immunoconjugates

Abstract

The invention provides immunoconjugates and methods of using the same.

Description

[0001] P39701-WQ-1

[0002] IMMUNOCONJUGATES

[0003] TECHNICAL FIELD

[0004] The present invention generally relates to immunoconjugates, particularly immunoconjugates comprising a mutant interferon alpha 2a polypeptide and an antibody that binds to PD-L1. In addition, the invention relates to polynucleotide molecules encoding the immunoconjugates, and vectors and host cells comprising such polynucleotide molecules. The invention further relates to methods for producing the mutant immunoconjugates, pharmaceutical compositions comprising the same, and uses thereof.

[0005] BACKGROUND

[0006] The type-I interferons (IFN) are a family of cytokines that play a key role in inflammation, immunoregulation, tumor cell recognition, and T-cell response. Interferon alpha (IFN-a) is a type-I interferon that is naturally produced by the immune system to attack viruses and cancer cells. It has been extensively studied and found to be effective in the treatment of certain types of cancer, such as melanoma, renal cell carcinoma, and chronic myeloid leukemia. The mechanism of action of IFN-a involves inhibition of the tumor cell growth and boosting of the immune system’s ability to recognize and destroy cancer cells.

[0007] The term “IFN-a” stands for a family of cytokines that comprises multiple subtypes, including IFN-al, IFN-a2 and several others. IFN-al and IFN-a2 differ in their amino acid sequence, but both have similar biological activities and are used clinically for the treatment of cancer and viral infections. IFN-a2 has been shown to be more potent than IFN-al in terms of its antiviral and antiproliferative activities.

[0008] The mode of action of IFN-a involves binding to its receptor IFNAR, which is composed of the IFNAR1 and IFNAR2 subunits, a crucial step in the activation of the JAK-STAT signaling pathway. This leads to the transcription of genes involved in immune cell activation and apoptosis of cancer cells. The binding of IFN-a to its receptor is a key molecular event that underlies the therapeutic efficacy of IFN-a in the treatment of cancer.

[0009] The therapeutic use of IFN-a can have significant side effects, including flu-like symptoms such as fever, chills, and fatigue. For this reason, the targeting of IFN-a to tumor cells and to immune cells in the tumor microenvironment (TME) is a highly promising approach. Targeted therapies use agents such as antibodies or antigen-binding domains to specifically target cancer cells based on their unique molecular characteristics, such as overexpression of certain tumor markers on their surface. By selectively targeting cancer cells and immune cells in the TME, damage of healthy cells and / or tissues is avoided, reducing the incidence and severity of undesirable side effects. Furthermore, due to their specificity, targeted therapies can achieve high response rates and improved outcomes in patients. Overall, the benefits of targeted tumor therapy suggest that this approach has the potential to significantly improve outcomes for patients with cancer, while minimizing the toxicity and inconvenience associated with traditional chemotherapy.

[0010] In the case of IFN-a, however, targeting alone may not be sufficient to prevent IFN-a from exercising its activity also in the periphery, i.e. in healthy cells and tissues. Thus, novel therapeutic agents are needed that are capable of preventing IFN-a from acting on its receptor unless the molecule is in the proximity of a tumor cell.

[0011] WO2017 / 134302 discloses chimeric proteins that utilize targeting moieties that specifically bind to PD-L1 and modified signaling agents with mutations for improved safety and efficacy in treating various diseases, particularly cancer.

[0012] W02019 / 201161 discloses an IFN-anti-PD-Ll fusion protein targeting PD-L1 and IFN receptors to enhance immune response against tumors by promoting T cell reactivation and antigen presentation.

[0013] WO2020 / 198654 discloses a chimeric protein system utilizing IFN-al and targeting moieties to selectively activate IFN AR signaling at target cells, offering improved therapeutic potential for cancer, autoimmune diseases, and infections.

[0014] WO2022 / 140797 discloses immunocytokines which contain an IgG-like antigen-binding protein that recognizes a target antigen, wherein the cytokine is located at hinge region of the antigen-binding protein. These molecules are designed to enhance cytokine activity in the presence of a target antigen, potentially improving treatment efficacy. In some embodiments, an IFN-a (e.g., IFN-a2b) / anti-PD-Ll immunocytokine is provided. I. SUMMARY

[0015] The present invention provides a novel approach of targeting a mutant form of IFN-a2 with advantageous properties for immunotherapy directly to cancer cells and immune cells in the tumor micro environment, thus avoiding damaging of healthy cells and / or tissues and reducing the incidence and severity of undesirable side effects. Targeting to cancer and immune effector cells is achieved by conjugation of the mutant IFN-a2 molecule to an antibody that binds to PD-L1.

[0016] The IFN-a2 mutant used in the present invention has been designed to overcome the problems associated with IFN-a2 immunotherapy, in particular toxicity caused by off-target IFN-a activity. By using an antibody that binds to PD-L1, the suppression of T-cell activity induced by the interaction of PD1 with its ligand PD-L1 may additionally be reversed, thus further enhancing the immune response.

[0017] Of note, the immunoconjugate of the invention, comprising an antibody that binds to PD-L1, shows significantly superior anti-tumor efficacy in vivo as compared to a similar immunoconjugate targeting PD-L1 (see the Examples herein below). They further demonstrate low cytokine induction as measured in a human whole blood assay, in human NK-92 cells and in human primary dendritic cell subsets, and reduced stimulation of human primary T cells, indicating an improved safety profile.

[0018] In a general aspect, the invention provides an immunoconjugate comprising an antibody that binds to PD-L1 and a polypeptide that signals through IFNAR. The polypeptide signaling through IFNAR is particularly an IFN-a2 polypeptide. In a first aspect, the invention provides an immunoconjugate comprising a mutant IFN-a2 polypeptide and an antibody that binds to PD-L1, wherein the mutant IFN-a2 polypeptide is a human IFN-a2 molecule comprising at last one amino acid substitution selected from the group consisting of L30A, L30H, and L153D (numbering relative to the mature human IFN-a2 sequence SEQ ID NO: 17). In a particular aspect, the invention provides an immunoconjugate comprising a mutant IFN-a2 polypeptide and an antibody that binds to PD-L1, wherein the mutant IFN-a2 polypeptide is a human IFN-a2 molecule comprising the amino acid substitution L30A (numbering relative to the mature human IFN-a2 sequence SEQ ID NO: 17). In a further aspect, the invention provides an immunoconjugate comprising a mutant IFN-a2 polypeptide and an antibody that binds to PD-L1, wherein the mutant IFN-a2 polypeptide is a human IFN-a2 molecule comprising at last one amino acid substitution selected from the group consisting of L30A, L30H, and L153D, in particular at least the amino acid substitution L30A (numbering relative to the human IFN-a2 sequence SEQ ID NO: 17); and

[0019] wherein the antibody comprises (a) a heavy chain variable region (VH) comprising a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and (b) a light chain variable region (VL) comprising a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6, or

[0020] wherein the antibody comprises (a) a heavy chain variable region (VH) comprising a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9, a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11, and (b) a light chain variable region (VL) comprising a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12, a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14.

[0021] In another aspect, the invention provides an immunoconjugate comprising a mutant IFN-a2 polypeptide and an antibody that binds to PD-L1, wherein the mutant IFN-a2 polypeptide is a human IFN-a2 molecule comprising at last one amino acid substitution selected from the group consisting of L30A, L30H, and L153D, in particular at least the amino acid substitution L30A (numbering relative to the human IFN-a2 sequence SEQ ID NO: 17); and

[0022] wherein the antibody comprises (a) a heavy chain variable region (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 7, and (b) a light chain variable region (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 8, or

[0023] wherein the antibody comprises (a) a heavy chain variable region (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 15, and (b) a light chain variable region (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 16.

[0024] In some embodiments, the mutant IFN-a2 polypeptide comprises a sequence selected from the group consisting of SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24. In some embodiments, the mutant IFN-a2 polypeptide comprises the sequence of SEQ ID NO: 22. In some embodiments, the immunoconjugate comprises not more than one mutant IFN-a2 polypeptide. In some embodiments, the immunoconjugate comprises an anti-PD-Ll antibody that is an antibody fragment selected from the group consisting of a Fv, a Fab, a Fab’, a scFv, and a F(ab’)2. In some embodiments the Fab is a DutaFab. In some embodiments, the antibody comprises an Fc domain composed of a first and a second subunit. In some such embodiments, the Fc domain is an IgG class, particularly an IgGi subclass, Fc domain, and / or the Fc domain is a human Fc domain. In some embodiments, the antibody is an IgG class, particularly an IgGi subclass immunoglobulin.

[0025] In some embodiments wherein the immunoconjugate comprises an Fc domain, the Fc domain comprises a modification promoting the association of the first and the second subunit of the Fc domain. In some embodiments, in the CH3 domain of the first subunit of the Fc domain an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain of the first subunit which is positionable in a cavity within the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit is positionable. In some embodiments, in the first subunit of the Fc domain the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the second subunit of the Fc domain the tyrosine residue at position 407 is replaced with a valine residue (Y407V) and optionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numberings according to Kabat EU index). In some such embodiments, in the first subunit of the Fc domain additionally the serine residue at position 354 is replaced with a cysteine residue (S354C) or the glutamic acid residue at position 356 is replaced with a cysteine residue (E356C), and in the second subunit of the Fc domain additionally the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C) (numberings according to Kabat EU index). In some embodiments, the mutant IFN-a2 polypeptide is fused at its amino-terminal amino acid to the carboxy -terminal amino acid of one of the subunits of the Fc domain, particularly the first subunit of the Fc domain, optionally through a linker peptide. In some such embodiments, the linker peptide has the amino acid sequence of SEQ ID NO: 25.

[0026] In some embodiments wherein the immunoconjugate comprises an Fc domain, the Fc domain comprises one or more amino acid substitution that reduces binding to an Fc receptor, particularly an Fey receptor, and / or effector function, particularly antibody-dependent cell-mediated cytotoxicity (ADCC). In some such embodiments, said one or more amino acid substitution is at one or more position selected from the group of L234, L235, and P329 (Kabat EU index numbering). In some embodiments, each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A and P329G (Kabat EU index numbering).

[0027] In some embodiments, the immunoconjugate according to the invention comprises a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 33, SEQ ID NO: 36, and SEQ ID NO: 37, a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 34, and a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 35. In some embodiments, the immunoconjugate according to the invention comprises a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 38, a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 39, and a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 40. In some embodiments, the immunoconjugate essentially consists of a mutant IFN-a2 polypeptide and an IgGi immunoglobulin molecule, joined by a linker sequence.

[0028] The invention further provides one or more isolated polynucleotide encoding an immunoconjugate of the invention, one or more vector (particularly expression vector) comprising said polynucleotides, and host cells comprising said polynucleotide(s) or said vector(s).

[0029] Also provided is a method of producing an immunoconjugate comprising a mutant IFN-a2 polypeptide and an antibody that binds to PD-L1, comprising (a) culturing the host cell of the invention under conditions suitable for the expression of the immunoconjugate, and optionally (b) recovering the immunoconjugate. Also provided by the invention is an immunoconjugate comprising a mutant IFN-a2 polypeptide and an antibody that binds to PD-L1, produced by said method.

[0030] The invention further provides a pharmaceutical composition comprising an immunoconjugate of the invention and a pharmaceutically acceptable carrier, and methods of using an immunoconjugate of the invention.

[0031] In particular, the invention encompasses an immunoconjugate according to the invention for use as a medicament, and for use in the treatment of a disease. In a particular embodiment, said disease is cancer.

[0032] Also encompassed by the invention is the use of an immunoconjugate according to the invention in the manufacture of a medicament for the treatment of a disease. In a particular embodiment, said disease is cancer.

[0033] Further provided is a method of treating disease in an individual, comprising administering to said individual a therapeutically effective amount of a composition comprising an immunoconjugate according to the invention in a pharmaceutically acceptable form. In a particular embodiment, said disease is cancer.

[0034] Also provided is a method of stimulating the immune system of an individual, comprising administering to said individual an effective amount of a composition comprising an immunoconjugate according to the invention in a pharmaceutically acceptable form.

[0035] II. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Schematic illustration of the immunoconjugate used in the Examples (Figure 1

[0037] A), comprising an antibody that binds to PD-L1 and an IFN-a2 mutant polypeptide. The anti-PD-Ll antibody is an IgG class immunoglobulin and comprises a human IgGi Fc domain composed of a first and a second subunit which comprise modifications in the Fc domain promoting heterodimerization. In the first subunit of the Fc domain the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the second subunit of the Fc domain the tyrosine residue at position 407 is replaced with a valine residue (Y407V) and optionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numberings according to Kabat EU index). Further, each subunit of the Fc domain comprises the “P329G LALA” amino acid substitutions (i.e. L234A, L235A and P329G (Kabat EU index numbering)) in order to reduce Fey receptor (as well as complement) binding of the human IgGi Fc domain.

[0038] The mutant IFN-a2 polypeptide here is fused at its amino-terminal amino acid to the carboxy-terminal amino acid of the first subunit of the Fc domain, e.g. through the linker peptide of SEQ ID NO: 25. (Black dot: modification in the Fc domain promoting heterodimerization.)

[0039] Figure IB shows schematic representations of different formats of the immunoconjugates of the invention that were used in the examples. On the very left is the form also embodied by Figure 1 A wherein the IFN-a2 mutant is fused by its N-terminus to the C-terminal end of the first subunit of the Fc domain. In the middle, a one-armed format is shown wherein the anti-PD-Ll antibody is a Fab fused by its C-terminus to the N-terminus of the second subunit of the Fc domain while the IFN-a2 mutant is fused via its N-terminus and via a peptide linker to the C-terminus of the first subunit of the Fc domain. On the right side, an untargeted Fc-linked IFN-a2 mutant is shown which was used as a control in some of the examples included herein.

[0040] Figure 2 Results of an SPR assay demonstrating lack of competitive binding of various tested anti-PD-Ll Fabs with Atezolizumab. The tested anti-PD-Ll Fabs were derived from the anti-PD-Ll binding CDRs (H-side) of the binder M14H (parental), as described in Example 1 b. The sensorgram shows that the tested Fabs did not compete with Atezolizumab binding to PD-L1. Figure 3 Results of an SPR assay demonstrating the inhibition of PD1 / PD-L1 interaction by various anti-PD-Ll Fabs. The two tested anti-PD-Ll Fabs (P1AI4475 “M14” and P1AI4392) were derived from the binder M14 (parental), as described in Example 1 b. The sensorgrams show that the two M14H (parental)-derived anti- PD-Ll Fabs block binding of PD-L1 to PD1, like the Atezolizumab-based anti- PD-Ll Fab (P1AI4841) which was used as a positive control. P1AH0183 is a PD-L1 -specific antibody, which does not block PD-L1 / PD1 interaction, and was used here as a control.

[0041] Figure 4 Results of a PD1 / PD-L1 blockade bioassay, demonstrating that M14 (parental- derived anti-PD-Ll Fabs can block PD1 / PD-L1 interaction also in a cellular context, whereas monovalent M14(parental) derived anti-PD-Ll Fabs show nearly no blockage.

[0042] Figure 5 Schematic illustration of the activity assay testing immunoconjugates on low and high expressing PD-L1 HEK-Blue IFN-a / p reporter cells. When not enough PD-L1 molecules are present on the cell surface, the affinity of the IFN-a2 mutant is too low to bind to its cell surface receptor IFNAR1 / 2. No activation of the reporter cells and consequently also no reporter gene expression is achieved. When sufficient PD-L1 is expressed on the cell surface, the antibody of the immunoconjugate binds to PD-L1 on the cell surface. The “apparent” concentration of the IFN-a2 mutant on the surface of the reporter cell is now sufficiently increased to overcome its low affinity for the IFNAR1 / 2 receptor and to trigger detectable SEAP production of the reporter cells (the figure was created with BioRender.com).

[0043] Figure 6 In vitro activation of PD-L1 transfected HEK-Blue IFN-a / p cells ("Clone 045") by different immunoconjugates having IFN-a2 mutants with different linker lengths (+: antibody with 15 aa linker; *: antibody with 60 aa linker) between the IFN-a2 mutant and the Fc domain.

[0044] Figure 7 HMW and LMW formation for stressed immunoconjugate samples dependent on linker length. An unstressed reference was used as control. Figure 8 In vitro activation of parental HEK-Blue IFN-a / p cells ("parental") or PD-L1 transfected HEK-Blue IFN-a / p cells ("Clone 045") by immunoconjugates having IFN-a2 mutants with different amino acid substitutions, or recombinant human IFN-a2a, after 24 hours of incubation. Some HEK cells were preincubated with the PD-L1 blocking antibody Atezolizumab to evaluate the potential for PD-L1 -independent activation by the different molecules. Conditions with anti-PD-Ll antibody pre-incubation are indicated in the figure by “molecule ID + PD-L1”. The figures display the relative luminescence units (RLU), which indicate the activity of the reporter gene alkaline phosphatase (SEAP). n = 4.

[0045] Figure 9 In vitro activation of parental HEK-Blue IFN-a / p cells ("parental", Figure 9 A)

[0046] or PD-L1 transfected HEK-Blue IFN-a / p cells ("Clone 045", Figure 9 B) by immunoconjugates having IFN-a2 mutants with different amino acid substitutions, either in a two-armed format (with a bivalent anti-PD-Ll IgGl antibody) as shown in Figure 1 A, or as a one armed format (with one monovalent Fab fused to an Fc domain as shown in Figure 1 B, in the middle), after 24 hours of incubation. Recombinant human IFN-a2a was used as a control. The figures display the relative luminescence units (RLU), which indicate the activity of the reporter gene alkaline phosphatase (SEAP). n = 4.

[0047] Figure 10 ECso values calculated from dose-response curves of parental HEK-Blue IFN-a / p cells (-300 PD-L1 per cell, Figure 10 A) or transfected with PD-L1 (-25,000 or -200,000 PD-L1 per cell, Figure 10 B and Figure 10 C) and treated with different concentrations of monovalent and bivalent immunoconjugates or recombinant IFN-a2a for 24 h. n = 4.

[0048] Figure 11 Relative ECso values of the in vitro activation of parental HEK-Blue IFN-a / p cells versus PD-L1 transfected (“cl045” = clone 045 or “cl39” = clone 039) HEK-Blue IFN-a / p cells for the tested immunoconjugate P1AI4283 after 24 hours of incubation, compared to the reference molecule (P1AF8334). Recombinant human IFN-a2a was used as a control.

[0049] Figure 12 Direct comparison of in vitro activation of parental HEK-Blue IFN-a / p cells ("parental", Figure 12 A) or PD-L1 transfected HEK-Blue IFN-a / p cells ("Clone 045", Figure 12 B) between the immunoconjugate (Molecule ID: Pl A4283) and the reference molecule (Molecule ID: Pl AF8334) after 24 hours of incubation. Recombinant human IFN-a2a was used as a control. The figures display the optical density (OD) at a wavelength of 620 nm, which correlates with the activity of the reporter gene alkaline phosphatase (SEAP). n = 4. Recombinant human IFN-a2a was used as a control.

[0050] Figure 13 Cytokine response (Figure 13 A: IL6, Figure 13 B: TNFa, Figure 13 C: IFN- y) of human PBMCs after 24 hours in vitro stimulation with immunoconjugates with targeted attenuated IFN-a2a molecules. Recombinant human IFN-a2a (labelled “hu rec IFNa”) was used as a positive control. Recombinant human IFN-a2a was used as a control.

[0051] Figure 14: Results of IFN-y ELISPOT analysis. In vitro IFN-y secretion induced by different immunoconjugates or recombinant IFN-a2a was determined in human PBMCs after 24 hours incubation. The figure shows the number of spot forming cells dependent on the antibody concentration in nM, n= 2; error bars represent standard deviation.

[0052] Figure 15 Inhibition of human tumor cell proliferation in vitro by 10 or 100 nM of the immunoconjugate (Molecule ID: Pl AI4283), the reference molecule (Molecule ID: P1AF8334) or recombinant IFN-a2a in different tumor cell lines (Figure 15 A: HCC1954, Figure 15 B: LOVO, Figure 15 C: OVCAR-3). The confluence of the cells was measured every 4 hours for up to ten days, n=2.

[0053] Figure 16 CXCL10 (IP-10) induction by the immunoconjugate (P1AI4283), the reference molecule (Molecule ID: P1AF8334) or recombinant IFN-a2a in vitro in different human tumor cell lines (HCC1954, OVCAR-3). Error bars represent standard deviation. The levels of CXCL10 in the supernatant were measured by ELISA after 24 hours incubation, n=2. Figure 16 A: HCC1954: 30,000 cells.

[0054] Figure 16 B shows a direct comparison of the ECso values calculated for the curves shown in Figure 16 A. Figure 16 C: OVCAR-3: 30,000 cells.

[0055] Figure 17 Induction of MHCI (Figure 17A) and PD-L1 (Figure 17B) expression of HCC1954 cells over time with immunoconjugate (Molecule ID: P1AI4283), the reference molecule (Molecule ID: Pl AF8334) and recombinant human IFN-a2.

[0056] Figure 17 A shows the MHCI and Figure 17 B shows the green PD-L1 object count per image over time. Of note, in contrast to Pl AI4283, Pl AF8334 blocks the binding of the anti-PD-Ll staining antibody. Thus, for P1AF8334 PD-L1 expression over time cannot be measured.

[0057] Figure 18 In vitro IFN-y secretion induced by immunoconjugate (Molecule ID:

[0058] P1AI4283), the reference molecule (Molecule ID: P1AF8334) or recombinant IFN-a2a in human NK-92 cells after 48 hours incubation. The figure shows pg / ml IFN-y secretion dependent on the antibody concentration in nM, n= 2; error bars represent standard deviation.

[0059] Figure 19 In vitro human CD80 expression of pDCs (Figure 19A), DC1 (Figure 19B)

[0060] and DC2 (Figure 19C) treated with immunoconjugate (Molecule ID: P1AI4283), the reference molecule (Molecule ID: P1AF8334), an untargeted Fc-fused mutant IFN-a2a (Molecule ID: P1AG0745), or recombinant IFN-a2a measured after 24h incubation. The figures are showing the geometric mean of CD80 expression on different DC subsets treated with 10 nM of indicated antibodies or recombinant IFN-a2a.

[0061] Figure 20 T-cell activation after 24 hours incubation with 10 nM immunoconjugate (Molecule ID: Pl AI4283), the reference molecule (Molecule ID: P1AF8334) or recombinant IFN-a2a. The figures show the percentage of CD69 expressing CD4+ (Figure 20A) or CD8+ (Figure 20B) T cells. Error bars represent standard deviation.

[0062] Figure 21 In vitro CXCL10 (Figure 21 A) and IL6 (Figure 21B) secretion in pg / ml of fresh human whole blood incubated with indicated immunoconjugate (Molecule ID: P1AI4283), reference molecule (Molecule ID: P1AF8334) or recombinant human IFN-a2a at a concentration of 4 nM for 24 h. Shown are three independent experiments with a total number of 11 donors. Error bars represent standard deviation, p-values indicated by asterisks are ****■. p<0.0001 and *: p<0.05. Figure 22 Internalization of 10 nM immunoconjugate (Molecule ID: P1AI4283) and reference molecule (Molecule ID: P1AF8334), which were conjugated to human / mouse Fabfluor pH Red Labeling reagent, into the PD-L1 expressing human tumor cell line HCC1954. The figure shows the red object count per image within 48 h incubation. A higher signal correlates with an increased internalization rate. Error bars represent standard deviation.

[0063] Figure 23 Results of the efficacy study of immunoconjugate (P1AI4283) in MDA-MB- 231 tumor-bearing humanized NSG mice. Tumor volume was measured by caliper 2 times a week. Figure 23 shows a direct comparison of the results of all subgroups, over a period from day 22 (start of treatment) to day 51 (study termination). Data is shown as tumor volume (Median + / -IQR) over time.

[0064] Figure 24 Sequence of wildtype mature human Interferon alpha 2a.

[0065] III. DEFINITIONS

[0066] 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.

[0067] Unless otherwise defined herein the term “comprising of’ shall include the term “consisting of’.

[0068] The term “about” as used herein in connection with a specific value (e.g. temperature, concentration, time and others) shall refer to a variation of + / - 1 % of the specific value that the term “about” refers to.

[0069] The terms “Interferon alpha”, “Interferon a”, “IFN alpha” or “IFN-a”, as used herein, refers to any native Interferon alpha from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length”, unprocessed Interferon alpha as well as any form of Interferon alpha that results from processing in the cell, unless otherwise indicated. The term also encompasses naturally occurring variants of Interferon alpha, e.g., splice variants or allelic variants. Interferons (IFNs) are a family of proteins that were originally named for their ability to interfere with viral replication and propagation. To date, it is known that interferons are also involved in combating bacterial and parasitic infections, inhibit cell division, and promote or impede the differentiation of cells. The interferons are classified based on their receptor specificity into three types of interferons: type I, type II and type III. The interferons of type I are monomeric proteins and include IFN alpha, IFN beta and IFN omega that are products of leukocytes and fibroblasts, IFN kappa that is expressed by human keratinocytes, IFN epsilon that is exclusively expressed in lung, brain, small intestine and the reproductive tissue and IFN tau that has been described only in ruminants. The term "type I interferon" as used herein is intended to refer to members of the type I interferon family of molecules that are ligands for IFNAR-I (i.e., members of the type I interferon family of molecules that are capable of binding IFNAR-I). Examples of type I interferon ligands are interferon alpha 1, 2a, 2b, 4, 5, 6, 7, 8, 10, 14, 16, 17, 21, interferon beta and interferon omega.

[0070] The interferon alpha family is composed of 13 intron-less fully translated genes (excluding pseudogenes). Each member includes mature proteins of 165 or 166 amino acid residues, with two conserved disulfide bonds: Cysl-Cys98 and Cys29-Cysl38. A high level of sequence homology (70-99%) is displayed among the various interferon alpha subtypes, and about 35% homology exists between these subtypes and IFN beta. Despite the high homology, a shared 3D core structure and a shared receptor of the different subtypes, their biological activities, among them anti-proliferative, antiviral, and immunomodulation, differ notably.

[0071] Of the known IFN alpha subtypes, only interferon alpha 2 (IFN-a2) has been extensively studied for its pharmaceutical potential. IFN-a2 is known to have anti-cancer effects. It is mainly used in second line adjunct therapy of hematopoietic cancers. However, this treatment is not always effective and sometimes results in intolerable side effects related to the dosage and duration of therapy. Three alleles exist: Interferon alpha-2A, Interferon alpha-2B and Interferon alpha-2C. In nature, allele alpha-2B is the predominant allele while allele alpha-2A is less predominant and alpha-2C only a minor allelic variant. The terms “human IFN-a”, “human IFN-a polypeptide”, “human IFN-a2”or “human IFN-a2 polypeptide” may for ease of reading also be referred to as “huIFN-a”, “huIFN-a polypeptide”, “huIFN-a2” or “huIFN-a2 polypeptide” herein. The terms “human Interferonalpha 2”, “human Interferon-a 2”, “human IFN-alpha 2”, or “human IFN-a2”, as used herein, relate to mature human interferon alpha 2 either of the human IFN-a2a allele, having the amino acid sequence set forth in SEQ ID NO: 17, or of the mature human IFN-a2b allele, having the amino acid sequence set forth in SEQ ID NO: 20. They relate preferably to human IFN-a2 of the human IFN-a2a allele having the amino acid sequence set forth in SEQ ID NO: 17. They may also relate to the full-length precursors of the respective human IFN-a2 alleles, i.e. IFN-a2a or IFN-a2b comprising an N-terminal signaling peptide of SEQ ID NO: 18. In one aspect, the mature human IFN-a2 has the sequence of the human IFN-a2a precursor of SEQ ID NO: 19. In one aspect, the human IFN-a2 has the sequence of the human IFN-a2b precursor of SEQ ID NO: 21.

[0072] The terms may further relate to functional variants of these proteins. Such functional variants may be homologues of human IFN-a2 that have typically at least one amino acid exchange that does not significantly impair functionality of the protein, i.e. binding to and / or activating of the human IFNAR1 / 2 receptor. The sequence identity for such variants is thus typically higher than 95%, often more than 98%. In one embodiment, such a variant comprises an amino acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the human IFN-a2 sequence of SEQ ID NO: 17. Particularly, the sequence identity is at least about 95%, more particularly at least about 98%. In particular embodiments, the human IFN-a2 molecule is a full-length IFN-a2a molecule. Functional variants of human IFN-a2 may also comprise one or more amino acid exchanges that prevent glycosylation of the human IFN-a2 as disclosed in W02016 / 065409A1. It is understood that the polypeptides described herein may also comprise additional amino acid sequences on the N- or C-terminus, such as a signal peptide, which is also present in naturally occurring human IFN-a2 prior to post-translational processing. Other elements that may be present include various tags or markers that facilitate expression, purification and / or detection, as well as protease recognition sites that allow cleavage of such additional sequence elements. In an embodiment, the human IFN-a2 binds to and / or activates the IFN-a / p receptor (IFNAR), i.e., IFNAR1 and / or IFNAR2. The terms “IFNAR” and “IFNAR1 / 2” are used interchangeably herein and refer to the interferon-a / p receptor, a membrane receptor which binds endogenous type I interferons and consists of the two subunits IFNAR1 and IFNAR2.

[0073] The term "IFN-a2 mutant" or "mutant IFN-a2 polypeptide" as used herein is intended to encompass any mutant form of various forms of the IFN-a2 molecule including full-length IFN-a2, truncated forms of IFN-a2 and forms where IFN-a2 is linked to another molecule such as by fusion or chemical conjugation. "Full-length" when used in reference to IFN-a2 is intended to mean the mature, natural length IFN-a2 molecule. For example, full-length human IFN-a2a refers to a molecule that has 165 amino acids (see e.g. SEQ ID NO: 17). The various forms of IFN-a2 mutants are characterized in having at least one amino acid mutation affecting the interaction of IFN-a2 with IFNAR, in particular reducing their affinity for the IFNAR1 and IFNAR2 receptor complex (IFNAR), particularly the IFNAR2 receptor complex. In particular, they have reduced or abolished ability to activate IFNAR expressing cells while still retaining some ability to bind IFNAR and activate the IFNAR receptor complex. This mutation may involve substitution, deletion, truncation or modification of the wild-type amino acid residue normally located at that position. Mutants obtained by amino acid substitution are preferred. Unless otherwise indicated, an IFN-a2 mutant may be referred to herein as a mutant IFN-a2 peptide sequence, a mutant IFN-a2 polypeptide, a mutant IFN-a2 protein or a mutant IFN-a2 analog. In some instances, they may also be referred to as “attenuated huIFN-a2” herein.

[0074] Designation of various forms of IFN-a2 is herein made with respect to the sequence shown in SEQ ID NO: 17 (see also Figure 24). Various designations may be used herein to indicate the same mutation. For example, a mutation from leucine at position 30 to alanine can be indicated as 30 A, A30, A30, L30A, or Leu30Ala.

[0075] In some aspects, the IFN-a2 mutant has a biological activity selected from less than 70% less than 60% less than 50% less than 40% less than 30% less than 20% or less than 10% of the biological activity of the wild-type huIFN-a2 of which it is deduced (i.e., the wild-type huIFN-a2 of which the coding sequence has been mutated to obtain the mutant IFN). IFN-a2 mutants confer reduced biological activity and thus reduced off-target activity and off-target toxicity, to the fusion proteins described herein. The targeting of the IFN-a2 mutants achieved by the antibody of the immunoconjugate restores the activity of the IFN-a2 mutant with the degree of activity restoration apparently correlated with the level of targeting biologic on the cells.

[0076] The term “amino acid mutation” as used herein is meant to encompass amino acid substitutions, deletions, insertions, and modifications. Any combination of substitution, deletion, insertion, and modification can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g. reduced binding to IFNAR. Amino acid sequence deletions and insertions include amino- and / or carboxy-terminal deletions and insertions of amino acids. An example of a terminal deletion is the deletion of the alanine residue in position 1 of full-length human IFN-a2. Preferred amino acid mutations are amino acid substitutions. For the purpose of altering e.g. the binding characteristics of an IFN-a2 polypeptide, non-conservative amino acid substitutions, i.e. replacing one amino acid with another amino acid having different structural and / or chemical properties, are particularly preferred. Preferred amino acid substitutions include replacing a hydrophobic by a hydrophilic amino acid. Amino acid substitutions include replacement by non-naturally occurring amino acids or by naturally occurring amino acid derivatives of the twenty standard amino acids (e.g. 4-hydroxyproline, 3 -methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis and the like. It is contemplated that methods of altering the side chain group of an amino acid by methods other than genetic engineering, such as chemical modification, may also be useful.

[0077] As used herein, a “wild-type” form of IFN-a2 is a form of IFN-a2 that is otherwise the same as the mutant IFN-a2 polypeptide except that the wild-type form has a wild-type amino acid at each amino acid position of the mutant IFN-a2 polypeptide. For example, if the IFN-a2 mutant is the full-length IFN-a2 (i.e. IFN-a2 not fused or conjugated to any other molecule), the wild-type form of this mutant is full-length native IFN-a2. If the IFN-a2 mutant is a fusion between IFN-a2 and another polypeptide encoded downstream of IFN-a2 (e.g. an antibody chain) the wild-type form of this IFN-a2 mutant is IFN-a2 with a wild-type amino acid sequence, fused to the same downstream polypeptide. Furthermore, if the IFN-a2 mutant is a truncated form of IFN-a2 (the mutated or modified sequence within the non-truncated portion of IFN-a2) then the wild-type form of this IFN-a2 mutant is a similarly truncated IFN-a2 that has a wild-type sequence. For the purpose of comparing IFN-a2 receptor binding affinity or biological activity of various forms of IFN-a2 mutants to the corresponding wild-type form of IFN-a2, the term wild-type encompasses forms of IFN-a2 comprising one or more amino acid mutation that does not affect IFN-a2 receptor binding compared to the naturally occurring, native IFN-a2. In certain embodiments according to the invention the wild-type IFN-a2 polypeptide to which the mutant IFN-a2 polypeptide is compared comprises the amino acid sequence of SEQ ID NO: 17. In other embodiments the wild-type IFN-a2 polypeptide to which the mutant IFN-a2 polypeptide is compared comprises the amino acid sequence of SEQ ID NO: 20.

[0078] “Affinity” refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., a receptor) and its binding partner (e.g., a ligand). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., an antigen binding moiety and an antigen, or a receptor and its ligand). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD), which is the ratio of dissociation and association rate constants (k0fr and kon, respectively). Thus, equivalent affinities may comprise different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured by well established methods known in the art, including those described herein. A particular method for measuring affinity is Surface Plasmon Resonance (SPR).

[0079] The affinity of the mutant or wild-type IFN-a2 polypeptide for IFNAR can be determined in accordance with the method set forth in the WO 2012 / 107417 by surface plasmon resonance (SPR), using standard instrumentation such as a BIAcore instrument (GE Healthcare) and receptor subunits such as may be obtained by recombinant expression (see e.g. Shanafelt et al., Nature Biotechnol 18, 1197-1202 (2000)). Alternatively, binding affinity of IFN-a2 mutants for IFNAR may be evaluated using cell lines known to express one or the other such form of the receptor. Specific illustrative and exemplary embodiments for measuring binding affinity are described hereinafter.

[0080] As used herein, the term “effector cells” refers to a population of lymphocytes that mediate the cytotoxic effects of IFN-a2. Effector cells include effector T cells such as CD8+cytotoxic T cells, NK cells, lymphokine-activated killer (LAK) cells and macrophages / monocytes as well as dendritic cells.

[0081] As used herein, the term “PDL1”, “human PDL1”, “PD-L1” or “human PD-L1” (also known as Programmed death ligand 1, cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-Hl) refers to the human protein PD-L1 (SEQ ID NO: 45). As used herein, an antibody "binding to PD-L1”, "specifically binding to PD-L1”, “that binds to PD-L1” or “anti-PD-Ll antibody” refers to an antibody that is capable of binding PD-L1, especially a PD-L1 polypeptide expressed on a cell surface, with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting PD-L1. In one embodiment, the extent of binding of an anti-PD-Ll antibody to an unrelated, non-PD-Ll protein is less than about 10% of the binding of the antibody to PD-L1 as measured, e.g., by radioimmunoassay (RIA) or flow cytometry (FACS) or by a Surface Plasmon Resonance assay using a biosensor system such as a Biacore® system. In certain embodiments, an antibody that binds to PD-L1 has a KD value of the binding affinity for binding to human PD-L1 of < 1 pM, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g. 10'8M or less, e.g. from 10'8M to 10'13M, e.g., from 10'9M to 10'13M).

[0082] By "specific binding" is meant that the binding is selective for the antigen and can be discriminated from unwanted or non-specific interactions. The ability of an antibody to bind to a specific antigen (e.g. PD-L1) can be measured either through an enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to one of skill in the art, e.g. surface plasmon resonance (SPR) technique (analyzed e.g. on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)), and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the extent of binding of an antibody to an unrelated protein is less than about 10% of the binding of the antibody to the antigen as measured, e.g., by SPR. The antibody comprised in the immunoconjugate described herein specifically binds to PD-L1.

[0083] As used herein, term "polypeptide" refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain of two or more amino acids and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "protein", "amino acid chain", or any other term used to refer to a chain of two or more amino acids, are included within the definition of "polypeptide", and the term "polypeptide" may be used instead of, or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide may be derived from a natural biological source or produced by recombinant technology but is not necessarily translated from a designated nucleic acid sequence. It may be generated in any manner, including by chemical synthesis. Polypeptides may have a defined three-dimensional structure, although they do not necessarily have such structure. Polypeptides with a defined three-dimensional structure are referred to as folded, and polypeptides which do not possess a defined three-dimensional structure but rather can adopt a large number of different conformations, and are referred to as unfolded.

[0084] By an "isolated" polypeptide or a variant, or derivative thereof is intended a polypeptide that is not in its natural milieu. No particular level of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purpose of the invention, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0085] “Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or the FASTA program package. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the ggsearch program of the FASTA package version 36.3.8c or later with aBLOSUM50 comparison matrix. The FASTA program package was authored by W. R. Pearson and D. J. Lipman (1988), “Improved Tools for Biological Sequence Analysis”, PNAS 85:2444-2448; W. R. Pearson (1996) “Effective protein sequence comparison” Meth. Enzymol. 266:227- 258; and Pearson et. al. (1997) Genomics 46:24-36, and is publicly available from

[0086] http: / / fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml.

[0087] Alternatively, a public server accessible at

[0088] http: / / fasta.bioch.virginia.edu / fasta_www2 / index.cgi

[0089] can be used to compare the sequences, using the ggsearch (global protein:protein) program and default options (BLOSUM50; open: -10; ext: -2; Ktup = 2) to ensure a global, rather than local, alignment is performed. Percent amino acid identity is given in the output alignment header.

[0090] The term "polynucleotide" refers to an isolated nucleic acid molecule or construct, e.g. messenger RNA (mRNA), virally-derived RNA, or plasmid DNA (pDNA). A polynucleotide may comprise a conventional phosphodiester bond or a non-conventional bond (e.g. an amide bond, such as found in peptide nucleic acids (PNA). The term "nucleic acid molecule" refers to any one or more nucleic acid segments, e.g. DNA or RNA fragments, present in a polynucleotide.

[0091] By "isolated" nucleic acid molecule or polynucleotide is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment. For example, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered isolated for the purposes of the present invention. Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution. An isolated polynucleotide includes a polynucleotide molecule contained in cells that ordinarily contain the polynucleotide molecule, but the polynucleotide molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the present invention, as well as positive and negative strand forms, and double-stranded forms. Isolated polynucleotides or nucleic acids according to the present invention further include such molecules produced synthetically. In addition, a polynucleotide or a nucleic acid may be or may include a regulatory element such as a promoter, ribosome binding site, or a transcription terminator. “Isolated polynucleotide (or nucleic acid) encoding [e.g. an immunoconjugate of the invention]” refers to one or more polynucleotide molecules encoding antibody heavy and light chains and / or IFN-a2 polypeptides (or fragments thereof), including such polynucleotide molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.

[0092] The term "expression cassette" refers to a polynucleotide generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a target cell. The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter. In certain embodiments, the expression cassette comprises polynucleotide sequences that encode immunoconjugates of the invention or fragments thereof.

[0093] The term “vector” or "expression vector" refers to a DNA molecule that is used to introduce and direct the expression of a specific gene to which it is operably associated in a cell. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. The expression vector of the present invention comprises an expression cassette. Expression vectors allow transcription of large amounts of stable mRNA. Once the expression vector is inside the cell, the ribonucleic acid molecule or protein that is encoded by the gene is produced by the cellular transcription and / or translation machinery. In one embodiment, the expression vector of the invention comprises an expression cassette that comprises polynucleotide sequences that encode immunoconjugates of the invention or fragments thereof.

[0094] 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. A host cell is any type of cellular system that can be used to generate the immunoconjugates of the present invention. Host cells include cultured cells, e.g. mammalian cultured cells, such as HEK cells, CHO cells, BHK cells, NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, insect cells, and plant cells, to name only a few, but also cells comprised within a transgenic animal, transgenic plant or cultured plant or animal tissue.

[0095] The term "antibody" herein 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 antigen binding activity.

[0096] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e. the individual antibodies comprised in the population are identical and / or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.

[0097] An "isolated" antibody is one which has been separated from a component of its natural environment, i.e. that is not in its natural milieu. No particular level of purification is required. For example, an isolated antibody can be removed from its native or natural environment. Recombinantly produced antibodies expressed in host cells are considered isolated for the purpose of the invention, as are native or recombinant antibodies which have been separated, fractionated, or partially or substantially purified by any suitable technique. As such, the immunoconjugates of the present invention are isolated. In some embodiments, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) methods. For review of methods for assessment of antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0098] The terms “full-length antibody,” “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure.

[0099] 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), and single-domain antibodies. For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005). For a review of scFv fragments, see e.g. Pliickthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Patent Nos.

[0100] 5,571,894 and 5,587,458. For discussion of Fab and F(ab')2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Patent No.

[0101] 5,869,046. Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, EP 404,097; WO 1993 / 01161; Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat Med 9, 129-134 (2003). Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see e.g. U.S. Patent No. 6,248,516 Bl). Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g. E. coli or phage), as described herein.

[0102] A “DutaFab” as used herein is a bispecific antibody fragment as disclosed in WO2012 / 163520. In a DutaFab, a single pair of a VH domain and a VL domain specifically binds to two different epitopes, wherein one paratope comprises amino acid residues from CDR-H2, CDR-L1 and CDR-L3 and the other paratope comprises amino residues from CDR-Hl, CDR-H3 and CDR-L2. DutaFabs comprise two non-overlapping paratopes within a cognate VH / VL pair. DutaFabs and methods for their generation by screening of libraries comprising monospecific Fab fragments are disclosed in WO2012 / 163520. Herein, the term “DutaFab” may particularly refer to a (bispecific) Fab which comprises a single pair of a VH domain and a VL domain and which specifically binds to two different epitopes, wherein one paratope comprises amino acid residues from CDR-H2, CDR-L1 and CDR-L3 and the other paratope comprises amino residues from CDR-H1, CDR-H3 and CDR-L2. DutaFabs have been known in the art for being engineered therapeutic Fab fragments that may bind two targets simultaneously (Beckmann R et al. Nat Commun. 2021 Jan 29;12(l):708. doi: 10.1038 / s41467-021-20949-3). However, by modifying one of the two paratopes of the DutaFab in such a way that it no longer binds to a specific target in the target organism, a DutaFab can also be made into a monospecific binder. Both bispecific and monospecific DutaFabs may be comprised in the immunoconjugates described herein.

[0103] The term “immunoglobulin molecule” refers to a protein having the structure of a naturally occurring antibody. For example, immunoglobulins of the IgG class are heterotetrameric glycoproteins of about 150,000 daltons, composed of two light chains and two heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable domain (VH), also called a variable heavy domain or a heavy chain variable region, followed by three constant domains (CHI, CH2, and CH3), also called a heavy chain constant region. Similarly, from N- to C-terminus, each light chain has a variable domain (VL), also called a variable light domain or a light chain variable region, followed by a constant light (CL) domain, also called a light chain constant region. The heavy chain of an immunoglobulin may be assigned to one of five types, called a (IgA), 5 (IgD), a (IgE), y (IgG), or p (IgM), some of which may be further divided into subtypes, e.g. yi (IgGi), 72 (IgG?), 73 (IgGs), 74 (IgG4), ai (IgAi) and a? (IgA?). The light chain of an immunoglobulin may be assigned to one of two types, called kappa (K) and lambda (X), based on the amino acid sequence of its constant domain. An immunoglobulin essentially consists of two Fab molecules and an Fc domain, linked via the immunoglobulin hinge region.

[0104] The term "antigen binding domain" refers to the part of an antibody that comprises the area which specifically binds to and is complementary to part or all of an antigen. An antigen binding domain may be provided by, for example, one or more antibody variable domains (also called antibody variable regions). Particularly, an antigen binding domain comprises an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).

[0105] 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 hypervariable regions (HVRs). See, e.g., Kindt et al., Kuby Immunology, 6thed., W.H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. As used herein in connection with variable region sequences, "Kabat numbering" refers to the numbering system set forth by Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0106] 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), referred to as “numbering according to Kabat” or “Kabat numbering” 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 heavy chain constant domains (CHI, Hinge, CH2 and CH3), which is herein further clarified by referring to “numbering according to Kabat EU index” in this case.

[0107] The term “hypervariable region” or “HVR”, as used herein, refers to each of the regions of an antibody variable domain which are hypervariable in sequence (“complementarity determining regions” or “CDRs”) and / or form structurally defined loops (“hypervariable loops”) and / or contain the antigen-contacting residues (“antigen contacts”). Generally, antibodies comprise six HVRs; three in the VH (Hl, H2, H3), and three in the VL (LI, L2, L3). Exemplary HVRs herein include: (a) hypervariable loops occurring at amino acid residues 26-32 (LI), 50-52 (L2), 91-96 (L3), 26-32 (Hl), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));

[0108] (b) CDRs occurring at amino acid residues 24-34 (LI), 50-56 (L2), 89-97 (L3), 31-35b (Hl), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991));

[0109] (c) antigen contacts occurring at amino acid residues 27c-36 (LI), 46-55 (L2), 89-96 (L3), 30-35b (Hl), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and

[0110] (d) combinations of (a), (b), and / or (c), including HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (Hl), 26-35b (Hl), 49-65 (H2), 93-102 (H3), and 94-102 (H3).

[0111] Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

[0112] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following order in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0113] A “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. Such variable domains are referred to herein as “humanized variable region”. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity. A “humanized form” of an antibody, e.g. of a non-human antibody, refers to an antibody that has undergone humanization. Other forms of "humanized antibodies" encompassed by the present invention are those in which the constant region has been additionally modified or changed from that of the original antibody to generate the properties according to the invention, especially in regard to Clq binding and / or Fc receptor (FcR) binding.

[0114] A “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues. In certain embodiments, a human antibody is derived from a non-human transgenic mammal, for example a mouse, a rat, or a rabbit. In certain embodiments, a human antibody is derived from a hybridoma cell line. Antibodies or antibody fragments isolated from human antibody libraries are also considered human antibodies or human antibody fragments herein.

[0115] The “class” of an antibody or immunoglobulin refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGi, IgG2, IgGs, IgG4, IgAi, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, 5, a, y, and p, respectively.

[0116] The term “Fc domain” or “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. Although the boundaries of the Fc region of an IgG heavy chain might vary slightly, the human IgG heavy chain Fc region is usually defined to extend from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Therefore, an antibody produced by a host cell by expression of a specific nucleic acid molecule encoding a full-length heavy chain may include the full-length heavy chain, or it may include a cleaved variant of the full-length heavy chain (also referred to herein as a “cleaved variant heavy chain”). This may be the case where the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering according to Kabat EU index). Therefore, the C-terminal lysine (Lys447), or the C-terminal glycine (Gly446) and lysine (K447), of the Fc region may or may not be present. Amino acid sequences of heavy chains including Fc domains (or a subunit of an Fc domain as defined herein) are denoted herein without C-terminal glycine-lysine dipeptide if not indicated otherwise. In one embodiment of the invention, a heavy chain including a subunit of an Fc domain as specified herein, comprised in an immunoconjugate according to the invention, comprises an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to EU index of Kabat). In one embodiment of the invention, a heavy chain including a subunit of an Fc domain as specified herein, comprised in an immunoconjugate according to the invention, comprises an additional C-terminal glycine residue (G446, numbering according to EU index of Kabat). Compositions of the invention, such as the pharmaceutical compositions described herein, comprise a population of immunoconjugates of the invention. The population of immunoconjugates may comprise molecules having a full-length heavy chain and molecules having a cleaved variant heavy chain. The population of immunoconjugates may consist of a mixture of molecules having a full-length heavy chain and molecules having a cleaved variant heavy chain, wherein at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the immunoconjugates have a cleaved variant heavy chain. In one embodiment of the invention, a composition comprising a population of immunoconjugates of the invention comprises an immunoconjugate comprising a heavy chain including a subunit of an Fc domain as specified herein with an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to EU index of Kabat). In one embodiment of the invention, a composition comprising a population of immunoconjugates of the invention comprises an immunoconjugate comprising a heavy chain including a subunit of an Fc domain as specified herein with an additional C-terminal glycine residue (G446, numbering according to EU index of Kabat). In one embodiment of the invention, such a composition comprises a population of immunoconjugates comprised of molecules comprising a heavy chain including a subunit of an Fc domain as specified herein; molecules comprising a heavy chain including a subunit of a Fc domain as specified herein with an additional C-terminal glycine residue (G446, numbering according to EU index of Kabat); and molecules comprising a heavy chain including a subunit of an Fc domain as specified herein with an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to EU index of Kabat). 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 (see also above). A “subunit” of an Fc domain as used herein refers to one of the two polypeptides forming the dimeric Fc domain, i.e. a polypeptide comprising C-terminal constant regions of an immunoglobulin heavy chain, capable of stable self-association. For example, a subunit of an IgG Fc domain comprises an IgG CH2 and an IgG CH3 constant domain.

[0117] A “modification promoting the association of the first and the second subunit of the Fc domain” is a manipulation of the peptide backbone or the post-translational modifications of an Fc domain subunit that reduces or prevents the association of a polypeptide comprising the Fc domain subunit with an identical polypeptide to form a homodimer. A modification promoting association as used herein particularly includes separate modifications made to each of the two Fc domain subunits desired to associate (i.e. the first and the second subunit of the Fc domain), wherein the modifications are complementary to each other so as to promote association of the two Fc domain subunits. For example, a modification promoting association may alter the structure or charge of one or both of the Fc domain subunits so as to make their association sterically or electrostatically favorable, respectively. Thus, (hetero)dimerization occurs between a polypeptide comprising the first Fc domain subunit and a polypeptide comprising the second Fc domain subunit, which might be non-identical in the sense that further components fused to each of the subunits (e.g. antigen binding moieties) are not the same. In some embodiments, the modification promoting association comprises an amino acid mutation in the Fc domain, specifically an amino acid substitution. In a particular embodiment, the modification promoting association comprises a separate amino acid mutation, specifically an amino acid substitution, in each of the two subunits of the Fc domain.

[0118] The term “effector functions” when used in reference to antibodies refers to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen presenting cells, down regulation of cell surface receptors (e.g. B cell receptor), and B cell activation. Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism leading to the lysis of antibody-coated target cells by immune effector cells. The target cells are cells to which antibodies or derivatives thereof comprising an Fc region specifically bind, generally via the protein part that is N-terminal to the Fc region. As used herein, the term “reduced ADCC” is defined as either a reduction in the number of target cells that are lysed in a given time, at a given concentration of antibody in the medium surrounding the target cells, by the mechanism of ADCC defined above, and / or an increase in the concentration of antibody in the medium surrounding the target cells, required to achieve the lysis of a given number of target cells in a given time, by the mechanism of ADCC. The reduction in ADCC is relative to the ADCC mediated by the same antibody produced by the same type of host cells, using the same standard production, purification, formulation and storage methods (which are known to those skilled in the art), but that has not been engineered. For example, the reduction in ADCC mediated by an antibody comprising in its Fc domain an amino acid substitution that reduces ADCC, is relative to the ADCC mediated by the same antibody without this amino acid substitution in the Fc domain. Suitable assays to measure ADCC are well known in the art (see e.g. PCT publication no. WO 2006 / 082515 or PCT publication no. WO 2012 / 130831).

[0119] The terms “target cell” and “target tissue” herein refer to a specific type of cell or tissue, respectively, which is the intended recipient of the fusion proteins, antibodies or treatments described herein. In particular, they may be a cell or tissue that exhibits a particular receptor or characteristic that makes it susceptible or responsive to the intended effect of the fusion protein, antibody or treatment. In the context of the immunoconjugates described herein, the receptor present on the target cell or in the target tissue may particularly be PD-L1.

[0120] An “activating Fc receptor” is an Fc receptor that following engagement by an Fc domain of an antibody elicits signaling events that stimulate the receptor-bearing cell to perform effector functions. Human activating Fc receptors include FcyRIIIa (CD16a), FcyRI (CD64), FcyRIIa (CD32), and FcaRI (CD89).

[0121] As used herein, the terms “engineer, engineered, engineering”, are considered to include any manipulation of the peptide backbone or the post-translational modifications of a naturally occurring or recombinant polypeptide or fragment thereof. Engineering includes modifications of the amino acid sequence, of the glycosylation pattern, or of the side chain group of individual amino acids, as well as combinations of these approaches.

[0122] “Reduced binding”, for example, reduced binding to an Fc receptor, refers to a decrease in affinity for the respective interaction, as measured for example by SPR. For clarity, the term includes also reduction of the affinity to zero (or below the detection limit of the analytic method), i.e. complete abolishment of the interaction. Conversely, “increased binding” refers to an increase in binding affinity for the respective interaction.

[0123] As used herein, the term "immunoconjugate" refers to a polypeptide molecule that includes at least one IFN-a2 molecule and at least one antibody. The IFN-a2 molecule can be joined to the antibody by a variety of interactions and in a variety of configurations as described herein. In particular embodiments, the IFN-a2 molecule is fused to the antibody via a peptide linker. Particular immunoconjugates according to the invention essentially consist of one IFN-a2 molecule and an antibody joined by one or more linker sequences.

[0124] By “fused” is meant that the components (e.g. an antibody and an IFN-a2 molecule) are linked by peptide bonds, either directly or via one or more peptide linkers.

[0125] The terms "linker", “linker peptide”, “peptide linker” or “peptidic linker” are used herein to refer to a peptide comprising one or more amino acids, typically about 2 to 20 amino acids. Peptide linkers are known in the art or are described herein. Suitable, non-immunogenic peptide linkers are, for example, (GsS)n (SEQ ID NO: 27) or (G4S)n(SEQ ID NO: 31) peptide linkers, wherein “n” is generally a number between 1 and 10, typically between 2 and 4, in particular 2. Peptide linkers of particular interest are (GSGGS)n(SEQ ID NO: 26), (GGGS)n(SEQ ID NO: 27), (GSGGG)n(SEQ ID NO: 28), (GGGSG)n(SEQ ID NO: 29), (GSSSG)n(SEQ ID NO: 30), (GGGGS)n(SEQ ID NO: 31) and (GGSGG)n(SEQ ID NO: 32), where n represents an integer of at least 1, preferably from 4 to 6. In one embodiment, the linker has the sequence of SEQ ID NO: 25.

[0126] As used herein, the terms "first" and "second" with respect to Fc domain subunits etc., are used for convenience of distinguishing when there is more than one of each type of moiety. Use of these terms is not intended to confer a specific order or orientation of the immunoconjugate unless explicitly so stated. Glutamine or glutamate residues at the N-terminus of antibody heavy or light chains may be converted to pyro-glutamate spontaneously (see e.g. Liu et al., Journal of Pharmaceutical Sciences 97, 2426-2447 (2008), Rehder et al., Journal of Chromatography A 1102, 164-175 (2006), Chelius et al., Anal Chem 78, 2370-2376 (2006)). Hence, variable domains disclosed herein which comprise either a glutamine (Q) or a glutamate (E) amino acid residue at the N-terminus of an the antibody heavy or light chain, may comprise an N-terminal pyro-glutamate (pyroE) residue instead of the N-terminal Q or E residue. Likewise, antibody heavy chains or light chains disclosed herein which comprise either a glutamine (Q) or a glutamate (E) amino acid residue at the N-terminus, may comprise an N-terminal pyro-glutamate (pyroE) residue instead of the N-terminal Q or E residue. Accordingly, for each antibody heavy chain, light chain, or variable domain sequence disclosed herein that contains an N-terminal Q or E residue, the corresponding sequence with an N-terminal pyroE residue is also encompassed.

[0127] An "effective amount" of an agent refers to the amount that is necessary to result in a physiological change in the cell or tissue to which it is administered.

[0128] A "therapeutically 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. A therapeutically effective amount of an agent for example eliminates, decreases, delays, minimizes or prevents adverse effects of a disease.

[0129] 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). Particularly, the individual or subject is a human.

[0130] 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.

[0131] A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical composition, 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. As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to clinical intervention in an attempt to alter the natural course of a disease in 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 some embodiments, immunoconjugates of the invention are used to delay development of a disease or to slow the progression of a disease.

[0132] IV. SPECIFIC EMBODIMENTS

[0133] 1. Mutant IFN-a2 polypeptide

[0134] The immunoconjugates according to the present invention comprise a mutant IFN-a2 polypeptide having advantageous properties for immunotherapy. In particular, pharmacological properties of IFN-a2 that contribute to toxicity but are not essential for efficacy of IFN-a2 are eliminated in the mutant IFN-a2 polypeptide. Thus, without wishing to be bound by theory, reducing or abolishing the affinity of IFN-a2 to the IFNAR should improve the overall safety profile of the immunoconjugates of the present invention. In particular, the immunoconjugates of the present invention demonstrate low cytokine induction as measured in a human whole blood assay and in human NK-92 cells. They further show reduced stimulation of human dendritic cell subsets and primary T cells, indicating an improved safety profile.

[0135] The mutant interferon alpha 2 (IFN-a2) polypeptide comprised in the immunoconjugate according to the invention comprises at least one amino acid mutation that abolishes or reduces affinity of the mutant IFN-a2 polypeptide to the IFNAR receptor compared to a wildtype IFN-a2 polypeptide. In particular, mutant interferon alpha 2 (IFN-a2) polypeptide comprised in the immunoconjugate according to the invention comprises at least one amino acid substitution selected from the group consisting of the amino acid substitutions L30A, L30H, and L153D. Whenever amino acid substitutions of mutant IFN-a2 are described herein, the numbering of the amino acids given is relative to the mature human IFN-a2 sequence of SEQ ID NO: 17, unless stated otherwise. Mutants of human IFN-a2 (huIFN-a2) with decreased affinity to IFNAR may for example be generated by amino acid substitution at amino acid position L15, A19, R22, R23, L26, F27, L30, L30, K31, D32, R33, R33, R33, H34, D35, Q40, F64, N65, T69, H57, E58, Q61, L80, Y85, Y89, D114, L117, R120, R120, R125, K121, K133, K134, R144, A145, A145, R149, M148, S152, L153, or N156, or combinations thereof (numbering relative to the human IFN-a2 sequence SEQ ID NO: 17). Exemplary amino acid substitutions include L15A, A19W, R22A, R23A, L26A, F27A, L30A, L30V, K31A, D32A, R33K, R33A, R33Q, H34A, D35A, Q40A, F64A, N65A, T69A, H57Y, E58N, Q61S, L80A, Y85A, Y89A, D114R, L117A, R120E, R120A, R125A, K121E, K133A, K134A, R144A, A145G, A145M, R149A, M148A, S152A, LI 53 A, N156A. The particular IFN-a2 mutants comprised in the immunoconjugates of the invention comprise an amino acid mutation at a position corresponding to residue 30 or 153 of human IFN-a2, or a combination thereof. More specifically, the amino acid substitution is selected from the group consisting of the amino acid substitutions L30A, L30H, and L153D. These mutants have substantially reduced affinity to the IFNAR receptor compared to a wild-type form of the IFN-a2 mutant. The IFN-a2 mutants comprised in the immunoconjugates of the invention may comprise additional amino acid substitutions. In some embodiments, they comprise one or more of the amino acid substitutions disclosed above.

[0136] In some embodiments, the IFN-a2 mutants comprised in the immunoconjugates of the invention further comprise an amino acid substitution or deletion in position T106 (numbering relative to the mature human IFN-a2 sequence of SEQ ID NO: 17). In particular embodiments, the IFN-a2 mutants comprise an amino acid substitution in the position T106 wherein T106 is substituted with an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, and Y. In certain embodiments, T106 is deleted or substituted with an amino acid selected from the group consisting of A, E, S, and V. Deletion or substitution of T 106 removes the O-linked glycosylation site found in natural IFN-a2 and thus reduces the heterogeneity of the molecule as measured in IEF gels, when expressed in mammalian cells such as CHO or HEK cells, whilst at least substantially maintaining the activity of the attenuated IFN-a2 to bind to IFNAR and to initiate downstream signaling.

[0137] IFN-a2 mutants useful in the invention, in addition to having mutations in the region of IFN-a2 that forms the interface of IFN-a2 with IFNAR or at the glycosylation site, also may have one or more mutations in the amino acid sequence outside these regions. Such additional mutations in human IFN-a2 may provide additional advantages, such as reduced immunogenicity. For example, the IFN-a2 mutant may comprise one or more amino acid substitutions in any of the positions selected from the group consisting of 9, 17, 47, 65, 66, 117, 123, 128, 147, and 157 (numbering relative to the human IFN-a2 sequence SEQ ID NO: 17); in particular, wherein said substitution comprises the change of the amino acid of said position to alanine, glycine, or threonine, as described in WO2021 / 126929. As described therein, the IFN-a2 mutant may comprise the amino acid substitutions L9A, F47A, L117A, F123A, and L128A; the amino acid substitutions I147T and L157A; the amino acid substitutions N65A and L66A; the amino acid substitutions L17A, I147T, and L157A; the amino acid substitutions 9A, F47A, L117A, F123A, and L128A; the amino acid substitutions I147T and L157A; or the amino acid substitutions N65A and L66A.

[0138] Alternatively, or conjunctively, the IFN-a2 mutant may include amino acid substitutions at one or more positions selected from the group consisting of positions 24, 26, 27, 38, 55, 63, 64, 66, 61, 16, 84, 85, 89, 103, 110, 111, 116, 117, 119, 122, 123, 126, 128, 129, 130, and 153. In some embodiments, the amino acid substitutions may be selected from the group consisting of L26P, F27S, F38E, I63T, Y85S, Y89D, Y89E, Y89N, N103E, L110G, Mil IT, M111S, Ml HE, I116S, I116Q, L117G, L117A, Y122E, Y122Q, F123H, I126A, L128A, and L153S (see WO02 / 85941). The resulting mutants may be used in conjunction with the particular IFN-a2 mutations of the invention.

[0139] The IFN-a2 mutant may further include amino acid substitutions that result in improved thermostability, as described in FR2905375. For example, the IFN-a2 mutant may have one or more amino acid substitutions selected from the group consisting of D2S, D2N, D2M, D2L, L3M, Q5S, Q5I, H7S, H7T, H7L, S8I, S8C, SUM, R12T, M16S, M21I, R22G, R22T, R22Y, R23A, I24L, I24D, L26T, K31G, P39H, E41M, E41V, E41T, E42V, F43A, N45T, Q46R, F47Y, F47V, F47I, Q48P, I53D, P54L, L56P, L56H, M59F, Q61L, N65F, L66A, F67S, F67L, S72Y, W76G, T79S, F84S, Y89C, L92N, L95D, A97M, Q101M, Q101Y, Q101R, G102V, P109L, M111N, M111P, E113Y, D114S, A118S, A118G, K121W, Y122P, Y122S, F123P, F123Y, Q124P, R1251, S136A, W140L, W140T, V142D, V142M, V142L, V142E, V143A, R144S, E146M, S150P, F151S, L157T, S160R, R162Q, and S163T (numbering relative to the human IFN-a2 sequence SEQ ID NO: 17). Particularly, the IFN-a2 mutant has a single substitution selected from the group consisting of D2N, D2M, L3M, H7T, H7L, N45T, M59F, T79S, Y89C, F123Y, R1251, R162Q, and V143A. Other characteristics of useful IFN-a2 mutants may include the ability to induce proliferation of IFN-a2 receptor-bearing T and / or NK cells, the ability to induce IFN-a2 signaling in IFN-a2 receptor-bearing T and / or NK cells, the ability to generate interferon (IFN)-y as a secondary cytokine by NK cells, a reduced ability to induce elaboration of secondary cytokines - particularly IL-10 and TNF-a - by peripheral blood mononuclear cells (PBMCs), a reduced ability to activate regulatory T cells, a reduced ability to induce apoptosis in T cells, and a reduced toxicity profile in vivo.

[0140] In certain embodiments said amino acid mutation reduces the affinity of the mutant IFN-a2 polypeptide to the a-subunit of the IFN-a2 receptor by at least 5-fold, specifically at least 10-fold, more specifically at least 25-fold. In embodiments where there is more than one amino acid mutation that reduces the affinity of the mutant IFN-a2 polypeptide to the a-subunit of the IFN-a2 receptor, the combination of these amino acid mutations may reduce the affinity of the mutant IFN-a2 polypeptide to the IFNAR by at least 30-fold, at least 50-fold, or even at least 100-fold. In one embodiment, said amino acid mutation or combination of amino acid mutations abolishes the affinity of the mutant IFN-a2 polypeptide to the a-subunit of the IFN-a2 receptor so that no binding is detectable by surface plasmon resonance.

[0141] In certain embodiments, the mutant IFN-a2 polypeptide is essentially a full-length IFN-a2 molecule. In certain embodiments, the mutant IFN-a2 polypeptide is a human IFN-a2a molecule. In one embodiment the mutant IFN-a2 polypeptide comprises the sequence of human IFN-a2a comprising SEQ ID NO: 17 with at least one amino acid mutation selected from the amino acid substitutions L30A, L30H, or L153D that abolishes or reduces affinity of the mutant IFN-a2 polypeptide to the IFNAR, compared to an IFN-a2a polypeptide comprising SEQ ID NO: 17 without said mutation. In another embodiment, the mutant IFN-a2 polypeptide comprises the sequence of SEQ ID NO: 20 with at least one amino acid mutation selected from the group consisting of amino acid substitutions L30A, L30H, and L153D that abolishes or reduces affinity of the mutant IFN-a2 polypeptide to IFNAR, compared to an IFN-a2 polypeptide comprising SEQ ID NO: 20 without said mutation. In a particular embodiment, the mutant IFN-a2 polypeptide comprises the sequence of human IFN-a2a comprising SEQ ID NO: 17 with at least the amino acid mutation L30A. In one embodiment, the mutant IFN-a2 polypeptide comprises the sequence of human IFN-a2a comprising SEQ ID NO: 17 with exactly one amino acid mutation, which is L30A. In a specific embodiment, the mutant IFN-a2 polypeptide can elicit one or more of the cellular responses selected from the group consisting of: inhibition of tumor cell proliferation, inducing apoptosis in tumor cells, increase of tumor cell immunogenicity by MHC upregulation, proliferation in an activated T lymphocyte cell, differentiation in an activated T lymphocyte cell, cytotoxic T cell (CTL) activity, proliferation in an activated B cell, differentiation in an activated dendritic cell (DC), proliferation in an activated DC, differentiation in an activated DC, proliferation in a natural killer (NK) cell, differentiation in a NK cell, cytokine secretion by an activated T cell or an NK cell, and NK / lymphocyte activated killer (LAK) antitumor cytotoxicity.

[0142] In one embodiment the mutant IFN-a2 polypeptide has a reduced ability to induce IFN-a2 signaling in regulatory T cells, compared to a wild-type IFN-a2 polypeptide. In one embodiment the mutant IFN-a2 polypeptide induces less activation-induced cell death (AICD) in T cells, compared to a wild-type IFN-a2 polypeptide. In one embodiment, the mutant IFN-a2 polypeptide has a reduced toxicity profile in vivo, compared to a wild-type IFN-a2 polypeptide. In one embodiment, the mutant IFN-a2 polypeptide has a prolonged serum half-life, compared to a wild-type IFN-a2 polypeptide.

[0143] IFN-a2 mutants useful in the invention, in addition to having mutations in the region of IFN-a2 that forms the interface of IFN-a2 with IFNAR or the glycosylation site, also may have one or more mutations in the amino acid sequence outside these regions. Such additional mutations in human IFN-a2 may provide additional advantages such as increased expression or stability.

[0144] The skilled person will be able to determine which additional mutations may provide additional advantages for the purpose of the invention.

[0145] In one embodiment, the mutant IFN-a2 polypeptide comprises no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 amino acid mutation(s) as compared to the corresponding wild-type IFN-a2 sequence, e.g. the human IFN-a2 sequence of SEQ ID NO: 17. In a particular embodiment, the mutant IFN-a2 polypeptide comprises no more than 1 amino acid mutation as compared to the corresponding wild-type IFN-a2 sequence, e.g. the human IFN-a2 sequence of SEQ ID NO: 17. In one embodiment, the mutant IFN-a2 polypeptide comprises the sequence of SEQ ID NO: 22. In one embodiment, the mutant IFN-a2 polypeptide consists of the sequence of SEQ ID NO: 23. In one embodiment, the mutant IFN-a2 polypeptide consists of the sequence of SEQ ID NO: 24.

[0146] 2. Immunoconjugates

[0147] Immunoconjugates as described herein comprise an IFN-a2-molecule and an antibody. Such immunoconjugates significantly increase the efficacy of IFN-a2 therapy by directly targeting IFN-a2 e.g. into a tumor microenvironment. According to the invention, an antibody comprised in the immunoconjugate can be a whole antibody or immunoglobulin, or a portion or variant thereof that has a biological function such as antigen specific binding affinity.

[0148] The general benefits of immunoconjugate therapy are readily apparent. For example, an antibody comprised in an immunoconjugate recognizes a tumor-specific epitope and results in targeting of the immunoconjugate molecule to the tumor site. Therefore, high concentrations of IFN-a2 can be delivered into the tumor microenvironment, thereby resulting in activation and proliferation of a variety of immune effector cells mentioned herein using a much lower dose of the immunoconjugate than would be required for unconjugated IFN-a2. Moreover, since application of IFN-a2 in form of immunoconjugates allows lower doses of the cytokine itself, the potential for undesirable side effects of IFN-a2 is restricted, and targeting the IFN-a2 to a specific site in the body by means of an immunoconjugate may also result in a reduction of systemic exposure and thus less side effects than obtained with unconjugated IFN-a2.

[0149] In addition, the increased circulating half-life of an immunoconjugate compared to unconjugated IFN-a2 contributes to the efficacy of the immunoconjugate. However, this characteristic of IFN-a2 immunoconjugates may again aggravate potential side effects of the IFN-a2 molecule: Because of the significantly longer circulating half-life of IFN-a2 immunoconjugate in the bloodstream relative to unconjugated IFN-a2, the probability for IFN-a2 or other portions of the fusion protein molecule to activate components generally present in the vasculature is increased. The same concern applies to other fusion proteins that contain IFN-a2 fused to another moiety such as Fc or albumin, resulting in an extended halflife of IFN-a2 in the circulation. Therefore, an immunoconjugate comprising a mutant IFN- a2 polypeptide as described herein, with reduced toxicity compared to wild-type forms of IFN-a2, is particularly advantageous.

[0150] As described hereinabove, targeting IFN-a2 directly to immune effector cells rather than tumor cells may be advantageous for IFN-a2 immunotherapy.

[0151] Accordingly, the invention provides a mutant IFN-a2 polypeptide as described hereinbefore, and an antibody that binds to PD-L1. In one embodiment, the mutant IFN-a2 polypeptide and the antibody form a fusion protein, i.e. the mutant IFN-a2 polypeptide shares a peptide bond with the antibody. In some embodiments, the antibody comprises an Fc domain composed of a first and a second subunit. In a specific embodiment, the mutant IFN-a2 polypeptide is fused at its amino-terminal amino acid to the carboxy -terminal amino acid of one of the subunits of the Fc domain, optionally through a linker peptide. In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is an immunoglobulin molecule, particularly an IgG class immunoglobulin molecule, more particularly an IgGi subclass immunoglobulin molecule. In one such embodiment, the mutant IFN-a2 polypeptide shares an amino-terminal peptide bond with one of the immunoglobulin heavy chains. In certain embodiments, the antibody is an antibody fragment. In some embodiments, the antibody is a Fab molecule or a scFv molecule. In one embodiment, the antibody is a Fab molecule. In another embodiment, the antibody is a scFv molecule. In yet another embodiment, the antibody is a DutaFab. The immunoconjugate may also comprise more than one antibody. Where more than one antibody is comprised in the immunoconjugate, e.g. a first and a second antibody, each antibody can be independently selected from various forms of antibodies and antibody fragments. For example, the first antibody can be a Fab molecule and the second antibody can be a scFv molecule. In a specific embodiment, each of said first and said second antibodies is a scFv molecule or each of said first and said second antibodies is a Fab molecule. In one embodiment, each of said first and said second antibodies is a Fab molecule. In a particular embodiment, each of said first and said second Fab molecules is a DutaFab. In one embodiment, each of said first and said second antibodies binds to PD-L1.

[0152] 3. Immunoconjugate Formats

[0153] Exemplary immunoconjugate formats are described in PCT publication no. WO 2011 / 020783, which is incorporated herein by reference in its entirety. These immunoconjugates comprise at least two antibodies. Thus, in one embodiment, the immunoconjugate according to the present invention comprises a mutant IFN-a2 polypeptide as described herein, and at least a first and a second antibody. In a particular embodiment, said first and second antibody are independently selected from the group consisting of an Fv molecule, particularly a scFv molecule, and a Fab molecule. In a specific embodiment, said mutant IFN-a2 polypeptide shares an amino- or carboxy-terminal peptide bond with said first antibody and said second antibody shares an amino- or carboxy-terminal peptide bond with either i) the mutant IFN-a2 polypeptide or ii) the first antibody. In a particular embodiment, the immunoconjugate consists essentially of a mutant IFN-a2 polypeptide and first and second antibodies, particularly Fab molecules, joined by one or more linker sequences. Such formats have the advantage that they bind with high affinity to the target antigen (PD-L1), but provide only monomeric binding to the IFN-a2 receptor, thus avoiding targeting the immunoconjugate to IFN-a2 receptor bearing immune cells at other locations than the target site. In a particular embodiment, a mutant IFN-a2 polypeptide shares a carboxy-terminal peptide bond with a first antibody, particularly a first Fab molecule, and further shares an amino-terminal peptide bond with a second antibody, particularly a second Fab molecule. In another embodiment, a first antibody, particularly a first Fab molecule, shares a carboxy-terminal peptide bond with a mutant IFN-a2 polypeptide, and further shares an amino-terminal peptide bond with a second antibody, particularly a second Fab molecule. In another embodiment, a first antibody, particularly a first Fab molecule, shares an amino-terminal peptide bond with a first mutant IFN-a2 polypeptide, and further shares a carboxy-terminal peptide with a second antibody, particularly a second Fab molecule. In a particular embodiment, a mutant IFN-a2 polypeptide shares a carboxy-terminal peptide bond with a first heavy chain variable region and further shares an amino-terminal peptide bond with a second heavy chain variable region. In another embodiment a mutant IFN-a2 polypeptide shares a carboxy-terminal peptide bond with a first light chain variable region and further shares an amino-terminal peptide bond with a second light chain variable region. In another embodiment, a first heavy or light chain variable region is joined by a carboxy-terminal peptide bond to a mutant IFN-a2 polypeptide and is further joined by an amino-terminal peptide bond to a second heavy or light chain variable region. In another embodiment, a first heavy or light chain variable region is joined by an aminoterminal peptide bond to a mutant IFN-a2 polypeptide and is further joined by a carboxy-terminal peptide bond to a second heavy or light chain variable region. In one embodiment, a mutant IFN-a2 polypeptide shares a carboxy-terminal peptide bond with a first Fab heavy or light chain and further shares an amino-terminal peptide bond with a second Fab heavy or light chain. In another embodiment, a first Fab heavy or light chain shares a carboxy -terminal peptide bond with a mutant IFN-a2 polypeptide and further shares an amino-terminal peptide bond with a second Fab heavy or light chain. In other embodiments, a first Fab heavy or light chain shares an amino-terminal peptide bond with a mutant IFN-a2 polypeptide and further shares a carboxy-terminal peptide bond with a second Fab heavy or light chain. In one embodiment, the immunoconjugate comprises a mutant IFN-a2 polypeptide sharing an amino-terminal peptide bond with one or more scFv molecules and further sharing a carboxy-terminal peptide bond with one or more scFv molecules.

[0154] Particularly suitable formats for the immunoconjugates according to the present invention however comprise an immunoglobulin molecule as antibody. Such immunoconjugate formats are described in WO 2012 / 146628, which is incorporated herein by reference in its entirety.

[0155] Accordingly, in particular embodiments, the immunoconjugate comprises a mutant IFN-a2 polypeptide as described herein and an immunoglobulin molecule that binds to PD-L1, particularly an IgG molecule, more particularly an IgGi molecule. In one embodiment, the immunoconjugate comprises not more than one mutant IFN-a2 polypeptide. In one embodiment, the immunoglobulin molecule is human. In one embodiment, the immunoglobulin molecule comprises a human constant region, e.g. a human CHI, CH2, CH3 and / or CL domain. In one embodiment, the immunoglobulin comprises a human Fc domain, particularly a human IgGi Fc domain. In one embodiment, the mutant IFN-a2 polypeptide shares an amino- or carboxy-terminal peptide bond with the immunoglobulin molecule. In one embodiment, the immunoconjugate essentially consists of a mutant IFN-a2 polypeptide and an immunoglobulin molecule, particularly an IgG molecule, more particularly an IgGi molecule, joined by one or more linker sequences. In a specific embodiment, the mutant IFN-a2 polypeptide is fused at its amino-terminal amino acid to the carboxy-terminal amino acid of one of the immunoglobulin heavy chains, optionally through a linker peptide.

[0156] The mutant IFN-a2 polypeptide may be fused to the antibody directly or through a linker peptide, comprising one or more amino acids, typically about 2-20 amino acids. Linker peptides are known in the art and are described herein. Peptide linkers are known in the art or are described herein. Suitable, non-immunogenic peptide linkers are, for example, (GsS)n (SEQ ID NO: 27) or (G4S)n(SEQ ID NO: 31) peptide linkers, wherein “n” is generally an integer from 1 to 10, typically from 2 to 4, in particular 3. Peptide linkers of particular interest are (GSGGS)n(SEQ ID NO: 26), (GGGS)n(SEQ ID NO: 27), (GSGGG)n(SEQ ID NO: 28), (GGGSG)n (SEQ ID NO: 29), (GSSSG)n(SEQ ID NO: 30), (GGGGS)n(SEQ ID NO: 31) and (GGSGG)n (SEQ ID NO: 32), where n represents an integer of at least 1, preferably from 2 to 4. In one embodiment, the linker peptide has a length of at least 5 amino acids, in one embodiment a length of 5 to 100, in a further embodiment of 10 to 50 amino acids. In a particular embodiment, the linker peptide has a length of 15 amino acids. In one embodiment, the linker has the sequence of SEQ ID NO: 25.

[0157] In a particular embodiment, the immunoconjugate comprises a mutant IFN-a2 molecule and an immunoglobulin molecule, particularly an IgGi subclass immunoglobulin molecule, which binds to PD-L1, wherein the mutant IFN-a2 molecule is fused at its amino-terminal amino acid to the carboxy-terminal amino acid of one of the immunoglobulin heavy chains through the linker peptide of SEQ ID NO: 25.

[0158] In a particular embodiment, the immunoconjugate comprises a mutant IFN-a2 molecule and an antibody that binds to PD-L1, wherein the antibody comprises an Fc domain, particularly a human IgGi Fc domain, composed of a first and a second subunit, and the mutant IFN-a2 molecule is fused at its amino-terminal amino acid to the carboxy-terminal amino acid of one of the subunits of the Fc domain through the linker peptide of SEQ ID NO: 25.

[0159] 4. PD-L1 antibodies

[0160] The antibody comprised in the immunoconjugate of the invention binds to PD-L1, particularly human PD-L1, and is able to direct the mutant IFN-a2 polypeptide to a target site where PD-L1 is expressed, particularly to a T cell that expresses PD-L1, for example associated with a tumor.

[0161] The immunoconjugate of the invention may comprise two or more antibodies, which may bind to the same or to different antigens. In particular, embodiments, however, each of these antibodies binds to PD-L1. In one embodiment, the antibody comprised in the immunoconjugate of the invention is monospecific. In a particular embodiment, the immunoconjugate comprises a single, monospecific antibody, particularly a monospecific immunoglobulin molecule.

[0162] The antibody can be any type of antibody or fragment thereof that retains specific binding to PD-L1, particularly human PD-L1. Antibody fragments include, but are not limited to, Fv molecules, scFv molecule, Fab molecule, and F(ab')2 molecules. In particular embodiments, however, the antibody is a full-length antibody. In some embodiments, the antibody comprises an Fc domain, composed of a first and a second subunit. In some embodiments, the antibody is an immunoglobulin, particularly an IgG class, more particularly an IgGi subclass immunoglobulin.

[0163] In some embodiments, the antibody is a monoclonal antibody.

[0164] In some embodiments, the antibody comprises a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3, a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

[0165] In some embodiments, the antibody comprises (a) a heavy chain variable region (VH) comprising a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and (b) a light chain variable region (VL) comprising a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the heavy and / or light chain variable region is a humanized variable region. In some embodiments, the heavy and / or light chain variable region comprises human framework regions (FR).

[0166] In some embodiments, the antibody comprises a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9, a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10, a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11, a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12, a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14.

[0167] In some embodiments, the antibody comprises (a) a heavy chain variable region (VH) comprising a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9, a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11, and (b) a light chain variable region (VL) comprising a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12, a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the heavy and / or light chain variable region is a humanized variable region. In some embodiments, the heavy and / or light chain variable region comprises human framework regions (FR).

[0168] In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the antibody comprises a light chain variable region (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the antibody comprises (a) a heavy chain variable region (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 7, and (b) a light chain variable region (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 8.

[0169] In a particular embodiment, the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 7, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 8.

[0170] In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the antibody comprises a light chain variable region (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the antibody comprises (a) a heavy chain variable region (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 15, and (b) a light chain variable region (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 16.

[0171] In a particular embodiment, the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 15, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the antibody is a humanized antibody. In one embodiment, the antibody is an immunoglobulin molecule comprising a human constant region, particularly an IgG class immunoglobulin molecule comprising a human CHI, CH2, CH3 and / or CL domain. Exemplary sequences of human constant domains are given in SEQ ID NO: 42 and SEQ ID NO: 43 (human kappa and lambda CL domains, respectively) and SEQ ID NO: 44 (human IgGl heavy chain constant domains CH1-CH2-CH3). In some embodiments, the antibody comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO: 43, particularly the amino acid sequence of SEQ ID NO: 42. In some embodiments, the antibody comprises a heavy chain constant region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 44. Particularly, the heavy chain constant region may comprise amino acid mutations in the Fc domain as described herein.

[0172] 5. Fc domain

[0173] In particular embodiments, the antibody comprised in the immunoconjugates according to the invention comprises an Fc domain, composed of a first and a second subunit. The Fc domain of an antibody consists of a pair of polypeptide chains comprising heavy chain domains of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, each subunit of which comprises the CH2 and CH3 IgG heavy chain constant domains. The two subunits of the Fc domain are capable of stable association with each other. In one embodiment, the immunoconjugate of the invention comprises not more than one Fc domain.

[0174] In one embodiment, the Fc domain of the antibody comprised in the immunoconjugate is an IgG Fc domain. In a particular embodiment, the Fc domain is an IgGi Fc domain. In another embodiment, the Fc domain is an IgG4 Fc domain. In a more specific embodiment, the Fc domain is an IgG4 Fc domain comprising an amino acid substitution at position S228 (Kabat EU index numbering), particularly the amino acid substitution S228P. This amino acid substitution reduces in vivo Fab arm exchange of IgG4 antibodies (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). In a further particular embodiment, the Fc domain is a human Fc domain. In an even more particular embodiment, the Fc domain is a human IgGi Fc domain. An exemplary sequence of a human IgGi Fc region is given in SEQ ID NO: 41. 1. Fc domain modifications promoting heterodimerization

[0175] Immunoconjugates according to the invention comprise a mutant IFN-a2 polypeptide, particularly a single (not more than one) mutant IFN-a2 polypeptide, fused to one or the other of the two subunits of the Fc domain, thus the two subunits of the Fc domain are typically comprised in two non-identical polypeptide chains. Recombinant co-expression of these polypeptides and subsequent dimerization leads to several possible combinations of the two polypeptides. To improve the yield and purity of the immunoconjugate in recombinant production, it will thus be advantageous to introduce in the Fc domain of the antibody a modification promoting the association of the desired polypeptides.

[0176] Accordingly, in particular embodiments, the Fc domain of the antibody comprised in the immunoconjugate according to the invention comprises a modification promoting the association of the first and the second subunit of the Fc domain. The site of most extensive protein-protein interaction between the two subunits of a human IgG Fc domain is in the CH3 domain of the Fc domain. Thus, in one embodiment said modification is in the CH3 domain of the Fc domain.

[0177] There exist several approaches for modifications in the CH3 domain of the Fc domain in order to enforce heterodimerization, which are well described e.g. in WO 96 / 27011, WO 98 / 050431, EP 1870459, WO 2007 / 110205, WO 2007 / 147901, WO 2009 / 089004, WO 2010 / 129304, WO 2011 / 90754, WO 2011 / 143545, WO 2012058768, WO 2013157954, WO 2013096291. Typically, in all such approaches the CH3 domain of the first subunit of the Fc domain and the CH3 domain of the second subunit of the Fc domain are both engineered in a complementary manner so that each CH3 domain (or the heavy chain comprising it) can no longer homodimerize with itself but is forced to heterodimerize with the complementarily engineered other CH3 domain (so that the first and second CH3 domain heterodimerize and no homodimers between the two first or the two second CH3 domains are formed).

[0178] In a specific embodiment, said modification promoting the association of the first and the second subunit of the Fc domain is a so-called “knob-into-hole” modification, comprising a “knob” modification in one of the two subunits of the Fc domain and a “hole” modification in the other one of the two subunits of the Fc domain. The knob-into-hole technology is described e.g. in US 5,731,168; US 7,695,936; Ridgway et al., ProtEng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the method involves introducing a protuberance (“knob”) at the interface of a first polypeptide and a corresponding cavity (“hole”) in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation. Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g. tyrosine or tryptophan). Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine).

[0179] Accordingly, in a particular embodiment, in the CH3 domain of the first subunit of the Fc domain of the antibody comprised in the immunoconjugate an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain of the first subunit which is positionable in a cavity within the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit is positionable.

[0180] Preferably said amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W).

[0181] Preferably said amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V).

[0182] The protuberance and cavity can be made by altering the nucleic acid encoding the polypeptides, e.g. by site-specific mutagenesis, or by peptide synthesis.

[0183] In a specific embodiment, in the CH3 domain of the first subunit of the Fc domain (the “knobs” subunit) the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the CH3 domain of the second subunit of the Fc domain (the “hole” subunit) the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In one embodiment, in the second subunit of the Fc domain additionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numberings according to Kabat EU index).

[0184] In yet a further embodiment, in the first subunit of the Fc domain additionally the serine residue at position 354 is replaced with a cysteine residue (S354C) or the glutamic acid residue at position 356 is replaced with a cysteine residue (E356C) (particularly the serine residue at position 354 is replaced with a cysteine residue), and in the second subunit of the Fc domain additionally the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C) (numberings according to Kabat EU index). Introduction of these two cysteine residues results in formation of a disulfide bridge between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0185] In a particular embodiment, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W, and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (numbering according to Kabat EU index), or the first subunit of the Fc domain comprises the amino acid mutations Y349C, T366W and the second subunit of the Fc domain comprises the amino acid mutations S354C, T366S, L368A, Y407V (numbering according to Kabat EU index).

[0186] In some embodiments, the second subunit of the Fc domain additionally comprises the amino acid substitutions H435R and Y436F (numbering according to Kabat EU index).

[0187] In a particular embodiment the mutant IFN-a2 polypeptide is fused (optionally through a linker peptide) to the first subunit of the Fc domain (comprising the “knob” modification). Without wishing to be bound by theory, fusion of the mutant IFN-a2 polypeptide to the knobcontaining subunit of the Fc domain will (further) minimize the generation of immunoconjugates comprising two mutant IFN-a2 polypeptides (steric clash of two knobcontaining polypeptides).

[0188] Other techniques of CH3 -modification for enforcing the heterodimerization are contemplated as alternatives according to the invention and are described e.g. in WO 96 / 27011, WO 98 / 050431, EP 1870459, WO 2007 / 110205, WO 2007 / 147901, WO 2009 / 089004, WO 2010 / 129304, WO 2011 / 90754, WO 2011 / 143545, WO 2012 / 058768, WO 2013 / 157954, WO 2013 / 096291. In one embodiment the heterodimerization approach described in EP 1870459, is used alternatively. This approach is based on the introduction of charged amino acids with opposite charges at specific amino acid positions in the CH3 / CH3 domain interface between the two subunits of the Fc domain. One preferred embodiment for the antibody comprised in the immunoconjugate of the invention are amino acid mutations R409D; K370E in one of the two CH3 domains (of the Fc domain) and amino acid mutations D399K; E357K in the other one of the CH3 domains of the Fc domain (numbering according to Kabat EU index).

[0189] In another embodiment, the antibody comprised in the immunoconjugate of the invention comprises the amino acid mutation T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutations T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, and additionally amino acid mutations R409D; K370E in the CH3 domain of the first subunit of the Fc domain and amino acid mutations D399K; E357K in the CH3 domain of the second subunit of the Fc domain (numberings according to Kabat EU index).

[0190] In another embodiment, the antibody comprised in the immunoconjugate of the invention comprises amino acid mutations S354C, T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutations Y349C, T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, or said antibody comprises amino acid mutations Y349C, T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutations S354C, T366S, L368A, Y407V in the CH3 domains of the second subunit of the Fc domain and additionally amino acid mutations R409D; K370E in the CH3 domain of the first subunit of the Fc domain and amino acid mutations D399K; E357K in the CH3 domain of the second subunit of the Fc domain (all numberings according to Kabat EU index).

[0191] In one embodiment, the heterodimerization approach described in WO 2013 / 157953 is used alternatively. In one embodiment, a first CH3 domain comprises amino acid mutation T366K and a second CH3 domain comprises amino acid mutation L351D (numberings according to Kabat EU index). In a further embodiment, the first CH3 domain comprises further amino acid mutation L351K. In a further embodiment, the second CH3 domain comprises further an amino acid mutation selected from Y349E, Y349D and L368E (preferably L368E) (numberings according to Kabat EU index). In one embodiment, the heterodimerization approach described in WO 2012 / 058768 is used alternatively. In one embodiment, a first CH3 domain comprises amino acid mutations L351Y, Y407A and a second CH3 domain comprises amino acid mutations T366A, K409F. In a further embodiment, the second CH3 domain comprises a further amino acid mutation at position T411, D399, S400, F405, N390, or K392, e.g. selected from a) T411N, T411R, T41 IQ, T41 IK, T41 ID, T41 IE or T411W, b) D399R, D399W, D399Y or D399K, c) S400E, S400D, S400R, or S400K, d) F405I, F405M, F405T, F405S, F405V or F405W, e) N390R, N390K or N390D, f) K392V, K392M, K392R, K392L, K392F or K392E (numberings according to Kabat EU index). In a further embodiment, a first CH3 domain comprises amino acid mutations L351Y, Y407A and a second CH3 domain comprises amino acid mutations T366V, K409F. In a further embodiment, a first CH3 domain comprises amino acid mutation Y407A and a second CH3 domain comprises amino acid mutations T366A, K409F. In a further embodiment, the second CH3 domain further comprises amino acid mutations K392E, T41 IE, D399R and S400R (numberings according to Kabat EU index).

[0192] In one embodiment the heterodimerization approach described in WO 2011 / 143545 is used alternatively, e.g. with the amino acid modification at a position selected from the group consisting of 368, and 409 (numbering according to Kabat EU index).

[0193] In one embodiment, the heterodimerization approach described in WO 2011 / 090762, which also uses the knobs-into-holes technology described above, is used alternatively. In one embodiment, a first CH3 domain comprises amino acid mutation T366W and a second CH3 domain comprises amino acid mutation Y407A. In one embodiment, a first CH3 domain comprises amino acid mutation T366Y and a second CH3 domain comprises amino acid mutation Y407T (numberings according to Kabat EU index).

[0194] In one embodiment, the antibody comprised in the immunoconjugate or its Fc domain is of IgG2subclass and the heterodimerization approach described in WO 2010 / 129304 is used alternatively.

[0195] In an alternative embodiment, a modification promoting association of the first and the second subunit of the Fc domain comprises a modification mediating electrostatic steering effects, e.g. as described in PCT publication WO 2009 / 089004. Generally, this method involves replacement of one or more amino acid residues at the interface of the two Fc domain subunits by charged amino acid residues so that homodimer formation becomes electrostatically unfavorable but heterodimerization electrostatically favorable. In one such embodiment, a first CH3 domain comprises amino acid substitution of K392 or N392 with a negatively charged amino acid (e.g. glutamic acid (E), or aspartic acid (D), preferably K392D or N392D) and a second CH3 domain comprises amino acid substitution of D399, E356, D356, or E357 with a positively charged amino acid (e.g. lysine (K) or arginine (R), preferably D399K, E356K, D356K, or E357K, and more preferably D399K and E356K). In a further embodiment, the first CH3 domain further comprises amino acid substitution of K409 or R409 with a negatively charged amino acid (e.g. glutamic acid (E), or aspartic acid (D), preferably K409D or R409D). In a further embodiment, the first CH3 domain further or alternatively comprises amino acid substitution of K439 and / or K370 with a negatively charged amino acid (e.g. glutamic acid (E), or aspartic acid (D)) (all numberings according to Kabat EU index).

[0196] In yet a further embodiment, the heterodimerization approach described in WO 2007 / 147901 is used alternatively. In one embodiment, a first CH3 domain comprises amino acid mutations K253E, D282K, and K322D and a second CH3 domain comprises amino acid mutations D239K, E240K, and K292D (numberings according to Kabat EU index).

[0197] In still another embodiment, the heterodimerization approach described in WO 2007 / 110205 can be used alternatively.

[0198] In one embodiment, the first subunit of the Fc domain comprises amino acid substitutions K392D and K409D, and the second subunit of the Fc domain comprises amino acid substitutions D356K and D399K (numbering according to Kabat EU index).

[0199] 6. Fc domain modifications reducing Fc receptor binding and / or effector function

[0200] The Fc domain confers to the immunoconjugate favorable pharmacokinetic properties, including a long serum half-life which contributes to good accumulation in the target tissue and a favorable tissue-blood distribution ratio. At the same time, it may, however, lead to undesirable targeting of the immunoconjugate to cells expressing Fc receptors rather than to the preferred antigen-bearing cells. Moreover, the co-activation of Fc receptor signaling pathways may lead to cytokine release which, in combination with the IFN-a2 polypeptide and the long half-life of the immunoconjugate, results in excessive activation of cytokine receptors and severe side effects upon systemic administration. In line with this, conventional IgG-IFN-a2 immunoconjugates have been described to be associated with infusion reactions (see e.g. King et al., J Clin Oncol 22, 4463-4473 (2004)).

[0201] Accordingly, in particular embodiments, the Fc domain of the antibody comprised in the immunoconjugate according to the invention exhibits reduced binding affinity to an Fc receptor and / or reduced effector function, as compared to a native IgGi Fc domain. In one such embodiment the Fc domain (or the antibody comprising said Fc domain) exhibits less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5% of the binding affinity to an Fc receptor, as compared to a native IgGi Fc domain (or an antibody comprising a native IgGi Fc domain), and / or less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5% of the effector function, as compared to a native IgGi Fc domain (or an antibody comprising a native IgGi Fc domain). In one embodiment, the Fc domain (or an antibody comprising said Fc domain) does not substantially bind to an Fc receptor and / or induce effector function. In a particular embodiment, the Fc receptor is an Fey receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activating Fc receptor. In a specific embodiment, the Fc receptor is an activating human Fey receptor, more specifically human FcyRIIIa, FcyRI or FcyRIIa, most specifically human FcyRIIIa. In one embodiment, the effector function is one or more selected from the group of CDC, ADCC, ADCP, and cytokine secretion. In a particular embodiment, the effector function is ADCC. In one embodiment, the Fc domain exhibits substantially similar binding affinity to neonatal Fc receptor (FcRn), as compared to a native IgGi Fc domain. Substantially similar binding to FcRn is achieved when the Fc domain (or an antibody comprising said Fc domain) exhibits greater than about 70%, particularly greater than about 80%, more particularly greater than about 90% of the binding affinity of a native IgGi Fc domain (or an antibody comprising a native IgGi Fc domain) to FcRn.

[0202] In certain embodiments, the Fc domain is engineered to have reduced binding affinity to an Fc receptor and / or reduced effector function, as compared to a non-engineered Fc domain. In particular, embodiments, the Fc domain of the antibody comprised in the immunoconjugate comprises one or more amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor and / or effector function. Typically, the same one or more amino acid mutation is present in each of the two subunits of the Fc domain. In one embodiment, the amino acid mutation reduces the binding affinity of the Fc domain to an Fc receptor. In one embodiment, the amino acid mutation reduces the binding affinity of the Fc domain to an Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold. In embodiments where there is more than one amino acid mutation that reduces the binding affinity of the Fc domain to the Fc receptor, the combination of these amino acid mutations may reduce the binding affinity of the Fc domain to an Fc receptor by at least 10-fold, at least 20-fold, or even at least 50-fold. In one embodiment the antibody comprising an engineered Fc domain exhibits less than 20%, particularly less than 10%, more particularly less than 5% of the binding affinity to an Fc receptor as compared to an antibody comprising a non-engineered Fc domain. In a particular embodiment, the Fc receptor is an Fey receptor. In some embodiments, the Fc receptor is a human Fc receptor. In some embodiments, the Fc receptor is an activating Fc receptor. In a specific embodiment, the Fc receptor is an activating human Fey receptor, more specifically human FcyRIIIa, FcyRI or FcyRIIa, most specifically human FcyRIIIa. Preferably, binding to each of these receptors is reduced. In some embodiments binding affinity to a complement component, specifically binding affinity to Clq, is also reduced. In one embodiment, binding affinity to neonatal Fc receptor (FcRn) is not reduced. Substantially similar binding to FcRn, i.e. preservation of the binding affinity of the Fc domain to said receptor, is achieved when the Fc domain (or an antibody comprising said Fc domain) exhibits greater than about 70% of the binding affinity of a non-engineered form of the Fc domain (or an antibody comprising said non-engineered form of the Fc domain) to FcRn. The Fc domain, or antibody comprised in the immunoconjugate of the invention comprising said Fc domain, may exhibit greater than about 80% and even greater than about 90% of such affinity. In certain embodiments, the Fc domain of the antibody comprised in the immunoconjugate is engineered to have reduced effector function, as compared to a non-engineered Fc domain. The reduced effector function can include, but is not limited to, one or more of the following: reduced complement dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling inducing apoptosis, reduced crosslinking of target-bound antibodies, reduced dendritic cell maturation, or reduced T cell priming. In one embodiment, the reduced effector function is one or more selected from the group of reduced CDC, reduced ADCC, reduced ADCP, and reduced cytokine secretion. In a particular embodiment the reduced effector function is reduced ADCC. In one embodiment, the reduced ADCC is less than 20% of the ADCC induced by a non-engineered Fc domain (or an antibody comprising a non-engineered Fc domain).

[0203] In one embodiment, the amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor and / or effector function is an amino acid substitution. In one embodiment the Fc domain comprises an amino acid substitution at a position selected from the group of E233, L234, L235, N297, P331 and P329 (numberings according to Kabat EU index). In a more specific embodiment, the Fc domain comprises an amino acid substitution at a position selected from the group of L234, L235 and P329 (numberings according to Kabat EU index). In some embodiments, the Fc domain comprises the amino acid substitutions L234A and L235A (numberings according to Kabat EU index). In one such embodiment, the Fc domain is an IgGi Fc domain, particularly a human IgGi Fc domain. In one embodiment, the Fc domain comprises an amino acid substitution at position P329. In a more specific embodiment, the amino acid substitution is P329A or P329G, particularly P329G (numberings according to Kabat EU index). In one embodiment the Fc domain comprises an amino acid substitution at position P329 and a further amino acid substitution at a position selected from E233, L234, L235, N297 and P331 (numberings according to Kabat EU index). In a more specific embodiment, the further amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S. In particular embodiments, the Fc domain comprises amino acid substitutions at positions P329, L234 and L235 (numberings according to Kabat EU index). In more particular embodiments, the Fc domain comprises the amino acid mutations L234A, L235A and P329G (“P329G LALA”, “PGLALA” or “LALAPG”). Specifically, in particular embodiments, each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A andP329G (Kabat EU index numbering), i.e. in each of the first and the second subunit of the Fc domain the leucine residue at position 234 is replaced with an alanine residue (L234A), the leucine residue at position 235 is replaced with an alanine residue (L235A) and the proline residue at position 329 is replaced by a glycine residue (P329G) (numbering according to Kabat EU index). In one such embodiment, the Fc domain is an IgGi Fc domain, particularly a human IgGi Fc domain. The “P329G LALA” combination of amino acid substitutions almost completely abolishes Fey receptor (as well as complement) binding of a human IgGi Fc domain, as described in PCT publication no. WO 2012 / 130831, which is incorporated herein by reference in its entirety. WO 2012 / 130831 also describes methods of preparing such mutant Fc domains and methods for determining its properties such as Fc receptor binding or effector functions.

[0204] IgG4antibodies exhibit reduced binding affinity to Fc receptors and reduced effector functions as compared to IgGi antibodies. Hence, in some embodiments the Fc domain of the antibody comprised in the immunoconjugate of the invention is an IgG4Fc domain, particularly a human IgG4Fc domain. In one embodiment, the IgG4Fc domain comprises amino acid substitutions at position S228, specifically the amino acid substitution S228P (numberings according to Kabat EU index). To further reduce its binding affinity to an Fc receptor and / or its effector function, in one embodiment the IgG4Fc domain comprises an amino acid substitution at position L235, specifically the amino acid substitution L235E (numberings according to Kabat EU index). In another embodiment, the IgG4Fc domain comprises an amino acid substitution at position P329, specifically the amino acid substitution P329G (numberings according to Kabat EU index). In a particular embodiment, the IgG4Fc domain comprises amino acid substitutions at positions S228, L235 and P329, specifically amino acid substitutions S228P, L235E and P329G (numberings according to Kabat EU index). Such IgG4Fc domain mutants and their Fey receptor binding properties are described in PCT publication no. WO 2012 / 130831, incorporated herein by reference in its entirety.

[0205] In a particular embodiment, the Fc domain exhibiting reduced binding affinity to an Fc receptor and / or reduced effector function, as compared to a native IgGi Fc domain, is a human IgGi Fc domain comprising the amino acid substitutions L234A, L235A and optionally P329G, or a human IgG4Fc domain comprising the amino acid substitutions S228P, L235E and optionally P329G (numberings according to Kabat EU index).

[0206] In certain embodiments, N-glycosylation of the Fc domain has been eliminated. In one such embodiment, the Fc domain comprises an amino acid mutation at position N297, particularly an amino acid substitution replacing asparagine by alanine (N297A) or aspartic acid (N297D) (numberings according to Kabat EU index).

[0207] In addition to the Fc domains described hereinabove and in PCT publication no. WO 2012 / 130831, Fc domains with reduced Fc receptor binding and / or effector function also 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) (numberings according to Kabat EU index). Such Fc mutants include Fc 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).

[0208] Mutant Fc domains can be prepared by amino acid deletion, substitution, insertion or modification using genetic or chemical methods well known in the art. Genetic methods may include site-specific mutagenesis of the encoding DNA sequence, PCR, gene synthesis, and the like. The correct nucleotide changes can be verified for example by sequencing.

[0209] Binding to Fc receptors can be easily determined e.g. by ELISA, or by Surface Plasmon Resonance (SPR) using standard instrumentation such as a BIAcore instrument (GE Healthcare), and Fc receptors such as may be obtained by recombinant expression. Alternatively, binding affinity of Fc domains or antibodies comprising an Fc domain for Fc receptors may be evaluated using cell lines known to express particular Fc receptors, such as human NK cells expressing Fcyllla receptor.

[0210] Effector function of an Fc domain, or an antibody comprising an Fc domain, can be measured by methods known in the art. Examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362; Hellstrom et al. Proc Natl Acad Sci USA 83, 7059-7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502 (1985); U.S. Patent No. 5,821,337; Bruggemann 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 a animal model such as that disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).

[0211] In some embodiments, binding of the Fc domain to a complement component, specifically to Clq, is reduced. Accordingly, in some embodiments wherein the Fc domain is engineered to have reduced effector function, said reduced effector function includes reduced CDC. Clq binding assays may be carried out to determine whether the Fc domain, or antibody comprising the Fc domain, is able to bind Clq and hence has 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 et al., Blood 101, 1045-1052 (2003); and Cragg and Glennie, Blood 103, 2738-2743 (2004)).

[0212] 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).

[0213] V. PARTICULAR ASPECTS OF THE INVENTION

[0214] In one aspect, the invention provides an immunoconjugate comprising a mutant IFN-a2 polypeptide and an antibody that binds to PD-L1,

[0215] wherein the mutant IFN-a2 polypeptide is a human IFN-a2 molecule comprising the amino acid substitution L30A (numbering relative to the human IFN-a2 sequence SEQ ID NO: 17); and

[0216] wherein the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 7, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 8.

[0217] In one aspect, the invention provides an immunoconjugate comprising a mutant IFN-a2 polypeptide and an antibody that binds to PD-L1,

[0218] wherein the mutant IFN-a2 polypeptide is a human IFN-a2 molecule comprising the amino acid substitution L30A (numbering relative to the human IFN-a2 sequence SEQ ID NO: 17); and

[0219] wherein the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 15, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 16.

[0220] In one aspect, the invention provides an immunoconjugate comprising a mutant IFN-a2 polypeptide and an antibody that binds to PD-L1,

[0221] wherein the mutant IFN-a2 polypeptide comprises the amino acid sequence of SEQ ID NO: 22; and wherein the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 7, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 8.

[0222] In one aspect, the invention provides an immunoconjugate comprising a mutant IFN-a2 polypeptide and an antibody that binds to PD-L1,

[0223] wherein the mutant IFN-a2 polypeptide comprises the amino acid sequence of SEQ ID NO: 22; and

[0224] wherein the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 15, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 16.

[0225] In one embodiment according to any of the above aspects of the invention, the antibody is an IgG class immunoglobulin, comprising a human IgGi Fc domain composed of a first and a second subunit,

[0226] wherein in the first subunit of the Fc domain the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the second subunit of the Fc domain the tyrosine residue at position 407 is replaced with a valine residue (Y407V) and optionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numberings according to Kabat EU index),

[0227] and wherein further each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A and P329G (Kabat EU index numbering).

[0228] In this embodiment, the mutant IFN-a2 polypeptide may be fused at its amino-terminal amino acid to the carboxy -terminal amino acid of the first subunit of the Fc domain, through a linker peptide of SEQ ID NO: 25.

[0229] In one aspect, the invention provides an immunoconjugate comprising a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 33, SEQ ID NO: 36, and SEQ ID NO: 37, a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 34, and a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 35.

[0230] In one aspect, the invention provides an immunoconjugate comprising a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 38, a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 39, and a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 40.

[0231] In one particular aspect, the invention provides an immunoconjugate comprising a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 33, a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 34, and a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 35. In one such aspect, the immunoconjugate comprises a polypeptide comprising an amino acid sequence of SEQ ID NO: 33, a polypeptide comprising an amino acid sequence of SEQ ID NO: 34, and a polypeptide comprising an amino acid sequence of SEQ ID NO: 35.

[0232] 1. Polynucleotides

[0233] The invention further provides isolated polynucleotides encoding an immunoconjugate as described herein or a fragment thereof. In some embodiments, said fragment is an antigen binding fragment.

[0234] The polynucleotides encoding immunoconjugates of the invention may be expressed as a single polynucleotide that encodes the entire immunoconjugate or as multiple (e.g., two or more) polynucleotides that are co-expressed. Polypeptides encoded by polynucleotides that are co-expressed may associate through, e.g., disulfide bonds or other means to form a functional immunoconjugate. For example, the light chain portion of an antibody may be encoded by a separate polynucleotide from the portion of the immunoconjugate comprising the heavy chain portion of the antibody and the mutant IFN-a2 polypeptide. When coexpressed, the heavy chain polypeptides will associate with the light chain polypeptides to form the immunoconjugate. In another example, the portion of the immunoconjugate comprising one of the two Fc domain subunits and the mutant IFN-a2 polypeptide could be encoded by a separate polynucleotide from the portion of the immunoconjugate comprising the other of the two Fc domain subunits. When co-expressed, the Fc domain subunits will associate to form the Fc domain.

[0235] In some embodiments, the isolated polynucleotide encodes the entire immunoconjugate according to the invention as described herein. In other embodiments, the isolated polynucleotide encodes a polypeptide comprised in the immunoconjugate according to the invention as described herein.

[0236] In one embodiment, an isolated polynucleotide of the invention encodes the heavy chain of the antibody comprised in the immunoconjugate (e.g. an immunoglobulin heavy chain), and the mutant IFN-a2 polypeptide. In another embodiment, an isolated polynucleotide of the invention encodes the light chain of the antibody comprised in the immunoconjugate.

[0237] In certain embodiments, the polynucleotide or nucleic acid is DNA. In other embodiments, a polynucleotide of the present invention is RNA, for example, in the form of messenger RNA (mRNA). RNA of the present invention may be single stranded or double stranded.

[0238] 2. Recombinant Methods

[0239] Mutant IFN-a2 polypeptides useful in the invention can be prepared by deletion, substitution, insertion or modification using genetic or chemical methods well known in the art. Genetic methods may include site-specific mutagenesis of the encoding DNA sequence, PCR, gene synthesis, and the like. The correct nucleotide changes can be verified for example by sequencing. In this regard, the nucleotide sequence of native IFN-a2 has been described by Taniguchi et al. (Nature 302, 305-10 (1983)) and nucleic acid encoding human IFN-a2 is available from public depositories such as the American Type Culture Collection (Rockville MD). The sequence of native human IFN-a2 is shown in SEQ ID NO: 17, and also in Figure 24. Substitution or insertion may involve natural as well as non-natural amino acid residues. Amino acid modification includes well-known methods of chemical modification such as the addition of glycosylation sites or carbohydrate attachments, and the like.

[0240] Immunoconjugates of the invention may be obtained, for example, by solid-state peptide synthesis (e.g. Merrifield solid phase synthesis) or recombinant production. For recombinant production one or more polynucleotide encoding the immunoconjugate (fragment), e.g., as described above, is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such polynucleotide may be readily isolated and sequenced using conventional procedures. In one embodiment a vector, preferably an expression vector, comprising one or more of the polynucleotides of the invention is provided. Methods which are well known to those skilled in the art can be used to construct expression vectors containing the coding sequence of an immunoconjugate (fragment) along with appropriate transcriptional / translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination / genetic recombination. See, for example, the techniques described in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. (1989); and Ausubel etal., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N.Y (1989). The expression vector can be part of a plasmid, virus, or may be a nucleic acid fragment. The expression vector includes an expression cassette into which the polynucleotide encoding the immunoconjugate (fragment) (i.e. the coding region) is cloned in operable association with a promoter and / or other transcription or translation control elements. As used herein, a "coding region" is a portion of nucleic acid which consists of codons translated into amino acids. Although a "stop codon" (TAG, TGA, or TAA) is not translated into an amino acid, it may be considered to be part of a coding region, if present, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, 5' and 3' untranslated regions, and the like, are not part of a coding region. Two or more coding regions can be present in a single polynucleotide construct, e.g. on a single vector, or in separate polynucleotide constructs, e.g. on separate (different) vectors. Furthermore, any vector may contain a single coding region, or may comprise two or more coding regions, e.g. a vector of the present invention may encode one or more polypeptides, which are post- or co-translationally separated into the final proteins via proteolytic cleavage. In addition, a vector, polynucleotide, or nucleic acid of the invention may encode heterologous coding regions, either fused or unfused to a polynucleotide encoding the immunoconjugate of the invention, or variant or derivative thereof. Heterologous coding regions include without limitation specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain. An operable association is when a coding region for a gene product, e.g. a polypeptide, is associated with one or more regulatory sequences in such a way as to place expression of the gene product under the influence or control of the regulatory sequence(s). Two DNA fragments (such as a polypeptide coding region and a promoter associated therewith) are "operably associated" if induction of promoter function results in the transcription of mRNA encoding the desired gene product and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed. Thus, a promoter region would be operably associated with a nucleic acid encoding a polypeptide if the promoter was capable of effecting transcription of that nucleic acid. The promoter may be a cell-specific promoter that directs substantial transcription of the DNA only in predetermined cells. Other transcription control elements, besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can be operably associated with the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcription control regions are disclosed herein. A variety of transcription control regions are known to those skilled in the art. These include, without limitation, transcription control regions, which function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (e.g. the immediate early promoter, in conjunction with intron-A), simian virus 40 (e.g. the early promoter), and retroviruses (such as, e.g. Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit P-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissuespecific promoters and enhancers as well as inducible promoters (e.g. promoters inducible tetracycline). Similarly, a variety of translation control elements are known to those of ordinary skill in the art. These include, but are not limited to ribosome binding sites, translation initiation and termination codons, and elements derived from viral systems (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence). The expression cassette may also include other features such as an origin of replication, and / or chromosome integration elements such as retroviral long terminal repeats (LTRs), or adeno-associated viral (AAV) inverted terminal repeats (ITRs). Polynucleotide and nucleic acid coding regions of the present invention may be associated with additional coding regions which encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a polynucleotide of the present invention. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Those of ordinary skill in the art are aware that polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the translated polypeptide to produce a secreted or "mature" form of the polypeptide. For example, human IFN-a2 is translated with a 23 amino acid signal sequence at the N-terminus of the polypeptide, which is subsequently cleaved off to produce the mature, 165 amino acid human IFN-a2. In certain embodiments, the native signal peptide, e.g. the IFN-a2 signal peptide or an immunoglobulin heavy chain or light chain signal peptide is used, or a functional derivative of that sequence that retains the ability to direct the secretion of the polypeptide that is operably associated with it. Alternatively, a heterologous mammalian signal peptide, or a functional derivative thereof, may be used. For example, the wild-type leader sequence may be substituted with the leader sequence of human tissue plasminogen activator (TP A) or mouse P-glucuronidase.

[0241] DNA encoding a short protein sequence that could be used to facilitate later purification (e.g. a histidine tag) or assist in labeling the immunoconjugate may be included within or at the ends of the immunoconjugate (fragment) encoding polynucleotide.

[0242] In a further embodiment, a host cell comprising one or more polynucleotides of the invention is provided. In certain embodiments, a host cell comprising one or more vectors of the invention is provided. The polynucleotides and vectors may incorporate any of the features, singly or in combination, described herein in relation to polynucleotides and vectors, respectively. In one such embodiment a host cell comprises (e.g. has been transformed or transfected with) one or more vector comprising one or more polynucleotide that encodes the immunoconjugate of the invention. As used herein, the term "host cell" refers to any kind of cellular system which can be engineered to generate the immunoconjugates of the invention or fragments thereof. Host cells suitable for replicating and for supporting expression of immunoconjugates are well known in the art. Such cells may be transfected or transduced as appropriate with the particular expression vector and large quantities of vector containing cells can be grown for seeding large-scale fermenters to obtain sufficient quantities of the immunoconjugate for clinical applications. Suitable host cells include prokaryotic microorganisms, such as E. coli, or various eukaryotic cells, such as Chinese hamster ovary cells (CHO), insect cells, or the like. For example, polypeptides may be produced in bacteria in particular when glycosylation is not needed. After expression, the polypeptide may be isolated from the bacterial cell paste in a soluble fraction and can be further purified. 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, Nat Biotech 22, 1409-1414 (2004), and Li et al., Nat Biotech 24, 210-215 (2006). 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. Plant cell cultures can also be utilized as hosts. See e.g. US Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants). 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 et al., J Gen Virol 36, 59 (1977)), baby hamster kidney cells (BHK), mouse sertoli cells (TM4 cells as described, e.g., in Mather, Biol Reprod 23, 243-251 (1980)), 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 cells (MMT 060562), TRI cells (as described, e.g., in Mather et al., Annals N.Y. Acad Sci 383, 44-68 (1982)), MRC 5 cells, and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including dhfr' CHO cells (Urlaub et al., Proc Natl Acad Sci USA 77, 4216 (1980)); and myeloma cell lines such as YO, NS0, P3X63 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for protein production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). Host cells include cultured cells, e.g., mammalian cultured cells, yeast cells, insect cells, bacterial cells and plant cells, to name only a few, but also cells comprised within a transgenic animal, transgenic plant or cultured plant or animal tissue. In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell, such as a Chinese Hamster Ovary (CHO) cell, a human embryonic kidney (HEK) cell or a lymphoid cell (e.g., Y0, NSO, Sp20 cell).

[0243] Standard technologies are known in the art to express foreign genes in these systems. Cells expressing a mutant-IFN-a2 polypeptide fused to either the heavy or the light chain of an antibody may be engineered so as to also express the other of the antibody chains such that the expressed mutant IFN-a2 fusion product is an antibody that has both a heavy and a light chain.

[0244] In one embodiment, a method of producing an immunoconjugate according to the invention is provided, wherein the method comprises culturing a host cell comprising one or more polynucleotide encoding the immunoconjugate, as provided herein, under conditions suitable for expression of the immunoconjugate, and optionally recovering the immunoconjugate from the host cell (or host cell culture medium).

[0245] In the immunoconjugate of the invention, the mutant IFN-a2 polypeptide may be genetically fused to the antibody, or may be chemically conjugated to the antibody. Genetic fusion of the IFN-a2 polypeptide to the antibody can be designed such that the IFN-a2 sequence is fused directly to the polypeptide or indirectly through a linker sequence. The composition and length of the linker may be determined in accordance with methods well known in the art and may be tested for efficacy. Particular linker peptides are described herein. Additional sequences may also be included to incorporate a cleavage site to separate the individual components of the fusion if desired, for example an endopeptidase recognition sequence. In addition, an IFN-a2 fusion protein may also be synthesized chemically using methods of polypeptide synthesis as is well known in the art (e.g. Merrifield solid phase synthesis). Mutant IFN-a2 polypeptides may be chemically conjugated to other molecules, e.g. antibodies, using well-known chemical conjugation methods. Bi-functional cross-linking reagents such as homofunctional and heterofunctional cross-linking reagents well known in the art can be used for this purpose. The type of cross-linking reagent to use depends on the nature of the molecule to be coupled to IFN-a2 and can readily be identified by those skilled in the art. Alternatively, or in addition, mutant IFN-a2 and / or the molecule to which it is intended to be conjugated may be chemically derivatized such that the two can be conjugated in a separate reaction as is also well known in the art.

[0246] The immunoconjugates of the invention comprise an antibody. Methods to produce antibodies are well known in the art (see e.g. Harlow and Lane, "Antibodies, a laboratory manual", Cold Spring Harbor Laboratory, 1988). Non-naturally occurring antibodies can be constructed using solid phase-peptide synthesis, can be produced recombinantly (e.g. as described in U.S. patent No. 4,186,567) or can be obtained, for example, by screening combinatorial libraries comprising variable heavy chains and variable light chains (see e.g. U.S. Patent. No.

[0247] 5,969,108 to McCafferty). Immunoconjugates, antibodies, and methods for producing the same are also described in detail e.g. in PCT publication nos. WO 2011 / 020783, WO 2012 / 107417, and WO 2012 / 146628, each of which are incorporated herein by reference in their entirety.

[0248] Any animal species of antibody may be used in the immunoconjugates of the invention. Nonlimiting antibodies useful in the present invention can be of murine, primate, or human origin. If the immunoconjugate is intended for human use, a chimeric form of antibody may be used wherein the constant regions of the antibody are from a human. A humanized or fully human form of the antibody can also be prepared in accordance with methods well known in the art (see e.g. U.S. Patent No. 5,565,332 to Winter). Humanization may be achieved by various methods including, but not limited to (a) grafting the non-human (e.g., donor antibody) CDRs onto human (e.g. recipient antibody) framework and constant regions with or without retention of critical framework residues (e.g. those that are important for retaining good antigen binding affinity or antibody functions), (b) grafting only the non-human specificitydetermining regions (SDRs or a-CDRs; the residues critical for the antibody-antigen interaction) onto human framework and constant regions, or (c) transplanting the entire non-human variable domains, but "cloaking" them with a human-like section by replacement of surface residues. Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are further described, e.g., in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat’l Acad. Set. USA 86:10029-10033 (1989); US Patent Nos. 5, 821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing “resurfacing”); Dall’Acqua et al., Methods 36:43-60 (2005) (describing “FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing the “guided selection” approach to FR shuffling). Human framework regions that may be used for humanization include but are not limited to: framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); andPresta et al. J. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci.

[0249] 13:1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.

[0250] 271:22611-22618 (1996)).

[0251] Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel, Curr Opin Pharmacol 5, 368-74 (2001) and Lonberg, Curr Opin Immunol 20, 450-459 (2008). Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal’s chromosomes. In such transgenic mice, the endogenous immunoglobulin loci have generally been inactivated. For review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™ technology; U.S. Patent No. 5,770,429 describing HuMab® technology; U.S. Patent No. 7,041,870 describing K-M MOUSE® technology, and U.S. Patent Application Publication No. US 2007 / 0061900, describing VelociMouse® technology). Human variable regions from intact antibodies generated by such animals may be further modified, e.g., by combining with a different human constant region.

[0252] Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, e.g., Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Human antibodies generated via human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Set. USA, 103:3557-3562 (2006). Additional methods include those described, for example, in U.S. Patent No. 7,189,826 (describing production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3): 185-91 (2005).

[0253] Human antibodies may also be generated by isolation from human antibody libraries, as described herein.

[0254] Antibodies useful in the invention may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. Methods for screening combinatorial libraries are reviewed, e.g., in Lerner et al. in Nature Reviews 16:498-508 (2016). For example, a variety of methods is known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, e.g., in Frenzel et al. in MAbs 8:1177-1194 (2016); Bazan et al. in Human Vaccines and Immunotherapeutics 8:1817-1828 (2012) and Zhao et al. in Critical Reviews in Biotechnology 36:276-289 (2016) as well as in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O’Brien et al., ed., Human Press, Totowa, NJ, 2001) and in Marks and Bradbury in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003).

[0255] In certain phage display methods, repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al. in Annual Review of Immunology 12: 433-455 (1994). Phage typically display antibody fragments, either as singlechain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self-antigens without any immunization as described by Griffiths et al. in EMBO Journal 12: 725-734 (1993). Finally, naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom and Winter in Journal of Molecular Biology 227: 381-388 (1992). Patent publications describing human antibody phage libraries include, for example: US Patent Nos. 5,750,373; 7,985,840; 7,785,903 and 8,679,490 as well as US Patent Publication Nos. 2005 / 0079574, 2007 / 0117126, 2007 / 0237764 and 2007 / 0292936. Further examples of methods known in the art for screening combinatorial libraries for antibodies with a desired activity or activities include ribosome and mRNA display, as well as methods for antibody display and selection on bacteria, mammalian cells, insect cells or yeast cells. Methods for yeast surface display are reviewed, e.g., in Scholler et al. in Methods in Molecular Biology 503:135-56 (2012) and in Cherf et al. in Methods in Molecular biology 1319:155-175 (2015) as well as in the Zhao et al. in Methods in Molecular Biology 889:73-84 (2012). Methods for ribosome display are described, e.g., in He et al. in Nucleic Acids Research 25:5132-5134 (1997) and in Hanes et al. in PNAS 94:4937-4942 (1997).

[0256] Further chemical modification of the immunoconjugate of the invention may be desirable. For example, problems of immunogenicity and short half-life may be improved by conjugation to substantially straight chain polymers such as polyethylene glycol (PEG) or polypropylene glycol (PPG) (see e.g. WO 87 / 00056).

[0257] Immunoconjugates prepared as described herein may be purified by art-known techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity etc., and will be apparent to those having skill in the art. For affinity chromatography purification an antibody, ligand, receptor or antigen can be used to which the immunoconjugate binds. For example, an antibody which specifically binds the mutant IFN-a2 polypeptide may be used. For affinity chromatography purification of immunoconjugates of the invention, a matrix with protein A or protein G may be used. For example, sequential Protein A or G affinity chromatography and size exclusion chromatography can be used to isolate an immunoconjugate essentially as described in the Examples. The purity of the immunoconjugate can be determined by any of a variety of well-known analytical methods including gel electrophoresis, high-pressure liquid chromatography, and the like. 3. Compositions, Formulations, and Routes of Administration

[0258] In a further aspect, the invention provides pharmaceutical compositions comprising an immunoconjugate as described herein, e.g., for use in any of the below therapeutic methods. In one embodiment, a pharmaceutical composition comprises any of the immunoconjugates provided herein and a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical composition comprises any of the immunoconjugates provided herein and at least one additional therapeutic agent, e.g., as described below.

[0259] Further provided is a method of producing an immunoconjugate of the invention in a form suitable for administration in vivo, the method comprising (a) obtaining an immunoconjugate according to the invention, and (b) formulating the immunoconjugate with at least one pharmaceutically acceptable carrier, whereby a preparation of immunoconjugate is formulated for administration in vivo.

[0260] Pharmaceutical compositions of the present invention comprise a therapeutically effective amount of immunoconjugate dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that are generally non-toxic to recipients at the dosages and concentrations employed, i.e. do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of a pharmaceutical composition that contains immunoconjugate and optionally an additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards or corresponding authorities in other countries. Preferred compositions are lyophilized formulations or aqueous solutions. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives e.g. antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, antioxidants, proteins, drugs, drug stabilizers, polymers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.

[0261] An immunoconjugate 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.

[0262] Parenteral compositions include those designed for administration by injection, e.g. subcutaneous, intradermal, intralesional, intravenous, intraarterial intramuscular, intrathecal or intraperitoneal injection. For injection, the immunoconjugates of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer. The solution may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the immunoconjugates may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. Sterile injectable solutions are prepared by incorporating the immunoconjugates of the invention in the required amount in the appropriate solvent with various of the other ingredients enumerated below, as required. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and / or the other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsion, the preferred methods of preparation are vacuum-drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered liquid medium thereof. The liquid medium should be suitably buffered if necessary and the liquid diluent first rendered isotonic prior to injection with sufficient saline or glucose. The composition must be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less than 0.5 ng / mg protein. Suitable pharmaceutically acceptable carriers include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl 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). Aqueous injection suspensions may contain compounds which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, dextran, or the like. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl cleats or triglycerides, or liposomes.

[0263] Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatinmicrocapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (18th Ed. Mack Printing Company, 1990). Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, which matrices are in the form of shaped articles, e.g. films, or microcapsules. In particular embodiments, prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin or combinations thereof. In addition to the compositions described previously, the immunoconjugates may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the immunoconjugates may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

[0264] Pharmaceutical compositions comprising the immunoconjugates of the invention may be manufactured by means of conventional mixing, dissolving, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the proteins into preparations that can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

[0265] The immunoconjugates may be formulated into a composition in a free acid or base, neutral or salt form. Pharmaceutically acceptable salts are salts that substantially retain the biological activity of the free acid or base. These include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine. Pharmaceutical salts tend to be more soluble in aqueous and other protic solvents than are the corresponding free base forms.

[0266] 4. Therapeutic Methods and Compositions

[0267] Any of the immunoconjugates provided herein may be used in therapeutic methods. Immunoconjugates of the invention may be used as immunotherapeutic agents, for example in the treatment of cancers.

[0268] For use in therapeutic methods, immunoconjugates 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.

[0269] Immunoconjugates of the invention may be particularly useful in treating disease states where stimulation of the immune system of the host is beneficial, in particular conditions where an enhanced cellular immune response is desirable. These may include disease states where the host immune response is insufficient or deficient. Disease states for which the immunoconjugates of the invention may be administered comprise, for example, a tumor or infection where a cellular immune response would be a critical mechanism for specific immunity. The immunoconjugates of the invention may be administered per se or in any suitable pharmaceutical composition.

[0270] In one aspect, immunoconjugates of the invention for use as a medicament are provided. In further aspects, immunoconjugates of the invention for use in treating a disease are provided. In certain embodiments, immunoconjugates of the invention for use in a method of treatment are provided. In one embodiment, the invention provides an immunoconjugate as described herein for use in the treatment of a disease in an individual in need thereof. In certain embodiments, the invention provides an immunoconjugate for use in a method of treating an individual having a disease comprising administering to the individual a therapeutically effective amount of the immunoconjugate. In certain embodiments, the disease to be treated is a proliferative disorder. In a particular embodiment, the disease is cancer. In certain embodiments, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., an anti-cancer agent if the disease to be treated is cancer. In further embodiments, the invention provides an immunoconjugate for use in stimulating the immune system. In certain embodiments, the invention provides an immunoconjugate for use in a method of stimulating the immune system in an individual comprising administering to the individual an effective amount of the immunoconjugate to stimulate the immune system. An “individual” according to any of the above embodiments is a mammal, preferably a human. “Stimulation of the immune system” according to any of the above embodiments may include any one or more of a general increase in immune function, an increase in T cell function, an increase in B cell function, a restoration of lymphocyte function, an increase in the expression of IFN-a2 receptors, an increase in T cell responsiveness, an increase in natural killer cell activity or lymphokine-activated killer (LAK) cell activity, and the like.

[0271] In a further aspect, the invention provides for the use of an immunoconjugate of the invention in the manufacture or preparation of a medicament. In one embodiment, the medicament is for the treatment of a disease in an individual in need thereof. In one embodiment, the medicament is for use in a method of treating a disease comprising administering to an individual having the disease a therapeutically effective amount of the medicament. In certain embodiments, the disease to be treated is a proliferative disorder. In a particular embodiment, the disease is cancer. In one embodiment, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., an anti-cancer agent if the disease to be treated is cancer. In a further embodiment, the medicament is for stimulating the immune system. In a further embodiment, the medicament is for use in a method of stimulating the immune system in an individual comprising administering to the individual an effective amount of the medicament to stimulate the immune system. An “individual” according to any of the above embodiments may be a mammal, preferably a human. “Stimulation of the immune system” according to any of the above embodiments may include any one or more of a general increase in immune function, an increase in T cell function, an increase in B cell function, a restoration of lymphocyte function, an increase in the expression of IFN-a2 receptors, an increase in T cell responsiveness, an increase in natural killer cell activity or lymphokine-activated killer (LAK) cell activity, and the like.

[0272] In a further aspect, the invention provides a method for treating a disease in an individual. In one embodiment, the method comprises administering to an individual having such disease a therapeutically effective amount of an immunoconjugate of the invention. In one embodiment, a composition is administered to said individual, comprising the immunoconjugate of the invention in a pharmaceutically acceptable form. In certain embodiments, the disease to be treated is a proliferative disorder. In a particular embodiment, the disease is cancer. In certain embodiments, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., an anti-cancer agent if the disease to be treated is cancer. In a further aspect, the invention provides a method for stimulating the immune system in an individual, comprising administering to the individual an effective amount of an immunoconjugate to stimulate the immune system. An “individual” -n - according to any of the above embodiments may be a mammal, preferably a human. “Stimulation of the immune system” according to any of the above embodiments may include any one or more of a general increase in immune function, an increase in T cell function, an increase in B cell function, a restoration of lymphocyte function, an increase in the expression of IFN-a2 receptors, an increase in T cell responsiveness, an increase in natural killer cell activity or lymphokine-activated killer (LAK) cell activity, and the like.

[0273] In certain embodiments, the disease to be treated is a proliferative disorder, particularly cancer. Non-limiting examples of cancers include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer. Other cell proliferation disorders that may be treated using an immunoconjugate of the present invention include, but are not limited to neoplasms located in the: abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic region, and urogenital system. Also included are pre-cancerous conditions or lesions and cancer metastases. In certain embodiments, the cancer is chosen from the group consisting of kidney cancer, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer, head and neck cancer, prostate cancer and bladder cancer. A skilled artisan readily recognizes that in many cases the immunoconjugates may not provide a cure but may only provide partial benefit. In some embodiments, a physiological change having some benefit is also considered therapeutically beneficial. Thus, in some embodiments, an amount of immunoconjugate that provides a physiological change is considered an "effective amount" or a "therapeutically effective amount". The subject, patient, or individual in need of treatment is typically a mammal, more specifically a human.

[0274] In some embodiments, an effective amount of an immunoconjugate of the invention is administered to a cell. In other embodiments, a therapeutically effective amount of an immunoconjugates of the invention is administered to an individual for the treatment of disease. For the prevention or treatment of disease, the appropriate dosage of an immunoconjugate 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 route of administration, the body weight of the patient, the type of molecule (e.g. comprising an Fc domain or not), the severity and course of the disease, whether the immunoconjugate is administered for preventive or therapeutic purposes, previous or concurrent therapeutic interventions, the patient's clinical history and response to the immunoconjugate, and the discretion of the attending physician.. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.

[0275] The immunoconjugate is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 pg / kg to 15 mg / kg (e.g.

[0276] 0.1 mg / kg - 10 mg / kg) of immunoconjugate can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. One typical daily dosage might range from about 1 pg / kg to 100 mg / kg or more, depending on the factors mentioned above. 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. One exemplary dosage of the immunoconjugate would be in the range from about 0.005 mg / kg to about 10 mg / kg. In other non-limiting examples, a dose may also comprise from about 1 microgram / kg / body weight, about 5 microgram / kg / body weight, about 10 microgram / kg / body weight, about 50 microgram / kg / body weight, about 100 microgram / kg / body weight, about 200 microgram / kg / body weight, about 350 microgram / kg / body weight, about 500 microgram / kg / body weight, about 1 milligram / kg / body weight, about 5 milligram / kg / body weight, about 10 milligram / kg / body weight, about 50 milligram / kg / body weight, about 100 milligram / kg / body weight, about 200 milligram / kg / body weight, about 350 milligram / kg / body weight, about 500 milligram / kg / body weight, to about 1,000 mg / kg / body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg / kg / body weight to about 100 mg / kg / body weight, about 5 microgram / kg / body weight to about 500 milligram / kg / body weight, etc., can be administered, based on the numbers described above. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 5.0 mg / kg or 10 mg / kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, e.g. every week or every three weeks (e.g. such that the patient receives from about two to about twenty, or e.g. about six doses of the immunoconjugate). An initial higher loading dose, followed by one or more lower doses may be administered. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0277] The immunoconjugates of the invention will generally be used in an amount effective to achieve the intended purpose. For use to treat or prevent a disease condition, the immunoconjugates of the invention, or pharmaceutical compositions thereof, are administered or applied in a therapeutically effective amount. Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0278] For systemic administration, a therapeutically effective dose can be estimated initially from in vitro assays, such as cell culture assays. A dose can then be formulated in animal models to achieve a circulating concentration range that includes the IC50 as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.

[0279] Initial dosages can also be estimated from in vivo data, e.g., animal models, using techniques that are well known in the art. One having ordinary skill in the art could readily optimize administration to humans based on animal data.

[0280] Dosage amount and interval may be adjusted individually to provide plasma levels of the immunoconjugates which are sufficient to maintain therapeutic effect. Usual patient dosages for administration by injection range from about 0.1 to 50 mg / kg / day, typically from about 0.5 to 1 mg / kg / day. Therapeutically effective plasma levels may be achieved by administering multiple doses each day. Levels in plasma may be measured, for example, by HPLC.

[0281] In cases of local administration or selective uptake, the effective local concentration of the immunoconjugates may not be related to plasma concentration. One having skill in the art will be able to optimize therapeutically effective local dosages without undue experimentation. A therapeutically effective dose of the immunoconjugates described herein will generally provide therapeutic benefit without causing substantial toxicity. Toxicity and therapeutic efficacy of an immunoconjugate can be determined by standard pharmaceutical procedures in cell culture or experimental animals. Cell culture assays and animal studies can be used to determine the LD50 (the dose lethal to 50% of a population) and the ED50 (the dose therapeutically effective in 50% of a population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Immunoconjugates that exhibit large therapeutic indices are preferred. In one embodiment, the immunoconjugate according to the present invention exhibits a high therapeutic index. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosages suitable for use in humans. The dosage lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon a variety of factors, e.g., the dosage form employed, the route of administration utilized, the condition of the subject, and the like. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See, e.g., Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics, Ch. 1, p. 1, incorporated herein by reference in its entirety).

[0282] The attending physician for patients treated with immunoconjugates of the invention would know how and when to terminate, interrupt, or adjust administration due to toxicity, organ dysfunction, and the like. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity). The magnitude of an administered dose in the management of the disorder of interest will vary with the severity of the condition to be treated, with the route of administration, and the like. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency will also vary according to the age, body weight, and response of the individual patient.

[0283] The maximum therapeutic dose of an immunoconjugate comprising a mutant IFN-a2 polypeptide as described herein may be increased from those used for an immunoconjugate comprising wild-type IFN-a2.

[0284] 5. Other Agents and Treatments The immunoconjugates according to the invention may be administered in combination with one or more other agents in therapy. For instance, an immunoconjugate of the invention may be co-administered with at least one additional therapeutic agent. The term "therapeutic agent” encompasses any agent administered to treat a symptom or disease in an individual in need of such treatment. Such additional therapeutic agent may comprise any active ingredients suitable for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. In certain embodiments, an additional therapeutic agent is an immunomodulatory agent, a cytostatic agent, an inhibitor of cell adhesion, a cytotoxic agent, an activator of cell apoptosis, or an agent that increases the sensitivity of cells to apoptotic inducers. In a particular embodiment, the additional therapeutic agent is an anti-cancer agent, for example a microtubule disruptor, an antimetabolite, a topoisomerase inhibitor, a DNA intercalator, an alkylating agent, a hormonal therapy, a kinase inhibitor, a receptor antagonist, an activator of tumor cell apoptosis, or an anti angiogenic agent.

[0285] Such other agents are suitably present in combination in amounts that are effective for the purpose intended. The effective amount of such other agents depends on the amount of immunoconjugate used, the type of disorder or treatment, and other factors discussed above. The immunoconjugates 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.

[0286] Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate compositions), and separate administration, in which case, administration of the immunoconjugate of the invention can occur prior to, simultaneously, and / or following, administration of the additional therapeutic agent and / or adjuvant. Immunoconjugates of the invention may also be used in combination with radiation therapy.

[0287] 6. Articles of Manufacture

[0288] 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 which 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 an immunoconjugate 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 an immunoconjugate 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.

[0289] Embodiments of the invention

[0290] In the following, specific embodiments of the invention are listed:

[0291] 1. An immunoconjugate comprising a mutant IFN-a2 polypeptide and an antibody that binds to PD-L1,

[0292] wherein the mutant IFN-a2 polypeptide is a human IFN-a2 molecule comprising an amino acid substitution selected from the group consisting of L30A, L30H, and L153D (numbering relative to the human IFN-a2 sequence SEQ ID NO: 17).

[0293] 2. An immunoconjugate according to embodiment 1, wherein the human IFN-a2 molecule comprises the amino acid substitution L30A (numbering relative to the human IFN-a2 sequence SEQ ID NO: 17). 3. The immunoconjugate according to any one of embodiment 1 or 2, wherein the human IFN-a2 molecule comprises the amino acid substitution L30H (numbering relative to the human IFN-a2 sequence SEQ ID NO: 17).

[0294] 4. The immunoconjugate according to any one of embodiments 1 to 3, wherein the human IFN-a2 molecule is a human IFN-a2a molecule.

[0295] 5. The immunoconjugate according to any one of embodiments 1 to 4, wherein the mutant IFN-a2 polypeptide comprises a sequence selected from the group consisting of SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24.

[0296] 6. The immunoconjugate according to any one of embodiments 1 to 5, wherein the mutant IFN-a2 polypeptide comprises the sequence of SEQ ID NO: 22.

[0297] 7. An immunoconjugate according to any one of embodiments 1 to 6,

[0298] wherein the antibody comprises

[0299] (a) a heavy chain variable region (VH) comprising

[0300] a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1,

[0301] a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2,

[0302] a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and

[0303] a light chain variable region (VL) comprising

[0304] a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4,

[0305] a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and

[0306] a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6,

[0307] or

[0308] (b) a heavy chain variable region (VH) comprising

[0309] a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9,

[0310] a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10,

[0311] a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11, and

[0312] a light chain variable region (VL) comprising

[0313] a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12,

[0314] a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and

[0315] a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14.

[0316] 8. An immunoconjugate according to any one of embodiments 1 to 7, wherein the antibody comprises

[0317] (a) a heavy chain variable region (VH) comprising

[0318] a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1,

[0319] a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2,

[0320] a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and

[0321] a light chain variable region (VL) comprising

[0322] a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4,

[0323] a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and

[0324] a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

[0325] 9. An immunoconjugate according any one of embodiments 1 to 8; and

[0326] wherein the antibody comprises

[0327] (a) a heavy chain variable region (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 7, and a light chain variable region (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 8, or

[0328] (b) a heavy chain variable region (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 15, and a light chain variable region (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 16.

[0329] 10. An immunoconjugate according to any one of embodiments 1 to 9; and

[0330] wherein the antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 7, and a light chain variable region (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 8.

[0331] 11. The immunoconjugate according to any one of embodiments 1 to 10, comprising a) a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 33, SEQ ID NO: 36, and SEQ ID NO: 37, a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 34, and a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 35, or

[0332] b) a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 38, a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 39, and a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 40.

[0333] 12. The immunoconjugate according to any one of embodiments 1 to 11, comprising a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 33, SEQ ID NO: 36, and SEQ ID NO: 37, a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 34, and a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 35.

[0334] 13. The immunoconjugate according to any one of embodiments 1 to 12, comprising a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 33, a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 34, and a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 35.

[0335] 14. The immunoconjugate according to any one of embodiments 1 to 13, wherein the immunoconjugate comprises not more than one mutant IFN-a2 polypeptide. 15. The immunoconjugate according to embodiment 14, which is an antibody fragment selected from the group consisting of a Fv, a Fab, a Fab’, a scFv, and a F(ab’)2.

[0336] 16. The immunoconjugate according to any one of embodiments 1 to 15, wherein the antibody comprises an Fc domain composed of a first and a second subunit.

[0337] 17. The immunoconjugate according to embodiment 16 wherein the Fc domain is an IgG class, particularly an IgGl subclass, Fc domain.

[0338] 18. The immunoconjugate according to embodiment 16 or 17, wherein the Fc domain is a human Fc domain.

[0339] 19. The immunoconjugate according to any one of embodiments 16 to 18, wherein the Fc domain is a human IgG class Fc domain, particularly an human IgGl subclass Fc domain.

[0340] 20. The immunoconjugate according to any one of embodiments 1 to 19, wherein the antibody is a full-length antibody, particularly a full-length IgGl antibody.

[0341] 21. The immunoconjugate according to any one of embodiments 1 to 20, wherein the antibody is an IgG class, particularly an IgGl subclass immunoglobulin.

[0342] 22. The immunoconjugate according to any one of embodiments 16 to 21, wherein the Fc domain comprises a modification promoting the association of the first and the second subunit of the Fc domain.

[0343] 23. The immunoconjugate according to any one of embodiments 16 to 22, wherein in the first subunit of the Fc domain the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the second subunit of the Fc domain the tyrosine residue at position 407 is replaced with a valine residue (Y407V) and optionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numberings according to Kabat EU index).

[0344] 24. The immunoconjugate according to embodiment 23, wherein in the first subunit of the Fc domain additionally the serine residue at position 354 is replaced with a cysteine residue (S354C) or the glutamic acid residue at position 356 is replaced with a cysteine residue (E356C), and in the second subunit of the Fc domain additionally the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C) (numberings according to Kabat EU index).

[0345] 25. The immunoconjugate according to any one of embodiments 16 to 24, wherein the mutant IFN-a2 polypeptide is fused at its amino-terminal amino acid to the carboxy-terminal amino acid of one of the subunits of the Fc domain, particularly the first subunit of the Fc domain, optionally through a linker peptide.

[0346] 26. The immunoconjugate according to embodiment 25, wherein the linker peptide has the amino acid sequence of SEQ ID NO: 25.

[0347] 27. The immunoconjugate according to any one of embodiments 16 to 26, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor, particularly an Fey receptor, and / or effector function, particularly antibody-dependent cell-mediated cytotoxicity (ADCC).

[0348] 28. The immunoconjugate according to embodiment 27, wherein said one or more amino acid substitution is at one or more position selected from the group of L234, L235, and P329 (Kabat EU index numbering).

[0349] 29. The immunoconjugate according to embodiment 27 or 28, wherein each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A and P329G (Kabat EU index numbering).

[0350] 30. The immunoconjugate according to any one of embodiments 1 to 29, essentially consisting of a mutant IFN-a2 polypeptide and a full-length IgGl antibody, joined by a linker sequence.

[0351] 31. One or more isolated polynucleotides encoding the immunoconjugate according to any one of embodiments 1 to 30.

[0352] 32. One or more vector, particularly expression vector, comprising the polynucleotide(s) according to embodiment 31.

[0353] 33. A host cell comprising the polynucleotide(s) of embodiment 31 or the vector(s) of embodiment 32. 34. A method of producing an immunoconjugate comprising a mutant IFN-a2 polypeptide and an antibody that binds to PD-L1, comprising (a) culturing the host cell of embodiment 33 under conditions suitable for the expression of the immunoconjugate, and optionally (b) recovering the immunoconjugate.

[0354] 35. An immunoconjugate comprising a mutant IFN-a2 polypeptide and an antibody that binds to PD-L1, produced by the method of embodiment 34.

[0355] 36. A pharmaceutical composition comprising the immunoconjugate according to any one of embodiments 1-12, 14-30 or 35 and a pharmaceutically acceptable carrier.

[0356] 37. The immunoconjugate according to any one of embodiments 1-30 or 35 for use as a medicament.

[0357] 38. The immunoconjugate according to any one of embodiments 1-30 or 35 for use in the treatment of a disease.

[0358] 39. The immunoconjugate for use in the treatment of a disease of embodiment 38, wherein said disease is cancer.

[0359] 40. Use of the immunoconjugate according to any one of embodiments 1-30 or 35 in the manufacture of a medicament for the treatment of a disease.

[0360] 41. The use of embodiment 40, wherein said disease is cancer.

[0361] 42. A method of treating a disease in an individual, comprising administering to said individual a therapeutically effective amount of a composition comprising the immunoconjugate according to any one of embodiments 1-30 or 35 in a pharmaceutically acceptable form.

[0362] 43. The method of embodiment 42, wherein said disease is cancer.

[0363] 44. A method of stimulating the immune system of an individual, comprising administering to said individual an effective amount of a composition comprising the immunoconjugate according to any one of embodiments 1-30 or 35 in a pharmaceutically acceptable form. TABLE 1 : AMINO ACID SEQUENCES

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370]

[0371]

[0372]

[0373] VII. EXAMPLES

[0374] The following are examples of methods and compositions of the invention. It is understood that various other embodiments may be practiced, given the general description provided above.

[0375] Recombinant DNA

[0376]

[0377] Standard methods were used to manipulate DNA as described in Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. The molecular biological reagents were used according to the manufacturer's instructions.

[0378]

[0379] synthesis

[0380] Desired gene segments were prepared by chemical synthesis at Twist Bioscience (South San Francisco, USA). The synthesized gene fragments were cloned into an E. coli plasmid for propagation / amplification. The DNA sequences of subcloned gene fragments were verified by DNA sequencing. Alternatively, short synthetic DNA fragments were assembled by annealing chemically synthesized oligonucleotides or via PCR. The respective oligonucleotides were prepared by metabion GmbH (Planegg-Martinsried, Germany)

[0381]

[0382] of the basic / standard mammalian

[0383]

[0384] For the expression of a desired gene / protein (e.g. full length antibody or fusion protein heavy chain, full length antibody or fusion protein light chain, or an antigen used in the assays herein, e.g. PD-L1, IFN-a2 or others) a transcription unit comprising the following functional elements is used:

[0385] - the immediate early enhancer and promoter from the human cytomegalovirus (P-CMV) including intron A,

[0386] - a human heavy chain immunoglobulin 5 ’-untranslated region (5’UTR),

[0387] - a murine immunoglobulin heavy chain signal sequence,

[0388] - a gene / protein to be expressed (e.g. full length antibody or fusion protein heavy chain), and - the bovine growth hormone polyadenylation sequence (BGH pA). Beside the expression unit / cassette including the desired gene to be expressed, the basic / standard mammalian expression plasmid contains

[0389] - an origin of replication from the vector pUC18 which allows replication of this plasmid in E. coli, and

[0390] - a beta-lactamase gene which confers ampicillin resistance in E. coli.

[0391] Protein determination

[0392] The protein concentration of purified polypeptides was determined by determining the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence of the polypeptide.

[0393] Example 1: Generation and Characterization of the anti PD-L1 antigen binding domain M14 a) Generation of the anti-PD-Ll M14, DIO, E24, Hl 3 and J12 Fabs

[0394] The anti-PD-Ll binders used for targeting immunoconjugates comprising IFN-a2 mutants herein were generated during a campaign which had the goal to generate bispecific anti-PD-Ll / anti-IFN-a2a binders based on the DutaFab technology. DutaFabs are engineered therapeutic Fab fragments that can bind two distinct antigens. They comprise two binding sites with different specificities through the assignment of residues into two sides: the so-called H-side paratope encompassing CDR-H1, CDR-H3 and CDR-L2, and the L-side paratope encompassing CDR-L1, CDR-L3 and CDR-H2. Starting from DutaFabs, monospecific antigen binders can be generated by replacing one of the two paratopes with sequences that do not bind to a specific epitope (“dummy sequences”).

[0395] In an initial step of this campaign, DutaFabs binding to human PD-L1 were generated by phage display. For this, two sets of Roche in-house phage display libraries of synthetic Fab fragments were utilized. In the first set, the residues within the CDR-H1, CDR-H3, VHN-terminus and CDR-L2 regions of the Fab fragments (corresponding to the so-called H-side paratope) were diversified, whereas in the second set the residues within the CDR-L1, CDR-L3, VL N-terminus and CDR-H2 regions of the Fab fragments (corresponding to the so-called L-side paratope) were diversified. In each library set, the other three CDR regions were kept non-diversified as either invariant dummy sequence or in some contexts as CDRs specific for an irrelevant epitope. In both libraries, the CHI domains of the Fab fragments were fused via a linker to truncated gene-III protein to facilitate phage display.

[0396] Phage library panning was performed in four rounds, wherein the first round was performed with 250 nM in 1 ml of randomly S-S linker biotinylated PD-L1 ECD pre-immobilized on Dynabeads™ M-280 Streptavidin magnetic beads (Thermo Fisher catalog number 11206D), and rounds 2 to 4 were performed with 250 nM, 100 nM, and 100 nM in 1 ml of randomly S-S linker biotinylated PD-L1 ECD in solution, followed by capture of Fab-on-phage / target complexes on the beads. Only in round two, the Streptavidin beads were replaced by Neutravidin-coupled magnetic beads (Sera-Mag SpeedBeadNeutravidin-Coated Magnetic Particles, Cytiva: 78152104010350). In round one, captured phage clones bearing target-specific DutaFabs in combination with the beads were directly infected into log-phase TGI E. coli cells, and rescued using M13KO7 helper phage, according to standard protocols. In rounds 2 to 4, the DutaFab-bearing phages were eluted from the magnetic beads using either 100 mM DTT or 2.5 pM Atezolizumab prior to being used for infection.

[0397] For screening of selection outputs, a polyclonal plasmid miniprep of the respective selection round was prepared from the infected TGI E. coli cells. Plasmids were digested using BamHI restriction endonuclease, which cuts the phagemid pDuta4 upstream and downstream of the phage Fd gene 3 domain. Plasmids were recircularized by ligation, generating an in-frame fusion of a T7 tag at the C -terminus of the DutaFab CHI domain. The ligated polyclonal plasmids encoding T7-tagged DutaFabs were transformed into TGI E. coli cells, and single colonies were picked into microtiter plates. Soluble DutaFabs were expressed in microtiter plates and supernatants were clarified by centrifugation.

[0398] Human PD-L1 specific binders were identified by ELISA as follows: 20 pL mixture of biotinylated antigen huPD-Ll(ECD)-huFc fusion (P1AF6937; SEQ ID NO: 324 and SEQ ID NO: 325; produced as described in Example 1) (0.5 pg / mL final concentration in assay) and detection antibody anti-human Ig kappa chain specific antibody POD (peroxidase) (Millipore AP502, 1:6000 final concentration in assay) were mixed with 5 pL DutaFab-containing bacterial supernatant and added to streptavidin coated microtiter plates (Microcoat 384 SA, 11974998001). After an incubation for 60 min at RT, the plates were washed 6x with PBST, 0.1 % Tween20. The binding of DutaFabs to huPD-Ll(ECD)-huFc (P1AF6937) was detected by adding 30 pL TMB substrate to the wells. After an incubation of 5 min at RT, the absorbance was measured at 370 nm with an EnVision Reader (PerkinElmer).

[0399] Cynomolgus PD-L1 specific binders were identified by ELISA as follows: 20 pL mixture of biotinylated antigen cynoPD-Ll(ECD)-huFc (P1AF6938; SEQ ID NO: 326 and SEQ ID NO: 327; produced as described in Example 1) (0.5 pg / mL final in assay) and detection antibody anti-human Ig kappa chain specific antibody POD (Millipore AP502P, 1 :6000 final in assay) were mixed with 5 pL DutaFab-containing bacterial supernatant and added to streptavidin coated microtiter plates (Microcoat 384 SA, 11974998001). After an incubation for 60 min at room temperature, the plates were washed 6 times with PBST, 0.1 % Tween20. The binding of DutaFabs to cynoPD-Ll(ECD)-huFc (P1AF6938) was detected by adding 30 pL TMB substrate to the wells. After an incubation of 5 min at RT, the absorbance was measured at OD 370 nm with an EnVision Reader (PerkinElmer). ELISA-positive clones were bacterially expressed as soluble DutaFab fragments in 96-well format. Clones expressing PD-L1 -specific DutaFabs were identified, and the corresponding phagemids were sequenced and 115 unique sequences were discovered.

[0400] A selection of these unique clones was thereafter expressed in E. coli and purified via a one-step affinity capture using CaptureSelect™ IgG-CHl resin (Thermo Fisher; Catalog number: 19432001L) in order to be characterized. Binding to PD-L1 -positive cells was assessed via FACS. DutaFabs were examined using analytical size exclusion chromatography. SPR (Biacore 8K or 8K+) was used to determine binding kinetics to huPD-Ll-Fc (Pl AF6937), human PD-L1 (CD274) C -terminal extracellular domain (P1AF7352; SEQ ID NO: 328 and 329), cynomolgus PD-Ll-Fc (P1AF6938) and CD79B ECD dimer (P1AE1979; SEQ ID NO: 330) (negative control). Briefly, anti-human Fab antibody (Cytiva; Catalog number 28958325) was immobilized on a CM5 chip according to the manufacturer's instructions. 100 nM DutaFabs were captured 10 pl / min, 60 sec) and 0 nM, 10 nM, 50 nM and 150 nM of antigen was flown at 30 pl / min for 120 sec followed by a 240 second dissociation window at a flow rate of 30 pl / min. The surface was regenerated by injecting 10 mM glycine, pH 2, for 60s at a flow rate of 30 pl / min. Additionally; competitive binding to PD-L1 with Atezolizumab was investigated by SPR in a competitive binding assay setup. Table 2 shows affinity to PD-L1, epitope binding, competitive binding with Atezolizumab and blocking of PD-L1 / PD1 binding for several of the generated anti-PD-Ll DutaFabs. For this, the HEK-Blue IFN-a / p reporter gene assay was performed as described in Example 4 below, using the parental (Invivogen) HEK cell line with low PD-L1 expression on the cell surface (about 300 PD-L1 molecules / cell on the cell surface) and a modified cell line expressing recombinant PD-L1 on the cell surface (“clone 45”, about 25,000 PD-L1 molecules / cell on the cell surface), generated as described in Example 3. Notably, M14 (parental), a DutaFab with high affinity to PD-L1 bound to the C-terminus of PD-L1, thus not competing with Atezolizumab (which binds to the N-terminus of PD-L1) for PD-L1 binding, but it surprisingly blocked the interaction between PD-L1 and PD1. This was unexpected as usually only antibodies binding to the N-terminus of PD-L1 are able to block PD-L1 / PD1 interaction.

[0401] A selection of the identified unique PD-L1 binding clones was characterized further via analytical size exclusion chromatography, FACS (to determine binding to PD-L1 positive cells), and SPR (Biacore 8K or 8K+). Results from the SPR analysis are shown in Table 2.

[0402] Table 2: PD-L1 affinities of selected DutaFabs from phage display

[0403]

[0404] 5 huPD-Ll -binding DutaFabs E24, J12, H13, D10 and M14 covering a range of affinities and including DutaFabs binding to either PD-L1 N-terminus or C-terminus were selected for further experiments (see below). One of them, M14 parental, was also further affinity matured as part of the effort to generate a bispecific anti-PD-Ll anti-IFN-a2a antigen-binding domain, as described below.

[0405] b) Generation, Characterization and selection o f the Ml 4 Binder

[0406] In the context of another set of examples with the goal to create bispecific DutaFab, the M14 parental paratope was combined with an anti-IFN-a2a paratope into a bispecific anti-PD-Ll anti-IFN-a2a DutaFab. In order to optimize this molecule for improved PD-L1 binding, three phage display libraries were created by partial randomization of specific residues of the PD-L1 binding paratope. Four rounds of panning were performed with varying concentrations of biotinylated PD-Ll, leading to the identification of specific binders with enhanced affinity via ELISA testing. Selected clones, indicated by higher ELISA absorbance values, underwent recombinant expression and binding evaluation using SPR. Further optimization through mutagenesis and CDR shuffling refined the clone based on desired properties like yield, affinity, hydrophilicity, and thermal stability. These iterative processes produced binder variants with different PD-L1 binding affinities.

[0407] Since the resulting bispecific DutaFab clone showed good PD-L1 binding properties, it was then used to generate a monospecific, anti PD-L1 targeting arm. IFN-a2a binding was removed and potential immunogenicity reduced by replacing the L-side CDRs on the heavy and light chains that were specific for IFN-a2a (CDR-L1, CDR-L3 and CDR-H2) with amino acid stretches closer to those found in human antibody germlines. Generated molecules were variably analyzed using combinations of charge, hydrophilicity and immunogenicity predicting algorithms, SPR, analytical SEC, analytical HIC, and Differential Scanning Fluorimetry (DSF), and affinity-matured M14-derived binders selected based on these data.

[0408] Affinities for human and cynomolgus PD-L1 of these selected PD-L1 monospecific binders were analyzed. For this, Fab-fragments of corresponding binders were analyzed by SPR using Fc-tagged human and cynomolgus PD-L1 and His-tagged IFN-a, essentially as described in section a) of Example 2 f.

[0409] Affinity to human IFN-a2a was tested to ensure that any binding to IFN-a2a had been eradicated entirely by replacing the original L-side CDRs responsible for binding to IFN-a2a (CDR-L1, CDR-L3 and CDR-H2). As can be seen from the data shown in Table 3, all tested binders show binding to human as well as to cynomolgus PD-L1. Pl AI3436 and Pl AI3438 show particularly high affinity, both to human and to cynomolgus PD-L1. None of the tested Fabs showed binding to IFN-a2a. P1AI4260 was selected for creating immunoconjugates.

[0410] In order to describe the binding site of selected anti-PD-Ll Fabs to PD-L1 in more detail, it was tested whether the anti-PD LI Fabs can (1) bind to the C-terminal domain of PD-L1, (2) compete for binding with Atezolizumab (which bind to the N-terminal domain of PD-L1) and (3) inhibit binding of PD1 to PD LI by SPR experiments.

[0411] Table 3: Affinities of M14-derived anti-huPD-Ll Tabs for human or cynomolgus PD-L1

[0412]

[0413] Binding of M14 (parental)-derived anti-PD-Ll Fabs to the C-terminal domain of PD-L1 The SPR experiments were performed on a Biacore T200 at 25 °C with PBS-P+ as running and sample dilution buffer (0.2 M phosphate buffer with 27 mM KC1, 1.37 M NaCl and 0.5% Surfactant P20 (Tween 20), Cytiva).

[0414] Anti-Fab antibody (ThermoFischer) was directly immobilized on the second flow cell of a CM3 as described above. For the assay, the different anti-huPD-Ll Fabs had been expressed recombinantly as one-armed Fab-Fc fusion proteins. The Fab-Fc-fusion proteins were captured by injecting for 120 seconds at a flow rate of 10 pl / min.

[0415] As a second step PD-L1 (P1AF6939; SEQ ID NO: 331) or C-terminal domain of PD-L1 (P1AF7352) was injected for 120 sec at 30 pl / min. Binding curves were evaluated using Biacore T200 evaluation software 3.1 (Cytiva). Anti-huPD-Ll binding antibody Atezolizumab was run in parallel as a negative control, as it is known to bind to the extracellular domain of huPD-Ll, but not to its C-terminal domain.

[0416] Table 4: Affinities of selected M14 (parental)-derived anti-huPD-Ll Tabs to the extracellular domain or the C-terminal domain of huPD-Ll

[0417]

[0418] As shown in Table 4, the results of the assay confirm experimentally that the selected binder M14ic (P1AI4392; SEQ ID NO: 285, SEQ ID NO: 286 and SEQ ID NO: 287) and the binder (P1AI4475; SEQ ID NO: 291, SEQ ID NO: 292 and SEQ ID NO: 293) - like the parental binder they are both derived from - bind to the C-terminal domain of PD-L1. Atezolizumab, which is known to bind to the N-terminal domain of PD-L1, was used as a control.

[0419] SPR assays demonstrating lack of competitive binding with Atezolizumab

[0420] The SPR experiments were performed on a Biacore T200 at 25 °C with PBS-P+ (0.2 M phosphate buffer with 27 mM KC1, 1.37 M NaCl and 0.5% Surfactant P20 (Tween 20), Cytiva) as running and sample dilution buffer.

[0421] One-armed anti-PD-Ll Fab-Fc fusion (P1AI4841; SEQ ID NO: 315, SEQ ID NO: 316 and SEQ ID NO: 317) was directly immobilized on the second flow cell of a CM3 chip at pH 5.0 using the standard amine coupling kit (Cytiva) aiming for a sensor density of 500 RU. After activation of the sensor surface with a 1:1 mixture of 0.4 M l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and 0.1 M N-hydroxysuccinimide (NHS), 50nM P1AI4841 (diluted in 10 mM acetate pH 5.0) was injected with a flow rate of lOpl / min. After blocking with 1 M ethanolamine-HCl pH 8.5, the coupling procedure led to a sensor density of 500 RU. Recombinant one-armed human PD-Ll-Fc fusion (P1AF6937) at a concentration of 25 nM was captured by injecting for 120 seconds at a flow rate of 30 pl / min reaching a signal of approximately 30 RU. As a second step, the anti-PD-Ll Fab derived from M14 (parental) or a reference anti-PD-L1 or a control molecule was injected at a concentration of 75 nM for 120 sec at 30 pl / min. An increasing signal during the second injection phase indicates a different epitope because the binding epitope was not blocked by the capture PD-L1 binder on the surface. No additional binding indicates the same epitope region. The chip surface was regenerated after every cycle by injection of 0.85% H3PO4 for 60 seconds at a flow of lOpl / min. Bulk refractive index differences were corrected by subtracting the response obtained on the reference flow cell. Binding curves were evaluated using Biacore T200 evaluation software 3.1 (Cytiva).

[0422] The results of the experiment are shown in the sensorgram of Figure 2. The SPR experiments clearly demonstrated that M14 (parental)-derived anti-PD-Ll Fabs Pl AI4392, Pl AI4397 (SEQ ID NO: 288, SEQ ID NO: 289 and SEQ ID NO: 290), P1AI4475, P1AI4476 (SEQ ID NO: 294, SEQ ID NO: 295 and SEQ ID NO: 296), P1AI4478 (SEQ ID NO: 297, SEQ ID NO: 298 and SEQ ID NO: 299), P1AI4479 (SEQ ID NO: 300, SEQ ID NO: 301 and SEQ ID NO: 302), P1AI4480 (SEQ ID NO: 303, SEQ ID NO: 304 and SEQ ID NO: 305), P1AI4481 (SEQ ID NO: 306, SEQ ID NO: 307 and SEQ ID NO: 308), P1AI4483 (SEQ ID NO: 309, SEQ ID NO: 310 and SEQ ID NO: 311), P1AI4485 (SEQ ID NO: 312, SEQ ID NO: 313 and SEQ ID NO: 314), P1AI4903 (SEQ ID NO: 318, SEQ ID NO: 319 and SEQ ID NO: 320) and Pl AHO 179 (SEQ ID NO: 212, SEQ ID NO: 213 and SEQ ID NO: 214) did not compete with Atezolizumab binding to PD-L1. PD-Ll-Fc (P1AF6937) and anti-PD-Ll Fab based on Atezolizumab (P1AI4841) were used as negative controls.

[0423] Blocking the interaction of PD1 and PD-L1 by PD-L1 binding of M14 (parental)-derived Fab fragments

[0424] Antibodies that block interaction of human PD-L1 and human PD1 (e.g. Atezolizumab, BMS-936559, Avelumab, Durvalumab) usually bind to the N-terminal domain of PD-L1, which overlaps with the PDl-binding site of PD-L1. In contrast, the M14HH17L(parental)-derived anti-huPD-Ll monospecific Fabs and anti-PD-Ll / anti-IFN-a2a bispecific DutaFabs are directed against the C-terminal domain of PD-L1 but surprisingly are still capable to block the interaction of PD-L1 and PD1 in a SPR binding assay. To demonstrate this, SPR experiments were performed on a Biacore T200 at 25 °C with PBS-P+ as running and sample dilution buffer (0.2 M phosphate buffer with 27 mM KC1, 1.37 M NaCl and 0.5% Surfactant P20 (Tween 20), Cytiva). A CAP Chip (provided in the Biotin CAPture Kit, series S, Cytiva, 28920234) was hybridized with ssDNA-SA according manufacturer's instructions and loaded with biotinylated human PD1 extracellular domain (P1AF6937) for 180 seconds with a flow rate of 5 pL / min and at a concentration of 100 nM. Premixtures containing 50 nM of human PD-Ll_Fc fusion (P1AD8568; SEQ ID NO: 332) and 3-fold molar excess of M14HH17L(parental)-derived Fab-Fc were injected on both flow cells for 300 seconds at a flow speed of 5 pL / min. Dissociation time is set to 300 s. An increasing signal during the injection phase indicates that the binding of PD-L1 to PD1 is not blocked by the analyzed antibody, while no signal increase indicates blocking by the antibody.

[0425] As shown in the sensorgrams in Figure 3, the two M14 (parental)-derived anti-PD-Ll Fabs (P1AI4475 and P1AI4392) that were derived from the anti-PD LI binding CDRs (H-side) of the anti-PD-Ll / anti-IFN-a2a bispecific DutaFab M14 (parental), as described in above, block binding of PD-L1 to PD1, like the Atezolizumab-based anti-PD-Ll Fab (P1AI4841) which was used as a positive control. P1AH0183 is a PD-L1 -specific antibody which does not block PD-L1 / PD1 interaction (SEQ ID NO: 215, SEQ ID NO: 216 and SEQ ID NO: 217).

[0426] The PD1 / PD-L1 blockade bioassay (Promega) was also used to evaluate if the M14 (parental)-derived anti PD-L1 Fabs can also block PD1 / PD-L1 interaction in a cellular context. The principle of the assay is a bioluminescent cell-based assay that is based on the use of two different cell lines: a) Jurkat T cells genetically engineered to express human PD-1 and a luciferase reporter driven by an NF AT response element (NF AT -RE) (= PD-1 Effector Cells) and b) CHO-K1 cells genetically engineered to express human PD-L1 and an engineered cell surface protein designed to activate cognate TCRs in an antigen-independent manner (= PD-L1 aAPC / CHO-Kl Cells). When the two cell types are co-cultured, the PD-1 / PD-L1 interaction inhibits TCR signaling and NFAT-RE-mediated luminescence. Addition of either an anti-PD- 1 or an anti-PD-Ll antibody that blocks the PD-1 / PD-L1 interaction releases the inhibitory signal and results in TCR activation and NFAT-RE-mediated luminescence. The bioluminescent signal can be detected and quantified using the Bio-Gio™ Luciferase Assay System.

[0427] In this assay, monovalent or bivalent anti-PD-Ll binders were tested for blocking of PD1 / PD-L1 interaction. The assay was performed as described according to the manufacturer’s instructions. Atezolizumab which is known to block PD1 / PD-L1 interaction was used as a positive control. As can be seen in Figure 4, the parental (i.e. based on the parental M14 DutaFab) anti PD-L1 Fabs (P1AG5319 (monovalent; SEQ ID NO: 105, SEQ ID NO: 106 and SEQ ID NO: 107) and P1AG5176 (bivalent; SEQ ID NO: 93, SEQ ID NO: 94 and SEQ ID NO: 95) before affinity maturation and germlining- like the Atezolizumab-based anti-PD-Ll Fab (P1AI4841) - block binding of PD-L1 to PD1, even though they bind to the C-terminus of PD-L1 extracellular domain and therefore do not bind to the PD1 / PD-L1 interface region.

[0428] Example 2: Generation, purification and characterization of PD-L1 targeted immunoconjugates comprising IFN-«2 mutants

[0429] The following methods were used to produce and characterize the molecules described in the examples following thereafter.

[0430] a) Transient production of IgG-like proteins in Expi293F™ cells

[0431] Proteins, antibodies and cytokine IgG fusion proteins were expressed by transient transfection of Expi293F™ cells (ThermoFisher scientific, USA). Cells were seeded in Expi293™ medium (Gibco, Cat. N° 1435101) at a density of 2.5 x 106 / ml. Expression vectors and ExpiFectamine (Gibco, ExpiFectamine™ transfection kit, Catalog number 13385544) were separately mixed in OptiMEM™ reduced serum medium (Gibco, Catalog number 11520386). After 5 minutes, both solutions were combined, mixed by pipetting and incubated for 25 minutes at room temperature. Cells were added to the expression vector / ExpiFectamine solution and incubated for 24 hours at 37°C in a shaking incubator with a 5% CO2 atmosphere. One day post transfection, supplements (Transfection Enhancers 1 and 2, ExpiFectamine™ transfection kit) were added. Cell supernatants were harvested after 4-5 days by centrifugation and subsequent filtration (0.2 pm filter), and proteins were purified from the harvested supernatant by standard methods as indicated below. b) Protein puri fication

[0432] Recombinant immunoglobulin-like proteins were purified from cell culture supernatants by affinity chromatography using MabSelectSure-Sepharose™ (Cytiva, USA). Briefly, sterile filtered cell culture supernatants were captured on a Mab Select SuRe resin equilibrated with PBS buffer (10 mM sodium phosphate, 1 mM potassium phosphate, 137 mM sodium chloride and 2.7 mM potassium chloride, pH 7.4), washed with equilibration buffer, eluted with 100 mM sodium acetate, pH 3.0. After neutralization (= pH 5.5) with 1 M Tris pH 9.0, aggregated protein was separated from monomeric antibody species by size exclusion chromatography (Superdex 200, Cytiva) in 20 mM histidine, 140 mM NaCl, pH 6.0. Monomeric protein fractions were pooled, concentrated if required using e.g. a MILLIPORE Amicon Ultra (30KD MWCO) centrifugal concentrator and stored at -80°C. In some examples, an additional cation exchange chromatography (cIEX) purification step was performed before size exclusion chromatography. In this respect, the pH adjusted MabSelectSure eluate was diluted 1:1 with water and loaded on a Poros XS cation exchange resin equilibrated with 20 mM histidine pH 5.5 and eluted with 20 mM histidine, 500 mM NaCl, pH 5.5 applying a linear gradient.

[0433] c) Protein analytics by CE-SDS

[0434] Product purity and integrity were analyzed by CE-SDS using microfluidic Labchip technology (PerkinElmer, USA) under reducing and non-reducing conditions. For this purpose, 5 pl of sample solution was prepared using the HT Protein Express Reagent Kit according to the manufacturer's instructions and analyzed on LabChip GXII system using a HT Protein Express Chip. Data were analyzed using LabChip GX Software.

[0435] d) Protein analytics by SEC

[0436] Size exclusion chromatography (SEC) for the determination of the aggregation and oligomeric state of recombinant immunoglobulins was performed by HPLC chromatography. Briefly, purified product was applied to a TSKgel QC-PAK GFC 300 column (Tosoh Bioscience) or to a Tosoh TSKgel UP-SW3000 column in 250 mM KC1, 200 mM K2HPO4 / KH2PO4 buffer (pH 6.2) on a Dionex Ultimate® HPLC system (ThermoFischer Scientific, USA). The eluted antibody was quantified by UV absorbance and integration of peak areas. BioRad Gel Filtration Standard #151-1901 served as a gel filtration calibration standard.

[0437] e) Antibody characterization by UPLC coupled to UHR-ESI-QTOF mass spectrometry

[0438] The samples were analyzed by Ultimate 3000 UPLC (Thermo Fisher Scientific GmbH, Dreieich, Germany) on a PLRP-S 150 x 2.1 mm column (Agilent Technologies Deutschland GmbH, Waldbronn, Germany) using a 6.5 min gradient with acetonitrile with 0.1% formic acid (v / v) and water with 0.1% formic acid (v / v), respectively (both Thermo Fisher Scientific GmbH, Dreieich, Germany). For total mass determination, the UPLC was coupled to a UHR-ESI-QTOF maXis II ETD system (Bruker Daltonik, Bremen, Germany). Calibration was performed with sodium iodide (Honeywell, Morristown, NJ). For the human IgGl antibodies, data acquisition was done at 800-4000 m / z (ISCID: 85.0 eV). The raw mass spectra were evaluated and transformed into individual relative molar masses using an in-house developed software tool. f) PD-L1 A ffinity measurement by SPR

[0439] Affinities of different PD-L1 binders to human PD-L1 were assessed by surface plasmon resonance (SPR). The SPR experiments were performed on a Biacore T200 at 25 °C with PBS-P+ as running and sample dilution buffer (0.2 M phosphate buffer with 27 mM KC1, 1.37 M NaCl and 0.5% Surfactant P20 (Tween 20), Cytiva, Freiburg / Germany).

[0440] Anti-PGLALA antibody (in-house) or another appropriate capturing system was directly immobilized on a CM5 chip at pH 5.0 using the standard amine coupling kit (Cytiva, Freiburg / Germany). After activation of the sensor surface with a 1:1 mixture of 0.4 M l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and 0.1 MN-hydroxysuccinimide (NHS), 20 pg / ml anti-PGLALA (diluted in 10 mM acetate pH 5.0) was injected for 720 s with a flow rate of lOpl / min. After blocking with 1 M ethanolamine-HCl pH 8.5, the coupling procedure led to more than 12,000 capture surface density.

[0441] PD-L1 binders were captured for 60s at a flow rate of lOpl / min with a concentration of lOnM. Recombinant huPD-Ll (internal ID Pl AF6939) was injected at a concentration of 50 nM and then serially diluted with running buffer in 1 :3 ratio to 16.7 nM, 5.6 nM, 1.9 nM and 0.6 nM with a flow of 30 pl / min through the flow cells. Association and dissociation were monitored for 120s and 600 s respectively. The chip surface was regenerated after every cycle by using injection of lOmM NaOH (Cytiva, Freiburg / Germany) for 90 s at a flow of 30 pl / min. Bulk refractive index differences were corrected by subtracting the response obtained on reference flow cell. Binding curves were evaluated using Biacore T200 evaluation software 3.1 (Cytiva, Freiburg / Germany) and for the calculation of binding properties 1 : 1 Langmuir binding model was used.

[0442] ) IFNaR2 affinity measurement by SPR

[0443] Affinities of different IFN alpha variants to human IFN-a receptor 2 were assessed by surface plasmon resonance (SPR). The SPR experiments were performed on a Biacore T200 at 25 °C with PBS-P+ as running and sample dilution buffer (0.2 M phosphate buffer with 27 mM KC1, 1.37 M NaCl and 0.5% Surfactant P20 (Tween 20), Cytiva, Freiburg / Germany).

[0444] Anti -His antibody (Cytiva, Freiburg / Germany) was directly immobilized on a CM3 chip at pH 5.0 using the standard amine coupling kit (Cytiva, Freiburg / Germany). After activation of the sensor surface with a 1:1 mixture of 0.4 M l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and 0.1 M N-hydroxysuccinimide (NHS), 10 pg / ml anti-His (diluted in 10 mM acetate pH 5.0) was injected for 720 s with a flow rate of lOpl / min. After blocking with 1 M ethanolamine-HCl pH - Ill -

[0445] 8.5, the coupling procedure led to more than 5,000 RU anti-His surface density. Recombinant human His-tagged IFNaR2 extracellular domain (P1AD9385; SEQ ID NO: 333) was captured on the second flow cell for 60 s at a flow rate of 10 pl / min with a concentration of 50nM. IFN-a2a wildtype or Fc-IFN-a fusions were injected at a concentration of 500 nM and then serially diluted with running buffer in 1:3 ratio to 166.7 nM, 55.6 nM, 18.5 nM and 6 nM with a flow of 30pl / min through the flow cells. Association and dissociation were monitored for 300 s and 600 s respectively. The chip surface was regenerated after every cycle by using injection of Glycine pH1.5 (Cytiva, Freiburg / Germany) for 60 s at a flow of 30pl / min. Bulk refractive index differences were corrected by subtracting the response obtained on reference flow cell. Binding curves were evaluated using Biacore T200 evaluation software 3.1 (Cytiva, Freiburg / Germany) and for the calculation of binding properties 1 : 1 Langmuir binding model was used.

[0446] Example 3: Generation of PD-L1 expressing HEK-Blue IFN-a / p reporter cell line

[0447] HEK-Blue IFN-a / p reporter cell lines expressing different levels of PD-L1 were generated in order to test PD-L1 -dependent IFN-a activity of the immunoconjugates disclosed herein. Full-length cDNA encoding human PD-L1 was subcloned into a mammalian expression vector. The plasmid was transfected into HEK-Blue IFN-a / p (Invivogen, Catalog number hkb-ifnab) cells using Lipofectamine 3000 Reagent (Invitrogen, Catalog number L3000015) according to the manufacturer’s protocol. HEK-Blue IFN-a / p cells were maintained in DMEM media (PAN, #P04- 03596) supplemented with 10% FCS (Gibco, Catalog number 10500), 2 mM L-Glutamine (PAN, Catalog number P04-80100), 30 pg / mL Blasticidin (Gibco, Catalog number Al 113903) and 100 pg / mL Zeocin (Gibco, Catalog number R25001). Two days after transfection, Hygromycin (PAN, Catalog number P06-08020) was added to 200 pg / mL. After initial selection, the cells with the highest cell surface expression of PD-L1 were sorted by BD FACSAria III cell sorter (BD Biosciences) and cultured to establish stable cell clones. The expression level and stability was confirmed by FACS analysis using PE mouse anti-human CD274 (Clone MIH1) (BD Biosciences, #557924) over a period of 4 weeks. In addition, a QUANTIblue assay (Invivogen, Catalog number rep-qbs2) was performed according to the manufacturer's protocol in order to ensure that the reporter activation via the IFN-a pathway was not significantly affected by stable transfection of PD-L1. Both selected clones (clone 39 (RNCB accession IDCL022701) and clone 45 (RNCB accession IDCL022702)) showed comparable IFN-a stimulation to HEK-Blue-IFN-a / p wild-type cells. Quantification of the cell-surface PD-L1 using a bead based Quantification kit (BD, Catalog number 340495) revealed low PD-Ll-surface levels for HEK-Blue-IFN-a / p wildtype cells (~300 PD-L1 molecules / cell on cell surface) and high PD-Ll-surface levels of clone 45 (-25,000 PD-L1 molecules / cell on cell surface). The PD-L1 low expressing cells are hereinafter referred to as “parental” HEK-Blue-IFN-a / p cells, while the PD-L1 high expressing HEK-Blue-IFN-a / p cells are referred to as “clone 45” or “clone 39”, respectively.

[0448] Example 4: Determination of IFNAR activation by test molecules using the HEK-Blue Reporter cell assay

[0449] The cells obtained as described in Example 3 were used to determine IFN-a activity of test molecules, such as (attenuated) IFN-a2 mutants and IFN-a2 immunoconjugates. Upon binding of IFN-a2a to IFNAR1 / 2 receptors, the JAK / STAT / ISGF3 pathway was triggered, leading finally to the expression of the reporter gene which is under the control of the ISG54 promoter containing an IFN-stimulated response element (ISRE). The reporter gene, secreted embryonic alkaline phosphatase (SEAP), was produced by the cells and secreted into the medium. Its amount correlates with the extent of IFNAR1 / 2 receptor activation. The SEAP levels in the medium were measured by using a SEAP detection reagent like QUANTI-Blue™ and the color change of the detection reagent by the SEAP activity can be measured with a spectrophotometer.

[0450] Figure 5 shows a schematic illustration of the activity of PD-L1 targeted immunoconjugates comprising IFN-a2 mutants on low and high expressing PD-L1 HEK-Blue IFN-a / p reporter cells. When the cell surface density of PD-L1 is too low and PD-L1 and IFNAR1 / 2 receptors are not in close proximity, the PD-L1 targeted immunoconjugates comprising IFN-a2 mutants cannot activate its receptor. However, when sufficient PD-L1 is expressed on the cell surface, the chance that the IFN-a2a mutants comprised in the immunoconjugates bound to PD-L1 comes in close proximity to its receptor is higher and the IFNAR pathway will be activated, triggering detectable SEAP production of the reporter cells (the figure was created with BioRender.com).

[0451] For the assay, first, lOx titration series (8 steps) of the different PD-L1 -targeted immunoconjugates comprising IFN-a2a mutants were prepared, starting at 100 nM (final concentration in well between 10 nM and 0.000001 nM). 20 pL per construct of each concentration were transferred to 96-well flat bottom plates in duplicates. Subsequently, 4xlOA4 reporter cells / well were seeded in 180 pL medium (DMEM high glucose (4.5 g / L glucose) (PAN, Catalog number P04-03609) + 10 % heat-inactivated FBS (Anprotec, #AC-SM-0014Hi) + 2 mM L-glutamine (PAN, Catalog number P04-80100)). As positive control, recombinant IFN-a2a (PBL Assay Science, Catalog number 11101-2) was used in the same concentration range. After 24 h of incubation at 37°C, assay plates were centrifuged at 300 g for 5 min. Meanwhile, QUANTI-Blue solution (Invivogen, #rep-qbs) was prepared and 180 pL / well were distributed among wells of new 96-well flat bottom plates. 20 pL of supernatants of treated parental and hPD- L1 transfected HEK-Blue IFN-a / p cells were added. Substrate turnover by produced SEAP was allowed for 30 to 45 min before measuring the optical density (OD) at 640 nm.

[0452] In certain experiments, HEK-Blue reporter cells were pre-incubated with 5 pg / mL of Atezolizumab at 37°C for 30 minutes before the addition of test molecules. This pre-incubation aimed to block all PD-L1 molecules on the surface of the reporter cells and thereby assess the PD- L1 -independent activity of the test molecules.

[0453] Example 5: IFN-«2 fused to Fc via GS-linker leads to decreased affinity to IFNaR2 compared to untagged IFN alpha

[0454] All type I IFN-a molecules signal through interacting with the two IFNAR receptor subunits IFNaRl and 2, forming a trimeric complex. In order to evaluate whether fusing IFN-a2 N-terminally or C-terminally (via GS-linker) to an Fc region decreases the affinity to its receptor IFNAR, especially to its high affinity receptor subunit IFNaR2, we analyzed the affinity of IFN-a2a wildtype and IFN-a2a wildtype-Fc fusion proteins to IFNaR2 by surface plasmon resonance (SPR) as described in Example 2 g (results shown in Table 5).

[0455] Table 5: Affinities of IFN-«2a wildtype and IFN-«2a wildtype-Fc fusion proteins to IFNaR2 measured by surface plasmon resonance (SPR).

[0456]

[0457] The value measured for untagged IFN-a2a corresponds to data found in literature (J. Piehler et al, JBC, Vol. 275, No. 51, Issue of December 22, pp. 40425-40433, 2000). N- or C-terminal Fc fusions via GS-linker seem to lower the affinity already by a factor of around 6, compared to IFN-a2a.

[0458] The reduced affinity of the IFN-a_Fc fusions to IFNaR2 translates into reduced efficacy measured by HEK-Blue IFN-a / p reporter cell assay as described in Example 4 (results shown in Table 6).

[0459] Table 6 - EC50 values determined by HEK-Blue IFN-a / p reporter cell assay

[0460]

[0461] Example 6: Influence of linker length on efficacy of IFNAR1 / 2 activation by immunoconjugates comprising IFN-a2 mutants in HEK-Blue human IFNAR1 / 2 reporter cell in vitro assay

[0462] Attenuated (R149A) PD-L1 targeted IFN-a2a was fused to the Fc fragment via a (GnS)nlinker. We analyzed the impact of two different G4S-linker lengths 60 aa (20x GGS, P1AF8334, marked by *) vs 15 aa(PlAG2011, marked by+) on IFNaR2 affinity in a HEK-Blue assay. Both molecules are less potent on HEK cells with low PD-L1 expression (HEK parental) than rec. IFN-a but the potency of both molecules was comparable independent of the used linker length (results shown in Figure 6).

[0463] Table 7 - Molecules used for potency assay

[0464]

[0465] Example 7: Comparison of impact of different linker lengths on molecule degradation In order to test whether longer G4S linkers are more susceptible to degradation over time, we analyzed stabilities of three PD-L1 targeted IFN-a2a-Fc fusion molecules differing in the length of the G4S linker. Pl AG2011 (15aa linker) and P1AF8334 (60aa linker) as well as P1AF8337 (a construct comprising an anti-PD-Ll VHH fused N-terminally to one polypeptide of the Fc region and the IFN-a2a fused to the Fc region C-terminally with 60aa linker) were incubated in PBS at 37°C for two weeks at a concentration of 1 mg / ml. Subsequently, the stressed material was analyzed by analytical size exclusion chromatography as described in Example 2 d.

[0466] Table 8 - Tested molecules

[0467]

[0468] The results are shown in Figure 7. We detected significant LMW formation for the stressed samples with long linkers (9-14%; P1AF8334 and P1AF8337) and only a minor increase for the short linker (<3%; Pl AG2011) compared to the unstressed reference. Further analysis by LC-MS confirmed that LMW formation results from G4S linker degradation. No fragmentation of IFN-a was detected.

[0469] Example 8: Evaluation of the affinity of the IFN-a variants to its high affinity receptor IFNaR2

[0470] IFN-a2 signaling is mediated by the interaction with its receptors IFNaRl and IFNaR2 forming an active trimeric complex. Whereas IFNaR2 has a high affinity to IFN-a2a wildtype (around 3 nM), the affinity to IFNaRl lies only in the pM range. The specific activity of IFN-a2 seems to be directly proportional to its affinity for IFNAR1 and IFNAR2 (Nat Commun 5, 3016 (2014), Cell, Volume 146, Issue 4, 2011, Pages 621-632). In order to reduce the potency of IFN-a2a, we searched for IFN-a2a mutations reducing the affinity of IFN-a2a to IFNaR2. Based on data from available structural analysis (Cell, Volume 146, Issue 4, 2011, Pages 621-632) and mutational IFN- a analysis affecting IFN-a receptor interaction (JBC, Vol. 275, No. 51, Issue of December 22, pp.

[0471] 40425-40433, 2000) as well as own structural interpretations (using PDB ID 3S9D; 3SE3), several amino acid substitutions within IFN-a2a were generated.

[0472] PD-L1 -targeted Interferon alpha molecules were generated in IgG-like formats using Fc-LALAPG or Fc-LALAPG knob-into-hole modifications in the Fc region to create asymmetric constructs. IFN-a variants were fused to the C-terminus of one heavy chain via a (G4S)Xlinker. Further, the constructs may contain two Fab fragments (in the following “bivalent”, Figure 1 B, left), one Fab fragment (in the following “monovalent”; Figure 1 B, middle) or no Fab fragments (Figure 1 B, right).

[0473] In an initial round of experiments, 37 targeted IFN-a variants were generated to assess the impact of various amino acid substitutions in the IFN-a2 amino acid sequence on IFN-a2 activity. For this, monovalent IFN fusion molecules were generated by conjugating IFN-a2 molecules with different amino acid substitutions to create one armed Fc fusions (as shown in Figure 1 B, middle) that were based on the PD-L1 antibody atezolizumab (shown in Table 9) and were transiently expressed and purified as described above.

[0474] Table 9 - Tested IFN-a2a mutations

[0475] ""

[0476]

[0477] ""

[0478]

[0479] The modified IFN-a2a variants were produced as described in Example 2 a. Affinities of the IFN-a variants were evaluated by SPR as described in Example 2 g and variants with comparable or lower affinity (i.e. higher KD) as the variant R149A were selected for further evaluation. The results are shown in Table 9 (“low RU” indicates a low confidence in the KD calculation due to a weak SPR binding signal).

[0480] Example 9: Comparison of impact of different IFN-a2a mutants on IFNAR1 / 2 receptor activation in HEK-Blue IFN-a / reporter cell in vitro assay

[0481] The HEK-Blue IFN-a / p reporter cells described in Example 3 were used to characterize the IFNAR1 / 2 receptor binding of different attenuated IFN-a2a mutants. We were seeking to identify candidate molecules that can only activate IFNAR1 / 2 receptors when hPD-Ll is present in sufficient amounts on the cell surface. We aimed for molecules that had low residual IFNAR binding so that targeting of the IFN-a2a mutants to the PD Ll on the target cell surface is required to bring it into close proximity of the IFNAR receptor subunits on the cell surface so that it binds to the IFNAR1 / 2 receptor despite its decreased affinity for it. Accordingly, we strove to identify attenuated IFN-a2a mutants which showed at the same time low activation in the parental reporter cells (low PD-L1 levels) and high activation in the clone 45 cells (high PD-L1 levels). Such molecules are expected to demonstrate low undesired IFNAR activation in the periphery, but high IFNAR activation at the target cell or target tissue. For this purpose, the IFN-a2a with the mutations shown in Table 10 below were fused to the C-terminus of one heavy chain of an Fc region, and the N-terminus of the other subunit of the Fc region was fused to the C-terminus of the Atezolizumab Fab, as shown in Figure 1 B (middle). The assay was performed essentially as described in Example 4.

[0482] The results of the assay are shown in Table 10. Several single or double point mutations cause higher IFN-a2a activity decrease compared to R149A in the HEK-Blue IFN-a / p assay (parental & + Atezo preincubation). The molecules were rated on the one hand by how well they activated the IFNAR in cells with high PD Ll expression (clone 45) and on the other hand by the difference in activation between parental cells (low PD Ll expression) and clone 45 (high PD-L1 expression); the latter reflects the shift the therapeutic window either towards lower or higher immunoconjugate concentrations (+: stronger PD-DL1 independent activation than reference R149A; =: comparable activation as R149A; lower PD-L1 independent activation than R149A; — : slightly lower; — : much lower) (see Table 10). The HEK-Blue activation data for some of the molecules are also shown in Figure 8 A and Figure 8 B. Overall, L30A, L30H and L153D show the best balance for PD-L1 dependent IFN-a activation.

[0483] Table 10: Activity of different IFN-«2a variants on activity in a HEK-Blue IFN-a / p assay

[0484]

[0485]

[0486] As shown in Figure 8, the molecules with the L30A, L30H, L30V / L26A or L153D mutations show reduced IFN-a activation on PD-L1 low expressing cells compared to the control molecule Atezo monovalent_R149A (Hek parental). On PD-L1 high expressing cells (cl45), they show comparable activity to recombinant IFN-a2 but remain less potent than control molecule Atezo monovalent_R149A. Pre-incubating the reporter cells with the PD-L1 blocking antibody, Atezolizumab, before adding the immunoconjugates resulted in a significant reduction in IFNAR1 / 2 receptor activation across all molecules in both cell lines, except for the untargeted wild-type IFN-a2a. Table 11: Molecules used to generate the data shown in Figure 8

[0487]

[0488] Example 10: Impact of anti-PD-Ll targeting affinity and avidity on IFNAR1 / 2 activation of PD-L1 targeted immunoconjugates comprising IFN-«2a mutants in HEK-Blue human IFNAR1 / 2 reporter cell in vitro assay

[0489] As a next step, the newly generated anti PD-L1 binders M14, DIO, E24, H13 and J2 were tested for their ability to target attenuated IFN-a2a mutants to the cell surface and thus rescue IFN-a activity (for an overview of the molecules that were generated and tested see Table 12).

[0490] Parental and hPD-Ll transfected HEK-Blue IFN-a / p cells were used as reporter cell lines to analyze hPD-Ll dependent IFNAR1 / 2 receptor activation by PD-L1 -targeted immunoconjugates comprising IFN-a2a mutants (Figure 1 A and B, middle). Mono- and bivalent PD-L1 targeted immunoconjugates comprising IFN-a2a mutants were generated based on IFN-a2aRi49A, as shown in Figure 1 A and in Figure 1 B (left and middle), and different anti-PD-Ll binders (see Example 1) were used to evaluate the effect of PD-L1 affinities of the various targeting binders on IFN-a pathway activation. The HEK-Blue assays were performed essentially as described in Example 4. Table 12: Molecules used for testing effect of affinity and avidity in a HEK-Blue IFN-a / p assay

[0491]

[0492] Affinities of the different binders for PD-L1 determined by SPR essentially as described as in Example 2 f) are shown in Table 13.

[0493] The results are shown in Figure 9 A and B. Pl AG5176 with an affinity of about 4.4 nM delivered the best balance for PD-L1 dependent delivery of mutated IFN-a. At higher PD-L1 expression levels (clone 045, 25,000 receptors per cell), the new PD-L1 binders DIO, M14 and E24 show strong avidity effect (Figure 9 B). As shown in Figure 9 A, new PD-L1 binders DIO and M14 show strong avidity effect even at very low PD-L1 expression levels (parental; 300 receptors per cell). Table 13: Results of the HEK-Blue IFN-a / p assay

[0494]

[0495] Example 11: Comparison of selected attenuated mutant IFN-«2a variants using different targeting affinities and formats on IFNAR1 / 2 receptor activation in HEK-Blue human IFNAR1 / 2 reporter cell in vitro assay

[0496] Finally, the most promising anti-PD-Ll binders were combined with the best attenuated IFN-a2a mutants in different combinations (bivalent, monovalent) to identify molecules with suitable profile (Table 14).

[0497] Mono- and bivalent PD-L1 targeted immunoconjugates comprising IFN-a2a mutants were generated based on selected PD-L1 binder (Ml 4 / D 10) and selected IFN-a2a variants as shown in Figure 1 A and Table 14 and were used to identify the best balance for PD-L1 dependent IFN-a2a activation i.e. low IFN-a signaling at low PD-L1 cell surface level and high activation at high PD-L1 cell surface levels in a HEK Blue assay (conditions essentially as described in Example 4). The results of the HEK-Blue assays are shown in Figures 10 A - C. Molecules that are more potent on HEK cells with low PD-L1 expression (HEK parental; Figure 10 A) compared to Atezo(monovalent)-IFN-a2aR149A (benchmark) and molecules that are less potent on HEK cells with high PD-L1 expression (HEK clone 045, Figure 1 B) compared to rec. human IFN-a2a were not further tested.

[0498] Table 14 - Immunoconjugates generated to test impact of monovalent / bivalent targeting

[0499]

[0500] In Figure 11, the EC50 values measured for some of the molecules in the different cell lines have been set in relation to reference molecule P1AF8334. In general, it could be observed that reduced IFN-a activation on PD-L1 low expressing cells came along with less potency on PD-L1 high expressing cells. As can be seen from Figure 12, low PD-L1 expression levels are sufficient to restore the activity of PD-L1 (Atezo)-targeted R149A immunoconjugates comprising IFN-a2a mutants (P1AF8334), indicating a considerable risk for systemic toxicity. In contrast, the PD-L1 -targeted immunoconjugates comprising IFN-a2a L30A mutants (P1AI4283) show lower activity on PD-L1 low expressing cells (HEK-Blue parental - PD-L1 per cell: -300) than P1AF8334 (Figure 12 A) and still a considerable high activity on PD-L1 high expressing cells (HEK-Blue clone 045 -PD-L1 per cell: -25,000) (Figure 12 B), demonstrating a high PD-L1 dependency of IFN-a activity and potential lower risk for systemic toxicity.

[0501] Example 12: Cytokine release by human PBMCs stimulated in vitro with PD-L1 targeted immunoconjugates comprising IFN-«2a mutants

[0502] Human PBMCs were stimulated in vitro with indicated PD-L1 -targeted (M14 or D10) immunoconjugates comprising IFN-a2a mutants or recombinant human IFN-a2a at a concentration of 1 nM. Supernatants were collected and cytokines (IL6, TNF- a and IFN-y) were measured by using Luminex technology.

[0503] Therefore, blood was obtained from the blood donation service of the medical service Penzberg in 50 ml tubes (Greiner Bio-One, Cat.No. 227261) containing 0.5 mL Heparin (ratiopharm, Cat.No.

[0504] 03029843) and 0.5 mL 0,9 % Sodium Chloride solution (Fresenius Kabi, Cat.No. 0809078). In order to isolate peripheral blood mononuclear cells (PBMCs), the blood was diluted in the same volume of RPMH640 (PAN Biotech, Cat. No. P04-17000) and 30 ml of the blood mix were carefully poured into Pancoll tubes (PAN Biotech, Cat.No. P04-60225). The tubes were centrifuged at 800 g for 15 minutes at room temperature with low acceleration and without break. Afterwards the PBMCs were collected from the interface, washed twice with RPMI1640 and resuspended in 30-50 mL of RPMI1640. The cells were counted using a Neubauer chamber and 1:10 dilution of Trypan blue 0.4 % (Invitrogen, Cat.No. T10282) centrifuged and lxlOA7 cells per vial were frozen with 90 % FCS and 10 % DMSO. To perform the assay, frozen PBMCs were thawed by putting the cryovial shortly into a 37°C waterbath. Thawed cells were diluted 1:10 with medium and counted using a Neubauer chamber and 1:10 dilution of Trypan blue 0.4 %. Cells were centrifuged, the supernatant was discarded and the cells were resuspended with medium at a concentration of 0.27 cells / mL. 5xl0A5 PBMCs were seeded in 180 pl RPMH640 (PAN Biotech, Cat. No. P04-17000) medium containing 10 % FCS (Gibco, Cat.No.10500-064), 0,1 mM 2-Mercaptoethanol (Gibco, Cat.No. 31350-010), 2 mM L-Glutamine (Sigma, Cat.No. G7513)), 1 mMNaPyruvate (PAN, Cat.No. P04-43100), 100 pg / ml PenStrep (PAN, Cat.No. P06-07100), lx MEM NEAA (Pan, Cat.No. P08-32100) and 10 mM Hepes (Anprotech, Cat.No. AC-Ds-0007) into each well of a 96 well round-bottom plate and PD-L1 -targeted immunoconjugates comprising IFN-a2a mutants were added at a concentration of 10 nM in 20 pl of RPMI1640 with supplements described above . As positive control, rec. hu IFN-a2a (PBL Assay Science, Cat.No.11101-2) was used. As negative control, untargeted IFN-a2aRi49Awas used. In addition, P1AF8334 was used as reference.

[0505] After 24h of Incubation at 37°C and 5% CO2 supernatant was taken and used for Luminex Assay. The Assay was performed as described in the manufacturer’s protocol. In brief, supernatant, standard or quality control was added into the appropriate wells of a 96-well plate. Antibody-immobilized beads were pipetted into each well and the plate was incubated overnight at 2-8°C with shaking. Well contents were removed and Detection Antibody and additionally Streptavidin-Phycoerythrin was pipetted into each well. The readout was performed using Luminex 200. As shown in Figure 13, P1AI4283 (M14 bivalent, L30A) shows overall the lowest cytokine induction after treatment of human whole blood hinting towards an improved safety profile compared to free recombinant human IFN-a2a and the reference molecule P1AF8334 (Atezo monovalent, R149A). Moreover, the combination of the M14 binder with the L30A or L30H attenuation site is in this respect superior compared to other tested attenuation sites.

[0506] Additionally, IFNy ELISPOT analysis was performed. For this, human PBMCs were isolated as described before. The ELISPOT Assay was done according to manufacturer's instructions.

[0507] 2.5xlOA3 PBMCs in RPMI 1640 medium with supplements (see below) were seeded in each well of a 96 well ELISPOT plate and PD-L1 -targeted (M14 or D10) immunoconjugates comprising IFN-a2a mutants were added at a concentration of 100 nM to 0.0001 nM in RPMI1640 (PAN Biotech, Cat. No. P04-17000) medium containing 10 % FCS (Gibco, Cat.No.10500-064), 0,1 mM 2 -Mercaptoethanol (Gibco, Cat.No. 31350-010), 2 mM L-Glutamine (Sigma, Cat.No. G7513)), 1 mMNaPyruvate (PAN, Cat.No. P04-43100), 100 pg / ml PenStrep (PAN, Cat.No. P06-07100), lx MEM NEAA (Pan, Cat.No. P08-32100) and 10 mM Hepes (Anprotech, Cat.No. AC-Ds-0007). As positive control, recombinant huIFN-a2a (PBL Assay Science, Cat.No.11101-2) was used. As negative control, untargeted IFN-a2aRi49A was used. In addition, Pl AF8334 was used as reference. After 24 h of incubation at 37°C, well contents were removed. After Incubation with Detection Antibody (Biotinylated anti-IFNy), Streptavidin-Alkaline-Phosphatase mix was added. Additionally, substrate solution was added, and spot formation was monitored with ImmunoSpot Analyzer.

[0508] As shown in Figure 14, of the 6 selected molecules, M14 bivalent-IFN-a2aL30A and M14 bivalent- IFN-a2aL30H induce the lowest IFNy release by PBMCs (comparable to untargeted IFN-a2aRi49A). In summary, M14 bivalent-IFN-a2aL30A and M14 bivalent-fFN-a2aL30H show the best balance between low activity on PD-L1 low expressing cells (PBMCs) and high activity on PD-L1 high expressing cells (HEK clone 045 and 039).

[0509] Example 13: PD-Ll-targeted immunoconjugates comprising IFN-«2a mutants induced HCC1954 tumor growth inhibition and CXCL10 secretion

[0510] The effect of the PD-Ll-targeted immunoconjugate P1AI4283 in comparison to the reference molecule or recombinant Interferon alpha on the proliferation rate of the human tumor cell line HCC1954 was measured using the cell confluence application of the IncuCyte SX5 (Sartorius). HCC1954 cells were cultivated with 90 % RPMI 1640, 2 mM L-Glutamine, 1 mM Sodium Pyruvate, 10 mMHEPES, 4.5 g / L Glucose, 1.5 g / LNaHCO3 (PAN Biotech, Cat.No.P04- 18047) supplemented with 10 % Fetal Bovine Serum (Anprotec, Cat.No. AC-SM-0014Hi) and Penicillin / Streptomycin (Roche, Cat.no 11074440001). 7,500 HCC1954 tumor cells were seeded in 180 pl cultivation medium in a flat-bottom 96-well plate (Corning, Cat.No. 3585) and 20 pl of pre-diluted molecules (final cone. 10 nM) were added. The plate was transferred to the IncuCyte® and the confluence of the cells was measured every 4 hours for up to ten days (phase contrast, lOx objective). Data analysis was performed with the IncuCyte® Software 2022A (basic analysis). In addition to the HCC1954 breast cancer line the assay was repeated with the LoVo colon adenocarcinoma line and OVCAR-3 ovarian adenocarcinoma line. LoVo cells were cultured with Ham’s F-12 medium (Anprotec, Cat.No. AC-LM-0029) supplemented with 20 % Fetal Bovine Serum (Anprotec, Cat.No. AC-SM-0014Hi) and 2 mM L-Glutamine (PAN Biotech, Cat.No. P04-80100) and treated with 100 nM of constructs. The OVCAR-3 cells were cultured with RPMI 1640, 2 mM L-Glutamine, 1 mM Sodium Pyruvate, 10 mM HEPES, 4.5 g / L Glucose, 1.5 g / L NaHCO3 (PAN Biotech, Cat.No. P04- 18047) supplemented with 20 % Fetal Bovine Serum (Anprotec, Cat.No. AC-SM-0014Hi) and treated with 100 nM of constructs.

[0511] Next, the effect of the PD-Ll-targeted immunoconjugate Pl AI4283 in comparison to the reference molecule or recombinant Interferon alpha on the IP- 10 secretion of human HCC1954 breast cancer cells and on the IP- 10 secretion of human OVCAR-3 ovarian adenocarcinoma cells was measured by ELISA. To analyze the effect on HCC1954 cells, 30,000 cells / well were seeded in 150 pl / well cultivation medium and 50 pl of respective constructs or recombinant human IFN-a2a (PBL Assay Science #11101-2, Lot#7191) dilutions (10 nM to 0.001 nM final concentration / well, 1:10 titration) were added per well. To analyze the effect on OVCAR-3 cells, 30,000 cells / well were seeded in 180 pL / well cultivation medium and 20 pL of respective constructs or recombinant human IFN-a2a dilutions (100 nM to 0.01 nM final concentration / well, 1:10 titration) were added per well. After 24 h of incubation, plates of both cell lines were centrifuged and supernatants for an IP- 10 ELISA (Human CXCL10 / IP-10 DuoSet ELISA, R&D # DY266 and DuoSet Ancillary Reagent Kit2, R&D # DY008) were collected. The IP- 10 ELISA was performed as described in the protocol provided by the manufacturer. A day before, ELISA plates were coated with 100 pl capture antibody (2 pg / ml in PBS without carrier) and incubated overnight at room temperature. The next day, the plates were washed, blocked and 100 pl of 1:2 diluted samples or 100 pl of standards (2,000 pg / ml - 31.2 pg / ml, diluted in Reagent Diluent, 1:2 titration) were applied to the ELISA plates for two hours at room temperature. Afterwards the supernatants were aspirated, wells were washed and 100 pl detection antibody (12.5 ng / ml diluted in Reagent Diluent) were added to each well and incubated for 2 hours at room temperature. After another aspiration and wash step, 100 pl streptavidin-HRP (40-fold dilution in Reagent Diluent) were added to each well and incubated for 20 minutes at room temperature. The aspiration and wash step was then repeated.

[0512] 100 pl of Substrate Solution (equal volumes of Color Reagent A and B) were added to each well and incubated for 20 minutes in the dark at room temperature. Finally, 50 pl stop solution was added to each well and the optical density was determined by using a microplate reader (set to 450 nm, wavelength correction 570 nm).

[0513] As can be seen from Figure 15, the PD-Ll(M14)-targeted immunoconjugate substantially decreases the proliferation rate of human HCC1954 tumor cells (breast cancer cell line) (Figure 15 A) and induce high CXCL10 secretion (key chemoattractant for T cells) (Figure 16 A).

[0514] Similarly, PD-Ll(M14)-targeted immunoconjugates comprising IFN-a2a mutants led to a considerable decrease of the proliferation rate of LoVo colon adenocarcinoma cells (Figure 15 B).

[0515] The proliferation of OVCAR-3 ovarian adenocarcinoma cells was diminished when treated with PD-Ll(M14)-targeted immunoconjugate, however not as strong as compared to the recombinant human IFN-a2a positive control which completely inhibits cell growth (Figure 15 C). Moreover, the IP- 10 secretion of OVCAR-3 cells was strongly upregulated upon recombinant human IFN- a2a treatment but only minor upon PD-Ll(M14)-targeted immunoconjugate treatment (Figure 16 A).

[0516] The effect of PD-Ll(M14)-targeted immunoconjugate was not as significant as the effect of recombinant human IFN-a2a on OVCAR-3 cells due to the low PD-L1 receptor amount on these cells. These results show the desired mode of action of the molecule to act predominantly on PD-L1 high expressing cells like HCC1954 (~ 20,000 PD-L1 molecules per cell) and to have reduced effect on PD-L1 lower expressing cells such as LoVO (-400 PD-L1 molecules per cell) and to have almost no effects on PD-L1 low expressing cells like OVCAR-3 (around 100 PD-L1 molecules per cell).

[0517] Example 14: PD-Ll-targeted IFN-«2a induced MHC I and PD-L1 expression in human HCC1954 tumor cells (breast cancer cell line)

[0518] MHC1 and PD-L1 expression in HCC1954 breast cancer cell line was measured using the live cell imaging of the Incucyte SX5 (Sartorius). Cells were cultivated with 90% RPMI 1640, 2mM L-Glutamine, ImM Sodium Pyruvate, lOmM HEPES, 4.5g / L Glucose, 1.5 g / L NaHCCh (PAN Biotech, Cat.No.P04- 18047) supplemented with 10% Fetal Bovine Serum (Anprotec, Cat.No. AC-SM-0014Hi)) and Penicillin / Streptomycin (Roche, Cat.no 11074440001). 10,000 tumor cells were seeded in 50pl cultivation medium in a flat-bottom 96-well plate (Corning, Cat.No. 3585). The Incucyte Mouse IgGl Fabfluor-488 Antibody Labeling Dye (Sartorius, Cat.No. 4745) and the Incucyte Mouse IgG2a Fabfluor-594 Antibody Labeling Dye (Sartorius, Cat.No. BA-04863) were mixed with a molar ratio of 1:3 with a final cone. of Ipg / ml Ultra-LEAF™ Purified anti-human CD274 (B7-H1, PD-L1, Isotyp Mouse IgGl, Biolegend Clone M1H2, Cat.No. 393602) Antibody and Ultra-LEAF™ Purified anti-human HLA-A,B,C Antibody (Clone W6 / 32, Isotype Mouse IgG2a, Biolegend Cat.No. 311428) and incubated 15min. to allow conjugation. 50pl Opti-Green background suppressor (final cone. 0.5pM) and 50pl of PD-Ll-IFN-a molecules (final cone. lOnM) were added to the plate. The plate was transferred to the IncuCyte and green and red fluorescence was measured every 2h (lOx objective). Data analysis was performed with the Incucyte® Software 2022A (basic analysis, Spectral unmixing %Red contributes to Green value: 2.8%).

[0519] As can be seen from Figures 17 A and B, the PD-Ll-targeted attenuated IFN-a2a molecule showed a strong inducement of MHCI and PD-L1 expression of HCC1954 tumor cells, indicating increased tumor immune recognition and enhanced PD-L1 -targeting. Since the PD-L1 -targeting domains of the reference molecule block the binding of the anti-PD-Ll staining antibody, PD-L1 expression levels upon treatment with the reference molecule could not be detected in this assay.

[0520] Example 15: PD-Ll-targeted IFN-«2a human NK cell activation assay

[0521] In order to compare the IFN-a activity of the PD-Ll-targeted immunoconjugate P1AI4283 and reference molecule P1AF8334 on human NK cells, the NK-92 cell line (Roche cell bank Specimen- ID: CLPZ05487) was cultivated in 80 % MEM alpha w / o nucleosides (Gibco #12561-056), 12.5 % FCS (Gibco #10500-064), 12.5 % Horse Serum (Anprotech AC-SM-0059), 2 mM L-Glutamine (Anprotech #AN- 182008), 0.1 mM 2-Mercaptoethanol (Gibco #31350-010), 0.2 mM Myo-Inositol (Sigma #7508), 0.02 mM Folic Acid (Sigma #8758) and 100 U / ml IL-2 (Roche #11147528001). 100,000 NK-92 cells / well were seeded in 150 pL corresponding medium without IL-2 and 50 pl of respective construct or recombinant human IFN-a2a (PBL Assay Science #11101-2) titrations (100 nM to 0.001 nM final concentration, 1:10 titration) were added.

[0522] After 48 h of incubation, plates were centrifuged and supernatants for an IFN-y ELISA (Human IFN-y DuoSet ELISA, R&D # DY285B and DuoSet Ancillary Reagent Kit2, R&D # DY008) were collected. The IFN-y ELISA was performed as described in the protocol provided by the manufacturer. A day before ELISA plates were coated with 100 pL / well Capture antibody (2 pg / ml in PBS without carrier protein) and incubated overnight at room temperature. The next day the plates were washed and blocked with 300 pL Reagent Diluent for 1 h at room temperature. The plates were washed and 100 pL of undiluted samples or 100 pL of standards (600 pg / ml - 9.38 pg / ml diluted in Reagent Diluent, 1:2 titration) were applied for two hours at room temperature. Afterwards the supernatants were aspirated, wells were washed and 100 pL detection antibody (200 ng / ml diluted in Reagent Diluent) were added to each well and incubated for 2 hours at room temperature. After another aspiration and wash step, 100 pL streptavidin-HRP (40-fold dilution in Reagent Diluent) were added to each well and incubated for 20 minutes at room temperature. The aspiration and wash step was then repeated. 100 pL of Substrate Solution (equal volumes of Color Reagent A and B) were added to each well and incubated for 20 minutes in the dark at room temperature. Finally, 50 pL of the stop solution were added to each well and the optical density using a microplate reader (set to 450 nm, wavelength correction 570 nm) was determined.

[0523] Since NK-92 cells have with about 100 PD-L1 molecules per cell a low PD-L1 expression, the PD-Ll-targeted attenuated immunoconjugate induces lower IFNy secretion of human NK-92 cells compared to recombinant IFN-a2a as can be seen from Figure 18. In addition, Pl AI4283 induced less IFNy secretion than the reference molecule P1AF8334. This indicates lower periphery NK cell activation, hinting towards an improved safety profile.

[0524] Example 16: PD-Ll-targeted IFN-a2a shows reduced stimulation of human primary DC subsets

[0525] To test the effect of PD-L1 targeted immunoconjugates comprising IFN-a2a mutants on human primary dendritic cell (DC) subsets, primary DCs were isolated from human whole blood from healthy donors and analyzed upon PD-Ll-targeted immunoconjugates comprising IFN-a2a mutants or recombinant human IFN-a2a treatments for activation markers. Therefore, blood was obtained from the blood donation service of the medical services at Roche Penzberg in 50 ml tubes (Greiner Bio-One, Cat.No. 227261) containing 0.5 mL ofHeparin (ratiopharm, Cat.No. 03029843) and 0.5 mL of 0,9 % Sodium Chloride solution (Fresenius Kabi, Cat.No. 0809078). In order to isolate peripheral blood mononuclear cells (PBMCs), the blood was diluted in the same volume of RPMI1640 (PAN Biotech, Cat. No. P04-17000) and 30 ml of the blood mix were carefully poured into Pancoll tubes (PAN Biotech, Cat.No. P04-60225). The tubes were centrifuged at 800 g for 15 minutes at room temperature with low acceleration and without break. Afterwards the PBMCs were collected from the interface, washed twice with RPMI1640 and resuspended in 30-50 mL of RPMI1640. The cells were counted and applied to the human Pan-DC Enrichment Kit (Miltenyi #130-100-777) according to the manufacturer’s protocol. In brief, the cells were centrifuged (300 g, 10 min at 4°C) and resuspended in MACS Buffer (350 JJ.1 / 10A8 cells). FcR Blocking Reagent (50 JJ.1 / 10A8 cells) and Pan DC Biotin-Antibody Cocktail (100 JJ.1 / 10A8cells) were added for 5 min at RT. MACS buffer (400 JJ.1 / 10A8cells) and Pan DC MicroBead-Antibody Cocktail (100 JJ.1 / 10A8 cells) were added for 5 min at RT. Afterwards, the cells were washed with MACS Buffer (10 ml / 10A8 cells), centrifuged and resuspended in MACS Buffer (500 JJ.1 / 10A8 cells). For the Magnetic separation LS columns (Miltenyi #130-042-401) were prepared by placing them into a magnet and rinsing them with 3 ml MACS Buffer. After applying the cell suspension onto the columns, they were washed once with 3 ml MACS Buffer. Unlabeled cells (enriched dendritic cell fraction) that pass through were collected, centrifuged and resuspended for counting.

[0526] Isolated dendritic cells were seeded in 96-well round bottom plates (Costar #3799) with lxl0A5 cells / well in 180 pL medium containing RPMI 1640 (PAN #P04-17500), 10 % FCS (Anprotech, Cat:AC-SM-0014Hi), 2 mM L-Glutamine (Sigma, CatNr: G7513), 0.1 mM 2-Mercaptoethanol (Gibco #31350-010), 1 mM Na-Pyruvate (Anprotech Cat: AC-DS-0023), lx MEM NEAA (PAN Cat# P08-32100), lx MEM vitamin solution (PAN #P08-41100), 10 mM Hepes (Anprotech #AC-DS-0007), 100 pg / ml Penicillin / Streptomycin (PAN #P06-07100- 100ml). PD-L1 -targeted immunoconjugates comprising IFN-a2a mutants or reference molecule were added at a final concentration of 10 nM in 20 pl of medium. As controls, 10 nM recombinant human IFN-a2a (PBL Assay Science #11101-2) was applied as positive control and an attenuated huIFN-a R149 fused C-terminally to an effector silent Fc region carrying the LALAPG mutation (P1AG0745) was used as a negative control.

[0527] After 24 h of incubation, cells were stained for flow cytometry analysis. Therefore, cells were transferred to a 96-well V-bottom plate (Costar #3357), centrifuged (5 min, 400 g, RT) and washed twice with 200 pl PBS (Anprotec #AC-BS-0002). Zombie UV dye (Biolegend #423107) was diluted in PBS (1:400) and pellets were resuspended in 100 pl. After a 20 min incubation time at 4°C in the dark, 150 pl FACS buffer containing PBS, 3 % FCS (Gibco #10500-064) and 2 mM EDTA (Gibco #15575) were added. Then cells were centrifuged and washed with 250 pl FACS-buffer. Pellets were resuspended in 50 pl of 1:50 diluted human Fc Block (BD Pharmingen, Cat.No. 564219) in FACS buffer and incubated for 10 min at RT. Afterwards, 50 pl / well Mastermix of extracellular staining antibodies (2x antibody concentration) were added containing FITC anti-human Lineage Cocktail (CD3, 14, 16, 19,20,56) (Biolegend #348801), BV605 antihuman CD141 (Biolegend #344118), BUV395 anti-human CDllc (BD Biosciences #748289), BV421 anti-human CDlc (Biolegend #331526), BUV661 anti-human CD123 (BD Biosciences #741541), BUV661 anti-human CD 123 (BD Biosciences #741541), APC anti-human CD303 (Biolegend #354206), PE / Cy7 anti -human PD-L1 (Biolegend #329718), BV785 anti-human CD86 (Biolegend #305442), BV711 anti-human CD80 (Biolegend #305236) and PE anti-human CLEC9A (Biolegend #353804). All antibodies were diluted 1 : 100 in FACS buffer, except PE antihuman CLEC9A, which was diluted 1:50. As control, an isotype cocktail was used containing PE / Cy7 mouse IgG2b from Biolegend #400326, BV785 mouse IgG2b from Biolegend #400356, BV711 mouse IgGl from Biolegend #400168 and targeted lineage antibodies from the extracellular staining antibody mastermix). Staining was performed for 20 min. at 4°C in the dark. Afterwards cells were washed with 150 pl / well FACS-buffer, centrifuged and collected in 200 pl FACS-buffer for measurement at 5-laser LSR-Fortessa (BD Bioscience with DIVA software). As can be seen from Figure 19, CD80 expression is upregulated only upon recombinant human IFN-a2a treatment. For the PD-L1 -targeted immunoconjugate, CD80 expression is comparable to the medium control. Since DCs have about 100 PD-L1 molecules per cell (= low PD-L1 expression), low DC activation by the PD-L1 -targeted immunoconjugate is expected. In pDCs, P1AI4283 upregulated CD80 but to a lower extent compared to recombinant human IFN-a2a, indicating lower periphery DC activation compared to rec hu IFN-a2a, translating into improved safety compared to the recombinant human IFN-a2a used as control here.

[0528] Example 17: PD-Ll-targeted IFN-a2a shows reduced stimulation of human primary T cells Human primary CD4 and CD8 T cells (characterized by low surface PD-L1 expression levels, 50 - 200 PD-L1 proteins per cell) were stimulated with PD-Ll-targeted immunoconjugates comprising IFN-a2a mutants or recombinant human IFN-a2a for 24 h, and activation marker CD69 was measured by flow cytometry upon treatment. Therefore, blood was obtained from the blood donation service of the medical service Penzberg in 50 ml tubes (Greiner Bio-One, Cat.No.

[0529] 227261) containing 0.5 ml of Heparin (ratiopharm, Cat.No. 03029843) and 0.5 ml of 0,9 % Sodium Chloride solution (Fresenius Kabi, Cat.No. 0809078). In order to isolate peripheral blood mononuclear cells (PBMCs), the blood was diluted in the same volume of RPMI1640 (PAN Biotech, Cat. No. P04-17000) and 30 ml of the blood mix were carefully poured into Pancoll tubes (PAN Biotech, Cat.No. P04-60225). The tubes were centrifuged at 800 g for 15 minutes at room temperature with low acceleration and without break. Afterwards the PBMCs were collected from the interface, washed twice with RPMH640 and resuspended in 30-50 mL of RPMH640. The cells were counted using a Neubauer chamber and 1:10 dilution of Trypan blue 0.4 % (Invitrogen, Cat.No. T10282). Subsequently, T cells were isolated from the PBMCs with the Miltenyi Pan T cell isolation kit (Cat.No. 130-095-535) according to manufacturer's instructions. In brief, nontarget cells were labeled by using a cocktail of biotin-conjugated antibodies. Subsequently, nontarget cells were magnetically labelled with the Pan T Cell MicroBead Cocktail. Isolation of T cells was achieved by depletion of magnetically labelled cells. T cells were frozen with 90 % FCS and 10 % DMSO. After thawing, 50,000 T-cells were seeded in 180pl into a 96 well round-bottom plate and PD-Ll-targeted immunoconjugate, recombinant human IFN-a2a or reference molecule were added at a final concentration of 10 nM in 20 pl of RPMI1640 (PAN Biotech, Cat. No. P04-17000) medium containing 10 % FCS (Gibco, Cat.No.10500-064), 0,1 mM 2-Mercaptoethanol (Gibco, Cat.No. 31350-010), 2 mM L-Glutamine (Sigma, Cat.No. G7513), 1 mM NaPyruvate (PAN, Cat.No. P04-43100), 100 pg / ml PenStrep (PAN, Cat.No. P06-07100), lx MEM NEAA (Pan, Cat.No. P08-32100) and 10 mM Hepes (Anprotech, Cat.No. AC-Ds-0007). As positive control, rec. hu IFN-a2a (PBL Assay Science, Cat.No.11101-2) was used. After 72 hours cells were washed once with FACS buffer (PBS (Anprotec #AC-BS-0002) containing 3 % FCS , 2 mM EDTA (Gibco #15575)) and incubated with 20 pl of 1:50 diluted human Fc Block (BD Pharmingen, Cat.No. 564219) in FACS buffer. After 15 minutes of incubation at 4°C, cells were washed with FACS buffer and 20 pl of a mixture of fluorescently labeled antibodies in FACS buffer were added to the cells. The following fluorescently labeled antibodies were used: FITC anti-human CD3 (Biolegend, Cat. No.100204), PE-Cy7 anti-human CD8 (Biolegend, Cat. No. 980910), BUV395 anti-human CD4 (BD Biosciences, Cat.No. 564724), APC anti -human CD69 (Biolegend, Cat. No. 310910). After 20 minutes of incubation at 4°C, cells were washed twice with FACS buffer and then resuspended in 200 pl of FACS buffer containing the viability dye DAPI to distinguish between live and dead cells.

[0530] Cells were analyzed the same day using 5-laser LSR-Fortessa (BD Bioscience with DIVA software). Data analysis was performed using the Flow Jo version 10 software (FlowJo LLC). Live (DAPI negative) cells, positive for CD3 and positive for CD4 or CD8 cells were analyzed for CD69 expression.

[0531] As can be seen from Figure 20 A and B, the PD-L1 -targeted immunoconjugate induces lower levels of CD69 expression on naive CD4 and CD8 T cells compared to recombinant IFN-a2a. Since peripheral T cells have about 100-200 PD-L1 molecules per cell (= low PD-L1 expression), low T cell activation by the PD-L1 -targeted immunoconjugate is expected. This lower periphery T cell activation potentially can result in an improved safety profile.

[0532] Example 18: PD-Ll-targeted IFN-«2a whole blood cytokine assay

[0533] Fresh human whole blood from healthy donors (low PD-L1 expression) was incubated with indicated PD-Ll-targeted IFN-a2a molecules at a concentration of 4 nM for 24 h, and IL-6 and CXCL10 levels were measured upon treatment.

[0534] Blood was obtained from the blood donation service of the medical service Penzberg. 100 pl blood were transferred into a 96 well round-bottom plate containing 80 pl RPMI1640 (PAN Biotech, Cat. No. P04-17000) medium supplemented with 10 % FCS (Gibco, Cat.No.10500-064), 0.1 mM 2 -Mercaptoethanol (Gibco, Cat.No. 31350-010), 2 mM L-Glutamine (Sigma, Cat.No. G7513)), 1 mM Pyruvate (PAN, Cat.No. P04-43100), 100 pg / ml PenStrep (PAN, Cat.No. P06-07100), lx MEM NEAA (Pan, Cat.No. P08-32100) and 10 mM Hepes (Anprotech, Cat.No. AC-Ds-0007) per well. PD-Ll-targeted immunoconjugate, recombinant human IFN-a2a or reference molecule were added at a final concentration of 4 nM in 20 pl of RPMI1640 with supplements. As positive control, rec. hu IFN-a2a (PBL Assay Science, Cat.No.11101-2) was used. After 24 h, plates were centrifuged and supernatants were collected for the CXCL10 or IL-6 ELISA (R&D, Cat.No. DY266 , Cat.No.DY206, Cat.No. DY008). Supernatants for the CXCL10 ELISA were diluted 1:10, for the IL-6 ELISA 1:2. The two ELISAs were performed as described in the manufacturer’s protocol. The optical density was determined using a microplate reader (set to 450 nm, wavelength correction 570 nm).

[0535] As can be seen in Figure 21 A and B, the PD-L1 -targeted immunoconjugate P1AI4283 induces significantly lower CXCL10 release compared to recombinant IFN-a2a (“rec. IFN-a”) and the reference molecule (P1AI8334) in vitro in human whole blood, potentially indicating lower periphery activity and thus improved safety. The result was considered statistically significant based on an unpaired t-test, with a p-value of p= 0.0193 for IL6 levels of rec hu IFN-a2a vs P1AI4283 and p<0.0001 for CXCL10 levels of rec hu IFN-a2a vs P1AI4283.

[0536] Example 19: PD-Ll-targeted IFN-«2a internalization assay

[0537] Internalization of PD-L1 targeted IFN-a2 by HCC1954 cells was measured using the live cell imaging of the Incucyte SX5 (Sartorius). Cells were cultivated with 90 % RPMI 1640, 2 mM L- Glutamine, 1 mM Sodium Pyruvate, 10 mM HEPES, 4.5 g / L Glucose, 1.5 g / L NaHCCf (PAN Biotech, Cat.No.P04-18047) supplemented with 10 % Fetal Bovine Serum (Anprotec, Cat.No. AC-SM-0014Hi)) and Penicillin / Streptomycin (Roche, Cat.no 11074440001). 15,000 tumor cells were seeded in 200 pl cultivation medium in a flat-bottom 96-well plate (Corning, Cat.No. 3585) and incubated overnight at 37°C to adhere. 10 nM of PD-L1 targeted IFN-a antibodies and 30 nM of the Incucyte Human / Mouse Fabfluor-pH Red Antibody Labeling Reagent (Sartorius, Cat.No.

[0538] 4722 / 4750) were mixed in media and incubated for 15 min at 37°C to allow conjugation. The supernatant of the cells were removed and lOOpl of the Antibody -Fabfluor Mix was added. The plate was transferred to the Incucyte and imaged every 2 hours (lOx objective). Data analysis was performed with the Incucyte® Software 2022A (Basic analysis, Red object count per Image). As can be seen from Figure 22, the PD-Ll-targeted immunoconjugate shows lower HCC1954 internalization rate over time compared to the reference molecule, leadi...

Claims

WHAT IS CLAIMED IS:

1. An immunoconjugate comprising a mutant IFN-a2 polypeptide and an antibody that binds to PD-L1, wherein the mutant IFN-a2 polypeptide is a human IFN-a2 molecule comprising an amino acid substitution selected from the group consisting of L30A, L30H, and L153D, in particular the amino acid substitution L30A (numbering relative to the human IFN-a2 sequence SEQ ID NO: 17).

2. The immunoconjugate according to claim 1, wherein the mutant IFN-a2 polypeptide comprises the sequence of SEQ ID NO: 22.

3. An immunoconjugate according to claim 1 or 2, wherein the antibody comprises (a) a heavy chain variable region (VH) comprisinga HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1,a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2,a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3, anda light chain variable region (VL) comprisinga HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4,a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, anda HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6,or(b) a heavy chain variable region (VH) comprisinga HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9,a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10,a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11, anda light chain variable region (VL) comprisinga HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12,a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13, anda HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14.

4. An immunoconjugate according any one of claims 1 to 3, wherein the antibody comprises(a) a heavy chain variable region (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 7, and a light chain variable region (VL) comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 8,or(b) a heavy chain variable region (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 15, and a light chain variable region (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 16.

5. An immunoconjugate according to any one of claims 1 to 4, wherein the antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 7, and a light chain variable region (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 8.

6. An immunoconjugate according to any one of claims 1 to 5, wherein the antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 7, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 8.

7. The immunoconjugate according to any one of claims 1 to 6, comprisinga) a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 33, SEQ ID NO: 36, and SEQ ID NO: 37, a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 34, and a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 35,orb) a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 38, a polypeptide comprising an amino acid sequence that is at least about 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 39, and a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 40.

8. The immunoconjugate according to any one of claims 1 to 7, comprising a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 33, a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 34, and a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 35.

9. The immunoconjugate according to any one of claims 1 to 8, wherein the immunoconjugate comprises not more than one mutant IFN-a2 polypeptide.

10. The immunoconjugate according to any one of claims 1 to 9, wherein the antibody is a) an antibody fragment selected from the group consisting of a Fv, a Fab, a Fab’, a scFv, and a F(ab’)2, orb) a full-length antibody, particularly a full-length IgGl antibody.

11. The immunoconjugate according to any one of claims 1 to 10, wherein the antibody comprises an IgG class Fc domain, particularly an IgGl subclass, Fc domain composed of a first and a second subunit.

12. The immunoconjugate according to claim 11, wherein in the first subunit of the Fc domain the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the second subunit of the Fc domain the tyrosine residue at position 407 is replaced with a valine residue (Y407V) and optionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numberings according to Kabat EU index).

13. The immunoconjugate according to claim 12, wherein in the first subunit of the Fc domain additionally the serine residue at position 354 is replaced with a cysteine residue (S354C) or the glutamic acid residue at position 356 is replaced with a cysteine residue(E356C), and in the second subunit of the Fc domain additionally the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C) (numberings according to Kabat EU index).

14. The immunoconjugate according to any one of claims 11 to 13, wherein the mutant IFN-a2 polypeptide is fused at its amino-terminal amino acid to the carboxy-terminal amino acid of one of the subunits of the Fc domain, particularly the first subunit of the Fc domain, optionally wherein the mutant IFN-a2 polypeptide is fused to the Fc domain via a linker peptide, particularly a linker peptide of the amino acid sequence of SEQ ID NO: 25.

15. One or more isolated polynucleotides encoding the immunoconjugate according to any one of claims 1 to 14.

16. A host cell comprising the polynucleotide(s) of claim 15.

17. A method of producing an immunoconjugate comprising a mutant IFN-a2 polypeptide and an antibody that binds to PD-L1, comprising (a) culturing the host cell of claim 16 under conditions suitable for the expression of the immunoconjugate, and optionally (b) recovering the immunoconjugate.

18. A pharmaceutical composition comprising the immunoconjugate according to any one of claims 1 to 14 and a pharmaceutically acceptable carrier.

19. The immunoconjugate according to any one of claims 1 to 14 for use as a medicament.

20. The immunoconjugate according to any one of claims 1 to 14 for use in the treatment of a disease, in particular cancer.

21. Use of the immunoconjugate according to any one of claims 1 to 14 in the manufacture of a medicament for the treatment of a disease, in particular cancer.

22. A method of treating a disease, in particular cancer, in an individual, comprising administering to said individual a therapeutically effective amount of a composition comprising the immunoconjugate according to any one of claims 1 to 14 in a pharmaceutically acceptable form.

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