IL-2 fusion protein

Abstract

The present invention relates to PD-1:PD-L1 non-blocking immunoconjugates comprising an IL-2 polypeptide that provides a biased signal to the dimeric IL-2 receptor and a PD-1 targeting domain, and their use to treat cancer.

Description

[Technical Field]

[0001] The present invention relates to IL-2 fusion proteins designed to target exhausted T cells that express PD-1. The invention further relates to pharmaceutical compositions comprising the IL-2 fusion proteins and methods of treating cancer patients with the same, optionally in combination with PD-1 or PD-L1 antagonist antibodies. [Background technology]

[0002] Recombinant IL-2 (aldesleukin) is a promising immunotherapy that activates the IL-2 receptor expressed on effector T cell subsets, including CD8+ T cells and NK cells. In its native form, IL-2 is a 15-kDa protein that can signal through the high-affinity trimeric receptor IL-2R alpha / beta / gamma [composed of the IL-2R alpha chain (CD25), the IL-2R beta chain (CD122), and the IL-2R gamma chain (CD132)] or the intermediate-affinity dimeric receptor IL-2R beta / gamma. The CD122 and CD132 chains bind to the JAK and PI3K kinase families, enabling intracellular signaling. Meanwhile, CD25, although not involved in signal transduction, enhances receptor affinity for IL-2 by 10- to 100-fold. Regulatory T cells (Tregs) and pulmonary endothelial cells express CD25 and primarily utilize high-affinity trimeric receptors, whereas CD8+ T cells and NK cells (effector cells) do not express CD25 at steady state and instead display dimeric receptors on their cell surface. This receptor expression profile favors preferential stimulation of immunosuppressive Tregs over CD8+ T cells and NK cells, limiting the potential for antitumor responses induced by aldesleukin-mediated treatment. IL-2R-mediated activation of pulmonary endothelial cells contributes to the potent IL-2-associated side effects, such as hypotension and vascular leak syndrome.

[0003] One approach to improving the efficacy of aldesleukin is to develop molecules that bias signaling toward the intermediate-affinity IL-2 receptor expressed by effector cells and limit activation of the high-affinity IL-2 receptor expressed by Tregs, which can suppress antitumor immunity (Noverwijk WW et al. 2021, Ann. Rev. Med. 72:281). The antitumor activity of such receptor-biased IL-2 molecules can be further enhanced by targeting them to the tumor or tumor microenvironment, for example, by fusing the cytokine to an antibody that binds to a tumor antigen. These molecules present IL-2 to the entire cell and are often referred to as trans-signaling IL-2 bispecifics. IL-2 constructs targeted to tumor tissue have been shown to have synergistic effects when used in cancer models in combination with checkpoint inhibitor antibodies targeting PD-L1 (Klein C. 2017 Oncoimmunology 6:e1277306). Alternatively, receptor-biased IL-2 molecules can be targeted directly to CD8 T cells using targeting antibodies that bind to CD8, PD-1, or other antigens expressed on the surface of CD8 T cells. Because these constructs present IL-2 on the same T cell surface, they are often referred to as cis-signaling IL-2 bispecifics. Such cis-signaling fusion proteins have been generated using anti-PD-1 / IL-2 bispecifics (WO2018184964A1, Deak LC et al. 2022, Nature 610:161) and demonstrated excellent efficacy in preclinical mouse models. In all known anti-PD-1 / IL-2 constructs, the targeting arm consists of a blocking anti-PD-1 antibody (blocking the PD-1 / PD-1L interaction). Such a targeting arm has the potential advantage of relieving T cell suppression by PD-L1 and delivering a proliferative IL-2 signal to the same effector cells. However, there are inherent challenges in pharmacologically administering blocking therapeutics (which require complete target coverage) and agonistic therapeutics (which require agonistic doses and frequency).Specifically, in the case of PD-1 and IL-2 therapeutics, anti-PD-1 antibodies are administered at mg / kg doses to ensure complete target coverage, while IL-2 antibodies are administered at microgram / kg doses to avoid overstimulation and on-target toxicity. This discrepancy can be addressed by mutating the IL-2 portion of the molecule to reduce its affinity for its receptor, thereby enabling higher doses of the bispecific. However, mutations in the IL-2 construct can lead to the generation of anti-drug antibodies, leading to treatment failure and toxicity. Furthermore, in patients receiving PD-1 checkpoint inhibitor (CPi) therapy, the epitope targeted by the PD-1 / IL-2 fusion protein is already occupied by the CPi, preventing the fusion protein from binding to target cells. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] WO 2018184964(A1) [Non-patent literature]

[0005] [Non-Patent Document 1] Noverwijk WW et al. 2021, Ann.Rev.Med.72:281 [Non-patent document 2] Klein C. 2017 Oncoimmunology 6:e1277306 [Non-patent document 3] Deak LC et al. 2022, Nature 610:161 Summary of the Invention [Problem to be solved by the invention]

[0006] Based on the above-mentioned prior art, it is an object of the present invention to provide an IL-2 molecule with improved therapeutic properties. [Means for solving the problem]

[0007] This object is achieved by the subject matter of the independent claims herein, with further advantageous embodiments described in the dependent claims herein, the examples, the figures and the general description.

[0008] Summary of the Invention The present inventors aimed to develop a PD-1-targeted therapeutic IL-2 molecule that does not inhibit the binding and antagonism of conventional PD-1- or PD-L1-targeting checkpoint inhibitor antibodies. We engineered a non-blocking anti-PD-1 antibody to target IL-2 to PD-1-expressing cells. The anti-PD-1 binding domain of the non-blocking antibody was used as a targeting moiety for immunoconjugates further comprising an IL-2 polypeptide, delivering IL-2 signaling to appropriate exhausted CD8 T cells and enabling optimal combination with existing anti-PD1 therapeutics. The resulting compound provides highly targeted IL-2 signaling to exhausted T cells and can be more flexibly combined with existing CPi treatment regimens targeting the PD-1 / PD-L1 signaling axis. The result is an immunoconjugate compound that effectively delivers IL-2 stimulation while simultaneously allowing for the adjustment of the dosage of potent active agents, resulting in safe and effective cancer treatment.

[0009] One embodiment of the present invention is an IL-2 fusion protein immunoconjugate comprising an anti-PD-1 binding domain and an anti-IL-2 antigen binding domain. The anti-PD-1 binding domain comprises heavy and light chain polypeptides derived from an anti-PD-1 antibody (an antibody capable of specifically binding to the extracellular domain of human PD-1 protein). The anti-IL-2 binding domain comprises heavy and light chain polypeptides derived from an anti-IL-2 antibody (an antibody capable of specifically binding to human IL-2 protein). One of the antibody variable domains within the heavy or light chain of the anti-IL-2 antigen binding domain is optionally linked to an IL-2 polypeptide by one or two peptide linkers.

[0010] In certain embodiments, the IL-2 is human IL-2, an artificial IL-2 variant polypeptide such as Proleukin, or an IL-2 mutein polypeptide. In certain embodiments, the IL-2 polypeptide is a circularly permuted IL-2 polypeptide (IL2CP), characterized by amino acid residues rearranged relative to the human IL-2 sequence. Together, the anti-IL-2 binding domain bound to the IL-2 polypeptide biases IL-2 signaling toward the dimeric IL-2 receptor expressed on the target cell.

[0011] In certain embodiments, the anti-PD-1 binding domain incorporated into an immunoconjugate of the invention is derived from a non-blocking PD-1 antibody. That is, binding of the immunoconjugate to cells expressing PD-1 does not significantly inhibit the binding of PD-1 to its natural ligand PD-L1 or to PD-1 antagonist compounds, such as the antibodies nivolumab or pembrolizumab. This feature allows the immunoconjugate of the invention to be used as a pharmaceutical in combination with CPi compounds that bind to either PD-1 or PD-L1 and inhibit checkpoint blockade.

[0012] In a specific embodiment of the immunoconjugate according to the invention, the IL-2 polypeptide is IL2CP incorporated within the variable domain of the heavy or light chain of the anti-IL-2 binding domain, providing a single fusion polypeptide. In an alternative embodiment, the IL-2 polypeptide is linked by a peptide linker to the N'-terminal domain of either the heavy or light chain variable domain of the anti-IL-2 binding domain, providing a single fusion polypeptide.

[0013] In certain embodiments of the immunoconjugate according to the invention, it comprises an Fc portion, in particular an IgG isotype Fc portion, which in certain embodiments is characterized by amino acid modifications to enhance correct heavy chain pairing and / or to optimize the level of Fc effector function.

[0014] In a more specific embodiment, an immunoconjugate according to the invention is a heterotetrameric IgG (e.g., Crossmab, or kappa / lambda format) in which one antibody arm comprises an anti-PD-1 binding domain and one antibody arm comprises an anti-IL-2 antibody binding domain linked to an IL-2 polypeptide.

[0015] In certain embodiments, the immunoconjugate is an anti-PD-1 IgG in which both antibody arms (each arm comprising a heavy chain polypeptide associated with a light chain polypeptide) are characterized by an anti-PD-1 binding domain. In such embodiments, IL-2 signaling occurs through the fusion of an anti-IL-2 antibody binding domain in the form of an scFv, the scFv incorporating or linking an IL-2 polypeptide. In certain embodiments of the immunoconjugate, the C'-terminal residue of the anti-IL-2 binding domain scFv is linked to the N-terminal domain of an anti-PD-1 antibody.

[0016] A further aspect of the invention relates to a pharmaceutical composition comprising the immunoconjugate, a nucleic acid encoding the immunoconjugate, or a host cell engineered to contain said nucleic acid. The invention further encompasses an immunoconjugate according to the invention for use in the treatment of cancer, and a method of treating a cancer patient by administering a therapeutically effective amount of an immunoconjugate according to the invention. Terms and Definitions

[0017] For the purposes of interpreting this specification, the following definitions shall apply, and where appropriate, terms used in the singular shall also include the plural and vice versa. In the event that a definition set forth below conflicts with any document incorporated herein by reference, the definition set forth herein shall control.

[0018] As used herein, the terms "comprising," "having," "containing," "including," and other similar forms, and their grammatical equivalents, are intended to be equivalent in meaning and to be open-ended in that the listing of one or more items following any one of these words does not imply an exhaustive listing of such one or more items, or that it is limited to only the listed item or items. For example, an item "comprising" components A, B, and C can consist of components A, B, and C (i.e., contain only components A, B, and C), or it can include not only components A, B, and C, but also one or more other components. Thus, "comprises" and its similar forms, and its grammatical equivalents, are intended and understood to include disclosure of "consisting essentially of" or "consisting of" embodiments.

[0019] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of that range, and any other stated or intervening value within that stated range, is encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where one or both of the limits are included in the stated range, ranges excluding either or both of those included limits are also included in the disclosure.

[0020] As used herein, reference to "about" a value or parameter includes (and describes) a variation on the value or parameter itself. For example, a statement referring to "about X" also includes the statement "X."

[0021] As used in this specification, including the appended claims, the singular forms "a," "or," and "the" include plural references unless the context clearly dictates otherwise.

[0022] As used herein, "and / or" is considered to specifically describe each of the two specified features or components with or without the other features or components. Thus, the term "and / or" used in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A alone," and "B alone." Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A alone; B alone; and C alone.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry, organic synthesis). Standard techniques are used for molecular, genetic, and biochemical techniques (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY and Ausubel et al., Short Protocols in Molecular Biology (2002) 5th Ed., John Wiley & Sons, Inc.) and chemical techniques.

[0024] The term "PD-1" in the context of the present specification relates to the human PD-1 protein, which is sometimes also called CD279 (Uniprot Q15116), encoded by the "PDCD1" gene.

[0025] The term "PD-1" in the context of the present specification relates to the human PD-L1 protein, which is sometimes also called CD274 (Uniprot Q9NZQ7), encoded by the gene "CD274".

[0026] The term "immunoconjugate" in the context of this specification refers to a recombinant polypeptide molecule comprising at least one anti-PD-1 antibody binding domain disclosed herein covalently linked to at least one additional IL-2 signaling moiety. The IL-2 signaling moiety comprises both an IL-2 polypeptide (e.g., human IL-2 or IL-2CP) linked to the antigen-binding domain of an anti-IL-2-specific antibody, optionally by one or more short peptide linkers. The IL-2 moiety of the immunoconjugate can bind to the IL-2 receptor expressed on the cell surface of immune cells and initiate downstream effects. That is, the anti-PD-1 binding domain of the "immunoconjugate" functions as a targeting domain, and binding of the second IL-2-containing moiety to the cell initiates downstream functions in the target immune cell. The IL-2 signaling moieties provided herein exhibit preferentially biased signaling through the dimeric IL-2 receptor.

[0027] The term "IL-2" in the context of this specification relates to human IL-2 and functional variants thereof, such as the wild-type human amino acid sequence (Uniprot P60568), and variant proteins as set out in Table 2.

[0028] The term "IL2CP" or "IL2-CP" in the context of this specification refers to a circularly permuted IL-2 polypeptide created by "opening up" the IL-2 polypeptide sequence and fusing the native N- and C-terminal amino acid residues to create new N'- and C'-termini. This generates a rearranged IL2CP polypeptide while retaining the critical tertiary structure of the cytokine that enables it to signal through its dedicated receptor.

[0029] The term "dimeric IL-2 receptor" in the present context relates to a heterodimeric receptor comprising two receptor chains CD122 and CD132.

[0030] Any patent documents cited herein are deemed to be incorporated herein by reference in their entirety. array

[0031] Sequences similar or identical (e.g., at least about 70% sequence identity) to the sequences disclosed herein are also part of the present invention. In some embodiments, sequence identity at the amino acid level can be about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more. At the nucleic acid level, sequence identity can be about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more. Alternatively, substantial identity exists when the nucleic acid segment hybridizes under selective hybridization conditions (e.g., under very high stringency hybridization conditions), to the complement of the strand. The nucleic acid can be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form.

[0032] In the present context, the terms "sequence identity" and "percentage of sequence identity" refer to a quantitative parameter that represents the results of sequence comparison, which is determined by comparing the two aligned sequences position by position. Methods for aligning sequences for comparison are well known to those skilled in the art. Sequence alignment for comparison can be performed by Smith & Waterman's local homology algorithm, Adv. Appl. Math. 2: 482 (1981), Needleman & Wunsch's global alignment algorithm, J. Mol. Biol. 48: 443 (1970), Pearson & Lipman's similarity search method, Proc. Nat. Acad. Sci. 85: 2444 (1988), or computerized implementations of these algorithms, including but not limited to CLUSTAL, GAP, BESTFIT, BLAST, FASTA, and TFASTA. Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information (http: / / blast.ncbi.nlm.nih.gov / ) and other sources.

[0033] An example of an amino acid sequence comparison is the BLASTP algorithm using default settings: Expectation threshold: 10; Word size: 3; Maximum matches within query range: 0; Matrix: BLOSUM62; Gap cost: Existence 11, Extension 1; Composition adjustment: Conditional composition score matrix adjustment. An example of such a comparison of nucleic acid sequences is the BLASTN algorithm using default settings: Expectation threshold: 0; Word size: 28; Maximum matches within query range: 0; Match / Mismatch score: 1-2; Gap cost: Linear. Unless otherwise specified, the sequence identity values ​​provided herein refer to values ​​obtained using the BLAST series of programs (Altschul et al., J. Mol. Biol. 215: 403-410 (1990)) using the default parameters identified above for protein and nucleic acid comparisons, respectively.

[0034] Reference to identical sequences without specifying a percentage value means 100% identical sequences (ie, the same sequence).

[0035] General Biochemistry: Peptides, Amino Acid Sequences The term "polypeptide" in the context of this specification refers to a molecule consisting of 50 or more amino acids forming a linear chain, where the amino acids are connected by peptide bonds. The amino acid sequence of a polypeptide may represent the amino acid sequence of an entire protein (as found physiologically) or a fragment thereof. The terms "polypeptide" and "protein" are used interchangeably herein and include proteins and fragments thereof. Polypeptides are disclosed herein as amino acid residue sequences.

[0036] The term "peptide" in the present context relates to a molecule consisting of up to 50 amino acids, in particular 8 to 30 amino acids, more in particular 8 to 15 amino acids, which form a linear chain in which the amino acids are connected by peptide bonds.

[0037] The sequence of amino acid residues is written from the amino terminus to the carboxyl terminus. Capital letters at sequence positions refer to L-amino acids using the single-letter code (Stryer, Biochemistry, vol. 3, p. 21). Lowercase letters at amino acid sequence positions refer to the corresponding D- or (2R)-amino acids. The sequence is written from left to right from the amino terminus to the carboxyl terminus. According to standard nomenclature, amino acid residue sequences are represented by either three-letter or one-letter codes as follows: alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).

[0038] The term "variant" refers to a polypeptide that differs from a reference polypeptide but retains essential properties. A typical variant of a polypeptide differs from another, reference polypeptide in its primary amino acid sequence. Generally, differences are limited so that the sequences of the reference and variant are similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and / or deletions). A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A polypeptide variant may be naturally occurring, such as an allelic variant, or it may be a variant that is not known to occur naturally.

[0039] In the present context, the term "dimer" refers to a unit consisting of two subunits. In the present context, the term "heterodimer" refers to a dimer composed of two non-identical subunits.

[0040] In the present context, the term "amino acid linker" or "peptide linker" refers to a polypeptide of variable length that is used to join two polypeptides to produce a single polypeptide chain. Exemplary embodiments of linkers useful in practicing the invention defined herein are oligopeptide chains of 1, 2, 3, 4, 5, 10, 20, 30, 40, or 50 amino acids.

[0041] There are no restrictions on the amino acid composition of the linker. In one embodiment, the linker consists of amino acids selected from the group G, S, A, and D. Important features of the conjugated peptide linkers defined herein are low immunogenicity and a peptide length that allows the domains connected by the linker to interact and form functional entities as disclosed herein. In particularly desirable embodiments of the domain peptide linkers defined above, the sequences are primarily composed of stretches of small polar amino acids, such as glycine (G) and serine (S).

[0042] In one embodiment, the peptide linker is ≧15 amino acids in length, particularly 15-30 amino acids in length, and the amino acids are selected from G, S, A and D.

[0043] Non-limiting examples of amino acid linkers are monomers, dimers, trimers or tetramers of a peptide motif consisting of three or four glycines and one serine.

[0044] Any of the embodiments of peptide linkers disclosed herein encompass structures in which amino acids with similar properties can be substituted, e.g., amino acids V, L, I, P, S, C, or M can be substituted with G, S, or S, and D can be substituted with E.

[0045] Specific non-limiting examples of linkers are provided by SEQ ID NOs: 014, 015, 016, 017, 018, 019, 020, 021, 022, 023, and 024.

[0046] General molecular biology: nucleic acid sequence, expression The term "recombinant" in the context of the present specification relates to a nucleic acid that is the product of one or more steps of cloning, restriction and / or ligation and that differs from a naturally occurring nucleic acid. A recombinant viral particle comprises a recombinant nucleic acid.

[0047] The terms "gene expression" or "expression," or "gene product," may refer to either or both the process—and product—of producing a nucleic acid (RNA) or producing a peptide or polypeptide, also referred to as transcription and translation, respectively, or any intermediate process that regulates the processing of genetic information to produce a polypeptide product. The term "gene expression" can also apply to the transcription and processing of an RNA gene product, such as a regulatory RNA or a structural (e.g., ribosomal) RNA. When the expressed polynucleotide is derived from genomic DNA, expression can include splicing of mRNA in eukaryotic cells. Expression can be assessed at both the level of transcription and translation, i.e., the mRNA and / or the protein product.

[0048] In the context of this specification, the term "transmissible expression vector" refers to a plasmid, viral genome, or RNA used to transfect (in the case of a plasmid or RNA) or transduce (in the case of a viral genome) a gene of interest into a target cell, or, in the case of a transfected RNA construct, to translate the corresponding protein of interest from the transfected mRNA. In vectors that operate at the level of transcription and subsequent translation, the gene of interest is under the control of a promoter sequence that is operable in the target cell so that the gene of interest is transcribed constitutively, in response to a stimulus, or depending on the state of the cell. In certain embodiments, the viral genome is packaged into a capsid, resulting in a viral vector that can transduce target cells.

[0049] Binding; binders, ligands, antibodies: The term "specific binding" in the context of the present invention refers to the property of a ligand to bind to a target with a particular affinity and target specificity. The affinity of such a ligand is indicated by the dissociation constant of the ligand. A specifically reactive ligand has a dissociation constant of 10 or less when bound to a target. -7 mol / L or less (especially 10 -9mol / L or less), but when interacting with a molecule that has nearly the same chemical composition as the target but a different conformation, the dissociation constant is at least three orders of magnitude higher.

[0050] In the context of this specification, the term "dissociation constant (K D The term "equilibrium constant" is used in the sense known in the arts of chemistry and physics; it refers to the equilibrium constant that measures the tendency of a complex consisting of [often two] different components to reversibly dissociate into its constituent components. The complex may be, for example, an antibody-antigen complex AbAg consisting of an antibody Ab and an antigen Ag. D is expressed in molar concentration [mol / l] and corresponds to the concentration of [Ab] at which half of the binding sites for [Ag] are occupied, i.e., the concentration of unbound [Ab] is equal to the concentration of the [AbAg] complex. The dissociation constant can be calculated using the following formula:

number

[0051] In the context of this specification, "off-rate (K off ;[1 / s])" and "on-rate (K on ;[L / (sec * The terms "antibody dissociation (K mol)" and "antibody dissociation (K mol)" are used in the sense known in the art of chemistry and physics; they refer to the dissociation of an antibody from its target antigen (K mol). off ) or meeting (K on ) refers to the rate constant that measures the off and K. on can be determined experimentally using well-established methods by those skilled in the art. off and K. on Surface plasmon resonance is employed as a method for measuring the dissociation constant K, which is the principle behind biosensor systems such as the Biacore® or ProteOn® systems. These also measure the dissociation constant K using the following equation: D can be used to find:

number

[0052] Binding rate K on The natural upper limit of 9 L / (seconds * mol).

[0053] In the present context, the term "antibody" relates to whole antibodies, including but not limited to immunoglobulin type G (IgG), type A (IgA), type D (IgD), type E (IgE) or type M (IgM), any antigen-binding fragment or single chain thereof, and related or derived constructs. A whole antibody is a glycoprotein comprising at least two heavy (H) and two light (L) chains inter-connected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (V H ) and heavy chain constant region (C H The heavy chain constant region of IgG is composed of C H 1. C H 2 and C H Each light chain is composed of three domains: a light chain variable region (referred to herein as V L ) and the light chain constant region (C L The light chain constant region consists of one domain, C L The variable regions of the heavy and light chains contain the binding domains that interact with antigens. The binding specificity of an antibody is primarily determined by its complementarity-determining regions (CDRs). The numbering system used herein to identify the amino acid residues in the CDR regions is shown in Figure 1. The constant regions of an antibody may mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system. Similarly, this term encompasses so-called nanobodies or single domain antibodies, antibody fragments consisting of a single monomeric variable antibody domain.

[0054] In the context of this specification, the term "fragment crystallizable (Fc) region" is used in the sense known in the art of cell biology and immunology; when applied to IgG, it refers to the C region covalently linked by disulfide bonds. H 2 and C H Refers to the portion of an antibody that contains two identical heavy chain fragments consisting of three domains.

[0055] In the present context, the term "single-chain variable fragment (scFv)" refers to a fusion protein in which the variable region of the light chain of a Fab fragment of a monoclonal antibody and the variable region of the heavy chain variable domain are linked by a flexible peptide linker. Multimeric "scFv"s can be produced by linking two or more pairs of heavy and light chain domains with a peptide linker.

[0056] In the context of this specification, the term "humanized antibody" refers to an antibody originally produced by immune cells of a non-human species whose protein sequence has been modified to increase its similarity to antibody variants naturally produced in humans. As used herein, the term "humanized antibody" includes antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences as well as within the CDR sequences derived from the germline of another mammalian species.

[0057] (Cancer) Immunotherapy In the context of this specification, the term "anti-PD-1 antagonist antibody" is intended to encompass agents, in particular antibodies (or antibody-like molecules), that are capable of interrupting the signal cascade that leads to T cell inhibition after T cell activation as part of an immune checkpoint mechanism known to those skilled in the art. Non-limiting examples include clinically available antibody drugs such as nivolumab (Bristol-Myers Squibb; CAS No. 946414-94-4), pembrolizumab (Merck; CAS No. 1374853-91-4), dostallimab (Tesaro; CAS No. 2022215-59-2), and sintilimab (Eli Lilly, InnoVenture). and antibodies against PD-1 (Uniprot Q15116), exemplified by tislelizumab (BeiGene; CAS No. 1858168-59-8), cemiplimab (CAS No. 1801342-60-8), cetrelimab (CAS No. 2050478-92-5), southernlimab (CAS No. 2206792-50-7), toripalimab (CAS No. 1924598-82-2), zelvalimab (CAS No. 2315361-37-4), or ezabenlimab (CAS No. 2249882-54-8).

[0058] As used herein, the term "pharmaceutical composition" refers to a compound of the present invention or a pharmaceutically acceptable salt thereof, together with at least one pharmaceutically acceptable carrier. In certain embodiments, a pharmaceutical composition according to the present invention is provided in a form suitable for topical, parenteral, or injectable administration.

[0059] As used herein, the term "pharmaceutically acceptable carrier" includes any solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial, antifungal), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, and the like, and combinations thereof, as known to those of skill in the art (see, e.g., Remington: The Science and Practice of Pharmacy, ISBN 0857110624).

[0060] The term "cancer" as used in the context of this specification relates to malignant neoplastic diseases; the terms "cancer" and "malignant neoplastic diseases" are used interchangeably herein. Specific examples include carcinomas (cancers of epithelial origin), sarcomas (cancers of connective tissue origin), lymphomas and leukemias, germ cell-derived tumors, and blastomas. Certain alternatives of any of the aspects and embodiments disclosed herein are directed to the use of the compounds and compositions of the present invention in the treatment of solid tumors. Other alternatives of any of the aspects and embodiments disclosed herein are directed to the use of the combinations of the present invention in the treatment of liquid cancers such as myeloid or granulocytic leukemias, particularly AML, lymphocytic, lymphocytic, or lymphoblastic leukemias and lymphomas, polycythemia vera, or erythrocytes.

[0061] As used herein, the term "treating" or "treatment" of any disease or disorder (e.g., cancer) refers, in one embodiment, to ameliorating the disease or disorder (e.g., slowing, halting, or alleviating the progression of the disease or at least one of its clinical symptoms). In another embodiment, "treating" or "treatment" refers to alleviating or improving at least one physical parameter, including those that may not be discernible to the patient. In yet another embodiment, "treating" or "treatment" refers to modulating the disease or disorder, either physically (e.g., stabilization of discernible symptoms), physiologically (e.g., stabilization of physical parameters), or both. Methods for assessing the treatment and / or prevention of disease are generally known to those of skill in the art, unless otherwise specified herein below. DETAILED DESCRIPTION OF THE INVENTION

[0062] Detailed Description of the Invention A first aspect of the present invention is an immunoconjugate comprising an anti-PD-1 antibody binding domain, an anti-IL-2 antibody antigen-binding domain, and an IL-2 polypeptide. The IL-2 polypeptide is covalently linked to amino acid residues of the anti-IL-2 binding domain, optionally by one or two peptide linkers, to provide a single, contiguous recombinant polypeptide, resulting in an immunoconjugate that provides biased IL-2 signaling to the dimeric IL-2 receptor. The characteristics of each of these functional elements are described in detail in the following sections.

[0063] Anti-PD1 binding domain The anti-PD1 binding domain of the immunoconjugate of the present invention is the antigen-binding domain portion of an anti-PD-1 antibody, i.e., it comprises heavy and light chain polypeptides derived from the antibody that can specifically bind to the extracellular domain of human PD-1 protein. PD-1 specificity is conferred by the antibody heavy chain variable domain polypeptide (PD1-VH) and the antibody light chain variable domain polypeptide (PD1-VL) comprising the antibody CDRs and surrounding framework residues.

[0064] The anti-PD-1 binding domain, and thus the immunoconjugate as a whole, does not block the interaction between PD-1 and the antagonist CPi antibodies that target PD-1 currently in commercial / clinical use, including pembrolizumab and nivolumab. The non-blocking nature of the immunoconjugate allows the compound to be administered in combination with existing regulatory-approved anti-PD-1 checkpoint inhibitor antibodies as therapeutics targeting PD-1+ cells. The immunoconjugate targets PD-1+ cells and delivers IL-2 signaling to exhausted PD-1+ cells without interfering with the effects of co-administered CPi compounds (Table 17, Figure 3). Example 13 further demonstrates that the immunoconjugate does not inhibit the binding of PD-1's natural ligand, PD-L1, to PD-1.

[0065] Significant inhibition of PD-1 antagonist antibody binding by immunoconjugates to the cells can be determined by measuring the percentage of immunoconjugate bound to a sample of human PD-1+ cells (e.g., Jurkat PD-1+ cells) after preincubation with a 100-fold molar excess of pembrolizumab or nivolumab. The mean fluorescence intensity (MFI) of the immunoconjugate in the pembrolizumab or nivolumab pre-blocked samples is lower than that of the control untreated samples, being 20% ​​or less, particularly 15% or less.

[0066] In one embodiment, binding of the immunoconjugate to PD-1+ cells is not significantly inhibited by a PD-1 antagonist antibody, particularly pembrolizumab or nivolumab.

[0067] In one embodiment, binding of the immunoconjugate to recombinant PD-1 does not inhibit binding of recombinant PD-L1 to PD-1.

[0068] The inventors used several strategies to obtain suitable anti-PD-1 binding domains according to the present invention. First, the affinity, clinical safety (e.g., cross-reactivity), and pharmacokinetic properties (e.g., stability) of non-blocking antibodies were optimized to develop antibodies with more favorable clinical properties. Second, suitable novel monoclonal antibodies were developed by screening hybridomas for the non-blocking properties of pembrolizumab or nivolumab, as well as multiple parameters such as PD-1 affinity, tissue cross-reactivity, heavy and light chain liability, and immunogenicity.

[0069] In some embodiments of immunoconjugates according to the invention, the anti-PD-1 binding domain is derived from one of the antibodies disclosed herein as optimized clones derived from the parent clone z2-XVT458, assigned as z2-XVT458m1-m6 (SEQ ID NOS: 057-068). In alternative embodiments, the anti-PD-1 binding domain is derived from a clone selected from those disclosed in Tables 14, 15, or 16. Such antibodies exhibit favorable high-affinity binding to PD-1 and cross-reactivity to human and primate PD-1, allowing for clinical trials in primate models.

[0070] In some embodiments of the immunoconjugates according to the invention, the PD1-VH comprises an HCDR1 having the sequence GFTFSINAMT (SEQ ID NO: 118), an HCDR2 having the sequence TISGSGFSTYYADSLKGR (SEQ ID NO: 119), and an HCDR3 having the sequence EVYGDY (SEQ ID NO: 120). 1 SSNIGSX 2 X 3 VF (SEQ ID NO: 121) 1 is N, S, Q, or A, and X 2 X 3 In certain embodiments, the LCDR1 has the sequence SNNQRPS (SEQ ID NO: 122), and the LCDR3 has the sequence AAWDDSLSIWVF (SEQ ID NO: 123). 1 is selected from the list consisting of S, Q, or A; X 2 X 3 is a pair of amino acid residues selected from QS, SS, or NA.

[0071] This includes the PD-1 binding domain tested in the examples that has higher affinity for PD-1 with modified deamination and glycosylation sites compared to the LCDR1 of the high-affinity anti-PD-1 parent clone 21A08 (see SEQ ID NOs: 071, 072).

[0072] In some embodiments of the immunoconjugates according to the invention, the PD1-VH comprises an HCDR1 having the sequence GFTFSINAMT (SEQ ID NO: 118), an HCDR2 having the sequence TISGSGFSTYYADSLKGR (SEQ ID NO: 119), and an HCDR3 having the sequence EVYGDY (SEQ ID NO: 120). QS VF (underlined parts are in bold) ) (SEQ ID NO: 124), LCDR2 having the sequence SNNQRPS (SEQ ID NO: 122), and LCDR3 having the sequence AAWDDSLSIWVF (SEQ ID NO: 123). Such embodiments relate to anti-PD-1 binding domains having the CDRs of clone 21A08Ap1, which is a high-affinity, non-blocking anti-PD-1 antibody that binds to both human and primate PD-1 with little cross-reactivity to other proteins. In specific embodiments, the complete heavy and light chain variable domains are identical to those of 21A8Ap1, and the PD1-VH comprises the polypeptide having SEQ ID NO: 085 and the PD1-VL comprises the polypeptide having SEQ ID NO: 086. In more specific embodiments, the PD1-VH consists of SEQ ID NO: 085 and the PD1-VL consists of SEQ ID NO: 086.

[0073] In some embodiments of the immunoconjugates according to the invention, the PD1-VH comprises an HCDR1 having the sequence GFTFSINAMT (SEQ ID NO: 118), an HCDR2 having the sequence TISGSGFSTYYADSLKGR (SEQ ID NO: 119), and an HCDR3 having the sequence EVYGDY (SEQ ID NO: 120). SSThe PD1-VH comprises an LCDR1 having the sequence SNNQRPS (SEQ ID NO: 122), an LCDR2 having the sequence AAWDDSLSIWVF (SEQ ID NO: 123), and an LCDR3 having the sequence AAWDDSLSIWVF (SEQ ID NO: 124). This embodiment relates to an anti-PD-1 binding domain comprising the CDRs of clone 21A08Ap2, a high-affinity, non-blocking anti-PD-1 antibody that exhibits minimal cross-reactivity with other proteins and binds to both human and primate PD-1. In a specific embodiment, the complete heavy and light chain variable domains are identical to those of 21A8Ap2, with PD1-VH comprising the polypeptide having SEQ ID NO: 085 and PD1-VL comprising the polypeptide having SEQ ID NO: 087. In a more specific embodiment, PD1-VH consists of SEQ ID NO: 085 and PD1-VL consists of SEQ ID NO: 087.

[0074] In some embodiments of the immunoconjugates according to the invention, the PD1-VH comprises an HCDR1 having the sequence GFTFSINAMT (SEQ ID NO: 118), an HCDR2 having the sequence TISGSGFSTYYADSLKGR (SEQ ID NO: 119), and an HCDR3 having the sequence EVYGDY (SEQ ID NO: 120). NA The PD1-VH comprises an LCDR1 having the sequence SNNQRPS (SEQ ID NO: 122), an LCDR2 having the sequence AAWDDSLSIWVF (SEQ ID NO: 123), and an LCDR3 having the sequence AAWDDSLSIWVF (SEQ ID NO: 124). This embodiment relates to an anti-PD-1 binding domain comprising the CDRs of clone 21A08Ap3, a high-affinity, non-blocking anti-PD-1 antibody that binds to both human and primate PD-1. In a specific embodiment, the complete heavy and light chain variable domains are identical to those of 21A8Ap3, and the PD1-VH comprises the polypeptide having SEQ ID NO: 085 and the PD1-VL comprises the polypeptide having SEQ ID NO: 091. In a more specific embodiment, the PD1-VH consists of SEQ ID NO: 085 and the PD1-VL consists of SEQ ID NO: 091.

[0075] In some embodiments of the immunoconjugates according to the invention, the PD1-VH comprises an HCDR1 having the sequence GFTFSISAMT (SEQ ID NO: 135), an HCDR2 having the sequence GFTFSISAMT (SEQ ID NO: 136), an HCDR3 having the sequence GFTFSISAMT (SEQ ID NO: 137), an HCDR4 having the sequence GFTFSISAMT (SEQ ID NO: 138), an HCDR5 having the sequence TISGSGGSTYYSDSVKG (SEQ ID NO: 136), and HCDR3 having the sequence EVYGDY (SEQ ID NO: 120). PD1-VL also contains an HCDR2 having the sequence SGSSSNIGSNS (SEQ ID NO: 126), LCDR2 having the sequence SNNQRPS (SEQ ID NO: 122), and LCDR3 having the sequence AAWDDSLSIWVF (SEQ ID NO: 123). Such embodiments relate to anti-PD-1 binding domains comprising the CDRs of clone 22F13, a high-affinity, anti-PD-1, non-blocking antibody that binds to both human and primate PD-1.

[0076] In some embodiments of the immunoconjugates according to the invention, the PD1-VH comprises the sequence GFTFSTYAMS HCDR1 having the sequence (SEQ ID NO: 138) TISGTGYSTYFADSVKG (SEQ ID NO: 139), and HCDR3 having the sequence EVYGDY (SEQ ID NO: 120). PD1-VL also contains an HCDR2 having the sequence SGRSSNIGSNS (SEQ ID NO: 140), LCDR2 having the sequence SNNQRPS (SEQ ID NO: 122), and the sequence AAWDDSLSGWV (SEQ ID NO: 141). One such embodiment relates to an anti-PD-1 binding domain having the CDRs of clone 25I20, a high-affinity, non-blocking anti-PD-1 antibody that binds to both human and primate PD-1.

[0077] In some embodiments of the immunoconjugates according to the invention, the PD1-VH comprises the sequence GFTFSSYS HCDR1 having the sequence (SEQ ID NO: 142) FISSSSPTLYYADSVKG (SEQ ID NO: 143), and HCDR2 having the sequence ARQGLTPFDY (SEQ ID NO: 144). PD1-VL also contains an HCDR3 having the sequence RASQSVSSYLA LCDR1 having the sequence (SEQ ID NO: 145), GASTRAT (SEQ ID NO: 146), and LCDR2 having the sequence QQYNNWPYT(SEQ ID NO: 147). One such embodiment relates to an anti-PD-1 binding domain having the CDRs of clone 20H02, a high-affinity, non-blocking anti-PD-1 antibody that binds to both human and primate PD-1.

[0078] In some embodiments of the immunoconjugates according to the invention, the PD1-VH comprises the sequence GFTFSNYGMH HCDR1 having the sequence (SEQ ID NO: 148) VIWYDGSKKYYADSVKG (SEQ ID NO: 149), and HCDR2 having the sequence NSGH (SEQ ID NO: 150). PD1-VL also contains an HCDR3 having the sequence RSSQSLLDSDDGNTYLD LCDR1 having the sequence (SEQ ID NO: 151), TLSYRAS (SEQ ID NO: 152), and LCDR2 having the sequence MQCIEFPHT (SEQ ID NO: 153). One such embodiment relates to an anti-PD-1 binding domain having the CDRs of clone 39F23, a high-affinity, non-blocking anti-PD-1 antibody that binds to both human and primate PD-1.

[0079] In some embodiments of the immunoconjugates according to the invention, the PD1-VH comprises the sequence GFTFSSYSMN HCDR1 having the sequence (SEQ ID NO: 154) YITSSSNTMYYADSVKG (SEQ ID NO: 155), and HCDR2 having the sequence IVGAPFDY (SEQ ID NO: 156). PD1-VL also contains an HCDR3 having the sequence RASQSVSSSLA LCDR1 having the sequence (SEQ ID NO: 157), GASTRAT (SEQ ID NO: 146), and LCDR2 having the sequence QQYNNWPFT (SEQ ID NO: 158). One such embodiment relates to an anti-PD-1 binding domain having the CDRs of clone 40B20, a high-affinity, non-blocking anti-PD-1 antibody that binds to both human and primate PD-1.

[0080] In some embodiments of the immunoconjugates according to the invention, the PD1-VH comprises the sequence GYTFTSYFMH HCDR1 having the sequence (SEQ ID NO: 159) LINPDGGNTDYAQKFQG (SEQ ID NO: 160), and HCDR2 having the sequence DGNYYDSSGYYYPDAFDI (SEQ ID NO: 161). PD1-VL also contains an HCDR3 having the sequence QGDSLRRFYAS (SEQ ID NO: 162), LCDR2 having the sequence GKDNRPS (SEQ ID NO: 163), and the sequence NSRDSSGTHVV (SEQ ID NO: 164). One such embodiment relates to an anti-PD-1 binding domain having the CDRs of clone 56H02, a high-affinity, non-blocking anti-PD-1 antibody that binds to both human and primate PD-1.

[0081] In further embodiments of the immunoconjugates of the invention, the anti-PD-1 binding domain is derived from the parent clone z2-XVT458, which has high affinity for both human and primate PD-1. In addition to the CDR mutations, the sequences of z2-XVT458-m1, z2-XVT458-m3, and z2-XVT458-m6 contain a G57D mutation immediately downstream of CDRL2, which may contribute to the increased affinity of these clones.

[0082] In one embodiment of the immunoconjugate according to the invention, the PD1-VH comprises an HCDR1 having the sequence NFYIH (SEQ ID NO: 127), an HCDR2 having the sequence NFYIH (SEQ ID NO: 127), an HCDR3 having the sequence NFYIH (SEQ ID NO: 127), an HCDR4 having the sequence NFYIH (SEQ ID NO: 127), an HCDR5 having the sequence R IYPNYG I PD1-VL also contains an HCDR2 with the sequence TAYNQKFKD (underlined in bold) (SEQ ID NO: 128), and an HCDR3 with the sequence GYSYAMDY (SEQ ID NO: 129). PD1-VL also contains an LCDR1 with the sequence SASQGISGDLN (SEQ ID NO: 130), an HCDR3 with the sequence HTS QR LCDR2 with HS (underlined in bold) (SEQ ID NO: 131), and sequence Q GYSKDLLT (underlined in bold) (SEQ ID NO: 132). Such embodiments encompass anti-PD-1 binding domains having the CDRs of clone XVT458-Z2-M3. This clone has high affinity for binding to human and primate PD-1 with little cross-reactivity with other human protein antigens. In specific embodiments, the complete heavy and light chain variable domains are identical to those of XVT458-Z2-M3, the PD1-VH comprises or consists of a polypeptide having SEQ ID NO: 061, and the PD1-VL comprises or consists of a polypeptide having SEQ ID NO: 062.

[0083] In one embodiment of an immunoconjugate according to the invention, the PD1-VH comprises an HCDR1 having the sequence NFYIH (SEQ ID NO: 127), an HCDR2 having the sequence SIYPNYG I PD1-VL also contains an HCDR2 with the sequence TAYNQKFKD (underlined in bold) (SEQ ID NO: 133), and an HCDR3 with the sequence GYSYAMDY (SEQ ID NO: 129). PD1-VL also contains an LCDR1 with the sequence SASQGISGDLN (SEQ ID NO: 130), an HCDR3 with the sequence HTS Q LCDR2 with LHS (underlined in bold) (SEQ ID NO: 134), and sequence Q G YSKDLLT (underlined in bold) (SEQ ID NO: 132). Such embodiments encompass anti-PD-1 binding domains having the CDRs of clone XVT458-Z2-m6. This clone has high affinity for binding to human and primate PD-1 with little cross-reactivity with other human protein antigens. In specific embodiments, the complete heavy and light chain variable domains are identical to those of XVT458-Z2-m6, the PD1-VH comprises or consists of a polypeptide having SEQ ID NO: 067, and the PD1-VL comprises or consists of a polypeptide having SEQ ID NO: 068.

[0084] In certain embodiments of the immunoconjugates according to the invention, the affinity constant (K D ) is 4.0 × 10 -9 ~1×10 -11In certain embodiments, the K D is 2.0 x 10 -9 ~1.5×10 -11 In certain embodiments, the anti-PD-1 domain confers very high affinity to PD-1, with a K D is 2 x 10 -9 ~2×10 -11 mol / L range.

[0085] In certain other embodiments of the immunoconjugates according to the invention, the affinity constant (K D ) is 4.0 × 10 -8 ~1.5×10 -10 In certain embodiments, the K D is 2.0 x 10 -8 ~1.5×10 -10 mol / L.

[0086] In certain embodiments, the anti-PD1 antigen-binding domain is a variant of 21A08. In certain embodiments thereof, the PD1-VH comprises or consists of a polypeptide at least 95% or more, 98% or more, or 99% or more similar to SEQ ID NO:085 (VH 21A08), and the PD1-VL comprises or consists of a polypeptide at least 95% or more, 98% or more, or 99% or more similar to SEQ ID NO:086 (VL 21A08Ap1). In certain embodiments, the PD1-VH comprises or consists of a polypeptide at least 95% or more, 98% or more, or 99% or more similar to SEQ ID NO:085 (VH 21A08), and the PD1-VL comprises or consists of a polypeptide at least 95% or more, 98% or more, or 99% or more similar to SEQ ID NO:087 (VL 21A08Ap2). In specific embodiments, PD1-VH comprises or consists of a polypeptide that is 95% or more, 98% or more, or 99% or more similar to SEQ ID NO: 085 (VH 21A08), and PD1-VL comprises or consists of a polypeptide that is 95% or more, 98% or more, or 99% or more similar to SEQ ID NO: 091 (VL 21A08Ap3). These variants of 21A08 exhibit increased K-like activity against PD-1. D is 1.0×10 -9 ~1.5×10 -11 It is characterized by being.

[0087] In certain embodiments, the anti-PD1 antigen-binding domain is a variant of XVT458. In certain embodiments thereof, the PD1-VH comprises or consists of a polypeptide 95% or more, 98% or more, or 99% or more similar to SEQ ID NO:061 (VH XVT458-Z2-M3), and the PD1-VL comprises or consists of a polypeptide 95% or more, 98% or more, or 99% or more similar to SEQ ID NO:062 (VL XVT458-Z2-M3). In certain embodiments, the PD1-VH comprises or consists of a polypeptide 95% or more, 98% or more, or 99% or more similar to SEQ ID NO:067 (VH XVT458-Z2-M6), and the PD1-VL comprises or consists of a polypeptide 95% or more, 98% or more, or 99% or more similar to SEQ ID NO:068 (VL XVT458-Z2-M6). These variants of XVT458 inhibit the K-antibody against PD-1. D is 2.0×10 -9 ~1.0×10 -10 It is characterized by:

[0088] IL-2 polypeptides and anti-IL-2 binding domains The anti-IL-2 binding domain comprises heavy and light chain polypeptides derived from an anti-IL-2 antibody capable of specifically binding to human IL-2 protein. The IL-2 polypeptide is covalently linked to amino acid residues of the anti-IL-2 binding domain, optionally by one or two peptide linkers, to provide a single, contiguous recombinant polypeptide that provides biased signaling to the dimeric IL-2 receptor.

[0089] The anti-IL-2 binding domain first comprises an antibody heavy chain variable domain polypeptide (IL2-VH) having, from N'-terminus to C'-terminus, the following domains: a first framework region (IL2VH1), a first complementarity-determining region (IL2HCDR1), a second framework region (IL2VH2), a second complementarity-determining region (IL2HCDR2), a third framework region (IL2VH3), a third complementarity-determining region (IL2HCDR3), and a fourth framework region (IL2VH4). The anti-IL-2 binding domain further comprises an antibody light chain variable domain polypeptide (IL2-VL) having, from the N' to C' terminus, the following domains: a first framework region (IL2VL1), a first complementarity determining region (IL2LCDR1), a second framework region (IL2VL2), a second complementarity determining region (IL2LCDR2), a third framework region (IL2VL3), a third complementarity determining region (IL2LCDR3), and a fourth framework region (IL2VL4).

[0090]

[0023] Embodiments of the IL-2 polypeptide portion of an immunoconjugate according to the invention include human IL-2; an artificial IL-2 variant polypeptide, such as Proleukin; or an IL-2 mutein as provided in Table 2. IL-2 polypeptides according to the invention lack the signal peptide M1-S21 of native IL-2 SEQ ID NO:005. IL-2 polypeptides comprise the following functional regions, as shown in Table 2: the first alpha helix (A), the second alpha helix (B), the third alpha helix (C), and the fourth alpha helix (D). In some embodiments, the IL-2 polypeptide is a mutein, which is a recombinant IL-2 protein containing one or more amino acid substitutions or deletions to modify function, such as reduced binding to CD25. In certain embodiments, the immunoconjugate comprises a mutein selected from those listed in Table 2. Mutein or IL-2 variant polypeptides according to the invention are greater than 90%, particularly 95%, or 98% similar to SEQ ID NO:005 and have equivalent biological function to SEQ ID NO:005. Non-limiting examples of variants and muteins are disclosed as SEQ ID NOs: 006 and 007. The biological function of an IL-2 polypeptide is determined, according to the present invention, by the K of an immunoconjugate to the dimeric IL-2 receptor as measured by surface plasmon resonance (SPR). D is in the range of 500 nM to 0.1 nM, particularly 100 nM to 0.1 nM, more particularly 10 nM to 0.1 nM, and even more particularly K D is defined as the affinity for the dimeric IL-2 receptor, with a value in the range of 1 nM to 0.1 nM.

[0091] Circularly permuted IL-2 polypeptide In particular embodiments of immunoconjugates according to the invention, the IL-2 polypeptide is a circularly permuted IL-2 (IL2CP) having functional domains rearranged relative to the domain positions in the wild-type sequence of SEQ ID NO: 005, as in the example provided in Table 3. Particular embodiments of IL-2 polypeptide starting sequences amenable to circular permutation are wild-type (WT) SEQ ID NO: 005, SEQ ID NO: 006, or a mutein as set forth in Table 2.

[0092] Circularly permuted IL-2 polypeptides are generated by "opening" an IL-2 polypeptide sequence, such as SEQ ID NO: 005 or 006, to create new N'- and C'-termini and combine the native N'- and C'-terminal amino acid residues (see SEQ ID NOs: 010-013 for examples of the resulting circularly permuted interleukin sequences). This provides a permuted IL2CP polypeptide that retains the tertiary structure of human IL-2 (see, e.g., WO2013184942A1). In some embodiments, the IL-2 polypeptide is opened between two consecutive amino acids. In other embodiments, the IL-2 polypeptide is opened between two positions within a single region (thus potentially truncating one or several amino acids of the IL-2 polypeptide, such as the structure disclosed in WO2013184942A1). In certain embodiments, the N- and C-termini of the new IL2CP are amino acids that are no more than two amino acids apart in the original IL-2 polypeptide sequence. In a more specific embodiment, the C-terminus and N-terminus of IL2CP are adjacent residues in a human IL-2 polypeptide or mutein, and the IL2CP contains all amino acid residues of the original IL-2 polypeptide sequence, none of which have been truncated.

[0093] In particular embodiments of the immunoconjugate, the anti-IL-2 binding domain is linked to an IL2CP in which the N-terminal amino acid of a human IL-2 polypeptide or variant thereof, particularly an N-terminal amino acid selected from A21, P22, T23, S24, S25, or S26, is linked to a C-terminal amino acid of a human IL-2 polypeptide or variant thereof selected from T150, L152, and T153. In a more particular embodiment, IL-2 residue P22 is linked to T153.

[0094] Importantly, IL2CP is opened at a position that does not disrupt the alpha-helical structure. The inventors have identified such regions between the C and B helices, between the A and B helices, and between the C and D helices. In certain embodiments, both the new N- and C-termini are located within the region V89-D104, particularly S95-N97, more particularly K96 and N97. This provides for IL2CP cleavage between C / B, where the alpha helices of IL2CP are in the order CDAB. In some embodiments, both the new N- and C-termini are located within the region G47-E72, particularly L56-E72, more particularly K63-F64. This provides for IL2CP cleavage between A / B, where the alpha helices of IL2CP are in the order BCDA. In some embodiments, both the new N- and C-termini are located within the region G118-I134, particularly S95-N97, more particularly S119-A132. This provides for IL2CP cleavage between C / D, where the alpha helices of IL2CP are in the order DABC.

[0095] Fusion of an anti-IL-2 binding domain with an IL-2 polypeptide In the immunoconjugates of the present invention, one of the antibody variable domains within the heavy or light chain of the anti-IL-2 antigen-binding domain is optionally linked to an IL-2 polypeptide by one or two peptide linkers. Together, the anti-IL-2 binding domain linked to the IL-2 polypeptide provides biased IL-2 signaling to the dimeric IL-2 receptor expressed on exhausted CD8+ T cells. In some embodiments, the N-terminus of IL2CP or the C-terminus of IL2CP is optionally covalently linked to IL2-VL or IL2-VH by one or two peptide linkers. In some embodiments, the N-terminus of IL2CP or the C-terminus of IL2CP is optionally covalently linked to IL2-VL and IL2-VH by one or two peptide linkers, i.e., IL2CP is incorporated within IL2-VL or IL2-VH.

[0096] In some embodiments of the immunoconjugate according to the invention, both the N'-terminus of IL2CP and the C'-terminus of IL2CP are linked, optionally by one or two peptide linkers, to the IL2HCDR1, IL2HCDR2, IL2VH 3、 The IL2CP domain is covalently linked to an amino acid residue within a domain selected from the list consisting of IL2HCDR3, IL2LCDR1, IL2LCDR2, IL2VL3, or IL2LCDR3. Our crystal structure analysis suggests that these are particularly useful sites for incorporating IL2CP to ensure optimal positioning of the cytokine relative to the anti-IL-2 binding domain and maintain biased IL-2 signaling to the IL-2 receptor.

[0097] In some embodiments of immunoconjugates according to the invention, the anti-IL-2 binding domain is fused to an IL2CP that is at least 98% similar to SEQ ID NO: 010, CDAB variant 1 of IL2CP validated in Example 1. In some embodiments of immunoconjugates according to the invention, the anti-IL-2 binding domain is fused to an IL2CP that is at least 98% similar to SEQ ID NO: 011, CDAB variant 2 of IL2CP validated in Example 1. In these embodiments, the N-terminus of IL2CP and the C-terminus of IL2CP are covalently linked, directly or by one or two peptide linkers, to amino acid residues within a domain selected from the list consisting of IL2LCDR1, IL2LCDR2, IL2HCDR1, or IL2HCDR3, particularly IL2LCDR1 or IL2HCDR3. In certain embodiments, the CDAB IL2CP polypeptide is incorporated within IL2LCDR1.

[0098] In some embodiments of immunoconjugates according to the invention, the anti-IL-2 binding domain is linked to an IL2CP that is at least 98% similar to SEQ ID NO: 012, IL2CP variant 3 BCDA. In these embodiments, the N-terminus of IL2CP and the C-terminus of IL2CP are covalently linked, optionally by one or two peptide linkers, to amino acid residues within a domain selected from the list consisting of IL2LCDR1, IL2LCDR3, IL2HCDR1, IL2HCDR2, and IL2VH3. In certain embodiments, the N-terminus of IL2CP and the C-terminus of IL2CP are linked to IL2HCDR2. IL2CP in BCDA format may also be linked to the anti-IL-2 binding domain at the end of IL2VL4 by a flexible linker.

[0099] In some embodiments of the immunoconjugates according to the invention, the anti-IL-2 binding domain is linked to an IL2CP that is at least 98% similar to IL2CP variant 3 DABC, SEQ ID NO: 013. In these embodiments, the N-terminus of IL2CP and the C-terminus of IL2CP are preferably covalently linked, optionally by one or two peptide linkers, to amino acid residues within a domain selected from the list consisting of IL2HCDR2 and IL2VH3.

[0100] In a particular embodiment of the immunoconjugate according to the invention, IL2HCDR1, IL2HCDR2, IL2VH 3、In a domain selected from the list consisting of IL2HCDR3, IL2LCDR1, IL2LCDR2, IL2VL3, or IL2LCDR3, 0 (no amino acids), or 1, 2, 3, or 4 consecutive amino acids, in particular 2 consecutive amino acids, are replaced by a polypeptide consisting of, listed N to C, the following: a first peptide linker, an IL2CP as described in the previous five paragraphs, and a second peptide linker. For this aspect of the invention, "0" means that the sequence is inserted into the IL2-VH or IL2-VL region, but no amino acids are lost from the anti-IL-2 binding domain antibody portion; "1" means that IL2CP is inserted in place of the one missing amino acid, "2" means similar, but two amino acids have been lost from IL-2-VH or VL or replaced by IL2CP, etc.

[0101] In a specific embodiment of the immunoconjugate according to the invention, it comprises IL2CP incorporated within IL-2-VH or IL-2-VL, with the respective termini flanked or connected by a short peptide linker. In a more specific embodiment, both the first and second peptide linkers are 1 to 20 amino acids in length. In an even more specific embodiment, both the first and second peptide linkers are 2 to 7 amino acids in length. In a further specific embodiment of the immunoconjugate according to the invention, any peptide linker flanking IL2CP is composed of glycine (G), or G and serine (S) residues. In a more specific embodiment, the peptide linker connecting the C-terminal or N-terminal residue of the IL2 polypeptide to the amino acids of IL-2VH or IL-VL has a sequence selected from the sequences assigned as SEQ ID NOs: 014 to 024.

[0102] N-terminally linked IL-2 or IL2CP In an alternative embodiment of the immunoconjugate according to the invention, the IL-2 polypeptide (optionally IL2CP) is covalently linked to the N-terminal amino acid residue of the IL2VL4 domain or the N-terminal amino acid residue of the IL2VH4 domain by a peptide linker. In a specific embodiment, the N-terminal peptide linker is 10 to 30, more particularly 21 or 22 amino acids in length. The cytokine can be linked to the antibody-binding domain when combined with a medium-length binding peptide, which allows for flexibility in the positioning of the cytokine and antibody-binding domain, although longer peptide linkers may allow for intermolecular "trans" interactions, which may lead to the formation of aggregates.

[0103] Receptor-biased L-2 signaling In certain embodiments of the immunoconjugate according to the invention, the immunoconjugate has a K D is in the range of 500nM to 0.1nM, 100nM to 0.1nM, and 10nM to 0.1nM, and especially K D The binding affinity according to this embodiment of the invention is characterized by a binding affinity in the range of 1 nM to 0.1 nM. Binding affinity according to this embodiment of the invention is determined using surface plasmon resonance. The binding of the Fab fragment to CD122:CD132 of a representative compound (QTY065) comprising an IL-2 anti-IL-2 binding domain fusion according to the invention is 0.322 nM as measured by SPR at 200 nM in the Examples. A further embodiment of receptor-biased binding is characterized as an immunoconjugate with no detectable binding to CD25 as measured by SPR at 200 nM.

[0104] Anti-IL-2 binding domain It is understood that the CDR designations provided in the embodiments of the anti-IL-2 binding domains provided herein are based on uninterrupted (contiguous) antibody sequences, which may be interrupted by IL2CP fused within the IL2-VH or IL2-VL, as defined herein. It is clear that alignment to the canonical CDR sequence would not be recognized otherwise. Thus, the term CDR according to the present invention does not simply encompass the canonical CDR amino acids, but may also encompass a longer recombinant polypeptide in which those residues aligned to the canonical CDR residues are interrupted by IL2CP, and optionally, a peptide linker connecting the antibody binding region and the IL-2 polypeptide domain of the immunoconjugate.

[0105] In certain embodiments of the immunoconjugates according to the invention, the IL2-VH is more than 95%, in particular more than 98% or 99% identical to SEQ ID NO: 043 (VH antibody A) and the IL2-VL is more than 95%, in particular more than 98% or 99% identical to SEQ ID NO: 042 (VL antibody A) and has a similar biological function to antibody A (provided that, for example, the IL-2 polypeptide according to claim 2 or a peptide linker optionally incorporated into one of the variable chains is excluded from the amino acid alignment / sequence identity). According to these embodiments, the affinity constant (K) of an antibody characterized by IL-2VH and IL2-VL for an IL-2 polypeptide (Proleukin) having SEQ ID NO: 006 is D ) is in the range of 500 nM to 2 nM, particularly 270 nM to 2 nM, more particularly about 2 nM. D ) refers to the interaction of the corresponding free antibody employed as an anti-IL-2 binding domain according to the present invention with free IL-2 (not the immunoconjugate in which these two entities are bound).

[0106] In certain embodiments of the immunoconjugates according to the invention, the anti-IL-2 antigen-binding domain comprises or consists of an IL2-VH having the sequence of SEQ ID NO: 043 (antibody A VH) associated with an IL2-VL selected from SEQ ID NOs: 025-031. Such embodiments include the LCDR1 IL2CP-incorporating antibody A constructs EAD409, XFO227, QTY065, FJC828, PG035, DRV470, and XUB802. In specific embodiments, the anti-IL-2 binding domain comprises or consists of SEQ ID NO: 043 and SEQ ID NO: 027.

[0107] In one embodiment of an immunoconjugate according to the invention, the anti-IL-2 antigen binding domain comprises or consists of an IL2-VL having the sequence of SEQ ID NO: 042 (Antibody A VL) associated with an IL2-VH selected from SEQ ID NO: 046 or 047. Such embodiments relate to constructs incorporating an Antibody A-based CDRH3 (VBE401) or CDRH2 (LI707) IL2CP, each of which contains a separate validated IL2CP sequence.

[0108] In a further embodiment of the immunoconjugate according to the invention, the IL2-VH is more than 95%, particularly more than 98% or more than 99% identical to SEQ ID NO: 044 (VH antibody B) and the IL2-VL is more than 95%, particularly more than 98% or more than 99% identical to SEQ ID NO: 050 (VL antibody B). In such an embodiment, the affinity constant (K D ) is in the range of 500 nM to 2 nM, particularly 270 nM to 2 nM.

[0109] In certain embodiments of the immunoconjugates according to the invention, the anti-IL-2 antigen-binding domain comprises or consists of an IL2-VH having the sequence of SEQ ID NO: 044 (antibody B VH) associated with an IL2-VL selected from SEQ ID NOs: 032-036. These embodiments relate to IL2CPs incorporated into the LCDR1 of antibody B, EPK959, GYG794, DRO069, or ECV200, and BFC885, which showed little binding to CD25 as measured by SPR (Table 5). These validated alternatives for IL2CP composition and conjugation incorporation provide biased signaling toward the dimeric IL-2 receptor according to the invention.

[0110] Fc part In certain embodiments of the immunoconjugates according to the invention, the immunoconjugate comprises an Fc portion, which increases its half-life in vivo. In a specific embodiment, the Ig Fc is the Fc portion of an IgG.

[0111] In certain embodiments of the immunoconjugate, the IgG Fc portion is characterized by the presence of one or more modifications in the constant region of the heavy chain to enhance correct heavy chain pairing. In certain embodiments, the modifications are selected from the following knob and hole paired mutations to enhance heavy chain pairing: - Knob: S354C, T366W & Hole: Y349C, T366S, L368A, Y407V; - Knob: T366Y, and hole Y407T; - Knob: Y349C T366W, and hole: S354C, T366S, L368A, Y407V; - Knob: T366W, and holes: Y407A, T366S, L368A.

[0112] In certain embodiments of the immunoconjugates of the invention, the IgG Fc portion is characterized by the presence of one or more modifications in the constant region of the heavy chain to reduce effector function of the Fc portion. In certain embodiments, the one or more modifications are selected from L234A, L235A (LALA), L234A, L235A, P329G (LALA-PG), L234A, L235A, P329A (LALA-PA), N297A, N297Q, N297G, and D265A, N297G (DANG). In certain embodiments, the immunoconjugate comprises P329A (LALA-PA).

[0113] Immunoconjugate Format In a specific embodiment of an immunoconjugate according to the invention, the immunoconjugate is a heterotetrameric IgG consisting of a first heterodimer comprising an anti-PD-1 binding domain and a second heterodimer comprising an anti-IL2 binding domain. This format is shown in Figure 4 and includes formats such as the heterotetrameric kappa / lambda IgG format and the Crossmab format (Figure 4C, WO2009 / 080253).

[0114] In certain embodiments of immunoconjugates according to the invention, the immunoconjugate is in a heterotetrameric kappa / lambda IgG format, with an anti-PD-1 binding domain characterized by a lambda light chain derived from, for example, the 21A08 antibody clone listed herein (Figure 4F).

[0115] In certain embodiments of an immunoconjugate according to the invention, the immunoconjugate comprises or consists of polypeptides having the sequences of SEQ ID NO: 011, SEQ ID NO: 095, SEQ ID NO: 112, and SEQ ID NO: 052 (NZA596).

[0116] In certain embodiments of an immunoconjugate according to the invention, the immunoconjugate comprises or consists of polypeptides having the sequences of SEQ ID NO:094, SEQ ID NO:095, SEQ ID NO:100, and SEQ ID NO:052 (XWY176).

[0117] In certain embodiments of an immunoconjugate according to the invention, the immunoconjugate comprises or consists of polypeptides having the sequences of SEQ ID NO: 097, SEQ ID NO: 098, SEQ ID NO: 100, and SEQ ID NO: 052 (GQM289).

[0118] In certain embodiments of an immunoconjugate according to the invention, the immunoconjugate comprises or consists of polypeptides having the sequences of SEQ ID NO:117, SEQ ID NO:104, SEQ ID NO:112, and SEQ ID NO:052 (BGY642).

[0119] In an alternative embodiment of an immunoconjugate according to the invention, the immunoconjugate is in an immunoglobulin ScFv format, as shown in Figures 4D and 4E. This format comprises an anti-PD1 antibody comprising a first and second anti-PD-1 binding domain. Both the first and second binding domains are characterized by an antibody heavy chain comprising a PD1-VH and an antibody light chain comprising a PD1-VL, as defined in the "Anti-PD1 Binding Domain" section. This format further comprises an anti-IL-2 ScFv comprising an anti-IL-2 binding domain, as defined in the "Anti-IL-2 Binding Domain" section. In the immunoglobulin ScFv format, the C-terminal residue of the anti-IL-2 ScFv IL2-VH or IL2-VL domain is linked to the N-terminal residue of the anti-PD1 antibody heavy or light chain via a peptide linker. In a specific embodiment, the peptide linker is 10 to 30 amino acids in length. In an even more specific embodiment, the peptide linker has the sequence (G4S)2 or SEQ ID NO: 024.

[0120] In certain embodiments of immunoconjugates according to the invention, the immunoconjugate comprises or consists of polypeptides having the sequences of SEQ ID NOs: 094, 095, and 096. In certain embodiments of immunoconjugates according to the invention, the immunoconjugate comprises or consists of polypeptides having the sequences of SEQ ID NOs: 097, 098, and 099. In certain embodiments of immunoconjugates according to the invention, the immunoconjugate comprises or consists of polypeptides having the sequences of SEQ ID NOs: 105, 106, and 107. In certain embodiments of immunoconjugates according to the invention, the immunoconjugate comprises or consists of polypeptides having the sequences of SEQ ID NOs: 108, 109, and 110. In certain embodiments of immunoconjugates according to the invention, the immunoconjugate comprises or consists of polypeptides having the sequences of SEQ ID NOs: 113, 106, and 114. In certain embodiments of immunoconjugates according to the invention, the immunoconjugate comprises or consists of polypeptides having the sequences of SEQ ID NOs: 115, 109, and 116. All of these can be represented in the format of Figure 4E.

[0121] Further aspects of the invention Another aspect of the invention relates to an isolated nucleic acid encoding an immunoconjugate according to any one of the immunoconjugate aspects of the invention. Another aspect of the invention relates to an expression vector comprising the isolated nucleic acid. The invention further encompasses a host cell comprising the nucleic acid or the expression vector.

[0122] Another aspect of the present invention is a method for producing a pharmaceutical composition comprising: i) an immunoconjugate as defined in any one of the aspects or embodiments of the invention herein, and ii) Anti-PD-1 antagonist antibody The present invention relates to a combination medicine comprising:

[0123] In certain embodiments, the anti-PD1 antagonist antibody is selected from the list consisting of nivolumab, pembrolizumab, dostallimab, sintilimab, tislelizumab, cemiplimab, cetrelimab, southernlimab, toripalimab, zetrelimab, or ezabenlimab. In more particular embodiments, the antibody is cemiplimab, dostallimab, zetrelimab, tislelizumab, ezabenlimab, toripalimab, cetrelimab, nivolumab, or pembrolizumab. In even more particular embodiments, the antibody is nivolumab or pembrolizumab.

[0124] Treatment and medical uses Also within the scope of the present invention is a method of treating cancer in a patient in need thereof, comprising administering to the patient an immunoconjugate according to the above-described aspects and embodiments.

[0125] In some embodiments, the immunoconjugate is a heterotetrameric immunoglobulin characterized by two heavy chains and two light chains, one pair of which forms an IL-2-binding ligand and is fused to an IL-2 polypeptide (as in Figure 4C or E). In some embodiments, the immunoconjugate consists of an anti-PD-1 antibody that is a heterodimer of an associated pair of heavy and light chains, linked by a peptide linker to an scFv comprising the heavy and light chain variable domains of an anti-IL-2 antibody, one of which anti-IL-2 domains is linked (optionally by a peptide linker) to an IL-2 polypeptide (as in Figure 4D or E).

[0126] Another aspect of the present invention is an immunoconjugate comprising a non-blocking anti-PD-1 binding domain according to the present invention for use in a patient receiving an anti-PD-1 antagonist antibody. In a specific embodiment, the anti-PD-1 antibody is selected from the list consisting of nivolumab, pembrolizumab, dostallimab, sintilimab, tislelizumab, cemiplimab, cetrelimab, southernlimab, toripalimab, zelvalimab, and ezabenlimab. In a more specific embodiment, the antibody is cemiplimab, dostallimab, zelvalimab, tislelizumab, ezabenlimab, toripalimab, cetrelimab, nivolumab, or pembrolizumab.

[0127] In some embodiments, an immunoconjugate according to the invention is administered to a patient who has received or will receive treatment with an anti-PD-1 antagonist antibody within 6 weeks, particularly within 3 weeks, of the immunoconjugate.

[0128] Another aspect of the present invention is an anti-PD1 antagonist antibody for use in a patient receiving an immunoconjugate according to the invention. In certain embodiments, the anti-PD-1 antibody is selected from the list consisting of nivolumab, pembrolizumab, dostallimab, sintilimab, tisulizumab, cemiplimab, cetrelimab, southernlimab, toripalimab, zelvalimab, and ezabenlimab. In certain embodiments, the antibody is used in a patient who has received an immunoconjugate according to the invention within the past month. In some embodiments, the patient has been treated with an immunoconjugate according to any one of the embodiments of the invention within the past six weeks or is scheduled to be treated with an immunoconjugate in the next six weeks.

[0129] The invention further includes nivolumab for use in a patient receiving an immunoconjugate according to the invention.The invention further includes pembrolizumab for use in a patient receiving an immunoconjugate according to the invention.

[0130] Similarly, the present invention encompasses a method for treating a patient diagnosed with cancer. The method involves administering to the patient an effective amount of an immunoconjugate as specified herein, or a pharmaceutically acceptable salt thereof as defined herein, optionally in combination with an anti-PD-1 antagonist antibody, as defined in detail herein. In certain embodiments, the immunoconjugate and the anti-PD-1 antagonist antibody are co-administered to the same subject within a medically relevant therapeutic window.

[0131] Pharmaceutical Compositions, Dosage Forms / Dosage Forms and Salts According to one aspect of the compounds according to the invention, the immunoconjugate according to the invention is provided as a pharmaceutical composition, pharmaceutical dosage form, or pharmaceutical dosage form, said pharmaceutical composition, pharmaceutical dosage form, or pharmaceutical dosage form comprising the immunoconjugate of the invention and at least one pharmaceutically acceptable carrier, diluent, or excipient.

[0132] In certain embodiments of the invention, the immunoconjugates of the invention are typically formulated into pharmaceutical formulations to provide easily controlled dosage of the drug and to provide the patient with a clear, easy-to-use product.

[0133] The present invention further encompasses pharmaceutical compositions comprising an immunoconjugate of the invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. In a further embodiment, the composition comprises at least two pharmaceutically acceptable carriers, such as those described herein.

[0134] Certain embodiments of the present invention relate to dosage forms for parenteral administration, such as subcutaneous, intravenous, intrahepatic or intramuscular injection forms. Optionally, pharmaceutically acceptable carriers and / or excipients may be present.

[0135] The pharmaceutical compositions of the present invention may be subjected to conventional pharmaceutical procedures such as sterilization, and / or may contain conventional inert diluents, lubricants, or buffers, as well as adjuvants such as preservatives, stabilizers, wetting agents, emulsifiers, and buffers. They may be manufactured by standard processes, such as conventional mixing, granulation, dissolution, or lyophilization processes. Many such procedures and methods for preparing pharmaceutical compositions are known in the art; see, for example, L. Lachman et al., The Theory and Practice of Industrial Pharmacy, 4th ed., 2013 (ISBN 8123922892).

[0136] Manufacturing and treatment methods according to the present invention The present invention further encompasses, as a further aspect, the use of an immunoconjugate as defined in detail above for use in a method for the manufacture of a medicament for the treatment or prevention of cancer.

[0137] Similarly, the present invention encompasses a method of treating a patient diagnosed with cancer, which method involves administering to the patient an effective amount of an immunoconjugate as specified herein, or a pharmaceutically acceptable salt thereof as defined herein, optionally in combination with an anti-PD-1 antagonist antibody.

[0138] For example, wherever alternatives of a single separable feature, such as an isotype protein or coding sequence, or cancer, are positioned herein as "embodiments," it will be understood that such alternatives may be freely combined to form individual embodiments of the invention disclosed herein. Thus, any of the alternative embodiments of an immunoconjugate may be combined with any of the alternative embodiments of an anti-PD-1 antagonist compound, and these combinations may be combined with any of the medical indications described herein.

[0139] The present invention further encompasses the following: A. Immunoconjugates comprising or consisting of polypeptides having the sequences of SEQ ID NO:111, SEQ ID NO:095, SEQ ID NO:112, and SEQ ID NO:052 (NZA596). B. Immunoconjugates comprising or consisting of polypeptides having the sequences of SEQ ID NO: 094, SEQ ID NO: 095, SEQ ID NO: 100, and SEQ ID NO: 052 (XWY176). C. Immunoconjugates comprising or consisting of polypeptides having the sequences of SEQ ID NO: 097, SEQ ID NO: 098, SEQ ID NO: 100, and SEQ ID NO: 052 (GQM289). D. Immunoconjugates comprising or consisting of polypeptides having the sequences of SEQ ID NO:117, SEQ ID NO:104, SEQ ID NO:112, and SEQ ID NO:052 (BGY642). E. An immunoconjugate comprising or consisting of a polypeptide having the sequence of SEQ ID NO:094, SEQ ID NO:095, and SEQ ID NO:096 (LTJ498). F. An immunoconjugate comprising or consisting of a polypeptide having the sequences of SEQ ID NO: 097, SEQ ID NO: 098, and SEQ ID NO: 099 (JLI141). G. An immunoconjugate comprising or consisting of polypeptides having the sequences of SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107 (TMU471). H. An immunoconjugate comprising or consisting of a polypeptide having the sequence of SEQ ID NO: 108, SEQ ID NO: 109, and SEQ ID NO: 110 (MDS446). I. Immunoconjugates comprising or consisting of polypeptides having the sequences of SEQ ID NO: 113, SEQ ID NO: 106, and SEQ ID NO: 114 (KTX917). J. Immunoconjugates comprising or consisting of polypeptides having the sequences of SEQ ID NO: 115, SEQ ID NO: 109, and SEQ ID NO: 116 (CIT348). K. An immunoconjugate comprising or consisting of a polypeptide having the sequences of SEQ ID NO: 103, SEQ ID NO: 104, and SEQ ID NO: 052 (QAB373). L. An immunoconjugate comprising or consisting of a polypeptide having the sequence of SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:090 and SEQ ID NO:052 (RIB426).

[0140] The following items are further encompassed by the present invention: Item 1. i. an anti-PD1 antigen-binding domain; - an antibody heavy chain variable domain polypeptide (PD1-VH), and - comprising an antibody light chain variable domain polypeptide (PD1-VL), the anti-PD1 antigen-binding domain; ii. an anti-IL-2 antigen binding domain; an antibody heavy chain variable domain polypeptide (IL2-VH) having, from the N' to C' terminus, the following domains: a first framework region (IL2VH1), a first complementarity determining region (IL2HCDR1), a second framework region (IL2VH2), a second complementarity determining region (IL2HCDR2), a third framework region (IL2VH3), a third complementarity determining region (IL2HCDR3), and a fourth framework region (IL2VH4); and - an antibody light chain variable domain polypeptide (IL2-VL) having from the N' to C' terminus the following domains: a first framework region (IL2VL1), a first complementarity determining region (IL2LCDR1), a second framework region (IL2VL2), a second complementarity determining region (IL2LCDR2), a third framework region (IL2VL3), a third complementarity determining region (IL2LCDR3), and a fourth framework region (IL2VL4). the anti-IL-2 antigen binding domain comprising: iii. IL-2 polypeptide 1. An immunoconjugate comprising: wherein a single contiguous recombinant polypeptide is provided, covalently linked to IL2-VL or IL2-VH, The immunoconjugate, wherein the binding of pembrolizumab or nivolumab to PD-1+ cells does not significantly inhibit the binding of the immunoconjugate to said cells.

[0141] Item 2. - PD1-VH comprises an HCDR1 having the sequence GFTFSINAMT (SEQ ID NO: 118), an HCDR2 having the sequence TISGSGFSTYYADSLKGR (SEQ ID NO: 119), and an HCDR3 having the sequence EVYGDY (SEQ ID NO: 120); and - PD1-VL is the sequence SGX 1 SSNIGSX 2 X 3 VF (SEQ ID NO: 121), an LCDR2 having the sequence SNNQRPS (SEQ ID NO: 122), and an LCDR3 having the sequence AAWDDSLSIWVF (SEQ ID NO: 123); where X 1 is N, S, Q or A, in particular X 1 is S, Q, or A; and X 2 X 3 is NS, QS, SS, or NA, and in particular, X 2 X 3 is QS, SS, or NA, Item 1. The immunoconjugate according to item 1.

[0142] Item 3. PD1-VL has the sequence SGASSNIGS QS 3. The immunoconjugate of item 1 or 2, comprising an LCDR1 having the sequence VF (underlined in bold) (SEQ ID NO: 124).

[0143] Item 4. The immunoconjugate of Item 3, wherein the PD1-VH comprises or consists of a polypeptide having the sequence of SEQ ID NO: 085, and the PD1-VL comprises or consists of a polypeptide having the sequence of SEQ ID NO: 086.

[0144] Item 5. PD1-VL has the sequence SGASSNIGS SS 3. The immunoconjugate of item 1 or 2, comprising LCDR1 having VF (underlined in bold) (SEQ ID NO: 125).

[0145] Item 6. The immunoconjugate of Item 5, wherein the PD1-VH comprises or consists of a polypeptide having the sequence of SEQ ID NO: 085, and the PD1-VL comprises or consists of a polypeptide having the sequence of SEQ ID NO: 087.

[0146] Item 7. PD1-VL is a gene encoding the sequence SGASSNIGS NA 3. The immunoconjugate of item 1 or 2, comprising LCDR1 having VF (underlined in bold) (SEQ ID NO: 126).

[0147] Item 8. The immunoconjugate of Item 7, wherein the PD1-VH comprises or consists of a polypeptide having VH 21A08 of SEQ ID NO: 085, and the PD1-VL comprises or consists of a polypeptide having SEQ ID NO: 91.

[0148] Item 9. - PD1-VH comprises or consists of a polypeptide at least (≧) 95% or more, 98% or more, 99% or more similar to SEQ ID NO: 085, and PD1-VL comprises or consists of a polypeptide at least (≧) 95% or more, 98% or more, 99% or more similar to SEQ ID NO: 086; - PD1-VH comprises or consists of a polypeptide that is ≥ 95%, ≥ 98%, ≥ 99% similar to SEQ ID NO: 085, and PD1-VL comprises or consists of a polypeptide that is ≥ 95%, ≥ 98%, ≥ 99% similar to SEQ ID NO: 087; - PD1-VH comprises or consists of a polypeptide that is ≥ 95%, ≥ 98%, ≥ 99% similar to SEQ ID NO: 085, and PD1-VL comprises or consists of a polypeptide that is ≥ 95%, ≥ 98%, ≥ 99% similar to SEQ ID NO: 091; where the affinity constant of the immune complex for PD-1 (K D ) is 1.0 x 10, as measured according to the protocol provided in Example 2. -9 ~1.5×10 -11 in the mol / L range, 9. The immunoconjugate according to any one of items 1 to 8.

[0149] Item 10. - PD1-VH comprises an HCDR1 having the sequence NFYIH (SEQ ID NO: 127), an HCDR2 having the sequence RIYPNYGITAYNQKFKD (SEQ ID NO: 128), and an HCDR3 having the sequence GYSYAMDY (SEQ ID NO: 129); and - PD1-VL comprises an LCDR1 having the sequence SASQGISGDLN (SEQ ID NO: 130), an LCDR2 having the sequence HTSQRHS (SEQ ID NO: 131), and an LCDR3 having the sequence QGYSKDLLT (SEQ ID NO: 132); Item 1. The immunoconjugate according to item 1.

[0150] Item 11. The immunoconjugate of Item 10, wherein the PD1-VH comprises or consists of a polypeptide having SEQ ID NO: 061, and the PD1-VL comprises or consists of a polypeptide having SEQ ID NO: 062.

[0151] Item 12. - PD1-VH has HCDR1 with the sequence NFYIH (SEQ ID NO: 127), I comprising an HCDR2 having the sequence TAYNQKFKD (underlined in bold) (SEQ ID NO: 133), and an HCDR3 having the sequence GYSYAMDY (SEQ ID NO: 129); and - PD1-VL has LCDR1 with the sequence SASQGISGDLN (SEQ ID NO: 130), the sequence HTS Q LCDR2 with LHS (underlined in bold) (SEQ ID NO: 134), and sequence Q G containing an LCDR3 with YSKDLLT (underlined in bold) (SEQ ID NO: 132); Item 1. The immunoconjugate according to item 1.

[0152] Item 13. The immunoconjugate of Item 12, wherein the PD1-VH comprises or consists of a polypeptide having SEQ ID NO: 067, and the PD1-VL comprises or consists of a polypeptide having SEQ ID NO: 068.

[0153] Item 14. - PD1-VH comprises or consists of a polypeptide that is >= 95%, >= 98%, >= 99% similar to SEQ ID NO: 061, and PD1-VL comprises or consists of a polypeptide that is >= 95%, >= 98%, >= 99% similar to SEQ ID NO: 062; or - PD1-VH comprises or consists of a polypeptide that is >= 95%, >= 98%, >= 99% similar to SEQ ID NO: 067, and PD1-VL comprises or consists of a polypeptide that is >= 95%, >= 98%, >= 99% similar to SEQ ID NO: 068; and The affinity constant (Kd) of the immunoconjugate for PD-1 was 2×10, as measured by the protocol provided in Example 2. -9 ~2×10 -11 in the mol / L range, The immunoconjugate according to any one of items 1 and 10 to 13.

[0154] Item 15. The IL-2 polypeptide is a circularly permuted IL-2 (IL2CP) polypeptide; Compared to a human IL-2 polypeptide or a variant thereof, the IL2CP: - the N-terminal amino acid of the human IL-2 polypeptide or variant thereof is linked to the C-terminal amino acid of the human IL-2 polypeptide or variant thereof; and - the IL-2 polypeptide or variant thereof is opened at a position that does not disrupt the alpha helix structure to provide an IL2CP N-terminus and an IL2CP C-terminus; and - the IL2CP N-terminus and / or the IL2CP C-terminus are covalently linked to an IL2-VL or IL2-VH, 15. The immunoconjugate according to any one of items 1 to 14.

[0155] Item 16. The immunoconjugate of Item 15, wherein an N-terminal amino acid selected from A21, P22, T23, S24, S25, or S26 of a human IL-2 polypeptide or a variant thereof is linked to a C-terminal amino acid of a human IL-2 polypeptide or a variant thereof selected from T150, L152, and T153.

[0156] Item 17. The immunoconjugate according to Item 16, wherein the N-terminal amino acid is P22 and the C-terminal amino acid is T153.

[0157] Item 18. The immunoconjugate according to any one of Items 15 to 17, wherein both the IL2CP N-terminus and the IL2CP C-terminus are located within V89 to D104, particularly within S95 to N97, more particularly within K96 and N97.

[0158] Item 19. The immunoconjugate according to any one of items 15 to 17, wherein both the IL2CP N-terminus and the IL2CP C-terminus are located within G47 to E72, particularly within L56 to position E72, more particularly within K63 to F64.

[0159] Item 20. The immunoconjugate according to any one of Items 15 to 17, wherein both the IL2CP N-terminus and the IL2CP C-terminus are located within G118 to I134, particularly within S119 to A132.

[0160] Item 21. Both the IL2CP N'-terminus and the IL2CP C'-terminus are optionally linked by one or two peptide linkers to IL2HCDR1, IL2HCDR2, IL2VH 3、 21. The immunoconjugate according to any one of items 15 to 20, wherein the immunoconjugate is covalently linked to an amino acid residue within a domain selected from the list consisting of IL2HCDR3, IL2LCDR1, IL2LCDR2, IL2VL3 or IL2LCDR3.

[0161] Item 22. IL2CP is more than (>)98% similar to SEQ ID NO:010 or SEQ ID NO:011; and 21. The immunoconjugate according to any one of items 15 to 20, wherein the IL2CP N'-terminus and the IL2CP C'-terminus are covalently linked, optionally by one or two peptide linkers, to amino acid residues within a domain selected from the list consisting of IL2LCDR1, IL2LCDR2, IL2HCDR1, or IL2HCDR3, in particular IL2LCDR1 or IL2HCDR3.

[0162] Item 23. IL2CP is more than (>)98% similar to SEQ ID NO:012; and 21. The immunoconjugate according to any one of items 15 to 20, wherein the IL2CP N'-terminus and the IL2CP C'-terminus are covalently linked, optionally by one or two peptide linkers, to amino acid residues within a domain selected from the list consisting of IL2LCDR1, IL2LCDR3, IL2HCDR1, IL2HCDR2, IL2VH3, IL2VL4, in particular IL2HCDR2.

[0163] Item 24. IL2CP is more than (>)98% similar to SEQ ID NO:013; and 21. The immunoconjugate according to any one of items 15 to 20, wherein the IL2CP N'-terminus and the IL2CP C'-terminus are covalently linked, optionally by one or two peptide linkers, to amino acid residues within a domain selected from the list consisting of IL2HCDR2, or IL2VH3.

[0164] Item 25. 0, 1, 2, 3, or 4, particularly 2, consecutive amino acids in a domain selected from the list consisting of IL2HCDR1, IL2HCDR2, IL2VH3, IL2HCDR3, IL2LCDR1, IL2LCDR2, IL2VL3, or IL2LCDR3 are: - a first peptide linker, - IL2CP, - a second peptide linker; and is replaced by a polypeptide consisting of wherein both the first and second peptide linkers are 1 to 20 amino acids in length, and particularly, both the first and second peptide linkers are between 2 and 7 amino acids in length. 25. The immunoconjugate according to any one of items 15 to 24.

[0165] Item 26. The immunoconjugate according to any one of Items 1 to 14, wherein the IL-2 polypeptide is covalently linked to the N-terminal amino acid residue of the IL2VL4 domain or the N'-terminal amino acid residue of the IL2VH4 domain via a peptide linker, and particularly the peptide linker is 10 to 30, more particularly 21 or 22, amino acids in length.

[0166] Item 27. Immunoconjugates - Affinity constant (K) of the immunoconjugate for the dimeric IL-2 receptor D ) is in the range of 500 nM to 0.1 nM, particularly 100 nM to 0.1 nM, more particularly 10 nM to 0.1 nM, and even more particularly K D is in the range of 1 nM to 0.1 nM; and - has no detectable binding to CD25 27. The immunoconjugate according to any one of items 1 to 26, characterized in that

[0167] Item 28. IL2-VH is more than (>) 95%, particularly more than 98% or more than 99% identical to SEQ ID NO: 043, and IL2-VL is more than (>) 95%, particularly more than 98% or more than 99% identical to SEQ ID NO: 042; and K of an antibody characterized by IL-2VH and IL2-VL against an IL-2 polypeptide having SEQ ID NO: 006 D is in the range of 500 nM to 2 nM, particularly 270 nM to 2 nM, more particularly about 2 nM; 28. The immunoconjugate according to any one of items 1 to 27.

[0168] Item 29. The immunoconjugate according to any one of Items 1 to 27, wherein the anti-IL-2 antigen-binding domain comprises or consists of an IL2-VH having the sequence of SEQ ID NO: 043 associated with an IL2-VL selected from SEQ ID NOs: 025 to 031.

[0169] Item 30. The immunoconjugate of any one of Items 1 to 27, wherein the anti-IL-2 antigen-binding domain comprises or consists of an IL2-VL having the sequence of SEQ ID NO: 042 associated with an IL2-VH selected from SEQ ID NO: 046 or 047.

[0170] Item 31. IL2-VH is more than 95%, particularly more than 98% or more than 99% identical to SEQ ID NO: 044, and IL2-VL is more than 95%, particularly more than 98% or more than 99% identical to SEQ ID NO: 050; and K of an antibody characterized by IL-2VH and IL2-VL against an IL-2 polypeptide having SEQ ID NO: 006 D is in the range of 500nM to 2nM, particularly 270nM to 2nM; 28. The immunoconjugate according to any one of items 1 to 27.

[0171] Item 32. The immunoconjugate of any one of Items 1 to 27, wherein the anti-IL-2 antigen-binding domain comprises or consists of an IL2-VH having the sequence of SEQ ID NO: 044 associated with an IL2-VL selected from SEQ ID NOs: 032 to 036.

[0172] Item 33. The immunoconjugate according to any one of items 1 to 32, wherein the immunoconjugate comprises an Fc portion, particularly an IgG Fc portion.

[0173] Item 34. The IgG Fc portion is characterized by the presence of one or more modifications to the constant region of the heavy chain to enhance correct heavy chain pairing, in particular: - Knob: S354C, T366W & Hole: Y349C, T366S, L368A, Y407V; - Knob: T366Y, and hole Y407T; - Knob: Y349C, T366W, and holes S354C, T366S, L368A, Y407V; or - Knob: T366W, and holes: Y407A, T366S, L368A a set of knob and hole modifications selected from 34. The immunoconjugate according to item 33.

[0174] Item 35. The IgG Fc portion is characterized by the presence of one or more modifications to the constant region of the heavy chain to reduce the effector function of the Fc portion; In particular, characterized by modifications to the constant region of the heavy chain selected from L234A, L235A (LALA), L234A, L235A, P329G (LALA-PG), L234A, L235A, P329A (LALA-PA), N297A, N297Q, N297G and D265A, N297G (DANG), more particularly P329A (LALA-PA), 35. The immunoconjugate according to any one of items 33 or 34.

[0175] Item 36. Immunoconjugates are - a first heterodimer comprising an anti-PD-1 binding domain as defined in any one of paragraphs 2 to 14; and - a second heterodimer comprising an anti-IL2 binding domain, as defined in any one of items 15 to 32. 36. The immunoconjugate according to any one of items 1 to 35, which is a heterotetrameric IgG consisting of:

[0176] Item 37. The immunoconjugate according to Item 36, wherein the heterotetrameric IgG format is a heterotetrameric kappa / lambda IgG format.

[0177] Item 38. The immunoconjugate according to Item 36, wherein the heterotetrameric IgG format is a crossmab format.

[0178] Item 39. The immunoconjugate of Item 36 or 37, wherein the heterotetrameric IgG format is a heterotetrameric kappa / lambda IgG format and the anti-PD-1 binding domain is characterized by a lambda light chain.

[0179] Item 40. The immunoconjugate according to any one of items 1 to 39, wherein the immunoconjugate comprises or consists of a polypeptide having the sequence of SEQ ID NO: 111, SEQ ID NO: 095, SEQ ID NO: 112, and SEQ ID NO: 052 (NZA596).

[0180] Item 41. The immunoconjugate according to any one of items 1 to 39, wherein the immunoconjugate comprises or consists of a polypeptide having the sequence of SEQ ID NO: 117, SEQ ID NO: 104, SEQ ID NO: 112, and SEQ ID NO: 091 (BGY642).

[0181] Item 42. The immunoconjugate according to any one of items 1 to 39, wherein the immunoconjugate comprises or consists of a polypeptide having the sequence of SEQ ID NOs: 113, 106, and 114 (KTX917).

[0182] Item 43. The immunoconjugate according to any one of items 1 to 39, wherein the immunoconjugate comprises or consists of a polypeptide having the sequence of SEQ ID NOs: 115, 109, and 116 (CIT348).

[0183] Item 44. The immunoconjugate according to any one of items 1 to 39, wherein the immunoconjugate comprises or consists of a polypeptide having the sequence of SEQ ID NO: 094, SEQ ID NO: 095, SEQ ID NO: 100, and SEQ ID NO: 052 (XWY176).

[0184] Item 45. The immunoconjugate according to any one of items 1 to 39, wherein the immunoconjugate comprises or consists of a polypeptide having the sequence of SEQ ID NO: 097, SEQ ID NO: 098, SEQ ID NO: 100, and SEQ ID NO: 052 (GQM289).

[0185] Item 46. Immunoconjugates are: - an anti-PD1 antibody comprising a first and a second anti-PD-1 binding domain, wherein both the first and second binding domains are characterized by an antibody light chain comprising a PD1-VH and a PD1-VL as defined in any one of items 2 to 14; and - an anti-IL-2 scFv comprising an anti-IL-2 binding domain as defined in any one of items 15 to 21; an immunoglobulin scFv format comprising wherein the C-terminal residue of the anti-IL-2 scFv IL2-VH or IL2-VL domain is linked to the N-terminal residue of the heavy chain or light chain of the anti-PD1 antibody via a peptide linker, particularly the peptide linker is 10 to 30 amino acids in length, and even more particularly the peptide linker is (G4S )2 or the immunoconjugate according to any one of items 1 to 35, having the sequence of SEQ ID NO: 024.

[0186] Item 47. The immunoconjugate according to Item 46, wherein the immunoconjugate comprises or consists of polypeptides having the sequences of SEQ ID NOs: 094, 095, and 096.

[0187] Item 48. The immunoconjugate according to Item 46, wherein the immunoconjugate comprises or consists of polypeptides having the sequences of SEQ ID NOs: 097, 098, and 099.

[0188] Item 49. The immunoconjugate according to Item 46, wherein the immunoconjugate comprises or consists of polypeptides having the sequences of SEQ ID NOs: 105, 106, and 107.

[0189] Item 50. The immunoconjugate according to Item 46, wherein the immunoconjugate comprises or consists of polypeptides having the sequences of SEQ ID NOs: 108, 109, and 110.

[0190] Item 51. The immunoconjugate according to Item 46, wherein the immunoconjugate comprises or consists of polypeptides having the sequences of SEQ ID NOs: 113, 106, and 114.

[0191] Item 52. The immunoconjugate according to Item 46, wherein the immunoconjugate comprises or consists of polypeptides having the sequences of SEQ ID NOs: 115, 109, and 116.

[0192] Item 53. An isolated nucleic acid encoding the immunoconjugate of any one of Items 1 to 52.

[0193] Item 54. An expression vector comprising the isolated nucleic acid of Item 53.

[0194] Item 55. A host cell comprising the nucleic acid according to Item 53 or the expression vector according to Item 54.

[0195] Item 56. An immunoconjugate as defined in any one of items 1 to 52, and anti-PD-1 antagonist antibodies, in particular anti-PD-1 antagonist antibodies selected from the list consisting of nivolumab, pembrolizumab, dostallimab, sintilimab, tislelizumab, cemiplimab, cetrelimab, southernlimab, in particular nivolumab or pembrolizumab, A combination medicine comprising:

[0196] Item 57. A pharmaceutical composition comprising the immunoconjugate according to any one of Items 1 to 52, the isolated nucleic acid according to Item 53, the expression vector according to Item 54, the host cell according to Item 55, and a pharmaceutically acceptable excipient.

[0197] Item 58. The immunoconjugate according to any one of Items 1 to 52, the isolated nucleic acid according to Item 53, the expression vector according to Item 54, the host cell according to Item 55, or the combination drug according to Item 56, or the pharmaceutical composition according to Item 57, for use as a pharmaceutical.

[0198] Item 59. A drug selected from the immunoconjugate according to any one of Items 1 to 52, the isolated nucleic acid according to Item 53, the expression vector according to Item 54, the host cell according to Item 55, the combined pharmaceutical composition according to Item 56, or the pharmaceutical composition according to Item 57, for use in treating cancer.

[0199] Item 60. i) selecting a suitable patient; and ii) administering a therapeutically effective amount of the immunoconjugate according to any one of Items 1 to 52, the isolated nucleic acid according to Item 53, the expression vector according to Item 54, the host cell according to Item 55, or the combination drug according to Item 56, or the pharmaceutical composition according to Item 57. A method of treatment comprising:

[0200] Item 61. The agent for use according to Item 59, wherein the agent is administered within 6 weeks before or after treatment with an anti-PD-1 antagonist antibody.

[0201] Item 62. The anti-PD1 antagonist antibody is selected from the list consisting of nivolumab, pembrolizumab, dostallimab, sintilimab, tislelizumab, cemiplimab, southernlimab, zelvalimab, ezabenlimab, toripalimab, or cetrelimab; 62. The drug for use according to item 61, wherein the anti-PD1 antagonist antibody is selected from the list consisting of nivolumab, pembrolizumab, dostallimab, tislelizumab, cemiplimab, zelvalimab, ezabenlimab, toripalimab, or cetrelimab.

[0202] Item 63. An anti-PD-1 antagonist antibody for use in the treatment of cancer, wherein the anti-PD-1 antagonist antibody is administered within 6 weeks before or after treatment with the agent defined in Item 59.

[0203] Item 64. The anti-PD-1 antagonist antibody is selected from the list consisting of nivolumab, pembrolizumab, dostallimab, sintilimab, tislelizumab, cemiplimab, southernlimab, zelvalimab, ezabenlimab, toripalimab, or cetrelimab; 64. The anti-PD-1 antagonist antibody for use according to item 63, wherein the anti-PD-1 antagonist antibody is selected from the list consisting of nivolumab, pembrolizumab, dostallimab, tislelizumab, cemiplimab, zelvalimab, ezabenlimab, toripalimab, or cetrelimab.

[0204] The present invention is further explained by the following examples and figures, from which further embodiments and advantages can be derived, which are intended to illustrate the invention without limiting its scope. [Brief explanation of the drawings]

[0205] [Figure 1-1] FIG. 1 shows the labeling of antibody residues in a recognized format using the standard numbering and Kabat numbering systems. [Figure 1-2] FIG. 1 shows the labeling of antibody residues in a recognized format using the standard numbering and Kabat numbering systems. [Figure 2] Figure 2 shows how the IL2-CP format can be altered to suit incorporation into various variable domain sites: (Left) Diagram of IL-2 in the correct orientation for binding to antibody A or B; (Center) Diagram of LCDR1 with CDAB IL-2CP incorporated into antibody A, B, or C; (Right) HCDR3 with BCDA IL-2CP incorporated. [Figure 3] SPR sensorgrams (response units, RU per time (sec)) of PD-1 premixed with antibody or buffer performed on immobilized PD-L1 as analyte. [Figure 4] Figure 4 shows testing of major formats with various PD-1 binding moiety configurations, including A. dual-scFv fusion, B. Fab-dual-scFv, C. heterotetrameric IgG (IgG CrossMab), D-E. bivalent PD-1 IgG antibody with two binding sites of anti-hIL-2 ScFv, and F. heterotetrameric IgG (kappa / gamma). [Figure 5] Figure 5 shows cell proliferation analyzed by flow cytometry, detecting Ki67+ cells in CD8 T cells, NK cells, and Treg cells. 0.2 mg / kg of the bispecific compound was administered intravenously to wild-type (WT) C57BL / 6 mice. Blood was collected before injection and on days 3 and 6 after compound administration. [Figure 6-1]A) The top shows a representative image of the NZA596 compound digested to obtain two Fab fragments and the Fc domain. The bottom shows the deconvoluted MS profile of the detected Fab fragment, showing a single species with the expected molecular weight. [Figure 6-2] B) The top shows a representative image of the BGY642 compound digested to obtain two Fab fragments and the Fc domain. The bottom shows the deconvoluted MS profile of the detected Fab fragment, showing a single species with the expected molecular weight. [Figure 7] Top) Representative images of cis-signaling on unblocked CTV+ cells and blocked CSFE+ cells. Bottom) Percentage of pSTAT5+ cells plotted against NZA596 compound concentration in nM. CFSE+ cells were pre-incubated with 21A08Ap1 antibody, pembrolizumab, or nivolumab (n=3). [Figure 8] Figure 8 shows the number of unique TCR rearrangements in TILs from mice treated intravenously with vehicle, NZA594, or QTY065 at 0.2 mg / kg on days 0 and 3. Genomic DNA was extracted from tumors of mice sacrificed on study day 5 (n=8). Statistical analysis was performed by unpaired Wilcox test. [Example]

[0206] Example 1: Design and production of anti-IL-2 antibody IL-2 cytokine fusion protein Antibodies that bind to IL-2 were derived, isolated, and structurally analyzed using methods well known to those skilled in the art. Antibody A (HC SEQ ID NO: 001, LC SEQ ID NO: 002) is a high-affinity anti-IL-2 antibody, and antibody B (HC SEQ ID NO: 003, LC SEQ ID NO: 004) is a low-affinity anti-IL-2 antibody. Antibody C has no IL-2 affinity and is included as a control. Genes encoding VL-CL (light chain) and VH-CH1-CH2-CH3 (heavy chain) were cloned into separate plasmids in the mammalian expression vector pcDNA3.4. Antibodies were produced using transient gene expression in Expi293 cells (Gibco, A14527) according to the standard protocol provided by the vendor. Plasmid DNA (HC / LC ratio 1:2 w / w) was transfected using ExpiFectamine™ 293 reagent. Cells were maintained at 37°C, 8% CO2, and on an orbital shaker (150 rpm) for 6 days. Antibodies were purified to homogeneity from the supernatant by Protein A Chromatography (MabSelect™ SuRe™, GE17-5438-01). Protein quality control was performed by SDS-PAGE (NuPAGE™ 4-12% Bis-Tris Protein Gels, ThermoFisher) and analytical size exclusion chromatography (SEC) (GE lifesciences, Superdex 200 increase 10 / 300). Yields and purity are reported in Table 1. All antibodies were obtained as pure protein products and eluted as a single peak in SEC-HPLC analysis.

[0207] Binding to IL-2 was first tested by ELISA using serial dilutions of the antibody on coated IL-2. Recombinant human IL-2 was coated onto Maxisorp ELISA plates (Invitrogen, 44-2404-21) at 5 μg / ml overnight at 4°C. After 2 hours of blocking, antibodies A, B, and C were incubated in serial dilutions starting at 10 μg / ml in assay buffer. Antibodies were detected with anti-human IgG-peroxidase (Sigma, A0170). After addition of chemiluminescent substrate and blocking, absorbance was read (A on a Spectramax iD3 plate reader).450 ~A 570 ). EC 50 Values ​​were obtained by plotting absorbance versus log(concentration) and performing sigmoidal 4-PL fitting using GraphPad Prism (Table 1). Only antibody A showed binding to IL-2 by ELISA, which could be detected on the Fc portion with an anti-human IgG antibody conjugated with HRP. The binding affinities of antibodies A, B, and C of the present invention to recombinant human IL-2 (rhIL-2) were then measured using the more sensitive biolayer interferometry (BLI) method. The antibodies were immobilized on an amine-reactive (second-generation) sensor (ForteBio, 18-5092). The association (600 s) and dissociation (900 s) of a dilution series of recombinant IL-2 (Acro Biosystems, IL2-H4113) were measured with the antibody-coated biosensor on the Octet-System (Octet RED, ForteBio). D Values ​​were obtained by fitting the kinetic data using ForteBio data analysis software (8.2). This assay confirmed that antibody A binds to rhIL-2 with high affinity. Binding of antibody B could not be measured by ELISA, but a weak affinity constant was measured by BLI. Antibody C showed no binding to rhIL-2 up to concentrations above 1000 nM (Table 1).

[0208] Table 1. Production yield by transient expression, purity and binding affinity to recombinant hIL-2 as measured by ELISA or BLI. [Table 1]

[0209] Fusion proteins comprising IL-2 polypeptides or IL-2 muteins containing amino acid substitutions with favorable pharmacokinetic properties (Table 2) have previously been demonstrated to provide IL-2 signaling when the cytokine is conjugated to an antibody chain by C-terminal attachment to an antibody variable chain domain (see, e.g., WO2018184964A1; Deak LC. et al., 2022, Nature 610:161; Gutbrodt KL. 2013 Sci. Trans. Med. 5:201; Gillies SD. (1992) PNAS 89(4):1428) or by N-terminal attachment to an anti-IL-2 antibody (see, e.g., WO2017122130A1).

[0210] Table 2. IL-2 Polypeptides [Table 2]

[0211] We analyzed the structure of IL-2 in complex with the antibody NARA1 (RCSB Protein Structure Data Bank 5LQB), from which humanized antibodies A and B were derived, and evaluated alternative formats for guiding the incorporation or fusion of IL-2 to maintain the cytokine's orientation relative to the antibody and similar receptor signaling qualities (Arena-Ramires et al., 2016 Sci. Trans. Med. 8:367). Based on the crystal structure of the NARA1 / IL-2 complex, we identified potential incorporation sites by examining the linker chain between different alpha-helical domains (here designated as AD) and their proximity to CDRs or framework regions on the antibody's VH or VL. We designed various combinations of circularly permuted IL-2 (IL-2CP) to preserve the cytokine's tertiary structure and orientation relative to its natural binding to the antibody when fused to the indicated heavy or light chain variable regions (Table 3, Figure 2). To maintain the same orientation on the antibody, IL-2 must be permuted (rearranged) differently depending on the junction site. To create a circular permutation in the IL-2 helical domain CDAB format, the crystal structure suggests that the region V89-D104 (see the Proleukin sequence) between the B and C helices should be opened to maintain the essential tertiary structure. To create a circular permutation in the IL-2 helical domain BCDA format, the crystal structure suggests that the region G47-E72 between the A and B helices could be opened. To create a circular permutation in the IL-2 helical domain DABC format, the crystal structure suggests that the region G118-I134 between the C and D helices could be targeted.

[0212] Table 3. Circularly permuted IL-2 polypeptides and optimal binding sites [Table 3]

[0213] Representative IL-2 fusion proteins were then designed according to several embodiments of the invention comprising antibody A, B, or C linked to IL-2 directly or via linkers of various lengths as shown in Table 4.

[0214] The LCDR1 of IL-2 antibody A, residues Y27d-D30 (or Y31-D34 structural numbering) and the region connecting IL-2 helix B and helix C between residues S95 and N97, were identified as promising regions for further design. The LCDR1 of each antibody A was opened between Y27d and D30 according to the Kabat definition, providing a new C-terminal residue for LCDR1 Y27d and a new N-terminal residue for LCDR1 D30. LCDR1 residue Y27d was linked to the N-terminus of IL-2CP, opened between K96 and N97 (SEQ ID NO: 10). The C-terminus of IL-2CP was linked to LCDR1 residue 30 according to the Kabat definition, and residues Q28 and G29 of the antibody LCDR1 were replaced by circulating IL-2CP (SEQ ID NO: 10), either directly or via peptide linkers of various lengths. This incorporation was repeated for the equivalent amino acid residues with the LCDR1 of the lower affinity IL-2 antibody B and antibody C, which has no affinity for IL-2 (Table 4).

[0215] Additional constructs were designed to enable the insertion of alternative IL-2CPs in the HDCR3 or HCDR2 regions. VBE401 was developed with IL-2 of SEQ ID NO: 011 opened between N97 and K96, and the cytokine inserted (N97 was removed, leaving F96 as the new N-terminus). SEQ ID NO: 011 was inserted between residues E98 and G99 in the HCDR3 of antibody A, following and preceding the linker GGG. Additionally, insertion of IL-2CP into HDRC2 was tested. A circularly permuted IL-2 was created by opening the sequence between K63 and F64, fusing the original N- and C-termini to provide SEQ ID NO: 012. SEQ ID NO: 012 was inserted between residues G53 and S54 in the HDCR2 of antibody A, following and preceding the linker GGG, as described above (LIZ707).

[0216] For each construct (Table 4), genes encoding VL-(IL-2)-CL, VL-CL, VH-CH1-CH2-CH3, and VH-(IL-2)-CH1-CH2-CH3 were cloned into separate plasmids in the mammalian expression vector pcDNA3.4. Antibody-IL-2 fusion proteins were produced using transient gene expression in Expi293 cells as described above for antibodies A, B, and C. All constructs were highly pure (>95%) and eluted as a single peak in SEC-HPLC analysis (Table 5).

[0217] Table 4. Antibody-IL-2 fusion proteins with an IL-2CP fusion site on each CDR of Antibody A, B, or C. The IL-2CP sequence is incorporated into the variable domain of the heavy or light chain of the antibody as indicated. All heavy chain constant regions were SEQ ID NO:048 and all light chain constant regions were SEQ ID NO:049. [Table 4]

[0218] Interaction of immunoconjugates with the CD132-CD122 heterodimeric receptor. The anti-IL-2 antibody clone 5344, which binds to the CD122 binding site on IL-2, was used to determine the correct folding of the IL-2 portion of the fusion protein. 50 The values ​​are reported in Table 6. The IL-2 portion of all constructs bound well to an anti-IL-2 antibody (clone 5344) in ELISA and could be detected on the Fc portion with an anti-human IgG antibody conjugated to HRP (Table 5).

[0219] The binding of one representative anti-IL-2 antibody / IL-2 fusion (QTY065) to the CD122 / CD132 complex (His-tagged, Acro Biosystem, Cat. # ILG-H5283) was further evaluated by SPR (BIAcore 3000, v4.1.2; GE Healthcare) and compared with Proleukin. CD122 / CD132 was captured on an NTA sensor chip (GE Healthcare). Proleukin or QTY065 Fab was used as the analyte. The calculated affinity constant of QTY065 Fab was 0.32 nM, comparable to the 0.42 nM obtained with Proleukin. The intermediate-affinity binding to the IL-2R complex (CD122 / CD132) remained unchanged for the IL-2CP antibody fusion protein.

[0220] Table 5. Purity of final protein by SEC-HPLC, functionality assessed by ELISA, and CD25 binding by SPR. [Table 5]

[0221] Biological effects of IL-2 tested by HEK Blue IL-2 reporter assay The functionality of the IL-2 fusion proteins was assessed using HEK-Blue™ IL-2 reporter cells, which express the trimeric IL-2 receptor (CD25 / CD122 / CD132) and were engineered to secrete secreted embryonic alkaline phosphatase activity (SEAP) upon IL-2-induced STAT5 activation. HEK-Blue™ IL-2 reporter cells (Invivogen) were incubated in growth medium with a dilution series of antibody-IL-2 fusion proteins (0.56 nM to 1:3 dilutions) for 20 hours at 37°C in 5% CO2. HEK-Blue™ IL-2 cell supernatants were diluted 1:10 in QUANTI-Blue™ solution and incubated for 3 hours at 37°C for colorimetric quantification of SEAP. Absorbance was read at 620 nm and plotted against protein concentration. E 50Values ​​were interpolated using GraphPad Prism. EC determined by colorimetric quantification of SEAP for each concentration of IL-2 antibody fusion protein. 50 The values ​​showed that the antibody-IL-2 fusion proteins induced STAT5 signaling with comparable potency on HEK Blue IL-2R reporter cells (Table 6).

[0222] Table 6. EC of immunoconjugate HEK Blue IL-2 reporter assay 50 value [Table 6]

[0223] Binding affinity to CD25 measured by surface plasmon resonance (SPR) analysis To provide optimally biased signaling to CD8 T cells, CD25 binding of the IL-2 fusion protein is preferably minimal. To understand whether the CD25-binding site on the antibody-IL-2 fusion protein is accessible, binding to CD25 was assessed by surface plasmon resonance (SPR) analysis (BIAcore 3000, GE Healthcare, 33-1140587-3682). His-tagged recombinant CD25 was captured via TrisNTA-biotin on an SA chip, and kinetic titrations of the IL-2 fusion protein of the present invention were performed at concentrations up to 500 nM. The IL-2 fusion protein against Antibody A showed no binding to CD25 at concentrations below 500 nM. Antibody A binds IL-2 with high affinity at the CD25-binding site, thereby blocking IL-2 binding to its receptor, CD25. Antibody B binds to IL-2 with low affinity and allows IL-2 to bind to CD25 only when fused to IL-2 with a long linker (13 or 14 amino acids or more). Antibody C has no affinity for IL-2 and allows IL-2 fused to it with any linker length to bind to CD25 (Tables 4 and 5).

[0224] pSTAT5 in mouse splenocytes (EC50 for Treg, CD8, and NK) STAT5 phosphorylation in mouse splenocytes was analyzed as a downstream signaling pathway of IL-2R activation. To assess the in vitro selectivity of antibody-IL-2 fusion proteins, pSTAT5 was measured in different cell populations after stimulation with Proleukin or compounds of the present invention. Freshly isolated mouse splenocytes from C57BL / 6 mice were incubated with serial dilutions of Proleukin or IL-2 antibody fusion proteins starting at 100 nM. Cells were immediately fixed and stained for surface markers (i.e., CD25, CD3, NK1.1, CD4, CD8). After permeabilization (Perm III buffer, BD Biosciences), intracellular staining was performed (FoxP3, pSTAT5) before acquisition by flow cytometry. CD8 + T cells, NK cells, CD4 + CD25 + FoxP3 + The percentage of pSTAT5+ Treg cells was plotted against the molar concentration of the IL-2-antibody fusion protein for each compound. EC50 values ​​were calculated using GraphPad Prism (Table 7). The potency of IL-2 fusion proteins with antibody A on Treg cells was significantly reduced compared to Proleukin, but the EC50 values ​​on NK and CD8 T cells were comparable. Fusion proteins with antibody B also showed reduced potency on Treg cells compared to Proleukin, but the bias effect decreased with increasing linker length (compound BFC885). IL-2 fused to antibody C signaled Treg cells with high activity comparable to Proleukin. The affinity of the antibody for the substituted IL-2 was required to effectively provide steric hindrance, thereby removing CD25 from the signaling complex (Table 7).

[0225] Table 7. EC50 of STAT5 activation in mouse splenocytes of IL-2-antibody fusion proteins on NK cells, CD8 cells, and Treg cells. [Table 7]

[0226] Applying the representative incorporation process to IL-2-specific antibody A or B yielded fusion proteins with equally favorable IL-2 CD25 binding (Table 5), stimulation of HEK Blue IL-2 reporter cells (Table 6), and reduced potency in STAT5 phosphorylation on Treg cells compared to Proleukin. This confirms that fusion to heavy or light chain variable regions is possible by adjusting the orientation of the circulating IL-2 protein to maintain the native binding orientation of the anti-IL-2 antibody and cytokine. Antibody B's low affinity for IL-2 (269 nM K D ) was sufficient to confer selective function to the resulting fusion protein. Peptide linkers up to 20 amino acids long retained the desired signaling qualities of IL-2 fusion proteins with antibody A, but shorter peptides were preferred in combination with lower affinity antibodies. Fusion proteins incorporating IL-2CP with antibody C did not eliminate CD25 binding (even with 0-1 amino acid linkers), indicating that an antibody affinity for IL-2 at least equivalent to that of antibody B is required.

[0227] Example 2: Generation of high-affinity, non-blocking anti-PD-1 antibodies Identification of PD-1 agonist and non-competitive anti-hPD-1 antibodies Three anti-human PD-1 antibodies were identified that were described as non-competitive with PD-1 antagonists or non-PD-L1 blocking (Table 8). These antibodies were tested for competition with commercially available PD-1 antagonist antibodies by flow cytometry using a 20-fold molar excess of competitor. Raji PD-1-expressing cells (Invitrogen) were incubated with serial dilutions of pembrolizumab and nivolumab starting at 81 μg / ml for 30 minutes at 4°C (1:3 serial dilutions). After washing the cells, biotin-labeled antibodies listed in column 1 of Table 8 were added at a constant concentration of 2 μg / ml. Bound antibodies were detected with Streptavidin-PE (Biolegend), and the MFI levels of bound compounds were compared to the MFI of samples without competitor to determine percent signal inhibition. The background of samples incubated with Streptavidin-PE alone was subtracted from all samples. Antibody XVT458 showed no competition with any of the PD-1 antagonists tested. Antibody ZJN296 showed partial competition with pembrolizumab, exhibiting a 47.4% reduced mean fluorescence intensity (MFI) compared to samples without competitor. Nevertheless, the signal did not show a concentration-dependent decrease, so the decrease in MFI in samples with a 20-fold higher concentration of competitor may be unrelated to the presence of pembrolizumab. Nivolumab did not significantly reduce the signal of antibody ZJN296. Antibody OVL714 completely competed with both pembrolizumab and nivolumab, indicating a possible shared epitope on the antigen.

[0228] Table 8. Antibodies listed as PD-1 antagonists or non-competitive with PD-L1 and their sources. Percent inhibition of binding signal to cells expressing human PD-1 upon pre-coating with a 20-fold molar excess of the indicated commercially available PD-1 antagonist antibodies. [Table 8]

[0229] Rehumanization and affinity maturation of antibody XVT458 The humanized VH sequence of XVT458, composed of frameworks shuffled from germline genes of different families, was rehumanized to obtain a VH with a matched germline (GH) sequence, resulting in z0-XVT458 (SEQ ID NOs: 053, 054). The rehumanization of the VH of XVT458 resulted in the human IGHV1-18 VH, which showed the highest homology with the framework regions of the parent construct. * 01 IGHJ6 * This was achieved by CDR-grafting onto the 01 framework. Mutations of Vernier zone residues were introduced to observe the effect on affinity for the hPD-1 antigen. Germline (GH wild-type or with point mutations) and parental humanized (uVH) were combined with humanized VL (uVL) in a full hIgG1 format (Table 9).

[0230] K D Decision: Binding of each antibody to PD-1 was assessed by SPR (Biacore 8K, GE Healthcare). Anti-human Fc IgG (Jackson) was immobilized on an amine-reactive CM5 chip (GE Healthcare) using 1x HBS-EP+ running buffer (GE Healthcare). Test antibodies were captured at a flow rate of 10 μl / min with a contact time of 30 seconds. Anti-PD-1 against human PD-1 (hPD-1) (AcroBiosystems, PD1-H82E4) and cynomolgus monkey (cyno) PD-1 (cynoPD-1) (AcroBiosystems, PD1-C5223) were run as analytes, diluted in 1x HBS-EP + running buffer (GE Healthcare) and subjected to a 240 s association and 600 s dissociation run at a flow rate of 30 μl / min. Regeneration was performed in 10 mM Glycine-HCl, pH 1.5, at a flow rate of 10 μL / min. This protocol is consistent with the K values ​​of the antibodies defined herein unless otherwise noted. D In all cases used to define K, adapted as necessary with respect to the nature of the interaction partners. D is used to determine the

[0231] Table 9. Anti-PD-1 antibody XVT458 and newly humanized VH derivatives and binding kinetics measured by SPR. [Table 9]

[0232] Binding of antibody candidates to cell surface-expressed antigens was confirmed by flow cytometry. CHO-S hPD-1 cells were incubated with 5-fold serial dilutions of the indicated humanized antibodies. Antibody binding was detected with goat anti-human IgG-PE. MFI was plotted against antibody concentration to obtain EC50 values. SPR binding kinetics, FACS EC50 binding values, and maximum MFI values ​​indicated that the antibodies bound to hPD-1-expressing cells, with the exception of z8-uIgGKV326 (Tables 9 and 10).

[0233] Table 10. Maximum MFI and EC50 values ​​of anti-PD-1 antibodies binding to hPD-1 expressed on CHO-S cells. [Table 10]

[0234] Humanized antibody z2-XVT458 (SEQ ID NOs: 055, 056) was affinity matured by inserting random mutations into the CDRs (parsimonious mutagenesis method). Each position in the antibody VH and VL-CDRs was mutated by PCR using mutagenesis primers containing the degenerate codon NNS at specific CDR positions, introducing all 20 amino acid combinations. The single mutant library was screened by scFv capture ELISA with a redundancy factor of 4 (96-well plate / position). Clones showing ELISA signals ≥2-fold higher than those of the wild-type scFv were sequenced, and clones with unique sequences were re-propagated, rescreened by capture ELISA, and ranked by dose-dependent ELISA against human and cyno antigens and FACS using human antigen-expressing cells. Mutants with improved affinity were used to design combinatorial libraries using the Kunkel method. The library was screened by scFv capture ELISA with a redundancy factor of 4 (4x the number of combinations). Clones showing ELISA signals ≥2-fold higher than those of wild-type scFv were sequenced. Clones with unique sequences were re-grown, re-screened by capture ELISA, and ranked by dose-dependent ELISA for human and cynoantigens, as well as FACS using human antigen-expressing cells. Primary screening by saturation mutagenesis of antibody CDRs identified 25 mutants at 10 CDR amino acid positions that showed capture ELISA signals for hPD-1 at least 2-fold higher than those of wild-type. Nine mutants showed improved binding to both recombinant hPD-1 and cynoPD-1, as well as to hPD-1 on CHO-S cells. A combinatorial library was created with these nine mutants, yielding 16 combinatorial mutants with improved affinity for recombinant and cell-expressed antigens. Based on the sequence of z2-XVT458 (variable domain SEQ ID NOs: 055 and 056), six subclones, z2-XVT458 m1 to m6 (SEQ ID NOs: 055 and 056), were selected for IgG conversion.In addition to the CDR mutations, the sequences of z2-XVT458-m1, z2-XVT458-m3, and z2-XVT458-m6 contain a G57D mutation immediately after CDRL2, which may contribute to the increased affinity of these clones. Clones selected in the IgG format showed significantly improved affinity for recombinant hPD-1, cynoPD-1, and hPD-1 on cells. Compared to the parent clone z2-XVT458, these clones (z2-XVT458m1-m6) showed improved affinity for both hPD-1 and cynoPD-1 as measured by SPR. off They also showed a 10-84 fold improvement in efficiency (Table 11; corresponding SEQ ID NOs: 057-068). For SPR, the same method as above was used.

[0235] Table 11. Parental XVT458, z2-XVT458 and subclonal affinity matured variants in IgG format. Binding affinities measured by flow cytometry, ELISA and SPR. [Table 11] JPEG2026501323000014.jpg108153

[0236] Humanization of antibody ZJN296 ZJN296 was cloned into the germline vector with the highest identity to the mouse framework (IGKV1-33 * 01 IGKJ2 and IGHV1-18 * 01 IGHJ6 * The VL and VL genes were humanized by CDR grafting using the IgG1A-specific CDR gene (Table 1). To increase the likelihood of retaining binding affinity to the target antigen, eight additional humanized VL genes and two humanized VH genes were designed by mutating human amino acids back to mouse. Such mutations maintain the original structure of the CDR loops of VH and VL, and can maintain antigen binding when these loops contact the antigen (Table 12).

[0237] Table 12. Mutations inserted into humanized ZJN296-0 VH (GH, SEQ ID NO: 069) and VL (SEQ ID NO: 070) and antibody variants of humanized ZJN296. [Table 12]

[0238] A recombinant humanized antibody variant of ZJN296 was expressed as human IgG1 in HEK293 cells. For SPR analysis (Biacore 8K, GE Healthcare), anti-human Fc IgG (Bethyl, A80-304P) was immobilized on a CM5 chip (Cytiva) using 1x HBS-EP + running buffer. Humanized anti-PD-1 antibodies were captured on the chip. hPD-1 and cynoPD-1 were run as analytes for a 180-second association phase followed by a 400-second dissociation phase using 1x HBS-EP + running buffer at a flow rate of 30 μL / min. 10 mM glycine (pH 1.5) was used as a regeneration buffer and injected into the flow cell following each dissociation phase.

[0239] Based on the SPR binding kinetics of human and cyno antigens and the binding data to cell-expressed hPD-1 tested in the supernatants, the five best candidates were recombinantly expressed and subjected to full SPR kinetics (Table 13). ZJN296-0 and ZJN296-6 were selected as candidates because ZJN296-0 has minimal back mutations and comparable binding avidity compared to the chimeric antibody, while ZJN296-6 has the best binding properties.

[0240] Table 13. SPR kinetic results of humanized purified ZJN296-derived antibodies [Table 13]

[0241] Screening for non-blocking antibody hybridomas A high-affinity binding antibody specific for human PD-1 (hPD-1), cross-reactive with cynomolgus monkey PD-1 (cynoPD-1), but not blocking PD-L1 or PD-1 antagonist antibodies, was generated by immunization of humanized mice (AlivaMab® mice) and hybridoma technology. Two separate immunization rounds (10 kappa-lambda mice in the first round, and 4 kappa and 4 lambda mice in the second round) were performed by immunization with the hPD-1 / cynoPD-1 combination. Primary functional screening was performed using HEK293 cells transfected with hPD-1 and cyPD-1. Binding was assessed by flow cytometry. Thirty-one 384-well plates were screened in the first round, and 15 384-well plates were screened in the second round. In the second round of screening, selected clones were first tested for competition with nivolumab and pembrolizumab from supernatants and then tested for binding to antigen-expressing cells by flow cytometry in the presence of competing antibodies and detected with an anti-mouse IgG antibody. Non-competitors were determined as MFI (+competition) / MFI (uncompetitor) > 0.6. Only non-competing clones that showed high affinity (KD < 1 nM) on BLI were expanded and purified. Of all screened clones, seven clones were identified that fit the desired binding profile (Table 14, CH 1、2、3 The VH and VL of the antibodies used were SEQ ID NOs: 071-084, with the corresponding CL (kappa) or CL (lambda). Table 14 summarizes the binding characteristics to human and cyno PD-1. EC50 binding to HEK293 cells transfected with hPD-1 and cyno PD-1 was measured by flow cytometry. Binding kinetics to recombinant antigens was measured by BLI. Association (220 seconds) and dissociation (480 seconds) of a dilution series of recombinant hPD-1 and cyno PD-1 (100, 25, 6.25 nM, AcroBio) were measured using an antibody-coated biosensor (anti-human-IgG CH1 Biosensor, ForteBio) on an Octet system (Octet RED, ForteBio). K DValues ​​were obtained by fitting the kinetic data using ForteBio data analysis software (8.2). PBS was used as the assay buffer (PBS 10 mM phosphate, 150 mM sodium chloride, pH 7.4). For dissociation, the biosensor was immersed in kinetic assay buffer (PBS 10 mM phosphate, 150 mM sodium chloride, 0.1% BSA, 0.02% Tween, pH 7.4). Sequencing of the final clones revealed that clones 21A08, 22F13, and 25I20 share the same HC / LC V-region and have the same CDR3. Clones 20H02, 39F23, 40B20, and 56H02 exhibited multiple developmental liabilities in the CDR regions. Clone 21A08 was selected as the best clone based on affinity and purity measured by SEC-HPLC compared with other sibling clones.

[0242] Table 14. EC50 by FACS binding to hPD-1 or cynoPD-1 expressing HEK293 cells, SPR binding kinetics to hPD-1 and cynoPD-1. [Table 14]

[0243] Mutations were introduced into the CDR-L1 of 21A08 to remove the N-glycosylation site present in CDR-L1. The first asparagine (Asp, N) in CDR-L1 was replaced with serine (Ser, S), glutamine (Gln, Q), or alanine (Ala, A). Binding of the 21A08 clone variants (21A08S, 21A08Q, and 21A08A) was tested by BLI and compared to the wild-type clone using supernatants from transiently transfected HEK293 cells and a positive control (pembrolizumab).

[0244] Table 15. Binding affinity (KD), dissociation constant (Koff) of antibody clone 21A08, deglycosylated variant. [Table 15]

[0245] Additionally, the deamidation site in CDR-L1 was removed by inserting the mutations shown in Table 16. The binding kinetics of 21A08A and the deamidation site-removed mutants were measured by SPR. For SPR analysis (Biacore 8K, GE Healthcare), anti-human Fc IgG (Bethyl, A80-304P) was immobilized on a CM5 chip (Cytiva), and the test anti-PD-1 antibodies were captured using 1x HBS-EP+ running buffer at a flow rate of 10 μL / min. hPD-1 and cynoPD-1 (His-tagged) were run as analytes diluted in 1x HBS-EP+ running buffer at a flow rate of 30 μL / min for a 250-second association phase followed by a 3600-second dissociation phase. 10 mM glycine (pH 1.5) was used as a regeneration buffer and injected into the flow cell following each dissociation phase. All clones lacking the N-glycosylation site retained binding to PD-1 (Table 15). None of the mutations in CDR-L1 significantly impaired the binding kinetics to hPD-1 or cynoPD-1.

[0246] Table 16. Antibody clone 21A08A and variants with removed deamidation site: Binding affinity and dissociation constants [Table 16]

[0247] Example 3: Binding Profiles of Non-Blocking Anti-PD-1 Antibodies Next, the binding profiles of non-blocking anti-PD-1 antibodies were evaluated using a human membrane protein cell array. The Retrogenix Cell Microarray Technology platform screened anti-PD-1 antibody candidates for cross-reactive binding to non-target proteins. Test antibodies were individually screened for binding to human HEK293 cells expressing 6018 full-length human membrane proteins, secreted and cell surface-associated human secreted proteins, and an additional 397 human heterodimers. For prescreening, 2 μg / mL of each test antibody or PBS alone was added to slides of fixed, non-transfected HEK293 cells. Binding to non-transfected cells was assessed using an AlexaFluor 647-labeled anti-human IgG Fc detection antibody (AF647 anti-hIgG Fc), followed by fluorescence imaging. Prescreening with antibody XVT458-z2-m5 detected high background. A second prescreening run at 0.5 mg / mL resulted in reduced background. For library screening, 6018 expression vectors encoding both ZsGreen1 and full-length human plasma membrane proteins or cell surface-tethered human secreted proteins were arrayed individually in duplicate on 17 microarray slides ("slide sets"). In addition, vectors encoding an additional 397 human heterodimers were co-arrayed on additional microarray slides. Human HEK293 cells were used for reverse transfection / expression. Test antibody XVT458-z2-m5 was added to each of the 18 slide sets at a final concentration of 0.5 μg / mL after cell fixation, while the remaining nine test antibodies were added at a final concentration of 2 μg / mL. Binding detection was performed using the same fluorescent secondary antibody (AF647 anti-hIgG Fc) used in prescreening. Fluorescent images were analyzed and quantified (for transfection) using ImageQuant software (GE Healthcare, version 8.2). Protein "hits" were defined as duplicate spots showing elevated signal compared to background levels.This was achieved by visual inspection using gridded images in ImageQuant software. Hits were classified as "strong, moderate, weak, or very weak" based on the intensity of the duplicate spots. 29 libraries were hit in the screening. After removing 12 interactions observed with the test antibodies and control treatments designated as nonspecific, and an additional 7 low-confidence interactions, 10 specific interactions for the test antibodies were identified (Table 17). All test antibodies showed a single specific interaction with PD-1 (PDCD1). Test antibodies ZJN296-0, ZJN296-6, XVT458-z2-m1, XVT458-z2-m2, XVT458-z2-m4, and XVT458-z2-m5 all showed moderate to weak interactions with other target proteins. Clones 21A08Ap1 (sequence numbers VH 085, VL 086), 21A08Ap2 (sequence numbers VH 085, VL 087), XVT458-z2-m3 (sequence numbers 061, 062), and XVT458-z2-m6 (sequence numbers 067, 068) showed a single specific interaction with the target of interest.

[0248] Table 17. Hits from the Retrogenix cell microarray screen. Protein type: plasma membrane (PM), secreted (S), tethered secreted (TS), heterodimer (HD), or evidence of plasma membrane (M). Hit number: very weak excess intensity. [Table 17]

[0249] Example 4: Non-competitive binding of anti-PD-1 antibodies to PD-L1 antagonists and PD-L1 Competition in hPD-1-expressing cells Antibodies 21A08Ap1 and 21A08Ap2 were tested for competition with commercially available PD-1 antagonist antibodies by flow cytometry. Jurkat PD-1-expressing cells were incubated with serial dilutions of pembrolizumab and nivolumab starting at 10 μM (1:3 serial dilutions) at 4°C for 30 minutes. Without washing the cells, biotin-labeled antibodies 21A08Ap1 and 21A08Ap2 were added at a constant concentration of 100 nM. Bound antibodies were detected with Streptavidin-PE (Biolegend), and the MFI levels of bound compounds were compared to the MFI of samples without competing compounds to determine percent signal inhibition. The background of samples incubated with Streptavidin-PE alone was subtracted from all samples. Inhibition was minimal at less than 20%, and neither pembrolizumab nor nivolumab significantly reduced the signal of antibodies 21A08Ap1 and 21A08Ap2 (Table 18).

[0250] Table 18. Competition of cell-expressed hPD-1 with commercially available PD-1 antagonists pembrolizumab and nivolumab. Percent signal inhibition by flow cytometry of test antibodies using a 100-fold molar excess of competitor. [Table 18]

[0251] Simultaneous binding of hPD-1 to 21A08Ap1 and PD-L1 Simultaneous binding of PD-1 (Fc-tagged) to anti-PD-1 antibodies and PD-L1 (His-tagged, Acrobiosystems, #H52H3) was assessed by SPR analysis using a Biacore T200 (Cytiva, #28975001). PD-L1 was captured on an anti-His-CM5 chip (Cytiva, chip: #29104988, His capture kit: #28995056). 1 μM PD-1 was premixed with 5 μM antibody (pembrolizumab, IgG1 isotype control antibody, or 21A08p1) or running buffer (Xantec, HBSTE:B HBSTE10) for at least 30 minutes before binding analysis (60 s association and dissociation). Data were analyzed using Biacore Insight Evaluation Software (Cytiva, V4.0.8 #29310606). Double reference subtraction was performed (surface without ligand and running buffer injection). Figure 3 shows the Biacore sensorgram. No binding (as a change in RU) was observed for PD-1 premixed with pembrolizumab, confirming that pembrolizumab blocks the PD-L1 binding site on PD-1. In contrast, the complex of PD-1 with antibody 21A08Ap1 was still able to bind to PD-L1, giving a signal of approximately 60 RU. Controls of PD-1 alone and PD-1 with an IgG1 isotype control showed an association signal of approximately 30 RU.

[0252] Example 5: Rational design of a non-blocking PD-1-targeted IL-2 fusion protein To determine the most suitable format for adding a non-blocking PD-1 targeting moiety to the fusion site of a CD122 / CD132 dimeric receptor-biased IL-2-anti-IL-2 antibody, proof-of-concept compounds were designed and characterized. The anti-PD-1 antibodies XVT458 or ZJN296 (Table 8) were combined with an antibody A-IL-2 fusion protein (QTY065, SEQ ID NOs: 051 and 052) to generate bispecific compounds that specifically deliver CD122-CD132-biased IL-2 to PD-1-expressing cells. Five formats were designed, differing in size and valency to the PD-1 antigen (Figure 4, Table 19). The dual-scFv fusions comprise the anti-PD-1 antibody XVT458 in scFv format, i.e., the VH and VL domains fused by a 15-amino acid glycine (G)-serine (S) linker, fused to the QTY065 IL-2 antibody fusion scFv fused by a G4S linker (Figure 4A). The Fab-dual-scFv consists of the QTY065 antibody IL-2 fusion as Fab fragments (IL-2-VL-CL and VH-CH1) fused at their C-termini to two anti-PD-1 scFv fragments of the XVT458 antibody (by the N-terminus VH-(G4S)3-VL (Figure 4B). The IgG CrossMab format is a heterotetrameric human IgG1 with HC1 and LC1 from the anti-PD-1 antibody XVT458 or ZJN296 and HC2 and LC2 from the antibody-IL-2 fusion QTY065 (Figure 4C). To ensure correct heavy chain pairing, knob-into-hole mutations (Y407T on HC1 and T366Y on HC2) were used. To improve correct light chain pairing, the CL and CH1 domains of HC1 and LC1 were swapped (WO2009080253A1). For Fc silencing of the IgG1 Fc region, mutations L234A, L235A, and P329G (WO2012130831A1) were inserted into both HC1 and HC2. The IgG-scFv fusion protein contains antibody XVT458 (consisting of HC1, identical LC(s), and HC2) as a fully human IgG1, with HC2 fused at either the N-terminus (4E) or C-terminus (4D) to the IL-2 antibody fusion QTY065 as an scFv (e.g., IL-2-VL). 抗体A -(G4S)3-VH抗体A -(G4S)2-HC2 XVT458 or HC2 XVT458 -(G4S) 2 / 4 -IL-2-VL 抗体A -(G4S)3-VH 抗体A ) Pairing to HC1 was improved by knob-into-hole mutations (as in CrossMab) and Fc silencing with L234A, L235A, and P329G mutations. The bispecific antibody was produced in Expi297 cells as described for the IL-2 fusion protein above.

[0253] The functionality of the bispecific antibodies was assessed by sandwich ELISA, which relies on binding to the target antigen (coated hPD-1) and the integrity of fusion IL-2 via a secondary antibody (anti-IL-2 clone 5344). 60 nM hPD-1 (ECD-His, in-house) was coated onto Maxisorp plates (Nunc) overnight at 4°C and blocked with 5% BSA in PBS. The bispecific antibodies were serially diluted in assay buffer and detected with biotinylated 5344 and streptavidin-HRP (BD Pharmingen, 554066). After the addition of TMB, the absorbance signal was read at 450 nm using a plate reader (Spectramax ID3). EC50 values ​​were determined by blotting absorbance versus concentration (Graphpad Prism, sigmoidal curve fit, 4PL, logarithmic scale). All compounds demonstrated binding to the bispecific site; the bivalent binding formats to hPD-1 exhibited lower EC50 values ​​compared to the monovalent formats (Table 19). Binding to hPD-1 expressed on the surface of Jurkat-PD-1 cells was confirmed by flow cytometry (Table 19). Binding to hPD-1 was maintained in all formats. As expected, compounds with two PD-1-binding domains exhibited increased binding to hPD-1 due to an avidity effect.

[0254] Table 19. Anti-PD-1, anti-hIL-2-IL-2 bispecific antibodies in various formats, valency, molecular weight (Mw), EC50 against hPD-1 measured by ELISA, and binding to hPD-1 expressed on Jurkat-hPD-1 cells measured by flow cytometry (fold MFI over background). [Table 19]

[0255] Example 6: Cell selectivity in vitro and in vivo To evaluate the functionality of the anti-IL-2 / IL-2 fusion protein arm of the bispecific compounds, STAT5 phosphorylation was analyzed in mouse splenocytes as a downstream signaling pathway of IL-2R activation. pSTAT5 was measured in different cell populations after stimulation with Proleukin or one of the bispecific compounds. Mouse splenocytes were incubated with serial dilutions of the bispecific compounds diluted in RPMI + 10% FBS. Starting at a concentration of 100 nM, the dilutions were 1:5, resulting in six total concentrations. Samples were incubated at 37°C for 15 minutes, and an equal volume of cytofix buffer (BD Biosciences, catalog no. 554655) was added per sample. The samples were then immediately fixed after 10 minutes of incubation at 37°C. After fixation, cells were stained with BV421 rat anti-mouse CD25 (clone PC61, BD Biosciences, 0.5 μL / sample), BV650 hamster anti-mouse CD3e (clone 145-2C11, BD Biosciences, 1 μL / sample), and BV711 mouse anti-mouse NK1.1 (clone PC136, BD Biosciences, 0.33 μL / sample) for 30 minutes at room temperature, followed by permeabilization with Perm Buffer III (BD Biosciences, catalog no. 558050) for 10 minutes on ice. The secondary staining included PE-CF594 rat anti-mouse CD4 (clone RM4-5, BD Biosciences, 0.25 μL / sample), APC-780 rat anti-mouse CD8b (clone H35-17.2, eBioscience, 0.167 μL / sample), AF488 rat anti-mouse FoxP3 (clone FJK-16, eBioscience, 0.5 μL / sample), and AF647 mouse anti-mouse pSTAT5 (clone pY694, BD Bioscience, 20 μL / sample). All samples were acquired using Cytek® Aurora's SpectroFlo® software. ".fcs" files were analyzed using FlowJo_v10.6.2. EC50 values ​​for the percentage of pSTAT5+ cells were obtained by sigmoidal 4PL fitting. The percentage of pSTAT5+ cells was plotted against the logarithmic concentration (M) for each cell population and each test compound.

[0256] The obtained EC50 values ​​are shown in Table 20. All formats except the IgG C-terminal scFv bispecific showed comparable (or up to 10-fold lower) potency than Proleukin in inducing STAT5 phosphorylation in NK and CD8 T cells (Table 20). EC50 values ​​for QWT744 and CUM013 could not be determined because the percentage of pSTAT5-positive cells with the highest concentration of test compound (100 nM) did not reach a maximal level. All compounds showed reduced potency in inducing pSTAT5 in Treg cells compared to Proleukin, indicating that signaling through the high-affinity trimeric IL-2R is inhibited in vitro (Table 20).

[0257] Table 20. EC50 values ​​of bispecific compounds, QTY065, and Proleukin against pSTAT5 positive parental NK, CD8, and Treg cells measured by flow cytometry of mouse splenocytes. [Table 20]

[0258] To confirm in vivo selectivity for CD122-CD132-expressing cells, the bispecific compound was administered intravenously at 0.2 mg / kg to wild-type C57BL / 6 mice. Blood samples were collected before injection and on days 3 and 6 after compound administration. Cell proliferation was analyzed by flow cytometry, and Ki67+ cells were detected in CD8 T cells, NK cells, and Treg cells (Figure 5). C57BL / 6 mice received a single intravenous injection of 0.2 mg / kg of the compound. Blood samples collected before compound administration and on days 3 and 6 after compound administration were processed and analyzed by flow cytometry. Cells were initially stained with CD25, CD3, NK1.1, CD4, CD8, and Zombie aqua fixable viability dye (Biolegend). After fixation and permeabilization (eBioscience), intracellular staining for Ki67 and FoxP3 was performed. All compounds showed only a slight increase in Ki67 expression in Treg cells on day 3, with levels decreasing on day 6 after compound administration. The proliferation (Ki67 expression) of CD8 T cells and NK cells in the blood of animals treated with bispecific compounds in IgG format (FQQ111 and ONG682) or IgG-N-ter scFv (YIM345) format was comparable to that of QTY065, reaching maximal levels of Ki67+ cells (>75%) on day 6 after administration. Compounds with low molecular weights (MW) and lacking the Fc domain, such as HRL470 and VNP090, showed Ki67+ levels of 22–25% in CD8 T cells and 33–40% in NK cells. The bispecific format of C-terminal fusion of anti-hIL-2 / IL-2 scFv on anti-PD-1 IgG (CUM013) showed lower potency compared to other Fc domain-bearing compounds with 47% Ki67+ NK cells and 25% Ki67+ CD8 T cells, which surprisingly suggests that IL-2 signaling is less potent when the cytokine is C-terminally linked to the target antibody domain (Figure 5).

[0259] Example 7: Antitumor effect in vivo To examine the additional protection conferred by a non-blocking PD-1 targeting moiety compared to an anti-IL-2 / IL-2 fusion protein lacking PD-1 targeting, we chose the B16F10 melanoma model, an aggressive tumor model resistant to immune checkpoint inhibitors (CPi). Immunophenotyping and immune cell proliferation of B16F10 subcutaneous tumors were performed in humanized PD-1 mice (C57BL / 6N-Pdcd1tm1 (huPDCD1-ICP11; Geno)), allowing assessment of both PD-1 targeting and IL-2 activity. Transgenic hPD-1 mice were inoculated with 1 × 10 6 B16F10 cells were injected subcutaneously. 3 When the tumor reached an average size of 100 mg / kg, the compound was administered intravenously at a dose of 0.2 mg / kg. Tumor volume was measured daily with a caliper and calculated using the formula (length x width x depth) / 2. At the end of the study, 6 days after the start of administration, the difference in tumor volume (%) relative to the vehicle was calculated. Tumor-infiltrating cells were analyzed by flow cytometry as described above using the antibodies listed in Table 21.

[0260] Table 21. Antibodies used for intracellular (IC) and extracellular (surface) staining for immunophenotyping of B16F10 tumors. [Table 21]

[0261] Administration of the nontargeted anti-IL-2 / IL-2 fusion protein QTY065 primarily led to an increase in intratumoral NK cells (8.2-fold compared to vehicle) in tumors of treated mice, while all PD-1-targeted compounds promoted a significant increase in CD8 T cells in B16F10 tumors. The smaller format HRL470 with a dual scFv fusion had the least effect (2.7-fold compared to vehicle), while IgG and IgG-scFv fusions led to a 4.6- to 8.1-fold increase in CD8 T cells compared to vehicle. Notably, the effect was enhanced within the target cell population, CD8+PD-1+ cells (Table 22). The C-terminal scFv compound CUM013, while not as potent in previous assays, demonstrated comparable efficacy to other bispecific formats in tumor-infiltrating lymphocyte (TIL) activity. Nevertheless, CUM013 was less effective at suppressing tumor growth than the other bispecifics (Table 23). Based on the results obtained, it was concluded that the heterotetrameric IgG (CrossMab format) and IgG-N'-scFv-IL-2 fusion format bispecific molecules are particularly effective in vivo, and the VNP090 and CUM013 formats were not pursued further and were shelved as potential back-ups.

[0262] Table 22. TILs from transgenic hPD-1 mice treated with non-targeted or PD-1-targeted IL-2 antibody fusion proteins as fold increase in cells / gram tumor relative to vehicle. Tumors analyzed on day 6 (n=4). [Table 22]

[0263] Table 23. Difference in tumor volume (%) compared to vehicle-treated mice on day 6. [Table 23]

[0264] In an additional study, tumor growth in hPD-1 transgenic mice was monitored after two doses of the bispecific compound compared with vehicle, a non-blocking PD-1 antibody lacking IL-2 binding (antibody XVT458 as an Fc-silenced human IgG1), or a non-targeting bivalent antibody-IL-2 fusion, QTY065. On day 0, tumors were 70-100 mm 3 This corresponds to the start of the study, when tumor volume reached 1000 mg / kg. On days 0 and 3, 1.25 nmole / kg of each compound was administered intravenously, and tumor growth was monitored. At the end of the study, the difference in tumor volume was compared to vehicle-treated mice (Table 24). Five days after treatment, tumors were treated and TILs were analyzed by flow cytometry using intracellular staining, as shown in Table 21. We determined the doubling of cell numbers per gram of tumor compared to vehicle for CD8 T cell subfamilies, including stem-like pre-exhausted CD8 T cells (CD8+PD-1+TCF1+), more effector-exhausted T cells (CD8+TCF1-CX3CR1+), and terminally exhausted T cells (CD8+TCF1-CX3CR1-), CD8 T cells, Treg cells, and NK cells. The control antibody XVT458 induced only a minimal increase in tumor cell infiltration. Bispecific compounds particularly induced an expansion of CD8+PD-1+ T cells and their derived subfamilies, with heterotetrameric IgG CrossMab and IgG-scFv N-terminal formats being the most effective compounds in terms of target cell targeting and antitumor potency (Tables 24 and 25).

[0265] Table 24. Difference (%) in tumor volume compared to vehicle-treated mice on day 5 (n=5-6). [Table 24]

[0266] Table 25. TILs from transgenic hPD-1 mice treated with non-targeted or PD-1-targeted IL-2 antibody fusion proteins as fold increase in cells per gram tumor relative to vehicle. Tumors analyzed 5 days after initiation of treatment. [Table 25]

[0267] Example 8: Increased in vitro potency of PD-1-targeted anti-hIL-2 / IL-2 fusion proteins Selected bispecific formats were then combined with fully functional non-blocking anti-hPD-1 antibodies (Table 26). Antibodies targeting the unrelated antigens MDC982 and KVC110 were used as non-targeting control antibodies. The anti-hIL-2 / IL-2 arm of QTY065 possesses a light chain of the kappa subfamily, which can be combined with a second arm of the lambda subfamily to form heterotetrameric compounds (bispecific antibodies with different specificities in each antigen-binding domain), eliminating the need for genetic engineering strategies such as CrossMab to ensure correct light chain pairing. The IL-2-anti-IL-2 fusion portion of QTY065 was combined with two representative non-blocking high-affinity PD-1 antibodies, 21A08Ap1 and 21A08Ap2 (both possessing lambda light chains), to design a bispecific in a heterotetrameric IgG kappa / lambda (Figure 4F) format. Constructs encoding the sequences disclosed in Table 26 were transfected into Expi293 cells and produced and purified as described above. The knob-in-hole mutations were Y407T on HC1 and T366Y on HC2, and the Fc-silencing mutations L234A, L235A, and P329A were incorporated.

[0268] Table 26. Heterodimeric combinations of QTY065 VH, VL(IL-2) and a second anti-hPD1 (or control) antibody in three different bispecific antibody formats. [Table 26]

[0269] Bispecific compounds were tested for their potency in inducing STAT5 phosphorylation in PD-1+ Jurkat cells expressing IL-2R CD122-CD132. Jurkat-PD1+ CD122+ cells were activated with serial dilutions of bispecific compounds for 15 min at 37°C and fixed by adding an equal volume of Cytofix buffer (BD Biosciences) for 10 min at 37°C. For staining of intracellular antigens, cells were permeabilized with ice-cold Perm buffer III (BD Biosciences) for 15 min on ice. Phosphorylated STAT5 was stained using anti-p-STAT5 pY694 antibody (clone 47 / Stat5, BD Biosciences). Flow cytometry was performed as previously described. EC50 values ​​were calculated using Graphpad Prism V9.3.1 using the equation Y = Bottom + (X^Hillslope). * Calculations were made using (Top-Bottom) / (X^HillSlope + EC50^HillSlope) ([antagonist] vs. response-variable slope (4 parameters)). PD-1-targeted bispecific compounds, with the exception of YPW986 and PXU588, showed increased potency and lower EC50 values ​​compared to the bivalent QTY065 and the non-targeted compound TSQ225 (Table 27). Binding ELISA assays demonstrated that YPW986 and PXU588 (i.e., heterotetramers in a CrossMab bispecific format, with one arm derived from QTY065 and one arm from either 2108Ap1 or 21A08Ap2) lost binding affinity to PD-1, in contrast to heterotetrameric kappa / lambda IgG formats using the same non-blocking anti-PD-1 clones XWY176 and GQM289, which retained the ability to stimulate the dimeric IL-2 receptor. In general, PD-1 anchoring to cells increased the IL-2 signaling potency of bispecific compounds. The heterotetrameric CrossMab bispecific IgG FQQ111 with a non-affinity-matured anti-PD-1 arm (XVT458) elicited a lower increase in potency upon targeting to PD-1, indicating that higher affinity binding is desirable.

[0270] Table 27. EC50 values ​​for STAT5 phosphorylation in Jurkat-PD-1+ CD122+ cells. [Table 27]

[0271] An additional functional effect of the fusion proteins of the present invention is the reduction of cell surface PD-1 by binding to IL-2R. Signaling through IL-2R leads to the internalization of dimeric or trimeric receptor complexes (Robb RJ. et al., J Exp. Med. (1987) doi:10.1084 / jem.165.4.1201). The reduction of cell surface hPD-1 and CD122 by the immunoconjugates of the present invention was tested by incubating stimulated PBMCs (i.e., PD-1+) with three different concentrations of the bispecific for 16 hours. As controls, the bivalent anti-hIL-2 / IL-2 compound QTY065 and a non-targeting IL-2 compound, IgG CrossMab, were used. Buffy coats or whole blood from healthy volunteers were received from the Blutspendezentrum SRK beider Basel or Aarau in accordance with the Swiss ethical committee, the Swiss Human Research Act (HRA; May 2021), and other applicable ethical regulations. PBMCs were isolated by density gradient centrifugation using Ficoll Plaque Plus (GE Healthcare). Frozen PBMCs were thaw-activated with plate-coated anti-CD3 (clone OKT3, BioLegend) and soluble anti-CD28 (clone CD28.2, BioLegend) for 3 days. After activation, PBMCs were incubated with the indicated bispecific compounds overnight at 37°C. Immediately after incubation, an equal volume of Cytofix buffer (BD Biosciences) was added, and cells were fixed for 10 minutes at 37°C. Surface markers were then stained using the antibodies listed in Table 28. Cells were acquired by flow cytometry as described above.

[0272] Table 28. Antibodies used for extracellular staining for cell surface expression experiments. [Table 28]

[0273] The difference in MFI of detected surface CD122 and PD-1 was calculated as the percentage reduction compared to PBMCs incubated with medium alone. Control compounds induced a reduction in surface CD122, but did not change PD-1 levels. PD-1-targeting compounds reduced both CD122 and PD-1 levels. This may indicate that binding to IL-2R is required for the reduction of cell surface PD-1 (Table 29).

[0274] Table 29. Percent reduction in CD122 and PD-1 MFI of CD8 T cells following incubation with control compounds or bispecific compounds compared to CD8 T cells incubated with media alone (n=2 donors). [Table 29]

[0275] Example 9: Improved PD-1 binders: in vivo antitumor efficacy and subcutaneous tumor immunophenotyping The improved bispecific compounds in Table 26 were tested in hPD-1 transgenic mice bearing subcutaneous B16F10 tumors. Transgenic hPD-1 mice (C57BL / 6N-Pdcd1tm1(huPDCD1-ICP11)Geno) were inoculated with 1x10 6 B16F10 cells were injected subcutaneously. 3When the tumors reached an average size of 1000 nmoles (day 0), the compound was administered intravenously at 1.25 nmoles / kg. A second dose was administered on day 3. Five days after the start of the study, mice were sacrificed and tumors were excised. Tumors were treated with a GentleMACS Octo Dissociator (Milteny), and cells were stained with NIR live dead stain (ThermoFisher, L10119). Cells were incubated in Fc block (TruStain FcX™, Biolegend, 101320) for 10 minutes before surface staining (see Table 21). Cells were fixed and permeabilized with FoxP3 Staining Buffer Set (ebioscience, #00-5523-00), followed by intracellular staining according to Table 21. All PD-1-targeted anti-IL-2 / IL-2 bispecific antibodies significantly increased intratumoral CD8 T cell numbers, particularly PD-1+ stem-like T cell and derivative numbers, compared to non-targeted compounds or vehicle (Table 30). The CD8 T cell / Treg ratio was increased in tumors from mice treated with bispecific compounds compared to vehicle or non-targeted bispecifics.

[0276] Table 30. TILs from transgenic hPD-1 mice treated with non-targeting or PD-1-targeting IL-2 antibody fusion proteins as fold increase in cells per gram tumor relative to vehicle. Tumors were analyzed on day 5. [Table 30]

[0277] In efficacy studies, transgenic hPD-1 mice (C57BL / 6N-Pdcd1tm1(huPDCD1-ICP11)Geno) were injected with 1 × 10 6 B16F10 cells were injected subcutaneously. 3When the tumors reached an average size of 100 mg / mL (day 0), 1.25 nmole / kg of compound was administered intravenously. Booster doses were administered on days 3 and 7. Tumors were measured daily, and the volume was calculated using the formula (length x width x depth) / 2. The difference in tumor volume between mice administered vehicle or each compound was reported as a percentage reduction compared to vehicle. All PD-1-targeted IL-2 fusion protein compounds effectively reduced tumor volume (Table 31).

[0278] Table 31. Day 7 tumor volume difference from vehicle (%). [Table 31]

[0279] Example 10: Stress test with anti-PD-1, anti-IL-2 / IL-2 bispecific antibody The heterotetrameric bispecific antibodies XWY176, TMU471, QAB373, and MDS446 were generated with modified knob-in-hole mutations (S354C, T366W / S354C, T366S, L368A, Y407V) and Fc silencing (Table 32).

[0280] Table 32. Bispecific compounds with knob-in-hole mutations and Fc silencing [Table 32]

[0281] The four bispecific antibodies in Table 32 were exposed to heat, pH, oxidation, and freeze-thaw stress conditions. The compounds were then tested by SEC-HPLC (Table 33), iCEIF (Table 34), and functional ELISA (Table 35). Post-translational modifications were analyzed by mass spectrometry. The bispecific antibodies XWY176, TMU471, QAB373, and MDS446 were exposed to different stress conditions: i) incubation at 40°C for 1 or 2 weeks, ii) three or four freeze-thaw cycles, iii) 0.1% (v / v) HO for 4 or 24 hours, iv) low pH 3.5 for 24 or 48 hours, and v) high pH 9.0 for 24 or 48 hours. To evaluate protein changes after the stress conditions, the compounds were tested by SEC-HPLC, iCEIF, and LC-MS. The functionality of the bispecific antibodies was assessed by a sandwich ELISA, which relies on binding to the target antigen (hPD-1) and the integrity of fusion IL-2 via a secondary antibody (anti-IL-2 clone 5344). 60 nM hPD-1 (ECD-His, in-house) was coated onto Maxisorp plates (Nunc) overnight at 4°C and blocked with 5% BSA in PBS. The bispecific antibodies were serially diluted in assay buffer and detected with biotinylated 5344 and streptavidin-HRP (BD Pharmingen, 554066). The end-of-absorbance signal after addition of TMB was read at 450 nm using a plate reader (Spectramax ID3). EC50 values ​​were determined by blotting absorbance versus concentration (Graphpad Prism, sigmoidal curve fit, 4PL, logarithmic scale). The change in EC50 value of unstressed samples (TO) was compared to stressed samples.

[0282] All constructs showed high potential for further development based on the criteria evaluated in this example. SEC-HPLC and iCIEF indicated that the conditions that induced the most protein changes were heat stress and high pH. The construct that showed a smaller increase in high molecular weight (HMW) species by SEC-HPLC was NZA596. The same construct also showed a smaller change in charged species by iCIEF. ELISA functionality assessment showed that all proteins retained at least 40% functionality after exposure to different stress conditions. Constructs NZA596 and CIT348 retained greater than 60% functionality under all conditions tested. PTM analysis showed oxidation of all four methionines under heat stress and oxidative conditions, with NZA596 performing slightly better.

[0283] Example 11: In vitro immunogenicity of anti-PD-1, anti-IL-2 / IL-2 bispecific antibodies The in vitro immunogenicity of NZA596, CIT348, BGY642, and KTX917 was tested by MHC-associated peptide proteomics (MAPPS) performed at Lonza. Monocyte-derived dendritic cells (DCs) from 10 different healthy donors (carrying mutations in HLA-DRB alleles) were incubated with 50 μg / ml of the protein of interest and then matured with LPS for 24 hours. After maturation, DCs were lysed, and the membrane fraction containing the HLA:peptide complexes was solubilized and incubated overnight at 4°C with Protein A Mag 66 epharose beads (Cytiva) coated with anti-HLA-DR antibody (Lonza). The following morning, the beads were washed with TBS, and peptides were eluted from the HLA-DR complexes with 0.1% TFA. Finally, peptides were purified through a 10 kDa molecular weight cutoff (MWCO) spin column and analyzed by MS. Peptide analysis revealed "immunogenic hotspots" that corresponded to HLA-presented peptides from two or more donors and were not present in the antibody's natural framework. Table 36 summarizes the immunogenic hotspots identified for each compound and the location of the peptides within the protein. The two non-blocking anti-PD-1 binding domains tested produced similar hotspot profiles. While all formats had acceptably low immunogenicity, those with fewer mutation regions and peptide linkers, most notably the kappa / lambda format, had the most favorable immunogenicity profiles.

[0284] Table 33. SEC-HPLC of bispecific compounds at time point (T0) or after different stress conditions: 1 week (1W) or 2 weeks (2W) at 40°C, freeze-thaw cycles, oxidative conditions (0.1% H2O2) for 4 or 24 hours, low pH (pH 3.5) and high pH (pH 9.0) for 4 or 24 hours. HMW: high molecular weight; LMW: low molecular weight. [Table 33]

[0285] Table 3. Capillary isoelectric focusing of bispecific compounds at time point 4.0 (T0) or after different stress conditions: 1 or 2 weeks at 40°C, freeze-thaw cycles, oxidizing conditions (0.1% H2O2) for 4 or 24 hours, low pH (pH 3.5) and high pH (pH 9.0) for 4 or 24 hours. [Table 34]

[0286] Table 3. Functional ELISA of bispecific compounds at 5.0 time point (T0) or after different stress conditions: EC50 values ​​(%) of stressed samples with full functionality at T0 set as 100% and EC50 at T0, 2 weeks at 40°C, 24 hours in oxidative conditions (0.1% H2O2), 24 hours at low pH (pH 3.5) and high pH (pH 9.0). [Table 35]

[0287] Table 36. Immunogenicity hot spots of bispecific compounds [Table 36]

[0288] Example 12: Mass spectrometry demonstrating correct light chain pairing To test for correct light chain pairing in purified bispecific constructs in the IgG CrossMab or IgG kappa / lambda formats, IgdE enzyme digestion was performed and the Fab fragments were analyzed by mass spectrometry. For each assayed Fab, a single peak corresponding to the theoretical molecular weight of the correctly assembled light and heavy chains was observed (Figure 6). No incorrect light chain pairing was detected.

[0289] Example 13: Cis-signaling in combination with PD-1 blocking antibodies An assay was established to assess whether exemplary bispecific constructs as disclosed herein transmit IL-2 to CD122-CD132 on PD-1-expressing cells bound by their anti-PD-1 arms (cis signaling) or to neighboring cells (trans signaling). Jurkat-PD-1+ CD122+ cells were labeled with either CFSE (Invitrogen, C34557) or CTV (Invitrogen, C34554). CFSE-labeled cells were exposed to 700 nm of the non-competitive parent antibodies against PD-1, pembrolizumab or nivolumab, to block the PD-1 epitope for 30 minutes at room temperature. After two washes, CTV-labeled and CFSE-labeled cells were mixed at a 1:1 ratio and activated with the bispecific compound NZA596 (1 nM) for 15 minutes at 37°C. Immediately after the stimulation period, cells were fixed with an equal volume of Cytofix buffer (BD Biosciences 554655) at 37°C for 10 minutes. For intracellular antigen staining, cells were permeabilized with ice-cold Perm buffer III (BD Biosciences) for 15 minutes on ice. Phosphorylated STAT5 was stained using anti-p-STAT5 pY694 antibody (clone 47 / Stat5, BD Biosciences). Immunoconjugate-mediated induction of STAT5 phosphorylation in these cells was analyzed. Cells preincubated with the parent antibody significantly reduced the efficacy of the immunoconjugate. This interference was not observed in cells pre-exposed to pembrolizumab or nivolumab (Table 37, Figure 7). Furthermore, the potency of the immunoconjugates on cells not previously exposed to any PD-1-binding antibody was the same for all samples (co-incubated with CFSE+ pre-blocked cells), indicating that the immunoconjugates signal in a cis manner on the same cells where PD-1 binding occurs (Table 37, Figure 7).

[0290] Table 37. Percentage of pSTAT5 compared to non-blocked cells after stimulation with 1 nM NZA596: [Table 37]

[0291] Example 14: Antitumor Efficacy of Bispecific Immunoconjugates in Combination with Pembrolizumab and Nivolumab in Mouse Models of Two Cancers The efficacy of an exemplary bispecific immunoconjugate (NZA596) as disclosed herein was tested as monotherapy or in combination with the commercially available PD-1 blockers pembrolizumab and nivolumab. hPD-1 transgenic C57BL / 6 mice were injected subcutaneously with B16F10 melanoma cells or MC38 colon tumor cells. Tumors were 70-100 mm 3 Mice were randomized when tumors reached an average size of 100 μg / mL, and treatment was initiated as shown in Table 38. Mice were dosed on day 0 (the day of randomization) and day 3. Pembrolizumab or nivolumab was also administered on day 7. Tumor growth inhibition was calculated relative to tumor volume in vehicle-treated mice on day 13 for mice bearing MC38 tumors and day 14 for mice bearing B16F10 tumors. In both tumor models, the bispecific immunoconjugate in combination with either of the checkpoint inhibitors induced significant tumor growth retardation compared to vehicle, even at low doses. The combination therapy resulted in more potent tumor growth inhibition compared to either monotherapy.

[0292] Table 38. Dosing schedule and doses for in vivo efficacy studies in hPD-1 transgenic mice bearing B16F10 or MC38 tumors. Tumor growth inhibition (TGI) was calculated as the percentage reduction in volume compared to the mean volume of the vehicle-treated group. * Dose 1: MC38 model: 0.1 mg / kg, B16F10 model: 0.2 mg / kg ** Dose 2: MC38 model: 0.2 mg / kg, B16F10 model: 2 mg / kg: 0.4 mg / kg. (n) = number of animals. [Table 38]

[0293] Example 15: Efficacy on pSTAT5 signaling in the presence of various existing PD-1 blockers STAT5 phosphorylation in Jurkat PD-1+ CD122+ cells was analyzed by flow cytometry after 15-minute incubation with a dilution series of an exemplary bispecific immunoconjugate (NZA596). Prior to stimulation, cells were incubated for 30–60 minutes with either growth medium alone, a pre-existing PD-1 blocker, or the parental anti-PD-1 antibody 21A08Ap1, all at saturating concentrations (200 nM). NZA596 was added to the cells without washing out the anti-PD-1 IgG, and pSTAT5+ cells were detected by flow cytometry. The EC50 of the bispecific immunoconjugate NZA596 on stimulated Jurkat PD-1+ CD122+ cells was calculated to be 0.85 nM by plotting the concentration against the percentage of pSTAT5+ cells. This value significantly increased when the parental anti-PD-1 antibody 21A08Ap1 was used to pre-block PD-1 in the cells. Preincubation of cells with pembrolizumab, nivolumab, cemiplamab, dostallimab, zelvalimab, tislelizumab, ezabenlimab, toripalimab, or cetrelimab did not alter the potency of NZA596 in inducing pSTAT5, demonstrating that these antibodies can block the PD-L1-PD-1 axis without altering the activity of the bispecific immunoconjugates described in this patent (Table 39). The difference in the binding epitope of PD-1 was also demonstrated by SPR, which showed simultaneous binding of NZA596 and a commercially available PD-1 blocking antibody on immobilized recombinant PD-1 (data not shown).

[0294] Table 39. List of anti-PD-1 antibodies pre-incubated with Jurkat PD-1+ CD122+ cells prior to stimulation with bispecific immunoconjugate NZA596. Potency (EC50) values ​​in nM and fold reduction based on pre-incubation with media. [Table 39]

[0295] Example 16: Bispecific immunoconjugates induce expansion of tumor-specific subsets of T cells in the tumor microenvironment In the context of tumors, PD-1 marks T cells chronically exposed to tumor antigens and thus marks a subset of T cells with relevant anti-tumor specificity. To gain a better understanding of the T cell repertoire expanded by the bispecific immunoconjugates of the present invention, we performed T cell receptor beta (TCRβ) chain sequencing of TILs from hPD-1 mice bearing B16F10 tumors to assess the diversity of the TCR repertoire. Transgenic hPD-1 mice were injected subcutaneously with B16F10 cells. Tumors were 50-100 mm. 3 On study day 0, when tumors reached a size of 100 μg, mice were randomized and intravenously administered 0.2 mg / kg of the bispecific immunoconjugate (targeted to PD-1), 0.2 mg / kg of the non-PD-1-targeted immunoconjugate (QTY065), or vehicle, followed by a second dose on day 3. Mice were sacrificed on day 5, and genomic DNA was extracted from tumors for sequencing of the TCRβ repertoire. Mice treated with the PD-1-targeted bispecific immunoconjugate exhibited a reduced TCR repertoire, likely resulting from the expansion of a subset of T cell clones derived from PD-1+ cells present before treatment that may be focused and tumor antigen-specific (Figure 8). In contrast, the non-PD-1-targeted immunoconjugate induced an increased enrichment of the TCR repertoire compared to vehicle, indicating that this non-targeted IL-2Rβ / γ antagonist expanded a more diverse set of T cell clones, including many specificities not associated with the tumor.

[0296] To quantify the effect of bispecific immunoconjugates on CD8+ T cells with defined tumor-neoantigen specificity, the kinetics of antigen-specific T cell responses in TILs from mice bearing MC38 or B16F10-OVA were analyzed by flow cytometry with MHC dextramers. Wild-type C57BL / 6 mice bearing B16F10-OVA or MC38 subcutaneous tumors were injected with 0.2 mg / kg of the murine surrogate bispecific immunoconjugate BGY642 (designated DSQ964, which targets mouse PD-1 with the antigen-binding domain of clone RMP1-30 and does not interfere with PD-1 binding to its inhibitory ligands), a non-targeting immunoconjugate (QTY065), or vehicle on day 0 post-randomization (tumor volumes 50–100 mm). 3 ) and 3 days after treatment. TILs were isolated on day 5 and analyzed by flow cytometry using dextramer, which specifically stains CD8+ T cells that recognize the ovalbumin neoantigen expressed by the B16F10-OVA tumor cell line and the p15E retroviral antigen expressed by the MC38 tumor cell line. In both tumor models, tumor neoantigen-specific T cells were increased in mice treated with DSQ964 compared with vehicle or a non-targeting IL-2 / anti-IL-2 fusion protein (Table 40). Furthermore, all tumor antigen-specific CD8 T cells were found to be PD-1+ (positive). Combined with the TCR sequencing data, these experiments confirmed that PD-1-targeted bispecific immunoconjugates increase the specific subset of CD8+ T cells that recognize tumor cells in mouse models of cancer.

[0297] Table 40. Fold increase in intratumoral Dextramer+CD8+PD-1+ T cells in mice treated with DSQ964 or QTY065 relative to mice treated with vehicle in B16F10-OVA (n=6 for vehicle, n=8 for DQS964, QTY065) or MC38 tumors (n=9). [Table 40]

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Claims

1. i. an anti-PD1 antigen-binding domain; an antibody heavy chain variable domain polypeptide (PD1-VH), and - antibody light chain variable domain polypeptide (PD1-VL) the anti-PD1 antigen-binding domain comprising: ii. an anti-IL-2 antigen binding domain; - from N' to C' terminus the following domains: first framework region (IL2VH １ ), the first complementarity determining region (IL2HCDR1), the second framework region (IL2VH ２ ), the second complementarity determining region (IL2HCDR2), the third framework region (IL2VH ３ ), the third complementarity determining region (IL2HCDR3), and the fourth framework region (IL2VH ４ an antibody heavy chain variable domain polypeptide (IL2-VH) having the following structure: - from N' to C' terminus the following domains: first framework region (IL2VL １ ), the first complementarity determining region (IL2LCDR1), the second framework region (IL2VL ２ ), the second complementarity determining region (IL2LCDR2), the third framework region (IL2VL ３ ), the third complementarity determining region (IL2LCDR3), and the fourth framework region (IL2VL ４ ), an antibody light chain variable domain polypeptide (IL2-VL) having the anti-IL-2 antigen binding domain comprising: iii. IL-2 polypeptide; 1. An immunoconjugate comprising: wherein the IL-2 polypeptide is covalently linked to an IL2-VL or an IL2-VH to provide a single contiguous recombinant polypeptide; The immunoconjugate, characterized in that the binding of pembrolizumab or nivolumab to PD-1+ cells does not significantly inhibit the binding of the immunoconjugate to said cells.

2. - PD1-VH comprises an HCDR1 having the sequence GFTFSINAMT (SEQ ID NO:118), an HCDR2 having the sequence TISGSGFSTYYADSLKGR (SEQ ID NO:119), and an HCDR3 having the sequence EVYGDY (SEQ ID NO:120); and - PD1-VL has the sequence SGX １ SSNIGSX ２ X ３ VF (SEQ ID NO: 121), LCDR1 having the sequence SNNQRPS (SEQ ID NO: 122), and LCDR3 having the sequence AAWDDSLSIWVF (SEQ ID NO: 123); Here, X １ is N, S, Q, or A; and X ２ X ３ The immunoconjugate of claim 1 , wherein is NS, QS, SS, or NA.

3. PD1-VL: a. SGASSNIGSQSVF (underlined in bold) (SEQ ID NO: 124); b. SGASSNIGSSSVF (underlined in bold) (SEQ ID NO: 125); or c. SGASSNIGSNAVF (underlined in bold) (SEQ ID NO: 126) The immunoconjugate of claim 2, comprising an LCDR1 having a sequence selected from the group consisting of:

4. a. the PD1-VH comprises or consists of a polypeptide having the sequence of SEQ ID NO: 085, and the PD1-VL comprises or consists of a polypeptide having the sequence of SEQ ID NO: 086; or b. the PD1-VH comprises or consists of a polypeptide having the sequence of SEQ ID NO: 085, and the PD1-VL comprises or consists of a polypeptide having the sequence of SEQ ID NO: 087; or c. The immunoconjugate of claim 2 or 3, wherein the PD1-VH comprises or consists of a polypeptide having the sequence of SEQ ID NO: 085 (VH) and the PD1-VL comprises or consists of a polypeptide having the sequence of SEQ ID NO:

91.

5. - PD1-VH comprises or consists of a polypeptide at least (≧) 95% or more, 98% or more, 99% or more similar to SEQ ID NO:085, and PD1-VL comprises or consists of a polypeptide at least 95% or more, 98% or more, 99% or more similar to SEQ ID NO:086; - PD1-VH comprises or consists of a polypeptide greater than 95%, greater than 98%, greater than 99% similar to SEQ ID NO:085, and PD1-VL comprises or consists of a polypeptide greater than 95%, greater than 98%, greater than 99% similar to SEQ ID NO:087; - PD1-VH comprises or consists of a polypeptide that is 95% or more, 98% or more, 99% or more similar to SEQ ID NO:085, and PD1-VL comprises or consists of a polypeptide that is 95% or more, 98% or more, 99% or more similar to SEQ ID NO:091; and The affinity constant (K Ｄ ) is 1.0 x 10 as measured by the protocol provided in Example 2. －９ ~1.5 x 10 －１１ The immunoconjugate according to any one of claims 1 to 4, wherein the immunoconjugate has a concentration in the range of 1000 mol / L.

6. a. PD1-VH comprises an HCDR1 having the sequence NFYIH (SEQ ID NO: 127), an HCDR2 having the sequence RIYPNYGITAYNQKFKD (underlined in bold) (SEQ ID NO: 128), and an HCDR3 having the sequence GYSYAMDY (SEQ ID NO: 129); and PD1-VL comprises an LCDR1 having the sequence SASQGISGDLN (SEQ ID NO: 130), an LCDR2 having the sequence HTSQRHS (underlined in bold) (SEQ ID NO: 131), and an LCDR3 having the sequence QGYSKDLLT (underlined in bold) (SEQ ID NO: 132); or b. The immunoconjugate of claim 1, wherein the PD1-VH comprises an HCDR1 having the sequence NFYIH (SEQ ID NO: 127), an HCDR2 having the sequence SIYPNYGITAYNQKFKD (underlined in bold) (SEQ ID NO: 133), and an HCDR3 having the sequence GYSYAMDY (SEQ ID NO: 129); and the PD1-VL comprises an LCDR1 having the sequence SASQGISGDLN (SEQ ID NO: 130), an LCDR2 having the sequence HTSQLHS (underlined in bold) (SEQ ID NO: 134), and an LCDR3 having the sequence QGYSKDLLT (underlined in bold) (SEQ ID NO: 132).

7. the IL-2 polypeptide is a circularly permuted IL-2 (IL2CP) polypeptide; 7. The immunoconjugate of any one of claims 1 to 6, wherein IL2CP is greater than (>) 98% similar to SEQ ID NO: 010 or SEQ ID NO: 011; and the N'-terminus of IL2CP and the C'-terminus of IL2CP are covalently linked, optionally by one or two peptide linkers, to amino acid residues within the IL2LCDR1 domain.

8. IL2-VH is (>) 98%, or more than 99% identical to SEQ ID NO: 043, and IL2-VL is (>) 98%, or more than 99% identical to SEQ ID NO: 042; and K of an antibody characterized by IL-2VH and IL-2VL against an IL-2 polypeptide having SEQ ID NO: 006 Ｄ The immunoconjugate of any one of claims 1 to 7, wherein the IgG antibody has a IgG binding affinity of 270 nM to 2 nM.

9. 9. The immunoconjugate of any one of claims 1 to 8, wherein the anti-IL-2 antigen binding domain comprises or consists of an IL2-VL having SEQ ID NO: 042 associated with an IL2-VH selected from SEQ ID NO: 046 or SEQ ID NO:

047.

10. The immunoconjugate of any one of claims 1 to 9, wherein the immunoconjugate comprises an IgG Fc portion.

11. The IgG Fc portion is characterized by the presence of one or more modifications to the constant region of the heavy chain to enhance correct heavy chain pairing, in particular: - Knob: S354C, T366W and hole: Y349C, T366S, L368A, Y407V; - Knob: T366Y, and hole: Y407T; - knob: Y349C, T366W and hole: S354C, T366S, L368A, Y407V; or - Knob: T366W, and holes: Y407A, T366S, L368A 11. The immunoconjugate of claim 10, characterized by a set of knob and hole modifications selected from:

12. The immunoconjugate is a first heterodimer comprising an anti-PD-1 binding domain as defined in any one of claims 1 to 6; and - a second heterodimer comprising an anti-IL2 binding domain as defined in any one of claims 1 or 8-9. The immunoconjugate according to any one of claims 1 to 11, which is a heterotetrameric IgG consisting of:

13. The immunoconjugate is a. SEQ ID NO:111, SEQ ID NO:095, SEQ ID NO:112, and SEQ ID NO:052; b. SEQ ID NO:117, SEQ ID NO:104, SEQ ID NO:112, and SEQ ID NO:091; c. SEQ ID NOs: 113, 106, and 114; d. SEQ ID NOs: 115, 109, and 116; e. SEQ ID NO:094, SEQ ID NO:095, SEQ ID NO:100, and SEQ ID NO:052; f. SEQ ID NO:097, SEQ ID NO:098, SEQ ID NO:100, and SEQ ID NO:052 13. The immunoconjugate according to any one of claims 1 to 12, comprising or consisting of a polypeptide having the sequence:

14. An isolated nucleic acid encoding the immunoconjugate of any one of claims 1 to 13, The isolated nucleic acid is contained in a mammalian expression vector under the control of a promoter operable in particular in mammalian cells.

15. - an immunoconjugate as defined in any one of claims 1 to 13, and an anti-PD-1 antagonist antibody selected from the list consisting of nivolumab, pembrolizumab, dostallimab, sintilimab, tislelizumab, cemiplimab, cetrelimab, southernlimab, in particular nivolumab or pembrolizumab; A combination medicine comprising:

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