approximately 0.25 mmol / g. The synthesis was carried out on a 0.588 mmol scale up to Ala73 by automated Fmoc SPPS using HCTU as the coupling reagent. Coupling of residue 72, Fmoc-Leu, was carried out using HATU as the coupling reagent. The coupling was repeated two more times at 45 °C to ensure complete coupling. Fmoc-5-oxaproline (3.00 equiv. relative to resin) was manually coupled to the free amine using HATU (2.95 equiv. relative to resin) and NMM (6.00 equiv. relative to resin) at room temperature for 2 h. The progress of the peptide synthesis was monitored by microcleavage and analysis using a mixture of (95:2.5:2.5) TFA:DODT:HO for 2 h. HPLC analysis was performed using a C18 column at 60 °C. The peptide was cleaved from the resin using a mixture of 95:2.5:2.5 TFA:DODT:HO (15 mL / g resin) for 2.0 h. Purification of the crude segment was performed by preparative HPLC using a Shiseido Capcell Pak C18 column (50 × 250 mm) preheated at 60 °C with a gradient of 20 to 75% CH3CN containing 0.1% TFA in 30 min. Pure product fractions were pooled and lyophilized to give Fmoc-Opr IL2(72-102)-Phe-α-keto acid in >98% purity (147.9 mg, 6% yield for synthesis, cleavage and purification steps). Analytical HPLC and ESI-HRMS were used to confirm the purity and accurate mass of the product. m / z calculated for C 184 H 285 N 47 O 53 [M]:4001.1051;measured 4001.1227.

[0399] Synthesis of Opr-IL2(105-133)

[0400] [ka] Opr-IL2(105-133) was synthesized on 2-chlorotrityl resin preloaded with Fmoc-Thr-OH with a substitution capacity of 0.25 mmol / g. After capping (diisopropylethylamine, methanol), the synthesis was carried out on a 0.34 mmol scale (1.5 g resin) by automated Fmoc SPPS up to Glu106. Cys(Acm)-OH (10 equiv. relative to resin) was used for coupling of Cys105 by the symmetric anhydride method using DIC (5 equiv. relative to resin) for 2 h at room temperature. Boc-5-oxaproline (2.00 equiv. relative to resin) was then coupled to the free amine on-resin using HATU (1.95 equiv.) and NMM (4 equiv.). The progress of the peptide synthesis was monitored by microcleavage and analysis using a mixture of (95:2.5:2.5) TFA:TIPS:HO for 1.5 h. HPLC analysis was performed using a C18 column at 60 °C. The peptide was cleaved from the resin using a mixture of 95:2.5:2.5 TFA:TIPS:H2O (15 mL / g resin) for 2.0 h. Purification of crude Opr-IL2(105–133) was performed by preparative HPLC using a Shiseido Capcell Pak C4 column (50 × 250 mm) preheated at 60 °C with a gradient of 10–65% CH3CN with 0.1% TFA in 10 min, then 65–95% CH3CN with 0.1% TFA in 20 min. Pure product fractions were pooled and lyophilized to give Opr-IL2(105–133) with >98% purity (108.5 mg, 9% yield for synthesis, cleavage and purification steps). Analytical HPLC and ESI-HRMS were used to confirm the purity and accurate mass of the product. m / z calculated for C 158 H 242 N 37 O 52 S[M+H]:3521.7145;found 3521.7140.

[0401] Synthesis of IL2-Seg12 of Compositions AB by KAHA Ligation

[0402] [ka] KAHA ligation: Seg1 (44 mg, 9.6 μmol, 1.2 equiv.) and Seg2 (40 mg, 8.0 μmol, 1 equiv.) were dissolved in DMSO:HO (9:1) containing 0.1 M oxalic acid (400 μL, 20 mM) and reacted at 60 °C for 20 h. The ligation vial was protected from light by wrapping in aluminum foil. The progress of the KAHA ligation was monitored by uHPLC at 60 °C using a Phenomenex C18 column (150 × 4.6 mm) with CH3CN / HO containing 0.1% TFA as the mobile phase with a gradient of 5 to 95% CH3CN in 7 min.

[0403] Photodeprotection and purification: After completion of ligation, the mixture was diluted approximately 20-fold (8 mL) with CH3CN / H2O (1:1) containing 0.1% TFA and irradiated at a wavelength of 365 nm for 1 h. Completion of the photolysis reaction was confirmed by injecting a sample into uHPLC using the method described previously. The photodeprotected sample was purified by preparative HPLC using a Shiseido Capcell Pack UG80 C18 column (50 × 250 mm) kept at 60 °C and a two-step gradient: dual gradient of CH3CN in water: 0.1% TFA: 10-35% in 5 min, then 35-65% in 35 min, at a flow rate of 40 mL / min using CH3CN and MQ-H2O containing 0.1% TFA as eluents. Fractions containing the product were pooled and lyophilized to give pure Seg12 (25.4 mg, 40% yield for the ligation and purification steps). m / z calculated for C 422 H 709 N 101 O 130 S[M]:9304.1694;measured 9304.1639.

[0404] KAHA Ligation for Preparation of IL2-Seg34 of Compositions AB by KAHA Ligation

[0405] [ka] Ligation: Seg3 (136 mg, 34 μmol, 1.2 equiv.) and Seg4 (100 mg, 28.40 μmol, 1 equiv.) were dissolved in DMSO / HO (9:1) containing 0.1 M oxalic acid (1.8 mL, 15 mM) and reacted for 16 h at 60 °C. The progress of the KAHA ligation was monitored by uHPLC at 60 °C using a Phenomenex C18 column (150 × 4.56 mm) with a gradient of 30 to 70% CH3CN in 7 min using CH3CN / HO containing 0.1% TFA as the mobile phase.

[0406] Fmoc deprotection and purification: After completion of ligation, the reaction mixture was diluted with DMSO (6 mL), 5% diethylamine (300 μL) was added, and the reaction mixture was shaken at room temperature for 7 min. To prepare the sample for purification, it was diluted with DMSO (4 mL) containing TFA (300 μL).

[0407] The sample was purified by preparative HPLC on a Shiseido Capcell Pack UG80 C18 column (50 × 250 mm) maintained at 60 °C using a gradient of 30 to 70% CH3CN in water containing 0.1% TFA in 35 min at a flow rate of 40 mL / min. Fractions containing the product were pooled and lyophilized to give pure Seg34 (43.4 mg, 21% yield after ligation and purification). Analytical HPLC and ESI-HRMS were used to confirm the purity and accurate mass of the product. m / z calculated for C 326 H 516 N 84 O 101 S[M]:7255.7545;measured:7255.7653.

[0408] Final KAHA ligation for preparation of IL2 linear protein composition AB by KAHA ligation Ligation: Seg12 (59.2 mg, 6.35 μmol, 1.2 equiv.) and Seg34 (38.5 mg, 5.3 μmol, 1 equiv.) were dissolved in DMSO / HO (9:1) containing 0.1 M oxalic acid (423 μL, 15 mM) and the ligation was allowed to proceed for 24 h at 60 °C. The progress of the KAHA ligation was monitored by analytical HPLC using a Shiseido Capcell Pak UG80 C18 column (250 × 4.6 mm) at 60 °C and CH3CN / HO containing 0.1% TFA as the mobile phase with a gradient of 30 to 95% CH3CN in 14 min.

[0409] Purification: After completion of ligation, the reaction mixture was diluted with 150 μL of DMSO and then further diluted with a mixture of (1:1) CH3CN:H2O containing 0.1% TFA (7 mL). The sample was purified by injection into a preparative HPLC using a Shiseido Capcell Pack UG80 C18 column (50 × 250 mm) preheated at 60 °C with a two-step gradient: 10-40% in 5 min and 40-80% in 35 min, flow rate: 40 mL / min with CH3CN and MQ-H2O containing 0.1% TFA as eluents. Fractions containing the product were pooled and lyophilized to obtain pure composition AB linear protein with Acm (42.3 mg, 48% yield for ligation and purification steps). Analytical HPLC and ESI-HRMS were used to confirm the purity and accurate mass of the product. m / z calculated for C 747 H 1225 N 185 O 229 S2[M]:16515.9340;measured 16515.9008.

[0410] Acm deprotection: Peptide IL2 linear protein with Acm (35.4 mg, 2.14 μmol) was dissolved in AcOH / HO (1:1) (8.6 mL, 0.25 mM) and 86 mg of AgOAc (1% m / v) was added to the solution. The mixture was protected from light and shaken at 50 °C for 2.5 h. After completion of the reaction as confirmed by HPLC, the sample was diluted with CHCN:HO (1:1) containing 0.1% TFA and purified by preparative HPLC using a Shiseido CapCell Pak UG80 C18 column (20 × 250 mm) maintained at 60 °C. A two-step gradient: 10-40% in 5 min and 40-95% in 30 min, flow rate: 10 mL / min, CH3CN containing 0.1% TFA and MQ-H2O as eluents were used for purification. The fractions containing the product were pooled and lyophilized to obtain pure IL2 linear protein (26.1 mg, 74% yield for the deprotection and purification steps). m / z calculated for C 741 H 1215 N 183 O 227 S2[M]:16373.8597;measured:16373.8253.

[0411] Synthesis of folded IL-2 composition AB

[0412] [ka] Reconstitution of linear protein: Linear protein (20 mg, 1.221 μmol) was dissolved in 6 M Gu·HCl aqueous solution containing 0.1 M Tris and 30 mM reduced glutathione (81 mL, 15 μM protein concentration), which was adjusted to pH 8.0 with 6 M aqueous HCl. The mixture was gently shaken at 50 °C for 2 h and monitored by analytical reversed-phase HPLC using a bioZen™ 3.6 μm intact C4 column (150 × 4.6 mm) at 25 °C with a gradient of 30 to 95% CH3CN in MQ-H2O containing 0.1% TFA in 18 min, flow rate: 1.0 mL / min.

[0413] Folding of the linear reconstituted protein: The previous solution was cooled to room temperature and diluted 3-fold to pH 8.0 with a second buffer (240 mL) containing 0.1 M Tris and 1.5 mM oxidized glutathione. The mixture was stored at room temperature and monitored by analytical HPLC using a bioZen™ 3.6 μm intact C4 column (150 × 4.6 mm) at 25 °C with a gradient of 30-95% acetonitrile with 0.1% TFA in 18 min at a flow rate of 1.0 mL / min. After 20 h, the folding solution was acidified to about pH 3 with 10% TFA in water and purified using preparative HPLC using a Shiseido Proteonavi C4 column (20 × 250 mm) with a two-step gradient of 5-40-95% acetonitrile with 0.1% TFA in 60 min at a flow rate of 10.0 mL / min. The fractions containing the folded IL2 protein were pooled together and lyophilized. The purity and identity of the pure folded protein (3.5 mg, 18% yield) was further confirmed by analytical RP-HPLC and high-resolution ESI mass spectrometry. m / z calculated for C 741 H 1213 N 183 O 227 S2[M]: 16371.8441; measured: 16371.8107, confirming successful synthesis of composition AB.

Claims

1. A composition comprising: a polypeptide that selectively binds to programmed cell death protein 1 (PD-1); a modified IL-2 polypeptide; a linker, wherein the linker has a first attachment point to the modified IL-2 polypeptide, and a second attachment point to the polypeptide that selectively binds to PD-1 and at least one of the first attachment point or the second attachment point is to a non-terminal residue of the polypeptide to which it is attached, the linker; A composition comprising the same.

2. The composition according to claim 1, wherein the first attachment point is at amino acid residue 42 or 45, and the amino acid residue numbering of the modified IL-2 polypeptide is based on SEQ ID NO: 1 as a reference sequence.

3. The composition according to claim 2, wherein the first attachment point is at amino acid residue F42Y or Y45.

4. The composition according to any one of claims 1 to 3, wherein the polypeptide that selectively binds to PD-1 is an anti-PD-1 antibody or an antigen-binding fragment thereof.

5. The composition according to claim 4, wherein the anti-PD-1 antibody or an antigen-binding fragment thereof comprises an Fc region.

6. The composition according to claim 5, wherein the second attachment point is at an amino acid residue in the Fc region.

7. The composition according to claim 6, wherein the second attachment point is in the Fc region at the position of amino acid residue K248, amino acid residue K288, amino acid residue K317, or a combination thereof (Eu numbering).

8. The composition according to claim 7, wherein the second attachment point is at the amino acid residue K248.

9. The composition according to claim 1, wherein the polypeptide that selectively binds to PD-1 is a monoclonal antibody.

10. The composition according to claim 9, wherein the polypeptide that selectively binds to PD-1 comprises IgG.

11. The composition according to claim 10, wherein the IgG is IgG1 or IgG4.

12. The composition according to claim 9, wherein the polypeptide that selectively binds to PD-1 comprises nivolumab, pembrolizumab, LZM-009 dostarlimab, sintilimab, spartalizumab, tislelizumab, or semaprilimab. **Claim 13**: The composition according to claim 1, wherein the linker comprises poly(alkylene oxide), polysaccharide, poly(vinylpyrrolidone), poly(vinyl alcohol), polyoxazoline, poly(acryloylmorpholine), or a combination thereof. **Claim 14**: The composition according to claim 1, wherein the modified IL-2 polypeptide comprises a non-linker polymer covalently attached thereto. **Claim 15**: The composition according to claim 14, wherein the non-linker polymer is attached to an N-terminal amino acid residue selected from the group consisting of amino acid residues 42 and 45. **Claim 16**: The composition according to claim 1, wherein the modified IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO:

3. **Claim 17**: A pharmaceutical composition comprising: a) the composition according to claim 1; and b) one or more pharmaceutically acceptable carriers or excipients. **Claim 18**: The composition according to claim 1 or the pharmaceutical composition according to claim 17 for use in treating cancer in a subject in need of treatment for cancer.

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