Splitted interleukin-2 prodrugs and uses thereof

Abstract

The invention relates to recombinant IL2 variant polypeptides suitable to generate IL2 prodrugs and the prodrugs comprising said variant polypeptides that may be activated at a target site.

Description

[0001] P39704-WQ-1

[0002] SPLITTED INTERLEUKIN-2 PRODRUGS AND USES THEREOF

[0003] The current invention relates to target-activatable split IL-2 prodrugs, suitable, e.g., for cancer immunotherapy.

[0004] BACKGROUND OF THE INVENTION

[0005] Interleukin-2 (IL-2), also known as T cell growth factor (TCGF), is a 15.5 kDa globular glycoprotein playing a central role in lymphocyte generation, survival and homeostasis. It has a length of 133 amino acids and consists of four antiparallel, amphiphatic a-helices that form a quaternary structure indispensable of its function (Smith, Science 240, 1169-76 (1988); Bazan, Science 257, 410-413 (1992)). Sequences of IL-2 from different species are found under NCBI RefSeq Nos. NP000577 (human), NP032392 (mouse), NP446288 (rat) or NP517425 (chimpanzee).

[0006] IL-2 mediates its action by binding to IL-2 receptors (IL-2R), which consist of up to three individual subunits, the different associations thereof can produce receptor forms that differ in their affinity to IL-2. Association of the a (CD25), P (CD122), and y (yc, CD132) subunits results in a trimeric, high-affinity receptor for IL-2. Dimeric IL-2 receptor consisting of the P and y subunits is termed intermediate-affinity IL-2R. The a subunit forms the monomeric low affinity IL-2 receptor. Although the dimeric intermediate-affinity IL-2 receptor binds IL-2 with approximately 100-fold lower affinity than the trimeric high-affinity receptor, both the dimeric and the trimeric IL-2 receptor variants are able to transmit signal upon IL-2 binding (Minami et al., Annu. Rev. Immunol. 11 (1993) 245-268). Hence, the a-subunit, CD25, is not essential for IL- 2 signaling. It confers high-affinity binding to its receptor, whereas the P subunit, CD 122, and the y-subunit are crucial for signal transduction (Krieg et al., Proc. Natl. Acad. Sci. 107, 11906-11 (2010)). Trimeric IL-2 receptors including CD25 are expressed by (resting) CD4+ forkhead box P3 (FoxP3)+ regulatory T (Treg) cells. They are also transiently induced on conventional activated T cells, whereas in the resting state these cells express only dimeric IL-2 receptors. Treg cells consistently express the highest level of CD25 in vivo (Fontenot et al., Nature Immunol 6, 1142- 51 (2005)).

[0007] IL-2 is synthesized mainly by activated T-cells, in particular CD4+ helper T cells. It stimulates the proliferation and differentiation of T cells, induces the generation of cytotoxic T lymphocytes (CTLs) and the differentiation of peripheral blood lymphocytes to cytotoxic cells and lymphokine- activated killer (LAK) cells, promotes cytokine and cytolytic molecule expression by T cells, facilitates the proliferation and differentiation of B-cells and the synthesis of immunoglobulin by B-cells, and stimulates the generation, proliferation and activation of natural killer (NK) cells (reviewed e.g. in Waldmann, Nat Rev Immunol 6, 595-601 (2009); Olejniczak andKasprzak, Med. Sci. Monit. 14, RA179-89 (2008); Malek, Annu Rev Immunol 26, 453-79 (2008)). Antibody-cytokine fusions can be used to deliver cytokines directly to tumors or immune cells and are in development for use in cancer immunotherapy [1,2]. In particular, antibody-fusions with IL- 2 have shown promise in stimulating T-cells and NK cells to recognize and kill cancer cells, with T-cell-targeted cis-activation approaches such as an anti-PD-1 antibody-IL2v conjugate showing great potential [2,3],

[0008] However, on-target, off-tumor effects can lead to systemic toxicity, and binding of targeted IL-2 to IL-2 receptors on peripheral cells negatively affects safety, pharmacokinetics and tumor targeting.

[0009] WO 2022 / 129313 discloses chain-exchange mediated reconstitution of a split type I cytokine, IL- 4, by fusing these split IL-4 counterparts to the C-termini of antibody-like molecules that undergo proximity-induced chain exchange and activate IL-4 functionality. The same document discloses activation of IL-2 by fusing an IL-2 and a blocking IL-2 receptor domain to the C-termini of antibody like molecules, whereby upon proximity induced chain exchange IL-2 functionality is restored.

[0010] Yet, there is a need for alternative and improved activatable prodrug molecules.

[0011] SUMMARY OF THE INVENTION

[0012] In one aspect the invention relates to a recombinant IL2 variant polypeptide (herein termed “IL2- v”), which is a splittable variant of human interleukin 2 (IL2), comprising the amino acid sequence of SEQ ID NO:34.

[0013] The invention also relates to splitted variants of the IL2-v polypeptide, comprising either helix A (these recombinant polypeptides are herein referred to as “IL2-v(A)”) or helices B, C and D these recombinant polypeptides are herein referred to as “IL2-v(BCD)”.

[0014] Thus, in another aspect the invention relates to a recombinant IL2 variant polypeptide (“IL2-v(A)”) that comprises SEQ ID NO: 92.

[0015] In another aspect the invention relates to a recombinant IL2-v(A) polypeptide comprising SEQ ID NO:92 with one amino acid substitution at position 11 of SEQ ID NO:92.

[0016] In another aspect the invention relates to a recombinant IL2-v(A) polypeptide comprising SEQ ID NO:92 with one or more amino acid substitutions, wherein the amino acid substitution is at position 16 and / or at position 20 of SEQ ID NO:92.

[0017] In another aspect the invention relates to a recombinant IL2-v(A) polypeptide comprising SEQ ID NO:92 with two or more amino acid substitutions, wherein the at least one of the amino acid substitutions is at position 16 and / or 20 of SEQ ID NO:92, and wherein at least one of the amino acid substitutions is at position 11, 14 and / or 25 of SEQ ID NO:92.

[0018] In certain embodiments the substitutions in SEQ ID NO:92 are selected from: a substitution at position 11 selected from QI IK or QI IE, a substitution at position 14 or L14A, a substitution at position 16 of H16L, a substitution at position 20 of D20T, and a substitution at position 25 of L25A.

[0019] In yet another aspect the invention relates to a recombinant IL2 variant polypeptide (“IL2- v(BCD)”) that comprises SEQ ID NO:93.

[0020] In another aspect the invention relates to a recombinant IL2-v(BCD) polypeptide comprising SEQ ID NO:93 with one amino acid substitution at position 102 of SEQ ID NO:93.

[0021] In certain embodiments the substitution at position 102 is T102D.

[0022] The invention also relates to an inactivated full length IL2-v polypeptide that comprises a destabilized interface between helices A and B, C and D. This polypeptide is herein referred to as “IL2-v(BCD-dA)”.

[0023] Thus, in another aspect the invention relates to a recombinant IL2 variant polypeptide (“IL2- v(BCD-dA)”) that comprises an inactivated splitted variant of human IL2 that comprises helix A of IL2 and a splitted variant of human IL2 that comprises helices B, C and D of IL2. The IL2- v(BCD-dA) polypeptide comprises a) an amino acid sequence of SEQ ID NO:92 with two or more amino acid substitutions, wherein the at least one of the amino acid substitutions is at position 16 and / or 20 of SEQ ID NO:92, and wherein at least one of the amino acid substitutions is at position 11, 14 and / or 25 of SEQ ID NO: 92, and b) an amino acid sequence of SEQ ID NO:93 with one amino acid substitution at position 102 of SEQ ID NO: 93.

[0024] The splited IL2 variants become functional upon association.

[0025] Thus, the invention further relates to a pharmaceutical composition comprising a set of recombinant polypeptides, comprising: a) the recombinant IL2-v(A) polypeptide of the invention, and b) the recombinant IL2-v(BCD-dA) polypeptide of the invention. The invention also relates to a composition comprising a first and a second precursor protein, wherein each precursor protein comprises an antibody binding site and an Fc domain, wherein the first precursor protein comprises the recombinant IL2-v(A) polypeptide of the invention, and wherein the second precursor protein comprises the the recombinant IL2-v(BCD-dA) polypeptide of the invention.

[0026] In certain embodiments the recombinant IL2-v(A) polypeptide is fused to the C-terminus of the Fc domain of the first precursor protein and the recombinant IL2-v(BCD-dA) polypeptide is fused to the C-terminus of the Fc domain of the second precursor protein.

[0027] In certain embodiments the recombinant IL2-v(A) polypeptide is fused via a peptidic linker to the antibody binding site or to the Fc domain of the first precursor protein, and wherein the recombinant IL2-v(BCD-dA) polypeptide is fused via a peptidic linker to the antibody binding site or to the Fc domain of the second precursor protein.

[0028] In certain embodiments the antibody binding sites of the first and the second precursor protein bind to a tumor associated antigen.

[0029] In certain embodiments the antibody binding sites of the first and the second precursor protein are Fab fragments.

[0030] The invention reports a pair of split IL-2 prodrugs, one of which is complemented with an inactive IL-2 dummy chain. These two IL-2 prodrugs are inactive, and can be fused to antibody-derived targeting molecules. The prodrugs bind to and subsequently reconstitute active IL-2 on target cells.

[0031] Using reporter cell lines and primary human T-cells it has been shown that pre-assembled split IL- 2v (without additional inactivation) retains activity and that inactivated IL-2v prodrugs can be generated by splitting IL-2v. These prodrugs become re-activated by a chain-exchange-based mechanism upon accumulation on target cells. Without being bound by this theory, it is assumed that this can trigger IL-2 receptor activation on the same cell (cis-activation of IL-2v on targetpositive cells such as activated T-cells), and can also signal in trans on neighboring cells (targeting tumor-associated antigens and activating T- and NK cells in close spatial proximity). For both settings, it was shown that IL-2v prodrug conversion occurs in an efficient manner with very limited non-specific activation. Therefore, split IL-2 prodrugs are a promising modality for safer IL-2 -based therapies.

[0032] DESCRIPTION OF THE FIGURES

[0033] Figure 1 IL-2v structure in complex with IL-2RPy [4] (PDB ID 5M5E). Helices A and C form the interaction surface with IL-2RP, while helix D interacts with IL-2Ry. Dashed lines indicate the 3+1 split interface which separates helix A from helices BCD.

[0034] Figure 2 Illustration of the 3+1 split IL-2v fragments consisting of IL-2v (A) and IL-2v (BCD), modified from PDB ID 5M5E, and their reconstitution into full IL-2v.

[0035] Figure 3 Illustration of the fusion protein PDl-IL2v, containing intact, non-split IL-2v fused to the C-terminus of a PD1 -targeting antibody via a flexible peptidic Gly- Ser linker.

[0036] Figure 4 Pre-formed split IL-2v was produced by fusion of the two split IL-2v fragments to the C-termini of heavy chains of a PD1 -targeting antibody. The fragments were fused via flexible peptidic Gly-Ser linkers.

[0037] Figure 5 IL-2 activity of preformed split IL-2v and full-length, non-split IL-2v fusion proteins was monitored with a HEK -Blue-IL-2 reporter assay using HEK-Blue- IL-2 cells engineered to overexpress human PD-1.

[0038] Figure 6 IL-2 activity of preformed split IL-2v and full-length, non-split IL-2v fusion proteins was monitored with a HEK-Blue-IL-2 reporter assay using standard HEK-Blue-IL-2 cells.

[0039] Figure 7 Fusion of an interleukin-2 fragment via a flexible peptidic Gly-Ser linker to the C-terminus of antibody -like molecules containing only one Fab (one-armed antibody).

[0040] Figure 8 Scheme of monoparatopic targeting strategy, involving binding of split IL-2v prodrugs to multiple copies of an antigen using the same binder.

[0041] Figure 9 Scheme of biparatopic targeting strategy, involving binding of split IL-2v prodrugs to two different epitopes on the same antigen.

[0042] Figure 10 Strategy to improve solubility and biophysical properties of split IL-2v (BCD)- fusion proteins. An inactive helix A from IL-2v, termed the “dummy-A” helix, is fused to the C-terminus of IL-2v (BCD) with a flexible peptidic Gly-Ser linker. The dummy-A helix acts as a chaperone and improves the solubility and biophysical properties of IL-2v (BCD), while the inactivating mutations abrogate IL-2 receptor heterodimerization.

[0043] Figure 11 The indicated mutations were introduced into helix A of IL-2v in the context of cytokine-PACE split IL-2v educts, with the methods as in

[0028] ,

[0044] Figure 12 Influence of indicated helix A mutations from molecules illustrated in Figure 11 on IL-2v activity, assessed by a HEK-Blue IL-2 reporter assay. rIL2: recombinant IL-2 (Miltenyi Biotec, IL-2 IS, premium grade). The H16L D20T mutations were selected for further characterization.

[0045] Figure 13 Trans-activation of PD-1 -targeted split IL-2v prodrugs using engineered CHO cells that overexpress PD-1. Detection of IL-2v activity was performed using cocultured HEK-Blue-IL-2 reporter cells that express secreted embryonic alkaline phosphatase (SEAP) upon engagement of the IL-2 receptor. Left / bottom: illustration of monoparatopic targeting principle, with both prodrugs containing the same PD-1 binder. Middle: illustration of biparatopic targeting principle using two different PD-1 binders that target different epitopes on PD-1. Right / top: split IL-2v activity in the absence of targeting was assessed by adding split IL-2v prodrugs directly to HEK-Blue IL-2 cells.

[0046] Figure 14 Efficient conversion of prodrugs into functional IL-2v can be observed with monoparatopic and biparatopic PD-1 -targeting split IL-2v (A) + IL-2v (BCD- dA) combinations in the presence of PD-1 CHO cells (targeted condition). Split IL-2v combinations in non-targeted conditions (in the absence of PD-1 CHO cells) only lead to IL-2v activity at the highest concentration tested. PD-1 0376 and PD-1 1040 refer to two different PD-1 binders, targeting distinct epitopes on PD-1. Individual split IL-2v prodrugs were tested in the targeted condition (in the presence of CHO-PD1 cells) and showed no activity in isolation.

[0047] Figure 15 Trans-activation concept of HER2 -targeted split IL-2v prodrugs on HER2- overexpressing SK-BR-3 cells. The experimental setup and detection of IL-2v activity is the same as in Figure 13.

[0048] Figure 16 Efficient conversion of split IL-2v prodrugs into functional IL-2v is observed with prodrugs targeting HER2 in the presence of SK-BR-3 cells (targeted condition), while non-targeted conditions (in the absence of SK-BR-3 cells) show activity only at the highest concentration tested. Individual split IL-2v prodrugs were tested in the targeted condition (in the presence of SK-BR-3 cells) and show no activity in isolation.

[0049] Figure 17 Format parameters that influence the prodrug conversion and activity of targeted split-IL-2v modules. Linkers that were varied in format-optimization experiments are indicated (red).

[0050] Figure 18 Trans-activation HEK-Blue IL-2 assay as in Figure 13, testing N-terminal and C-terminal PD-l-targeted split IL-2v prodrug formats. Left / bottom: Assay using CHO cells engineered to overexpress human PD-1. Right / top: assay using standard CHO cells, which lack detectable PD-1 expression

[0037] ,

[0051] Figure 19 Cis-activation concept and detection of IL-2 activity in HEK-Blue-IL-2 reporter cells that do not express PD-1 (right / top) or HEK-Blue IL-2 cells engineered to overexpress PD-1 (left / bottom).

[0052] Figure 20 Cis-activation of PD-l-targeted C-terminal split IL-2v prodrugs into functional entities can be observed in a dose-dependent manner (left / bottom). Activation of the IL-2 receptor is PD-1 specific, as almost no signal is observed using PD-1- negative cells (right / top). Figure 21 Cis-activation was monitored as in Figure 20 using PD-l-targeted split IL-2v prodrugs with engineered IL-2 interface mutations (see Table 3).

[0053] Figure 22 Cis-activation of IL-2 signaling on activated human CD4+ T-cells measured by quantification of STAT5 phosphorylation using FACS. In order to assess PD-1 specificity, half of the T-cell population was treated with an excess of PD-1 blocking antibody. The activity in that population corresponds to non- specific / non-targeted IL-2 signaling.

[0054] Figure 23 Overview of Granzyme B proliferation experiment. CTV: CellTrace Violet proliferation stain. DC: dendritic cells.

[0055] Figure 24 The percentage of Granzyme B-positive proliferating CD4+ T-cells after treatment with split IL-2v prodrugs or control constructs was quantified with FACS

[0056] Figure 25 Results of an in vivo efficacy experiment with PD-1 targeting split IL-2v molecules (combination of XVV194 + XVV173, see Table 3) and a human-PD- 1 -targeting murinized monoclonal antibody as single agents. The MC38 colorectal cell line was injected subcutaneously in Black 6-huPD-l transgenic mice to study tumor growth inhibition in a subcutaneous model. Tumor size was measured using a caliper. Therapy started when tumors reached 100 mm3. The dose injected per mouse was 3 mg / kg for all constructs, given once a week. The treatment lasted 3 weeks. The PD-1 -targeting split IL-2v molecule combination mediated superior efficacy in terms of tumor growth inhibition compared to Vehicle and huPD-1 murinized Mab single agent groups.

[0057] Figure 26 Expression of PD-1 by HEK-Blue IL-2 cells and engineered PD-1 HEK-Blue IL-2 cells was measured by flow cytometry using a phycoerythrin (PE)-labeled anti-PD-1 antibody. The data shows that HEK-Blue IL-2 cells do not express detectable levels of PD-1.

[0058] Figure 27 Expression of PD-1 by CHO-PD1 cells was measured by flow cytometry using a phycoerythrin (PE)-labeled anti-PD-1 antibody. For comparison, three cell lines derived from immortalized human T-cells engineered to express lower levels of PD-1 were stained with the same PE-labeled anti-PD-1 antibody. The data demonstrate that CHO-PD1 cells express a very high level of PD-1.

[0059] Figure 28 Expression of HER2 by HEK-Blue IL-2 cells and SK-BR-3 cells was measured by flow cytometry using a phycoerythrin (PE)-labeled anti-HER2 antibody. The data shows that HEK-Blue IL-2 cells express a very low level of HER2 compared to SK-BR-3 cells. DETAILED DESCRIPTION OF THE INVENTION

[0060] 1. DEFINITIONS

[0061] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art. Generally, nomenclatures used in connection with, and techniques of biochemistry, enzymology, molecular, and cellular biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art.

[0062] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth. As well, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein. It is also to be noted that the terms "comprising", "including", and "having" can be used interchangeably.

[0063] The term “about” denotes a range of + / - 20 % of the thereafter following numerical value. In one embodiment the term “about” denotes a range of + / - 10 % of the thereafter following numerical value. In one embodiment the term “about” denotes a range of + / - 5 % of the thereafter following numerical value.

[0064] The terms “comprise(s)”, “include(s)”, “having”, “has”, “can”, “contain(s)” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms or words that do not preclude the possibility of additional acts or structures. The term “comprising” also encompasses the term “consisting of’. The present disclosure also contemplates other embodiments “comprising”, “consisting of’ and “consisting essentially of’ the embodiments or elements presented herein, whether explicitly set forth or not.

[0065] The term "antibody" is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigenbinding activity.

[0066] The terms “binding site” or “antigen-binding site” as used herein denotes the region or regions of an antigen binding region to which the antigen actually binds. In case the antigen binding region is an antibody, the antigen-binding site includes antibody heavy chain variable domains (VH) and / or antibody light chain variable domains (VL), or pairs of VH / VL. Antigen-binding sites derived from antibodies that specifically bind to a target antigen can be derived a) from known antibodies specifically binding to the antigen or b) from new antibodies or antibody fragments obtained by de novo immunization methods using inter alia either the antigen protein or nucleic acid or fragments thereof or by phage display methods.

[0067] When being derived from an antibody, an antigen-binding site of an antibody according to the invention can contain six complementarity determining regions (CDRs) which contribute in varying degrees to the affinity of the binding site for antigen. There are three heavy chain variable domain CDRs (CDRH1, CDRH2 and CDRH3) and three light chain variable domain CDRs (CDRL1, CDRL2 and CDRL3). The extent of CDR and framework regions (FRs) is determined by comparison to a compiled database of amino acid sequences in which those regions have been defined according to variability among the sequences. Also included within the scope of the invention are functional antigen binding sites comprised of fewer CDRs (i.e., where binding specificity is determined by three, four or five CDRs). For example, less than a complete set of 6 CDRs may be sufficient for binding.

[0068] The term “valent” as used herein denotes the presence of a specified number of binding sites in an antibody molecule. A natural antibody for example has two binding sites and is bivalent. As such, the term “trivalenf ’ denotes the presence of three binding sites in an antibody molecule.

[0069] An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab, Fab-SH, F(ab)2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv, scFab); and multispecific antibodies formed from antibody fragments.

[0070] The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementary determining regions (CDRs). (See, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991). The term “constant domains” or “constant region” as used within the current application denotes the sum of the domains of an antibody other than the variable region. The constant region is not directly involved in binding of an antigen, but exhibits various effector functions.

[0071] Depending on the amino acid sequence of the constant region of their heavy chains, antibodies are divided in the “classes”: IgA, IgD, IgE, IgG and IgM, and several of these may are further divided into subclasses, such as IgGl, IgG2, IgG3, and IgG4, IgAl and IgA2. The heavy chain constant regions that correspond to the different classes of antibodies are called a, 5, 8, y and p, respectively. The light chain constant regions (CL) which can be found in all five antibody classes are called K (kappa) and X (lambda).

[0072] In wild type antibodies, the “hinge region” is a flexible amino acid stretch in the central part of the heavy chains of the IgG and IgA immunoglobulin classes, which links the two heavy chains by disulfide bonds, i.e. “interchain disulfide bonds” as they are formed between the two heavy chains. The hinge region of human IgGl is generally defined as stretching from about Glu216, or about Cys226, to about Pro230 of human IgGl (Burton, Molec. Immunol.22: 161-206 (1985)). By deleting cysteine residues in the hinge region or by substituting cysteine residues in the hinge region by other amino acids, such as serine, disulfide bond formation in the hinge region is avoided.

[0073] The “light chains” of antibodies from any vertebrate species can be assigned to one of two distinct types, called kappa (K) and lambda (X), based on the amino acid sequences of their constant domains. A wild type light chain typically contains two immunoglobulin domains, usually one variable domain (VL) that is important for binding to an antigen and a constant domain (CL).

[0074] Several different types of “heavy chains” exist that define the class or isotype of an antibody. A wild type heavy chain contains a series of immunoglobulin domains, usually with one variable domain (VH) that is important for binding antigen and several constant domains (CHI, CH2, CH3, etc.).

[0075] The term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. For example, the Fc-region comprised in therapeutics can bind to complement component Clq and Fc-gamma receptors (FcyR) to elicit cellular effector responses such as phagocytosis, cytokine release, and production of reactive oxygen species. See, e.g., X. Wang, et al., Protein & Cell 9: 63- 73 (2018); S.B. Mkaddem et al., Frontiers in Immunology, available at https: / / doi.org / 10.3389 / fimmu.2019.00811 (2019).

[0076] The “CH2 domain” of a human IgG Fc region usually extends from an amino acid residue at about position 231 to an amino acid residue at about position 340. The multispecific antibody is devoid of a CH2 domain. By “devoid of a CH2 domain” is meant that the antibodies according to the invention do not comprise a CH2 domain.

[0077] The “CH3 domain” comprises the stretch of residues C-terminal to a CH2 domain in an Fc region (i.e. from an amino acid residue at about position 341 to an amino acid residue at about position 447 of an IgG). The “CH3 domains” herein are variant CH3 domains, wherein the amino acid sequence of the natural CH3 domain was subjected to at least one distinct amino acid substitution (i.e. modification of the amino acid sequence of the CH3 domain) in order to promote heterodimerization of the two CH3 domains facing each other within the multispecific antibody.

[0078] The term “heavy chain Fab fragment” refers to a polypeptide comprising at least a heavy chain variable domain, with or without a leader sequence, and at least a portion of a heavy chain constant region 1 (CHI).

[0079] Amino acid “substitutions” or “replacements” or “mutations” (all terms are herein used interchangeably) within the polypeptide chains are prepared by introducing appropriate nucleotide changes into the antibody DNA, or by nucleotide synthesis. Such modifications can be performed, however, only in a very limited range. For example, the modifications do not alter the above mentioned antibody characteristics such as the IgG isotype and antigen binding, but may further improve the yield of the recombinant production, protein stability or facilitate the purification. In certain embodiments, antibody variants having one or more conservative amino acid substitutions are provided. A “double mutation” as referred herein means that both of the indicated amino acid substitutions are present in the respective polypeptide chain.

[0080] The term “amino acid” as used herein denotes an organic molecule possessing an amino moiety located at a-position to a carboxylic group. Examples of amino acids include: arginine, glycine, ornithine, lysine, histidine, glutamic acid, asparagic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophane, methionine, serine, proline. The amino acid employed is optionally in each case the L-form. The term “positively charged” or “negatively charged” amino acid refers to the amino acid side-chain charge at pH 7.4. Amino acids may be grouped according to common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He, Trp, Tyr, Phe;

[0081] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

[0082] (3) acidic or negatively charged: Asp, Glu;

[0083] (4) basic or positively charged: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro.

[0084] Table - Amino acids with specific properties

[0085] A “polypeptide chain exchange” between two proteins occurs, when two polypeptide chains from a first precursor protein dissociate and two polypeptide chains from a second precursor protein dissociate and a polypeptide chain derived from the first precursor protein pairs with a polypeptide chain derived from the second precursor protein. In consequence, a “product” protein is formed comprising a polypeptide chain from the first precursor polypeptide and a polypeptide chain from the second precursor polypeptide. Both polypeptide chains are associated via their dimerization domains in the product protein.

[0086] The term “nucleic acid” or “polynucleotide” includes any compound and / or substance that comprises a polymer of nucleotides. Each nucleotide is composed of a base, specifically a purine- or pyrimidine base (i.e. cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e. deoxyribose or ribose), and a phosphate group. Often, the nucleic acid molecule is described by the sequence of bases, whereby said bases represent the primary structure (linear structure) of a nucleic acid molecule. The sequence of bases is typically represented from 5’ to 3’. Herein, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA) including e.g., complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers comprising two or more of these molecules. The nucleic acid molecule may be linear or circular. In addition, the term nucleic acid molecule includes both, sense and antisense strands, as well as single stranded and double stranded forms. Moreover, the herein described nucleic acid molecule can contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugars or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules which are suitable as a vector for direct expression of an antibody of the invention in vitro and / or in vivo, e.g., in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and / or expression of the encoded molecule so that mRNA can be injected into a subject to generate the antibody in vivo (see e.g., Stadler et al, Nature Medicine 2017, published online 12 June 2017, doi: 10.1038 / nm.4356 or EP 2 101 823 Bl).

[0087] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment.

[0088] In more detail, the term "isolated" refers to material, which is substantially or essentially free from components that normally accompany the material as it is found in its native state. Thus, an “isolated plasmid” or “isolated plasmid DNA” does not contain materials normally associated with the plasmid or plasmid DNA in their in situ environment. For example, a nucleic acid or polynucleotide is said to be "isolated" when it is substantially separated from contaminant polynucleotides that correspond or are complementary to genes other than the target genes or that encode polypeptides other than the target gene product or fragments thereof. A skilled artisan can readily employ nucleic acid isolation procedures to obtain an isolated polynucleotide.

[0089] An “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g. cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0090] The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products.

[0091] The term “peptidic linker” or “peptide linker” as used herein denotes a peptide with amino acid sequences, which is in certain embodiments of synthetic origin. The peptidic linker is in certain embodiments a peptide with an amino acid sequence with a length of at least 30 amino acids, in one embodiment with a length of 32 to 50 amino acids. In one embodiment, the peptidic linker is a peptide with an amino acid sequence with a length of 32 to 40 amino acids. In one embodiment the peptidid linker is a glycine seriner linker. A “glycine serine linker” as referred to herein is a peptidic linker solely comprised og glycine and serine residues. In one embodiment the peptidic linker is (GxS)n with G = glycine, S = serine, (x = 3, n = 8, 9 or 10) or (x = 4 and n= 6, 7 or 8), in one embodiment with x = 4, n = 6 or 7, in one embodiment with x = 4, n = 7. In one preferred embodiment the peptidic linker is (G4S)6 (SEQ ID NO: 32) or (G4S)3G5 (SEQ ID NO: 33).

[0092] The term "pharmaceutical composition" refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the composition would be administered. A pharmaceutical composition of the present invention can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and / or mode of administration will vary depending upon the desired results. To administer an antibody according to the invention by certain routes of administration, it may be necessary to coat the antibody with, or co-administer the antibody with, a material to prevent its inactivation. For example, the heterodimeric polypeptide may be administered to a subject in an appropriate carrier, for example, liposomes, or a diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions.

[0093] A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

[0094] As used herein, the term “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In certain embodiments, an antibody according to the current invention is used to delay or prevent development of a disease or to slow the progression of a disease.

[0095] When a range of values is listed herein, it is intended to encompass the boundaries as well as each value and sub-range within that range. For example, “2 mg / kg to 6 mg / kg” is intended to encompass, for example, 2.0 mg / kg, 2.5 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 4.5 mg / kg, 5 mg / kg, 5.5 mg / kg, 6 mg / kg, 2.5 mg / kg to 3 mg / kg, 2.5 mg / kg to 4.5 mg / kg, 3 mg / kg to 4.5 mg / kg, 4.5 mg / kg to 6 mg / kg, 2.5 mg / kg to 4 mg / kg, and so forth.

[0096] An “effective amount” of an agent, e.g., a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0097] The terms “host cell”, “host cell line”, and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells”, which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0098] Human Interleukin-2 (IL2) as referred to herein is used with its standard meaning in the art known to be a type I cytokine and structurally comprising four helices: helix A, helix B, helix C and helix D. The amino acid sequence of (wild type) human IL2 is represented in SEQ ID NO:35.

[0099] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK PLEEVLNLAQSKNFHLRPRDLI SNINVIVLELKGSETTFMCEYADETAT IVEFLNRWI TFCQS I I STLT (SEQ ID NO: 35)

[0100] Human IL-2 consists of four alpha helices (denoted ABCD, Figure 1) connected in an “up-up- down-down” manner by long protruding loops [23,24], Binding studies have demonstrated that IL-2 likely binds to the hetero-dimeric receptor (IL2RPy) in a sequential fashion

[0025] , IL-2 first binds to IL-2RP with an intermediate affinity, and does not bind to IL2Ry in isolation. The IL- 2 / IL-2RP complex then binds to IL-2Ry with a high affinity (low-nM KD)

[0025] ,

[0101] A “recombinant IL2 variant polypeptide” refers to an amino acid sequence variant of human IL2 that comprises at least one of the typical helixes of human IL2. One variant of human IL2 that is reported herein is represented in SEQ ID NO:34, representing an amino acid sequence variant of human IL2 comprising all four helices (A to D) and being suitable for the generation of splitted versions thereof, particularly a first splitted polypeptide, which is herein also termed IL2-v(A), which comprises amino acids 1 to 31 of SEQ ID NO:34 and hence including helix A, and a second splitted polypeptide, which is herein also termed IL2-v(BCD), which comprises amino acids 32 to 133 of SEQ ID NO:34.

[0102] IL-2v:

[0103] APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFAMPKKATELKHLQCLEEELK PLEEVLNGAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQS I ISTLT (SEQ ID NO: 34)

[0104] IL2-v(A), without further amino acid substitutions:

[0105] APASSSTKKTQLQLEHLLLDLQMILNGINNY (SEQ ID NO:92)

[0106] IL2-v(BCD), without further amino acid substitutions:

[0107] KNPKLTRMLTAKFAMPKKATELKHLQCLEEELKPLEEVLNGAQSKNFHLRPRDLISNINVIVLE LKGSETTFMCEYADETATIVEFLNRWITFAQS I ISTLT (SEQ ID NO:93)

[0108] The recombinant IL2 variant polypeptides disclosed herein may comprise amino acid substitutions within the indicated amino acid sequence. The individual recombinant IL2 variant polypeptides may comprise up to 8 amino acid substitutions, in one embodiment up to 7 amino acid substitutions.

[0109] 2. CONCEPT OF THE INVENTION

[0110] The current invention is based, at least in part, on the finding that targeted split IL-2 receptor agonist prodrugs that become activated by chain-exchange / chain displacement and trigger IL-2- receptor-mediated signaling on or in proximity to target cells can be obtained by

[0111] (i) generating pairs of split IL-2 fusions with antibody derivatives, whose binding sites specifically bind to target cells,

[0112] (ii) modifying the two parts of these pairs in such a manner that each entity by itself does not trigger IL-2 receptor signaling,

[0113] (iii) using binding sites that target cell-surface antigens that lead to accumulation of prodrug pairs on target cells (iv) implementing into cell-surface-accumulated prodrug pairs means (sequences, structures) that enable chain-exchange between the two different entities of complementary prodrug pairs upon encountering each other on cell surfaces, but with low exchange propensities upon chance encounters at low concentrations or on non-target cells,

[0114] (v) selecting prodrug formats that favor chain-exchange-mediated conversion into functional IL-2v and thereby IL-2 receptor signaling on the same cell on which the prodrugs accumulate (cis-activation),

[0115] (vi) selecting prodrug formats that favor chain-exchange-mediated conversion into functional IL-2v on cells on which the prodrugs accumulate, and IL-2-receptor activation on IL-2 responsive cells in proximity to the targeted cells (trans-activation).

[0116] It has been shown with the provided experimental data that this prodrug approach enables selective reconstitution of IL-2 activity at or in close proximity to target cells. This ameliorates or prevents undesired effects such as toxicity, immunogenic propensity, and / or clearance / sink effects that are otherwise associated with systemic administration of constitutively active IL-2 and its derivatives.

[0117] ***

[0118] In addition to the various embodiments depicted and claimed, the disclosed subject matter is also directed to other embodiments having other combinations of the features disclosed or claimed herein. As such, the particular features presented herein can be combined with each other in other manners within the scope of the disclosed subject matter such that the disclosed subject matter includes any suitable combination of the features disclosed herein. The foregoing description of specific embodiments of the disclosed subject matter has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the disclosed subject matter to those embodiments disclosed.

[0119] ***

[0120] Herein is reported a pair of split IL-2 prodrugs, one of which is complemented with an inactive IL-2 dummy chain. These two IL-2 prodrugs are inactive, and can be fused to antibody-derived targeting molecules. The prodrugs bind to and subsequently reconstitute active IL-2 on target cells.

[0121] Useful methods and techniques for carrying out the current invention are described in e.g. Ausubel, F.M. (ed.), Current Protocols in Molecular Biology, Volumes I to III (1997); Glover, N.D., and Hames, B.D., ed., DNA Cloning: A Practical Approach, Volumes I and II (1985), Oxford University Press; Freshney, R.I. (ed.), Animal Cell Culture - a practical approach, IRL Press Limited (1986); Watson, J.D., et al., Recombinant DNA, Second Edition, CHSL Press (1992); Winnacker, E.L., From Genes to Clones; N.Y., VCH Publishers (1987); Celis, J., ed., Cell Biology, Second Edition, Academic Press (1998); Freshney, R.I., Culture of Animal Cells: A Manual of Basic Technique, second edition, Alan R. Liss, Inc., N.Y. (1987).

[0122] The use of recombinant DNA technology enables the generation of derivatives of a nucleic acid. Such derivatives can, for example, be modified in individual or several nucleotide positions by substitution, alteration, exchange, deletion or insertion. The modification or derivatization can, for example, be carried out by means of site directed mutagenesis. Such modifications can easily be carried out by a person skilled in the art (see e.g. Sambrook, J., et al., Molecular Cloning: A laboratory manual (1999) Cold Spring Harbor Laboratory Press, New York, USA; Hames, B.D., and Higgins, S.G., Nucleic acid hybridization - a practical approach (1985) IRL Press, Oxford, England).

[0123] General information regarding the nucleotide sequences of human immunoglobulins light and heavy chains is given in: Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0124] As used herein, the amino acid positions of all constant regions and domains of the heavy and light chain are numbered according to the Kabat numbering system described in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) and is referred to as “numbering according to Kabat” herein. Specifically, the Kabat numbering system (see pages 647-660) of Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) is used for the light chain constant domain CL of kappa and lambda isotype, and the Kabat EU index numbering system (see pages 661-723) is used for the constant heavy chain domains (CHI, Hinge, CH2 and CH3, which is herein further clarified by referring to “numbering according to Kabat EU index” in this case).

[0125] Likewise the hypervariable regions (HVRs) in the heavy and light chain variable domains of nonhuman and human antibodies are determined following Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). Accordingly, the HVRs of the antibodies according to the current invention have been determined according to Kabat and, thus, are denoted as “according to Kabat”. 3. EMBODIMENTS OF THE INVENTION

[0126] Splittable variant of human IL2 and variants thereof

[0127] In one aspect the invention relates to a recombinant IL2 variant polypeptide (herein termed “IL2- v”), which is a splittable variant of human interleukin 2 (IL2), comprising the amino acid sequence of SEQ ID NO:34. IL2v as disclosed herein differs from wild type human IL2 by 5 amino acid substitutions and has been shown to be suitable for the generation of splitted and inactived versions that may be combined to restore IL2 activity.

[0128] The IL2-v polypeptide can be splitted into two polypeptides, one comprising the helix A including amino acids 1 to 31 of SEQ ID NO:34 and another one comprising helices B, C and D comprising amino acids 32 to 133 of SEQ ID NO:34. The helix A comprising polypeptide is IL2-v(A) as referred to herein and the helices B, C and D comprising polypeptide is IL2-v(BCD) as referred to herein.

[0129] The amino acid sequence of the amino acids 32 to 133 of SEQ ID NO:34 are also represented in SEQ ID NO: 93.

[0130] Thus, in another aspect the invention relates to a recombinant IL2 variant polypeptide (“IL2- v(BCD)”) that comprises SEQ ID NO:93.

[0131] The amino acid sequence of the amino acids 1 to 31 of SEQ ID NO:34 are also represented in SEQ ID NO:92.

[0132] Thus, in another aspect the invention relates to a recombinant IL2 variant polypeptide (“IL2-v(A)”) that comprises SEQ ID NO: 92.

[0133] The invention also relates to variants of the IL2-v(A) polypeptide.

[0134] Inactivated IL2-v(A) polypeptide

[0135] The inventors have shown that IL2-v(A) may be inactivated by including amino acid substitutions at positions 16 and 20 of SEQ ID NO:92. Particularly, the inventors have shown that substitutions of H16L, D20T, H16L D20T or H16Y D20T were potent to inactivate the IL2-v(A) polypeptide.

[0136] Hence, in one embodiment, an IL2-v(A) polypeptide comprises the amino acid sequence of SEQ ID NO:92 with one or more amino acid substitutions, wherein the amino acid substitution is at position 16 and / or at position 20 of SEQ ID NO:92. In one embodiment the amino acid substitution at position 16 is L or Y, preferably L. In one embodiment the amino acid substitution at position 20 is T. In one embodiment the IL2-v(A) polypeptide comprises SEQ ID NO:92 with two amino acid substitutions: H16L or H16Y and D20T. In one embodiment the IL2-v(A) polypeptide comprises SEQ ID NO:92 with two amino acid substitutions: H16L and D20T.

[0137] Amino acid sequences of the indicated inactivated IL2-v(A) polypeptide are shown in SEQ ID NO:94 to 97:

[0138] Inactivated IL-2v( A) H16L

[0139] APAS S S TKKTQLQLELLLLDLQMI LNGINNY (SEQ ID NO:94)

[0140] Inactivated IL-2v(A) D20T

[0141] APAS S S TKKTQLQLEHLLLTLQMI LNGINNY (SEQ ID NO:95)

[0142] Inactivated IL-2v(A) H16L D20T

[0143] APAS S S TKKTQLQLELLLLTLQMI LNGINNY (SEQ ID NO:96)

[0144] Inactivated IL-2v(A) Hl 6Y D20T

[0145] APAS S S TKKTQLQLEYLLLTLQMI LNGINNY (SEQ ID NO:97)

[0146] In one embodiment, an IL2-v(A) polypeptide comprises an amino acid sequence selected from SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96 and SEQ ID NO:97. In one embodiment, an IL2- v(A) polypeptide comprises an amino acid sequence of SEQ ID NO:96.

[0147] The inventors have shown that the splitted variants of IL2-v, i.e. IL2-v(A) and IL2-v(BCD), may be combined to restore IL2-activity.

[0148] The splitted IL2 variants are suitable to generate inactive precursor proteins that may restore IL2 activity only at the target site.

[0149] Expression yield of IL2-v(BCD) may be improved by fusing IL2-v(BCD) to an inactivated IL2- v(A) polypeptide. This way, two precursor proteins can be generated that comprise inactive splitted IL2 variants that restore activity at a target site.

[0150] The inventors have shown that including destabilizing amino acid substitutions in a IL2-v(BCD) polypeptide that comprises an inactivated IL2-v(A) polypeptide may further improve restoring restoring of IL2 activity.

[0151] Destabilized IL2-v(A) polypeptide

[0152] Thus, in another aspect the invention relates to a recombinant IL2-v(A) polypeptide comprising SEQ ID NO:92 with one amino acid substitution at position 11 of SEQ ID NO:92. In another aspect the invention relates to a recombinant IL2-v(A) polypeptide comprising SEQ ID NO:92 with at least one one amino acid substitution at position 11, 14 and / or 25 of SEQ ID NO:92. In one embodiment, the IL2-v(A) polypeptide comprises amino acid substitutions at positions 14 and 25 of SEQ ID NO:92. In one embodiment, the IL2-v(A) polypeptide comprises amino acid substitutions at positions 11, 14 and 25 of SEQ ID NO:92.

[0153] In one embodiment, the substitution at position 11 is selected from QI IK or QI IE. In one embodiment the substitution at position 11 is Q 1 IK. In one embodiment the substitution at position 11 is QUE.

[0154] In one embodiment, the substitution at position 14 is L14A.

[0155] In one embodiment, the substitution at position 25 is L25A.

[0156] In another aspect the invention relates to a recombinant IL2-v(A) polypeptide comprising SEQ ID NO:92 with two or more amino acid substitutions, wherein the at least one of the amino acid substitutions is at position 16 and / or 20 of SEQ ID NO:92, and wherein at least one of the amino acid substitutions is at position 11, 14 and / or 25 of SEQ ID NO:92.

[0157] In one embodiment the IL2-v(A) polypeptide comprises SEQ ID NO:92 with substitions at position 11, 16 and 20.

[0158] In one embodiment the IL2-v(A) polypeptide comprises SEQ ID NO:92 with substitions at position 14, 25, 16 and 20.

[0159] In one embodiment the IL2-v(A) polypeptide comprises SEQ ID NO:92 with substitions at position 11, 14, 16, 20 and 25.

[0160] Amino acid sequences of the indicated inactivated IL2-v(A) polypeptide are shown in SEQ ID NO:98 to 101 :

[0161] Destabilized IL-2v(A) QI IK

[0162] APAS S S TKKTKLQLEHLLLDLQMI LNGINNY (SEQ ID NO:98)

[0163] Destabilized inactivated IL-2v(A) H16L D20T QI IE

[0164] APAS S S TKKTELQLELLLLTLQMI LNGINNY (SEQ ID NO:99)

[0165] Destabilized inactivated IL-2v(A) H16L D20T L14A L25A

[0166] APAS S S TKKTQLQAELLLLTLQMIANGINNY (SEQ ID NO:100)

[0167] Destabilized inactivated IL-2v(A) H16L D20T QI IE L14A L25A

[0168] APAS S S TKKTELQAELLLLTLQMIANGINNY (SEQ ID NO:101) In one embodiment, an IL2-v(A) polypeptide comprises an amino acid sequence selected from SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO: 100 and SEQ ID NO: 101. In one embodiment, an IL2-v(A) polypeptide comprises an amino acid sequence of SEQ ID NO:98. In one embodiment, an IL2-v(A) polypeptide comprises an amino acid sequence of SEQ ID NO:99. In one embodiment, an IL2-v(A) polypeptide comprises an amino acid sequence of SEQ ID NO: 100. In one embodiment, an IL2-v(A) polypeptide comprises an amino acid sequence of SEQ ID NO: 101.

[0169] Destabilized IL2-v(BCD) polypeptide and IL2-v(BCD-dA)

[0170] In another aspect the invention relates to a recombinant IL2-v(BCD) polypeptide comprising SEQ ID NO:93 with one amino acid substitution at position 102 of SEQ ID NO:93.

[0171] In certain embodiments the substitution at position 102 is T102D.

[0172] In the interest of clarity, it should be noted that the amino acid at position 102 of SEQ ID NO: 93 is the same as amino acid 133 of SEQ ID NO:34, as SEQ ID NO:93 is fully comprised in SEQ ID NO:34.

[0173] Destabilized IL2-v(BCD) polypeptide with a T102D substitution is represented in SEQ ID NO: 102

[0174] KNPKLTRMLTAKFAMPKKATELKHLQCLEEELKPLEEVLNGAQSKNFHLRPRDLISNINVIVLE LKGSETTFMCEYADETATIVEFLNRWITFAQS I ISTLD (SEQ ID NO: 102)

[0175] In one embodiment, an IL2-v(BCD) polypeptide comprises an amino acid sequence of SEQ ID NO:102.

[0176] As indicated above, the invention also relates to an inactivated full length IL2-v polypeptide that comprises a destabilized interface between helices A and B, C and D. This polypeptide is herein referred to as “IL2-v(BCD-dA)” and exhibits an improved expression profile over IL2-v(BCD) while also not exhibiting IL2 functionality.

[0177] Thus, in another aspect the invention relates to a recombinant IL2 variant polypeptide (“IL2- v(BCD-dA)”) that comprises an inactivated splitted variant of human IL2 that comprises helix A of IL2 and a splitted variant of human IL2 that comprises helices B, C and D of IL2. The IL2- v(BCD-dA) polypeptide comprises a) an amino acid sequence of SEQ ID NO:92 with two or more amino acid substitutions, wherein the at least one of the amino acid substitutions is at position 16 and / or 20 of SEQ ID NO:92, and wherein at least one of the amino acid substitutions is at position 11, 14 and / or 25 of SEQ ID NO: 92, and b) an amino acid sequence of SEQ ID NO:93 with one amino acid substitution at position 102 of SEQ ID NO: 93.

[0178] One destabilized IL2-v(BCD-dA) polypeptide with a T102D substitution is represented in SEQ ID NO:103:

[0179] APASSSTKKTELQAELLLLTLQMIANGINNYKNPKLTRMLTAKFAMPKKATELKHLQCLEEELK PLEEVLNGAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQS I ISTLD (SEQ ID NO:103)

[0180] In one embodiment, an IL2-v(BCD-dA) polypeptide comprises an amino acid sequence of SEQ ID NO: 103.

[0181] Compositions comprising two different recombinant split IL2 polypeptides

[0182] The invention further relates to a pharmaceutical composition comprising a set of recombinant polypeptides, comprising: a) the recombinant IL2-v(A) polypeptide of the invention, and b) the recombinant IL2-v(BCD-dA) polypeptide of the invention.

[0183] The invention also relates to a composition comprising a first and a second precursor protein, wherein each precursor protein comprises an antibody binding site and an Fc domain, wherein the first precursor protein comprises the recombinant IL2-v(A) polypeptide of the invention, and wherein the second precursor protein comprises the the recombinant IL2-v(BCD-dA) polypeptide of the invention.

[0184] In certain embodiments the recombinant IL2-v(A) polypeptide is fused to the C-terminus of the Fc domain of the first precursor protein and the recombinant IL2-v(BCD-dA) polypeptide is fused to the C-terminus of the Fc domain of the second precursor protein.

[0185] In certain embodiments the recombinant IL2-v(A) polypeptide is fused via a peptidic linker to the antibody binding site or to the Fc domain of the first precursor protein, and wherein the recombinant IL2-v(BCD-dA) polypeptide is fused via a peptidic linker to the antibody binding site or to the Fc domain of the second precursor protein.

[0186] Antigen binding sites

[0187] In certain embodiments the antibody binding sites of the first and the second precursor protein are antibody fragments. In one embodiment, the antibody binding sites of the first and the second precursor protein each comprise at least one pair of a VH and a VL domain. In one embodiment, the antibody binding sites of the first and the second precursor protein each comprise at least one Fab fragment.

[0188] In one embodiment, the first precursor protein is monovalent. In one embodiment, the first precursor protein is bivalent.

[0189] In one embodiment, the second precursor protein is monovalent. In one embodiment, the second precursor protein is bivalent.

[0190] In one embodiment, the first precursor protein and the second precursor protein are bivalent.

[0191] In certain embodiments the antibody binding sites of the first and the second precursor protein bind to a tumor associated antigen. In certain embodiments the antibody binding sites of the first and the second precursor protein bind to PD-1.

[0192] Fc domain

[0193] In certain embodiments the first and second precursor protein comprise an Fc domain of IgGl or IgG4 isotype. In one embodiment the first and second precursor protein comprise an Fc domain of IgGl isotype.

[0194] In certain embodiments, one or more amino acid modifications may be introduced into the Fc domain of the first and second precursor protein, thereby generating an Fc domain variant. The Fc domain variant may comprise a human Fc domain sequence (e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc domain) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0195] In certain aspects, the invention contemplates a polypeptide or complex variant that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half-life of the polypeptide in vivo is important yet certain effector functions (such as complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC)) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be conducted to confirm the reduction / depletion of CDC and / or ADCC activities. For example, Fc receptor (FcyR) binding assays can be conducted to ensure that the complex lacks FcyR binding (hence likely lacking ADCC activity), but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express FcyRIII only, whereas monocytes express FcyRI, FcyRII and FcyRIII. FcyR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat’l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat’l Acad. Sci. USA 82: 1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays methods may be employed (see, for example, ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat’l Acad. Sci. USA 95:652-656 (1998). Clq binding assays may also be carried out to confirm that the antibody is unable to bind Clq and hence lacks CDC activity. See, e.g., Clq and C3c binding ELISA in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano- Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M.S. et al., Blood 101 : 1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., Int’l. Immunol. 18(12): 1759-1769 (2006); WO 2013 / 120929 Al).

[0196] Antibodies with reduced effector function include those with substitution of one or more of Fc domain residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056). Such Fc domain mutants include Fc domain mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581).

[0197] Certain antibody variants with improved or diminished binding to FcyRs are described. (See, e.g., U.S. Patent No. 6,737,056; WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

[0198] In certain embodiments, a polypeptide comprises an Fc domain with one or more amino acid substitutions, which improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 of the Fc domain (EU numbering of residues).

[0199] In certain embodiments, a polypeptide comprises an Fc domain with one or more amino acid substitutions, which diminish FcyR binding, e.g., substitutions at positions 234 and 235 in the Fc domain polypeptide (EU numbering of residues). In one aspect, the substitutions are L234A and L235A in the Fc domain polypeptide (LALA). In certain aspects, the polypeptide or complex variant further comprises D265A and / or P329G in an Fc domain polypeptide derived from a human IgGl Fc domain polypeptide. In one embodiment, the substitutions are L234A, L235A and P329G (LALA-PG) in an Fc domain polypeptide derived from a human IgGl Fc domain polypeptide. (See, e.g., WO 2012 / 130831). In another embodiment, the substitutions are L234A, L235A and D265A (LALA-DA) in an Fc domain polypeptide derived from a human IgGl Fc - region polypeptide.

[0200] In some aspects, alterations are made in the Fc domain that result in altered (i.e., either improved or diminished) Clq binding and / or Complement Dependent Cytotoxicity (CDC), e.g., as described in US Patent No. 6, 194, 551 , WO 99 / 51642, and Idusogie et al . J. Immunol . 164 : 4178-4184 (2000).

[0201] Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in US2005 / 0014934 (Hinton et al.). Those antibodies comprise an Fc domain with one or more substitutions therein which improve binding of the Fc domain to FcRn. Such Fc domain polypeptide variants include those with substitutions at one or more of Fc domain polypeptide residues: 238, 252, 254, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc domain residue 434 (See, e.g., US Patent No. 7,371,826; Dall'Acqua, W.F., et al. J. Biol. Chem. 281 (2006) 23514-23524).

[0202] Fc domain residues critical to the mouse Fc-mouse FcRn interaction have been identified by site- directed mutagenesis (see e.g. Dall’Acqua, W.F., et al. J. Immunol 169 (2002) 5171-5180). Residues 1253, H310, H433, N434, and H435 (EU numbering of residues) are involved in the interaction (Medesan, C., et al., Eur. J. Immunol. 26 (1996) 2533; Firan, M., et al., Int. Immunol. 13 (2001) 993; Kim, J.K., et al., Eur. J. Immunol. 24 (1994) 542). Residues 1253, H310, and H435 were found to be critical for the interaction of human Fc with murine FcRn (Kim, J.K., et al., Eur. J. Immunol. 29 (1999) 2819). Studies of the human Fc-human FcRn complex have shown that residues 1253, S254, H435, and Y436 are crucial for the interaction (Firan, M., et al., Int. Immunol. 13 (2001) 993; Shields, R.L., et al., J. Biol. Chem. 276 (2001) 6591-6604). In Yeung, Y.A., et al. (J. Immunol. 182 (2009) 7667-7671) various mutants of residues 248 to 259 and 301 to 317 and 376 to 382 and 424 to 437 have been reported and examined (numbering according to Kabat EU index).

[0203] In certain embodiments, an Fc domain variant comprises an Fc domain with one or more amino acid substitutions, which reduce FcRn binding, e.g., substitutions at positions 253, and / or 310, and / or 435 of the Fc domain (EU numbering of residues). In certain embodiments, the Fc domain variant comprises an Fc domain with the amino acid substitutions at positions 253, 310 and 435. In one embodiment, the substitutions are 1253 A, H310A and H435A in an Fc domain derived from a human IgGl Fc domain. See, e.g., Grevys, A., et al., J. Immunol. 194 (2015) 5497-5508.

[0204] In certain embodiments, an Fc domain variant comprises an Fc domain with one or more amino acid substitutions, which reduce FcRn binding, e.g., substitutions at positions 310, and / or 433, and / or 436 of the Fc domain (EU numbering of residues). In certain embodiments, the Fc domain variant comprises an Fc domain with the amino acid substitutions at positions 310, 433 and 436. In one embodiment, the substitutions are H310A, H433A and Y436A in an Fc domain derived from a human IgGl Fc domain. (See, e.g., WO 2014 / 177460 Al).

[0205] In certain embodiments, an Fc domain variant comprises an Fc domain with one or more amino acid substitutions, which increase FcRn binding, e.g., substitutions at positions 252, and / or 254, and / or 256 of the Fc domain polypeptide (EU numbering of residues). In certain embodiments, the Fc domain variant comprises an Fc domain with amino acid substitutions at positions 252, 254, and 256. In one embodiment, the substitutions are M252Y, S254T and T256E in an Fc domain derived from a human IgGl Fc domain. See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351 concerning other examples of Fc domain variants.

[0206] The C-terminus of the Fc domain of the polypeptide according to the current invention can be a complete C-terminus ending with the amino acid residues PGK. The C-terminus of the Fc domain can be a shortened C-terminus in which one or two of the C-terminal amino acid residues have been removed. In one preferred embodiment, the C-terminus of the Fc domain is a shortened C- terminus ending with the dipeptide PG. In one embodiment, a polypeptide according to the current invention comprising an Fc domain including a C-terminal CH3 domain as specified herein, comprises the C-terminal glycine-lysine dipeptide (G446 and K447, EU index numbering of amino acid positions). In one embodiment, a polypeptide according to the current invention comprising an Fc domain including a C-terminal CH3 domain, as specified herein, comprises a C-terminal glycine residue (G446, EU index numbering of amino acid positions).

[0207] Peptidic linker

[0208] In the first and second precursor protein the splitted IL2 variants are fused to antibody domains, usually via a peptidic linker.

[0209] In one embodiment the recombinant IL2-v(A) polypeptide is fused via a peptidic linker to the antibody binding site or to the Fc domain of the first precursor protein, and the recombinant IL2- v(BCD-dA) polypeptide is fused via a peptidic linker to the antibody binding site or to the Fc domain of the second precursor protein.

[0210] In one embodiment the recombinant IL2-v(A) polypeptide is fused via a peptidic linker to the antibody binding site of the first precursor protein, and the recombinant IL2-v(BCD-dA) polypeptide is fused via a peptidic linker to the antibody binding site of the second precursor protein. In one embodiment the recombinant IL2-v(A) polypeptide is fused via a peptidic linker to the Fc domain of the first precursor protein, and the recombinant IL2-v(BCD-dA) polypeptide is fused via a peptidic linker to the the Fc domain of the second precursor protein.

[0211] In one embodiment the peptidic linker is a glycine serine linker. The peptidic linker is in certain embodiments a peptide with an amino acid sequence with a length of at least 30 amino acids, in one embodiment with a length of 32 to 50 amino acids. In one embodiment, the peptidic linker is a peptide with an amino acid sequence with a length of 32 to 40 amino acids. In one embodiment the peptidid linker is a glycine seriner linker. A “glycine serine linker” as referred to herein is a peptidic linker solely comprised og glycine and serine residues. In one embodiment the peptidic linker is (GxS)n with G = glycine, S = serine, (x = 3, n = 8, 9 or 10) or (x = 4 and n= 6, 7 or 8), in one embodiment with x = 4, n = 6 or 7, in one embodiment with x = 4, n = 7. In one preferred embodiment the peptidic linker is (G4S)6 (SEQ ID NO: 32) or (G4S)3G5 (SEQ ID NO: 33).

[0212] Recombinant Methods and Compositions

[0213] Antibodies and other polypeptides described herein may be produced using recombinant methods and compositions, e.g., as described in US 4,816,567. For these methods one or more isolated nucleic acid(s) encoding an antibody are provided.

[0214] In case of a native antibody or native antibody fragment two nucleic acids are required, one for the light chain or a fragment thereof and one for the heavy chain or a fragment thereof. Such nucleic acid(s) encode an amino acid sequence comprising the VL and / or an amino acid sequence comprising the VH of the antibody (e.g., the light and / or heavy chain(s) of the antibody). These nucleic acids can be on the same expression vector or on different expression vectors.

[0215] In case of a bispecific antibody with heterodimeric heavy chains four nucleic acids are required, one for the first light chain, one for the first heavy chain comprising the first heteromonomeric Fc- region polypeptide, one for the second light chain, and one for the second heavy chain comprising the second heteromonomeric Fc-region polypeptide. The four nucleic acids can be comprised in one or more nucleic acid molecules or expression vectors. Such nucleic acid(s) encode an amino acid sequence comprising the first VL and / or an amino acid sequence comprising the first VH including the first heteromonomeric Fc-region polypeptide and / or an amino acid sequence comprising the second VL and / or an amino acid sequence comprising the second VH including the second heteromonomeric Fc-region polypeptide of the antibody (e.g., the first and / or second light and / or the first and / or second heavy chains of the antibody). These nucleic acids can be on the same expression vector or on different expression vectors, normally these nucleic acids are located on two or three expression vectors, i.e. one vector can comprise more than one of these nucleic acids. Examples of these bispecific antibodies are CrossMabs (see, e.g., Schaefer, W. et al, PNAS, 108 (2011) 11187-1191). For example, one of the heteromonomeric heavy chain comprises the so-called “knob mutations” (T366W and optionally one of S354C or Y349C) and the other comprises the so-called “hole mutations” (T366S, L368A and Y407V and optionally Y349C or S354C) (see, e.g., Carter, P. et al., Immunotechnol. 2 (1996) 73) according to EU index numbering.

[0216] In one aspect, isolated nucleic acids encoding a polypeptide according to the current invention and as used in the methods as reported herein are provided.

[0217] In one aspect, a method of making a polypeptide according to the current invention is provided, wherein the method comprises culturing a host cell comprising nucleic acid(s) encoding the polypeptide according to the current invention, e.g., as provided above, under conditions suitable for expression of the polypeptide, and optionally recovering the polypeptide from the host cell (or host cell culture medium).

[0218] For recombinant production of a polypeptide according to the current invention, nucleic acids encoding the polypeptide, e.g., as described above, are isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the chains of the polypeptide) or produced by recombinant methods or obtained by chemical synthesis.

[0219] Suitable host cells for cloning or expression of polypeptide-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, antibodies may be produced in bacteria, in particular when glycosylation and Fc-region effector function are not needed. For expression of antibody fragments and polypeptides in bacteria, see, e.g., US 5,648,237, US 5,789,199, and US 5,840,523. (See also Charlton, K.A., In: Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, describing expression of antibody fragments in E. coli.) After expression, the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.

[0220] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for polypeptide-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been “humanized”, resulting in the production of a polypeptide with a partially or fully human glycosylation pattern. See Gerngross, T.U., Nat. Biotech. 22 (2004) 1409-1414; and Li, H. et al., Nat. Biotech. 24 (2006) 210-215.

[0221] Suitable host cells for the expression of (glycosylated) polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0222] Plant cell cultures can also be utilized as hosts. See, e.g., US 5,959,177, US 6,040,498, US 6,420,548, US 7,125,978, and US 6,417,429 (describing PLANTIBODIES(TM) technology for producing antibodies in transgenic plants).

[0223] Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293T cells as described, e.g., in Graham, F.L. et al., J. Gen Virol. 36 (1977) 59-74); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described, e.g., in Mather, J.P., Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells (as described, e.g., in Mather, J.P. et al., Annals N.Y. Acad. Sci. 383 (1982) 44-68); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR- CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220); and myeloma cell lines such as Y0, NS0 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki, P. and Wu, A.M., Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0224] In one aspect, the host cell is eukaryotic, e.g., a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell).

[0225] Pharmaceutical Compositions

[0226] In a further aspect, provided are pharmaceutical compositions comprising any of the polypeptides according to the current invention, e.g., for use in any of the below therapeutic methods. In certain embodiments, a pharmaceutical composition comprises any of the polypeptides according to the current invention and a pharmaceutically acceptable carrier. In certain embodiments, a pharmaceutical composition comprises any of the polypeptides according to the current invention and at least one additional therapeutic agent, e.g., as described below.

[0227] Pharmaceutical compositions (formulations) of a polypeptide according to the current invention can be prepared by combining the polypeptide with pharmaceutically acceptable carriers or excipients known to the skilled person. See, for example Remington's Pharmaceutical Sciences 16th edition, Oslo, A. Ed. (1980), Shire S., Monoclonal Antibodies: Meeting the Challenges in Manufacturing, Formulation, Delivery and Stability of Final Drug Product, 1st Ed., Woodhead Publishing (2015), §4 and Falconer R. J., Biotechnology Advances (2019), 37, 107412. Exemplary pharmaceutical compositions of a polypeptide according to the current invention are lyophilized, aqueous, frozen, etc.

[0228] Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as histidine, phosphate, citrate, acetate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyl dimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0229] The pharmaceutical composition according to the current invention may also contain more than one active ingredient as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended.

[0230] The pharmaceutical compositions according to the current invention are to be used for in vivo administration and are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.

[0231] Therapeutic Methods and Routes of Administration

[0232] Any of the polypeptides according to the current invention may be used in therapeutic methods.

[0233] In certain embodiments, a polypeptide according to the current invention for use as a medicament is provided. In further embodiments, a polypeptide according to the current invention for use in treating cancer is provided. In certain embodiments, a polypeptide according to the invention for use in a method of treatment is provided. In certain embodiments, the invention provides a polypeptide according to the invention for use in a method of treating an individual having cancer comprising administering to the individual an effective amount of the polypeptide according to the invention. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent (e.g., one, two, three, four, five, or six additional therapeutic agents). An “individual” according to any of the above aspects is preferably a human.

[0234] In certain embodiments, the invention provides for the use of a polypeptide according to the invention in the manufacture or preparation of a medicament. In one embodiment, the medicament is for treatment of cancer. In a further embodiment, the medicament is for use in a method of treating cancer comprising administering to an individual having cancer an effective amount of the medicament. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. An “individual” according to any of the above aspects may be a human.

[0235] In certain embodiments, the invention provides a method for treating cancer. In one embodiment, the method comprises administering to an individual having cancer an effective amount of a polypeptide according to the current invention. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. An “individual” according to any of the above aspects may be a human.

[0236] In certain embodiments, the invention provides pharmaceutical compositions comprising any of the polypeptides according to the invention, e.g., for use in any of the above therapeutic methods. In one embodiment, a pharmaceutical composition comprises any of the polypeptides according to the invention and a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical composition comprises any of the polypeptides according to the current invention and at least one additional therapeutic agent.

[0237] Polypeptides according to the current invention can be administered alone or used in a combination therapy. For instance, the combination therapy includes administering a polypeptide according to the invention and administering at least one additional therapeutic agent (e.g. one, two, three, four, five, or six additional therapeutic agents). In certain embodiments, the combination therapy comprises administering a polypeptide according to the invention and administering at least one additional therapeutic agent.

[0238] Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate pharmaceutical compositions), and separate administration, in which case, administration of the polypeptide of the invention can occur prior to, simultaneously, and / or following, administration of the additional therapeutic agent or agents. In one aspect, administration of the polypeptide according to the current invention and administration of an additional therapeutic agent occur within about one month, or within about one, two or three weeks, or within about one, two, three, four, five, or six days, of each other. In one aspect, the polypeptide according to the current invention and the additional therapeutic agent are administered to the patient on Day 1 of the treatment. Polypeptide of the invention can also be used in combination with radiation therapy.

[0239] A polypeptide of the invention (and any additional therapeutic agent) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g., by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.

[0240] Polypeptides of the invention would be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The polypeptide according to the invention need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of polypeptide present in the pharmaceutical composition, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically / clinically determined to be appropriate.

[0241] For the prevention or treatment of disease, the appropriate dosage of a polypeptide of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of polypeptide, the severity and course of the disease, whether the polypeptide is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the polypeptide, and the discretion of the attending physician. The polypeptide is suitably administered to the patient at one time or over a series of treatments. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays. Articles of Manufacture

[0242] In another aspect of the invention, an article of manufacture containing materials useful for the treatment, prevention and / or diagnosis of the disorders described above is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition that is by itself or combined with another composition effective for treating, preventing and / or diagnosing the condition and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is a polypeptide of the invention. The label or package insert indicates that the composition is used for treating the condition of choice. Moreover, the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises a polypeptide of the invention; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition. Alternatively, or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0243] ***

[0244] All publications, patents, and patent applications cited herein are hereby incorporated by reference herein in their entirety for all purposes to the same extent as if each individual publication, patent, and patent application were specifically and individually indicated to be so incorporated by reference. In the event that one or more of the incorporated literature and similar materials differs from or contradicts this application, including but not limited to defined terms, term usage, described techniques, or the like, this application controls.

[0245] ***

[0246] The following examples and figures as well as the sequences are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention. That is, although the disclosed teachings have been described with reference to various applications, methods, and compositions, it will be appreciated that various changes and modifications can be made without departing from the teachings herein and the claimed invention below. The examples are provided to better illustrate the disclosed teachings and are not intended to limit the scope of the teachings presented herein. While the present teachings have been described in terms of these exemplary embodiments, the skilled artisan will readily understand that numerous variations and modifications of these exemplary embodiments are possible without undue experimentation. All such variations and modifications are within the scope of the current teachings. DESCRIPTION OF THE AMINO ACID SEQUENCES

[0247] Figure 3-6:

[0248] Figure 11 and 12; Table 1 :

[0249] Figure 13-16:

[0250] Figure 17-18:

[0251] SEQ ID NO: 32 (G4S)6 linker

[0252] SEQ ID NO: 33 (G4S)3G5 linker

[0253] SEQ ID NO: 34 IL-2v amino acid sequence

[0254] SEQ ID NO: 35 Human IL-2 amino acid sequence

[0255] SEQ ID NO: 36-69 wild-type and variant heavy chain Fc-region amino acid sequences

[0256] SEQ ID NO: 70 (G4S)7 linker

[0257] SEQ ID NO: 71 (G4S)8 linker

[0258] SEQ ID NO: 72 (G4S)4 linker

[0259] SEQ ID NO: 73 (G4S)3 linker

[0260] SEQ ID NO: 74 G3SG2S linker

[0261] Figure 19-21, Table 3:

[0262] Figures 22-24:

[0263] Figure 25

[0264] SEQ ID NO: 91 XVV194 HC hole BCD only SEQ ID NO:92 IL2-v (A)

[0265] SEQ ID NO:93 IL2-v (BCD)

[0266] SEQ ID NO:94 IL2-v (A) H16L

[0267] SEQ ID NO:95 IL2-v (A) D20T

[0268] SEQ ID NO:96 IL2-v (A) H16L D20T

[0269] SEQ ID NO:97 IL2-v (A) H16Y D20T

[0270] SEQ ID NO:98 IL2-v (A) QI IK

[0271] SEQ ID NO:99 IL2-v (A) H16L D20T QI IE

[0272] SEQ ID NO: 100 IL2-v (A) H16L D20T L14A L25A

[0273] SEQ ID NO: 101 IL2-v (A) H16L D20T QI IE L14A L20A

[0274] SEQ ID NO:102 IL2-v(BCD) T102D

[0275] SEQ ID NO:103 IL2-v(BCD-dA) T102D

[0276] SPECIFIC EMBODIMENTS OF THE INVENTION

[0277] In the following specific embodiments of the invention are listed:

[0278] 1. A recombinant IL2 variant polypeptide (IL2-v), which is a splittable variant of human interleukin 2 (IL2), comprising the amino acid sequence of SEQ ID NO:34. 2. A recombinant IL2 variant polypeptide (IL2-v(A)), which comprises a splitted variant of human IL2 that comprises helix A of IL2, comprising SEQ ID NO:92.

[0279] 3. A recombinant IL2 variant polypeptide (IL2-v(A)) that comprises SEQ ID NO:92 with one amino acid substitution at position 11 of SEQ ID NO:92.

[0280] 4. A recombinant IL2 variant polypeptide (IL2-v(A)) that comprises SEQ ID NO:92 with one or more amino acid substitutions, wherein the amino acid substitution is at position 16 and / or at position 20 of SEQ ID NO:92.

[0281] 5. A recombinant IL2 variant polypeptide (IL2-v(A)) that comprises SEQ ID NO:92 with two or more amino acid substitutions, wherein the at least one of the amino acid substitutions is at position 16 and / or 20 of SEQ ID NO:92, and wherein at least one of the amino acid substitutions is at position 11, 14 and / or 25 of SEQ ID NO:92. 6. The recombinant polypeptide of one of embodiments 2 to 5, wherein the substitutions are selected from: a substitution at position 11 selected from QI IK or QI IE, a substitution at position 14 or L14A, a substitution at position 16 of H16L, a substitution at position 20 of D20T, and a substitution at position 25 of L25A.

[0282] 7. The recombinant polypeptide of one of embodiments 2 to 6 comprising an amino acid sequence selected from SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100 and SEQ ID NO: 101.

[0283] 8. The recombinant polypeptide of one of embodiments 2 to 7 comprising an amino acid sequence selected from SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96 and SEQ ID NO:97.

[0284] 9. A recombinant IL2 variant polypeptide (IL2-v(BCD)), which comprises a splitted variant of human IL2 that comprises helices B, C and D of IL2, comprising SEQ ID NO:93.

[0285] 10. A recombinant IL2 variant polypeptide (IL2-v(BCD)) comprising SEQ ID NO:93 with one amino acid substitution at position 102 of SEQ ID NO:93.

[0286] 11. The recombinant polypeptide of embodiment 10, wherein the substitution at position 102 is T102D.

[0287] 12. A recombinant IL2 variant polypeptide (IL2-v(BCD)) comprising SEQ ID NO: 102.

[0288] 13. A recombinant IL2 variant polypeptide (IL2-v(BCD-dA)), which comprises an inactivated splitted variant of human IL2 that comprises helix A of IL2 and a splitted variant of human IL2 that comprises helices B, C and D of IL2, comprising c) an IL2-v(A) polypeptide comprising SEQ ID NO:92 with one or more amino acid substitutions, wherein the amino acid substitution is at position 16 and / or at position 20 of SEQ ID NO:92, and d) an IL2-v(BCD) polypeptide.

[0289] 14. A recombinant IL2 variant polypeptide (IL2-v(BCD-dA)), which comprises an inactivated splitted variant of human IL2 that comprises helix A of IL2 and a splitted variant of human IL2 that comprises helices B, C and D of IL2, comprising e) an amino acid sequence of SEQ ID NO:92 with two or more amino acid substitutions, wherein the at least one of the amino acid substitutions is at position 16 and / or 20 of SEQ ID NO:92, and wherein at least one of the amino acid substitutions is at position 11, 14 and / or 25 of SEQ ID NO: 92, and f) an amino acid sequence of SEQ ID NO:93 with one amino acid substitution at position 102 of SEQ ID NO:93. The recombinant polypeptide of embodiment 13 or 14, wherein the substitutions in SEQ ID NO:92 are selected from: a substitution at position 11 selected from QI IK or QI IE, a substitution at position 14 or L14A, a substitution at position 16 of H16L, a substitution at position 20 of D20T, and a substitution at position 25 of L25, and wherein the substitution at position 102 of SEQ ID NO:93 is T102D. A pharmaceutical composition comprising a set of recombinant polypeptides, comprising: c) the recombinant IL2-v(A) polypeptide of one of embodiments 2 to 8, and d) the recombinant IL2-v(BCD-dA) polypeptide of one of embodiments 13 to 15. A composition comprising a first and a second precursor protein, wherein each precursor protein comprises an antibody binding site and an Fc domain, wherein the first precursor protein comprises the recombinant IL2-v(A) polypeptide of one of embodiments 2 to 8, and wherein the second precursor protein comprises the recombinant IL2-v(BCD-dA) polypeptide of one of embodiments 13 to 15. The composition of embodiment 17, wherein the recombinant IL2-v(A) polypeptide of embodiment 2 or 3 is fused to the C-terminus of the Fc domain of the first precursor protein and wherein the recombinant IL2-v(BCD-dA) polypeptide of embodiment 6 or 7 is fused to the C-terminus of the Fc domain of the second precursor protein. The composition of embodiment 17, wherein the recombinant IL2-v(A) polypeptide of embodiment 2 or 3 is fused to the N-terminus of the Fc domain of the first precursor protein and wherein the recombinant IL2-v(BCD-dA) polypeptide of embodiment 6 or 7 is fused to the N-terminus of the Fc domain of the second precursor protein. The composition of one of embodiments 17 to 19, wherein a) the first precursor protein comprises a recombinant IL2-v(A) polypeptide comprising SEQ ID NO:92 with a substitution at position 11 selected from QI IK or QI IE, and b) the second precursor protein comprises a recombinant IL2-v(BCD-dA) polypeptide comprising SEQ ID NO:34 with substitutions QI IK or QI IE, H16L, D20T, L14A and L25A and T133D. The composition of one of embodiments 17 to 20, wherein the recombinant IL2-v(A) polypeptide is fused via a peptidic linker to the antibody binding site or to the Fc domain of the first precursor protein, and wherein the recombinant IL2-v(BCD-dA) polypeptide is fused via a peptidic linker to the antibody binding site or to the Fc domain of the second precursor protein. The composition of embodiment 21, wherein the peptidic linker that has an amino acid sequence of (Gly4Ser)x3 G5 (SEQ ID NO: 33). The composition of one of embodiments 17 to 22, wherein the antibody binding sites of the first and the second precursor protein bind to a tumor associated antigen. The composition of one of embodiments 17 to 23 wherein the antibody binding sites of the first and the second precursor protein are Fab fragments. The composition of one of embodiments 17 to 24, wherein the first precursor protein binds to a first target and the second precursor protein binds to a second target, or wherein the first precursor protein binds to a first epitope on the target and the second precursor protein binds to a second, different epitope of the target. The composition of one of embodiments 17 to 25 further comprising a pharmaceutically acceptable carrier. A polypeptide comprising residues 1 to 31 of SEQ ID NO: 34 or SEQ ID NO: 35. The polypeptide according to embodiment 27, wherein the polypeptide comprises one or more mutations reducing the binding of the polypeptide to IL-2RP to abolish receptor heterodimerization. The polypeptide according to any one of embodiments 27 to 28, wherein the polypeptide comprises the mutation QI IK (numbering of residues according to SEQ ID NO: 34). The polypeptide according to any one of embodiments 27 to 29, wherein the polypeptide comprises the mutations L14A and L25A (numbering of residues according to SEQ ID NO: 34). A polypeptide comprising residues 32 to 133 of SEQ ID NO: 34 or SEQ ID NO: 35. The polypeptide according to embodiment 32, wherein the polypeptide comprises one or more mutations reducing the binding of the polypeptide to IL-2RP to abolish receptor heterodimerization. 33. The polypeptide according to any one of embodiments 31 to 32, wherein the polypeptide comprises the mutations T133D (numbering of residues according to SEQ ID NO: 34).

[0290] 34. The polypeptide according to any one of embodiments 27 to 33, wherein the polypeptide is conjugated to an Fc-region polypeptide.

[0291] 35. The polypeptide according to embodiment 34, wherein the polypeptide is conjugated to the C- terminus of the Fc-region polypeptide.

[0292] 36. The polypeptide according to any one of embodiments 1 to 35, wherein the Fc-region polypeptide is selected from an Fc-region polypeptide a) of the human subclass IgGl, or b) of the human subclass IgG4, or c) of the human subclass IgGl with the mutations L234A, L235A and P329G (numbering according to Kabat EU index), or d) of the human subclass IgG4 with the mutations S228P and L235E (numbering according to

[0293] Kabat EU index), or e) of the human subclass IgGl with the mutations L234A, L235A and P329G and the mutation

[0294] T366W (numbering according to Kabat EU index), or f) of the human subclass IgGl with the mutations T366S, L368A and Y407V (numbering according to Kabat EU index), or g) of the human subclass IgGl with the mutations L234A, L235A and P329G and the mutations i) T366W, and ii) S354C or Y349C (numbering according to Kabat EU index), or h) of the human subclass IgGl with the mutations L234A, L235A and P329G and the mutations i) T366S, L368A, and Y407V, and ii) Y349C or S354C (numbering according to Kabat EU index), i) of the human subclass IgG4 with the mutations S228P and L235E and the mutation T366W

[0295] (numbering according to Kabat EU index), or j) of the human subclass IgG4 with the mutations S228P and L235E and mutations T366S,

[0296] L368A and Y407V (numbering according to Kabat EU index), or k) of the human subclass IgG4 with the mutations S228P and L235E and the mutations i)

[0297] T366W, and ii) S354C or Y349C (numbering according to Kabat EU index), or l) of the human subclass IgG4 with the mutations S228P and L235E and the mutations i)

[0298] T366S, L368A, and Y407V, and ii) Y349C or S354C (numbering according to Kabat EU index), or m) of the human subclass IgGl with the mutations L234A, L235A, P329G, 1253 A, H310A and H435A and the mutation T366W (numbering according to Kabat EU index), or n) of the human subclass IgGl with the mutations L234A, L235A, P329G, 1253 A, H310A and

[0299] H435A and the mutations T366S, L368A and Y407V (numbering according to Kabat EU index), o) of the human subclass IgGl with the mutations L234A, L235A, P329G, 1253 A, H310A and

[0300] H435A and the mutations i) T366W, and ii) S354C or Y349C (numbering according to Kabat EU index), or p) of the human subclass IgGl with the mutations L234A, L235A, P329G, 1253 A, H310A and

[0301] H435A and the mutations i) T366S, L368A, and Y407V, and ii) Y349C or S354C (numbering according to Kabat EU index), or q) of the human subclass IgGl with the mutations L234A, L235A, P329G, M252Y, S254T and T256E and the mutation T366W (numbering according to Kabat EU index), or r) of the human subclass IgGl with the mutations L234A, L235A, P329G, M252Y, S254T and

[0302] T256E and the mutations T366S, L368A and Y407V (numbering according to Kabat EU index), s) of the human subclass IgGl with the mutations L234A, L235A, P329G, M252Y, S254T and

[0303] T256E and the mutations i) T366W, and ii) S354C or Y349C (numbering according to Kabat EU index), or t) of the human subclass IgGl with the mutations L234A, L235A, P329G, M252Y, S254T and

[0304] T256E and the mutations i) T366S, L368A, and Y407V, and ii) Y349C or S354C (numbering according to Kabat EU index), u) of the human subclass IgGl with the mutations L234A, L235A, P329G, H310A, H433A and Y436A and the mutation T366W (numbering according to Kabat EU index), or v) of the human subclass IgGl with the mutations L234A, L235A, P329G, H310A, H433A and Y436A and the mutations T366S, L368A and Y407V (numbering according to Kabat EU index), or w) of the human subclass IgGl with the mutations L234A, L235A, P329G, H310A, H433A and Y436A and the mutations i) T366W, and ii) S354C or Y349C (numbering according to Kabat EU index), or x) of the human subclass IgGl with the mutations L234A, L235A, P329G, H310A, H433A and Y436A and the mutations i) T366S, L368A, and Y407V, and ii) Y349C or S354C (numbering according to Kabat EU index).

[0305] 37. The polypeptide according to any one of embodiments 34 to 36, wherein the Fc-region polypeptide has an amino acid sequence selected from SEQ ID NO: 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66 and 67.

[0306] 38. The polypeptide according to any one of embodiments 34 to 37, wherein the polypeptide is conjugated to the Fc-region polypeptide via a peptidic linker of (Gly4Ser)x3 G5 (SEQ ID NO: 33).

[0307] 39. The polypeptide according to any one of embodiments 27 to 30 and 34 to 38, wherein the polypeptide comprises SEQ ID NO: 79.

[0308] 40. The polypeptide according to any one of embodiments 27 to 39, wherein the polypeptide is conjugated to the Fc-region polypeptide via a peptidic linker of (Gly4Ser)x6 (SEQ ID NO: 32)

[0309] 41. The polypeptide according to any one of embodiments 31 to 40, wherein the polypeptide comprises SEQ ID NO: 91.

[0310] 42. The polypeptide according to any one of embodiments 31 to 41, wherein the polypeptide comprises SEQ ID NO: 88.

[0311] 43. The polypeptide according to any one of embodiments 34 to 42, wherein the Fc-region polypeptide is conjugated at its N-terminus to a heavy chain Fab fragment.

[0312] 44. The polypeptide according to embodiment 43, wherein the polypeptide further comprises a light chain associated with the heavy chain Fab fragment.

[0313] 45. The polypeptide according to embodiment 44, wherein the Fab binds to a target.

[0314] 46. The polypeptide according to embodiment 45, wherein the target is a tumor associated antigen. 47. The polypeptide according to any one of embodiments 45 to 46, wherein the target is PD-1 or HER2.

[0315] 48. The polypeptide according to any one of embodiments 27 to 47, wherein the polypeptide is conjugated at its C-terminus via a second peptidic linker to a third polypeptide comprising residues 1-31 of SEQ ID NO: 34 or 35 with the mutations Hl 6L and D20T and / or the mutations QI IE, L14A and L25A (numbering of residues according to SEQ ID NO: 34).

[0316] 49. The polypeptide according to embodiment 48, wherein the second peptidic linker is (Gly4Ser)x6 (SEQ ID NO: 32).

[0317] 50. A composition comprising two polypeptides, wherein the first polypeptide is a polypeptide according to any one of embodiments 27 to 30 and 35 to 49 and the second polypeptide is a polypeptide according to any one of embodiments 31 to 49.

[0318] 51. The composition according to embodiment 50, wherein the first polypeptide binds to a first target and the second polypeptide binds to a second target, or wherein the first polypeptide binds to a first epitope on the target and the second polypeptide binds to a second, different epitope of the target.

[0319] 52. A complex comprising two polypeptides, wherein the first polypeptide is a polypeptide according to any one of embodiments 27 to 30 and 35 to 49 and the second polypeptide is a polypeptide according to any one of embodiments 31 to 49.

[0320] 53. The complex according to embodiment 52, wherein the first polypeptide binds to a first target and the second polypeptide binds to a second target, or wherein the first polypeptide binds to a first epitope on the target and the second polypeptide binds to a second, different epitope of the target.

[0321] 54. The complex according to any one of embodiments 52 to 53, wherein the formation of the complex of the surface of a target cell results in substantial STAT5 phosphorylation.

[0322] 55. The composition or the complex according to any one of embodiments 52 to 54, wherein a) the Fc-region polypeptide of the first and the second polypeptide are each of the human subclass IgGl, preferably of SEQ ID NO: 36, or b) the Fc-region polypeptide of the first and the second polypeptide are each of the human subclass IgG4, preferably of SEQ ID NO: 37, or c) the Fc-region polypeptide of the first and the second polypeptide are each of the human subclass IgGl each with the mutations L234A, L235A and P329G, preferably of SEQ ID NO: 38 (numbering according to Kabat EU index), or d) the Fc-region polypeptide of the first and the second polypeptide are each of the human subclass IgG4 with the mutations S228P and L235E, preferably of SEQ ID NO: 39 (numbering according to Kabat EU index), or e) the Fc-region polypeptide of the first and the second polypeptide are each of the human subclass IgGl with the mutations L234A, L235A and P329G and the mutation T366W in one Fc-region polypeptide and the mutations T366S, L368A and Y407V in the respective other Fc-region polypeptide, preferably of SEQ ID NO: 40 and 41 (numbering according to Kabat EU index), or f) the Fc-region polypeptide of the first and the second polypeptide are each of the human subclass IgGl with the mutations L234A, L235A and P329G and the mutations i) T366W, and ii) S354C or Y349C, in one Fc-region polypeptide and the mutations i) T366S, L368A, and Y407V, and ii) Y349C or S354C, in the respective other Fc-region polypeptide, preferably of SEQ ID NO: 42 and 43 or SEQ ID NO: 44 and 45 (numbering according to Kabat EU index), or g) the Fc-region polypeptide of the first and the second polypeptide are each of the human subclass IgG4 with the mutations S228P and L235E and the mutation T366W in one Fc- region polypeptide and the mutations T366S, L368A and Y407V in the respective other Fc-region polypeptide, preferably of SEQ ID NO: 46 and 47 (numbering according to Kabat EU index), or h) the Fc-region polypeptide of the first and the second polypeptide are each of the human subclass IgG4 with the mutations S228P and L235E and the mutations i) T366W, and ii) S354C or Y349C, in one Fc-region polypeptide and the mutations i) T366S, L368A, and Y407V, and ii) Y349C or S354C, in the respective other Fc-region polypeptide, preferably of SEQ ID NO: 48 and 49 or SEQ ID NO: 50 and 51 (numbering according to Kabat EU index), or i) the Fc-region polypeptide of the first and the second polypeptide are each of the human subclass IgGl with the mutations L234A, L235A, P329G, I253A, H310A and H435A and the mutation T366W in one Fc-region polypeptide and the mutations T366S, L368A and Y407V in the respective other Fc-region polypeptide, preferably of SEQ ID NO: 52 and 53 (numbering according to Kabat EU index), or j) the Fc-region polypeptide of the first and the second polypeptide are each of the human subclass IgGl with the mutations L234A, L235A, P329G, I253A, H310A and H435A and the mutations i) T366W, and ii) S354C or Y349C, in one Fc-region polypeptide and the mutations i) T366S, L368A, and Y407V, and ii) Y349C or S354C, in the respective other Fc-region polypeptide, preferably of SEQ ID NO: 54 and 55 or SEQ ID NO: 56 and 57 (numbering according to Kabat EU index), or k) the Fc-region polypeptide of the first and the second polypeptide are each of the human subclass IgGl with the mutations L234A, L235A, P329G, M252Y, S254T and T256E and the mutation T366W in one Fc-region polypeptide and the mutations T366S, L368A and Y407V in the respective other Fc-region polypeptide, preferably of SEQ ID NO: 58 and 59 (numbering according to Kabat EU index), or l) the Fc-region polypeptide of the first and the second polypeptide are each of the human subclass IgGl with the mutations L234A, L235A, P329G, M252Y, S254T and T256E and the mutations i) T366W, and ii) S354C or Y349C, in one Fc-region polypeptide and the mutations i) T366S, L368A, and Y407V, and ii) Y349C or S354C, in the respective other Fc-region polypeptide, preferably of SEQ ID NO: 60 and 61 or SEQ ID NO: 62 and 63 (numbering according to Kabat EU index), or m) the Fc-region polypeptide of the first and the second polypeptide are each of the human subclass IgGl with the mutations L234A, L235A, P329G, H310A, H433A and Y436A and the mutation T366W in one Fc-region polypeptide and the mutations T366S, L368A and Y407V in the respective other Fc-region polypeptide, preferably of SEQ ID NO: 64 and 65 (numbering according to Kabat EU index), or n) the Fc-region polypeptide of the first and the second polypeptide are each of the human subclass IgGl with the mutations L234A, L235A, P329G, H310A, H433A and Y436A and the mutations i) T366W, and ii) S354C or Y349C, in one Fc-region polypeptide and the mutations i) T366S, L368A, and Y407V, and ii) Y349C or S354C, in the respective other Fc-region polypeptide, preferably of SEQ ID NO: 66 and 67 or SEQ ID NO: 68 and 69 (numbering according to Kabat EU index). An isolated nucleic acid or a composition of nucleic acids encoding the polypeptide according to any one of embodiments 27 to 49. A host cell comprising the nucleic acid of embodiment 56. 58. A method of producing a polypeptide according to any one of the previous embodiments comprising culturing the host cell comprising a nucleic acid encoding the polypeptide under conditions suitable for the expression of the polypeptide.

[0323] 59. The method of any one of embodiments 59, further comprising recovering the polypeptide from the host cell or the cultivation medium.

[0324] 60. A polypeptide produced by the method of any one of embodiments 57 or 58.

[0325] 61. A pharmaceutical composition comprising the polypeptides according to any one of the previous embodiments and a pharmaceutically acceptable carrier.

[0326] 62. The polypeptides according to any one of the previous embodiments or the composition according to any one of the previous embodiments for use as a medicament.

[0327] 63. The polypeptides according to any one of the previous embodiments or the composition according to any one of the previous embodiments for use in the treatment of cancer.

[0328] 64. Use of the polypeptides according to any one of the previous embodiments or the composition according to any one of the previous embodiments in the manufacture of a medicament.

[0329] 65. The use according to embodiment 64, wherein the medicament is for treatment of cancer.

[0330] 66. A method of treatment of an individual in need of a treatment comprising the administration of the polypeptides according to any one of the previous embodiments or the composition according to any one of the previous embodiments in a therapeutically effective amount to the individual.

[0331] EXAMPLES

[0332] Rationale and outline o f experiments

[0333] Despite their promise in cancer immunotherapy, cytokines have many drawbacks, which limit their therapeutic potential. Due to their small size, they are rapidly cleared and have poor pharmacokinetic properties. As such, they need to be continuously infused at high doses, which can cause severe toxicity due to peripheral activity [9], To overcome these problems, cytokines such as IL-2 are being engineered to reduce their peripheral toxicity while also being attached to targeting moieties such as antibodies, in an attempt to deliver the cytokine directly to the tumor [1,2], Such engineering and targeting approaches have improved the safety profile of cytokines in cancer immunotherapy, however they also have significant drawbacks. One major issue behind targeted approaches is the lack of tumor-specific antigens. Cytokines are often targeted to tumor- associated antigens (TAAs), and while these are overexpressed in tumors, they are often also present in healthy tissue, which can lead to on-target, off-tumor toxicity [2, 12], Tumor penetration is also an issue, because targeted cytokines can still bind to cytokine receptors on peripheral cells, which can cause toxicity and reduce tumor penetration [1],

[0334] IL-2, which is a strong activator of CD8+ T-cells and NK cells, is approved for specific cancer indications but shows high toxicity and low efficacy (see below)

[0013] , Its development as a safe cancer immunotherapy agent faced the above-mentioned factors. New engineering approaches have shown increasing promise and have reignited the push for developing IL-2-based cancer therapeutics [2],

[0335] IL-2 binds with high affinity to the hetero-trimeric IL-2 receptor, consisting of IL-2Ra (CD25) and the class I cytokine receptors IL-2RP (CD122) and IL-2Ry (CD132)

[0014] , IL-2 also binds with intermediate affinity to the hetero-dimeric IL-2RP (CD122) and IL-2Ry (CD132) complex [7,14], The intermediate-affinity dimeric IL-2 receptor is predominantly expressed by resting CD8+ T- cells and NK cells, while the high-affinity hetero-trimeric receptor is expressed primarily by regulatory T-cells (Treg cells), but also transiently by newly activated T- and NK cells [14,15],

[0336] High-dose recombinant human IL-2 therapy was the first cancer immunotherapy treatment to show durable efficacy with complete response rates of 6-10 % in metastatic melanoma and renal cell carcinoma

[0016] , Despite its obvious potential, high-dose recombinant IL-2 therapy can result in severe systemic toxicity, causing capillary leak syndrome, hypotension, fever, chills, renal toxicity and other symptoms [17,18], The high therapeutic doses required combined with rapid clearance of recombinant IL-2 limits the use of IL-2 alone in cancer treatment

[0018] , To overcome these issues, targeted approaches such as antibody-IL-2 fusions have been developed, which aim to decrease systemic toxicity while also improving the pharmacokinetic behavior of IL-2 [1], To further reduce toxicity and to avoid the preferential stimulation of Tregs, which is thought to decrease efficacy in cancer immunotherapy, engineered versions of IL-2 were developed that abrogate binding to CD25 [3-5,19], One such variant (IL-2v), when fused to a PD-l-blocking antibody (PDl-IL2v), shows great promise as a cancer immunotherapy agent in preclinical studies [3].

[0337] Despite the promise of PDl-IL2v, there is still a need for a safer, conditionally active version of this therapy. PDl-IL2v, while primarily targeting IL-2v to PD-1 -expressing T-cells, still contains an unmasked IL-2v moiety that can bind to IL-2 receptors in the periphery. This can lead to toxicity, increased immunogenicity, and can narrow the therapeutic window.

[0338] Split antibody chain-exchange technologies, wherein functional T-cell engagers are reconstituted from inactive split CD3 binders, have been described

[0020] , In this split prodrug concept termed PACE, activation of split prodrugs occurs primarily on target cells, and is dependent on the antigen density. In contrast to other conditional activation approaches that rely on factors such as tumorspecific protease activity and pH differences [2], the split complementation approach discriminates between cancer cells and healthy cells based on the expression level of tumor-associated antigens. It has been demonstrated that splitting a type I cytokine is a viable method to create conditionally active targeted cytokines that reconstitute into functional entities upon accumulation on target cells [21,22].

[0339] The current invention is based, at least in part, on the finding that prodrugs consisting of a pair of antibody-fusion proteins - each member comprising one part of a split interleukin - can be used to target the inactive interleukin fragments to cells and reconstitute active interleukin directly on the surface of said cells. This occurs by a chain-exchange / chain-displacement-based mechanism upon accumulation on the target cells, wherein one of the interleukin fragments is stabilized with and shielded by an inactivated helix of said interleukin.

[0340] In contrast to previously known PACE- or cytokine-P ACE-derived approaches, activation is not driven by the PACE-mutations in the CH3 knob-into-hole interface [20,22], Instead, a chaindisplacement reaction occurs, which is driven by mutations within the interface between two helices of the interleukin. Using this approach, efficient reconstitution of interleukin activity on target-expressing cells, with very minimal non-specific activity can be achieved.

[0341] The current invention is exemplified in the following with a fusion of two inactive IL-2v fragments to antibodies targeting tumor-associated antigens or PD-1, and reconstitution of IL-2 signaling by targeted chain-displacement, which is driven by the increase in local concentration upon accumulation of the prodrugs on the cell surface. In the chain-displacement reaction, mutations within the helix A / C interface of IL-2v drive the exchange. Thereby efficient reconstitution of IL- 2v activity on target-expressing cells, with very minimal non-specific activity could be achieved. This is presented solely to exemplify the invention and shall not be constructed as a limitation. Based on the teaching put forward herein, a person skilled in the art can identify and generate comparable split interleukins of other classes. The true scope of the invention is set forth in the appended claims.

[0342] To create targeted IL-2 prodrugs, a 3+1 split approach was used. In this approach IL-2 was split directly downstream (C-terminal) of helix A (dashed line in Figure 1). The N-terminal fragment of the 3+1 split IL-2 (consisting of helix A) contains residues 1-31, while the complementary fragment (consisting of helices BCD) contains residues 32-133 of IL-2v (Figure 2). Without being bound by this theory, it is assumed that because helix A interacts primarily with IL-2RP and helices BCD interact with IL2RP and IL2Ry (see Figure 1), splitting IL-2v according to the current invention ensures that each fragment contains a substantial portion of the IL2RP-binding surface. Therefore, neither fragment in isolation should bind strongly enough to IL-2RP to trigger receptor heterodimerization.

[0343] In a first experiment, it was examined whether the act of splitting IL-2v into two fragments affects its intrinsic activity when re-assembled. To do so, the complementary split IL-2v fragments were fused with a flexible Gly-Ser linker to the C-terminus of an anti PD-1 antibody to generate a PD1- targeted, pre-assembled split IL-2v fusion protein (PDl-split-IL-2v; Figure 4; SEQ ID NO: 01, 04, 05), and compared IL-2 signaling activity with an intact, non-split IL-2v fusion protein (PDl-IL2v; Figure 3; SEQ ID NO: 01, 02, 03). These molecules were produced in transiently transfected HEK293 cells and were purified to homogeneity from cell culture supernatants.

[0344] The ability of these two molecules to trigger IL-2RPy-dependent signaling in HEK-Blue IL-2 reporter cells (Invivogen) was measured. The PD1 -targeted, pre-assembled split IL-2v fusion protein showed identical activity to PDl-IL2v on HEK-Blue-IL-2 cells engineered to overexpress human PD-1 (Figure 5). Using HEK-Blue IL-2 cells lacking PD-1 expression, the pre-assembled split IL-2v fusion protein retained signaling activity, with potency only slightly less than that of PDl-IL2v (Figure 6). Thus, it has been shown that split IL-2v, when pre-assembled to antibody heavy chains, assembles into the correct structure and activates the IL-2 receptor complex with a similar potency to intact, non-split IL-2v.

[0345] Reactivation of split IL-2v on target cells: complementation strategy and split IL-2v expression

[0346] In a second experiment, complementation of split IL-2v and reconstitution of IL-2v activity on target cells was accomplished via targeting the split IL-2v fragments to antigens expressed either on tumor cells or on T-cells. To target split IL-2v fragments, i.e. in not pre-assembled form, these were fused via a flexible Gly-Ser linker to the C-terminus of antibody-like molecules containing only one Fab arm (Figure 7). These monovalent, single-armed antibodies were designed to come into closer proximity than standard bivalent antibodies, which may be more sterically restricted once bound to the target antigen. Without being bound by this theory, it is assumed that split IL- 2v complementation is driven by the increase in local concentration upon targeted accumulation on the surface of the target cell.

[0347] Two complementation approaches for split IL-2v can be realized: Prodrugs with the same target binder (monoparatopic targeting) for use in situations when target antigen expression is high, as is the case with overexpressed tumor-associated antigens (Figure 8). For cis-activation approaches on T-cells, where antigen density can be low (such as in targeted delivery of IL-2v to PD-1 [3]), prodrugs that bind two different epitopes on the same antigen can be used (biparatopic targeting, Figure 9). All molecules, as well as those described in the following examples were produced in transiently transfected HEK293 cells and were purified to homogeneity from cell culture supernatants as previously described.

[0348] Splitting a protein exposes hydrophobic residues that would otherwise be buried in the protein core. This can lead to low expression, aggregation and instability

[0026] , Indeed, a low yield and a high aggregation propensity for the IL-2v (BCD) entity and fusion proteins thereof was observed. This behavior was despite the introduction of the K35E mutation, which has been reported to increase the yield and stability of IL-2 fusion proteins

[0027] (Figure 10). The other fragment, IL-2v (A), could be produced with high yield and had favorable biophysical properties (data not shown). It was found that in order to improve the solubility and yield of the IL-2v (BCD) fragment, a dummy helix needs to be added. Without being bound by this theory, it is assumed that this dummy helix acts as a chaperone and shields the hydrophobic core of IL-2v (BCD). The dummy helix is an engineered inactivated version of helix A from IL-2v. The dummy-A helix was inactivated through a series of point mutations that impair its interaction with IL-2|3R, thereby rendering the resulting IL-2v (BCD) - dummy helix A complex molecule inactive (Figure 11). In order to maintain sufficient flexibility of IL-2v (BCD) for complementation with the IL-2v (A) fragment, the dummy-A helix was attached to the C-terminus of IL-2v (BCD) with a flexible Gly-Ser linker. This molecule is henceforth referred to as split IL-2v (BCD-dA).

[0349] Antibody fusions harboring split IL-2v prodrugs - inactivation of helix A of IL-2v to create a dummy A helix

[0350] The PACE reaction generates active bispecific or trispecific antibodies from inactive prodrugs by antibody heavy-chain exchange

[0020] , It has been previously shown that these chain-exchange reactions can convert split IL-4 prodrugs into active IL-4 upon accumulation on target cells

[0028] , In the case of targeted split IL-2v complementation, a chain-displacement approach was utilized. In contrast to the previously known PACE-derived approaches, the chain-exchange according to the current invention is not driven by mutations in the CH3 knob-into-hole interface, but instead by mutations in the helix A / C interface in the split IL-2v (BCD-dA) molecule. For productive split IL-2v complementation, the active IL-2v (A) helix of one prodrug needs to displace the dummy- A helix in the IL-2v (BCD-dA) prodrug (Figure 10). To achieve this, helix A was first converted into a dummy helix A, so that it could be used as a shielding chaperone for IL-2v (BCD). To do so, mutations were introduced in the helix A / IL-2PR interface. These mutations need to fully inactivate IL-2v, so that the dummy A helix can be introduced as a chaperone into IL-2v (BCD) without rendering the split IL-2v (BCD) portion active.

[0351] Different mutations in helix A that interfere with the IL2PR interaction and thereby abrogate IL-2 signaling were tested. The mutations were introduced into the A helix of split IL-2v molecules in the context of cytokine-PACE educts

[0028] (Figure 11). In these molecules, the split IL-2v (A) and (BCD) portions are brought into close proximity through C-terminal fusion to the CH3 domains of the PACE educt. Figure 12 and Table 1 show the effects of different mutations in helix A of IL- 2v when paired with its split IL-2v (BCD) counterpart. It has now unexpectedly been found that the double mutations H16L D20T and H16Y D20T showed the strongest reduction in IL-2v activity. The H16L D20T combination is the preferred set of mutations for the dummy A helix in IL-2v (BCD-dA) prodrugs.

[0352] Table 1: Influence of helix A mutations on IL-2v signaling activity, assessed by a HEK Blue

[0353] IL-2 reporter assay. IL-2v activities from experiments as in Figure 12 are expressed as an approximate relative activity compared to split wild-type (wt) IL-2v molecules. Approximate relative activity is derived from EC50 values.

[0354] Targeted chain-displacement-mediated trans-activation of split IL-2v prodrugs

[0355] For target-specific prodrug activation, IL-2v (BCD-dA) and IL-2v (A) reactants need to undergo spontaneous chain-displacement when located in close spatial proximity, such as when bound to the cell surface (as previously shown for chain-exchange-enabled antibodies using the PACE technology

[0020] ). Without being bound by this theory, likewise to PACE, treatment of target- overexpressing cells with split IL-2v educts should lead to accumulation of the educts on target cells, subsequently leading to chain-exchange and reconstitution of active IL-2v. One specific application of split IL-2v prodrugs is the selective activation of T-cells and NK cells in the tumor. In this application, accumulation of split IL-2v educts on tumor cells overexpressing a tumor- associated antigen should generate active IL-2v on the tumor cells, which should result in activation of nearby T-cells and NK cells in trans (as in reference [4]). To assess this trans- activation functionality, CHO cells engineered to overexpress human PD-1 were treated with complementary PD-l-binding split IL-2v educts to assess target-specific, on-cell activation of IL- 2v (Figure 13). Split IL-2v educts with monoparatopic PD-1 targeting arms were compared with split IL-2v educts containing biparatopic PD-1 targeting arms (Figure 13). IL-2v activity was detected by co-culturing target cells with HEK-Blue IL-2 reporter cells (Invivogen), which report on IL-2 activity in a dose-dependent manner through the secretion of alkaline phosphatase. As a non-targeting control, PD-1 -targeting split IL-2v educts were added directly to HEK-Blue IL-2 reporter cells, as they do not express PD-1 (Figure 26). The results show dose-dependent IL-2v trans-activation in the presence of PD-1 -overexpressing target cells (Figure 14). In contrast, no IL- 2v activity is observed except at the highest concentration (200 nM) in the absence of PD-1- overexpressing target cells (Figure 14). This shows that efficient split IL-2v complementation is dependent on specific binding to target cells, which is in turn defined by the specificity of the targeting antibody. Furthermore, no activity of the single split IL-2v prodrugs in isolation was observed in the presence of target cells, confirming the inactivity of the individual prodrugs (Figure 14). No differences in IL-2 activity between monoparatopic and biparatopic PD-1 targeting were observed, likely due to the very high levels of PD-1 expressed by the PD-1 CHO target cells (Figure 27).

[0356] The trans-activation of the split IL-2v molecules on HER2-overexpressing SK-BR-3 breast cancer cells was likewise tested. SK-BR-3 cells were treated with complementary HER2 -binding split IL- 2v prodrugs in the monoparatopic targeting setting (Figure 15). IL-2v activity was detected by coculturing SK-BR-3 cells with HEK-Blue IL-2 reporter cells, as in Figures 13 and 14. As a control for non-targeted split IL-2v activation, the split IL-2v molecules were added directly to HEK-Blue IL-2 cells, which express very low levels of HER2 (Figure 28). The results show effective, dosedependent IL-2v trans-activation in the presence of HER2-overexpressing SK-BR-3 cells (Figure 16). In the absence of SK-BR-3 cells, activity is only observed at the highest concentration (200 nM) (Figure 16), similar to our observation with PD-1 -overexpressing cells (Figure 14). This confirms that efficient split IL-2v trans-activation depends on specific binding to and accumulation on target cells. Split IL-2v complementation is concentration-dependent, as evident by the nonspecific complementation observed at very high concentrations (Figure 14 and 16). No activity of the single split IL-2v prodrugs in isolation was observed in the presence of SK-BR-3 target cells, again confirming the inactivity of the individual prodrugs (Figure 16).

[0357] Influence of prodrug formats on activation of PDl-targeted split IL-2v prodrugs

[0358] The IL-2v prodrugs as reported herein and according to the current invention are converted by chain-exchange reactions into bispecific entities that need to bind two cell surface targets (target antigen and IL-2 receptor) to elicit desired functionality. Bispecific antibodies (bsAbs) also bind two targets, and it is known that the format of bsAbs can have a large influence on their functionalities [29,30], Format variables include attachment positions of fused entities, geometries of attachment, composition of fusion sites, linker sequences, and lengths of linkers between functional modules. Targeted split IL-2v prodrugs need to bind to cell-surface target antigens and need to be able to assemble into functional entities when bound to the cell surface. Therefore, the respective formats need to enable neighboring complementary prodrugs to come into close contact to initiate chain-exchange. Additionally, the active products of the exchange reaction must be able to access the IL-2 receptor while being bound to the cell-surface antigen. This requires sufficient flexibility, adequate spanning distances (for trans-activation of neighboring cells), and must also not be inhibited by steric constraints (e.g. in cis-activation).

[0359] As format requirements could be different for trans-activation (where a potentially greater distance is required) and cis-activation (where potential accessibility problems and steric hindrance might play a role), PD-l-IL-2v prodrugs were used to evaluate format parameters. The different formats and variants are shown in Figure 17, and their activities were analyzed in trans-activation and cis- activation experiments as shown in Figure 13 and 19. To compare activities of different formats, the EC50 values of the following split IL-2v prodrug pair was set to 100%: C-terminally attached split IL-2v (BCD-dA) with a linker length between BCD and dummy-A of (Gly4Ser)x6 (SEQ ID NO: 32), and C-terminally attached IL-2 (A) with a linker between CH3 and IL-2 (A) of (Gly4Ser)x3 G5 (SEQ ID NO: 33). The activities of the other formats were calculated relative to this value and are shown Table 2. Importantly, PD-l-targeted split IL-2v prodrugs in the N- terminal format retained their target-specificity, as trans-activation was observed only in the presence of CHO-K1 cells engineered to overexpress PD-1, and not in the presence of ordinary CHO-K1 cells, which lack detectable PD-1 expression

[0037] (Figure 18).

[0360] Table 2: Influence of format variants on the conversion of PD1 -targeted split IL-2v educts into functional IL-2v in trans-and cis-activation settings, as described above (Figure 13 for trans-assays, Figure 19 for cis-assays). n / a: not applicable

[0361] PD-l-targeted, chain-exchange-mediated cis-activation of split IL-2v prodrugs

[0362] As shown above, split IL-2v prodrugs can undergo spontaneous chain-exchange / chain- displacement when located in close spatial proximity, and thereby generate active IL-2v. This is not limited to only triggering IL-2 receptor activation on adjacent T-cells - they may also engage IL-2 receptors on the same cells. This provides the possibility to target IL-2v prodrugs to PD-1- expressing T-cells, and to activate the IL-2 receptor on these PD-1 -expressing T-cells in cis. This PD-l-targeted cis-activation approach has been demonstrated to result in better effector T-cells for cancer immunotherapy [3], The cis-targeting concept for split IL-2v and the corresponding assays, which utilize PD-1 overexpressing HEK-Blue IL-2 cells as well as PD-1 -negative HEK-Blue IL- 2 cells as controls, are illustrated in Figure 19. IL-2 activity generated by targeted prodrug conversion is detected by dose-dependent signals generated by the IL-2-responsive HEK-Blue reporter cell line. The results show effective cis-activation of IL-2 signaling when complementary prodrugs bind to PD-1 and accumulate on PD-1- and IL-2R-expressing reporter cells (Figure 20). In contrast, strongly reduced activity is seen with the split IL-2v prodrug combination with HEK Blue IL-2 cells, which lack PD-1 expression and hence do not enable cell-surface accumulation (Figure 20, see Figure 26 for PD-1 expression data). This further shows that IL-2v prodrug activation in the cis-setting is dependent on specific binding to target cells, which in turn is defined by the specificities of the targeting antibodies.

[0363] Antibody fusions harboring split IL-2v prodrugs - modulation of chain-exchange efficiency

[0364] The observed target-specific split IL-2v prodrug cis-activation on PD-1 -expressing cells was less potent (judging by EC50) than the PD-l-targeted pre-formed split IL-2v (Figure 20). Without being bound by this theory, it is assumed that this was due to the dummy A helix having too high of an affinity to the IL-2v (BCD) fragment. In order to provide a more efficient chain-exchange upon accumulation of complementary split IL-2v prodrugs on cell surfaces, the dummy-A / BCD interface was further destabilized. Without being bound by this theory, it is assumed that the temporary separation of the inactivating dummy-A helix from IL-2v (BCD) is thereby facilitated, and that binding of the functional IL-2 (A) helix to IL-2v (BCD), resulting in a functional complemented IL-2v, is improved. Thus, it has been found that the driving force behind the exchange reaction lies therefore in the dummy -A helix / BCD interaction, which needs to be weaker in the dummy-A helix compared to the functional IL-2 (A) helix. Without limitation, variables that influence said association are the introduction of asymmetrical mutations in the dummy-A helix, introduction of symmetrical mutations (repulsive charge mutations in dummy-A / BCD and attractive charge mutations in WT IL-2 A and BCD), engineering of environmental-triggered interactions (pH-dependent interactions), introduction of additional heterodimerization motifs, or truncations of the dummy-A helix to reduce its affinity to BCD. Therefore, interface variants of C-terminal split IL-2v prodrugs using the above-mentioned principles were produced and tested in a cis-activation assay as in Figure 19 and 20. Figure 21 and Table 3 show the results of this cis- activation assay. In Table 3, the effects of these changes on IL-2v prodrug activation are represented relative to cis-activation of unmodified split IL-2v entities in the C-terminal format.

[0365] Table 3: Influence of split IL-2v interface variants on the conversion of PD-1 -targeted C- terminal split IL-2v educts into functional PD-l-targeted IL-2v in cis-activation assays. IL-2 activity of the variants was assessed in cis with HEK-Blue IL-2 reporter cells engineered to overexpress human PD-1 (as in Figure 19) and is expressed as percentage activity (EC50) compared to the parental split IL-2v molecules (XVV140+XVV138).

[0366] The data showed that some mutations have a clear positive influence on split IL-2v complementation efficiency. It has been found that weakening the dummy-A helix-BCD hydrophobic core interactions through the mutations L14A L25A in dummy-A, as well as introduction of repulsive charge pairs into T133 of helix D and Ql l of dummy-A along with a corresponding attractive charge in IL-2 (A) boosted the complementation efficiency.

[0367] PD-l-specific activation of IL-2 receptor signaling in activated human T-cells

[0368] It has further been evaluated whether the prodrug variants with more efficient on-cell complementation could activate the IL-2 receptor on activated human T-cells, which upregulate PD-1 expression after activation [31,32], It was also tested whether the combination of the two most effective sets of mutations described above (L14A L25A in dummy-A and T133D QI IE in BCD-dA paired with QI IK in WT A) increases the potency of split IL-2v complementation in cis- activation. Therefore, purified CD4+ T-cells from human PBMCs were activated using immobilized anti-CD3 and anti-CD28 antibodies, which resulted in upregulated PD-1 expression. The activated T-cells were treated with the engineered PD-1 -targeting, C-terminal split IL-2v prodrugs according to the current invention, and STAT5 phosphorylation was monitored as a readout of IL-2 receptor activation. Figure 22 shows that the split IL-2v combination XVV194 + XVV173 resulted in dose-dependent STAT5 phosphorylation, consistent with its increased efficacy in HEK-Blue IL-2 cis-activation assays. XVV194 (IL-2v BCD-dA prodrug) contains the mutations T133D QI IE L14A L25A, which combines the effects of decreasing the strength of the dA-BCD hydrophobic interaction along with repulsive charges in the BCD-dA interface. XVV173 (IL-2v A prodrug) contains QI IK, which forms a salt bridge with T133D in the BCD portion of XVV194. Surprisingly, only this variant, and not the other variants tested, demonstrated substantial STAT5 phosphorylation. Without being bound by this theory, this may be due to the lower PD-1 expression level on activated T-cells compared to engineered PD-l-HEK-Blue IL-2 cells (data not shown). In order to test the PD-1 / target-cell specificity of split IL-2v complementation, half of the T cell population was treated with an excess of antibody that blocks the PD-1 receptor. In contrast to PDl-IL2v and the pre-assembled PD1 -split IL-2v, which activate the IL-2 receptor even on PD- 1-blocked T-cells, the combination of XVV194 + XVV173 resulted in extremely low STAT5 phosphorylation on PD-1 -blocked T cells (Figure 22). This shows that split IL-2v complementation is almost exclusively target-cell specific, which highlights its potential for an improved safety profile. An alternative dosing strategy was also tested: XVV173 (IL-2 A) was kept constant at the highest concentration while XVV194 (IL-2v BCD-dA) was titrated in at increasing concentrations. Thereby the local concentration of IL-2v (A) was kept constant, potentially increasing complementation efficiency at lower doses of IL-2v (BCD-dA). However, no difference in split IL-2v activity using this alternative dosing strategy was observed (Figure 22).

[0369] It was found that the combination of XVV 194 + XVV 173 did not reach the same plateau compared to pre-assembled split and non-split IL-2v (Figure 22). Without being bound by this theory, it is assumed that the kinetics of split IL-2v assembly might explain this behavior, as the pSTAT5 assay was analyzed after only 60 min. In order to test longer incubation times for split IL-2v complementation, the IL-2-mediated Granzyme B induction on CD4+ T cells was monitored after five days (Figure 23). It was found that the combination of split IL-2v constructs XVV173 + XVV194 increased the fraction of Granzyme B -proliferating T-cells to the same extent and with the same potency as pre-assembled split PDl-IL-2v (Figure 24). Without being bound by this theory, it is assumed that longer incubation times are potentially required for complete split IL-2v assembly, and that PD-1 -targeted split IL-2v is as active as the pre-assembled control when allowing for longer incubation times. Although the potency of PD1 -split IL-2v was lower than that of PDl-IL2v in both assays (based on EC50), it reached a plateau similar to PDl-IL2v in the Granzyme B assay (Figure 24), indicating that higher doses of split IL-2v prodrugs may achieve a similar overall efficacy compared to non-split PDl-IL2v.

[0370] Tumor-growth inhibition of PD-1 split IL-2v in a huPD-1 transgenic mouse model

[0371] The efficacy of PD-1 split IL-2v prodrugs (XVV173 + XVV194) was tested in an in vivo mouse tumor model. Tumor-bearing humanized PD-1 mice were treated with the PD-1 split IL-2v prodrugs. Tumor growth inhibition was compared with a murinized human PD-1 blocking antibody. It was found that the PD-1 split IL-2v prodrug combination resulted in superior tumor growth inhibition compared to both the vehicle group and the human PD-1 antibody treatment group (Figure 25). Importantly, no acute toxicity was observed, indicating that the PD-1 split IL- 2v prodrugs convert into active PD-1 IL-2v entities on the target cell only and are inactive in the periphery. However, some adverse events were observed after the third administration of the PD- 1 split IL-2v prodrugs, likely due to a humoral immune response against the PD-1 split IL-2v molecules as such. These results confirm the efficacy and safety of the PD-1 split IL-2v prodrugs in vivo.

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[0408] Detailed description of the experiments

[0409] Example 1

[0410] Protein design and engineering

[0411] For targeted split IL-2v prodrugs, antigen binding domains, i.e. pairs of cognate VH and VL sequences, of an anti-HER2 antibody (clone 4D5-8;

[0033] ) and anti-PD-1 antibodies (clones 0376 and 1040; [4]) were used.

[0412] For the screening of inactivating mutations in split IL-2v variants (see Figures 11-12), IL-2v was split into two parts and fused to the C-terminus of PACE antibody derivatives, as previously described for split IL-4 [20,28], In these constructs, VH and VL sequences from an anti- digoxigenin antibody (Digoxigenin (Dig)

[0034] ) and a non-binding antibody (Nadaceptin (Nada);

[0020] ) were used in the TriFab stem region, as previously described [20,28], Engineering of split IL-2v variants was performed using the crystal structure of IL-2v bound to the IL-2RPy complex [4], PDB ID 5M5E. Modeling and visualization of point mutations was performed using PyMOL (Schrodinger, Inc.), BIOVIA Discovery Studio (Dassault Systemes SE), and MOE software (Chemical Computing Group). Heavy chain heterodimerization was accomplished by introducing knob-into-hole mutations in the CH3 domains (knob: T366W, hole: T366S, L368A, Y407V)

[0035] ,

[0413] Example 2

[0414] Production of split IL-2v variants and antibody derivatives

[0415] All antibody derivatives and split IL-2v fusion constructs were expressed in Expi293 cells using the Expi293 Expression System (Thermo Fisher Scientific) according to the manufacturer’s instructions, as previously described [20,28], The cell culture supernatants were harvested by filtration. Proteins of interest containing an Fc-region were purified in two steps by Protein A chromatography with a HiTrap MabSelect SuRe column (Cytiva, Cat. No. 11003494) followed by size-exclusion chromatography with a Superdex 200 16 / 600 column (Cytiva, Cat. No. 28989335) or a Superdex 200 increase 10 / 300 column (Cytiva, Cat. No. 28990944). For constructs lacking an Fc-region, i.e. comprising only CH3 domains, proteins of interest were purified with a HiTrap KappaSelect column (Cytiva, Cat. No. 17545812). For all proteins, the fractions of interest were analyzed by SDS-PAGE and Coomassie Blue staining or by capillary electrophoresis (CE-SDS). Fractions containing the correct polypeptide chain composition were pooled, concentrated, and sterile-filtered. The final molecule purity was assessed by analytical SEC and CE-SDS. The purity and integrity of the final split IL-2v constructs used in the in vivo experiment (Figure 25) was confirmed by mass spectrometry.

[0416] Cell culture and flow cytometry

[0417] Cell lines HEK-Blue IL-2 (Invivogen, Cat. No. hkb-il2) and PDl-HEK-Blue IL-2 (derived from Invivogen hkb-il2, expressing PD-1) were cultured in Dulbecco’s Modified Eagle Medium, high glucose (DMEM-HG, Sigma-Aldrich, Cat. No. D5796) supplemented with 10 % FCS, 2 mM glutamine, IX HEK-Blue CLR selection (Invivogen, Cat. No. hb-csm), and 1 pg / ml puromycin. The cell lines CHO-K1 (ATCC CRL-9618) and PD1-CHO-K1 (derived from ATCC CRL-9618, expressing PD-1) were cultivated in Roswell Park Memorial Institute 1640 medium (RPMI-1640) supplemented with 10 % FCS and 2 mM glutamine. For the PD1-CHO-K1 cell line, 400 pg / ml G418 (Sigma-Aldrich, Cat. No. 4727878001) were added to the medium. All cell lines were cultivated at 37 °C, 80 % humidity, and 5 % CO2. For subculturing, cells were detached using Accutase solution (Sigma- Aldrich, A6964), and the viability and cell count were measured using a Vi-CELL XR cell viability analyzer (Beckman Coulter). Target-antigen expression levels were analyzed by flow cytometry for selected cell lines (Figures 26-28). Briefly, cells were resuspended in cold FACS buffer (PBS + 2% FCS), and 4 x 1E5 cells were seeded per well in 96-well U-bottom plates. The plates were centrifuged at 300 x g for 3 min. to pellet the cells and the supernatant was removed. Cells were resuspended in 95 pl of FACS buffer plus 5 pl of PE-labeled detection antibody, and were incubated for 1 hour on ice. The detection antibodies were as follows: PE antihuman CD279 (PD-1) (BioLegend, clone NAT105, cat. no. 367404), PE anti-human CD340 (erbB2 / HER-2) (BioLegend, clone 24D2, cat. no. 324406), and PE Mouse IgGl, K Isotype Control Antibody (BioLegend, clone MOPC-21, cat. no. 400112). The plate(s) was(were) centrifuged at 300 x g for 3 min to pellet the cells, and the supernatant was discarded. The cells were washed three times with 200 pl of cold FACS buffer, with a centrifugation step between each wash, as above. The cells were then resuspended in 100 pl of cold FACS buffer, and PE fluorescence was measured using a FACSCanto II instrument (BD Biosciences). A total of 1 x 1E4 events were collected per measurement. Data analysis and figure preparation was performed using FlowJo software (version 10.10, FlowJo LLC). Gating was performed using standard forward-scatter vs side-scatter analysis.

[0418] Example 4

[0419] HEK-Blue IL-2 reporter assays (Figure 12)

[0420] For testing mutations in the A helix of IL-2v, a HEK-Blue IL-2 reporter assay was performed (Invivogen hkb-il2). Activation of the IL-2 signaling pathway in HEK-Blue IL-2 cells results in the expression of secreted alkaline phosphatase (SEAP), which can be detected by an increase in absorbance at 640 nm in the presence of QUANTI-Blue™ solution (Invivogen, Cat. No. rep-qbs). Briefly, HEK-Blue IL-2 cells were detached with Accutase solution (Sigma-Aldrich, A6964), counted, and resuspended in DMEM-HG medium (Sigma-Aldrich, Cat. No. D5796). A total of 4 x 1E4 HEK-Blue IL-2 cells were seeded out per well in 96-well cell-culture plates, and the plates were incubated for 24 h at 37 °C with 5% CO2. Serial dilutions of IL-2v antibody derivatives were prepared in DMEM-HG medium and added to the HEK-Blue IL-2 cells. The plates were incubated for 18 h at 37 °C with 5 % CO2, and IL-2 activity was determined using QUANTI-Blue™ solution (Invivogen, Cat. No. rep-qbs). Briefly, the QUANTI-Blue working solution was prepared according to the manufacturer’s instructions, and 180 pl per well were pipetted into clear-bottom 96-well plates. Thereto 20 pl of cell culture supernatant from the HEK-Blue IL-2 plates were added and the plates were incubated for 15-30 min at 37 °C. Absorbance at 640 nm was measured using a Tecan Infinite F200 Pro plate reader. The data were analyzed and fitted with a three-parameter nonlinear regression using GraphPad Prism 8 software (GraphPad Software, Inc.).

[0421] Example 5

[0422] HEK-Blue IL-2 reporter assays (Figures 13-18)

[0423] In order to assess trans-activation of split IL-2v prodrugs (i.e. binding to a target cell and activating the IL-2R on a neighboring cell), a HEK-Blue IL-2 co-culture assay was performed. The target cells in this assay were either CHO-K1 cells engineered to overexpress PD-1 (PD1-CHO cells, see above and Figure 27), or SK-BR-3 cells, which express a high level of HER2 (Figure 28). The split IL-2v prodrugs contained either PD-1 binding sites or HER2 binding sites for targeting. The specificity and targeting window (EC50 in the presence of the target divided by EC50 in the absence of the target) of IL-2 trans-activation was determined by comparing the condition in the presence of target expression to the condition in the absence of target expression. For the nontarget controls in Figures 13-16, split IL-2v prodrugs were added directly to HEK-Blue IL-2 cells alone, which do not express detectable levels of PD-1 (Figure 26) and express only very minor levels of HER2 (Figure 28). For the non-target controls in Figures 17-18, split IL-2v prodrugs were added to CHO-K1 cells, which do not express PD-1

[0037] , Approximately 24 h before the assay, 1.5 x 1E4 PD1-CHO-K1 / CHO-K1 cells or 5 x 1E4 SK-BR-3 cells were seeded out per well in 96- well cell-culture plates. The plates were incubated at 37 °C with 5 % CO2. The following day, the medium was removed and 7.5 x 1E4 HEK-Blue IL-2 cells (resuspended in DMEM-HG medium) were added per well. In the absence of target cells in Figures 13-16, 7.5 x 1E4 HEK-Blue IL-2 cells were added directly to empty 96-well plates. Serial dilutions of split IL-2v prodrugs were prepared in DMEM-HG medium and added sequentially to the plates. This was performed to avoid pre-mixing of split IL-2v prodrugs at high concentrations, which might lead to premature insolution complementation. The plates were incubated for 21 h at 37°C with 5% CO2, and measurement of IL-2 activity was performed using QUANTLBlue solution, as described above. The data was analyzed and fitted with a three-parameter nonlinear regression using GraphPad Prism 8 software (GraphPad Software, Inc.).

[0424] Example 6

[0425] PDl-overexpressing HEK-Blue IL-2 reporter assays (Figures 19-21)

[0426] In order to assess the cis-activation potency of PD-1 -targeting split IL-2v prodrugs (i.e. binding to a target cell and activating the IL-2R on the same cell), a HEK-Blue IL-2 assay was performed using engineered HEK-Blue IL-2 cells that overexpress PD-1 (see Figure 26 for expression data). These engineered PD1 -HEK-Blue IL-2 cells serve as both the target cells and reporter cells in this assay. The specificity and targeting window of IL-2v cis-activation was determined by comparing the activity of split IL-2v prodrug pairs on PD1 -HEK-Blue IL-2 cells versus HEK-Blue IL-2 cells, which lack PD-1 expression (Figure 26). The assay protocol was as follows: 5 x 1E4 PD1-HEK- Blue IL-2 cells or 5 x 1E4 HEK-Blue IL-2 cells were added per well into 96-well cell-culture plates in DMEM-HG medium. Serial dilutions of split IL-2v prodrugs were prepared in DMEM- HG medium and added sequentially to the plates. This was performed to avoid pre-mixing of split IL-2v prodrugs at high concentrations, which might lead to premature in-solution complementation. The plates were incubated for 20-24 h at 37°C with 5% CO2, and measurement of IL-2 activity was performed using QUANTLBlue solution, as described above. The data was analyzed and fitted with a three-parameter nonlinear regression using GraphPad Prism 8 software (GraphPad Software, Inc.).

[0427] Example 7

[0428] IL-2R signaling (STAT5-P) in PD-1+ and PD-l-blocked human CD4+ T-cells (Figure 22)

[0429] Blood samples from healthy volunteers were obtained from a blood donation center (Zurich, Switzerland) with approval of the Cantonal Ethics Committee (Zurich). PBMCs were isolated from the blood of different healthy donors using density gradient centrifugation with Histopaque- 1077 (Sigma- Aldrich). All cells were cultured in RPML1640 medium (Gibco) supplemented with 10% heat-inactivated FBS (Gibco), GlutaMAX (Gibco), and 1% penicillin-streptomycin (Gibco, #15-140-122, 100X). Human CD4+ T-cells were isolated by using a CD4+ selection bead system following the manufacturer’s instructions (Miltenyi Biotec). CD4 positive cells (CD4+ cells) were activated at 37°C with 1 pg / mL of plate-bound anti-human CD3 antibody (BioLegend, #317326) and soluble anti-human CD28 antibody (BioLegend, #302934). After 3 days of in vitro activation, the cells were collected and washed multiple times to remove endogenous IL-2. Half of the cells was treated with 10 pg / mL of the non-IL-2v conjugated anti-PD-1 antibody for 30 min at room temperature to block the PD-1 epitope. Thereafter, unbound anti-PD-1 antibody was removed by washing. The other half of the cells were labeled with 5 pM of CellTrace Violet (#C34557, Thermo Fisher) and were not treated with anti-PD-1 antibody.

[0430] To assess IL-2R signaling (STAT5-P) and the cis / trans binding on CD4+ cells following treatment with PD-1 -targeting IL-2v prodrugs, both anti-PD-1 antibody-pretreated and untreated CD4+ cells were mixed 1 : 1 and incubated with increasing concentrations of the split IL-2v prodrug molecules for 60 min at 37°C. Directly after the incubation, cells were fixed with Phosphoflow Fix Buffer I (BD Biosciences, #557870) and incubated for 30 min at 37°C. Cells were then permeabilized overnight at -80 °C with Phosphoflow PermBuffer III (BD Biosciences, #558050) before being stained for 30 min at 4 °C with anti-STAT5-P-AF647 antibody (1 :20 dilution; BD Biosciences, #562076). Flow cytometry acquisition was performed on BD FACSymphony instrument (BD Biosciences) and analyzed using FlowJo software (FlowJo LLC). The percentage of cells expressing STAT5-P was measured on both CTV-positive cells (PD-1+ cells, unblocked) and CTV-negative cells (PD-l-blocked cells) (CTV: CellTrace Violet; Thermo Fisher, # C34557). CD4+ cells were identified based on size and granularity by forward versus side scatter (FSC vs SSC) gating. Thereafter, doublets were excluded based on FSC-A versus FSC-H gating. PD-1+ (unblocked) versus PD-l-blocked cells were differentiated based on CTV expression. The percentage of cells expressing STAT5-P (AF647+ cells) was determined on both PD-1+ (unblocked) and PD-l-blocked cells using statistical tools in FlowJo software (FlowJo LLC).

[0431] Example 8

[0432] Effect of split IL-2v prodrugs on cytotoxic Granzyme B secretion by allospecific human CD4+ T-cells co-cultured with allogeneic mature dendritic cells (Figures 23-24)

[0433] In order to further assess the PD-1 -targeting of split IL-2v prodrugs, CD4+ T-cells were exposed to allogeneic matured dendritic cells (DCs) to induce the generation of allospecific T-cells harboring different antigen-specificities and TCR-affinities. Importantly, allospecific T-cells express immune checkpoints like PD-1 and therefore are useful to measure the functional potency of anti-PD-1 antibodies, as they can unleash T-cell effector functions. To screen for the functionality and potency of PD-1 -targeting split IL-2v prodrugs in an allogeneic setting, freshly purified human CD4+ T-cells were co-cultured for 5 days in the presence of monocyte-derived allogeneic mature dendritic cells (mDCs). Monocytes were isolated from fresh PBMCs one week prior with CD14 beads (Miltenyi Biotec, #130-050-201). Immature DCs were generated from the monocytes by culturing them for 5 days in media containing GM-CSF (50 ng / mL; Peprotech, #300-03-20UG) and IL-4 (100 ng / mL; Peprotech, #200-04-50UG). To induce maturation of immature DCs (iDCs), TNF-a (50 ng / mL; Peprotech, #300-01A-50UG), IL-ip (50 ng / mL; Peprotech, #200-01B-50UG) and IL-6 (50 ng / mL; Peprotech, #200-06-50UG) were added to the culturing media for 2 additional days.

[0434] On the day of the minimal mixed lymphocyte reaction (mMLR), CD4+ T-cells were enriched via a MicroBead kit (Miltenyi Biotec) from PBMCs obtained from an unrelated donor. Prior to coculture, CD4+ T-cells were labeled with 5 pM of CellTrace Violet (CTV; Thermo Fisher, # C34557). 1 x 1E5 CD4+ T-cells were plated in flat-bottom 96-well plates together with mature allogeneic DCs (10: 1 ratio) for 5 days at 37°C, 5% CO2, in the presence of increasing concentrations of split IL-2v prodrugs (50 pL, 1 : 10 dilution steps, with the highest concentration being 66 nM). Five days later, the cells were incubated at 37°C for 5 additional hours in the presence of GolgiPlug (Brefeldin A; BD Biosciences, #555029) and GolgiStop (Monensin; BD Biosciences, #554724), before being washed, surface-stained with anti-human CD4 antibody and Fixable Viability Dye eFluor™ 780 (eBioscience, #65-05865-14), and finally fixed / permeabilized with Fix / Perm Buffer (BD Biosciences, #554722). The cells were then stained intracellularly for Granzyme B (BD Biosciences, #560212). The PD-1 -targeting split IL-2v prodrugs (XVV173 + XVV194) promoted T-cell secretion of Granzyme B (GrzB) in a concentration-dependent manner (Figure 24).

[0435] Example 9

[0436] In vivo tumor-growth inhibition in MC38 subcutaneous syngeneic tumor model (Figure 25)

[0437] PD-1 -targeting split IL-2v prodrugs (XVV173 + XVV194) were tested in the mouse colorectal adenocarcinoma cell line MC38, injected subcutaneously into human PD-1 -transgenic Black 6 mice. The MC38 colorectal tumor cells were routinely cultured in vitro in DMEM supplemented with 10% FCS, at 37°C in a water- saturated atmosphere at 5% CO2. 5 x 1E6 MC38 tumor cells per animal were injected subcutaneously in 200 pL of PBS into the flank of human PD-1- transgenic Black 6 mice using a 1 mL tuberculin syringe. Female human PD-1 -transgenic Black 6 mice, aged 5-6 weeks at the start of the experiment, were maintained under specific pathogen-free conditions with daily cycles of 12 hours light / 12 hours darkness, according to committed guidelines (GV-Solas; Felasa; TierschG). After arrival, the mice were maintained for one week to get accustomed to the new environment and for observation. Continuous health monitoring was carried out on a regular basis.

[0438] Mice were injected subcutaneously on study day 0 with 5 x 1E6 MC38 cells, and were randomized and weighed. Eight days after the tumor cell injection (tumor volume > 100 mm3), the mice were injected i.v. with either the first PD-l-split IL2v prodrug, non-conjugated, murinized anti-human PD-1 antibody, or a vehicle control. On day nine, the group that received the first PD-l-split IL2v prodrug was injected with the second, complementary PD-l-split IL2v prodrug. This schedule was repeated for two more weeks. All mice were injected i.v. with 200 pL of the appropriate solution. The mice in the vehicle control group were injected with histidine buffer. To obtain the proper amount of conjugate per 200 pL, the stock solutions were diluted with histidine buffer when necessary. Figure 25 shows that the PD-l-split IL2v constructs mediated superior efficacy in terms of tumor growth inhibition compared to vehicle and murinized anti-human PD1 monoclonal antibody single agent groups.

Claims

Patent Claims1. A recombinant IL2 variant polypeptide (IL2-v), which is a splittable variant of human interleukin 2 (IL2), comprising the amino acid sequence of SEQ ID NO:34.

2. A recombinant IL2 variant polypeptide (IL2-v(A)), which comprises a splitted variant of human IL2 that comprises helix A of IL2, comprising SEQ ID NO:92, or a variant thereof that comprises one amino acid substitution at position 11 of SEQ ID NO:92, or a variant thereof that comprises one or more amino acid substitutions, wherein the amino acid substitution is at position 16 and / or at position 20 of SEQ ID NO:92, or a variant thereof that comprises two or more amino acid substitutions, wherein the at least one of the amino acid substitutions is at position 16 and / or 20 of SEQ ID NO:92, and wherein at least one of the amino acid substitutions is at position 11, 14 and / or 25 of SEQ ID NO:92.

3. The recombinant polypeptide of claim 2, wherein the substitutions are selected from: a substitution at position 11 selected from QI IK or QI IE, a substitution at position 14 orL14A, a substitution at position 16 of H16L, a substitution at position 20 of D20T, and a substitution at position 25 of L25A.

4. A recombinant IL2 variant polypeptide (IL2-v(BCD)), which comprises a splitted variant of human IL2 that comprises helices B, C and D of IL2, comprising SEQ ID NO:93, or a variant thereof that comprises one amino acid substitution at position 102 of SEQ ID NO:93.

5. The recombinant polypeptide of claim 4, wherein the substitution at position 102 is T102D.

6. A recombinant IL2 variant polypeptide (IL2-v(BCD-dA)), which comprises an inactivated splitted variant of human IL2 that comprises helix A of IL2 and a splitted variant of human IL2 that comprises helices B, C and D of IL2, comprising a) an amino acid sequence of SEQ ID NO:92 with two or more amino acid substitutions, wherein the at least one of the amino acid substitutions is at position 16 and / or 20 of SEQ ID NO:92, and wherein at least one of the amino acid substitutions is at position 11, 14 and / or 25 of SEQ ID NO: 92, and b) an amino acid sequence of SEQ ID NO:93 with one amino acid substitution at position 102 of SEQ ID NO:93.

7. The recombinant polypeptide of claim 6, wherein the substitutions in SEQ ID NO:92 are selected from: a substitution at position 11 selected from QI IK or QI IE, a substitution at position 14 or L14A, a substitution at position 16 of H16L, a substitution at position 20 of D20T, and a substitution at position 25 of L25, and wherein the substitution at position 102 of SEQ ID NO:93 is T102D.

8. A pharmaceutical composition comprising a set of recombinant polypeptides, comprising: a) the recombinant IL2-v(A) polypeptide of claim 2 or 3, and b) the recombinant IL2-v(BCD-dA) polypeptide of claim 6 or 7.

9. A composition comprising a first and a second precursor protein, wherein each precursor protein comprises an antibody binding site and an Fc domain, wherein the first precursor protein comprises the recombinant IL2-v(A) polypeptide of claim 2 or 3, and wherein the second precursor protein comprises the the recombinant IL2-v(BCD-dA) polypeptide of claim 6 or 7.

10. The composition of claim 9, wherein the recombinant IL2-v(A) polypeptide of claim 2 or 3 is fused to the C-terminus of the Fc domain of the first precursor protein and wherein the recombinant IL2-v(BCD-dA) polypeptide of claim 6 or 7 is fused to the C-terminus of the Fc domain of the second precursor protein.

11. The composition of claim 9, wherein the recombinant IL2-v(A) polypeptide of claim 2 or 3 is fused to the N-terminus of the Fc domain of the first precursor protein and wherein the recombinant IL2-v(BCD-dA) polypeptide of claim 6 or 7 is fused to the N-terminus of the Fc domain of the second precursor protein.

12. The composition of one of claims 9 to 11, wherein c) the first precursor protein comprises a recombinant IL2-v(A) polypeptide comprising SEQ ID NO:92 with a substitution at position 11 selected from QI IK or QI IE, and d) the second precursor protein comprises a recombinant IL2-v(BCD-dA) polypeptide comprising SEQ ID NO:34 with substitutions QI IK or QI IE, H16L, D20T, L14A and L25A and T133D.

13. The composition of one of claims 9 to 12, wherein the recombinant IL2-v(A) polypeptide is fused via a peptidic linker to the antibody binding site or to the Fc domain of the first precursorprotein, and wherein the recombinant IL2-v(BCD-dA) polypeptide is fused via a peptidic linker to the antibody binding site or to the Fc domain of the second precursor protein.

14. The composition of claim 13, wherein the peptidic linker that has an amino acid sequence of (Gly4Ser)x3 G5 (SEQ ID NO: 33).

15. The composition of one of claims 9 to 14, wherein the antibody binding sites of the first and the second precursor protein bind to a tumor associated antigen.

16. The composition of one of claims 9 to 15, wherein the antibody binding sites of the first and the second precursor protein are Fab fragments.

17. The composition of one of claims 9 to 16, wherein the first precursor protein binds to a first target and the second precursor protein binds to a second target, or wherein the first precursor protein binds to a first epitope on the target and the second precursor protein binds to a second, different epitope of the target.

18. The composition of one of claims 9 to 17 further comprising a pharmaceutically acceptable carrier.

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