Interferon α2b variant

Abstract

The present invention provides a fusion polypeptide comprising a first domain and a second domain, wherein the first domain comprises a polypeptide ligand that binds to a cell surface-associated antigen, and the second domain comprises deglycosylated interferon α2b (IFNα2b), wherein the deglycosylated IFNα2b has a sequence of SEQ ID NO: 1 or SEQ ID NO: 2. The deglycosylated IFNα2b further comprises one or more amino acid substitutions or deletions that attenuate the activity of the deglycosylated IFNα2b.

Description

Field of the Invention

[0001] The present invention relates to polypeptides comprising a ligand targeting a cell surface antigen and deglycosylated interferon α2b (IFNα2b) and the use of these polypeptides in treating cancer. Background of the Invention

[0003] Many peptide and polypeptide molecules have been described that act by interacting with receptors on the cell surface, thereby stimulating, inhibiting or otherwise regulating biological responses, and are generally involved in signal transduction pathways within the cell carrying the receptor. Examples of these molecules include peptide and polypeptide hormones, cytokines, chemokines, growth factors, apoptosis-inducing factors, etc. These molecules can be soluble or can be attached to the surface of another cell.

[0004] Due to the biological activity of this type of molecule, some have potential uses as therapeutic agents. Several peptides or polypeptide molecules have been approved as therapeutic products by regulatory agencies, including for example human growth hormone, insulin, interferon IFN α 2b, IFN α 2a, IFN β, erythropoietin, G-CSF and GM-CSF. Many of these and other peptides have been shown to have potential in therapeutic applications, but also show toxicity when applied to human patients. One reason for toxicity is that most of these molecules trigger receptors on various cells (including cells other than those mediating therapeutic effects). For example, when IFN α 2b is used to treat multiple myeloma, its effectiveness is at least partially in combination with the type I interferon receptor on the myeloma cell, which then causes the proliferation of minimization and therefore limits disease progression. However, unfortunately, this IFN is also combined with many other normal cells in the body, triggering various other cell responses, some of which are harmful (for example flu-like symptoms, neutropenia, depression). The result of the "off target (off target)" activity of such peptides is that many peptides are not suitable as drug candidates. In this context, "off-target activity" refers to activity at the peptide's native receptor, but acting on the surface of cells other than the one mediating the therapeutically beneficial effect.

[0005] Although some peptides (such as IFNα2b) are approved for the treatment of medical conditions, they are poorly tolerated due to their "off-target" biological activity. This off-target activity and the associated poor tolerability also mean that some of these peptide-based drugs cannot be administered at doses high enough to have an optimal therapeutic effect on the target cells that mediate the therapeutic effect.

[0006] Similarly, known interferons, particularly IFN α, have been able to increase apoptosis and reduce proliferation of certain cancer cells since the mid-1980s. These biological activities are mediated by the type I interferon receptors on the cancer cell surface, and when stimulated, they trigger various signal transduction pathways that cause the proliferation and / or induction of terminal differentiation or apoptosis that are reduced. IFN α has been approved by the U.S. FDA for the treatment of several cancers, including melanoma, renal cell carcinoma, B-cell lymphoma, multiple myeloma, chronic myeloid leukemia (CML) and hairy cell leukemia. The "direct" effect of IFN α on tumor cells is mediated by the IFN α directly combined with the type I IFN receptors on these cells, and stimulates apoptosis, terminal differentiation or reduces proliferation. An "indirect" effect of IFN α on non-cancerous cells is to stimulate the immune system, which may produce additional anti-cancer effects by causing the immune system to reject tumors.

[0007] Unfortunately, type I interferon receptors are also present on most non-cancerous cells. Activation of this receptor on such cells by IFNα leads to the expression of numerous pro-inflammatory cytokines and chemokines, resulting in toxicity. This toxicity prevents the administration of IFNα to subjects at levels that maximize its anti-proliferative and pro-apoptotic activity against cancer cells.

[0008] Ozzello et al. (Breast Cancer Research and Treatment 25: 265-76, 1993) described the covalent attachment of human IFNα to tumor-targeting antibodies, thereby localizing the direct inhibitory activity of IFNα to tumors as a means of reducing tumor growth rate, and demonstrated that such conjugates have antitumor activity in xenograft models of human cancer. The mechanism of the observed anticancer activity was attributed to a direct effect of IFNα on cancer cells, as the human IFNα used in the experiments did not significantly interact with the murine type I IFN receptor, which could lead to an indirect anticancer effect. However, due to the lack of binding of human IFNα to murine cells, the authors were unable to assess the toxicity of the antibody-IFNα conjugate relative to free IFNα. These authors used chemical methods to link IFNα to the antibody.

[0009] Alkan et al. (Journal of Interferon Research, volume 4, number 3, p.355-63, 1984) demonstrated that connecting human IFNα to an antibody that binds to Epstein-Barr virus (EBV) membrane antigen (MA) will increase its anti-proliferative activity against cells expressing EBV-MA antigens. This increased efficacy depends on the antigen expression of the target cells and the binding specificity of the antibody. The cell line tested was the cancer cell line QIMR-WIL, a myeloid leukemia. The authors suggest that the connection of IFNα to antibodies can be used as a treatment for cancer because it can reduce tumor growth. Alkan et al. did not address the potential toxicity of these antibody-IFNα conjugates due to their interaction with normal antigen-negative cells.

[0010] It is also known that the connection between antibodies and IFNα can be achieved by preparing a fusion protein construct. For example, IDEC (WO01 / 97844) discloses a direct fusion of human IFNα to the C-terminus of the heavy chain of IgG targeting the tumor antigen CD20. Other groups have disclosed the use of various linkers between the C-terminus of the IgG heavy chain and IFNα. For example, US 7,456,257 discloses that the C-terminus of the constant region of the antibody heavy chain can be connected by the sequence (GGGGS) n The fusion protein constructs were linked to IFNα via a middle serine-glycine-rich (S / G) linker of , wherein n could be 1, 2, or 3, and there was no significant difference in the IFNα activity of the fusion protein constructs regardless of the linker length.

[0011] Morrison et al. (US2011 / 0104112A1; and Xuan C, Steward KK, Timmerman JM, Morrison SL.Targeted delivery of interferon-αvia fusion to anti-CD20results inpotent antitumor activity against B-cell lymphoma. Blood 2010; 115: 2864–71) also discloses IFNα and an intermediate S / G linker attached to the C-terminus of the heavy chain of an IgG antibody targeting cancer, and observed that the fusion of IgG and the linker to IFNα reduced the activity of IFNα on cells that did not express the corresponding antigen on the cell surface. When compared to human non-fusion protein IFNα (free IFNα) acting on human cells, the reduction in IFN activity of these fusion protein constructs was modest, but appeared to be more significant for mouse IFNα in mouse cells. The reduction in human IFNα activity observed by Morrison et al. and in US 7,456,257 upon fusion to the C-terminus of an antibody is modest and is generally considered a disadvantage because it reduces the efficacy of IFN. For example, Rossi et al. (Blood vol. 114, No. 18, pp 3864-71) pointed out this disadvantage and used an alternative strategy of linking IFNα to a tumor-targeting antibody, resulting in no observed loss of IFNα activity.

[0012] In general, the prior art teaches the use of effective IFNs and targeting them to cancer cells. While this approach results in increased IFN activity against cancer cells, it does not address the issue of IFN activity against normal, "off-target" cells. In the prior art examples described above, the human IFNα portion of the antibody-IFNα fusion protein retains a high proportion of native IFNα activity when exposed to human cells that do not express the corresponding antigen on their cell surface. This activity may result in toxicity caused by the IFNα portion of the fusion protein activating non-cancerous, normal ("off-target") cells. Therefore, it is necessary to reduce the "off-target" activity of IFN-based drugs while retaining the "on-target" therapeutic effects of these drugs. Maintaining the target-specific activity of these types of therapeutic agents while reducing their off-target toxicity would create a larger therapeutic concentration window for therapeutically useful peptides. For example, it would be ideal to use human IFNα in a form that allows its activity to be directed against cancer cells while minimizing its effect on normal human cells. Ideally, the type I interferon receptors on cancer cells would be stimulated to the maximum extent, while the same receptors on non-cancerous cells would experience minimal stimulation. Human IFNα needs to be targeted to cancer cells so that it has significantly more activity against cancer cells that display the antigen than against normal cells that do not. The same logic applies to other potential therapeutic molecules, such as other cytokines, peptide and polypeptide hormones, chemokines, growth factors, apoptosis-inducing factors, etc.

[0013] The logic of this approach has been demonstrated in WO 2013 / 059885 and WO 2014 / 178820, the disclosures of each of which are incorporated herein by cross-reference. SUMMARY OF THE INVENTION

[0015] In a first aspect, the present invention provides a fusion polypeptide comprising a first domain and a second domain, wherein the first domain comprises a polypeptide ligand that binds to a cell surface-associated antigen, and the second domain comprises deglycosylated interferon α2b (IFNα2b), the deglycosylated IFNα2b having a sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and wherein the deglycosylated IFNα2b further comprises one or more amino acid substitutions or deletions that reduce the activity of the deglycosylated IFNα2b.

[0016] In another aspect, the present invention provides a fusion polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs: 31, 61-77, 83 and 87 and a sequence selected from the group consisting of SEQ ID NOs: 81, 82 and 84.

[0017] In another aspect, the present invention provides a fusion polypeptide comprising SEQ ID NO: 87 and SEQ ID NO: 81.

[0018] In another aspect, the present invention provides a fusion polypeptide comprising SEQ ID NO: 79 and SEQ ID NO: 85.

[0019] In another aspect, the present invention provides a fusion polypeptide comprising SEQ ID NO: 80 and SEQ ID NO: 86.

[0020] In another aspect, the present invention provides a fusion polypeptide comprising SEQ ID NO:78.

[0021] In another aspect, the present invention provides a composition comprising the fusion polypeptide of the present invention and a pharmaceutically acceptable carrier or diluent.

[0022] In another aspect, the present invention provides a method of treating a tumor in a subject, comprising administering to the subject a fusion polypeptide of the present invention or a composition of the present invention, wherein the first domain of the fusion polypeptide binds to tumor cells.

[0023] In another aspect, the present invention provides use of the fusion polypeptide of the present invention in treating tumors, wherein the first domain of the fusion polypeptide binds to the tumor.

[0024] In another aspect, the present invention provides one or more isolated polynucleotides encoding one or more fusion polypeptides of the present invention.

[0025] In another aspect, the present invention provides a vector comprising one or more polynucleotides of the present invention.

[0026] In another aspect, the present invention provides a transformed cell comprising the vector of the present invention.

[0027] In another aspect, the present invention provides a method for producing a polypeptide ligand-attenuated IFNα2b fusion polypeptide in a mammalian cell, wherein the polypeptide ligand-attenuated IFNα2b fusion polypeptide has reduced heterogeneity and / or enhanced FcRn binding and / or improved target selectivity, the method comprising culturing a recombinant mammalian cell comprising a polynucleotide encoding the polypeptide ligand-attenuated IFNα2b fusion polypeptide, wherein T106 of the IFNα2b sequence is replaced by another amino acid or deleted, such that when expressed in the mammalian cell, the IFNα2b component of the fusion protein is aglycosylated.

[0028] BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 : Antiproliferative activity of (A) ARP1 and (B) NCI-H929 cells treated with anti-CD38-attenuated IFNα2b fusion proteins in the form of IgG1 or IgG4 with and without O-linked glycosylation of IFNα2b.

[0030] Figure 2A, B, C, D and E: Antiproliferative activity of anti-CD38-attenuated IFNα2b fusion proteins using different amino acid substitutions to remove the O-linked glycosylation site from attenuated IFNα2b.

[0031] Figure 3 : On-target activity of (A) A10.21 and (B) A10.43 anti-CD38-attenuated IFNα2b fusion proteins with (T106T) and without (T106A) O-linked glycosylation of IFNα2b.

[0032] Figure 4 : Off-target activities of (A) A10.21 and (B) A10.43 anti-CD38-attenuated IFNα2b fusion proteins with (T106T) and without (T106A) O-linked glycosylation of IFNα2b.

[0033] Figure 5 : Off-target activities of anti-CD38-attenuated IFNα2b fusion proteins with (T106T) and without (T106A or ΔT106) O-linked glycosylation of IFNα2b.

[0034] Figure 6: A, B, C, D, E and F: Off-target activities of anti-CD38-attenuated IFNα2b fusion proteins using different amino acid substitutions to remove the O-linked glycosylation site from attenuated IFNα2b.

[0035] Figure 7 : Off-target activity of A10.43 anti-CD38-attenuated IFNα2b fusion protein with (T106T) or without (T106A) O-linked glycosylation of IFNα2b.

[0036] Figure 8 : Efficacy of suboptimal doses of anti-CD38-attenuated IFNα2b fusion proteins with (T106T) or without (T106A) O-linked glycosylation in treating tumors in a murine model of multiple myeloma.

[0037] Figure 9 : The amount of charged species of A10.21 anti-CD38-attenuated IFNα2b fusion proteins with (T106T) or without (T106A, ΔT106, T106S, T106V, T106G, T106E) O-linked glycosylation of IFNα2b as assessed by the number of bands on IEF gels.

[0038] Figure 10: Amount of charged species of fusion proteins of A10.21 anti-CD38-attenuated IFNα2b with (T106T) or without (T106A) O-linked glycosylation of IFNα2b and with different Fc isotypes as assessed by the number of bands on IEF gels.

[0039] Figure 11 : The amount of charged species of A10.21 (IgG4 with S228P) anti-CD38-attenuated IFNα2b fusion proteins with (T106T) or without (T106A) O-linked glycosylation of IFNα2b in the presence of a YTE substitution in the antibody constant region, as assessed by the number of bands on IEF gels.

[0040] Figure 12 : Amount of charged species of A10.21 (IgG4 with S228P) anti-CD38-attenuated IFNα2b fusion protein with (T106T) or without (T106A) O-linked glycosylation of IFNα2b in the presence of various attenuating substitutions, as assessed by the number of bands on IEF gels.

[0041] Figure 13 : Antibodies with different target specificities; the amount of charged species of anti-CD138 antibodies, anti-HLA antibodies, and anti-CD38 antibodies (A02.12) (all IgG4 with S228P) fused to IFNα2b with reduced O-linked glycosylation with (T106T) or without (T106A) IFNα2b, as assessed by the number of bands on IEF gels.

[0042] Figure 14 : "On target" activity of anti-CD38-attenuated IFNα2b fusion proteins (A10.21IgG4(S228P)IFN(A145D)) with (T106T) and without (T106A, ΔT106, T106S, T106V, T106G, T106E) O-linked glycosylation of IFNα2b.

[0043] Figure 15 : "On-target" activity of two different anti-CD38 antibody-attenuated IFNα2b fusion proteins (A02.12 and A10.21, both IgG4 with S228P) that bind to different epitopes on CD38, with (T106T) or without (T106A) O-linked glycosylation of IFNα2b.

[0044] Figure 16: "On-target" activity of the A10.21 anti-CD38-attenuated IFNα2b fusion protein (A10.21IgG4(S228P)IFN) with (T106T) and without (T106A) O-linked glycosylation of IFNα2b, which has various IFN-attenuating substitutions (R33A, R144I, R145Q, A145K or A145G).

[0045] Figure 17 : "On-target" activity of antibodies with different target specificities; anti-CD138 antibody and anti-HLA antibody (both IgG4 with S228P) fused to IFNα2b with reduced O-linked glycosylation with (T106T) or without (T106A) IFNα2b.

[0046] Figure 18 : "On-target" activity of A10.21 anti-CD38-attenuated IFNα2b fusion protein (A10.21IgG4(S228P)IFN(A145D)) with (T106T) and without (T106A) O-linked glycosylation of IFNα2b in the presence of a YTE substitution in the antibody heavy chain.

[0047] Figure 19 : "Targeting" activity of A10.21 anti-CD38-attenuated IFNα2b (A145D) fusion proteins with (T106T) and without (T106A) O-linked glycosylated IFNα2b, which have various immunoglobulin Fc isotypes.

[0048] Figure 20 : Selectivity index of A10.21 anti-CD38-attenuated IFNα2b fusion proteins with and without O-linked glycosylation of IFNα2b in the presence of various amino acid substitutions to remove IFN, immunoglobulin constant region YTE substitutions to extend half-life, attenuated IFN and Fc isotype. Detailed Description of the Invention

[0050] Throughout the specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

[0051] Reference in this specification to any prior publication (or information derived therefrom) or any known matter is not and should not be taken as an acknowledgement or admission or any form of representation that the prior publication (or information derived therefrom) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

[0052] All publications mentioned in this specification are incorporated herein by reference in their entirety.

[0053] It must be noted that, as used in this specification, the singular forms "a," "an," and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "an agent" includes a single agent as well as two or more agents; reference to "a molecule" includes a single molecule as well as two or more molecules; and so forth.

[0054] The construct of the present invention is a deglycosylated IFNα2b fusion construct with a polypeptide ligand attenuated, which exhibits an antigen selectivity index relative to an activated signaling pathway elevated by the action of both the ligand targeting the cell surface receptor on the cell of interest and the attenuated IFNα2b with reduced affinity for the cell surface IFN receptor. These constructs are based on the findings outlined in WO 2013 / 059885, and in the context of antibody-IFN fusion constructs, the IFN portion can be mutated in such a way that the IFN activity on antigen-negative cells is significantly attenuated, while the IFN activity on antigen-positive cells is only moderately (if at all) attenuated. Compared to antigen-negative cells, such constructs show a stronger potency than free IFN on antigen-positive cells, i.e., 1, 2, 3, 4, or 5 orders of magnitude. In one embodiment, the antibody-attenuated IFN construct retains at least 1%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% of the potency of non-attenuated free (i.e., not linked to an antibody) IFN on antigen-positive cells. Furthermore, in one embodiment, the antibody-attenuated IFN construct retains at least 30%, at least 50%, at least 75% or at least 90% of the maximal activity of non-attenuated free (i.e., not linked to an antibody) IFN; in this context, "maximal activity" is understood to mean the amount of signaling activity (or its downstream effects) in the high plateau portion of the dose-response curve, where further additions of the agent do not further increase the response.

[0055] The present inventors have now discovered that unexpected advantages are achieved by using constructs comprising deglycosylated IFNα2b compared to constructs comprising O-glycosylated IFNα2b. In some embodiments, these advantages include one or more of the following: increased target activity, increased target selectivity, and enhanced affinity for FcRn, while providing a less heterogeneous product than O-glycosylated IFNα2b when produced in a mammalian cell expression system. Enhanced FcRn binding is desirable to improve the pK of biotherapeutics comprising an Fc region. Increased target selectivity is desirable because it potentially reduces off-target toxicity while substantially maintaining on-target activity. Reduced heterogeneity allows for increased yields of purified product from mammalian cell culture systems.

[0056] Therefore, in a first aspect, the present invention provides a fusion polypeptide comprising a first and a second domain, wherein the first domain comprises a polypeptide ligand that binds to a cell surface-associated antigen, and the second domain comprises deglycosylated interferon α2b (IFNα2b), the deglycosylated IFNα2b having a sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and wherein the deglycosylated IFNα2b further comprises one or more amino acid substitutions or deletions that reduce the activity of the deglycosylated IFNα2b.

[0057] In one embodiment of the invention, the sequence of the deglycosylated IFNα2b is SEQ ID NO: 1, wherein the residue at position 106 is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y. This represents a replacement of T106, which is normally present in human IFNα2b, with a naturally occurring amino acid that does not permit O-linked glycosylation when produced in mammalian cell culture. In another embodiment of the invention, the sequence of the deglycosylated IFNα2b is SEQ ID NO: 2. This represents a deletion of residue T106 found in normal human IFNα2b, which also removes the O-linked glycosylation site found in that molecule. As demonstrated herein, each substitution or deletion removes the O-glycosylation site from human attenuated IFNα2b and, when expressed in CHO cells, reduces the heterogeneity of the molecule as measured in IEF gels, while at least substantially maintaining the ability of attenuated IFNα2b to bind to cell surface IFN receptors and initiate downstream signaling.

[0058] In another embodiment, the sequence of the deglycosylated IFNα2b is SEQ ID NO: 1 modified by one or more attenuating mutations selected from the group consisting of L15A, R22A, R23A, S25A, L26A, F27A, L30A, L30V, K31A, D32A, R33A, R33K, R33Q, H34A, Q40A, D114R, L117A, R120A, R120E, R125A, R125E, K131A, E132A, K133A, K134A, M148A, R149A, S152A, L153A, N156A, (L30A, H57Y, E58N and Q61S), (M148A, H57Y, E58N and Q61S), (L153A, H57Y, E58N and Q61S), (R144A, H57Y, E58N and Q61S), (N65A, L80A, Y8 5A and Y89A), (N65A, L80A, Y85A, Y89A and D114A), (N65A, L80A, Y85A, Y89A and L117A), (N65A, L80A, Y85A, Y89A and R120A), (Y85A, Y89A and D114A), (D114A and R120A), (L117A and R120A), ( L117A, R120A and K121A), (R120A and K121A), (R120E and K121E), substitution of R at position 144 with A, D, E, G, H, I, K, L, N, Q, S, T, V or Y, substitution of A at position 145 with D, E, G, H, I, K, L, M, N, Q, S, T, V or Y, and deletion of residues L161-E165.

[0059] In another embodiment, the sequence of the deglycosylated IFNα2b is SEQ ID NO: 2 modified by one or more attenuating mutations selected from the group consisting of L15A, R22A, R23A, S25A, L26A, F27A, L30A, L30V, K31A, D32A, R33A, R33K, R33Q, H34A, Q40A, D113R, L116A, R119A, R119E, R124A, R124E, K130A, E131A, K132A, K133A, M147A, R148A, S149A, L152A, N155A, (L30A, H57Y, E58N and Q61S), (M147A, H57Y, E58N and Q61S), (L152A, H57Y, E58N and Q61S), (R143A, H57Y, E58N and Q61S), (N65A, L80A, Y8 5A and Y89A), (N65A, L80A, Y85A, Y89A and D113A), (N65A, L80A, Y85A, Y89A and L116A), (N65A, L80A, Y85A, Y89A and R119A), (Y85A, Y89A and D113A), (D113A and R119A), (L116A and R119A), ( L116A, R119A and K120A), (R119A and K120A), (R119E and K120E), substitution of R at position 143 with A, D, E, G, H, I, K, L, N, Q, S, T, V or Y, substitution of A at position 144 with D, E, G, H, I, K, L, M, N, Q, S, T, V or Y, and deletion of residues L160-E164.

[0060] In another embodiment, the sequence of the deglycosylated IFNα2b modified by an attenuating mutation is selected from the group consisting of SEQ ID NOs: 3-30 and SEQ ID NOs: 32-47.

[0061] In another embodiment, the cell surface associated antigen is selected from CD38, CD138, RANK ligand, HM1.24, CD56, CS1, CD20, CD74, IL-6R, Blys (BAFF), BCMA, HLA-SR, HLA-DR, kininogen, β2 microglobulin, FGFR3, ICAM-1, matriptase, CD52, EGFR, GM2, α4 integrin, IFG-1R, KIR, CD3, CD4, CD8, CD24, CD44, CD69, CD71, CD79, CD83, CD86, CD96, HLA, PD-1 , ICOS, CD33, CD115, CD11c, CD19, CD52, CD14, FSP1, FAP, PDGFRα, PDGFRβ, ASGR1, ASGR2, FSP1, RTI140 / Ti-α, HTI56, VEGF receptor, product of the CD241RCHE gene, CD117 (c-kit), CD71 (transferrin receptor), CD36 (thrombospondin receptor), CD34, CD45RO, CD45RA, CD115, CD168, CD235, CD236, CD237, CD238, CD239 and CD240.

[0062] In certain embodiments, the polypeptide ligand is an antibody or an antigen-binding portion thereof.

[0063] In another embodiment, the polypeptide ligand is an antibody that binds CD38. H The sequence is selected from SEQ ID No: 48-56 and 58, and the V L The sequence is selected from SEQ ID Nos: 81, 82 and 84.

[0064] In another embodiment, the polypeptide ligand is an antibody that binds to CD138. H The sequence is SEQ ID NO: 59, the V L The sequence is SEQ ID NO: 85.

[0065] In another embodiment, the polypeptide ligand binds to the RANK ligand. Preferably, the sequence of the polypeptide ligand is SEQ ID NO:57.

[0066] In another embodiment, the first domain is linked to the second domain via a peptide bond. The first domain can be directly linked to the second domain via a peptide bond (a "zero-length linker"), or it can be linked to the second domain via a peptide linker having a length of 1 to 20 amino acids. The linker can be (SGGGGS) n, wherein n is 1 to 3. Examples of linkers include SGGGGS and SGGGGSGGGGSGGGGS.

[0067] In another embodiment, the C-terminus of the first domain is linked to the N-terminus of the second domain.

[0068] In another embodiment, the amino acid sequence of the first domain is glycosylated.

[0069] In another aspect, the present invention provides a fusion polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs: 31, 61-77, 83 and 87 and a sequence selected from the group consisting of SEQ ID NOs: 81, 82 and 84.

[0070] In another aspect, the present invention provides a fusion polypeptide comprising SEQ ID NO: 87 and SEQ ID NO: 81.

[0071] In another aspect, the present invention provides a fusion polypeptide comprising SEQ ID NO: 79 and SEQ ID NO: 85.

[0072] In another aspect, the present invention provides a fusion polypeptide comprising SEQ ID NO: 80 and SEQ ID NO: 86.

[0073] In another aspect, the present invention provides a fusion polypeptide comprising SEQ ID NO:78.

[0074] It can be understood from the above discussion that the specific forms of the fusion polypeptide of the present invention are as follows:

[0075] a. A fusion polypeptide, wherein the sequence of deglycosylated IFNα2b is SEQ ID NO: 1, wherein the residue at position 106 is A.

[0076] b. A fusion polypeptide, wherein the sequence of the deglycosylated IFNα2b is SEQ ID NO: 1, wherein the residue at position 106 is C.

[0077] c. A fusion polypeptide, wherein the sequence of deglycosylated IFNα2b is SEQ ID NO: 1, wherein the residue at position 106 is D.

[0078] d. A fusion polypeptide, wherein the sequence of the deglycosylated IFNα2b is SEQ ID NO: 1, wherein the residue at position 106 is E.

[0079] e. A fusion polypeptide, wherein the sequence of deglycosylated IFNα2b is SEQ ID NO: 1, wherein the residue at position 106 is F.

[0080] f. A fusion polypeptide, wherein the sequence of deglycosylated IFNα2b is SEQ ID NO: 1, wherein the residue at position 106 is G.

[0081] g. A fusion polypeptide, wherein the sequence of deglycosylated IFNα2b is SEQ ID NO: 1, wherein the residue at position 106 is H.

[0082] h. A fusion polypeptide, wherein the sequence of deglycosylated IFNα2b is SEQ ID NO: 1, wherein the residue at position 106 is I.

[0083] i. A fusion polypeptide, wherein the sequence of deglycosylated IFNα2b is SEQ ID NO: 1, wherein the residue at position 106 is K.

[0084] j. A fusion polypeptide, wherein the sequence of deglycosylated IFNα2b is SEQ ID NO: 1, wherein the residue at position 106 is L.

[0085] k. A fusion polypeptide, wherein the sequence of deglycosylated IFNα2b is SEQ ID NO: 1, wherein the residue at position 106 is M.

[0086] 1. A fusion polypeptide, wherein the sequence of deglycosylated IFNα2b is SEQ ID NO: 1, wherein the residue at position 106 is N.

[0087] m. A fusion polypeptide, wherein the sequence of deglycosylated IFNα2b is SEQ ID NO: 1, wherein the residue at position 106 is P.

[0088] n. A fusion polypeptide, wherein the sequence of deglycosylated IFNα2b is SEQ ID NO: 1, wherein the residue at position 106 is Q.

[0089] o. A fusion polypeptide, wherein the sequence of deglycosylated IFNα2b is SEQ ID NO: 1, wherein the residue at position 106 is R.

[0090] p. A fusion polypeptide, wherein the sequence of deglycosylated IFNα2b is SEQ ID NO: 1, wherein the residue at position 106 is V.

[0091] q. A fusion polypeptide, wherein the sequence of deglycosylated IFNα2b is SEQ ID NO: 1, wherein the residue at position 106 is W.

[0092] r. A fusion polypeptide, wherein the sequence of the deglycosylated IFNα2b is SEQ ID NO: 1, wherein the residue at position 106 is Y.

[0093] s. A fusion polypeptide, wherein the sequence of deglycosylated IFNα2b is SEQ ID NO: 1 or SEQ ID NO: 2 modified by the attenuating mutation L15A.

[0094] t. A fusion polypeptide, wherein the sequence of the deglycosylated IFNα2b is SEQ ID NO: 1 or SEQ ID NO: 2 modified by the attenuating mutation A19W.

[0095] u. A fusion polypeptide, wherein the sequence of the deglycosylated IFNα2b is SEQ ID NO: 1 or SEQ ID NO: 2 modified by the attenuating mutation R22A.

[0096] v. A fusion polypeptide, wherein the sequence of the deglycosylated IFNα2b is SEQ ID NO: 1 or SEQ ID NO: 2 modified by the attenuating mutation R23A.

[0097] w. A fusion polypeptide, wherein the sequence of the deglycosylated IFNα2b is SEQ ID NO: 1 or SEQ ID NO: 2 modified by the attenuating mutation S25A.

[0098] x. A fusion polypeptide, wherein the sequence of the deglycosylated IFNα2b is SEQ ID NO: 1 or SEQ ID NO: 2 modified by the attenuating mutation L26A.

[0099] y. A fusion polypeptide, wherein the sequence of the deglycosylated IFNα2b is SEQ ID NO: 1 or SEQ ID NO: 2 modified by the attenuating mutation F27A.

[0100] z. A fusion polypeptide wherein the sequence of the deglycosylated IFNα2b is SEQ ID NO: 1 or SEQ ID NO: 2 modified by the attenuating mutation L30A or L30V.

[0101] aa. A fusion polypeptide, wherein the sequence of the deglycosylated IFNα2b is SEQ ID NO: 1 or SEQ ID NO: 2 modified by the attenuating mutation K31A.

[0102] bb. A fusion polypeptide, wherein the sequence of the deglycosylated IFNα2b is SEQ ID NO: 1 or SEQ ID NO: 2 modified by the attenuating mutation D32A.

[0103] cc. A fusion polypeptide, wherein the sequence of the deglycosylated IFNα2b is SEQ ID NO: 1 or SEQ ID NO: 2 modified by the attenuating mutation R33A, R33K or R33Q.

[0104] dd. A fusion polypeptide, wherein the sequence of the deglycosylated IFNα2b is SEQ ID NO: 1 or SEQ ID NO: 2 modified by the attenuating mutation H34A.

[0105] ee. A fusion polypeptide, wherein the sequence of the deglycosylated IFNα2b is SEQ ID NO: 1 or SEQ ID NO: 2 modified by the attenuating mutation Q40A.

[0106] ff. A fusion polypeptide, wherein the sequence of the deglycosylated IFNα2b is SEQ ID NO: 1 modified by the attenuating mutation D114R or SEQ ID NO: 2 modified by the attenuating mutation D113R.

[0107] gg. A fusion polypeptide, wherein the sequence of the deglycosylated IFNα2b is SEQ ID NO: 1 modified by the attenuating mutation L117A or SEQ ID NO: 2 modified by the attenuating mutation L116A.

[0108] hh. A fusion polypeptide, wherein the sequence of the deglycosylated IFNα2b is SEQ ID NO: 1 modified by the attenuating mutation R120A or R120E or SEQ ID NO: 2 modified by the attenuating mutation R119A or R119E.

[0109] ii. A fusion polypeptide, wherein the sequence of the deglycosylated IFNα2b is SEQ ID NO: 1 modified by the attenuating mutation R125A or R125E or SEQ ID NO: 2 modified by the attenuating mutation R124A or R124E.

[0110] jj. A fusion polypeptide, wherein the sequence of the deglycosylated IFNα2b is SEQ ID NO: 1 modified by the attenuating mutation K131A or SEQ ID NO: 2 modified by the attenuating mutation K130A.

[0111] kk. A fusion polypeptide, wherein the sequence of the deglycosylated IFNα2b is SEQ ID NO: 1 modified by the attenuating mutation E132A or SEQ ID NO: 2 modified by the attenuating mutation E131A.

[0112] 11. A fusion polypeptide, wherein the sequence of the deglycosylated IFNα2b is SEQ ID NO: 1 modified by the attenuating mutation K133A or SEQ ID NO: 2 modified by the attenuating mutation K132A.

[0113] mm. A fusion polypeptide, wherein the sequence of the deglycosylated IFNα2b is SEQ ID NO: 1 modified by the attenuating mutation K134A or SEQ ID NO: 2 modified by the attenuating mutation K133A.

[0114] nn. A fusion polypeptide, wherein the sequence of the deglycosylated IFNα2b is SEQ ID NO: 1 modified by the attenuating mutation M148A or SEQ ID NO: 2 modified by the attenuating mutation M147A.

[0115] oo. A fusion polypeptide, wherein the sequence of the deglycosylated IFNα2b is SEQ ID NO: 1 modified by the attenuating mutation R149A or SEQ ID NO: 2 modified by the attenuating mutation R148A.

[0116] pp. Fusion polypeptide, wherein the sequence of deglycosylated IFNα2b is SEQ ID NO: 1 modified by the attenuating mutation S152A or SEQ ID NO: 2 modified by the attenuating mutation S151A.

[0117] qq. A fusion polypeptide, wherein the sequence of the deglycosylated IFNα2b is SEQ ID NO: 1 modified by the attenuating mutation L153A or SEQ ID NO: 2 modified by the attenuating mutation L152A.

[0118] rr. A fusion polypeptide, wherein the sequence of the deglycosylated IFNα2b is SEQ ID NO: 1 modified by the attenuating mutation N156A or SEQ ID NO: 2 modified by the attenuating mutation N155A.

[0119] ss. A fusion polypeptide wherein the sequence of the deglycosylated IFNα2b is SEQ ID NO: 1 or SEQ ID NO: 2 modified by attenuating mutations L30A, H57Y, E58N and Q61S.

[0120] tt. A fusion polypeptide, wherein the sequence of the deglycosylated IFNα2b is SEQ ID NO: 1 modified by attenuating mutations M148A, H57Y, E58N and Q61S or SEQ ID NO: 2 modified by attenuating mutations M147A, H57Y, E58N and Q61S.

[0121] uu. A fusion polypeptide wherein the sequence of the deglycosylated IFNα2b is SEQ ID NO: 1 modified by attenuating mutations L153A, H57Y, E58N and Q61S or SEQ ID NO: 2 modified by attenuating mutations L152A, H57Y, E58N and Q61S.

[0122] vv. A fusion polypeptide wherein the sequence of the deglycosylated IFNα2b is SEQ ID NO: 1 modified by attenuating mutations R144A, H57Y, E58N and Q61S or SEQ ID NO: 2 modified by attenuating mutations R143A, H57Y, E58N and Q61S.

[0123] ww. A fusion polypeptide, wherein the sequence of the deglycosylated IFNα2b is SEQ ID NO: 1 or SEQ ID NO: 2 modified by attenuating mutations N65A, L80A, Y85A and Y89A.

[0124] xx. A fusion polypeptide wherein the sequence of the deglycosylated IFNα2b is SEQ ID NO: 1 modified by attenuating mutations N65A, L80A, Y85A, Y89A and D114A or SEQ ID NO: 2 modified by attenuating mutations N65A, L80A, Y85A, Y89A and D113A.

[0125] yy. A fusion polypeptide, wherein the sequence of the deglycosylated IFNα2b is SEQ ID NO: 1 modified by attenuating mutations N65A, L80A, Y85A, Y89A and L117A or SEQ ID NO: 2 modified by attenuating mutations N65A, L80A, Y85A, Y89A and L116A.

[0126] zz. A fusion polypeptide, wherein the sequence of the deglycosylated IFNα2b is SEQ ID NO: 1 modified by attenuating mutations N65A, L80A, Y85A, Y89A and R120A or SEQ ID NO: 2 modified by attenuating mutations N65A, L80A, Y85A, Y89A and R119A.

[0127] aaa. Fusion polypeptide, wherein the sequence of the deglycosylated IFNα2b is SEQ ID NO: 1 modified by attenuating mutations Y85A, Y89A and D114A or SEQ ID NO: 2 modified by attenuating mutations Y85A, Y89A and D113A.

[0128] bbb. A fusion polypeptide wherein the sequence of the deglycosylated IFNα2b is SEQ ID NO: 1 modified by attenuating mutations D114A and R120A or SEQ ID NO: 2 modified by attenuating mutations D113A and R119A.

[0129] ccc. A fusion polypeptide, wherein the sequence of the deglycosylated IFNα2b is SEQ ID NO: 1 modified by attenuating mutations L117A and R120A or SEQ ID NO: 2 modified by attenuating mutations L116A and R119A.

[0130] ddd. A fusion polypeptide wherein the sequence of the deglycosylated IFNα2b is SEQ ID NO: 1 modified by attenuating mutations L117A, R120A and K121A or SEQ ID NO: 2 modified by attenuating mutations L116A, R119A and K120A.

[0131] eee. A fusion polypeptide, wherein the sequence of the deglycosylated IFNα2b is SEQ ID NO: 1 modified by attenuating mutations R120A and K121A or SEQ ID NO: 2 modified by attenuating mutations R119A and K120A.

[0132] fff. A fusion polypeptide wherein the sequence of the deglycosylated IFNα2b is SEQ ID NO: 1 modified by attenuating mutations R120E and K121E or SEQ ID NO: 2 modified by attenuating mutations R119E and K120E.

[0133] ggg. A fusion polypeptide, wherein the sequence of deglycosylated IFNα2b of SEQ ID NO: 1 is modified by replacing R at position 144 with A, D, E, G, H, I, K, L, N, Q, S, T, V or Y, or SEQ ID NO: 2 is modified by replacing R at position 143 with A, D, E, G, H, I, K, L, N, Q, S, T, V or Y.

[0134] hhh. A fusion polypeptide, wherein the sequence of deglycosylated IFNα2b of SEQ ID NO: 1 is modified by replacing A at position 145 with D, E, G, H, I, K, L, M, N, Q, S, T, V, or Y, or SEQ ID NO: 2 is modified by replacing A at position 144 with D, E, G, H, I, K, L, M, N, Q, S, T, V, or Y.

[0135] iii. A fusion polypeptide, wherein the deglycosylated IFNα2b is SEQ ID NO: 1 modified by deletion of residues L161-E165 or SEQ ID NO: 2 modified by deletion of residues L161-E165.

[0136] jjj. The fusion polypeptide according to claim 1, wherein the sequence of the deglycosylated IFNα2b is selected from the group consisting of SEQ ID NOs: 3-30 and SEQ ID NOs: 32-47.

[0137] The term "antibody" as used herein refers broadly to any immunoglobulin (Ig) molecule composed of four polypeptide chains (two heavy (H) chains and two light (L) chains), or any functional fragment, mutant, variant or derivative thereof that retains the essential epitope binding characteristics of an Ig molecule. Such mutant, variant or derivative antibody forms are known in the art, and non-limiting embodiments thereof are discussed below.

[0138] In a full-length antibody, each heavy chain consists of a heavy chain variable region (abbreviated herein as HCVR or V H ) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as LCVR or V L ) and a light chain constant region. The light chain constant region consists of one domain, CL, which in humans can be either κ or λ. H and V L The V region can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). H and V L It consists of three CDRs and four FRs arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The immunoglobulin molecule can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0139] As used herein, an "antigen binding domain" or "antigen binding portion" of an antibody refers to one or more fragments of an antibody or protein that retains the ability to specifically bind to an antigen (e.g., CD38). It has been shown that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Such antibody embodiments can also be bispecific, dual specific, or multispecific, specifically binding to two or more different antigens. Examples of binding fragments encompassed within the term "antigen binding portion" of an antibody include (i) a Fab fragment, consisting of a V L 、V H 、C L and CH1 domains; (ii) F(ab')2 fragments, bivalent fragments consisting of two Fab fragments in addition to a portion of the hinge region, connected by a disulfide bridge in the hinge region; (iii) V H and CH1 domains; (iv) a V fragment consisting of a single-arm antibody L and V HFv fragments are composed of two domains, (v) domain antibodies (dAbs) (Ward et al. 1989 Nature 341 544-6, Winter et al., PCT publication WO 90 / 05144 A1, which is incorporated herein by reference), which contain a single variable domain; and (vi) isolated complementarity determining regions (CDRs). In addition, although the two domains V of the Fv fragment are L and V H The genes encoding the proteins are separated, but they can be joined using recombinant methods using synthetic linkers, allowing them to be made into a single protein chain, where V L and V H Single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. Other forms of single chain antibodies, such as diabodies, are also included. Diabodies are bivalent, bispecific antibodies in which V is the antigen-binding portion of the antibody. H and V L The domains are expressed on a single polypeptide chain, but a linker that is too short to allow pairing between the two domains on the same chain is used, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen-binding sites (see, e.g., Holliger, P. et al., 1993, Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak, RJ et al., 1994, Structure 2: 1121-1123). Such antibody binding portions are known in the art (Kontermann and Dubel, eds., Antibody Engineering 2001 Springer-Verlag. New York. 790 pp., ISBN 3-540-41354-5). In one embodiment, the antibody binding portion is a Fab fragment.

[0140] Antibodies as herein described can be humanized antibodies.Term " humanized antibody " should be construed as comprising the protein of human variable region, and it comprises from being transplanted onto the FR from human antibody or inserting therein the CDR (such antibody is also referred to as " CDR transplanted antibody ") of the antibody of non-human species (for example, mouse or rat or non-human primate).Humanized antibody also comprises wherein one or more residues of human protein are modified by one or more amino acid replacements and / or one or more FR residues of human protein are replaced with the protein of corresponding non-human residue.Humanized antibody can also be included in the residue that is not found in human antibody or non-human antibody.Any additional region (for example Fc district) of protein is usually people.Can use methods known in the art, for example US5,225,539, US6,054,297, US7,566,771 or US5,585,089 carry out humanization.Term " humanized antibody " also comprises superhumanized antibody, for example as described in US7,732,578.

[0141] The antibody herein can be human.Term " human antibody " used herein refers to the protein with variable antibody region and optionally constant antibody region found in human body, for example, in people's germline or somatic cell or from the library produced using these regions, find." human " antibody can include amino acid residues not encoded by human sequence, for example, the mutation (particularly relating to the conservative substitution or mutation of a small amount of residues in protein, for example 1,2,3,4 or 5 in protein residue) introduced by in vitro random mutation or site-directed mutagenesis.These " human antibodies " do not necessarily need to produce as the result of people's immune response, but can use recombinant means (for example, screening phage display library) and / or by transgenic animals (for example, mice), it comprises nucleic acid encoding human antibody constant region and / or variable region and / or uses guide selection (guide selection) (for example, as described in US5,565,332).The term also includes the affinity maturation form of this antibody. For purposes of this disclosure, human proteins will also be considered to include proteins comprising FRs from human antibodies or comprising FRs comprising a sequence derived from a human FR consensus sequence, wherein one or more CDRs are randomized or semi-randomized, e.g., as described in US 6,300,064 and / or US 6,248,516.

[0142] The antibody portion of the polypeptide of the present invention can be a full-length antibody of any class, preferably IgG1, IgG2, or IgG4. The constant domains of these antibodies are preferably human. The variable regions of such antibodies can be non-human, or preferably human or humanized. Antibody fragments can also be used instead of full-length antibodies.

[0143] The term "antibody" also includes engineered antibodies. As will be appreciated, there are many variants of engineered antibodies (e.g., mouse monoclonal antibodies, chimeric, humanized and human monoclonal antibodies, single-chain variable antibody fragments (scFv), miniantibodies, aptamers, and bispecific antibodies and diabodies as described above).

[0144] Single variable region domains (referred to as dAbs) are disclosed, for example, in (Ward et al., 1989, Nature 341: 544-546; Hamers-Casterman et al., 1993, Nature 363: 446-448; Davies & Riechmann, 1994, FEBS Lett. 339: 285-290).

[0145] Minibodies are small forms of whole antibodies that encode the essential elements of an entire antibody in a single chain. Suitably, the minicarrier consists of the VH and VL domains of a natural antibody fused to the hinge region and CH3 domain of an immunoglobulin molecule, such as disclosed in U.S. Patent No. 5,837,821.

[0146] In alternative embodiments, the engineered antibody may comprise a non-immunoglobulin derived protein framework. For example, reference may be made to (Ku & Schutz, 1995, Proc. Natl. Acad. Sci. USA 92: 6552-6556), which discloses a four-helix bundle protein cytochrome b562 with two loops randomized to generate CDRs, which has been selected for antigen binding.

[0147] There are a large number of non-antibody recognition proteins or protein domain scaffolds that can be used as antigen binding domains in the constructs of the present invention. These include scaffolds based on: cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) (Evibody; US7,166,697); human transferrin (Trans-body); a three-helix bundle from the Z-domain of protein A (Affibody); monomeric or trimeric human C-type lectin domain (Tetranectin); the tenth human fibronectin type III domain (AdNectin); the Kunitz-type domain of human or bovine trypsin inhibitor; insect defensin A (IICA29), APPI (Kuntiz domain); lipocalins, FABPs, Bilin-binding proteins, Apoloproptein D (Anticalins); human α-crystallin or ubiquitin molecules (Affilin); trypsin inhibitor II (microbody); α2p8 or ankyrin repeats (repeat motif proteins), charybdotoxin (scorpion toxin), Min-23, cellulose binding domains (Knottins); neocarcinomastatin, CBM4-2 and Tendamistat.

[0148] In addition to the scaffolds provided by antibody-derived domains or non-antibody folds as described above, naturally occurring ligand-binding proteins or protein domains can be used as ligand-binding domains in the present invention. For example, protein domains with ligand-binding properties include the extracellular domains of receptors, PDZ modules of signaling proteins such as Ras-binding protein AF-6, adhesion molecules, and enzymes.

[0149] In some embodiments, the present invention relates to an antibody or a polypeptide comprising the antibody of claim 1 , wherein the antibody or polypeptide comprises a polypeptide comprising ...

[0150] Examples of glycoengineering using the method described in Shinkawa T. et al., 2003 (J Biol Chem 278: 3466-73) method.

[0151] Many methods for affinity maturation of antibodies are known in the art. Many of these are based on the general strategy of generating a panel or library of variant proteins by mutagenesis, followed by selection and / or screening to improve affinity. Mutagenesis is typically performed at the DNA level, for example by error-prone PCR (Thie, Voedisch et al., 2009, Methods Mol Biol 525: 309-322), by gene shuffling (Kolkman and Stemmer 2001, Nat Biotechnol. May; 19(5): 423-8), by using mutagenic chemicals or irradiation, by using "variant" strains with error-prone replication mechanisms (Greener 1996, In Vitro Mutagenesis Protocols. Humana press, NJ) or by somatic hypermutation methods that exploit affinity maturation mechanisms (Peled, Kuang et al., 2008, Annu Rev Immunol. 26: 481-511). Mutagenesis can also be performed at the RNA level, for example by using Qβ replicase (Kopsidas, Roberts et al., 2006, Immunol Lett. 2006 Nov 15; 107(2): 163-8). Library-based methods that allow screening for improved variant proteins can be based on various display technologies, such as phage, yeast, ribosomes, bacteria or mammalian cells, and are well known in the art (Benhar 2007, Expert Opin Biol Ther. May; 7(5): 763-79). Affinity maturation can be achieved by more targeted / predictive methods, such as by site-directed mutagenesis or discovery-guided gene synthesis based on 3D protein modeling (see, for example, Queen, Schneider et al. 1989, PNAS, 86(24): 10029-33 or U.S. Pat. No. 6,180,370 or U.S. Pat. No. 5,225,539).

[0152] Methods of increasing ADCC have been described by: Ferrara, Brunker et al. 2006, Biotechnol Bioeng;93:851-61; Li, Sethuraman et al. 2006, Nat Biotechnol;24:210-5; Stavenhagen, Gorlatov et al. 2007, Cancer Res;67:8882-90; Shields, Namenuk et al. 2001, J Biol Chem;276:6591-604; Shinkawa, Nakamura et al. 2003, J Biol Chem;278:3466-73 and WO 2008 / 006554.

[0153] Methods for increasing CDC have been described by: Idusogie, Wong et al. 2001, J Immunol; 176:346-56; Dall'Acqua, Cook et al. 2006, J Biol Chem; 281:23514-24; Michaelsen, Aase et al. 1990, Scand J Immunol; 32:517-28; Brekke, Bremnes et al. 1993, Mol Immunol; 30:1419-25; Tan, Shopes et al. 1990, PNAS; 87:162-6; and Norderhaug, Brekke et al. 1991, Eur J Immunol; 21:2379-84.

[0154] References describing methods for increasing ADCC and CDC include Natsume, In et al. 2008, Cancer Res; 68:3863-72. The disclosures of each of these references are incorporated herein by cross-reference. In certain embodiments, it may be advantageous to reduce or eliminate the ADCC and CDC activities of the antibody component of the polypeptide of the present invention, such that IFNα2b activity is the primary activity of the polypeptide that regulates target cell survival.

[0155] Many methods for regulating antibody serum half-life and biodistribution are based on modifying the interaction between the antibody and the neonatal Fc receptor (FcRn), which has a key role in protecting IgG from catabolism and maintaining high serum antibody concentrations. Dall'Acqua et al. have described replacements in the Fc region of IgG1 that enhance binding affinity to FcRn, thereby increasing serum half-life (Dall'Acqua, Woods et al. 2002, J Immunol; 169: 5171-80), and further demonstrated enhanced bioavailability and regulation of ADCC activity with triple substitutions of M252Y / S254T / T256E (residue numbering according to EU Index) or M265Y / S267T / T269 (residue numbering according to the Kabat numbering system) (Dall'Acqua, Kiener et al. 2006, J Biol Chem; 279: 6213-6). See also U.S. Patent Nos. 6,277,375; 6821505; and 7,083,784. Hinton et al. have described constant domain amino acid substitutions at positions 250 and 428 that confer increased in vivo half-life (Hinton, Johlfs et al., 2004, J Biol Chem; 279: 6213-6; Hinton, Xiong et al., 2006, J Immunol; 176: 346-56). See also U.S. Patent No. 7,217,797. Petkova et al. have described constant domain amino acid substitutions at positions 307, 380, and 434 that confer increased in vivo half-life (Petkova, Akilesh et al., 2006, Int Immunol; 18: 1759-69). See also Shields et al., 2001, J Biol Chem; 276: 6591-604 and WO 2000 / 42072. Other examples of constant domain amino acid substitutions that modulate binding to Fc receptors and subsequent functions mediated by these receptors, including FcRn binding and serum half-life, are described in U.S. Patent Application Nos. 20090142340; 20090068175 and 20090092599. "S228P," numbered according to the EU Index as in Kabat, is also referred to herein as "S241P" according to Kabat et al. (1987 Sequences of proteins of immunological interest. United States Department of Health and Human Services, Washington DC.). This substitution stabilizes the hinge region of the IgG4 molecule, which has the effect of making the core sequence of the hinge region identical to that of antibodies of the IgG1 or IgG2 isotype.This results in a reduction in the spontaneous dissociation and reassociation of heavy chains, which normally leads to the production of heterodimeric IgG4 antibodies.

[0156] The glycans attached to the antibody molecule are known to affect the interaction of the antibody with Fc receptors and glycan receptors, thereby affecting the antibody activity, including serum half-life (Kaneko, Nimmerjahn et al. 2006, Science; 313: 670-3; Jones, Papac et al. 2007, Glycobiology; 17: 529-40; and Kanda, Yamada et al. 2007, Glycobiology; 17: 104-18). Therefore, certain glycoforms that modulate the activity of the desired antibody can confer therapeutic advantages. Methods for producing engineered glycoforms are known in the art and include, but are not limited to, those described in U.S. Patent Nos. US6,602,684; US7,326,681; US7,388,081 and WO 2008 / 006554.

[0157] Half-life extension by the addition of polyethylene glycol (PEG) has been widely used to extend the serum half-life of proteins as reviewed by Fishburn 2008, J Pharm Sci; 97:4167-83.

[0158] As will be appreciated, conservative amino acid substitutions can be made within the sequences of the present invention. "Conservative substitutions" refer to amino acids with similar properties. As used herein, the following groups of amino acids are considered conservative substitutions: H, R, and K; D, E, N, and Q; V, I, and L; C and M; S, T, P, A, and G; and F, Y, and W. However, it is not intended to make substitutions at sites of attenuation and / or glycosylation other than those specifically recited.

[0159] As used herein, the term "cell surface-associated antigen" refers broadly to any antigen expressed on the surface of a cell, including but not limited to a malignant cell or an infectious or foreign cell.

[0160] In certain aspects of the invention, the fusion polypeptide constructs or compositions of the invention can be used to treat patients with cancer. Cancers considered herein include: a group of diseases and conditions characterized by uncontrolled cell growth (e.g., tumor formation) without distinguishing these cells as specialized and distinct cells. These diseases and conditions include ABL1 proto-oncogene, AIDS-related cancers, acoustic neuroma, acute lymphocytic leukemia, acute myeloid leukemia, adenoid cystic carcinoma, adrenocortical carcinoma, carcinogenic myeloid transformation, alopecia, alveolar soft part sarcoma, anal cancer, angiosarcoma, aplastic anemia, astrocytoma, ataxia telangiectasia, basal cell carcinoma (skin), bladder cancer, bone cancer, intestinal cancer, brain stem glioma, brain and CNS tumors, breast cancer, carcinoid tumors, cervical cancer, childhood brain tumors, childhood cancers, childhood leukemia, childhood soft tissue sarcoma, chondrosarcoma, choriocarcinoma, chronic lymphocytic leukemia, chronic bone marrow Leukemia, colorectal cancer, cutaneous T-cell lymphoma, dermatofibrosarcoma protuberans, desmoplastic small round cell tumor, ductal carcinoma, endocrine cancer, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, extrahepatic bile duct cancer, eye cancer: melanoma, retinoblastoma, fallopian tube cancer, Fanconi anemia, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal cancer, gastrointestinal carcinoid tumors, genitourinary cancer, germ cell tumors, gestational trophoblastic disease, glioma, gynecologic cancer, hematologic malignancies, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, hereditary breast cancer, histiocytosis, Hodgkin's disease, human papillomavirus, hydatidiform mole, hypercalcemia , hypopharyngeal cancer, intraocular melanoma, islet cell carcinoma, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leiomyosarcoma, leukemia, Ley-Fraumeni syndrome, lip cancer, liposarcoma, liver cancer, lung cancer, lymphedema, lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, male breast cancer, malignant rhabdoid tumor of kidney, medulloblastoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic cancer, oral cancer, multiple endocrine neoplasia, mycosis fungoides, myelodysplastic syndrome, multiple myeloma, myeloproliferative disorders disease, nasal cancer, nasopharyngeal cancer, Wilms tumor, neuroblastoma, neurofibromatosis, Nijmegen breakage syndrome, non-melanoma skin cancer, non-small cell lung cancer (NSCLC), eye cancer, esophageal cancer, oral cancer, oropharyngeal cancer, osteosarcoma, anastomosis ovarian cancer, pancreatic cancer, paranasal cancer, parathyroid cancer, parotid cancer, penile cancer, peripheral neuroectodermal tumor, pituitary cancer, polycythemia vera, prostate cancer, rare cancers and related diseases, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, Rossman-Thomson syndrome, salivary gland cancer, sarcoma, schwannoma, Sézary syndrome, skin cancer, small cell lung cancer (SCLC), small intestine cancer, soft tissue sarcoma,Spinal cord tumors, squamous cell carcinoma (skin), gastric cancer, synovial sarcoma, testicular cancer, thymic cancer, thyroid cancer, transitional cell carcinoma (bladder), transitional cell carcinoma (kidney-renal pelvis- / -ureter), trophoblastic carcinoma, urethral cancer, urinary system cancer, urothelial carcinoma, uterine sarcoma, uterine cancer, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia and Wilms' tumor. In one embodiment, the tumor is selected from multiple myeloma or non-Hodgkin's lymphoma.

[0161] As intended for the treatment of cancer, the antibody portion of the fusion construct of the present invention can bind to a tumor-associated antigen, i.e., a cell surface antigen that is selectively expressed by cancer cells or overexpressed in cancer cells relative to the majority of normal cells. Many tumor-associated antigens (TAAs) are known in the art. Non-limiting examples of TAAs include the enzyme tyrosinase; the melanoma antigen GM2; alpha-fetoprotein (AFP); carcinoembryonic antigen (CEA); mucin 1 (MUC1); human epidermal growth factor receptor (Her2 / Neu); T-cell leukemia / lymphoma 1 (TCL1) oncoprotein. Exemplary TAAs associated with many different cancers are telomerase (hTERT); prostate-specific membrane antigen (PSMA); urokinase plasminogen activator and its receptor (uPA / uPAR); vascular endothelial growth factor and its receptor (VEGF / VEGFR); extracellular matrix metalloproteinase inducer (EMMPRIN / CD147); epidermal growth factor (EGFR); platelet-derived growth factor and its receptor (PDGF / PDGFR) and c-kit (CD117).

[0162] A list of other TAAs is provided in US 2010 / 0297076, the disclosure of which is incorporated herein by reference. Of particular interest are cell surface antigens associated with multiple myeloma leukemia or lymphoma cells, including but not limited to CD38, CD138, CD79, CS1, and HM1.24. In one embodiment, the antigen of the ligand-attenuated IFN construct, such as the antibody-attenuated interferon construct, is CD38.

[0163] CD38 is a 46 kDa type II transmembrane glycoprotein. It has a short N-terminal cytoplasmic tail of 20 amino acids, a single transmembrane helix, and a long extracellular domain of 256 amino acids (Bergsagel, P., Blood; 85:436, 1995 and Liu, Q., Structure, 13:1331, 2005). It is expressed on the surface of many immune cells, including CD4 and CD8 positive T cells, B cells, NK cells, monocytes, plasma cells, and a considerable proportion of normal bone marrow precursor cells (Malavasi, F., Hum. Immunol. 9:9, 1984). However, in lymphocytes, expression appears to depend on the differentiation and activation state of the cell. Resting T and B cells are negative, while immature and activated lymphocytes are mainly positive for CD38 expression (Funaro, A., J. Immunol. 145:2390, 1990). Additional studies have shown mRNA expression in non-hematopoietic organs such as pancreas, brain, spleen, and liver (Koguma, T., Biochim. Biophys. Acta 1223:160, 1994.)

[0164] CD38 is a multifunctional extracellular enzyme involved in transmembrane signaling and cell adhesion. It is also known as cyclic ADP-ribose hydrolase because it can convert NAD into ADP-ribose depending on the extracellular pH. + and NADP + Converted into cADPR, ADPR and NAADP. These products induce intracellular Ca 2+ Mobilization, which may lead to tyrosine phosphorylation and activation of cells. CD38 is also a receptor that can interact with its ligand CD31. Activation of the receptor by CD31 leads to intracellular events, including Ca 2+ Mobilization, cell activation, proliferation, differentiation, and migration (reviewed in Deaglio, S., Trends in Mol. Med. 14:210, 2008)

[0165] CD38 is expressed at high levels in multiple myeloma cells, in most cases in T- and B-lineage acute lymphoblastic leukemias, some acute myeloid leukemias, follicle-centered cell lymphomas, and T-lymphocytic lymphomas. (Malavasi, F., J. Clin Lab Res. 22:73, 1992). Recently, CD38 expression has become a reliable prognostic marker in B-lineage chronic lymphocytic leukemia (B-CLL) (Ibrahim, S., Blood. 98:181, 2001 and Durig, J., Leuk. Res. 25:927, 2002). Independent groups have demonstrated that CD38 expression in multiple myeloma cells is associated with a high prognostic value. +Patients with clonal B-CLL are characterized by an unfavorable clinical course with more advanced disease, poor response to chemotherapy and shorter survival (Morabito, F., Haematologica. 87:217, 2002).The consistent and enhanced expression of CD38 on lymphoid tumors makes it an attractive target for therapeutic antibody technology.

[0166] Preferred antigens for use in developing antibody-attenuated, deglycosylated IFNα2b fusion protein constructs targeting cancer are antigens that show selective or greater expression on cancer cells in vivo compared to most other non-transformed cells. Non-protein examples of such antigens include sphingolipids, ganglioside GD2 (Saleh et al., 1993, J. Immunol., 151, 3390-3398), ganglioside GD3 (Shitara et al., 1993, Cancer Immunol. Immunother. 36: 373-380), ganglioside GM2 (Livingston et al., 1994, J. Clin. Oncol. 12: 1036-1044), ganglioside GM3 (Hoon et al., 1993, Cancer Res. 53: 5244-5250), and Lewis antigens that can be displayed on proteins or glycolipids. x , lewis y and Lewis xyCarbohydrate antigens. Examples of protein antigens are HER-2 / neu, human papillomavirus-E6 or -E7, MUC-1; KS 1 / 4 pan-cancer antigen (Perez and Walker, 1990, J. Immunol. 142:3662-3667; Bumal, 1988, Hybridoma 7(4):407-415); ovarian cancer antigen CA125 (Yu et al., 1991, Cancer Res. 51(2):468-475); prostatic acid phosphate (Tailor et al., 1990, Nucl. Acids Res. 18(16):4928); prostate-specific antigen (Henttu and Vihko, 1989, Biochem. Biophys. Res. Comm. 160(2):903-910; Israeli et al., 1993, Cancer Res. 53: 227-230); melanoma-associated antigen p97 (Estin et al., 1989, J. Natl. Cancer Instit. 81(6): 445-446); melanoma antigen gp75 (Vijayasardahl et al., 1990, J. Exp. Med. 171(4): 1375-1380); prostate-specific membrane antigen; carcinoembryonic antigen (CEA) (Foon et al., 1994, Proc. Am. Soc. Clin. Oncol. 13: 294), MUC16 (antibodies include MJ-170, MJ-171, MJ-172 and MJ-173 [US 7,202,346], 3A5 [US 7,723,485]), NMB (US 8,039,593), malignant human lymphocyte antigen-APO-1 (Bernhard et al., 1989, Science 245:301-304); high molecular weight melanoma antigen (HMW-MAA) (Natali et al., 1987, Cancer 59:55-63; Mittelman et al., 1990, J. Clin. Invest. 86:2136-2144); Burkitt lymphoma antigen-38.13; CD19 (Ghetie et al., 1994, Blood 83:1329-1336); human B lymphoma antigen-CD20 (Reff et al., 1994, Blood 83:435-445); GICA 19-9 (Herlyn et al., 1982, J. Clin. Immunol. 2: 135), CTA-1 and LEA; CD33 (Sgouros et al., 1993, J. Nucl. Med.oncofetal antigens such as alpha-fetoprotein of hepatocarcinoma or carcinoembryonic antigen of bladder tumor (Hellstrom et al., 1985, Cancer. Res. 45:2210-2188); differentiation antigens such as human lung cancer antigens L6 or L20 (Hellstrom et al., 1986, Cancer Res. 46:3917-3923); fibrosarcoma antigens; human leukemia T cell antigen-Gp37 (Bhattacharya-Chatterjee et al., 1988, J. Immunol. 141:1398-1403); tumor-specific transplantable type cell surface antigens (TSTA), such as virus-induced tumor antigens, including T-type antigens, envelope antigens of DNA tumor viruses and RNA tumor viruses; pseudoglycoproteins, breast cancer antigens such as EGFR (epidermal growth factor receptor), polymorphic epithelial mucin (PEM) (Hilkens et al., 1992, Trends in Bio. Chem. Sci. 17:359); polymorphic epithelial mucin antigen; human milk fat globule antigen; colorectal tumor-associated antigens such as TAG-72 (Yokata et al., 1992, Cancer Res. 52:3402-3408), CO 17-1A (Ragnhammar et al., 1993, Int. J. Cancer 53:751-758); differentiation antigens found in gastric adenocarcinoma (Feizi, 1985, Nature 314:53-57) such as I (Ma), SSEA-1 found in bone marrow cells, VEP8, VEP9, My1, VIM-D5, M18 and M39 found in mammary epithelium, cancer D. 156-22 , TRA-1-85 (blood type H), C14 found in colon adenocarcinoma, F3 found in lung adenocarcinoma, AH6 found in gastric cancer, Y hapten found in embryonic carcinoma cells, TL5 (blood type A), E1 series (blood type B) antigens found in pancreatic cancer, FC10.2 found in embryonic carcinoma cells, gastric adenocarcinoma antigen, CO-514 (blood type Le a ), NS-10 found in adenocarcinoma, CO-43 (blood type Le b ), G49 found in A431 cells, 19.9 found in colon cancer; gastric cancer mucin; R found in melanoma 24 MH2 (blood type ALe) found in colon adenocarcinoma b / Le y ), 4.2, D1.1, OFA-1, G found in embryonic carcinoma cells M2, OFA-2 and M1:22:25:8, and SSEA-3 and SSEA-4. HMW-MAA (SEQ ID NO: 433) (also known as melanoma chondroitin sulfate proteoglycan) is a 2322-residue membrane-bound protein that is overexpressed in more than 90% of surgically removed benign nevi and melanoma lesions (Camploi et al., Crit Rev Immunol.; 24:267, 2004). Therefore, it may be a potential target cell surface-associated antigen.

[0167] Other exemplary cancer antigens for targeting with the fusion protein constructs of the present invention include (exemplary cancers are shown in parentheses): CD5 (T cell leukemia / lymphoma), CA15-3 (carcinoma), CA19-9 (carcinoma), L6 (carcinoma), CA 242 (colorectal cancer), placental alkaline phosphatase (cancer), prostatic acid phosphatase (prostate cancer), MAGE-1 (cancer), MAGE-2 (cancer), MAGE-3 (cancer), MAGE-4 (cancer), transferrin receptor (cancer), p97 (melanoma), MUC1 (breast cancer), MART1 (melanoma), CD20 (non-Hodgkin's lymphoma), CD52 (leukemia), CD33 (leukemia), human chorionic gonadotropin (cancer), CD38 (multiple myeloma), CD21 (B-cell lymphoma), CD22 (lymphoma), CD25 (B-cell lymphoma), CD37 (B-cell lymphoma), leukemia), CD45 (acute myeloid leukemia), HLA-DR (B-cell lymphoma), IL-2 receptor (T-cell leukemia and lymphoma), CD40 (lymphoma), CD79 (B-cell leukemia or lymphoma, Hodgkin lymphoma), various mucins (carcinoma), P21 (carcinoma), MPG (melanoma), Ep-CAM (epithelial tumors), folate receptor alpha (ovarian cancer), A33 (colorectal cancer), G250 (renal cancer), ferritin (Hodgkin lymphoma), de2-7 EGFR (glioblastoma, breast and lung cancer), fibroblast activation protein (epithelial), tenascin metalloproteinase (glioblastoma). Some particularly useful antibodies include, but are not limited to, BR64 (Trail et al., 1997, Cancer Research 57:100-105), BR96 mAb (Trail et al., 1993, Science 261:212-215), mAbs against the CD40 antigen, such as S2C6 mAb (Francisco et al., 2000, Cancer Res. 60:3225-3231), or other anti-CD40 antibodies, such as those disclosed in U.S. Patent Publication Nos. 2003-0211100 and 2002-0142358; mAbs against the CD30 antigen, such as AC10 (Bowen et al., 1993, J. Immunol. 151:5896-5906; Wahl et al., 2002, Cancer Res. 151:5906-5910); Res. 62(13):3736-42) or MDX-0060 (U.S. Patent Publication No. 2004-0006215) and mAbs against CD70 antigen, such as 1F6 mAb and 2F2 mAb (see, e.g., U.S. Patent Publication No. 2006-0083736) or antibodies 2H5, 10B4, 8B5, 18E7, 69A7 (U.S. Pat. No. 8,124,738).Other antibodies have been reviewed elsewhere (Franke et al., 2000, Cancer Biother. Radiopharm. 15:459 76; Murray, 2000, Semin. Oncol. 27:64 70; Breitling, F., and Dubel, S., Recombinant Antibodies, John Wiley, and Sons, New York, 1998).

[0168] In certain embodiments, useful antibodies can bind to receptors or receptor complexes expressed on target cells. The receptors or receptor complexes can include members of the immunoglobulin gene superfamily, major histocompatibility proteins, cytokine receptors, TNF receptor superfamily members, chemokine receptors, integrins, lectins, complement control proteins, growth factor receptors, hormone receptors, or neurotransmitter receptors. Non-limiting examples of suitable immunoglobulin superfamily members are CD2, CD3, CD4, CD8, CD19, CD22, CD79, CD90, CD152 / CTLA-4, PD-1, B7-H4, B7-H3, and ICOS. Non-limiting examples of suitable TNF receptor superfamily members are TACI, BCMA, CD27, CD40, CD95 / Fas, CD134 / OX40, CD137 / 4-1BB, TNFR1, TNFR2, RANK, osteoprotegerin, APO 3, Apo2 / TRAIL R1, TRAIL R2, TRAIL R3, and TRAIL R4. Non-limiting examples of suitable integrins are CD11a, CD11b, CD11c, CD18, CD29, CD41, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD103, and CD104. Non-limiting examples of suitable lectins are S-type, C-type, and I-type lectins. Examples of CEA antibodies are shown in Table 1.

[0169] Table 1

[0170]

[0171] Antibodies that bind to the CD22 antigen expressed on human B cells include, for example, HD6, RFB4, UV22-2, To15, 4KB128, and humanized anti-CD22 antibody (hLL2) (see, for example, Li et al. (1989) Cell. Immunol. 111:85-99; Mason et al. (1987) Blood 69:836-40; Behr et al. (1999) Clin. Cancer Res. 5:3304s-3314s; Bonardi et al. (1993) Cancer Res. 53:3015-3021).

[0172] Antibodies to CD33 include, for example, HuM195 (see, eg, Kossman et al. (1999) Clin. Cancer Res. 5:2748-2755; US5693761) and CMA-676 (see, eg, Sievers et al. (1999) Blood 93:3678-3684).

[0173] Exemplary anti-MUC-1 antibodies include, but are not limited to, Mc5 (see, e.g., Peterson et al. (1997) Cancer Res. 57: 1103-1108; Ozzello et al. (1993) Breast Cancer Res. Treat. 25: 265-276) and hCTMO1 (see, e.g., Van Hof et al. (1996) Cancer Res. 56: 5179-5185).

[0174] Exemplary anti-TAG-72 antibodies include, but are not limited to, CC49 (see, e.g., Pavlinkova et al. (1999) Clin. Cancer Res. 5:2613-2619), B72.3 (see, e.g., Divgi et al. (1994) Nucl. Med. Biol. 21:9-15), and those disclosed in U.S. Pat. No. 5,976,531.

[0175] Exemplary anti-HM1.24 antibodies include, but are not limited to, mouse monoclonal anti-HM1.24 and humanized anti-HM1.24 IgG1κ antibodies (see, eg, Ono et al. (1999) Mol. Immuno. 36:387-395).

[0176] In certain embodiments, the targeting moiety comprises an anti-Her2 antibody. The erBB 2 gene, more commonly known as (Her-2 / neu), is an oncogene that encodes a transmembrane receptor. Several antibodies against Her-2 / neu have been developed, some of which have been used clinically. These include trastuzumab (e.g., HERCEPTIN TM; Fornir et al. (1999) Oncology (Huntingt) 13:647-58), TAB-250 (Rosenblum et al. (1999) Clin. Cancer Res. 5:865-874), BACH-250 (supra), TA1 (Maier et al. (1991) Cancer Res. 51:5361-5369), and mAbs described in U.S. Pat. Nos. 5,772,997; 5,770,195 (mAb 4D5; ATCC CRL 10463); and U.S. Pat. No. 5,677,171.

[0177] Other fully human anti-Her2 / neu antibodies are well known to those skilled in the art. Such antibodies include, but are not limited to, C6 antibodies, such as C6.5, DPL5, G98A, C6MH3-B1, B1D2, C6VLB, C6VLD, C6VLE, C6VLF, C6MH3-D7, C6MH3-D6, C6MH3-D5, C6MH3-D3, C6MH3-D2, C6MH3-D1, C6MH3-C4, C6MH3-C3, C6MH3-B9, C6MH3-B5, C6MH3-B48, C6MH3-B47, C6 MH3-B46,C6MH3-B43,C6MH3-B41,C6MH3-B39,C6MH3-B34,C6MH3-B33,C6MH3-B31,C6MH3-B27,C6MH3-B25,C6M H3-B21, C6MH3-B20, C6MH3-B2, C6MH3-B16, C6MH3-B15, C6MH3-B11, C6MH3-B1, C6MH3-A3, C6MH3-A2, and C6ML3-9. These and other anti-HER2 / neu antibodies are described in US Patent Nos. 6,512,097 and 5,977,322, in PCT Publication No. WO 97 / 00271, in Schier et al. (1996) J Mol Biol 255:28-43, Schier et al. (1996) J Mol Biol 263:551-567, and others.

[0178] More generally, antibodies to various members of the epidermal growth factor receptor family are well suited for use as targeting antibodies or antigen-binding portions thereof in the constructs of the present invention. Such antibodies include, but are not limited to, anti-EGFR antibodies, as described in U.S. Patent Nos. 5,844,093 and 5,558,864 and European Patent No. 706,799A. Other illustrative anti-EGFR family antibodies include, but are not limited to, antibodies such as C6.5, C6ML3-9, C6MH3-B1, C6-B1D2, F5, HER3.A5, HER3.F4, HER3.H1, HER3.H3, HER3.E12, HER3.B12, EGFR.E12, EGFR.C10, EGFR.B11, EGFR.E8, HER4.B4, HER4.G4, HER4.F4, HER4.A8, HER4.B6, HER4.D4, HER4.D7, HER4.D11, HER4.D12, HER4.E3, HER4.E7, HER4.F8, and HER4.C7, among others (see, e.g., U.S. Patent Publications US 2006 / 0099205A1 and US 2004 / 0071696A1, which are incorporated herein by reference).

[0179] CD38 is particularly interesting as an antibody target for the fusion protein constructs of the present invention. Antibodies to CD38 include, for example, AT13 / 5 (see, for example, Ellis et al. (1995) J. Immunol. 155:925-937), HB7, and the like.

[0180] The present invention also provides compositions comprising the fusion polypeptides of the present invention. These compositions may further comprise any suitable adjuvant, such as, but not limited to, at least one of a diluent, a binder, a stabilizer, a buffer, a salt, a lipophilic solvent, a preservative, an adjuvant, and the like. Pharmaceutically acceptable adjuvants are preferred. Non-limiting examples and methods for preparing such sterile solutions are well known in the art, such as, but not limited to, Gennaro, Ed., Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co. (Easton, Pa.) 1990. A pharmaceutically acceptable carrier can be conventionally selected that is suitable for the mode of administration, solubility, and / or stability of the antibody composition as known in the art or as described herein.

[0181] Pharmaceutical excipients and additives that can be used in the compositions of the present invention include, but are not limited to, proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatized sugars such as sugar alcohols, aldonic acids, esterified sugars, and the like; and polysaccharides or sugar polymers), which may be present alone or in combination, comprising 1-99.99% weight or volume alone or in combination. Exemplary protein excipients include serum albumin, such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acids that can also act as buffers include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. A preferred amino acid is histidine. A second preferred amino acid is arginine.

[0182] Carbohydrate excipients suitable for use in the present invention include, for example, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, etc.; disaccharides such as lactose, sucrose, trehalose, cellobiose, etc.; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, etc.; and sugar alcohols such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), inositol, etc. Preferred carbohydrate excipients for use in the present invention are mannitol, trehalose, and raffinose.

[0183] The antibody composition may also include a buffer or pH adjuster; typically, a buffer is a salt prepared from an organic acid or base. Representative buffers include organic acid salts, such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris, tromethamine hydrochloride, phosphate buffer, or amino acid buffer. Preferred buffers for use in the compositions of the present invention are organic acid salts, such as citrate or amino acids.

[0184] Additionally, the compositions of the present invention may include polymeric excipients / additives such as polyvinylpyrrolidone, ficoll (polymerized sugar), dextrins (e.g., cyclodextrins such as 2-hydroxypropyl-β-cyclodextrin), polyethylene glycol, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, surfactants (e.g., polysorbates such as " 20" and " 80”), lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol), and chelating agents (e.g., EDTA).

[0185] These and other known pharmaceutical excipients and / or additives suitable for use in the antibody compositions of the present invention are known in the art and are listed, for example, in "Remington: The Science & Practice of Pharmacy", 19th ed., Williams & Williams, (1995) and "Physician's Desk Reference", 52nd ed., Medical Economics, Montvale, NJ (1998), the disclosures of which are incorporated herein by reference in their entirety. Preferred carrier or excipient materials are carbohydrates (e.g., sugars and sugar alcohols) and buffers (e.g., citrate) or polymeric agents.

[0186] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It should be understood that the present invention includes all such variations and modifications that fall within the spirit and scope of the invention. The present invention also includes all steps, features, compositions and compounds cited or indicated in this specification, either individually or collectively, and any and all combinations of any two or more of said steps or features.

[0187] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any materials and methods similar or equivalent to those herein can be used to practice or test the present invention, the preferred materials and methods are now described. Example

[0188] General approach

[0189] Production of antibody-fusion constructs in HEK-293E cells

[0190] The DNA sequences of the multiple domains in this article fusion polypeptide are provided in the sequence table incorporated into this article. DNA plasmids encoding protein constructs (antibody-attenuated IFNα2b fusion constructs) were prepared using HiSpeed ​​Plasmid Maxi Kit (Qiagen, Valencia, CA) and then transfected into HEK293E cells (CNRC, Montreal, Canada) using commercially available transfection reagents and OptiMEM culture medium (Invitrogen, Carlsbad, CA). These cells were grown in F17 synthetic culture medium supplemented with 0.45% (w / v) D-(+)-glucose (Sigma, Castle Hill, NSW), 25 μg / ml geneticin (Invitrogen, Carlsbad, CA) and 1 × GlutaMAX (Invitrogen, Carlsbad, CA). After expressing for 6 days in an incubator shaken at 5% CO2 and 120 rpm, the culture medium was separated and the protein A Mab Select SuRe was used. TM Affinity purification was performed using agarose beads (GE Healthcare, Piscataway, NJ). The purified protein construct was buffer exchanged into 0.2 M arginine HCl, 25 mM citric acid, 71.5 mM sodium hydroxide, pH 6.0 using a PD Midi-Trap G-25 column (GE Healthcare, Piscataway, NJ) or a HiPrep 26 / 10 desalting column (HiTrap Desalting HiPrep 26 / 10 Desalting). The purified protein construct was then concentrated using a 50 kDa Amicon Ultra centrifugal filter device (Millipore, Billerica, MA), and protein concentration was determined by reading the absorbance at 280 nm.

[0191] Production of antibody-fusion constructs in EXPI293 cells

[0192] DNA plasmids encoding protein constructs (antibody-IFNα2b related constructs) were prepared using the HiSpeed ​​Plasmid Maxi Kit (Qiagen, Valencia, CA) and then transfected into EXPI293 cells (Life Technologies, Carlsbad, CA) grown in EXPI expression medium (Life Technologies, Carlsbad, CA) using the transfection reagent and OptiMEM medium (Invitrogen, Carlsbad, CA) provided in the EXPI293 transfection kit. After 3 days of expression in an incubator with 5% CO2 and 125 rpm shaking, the culture medium was separated and the protein A Mab SelectSuRe was used. TM Affinity purification was performed using agarose beads (GE Healthcare, Piscataway, NJ). The purified protein construct was buffer-exchanged into 0.2 M arginine HCl, pH 6.0 using a PD Midi-Trap G-25 column (GE Healthcare, Piscataway, NJ) or a HiPrep 26 / 10 desalting column (HiTrap Desalting HiPrep 26 / 10 Desalting). The purified protein construct was then concentrated using a 50 kDa Amicon Ultra centrifugal filter device (Millipore, Billerica, MA), and protein concentration was determined by reading the absorbance at 280 nm.

[0193] Production of antibody-fusion constructs in CHO cells

[0194] DNA plasmids encoding protein constructs (antibody-IFNα2b related constructs) were prepared using the HiSpeed ​​Plasmid Maxi Kit (Qiagen, Valencia, CA), and then transfected using commercially available transfection reagents and OptiPro SFM. TM Culture medium (Invitrogen, Carlsbad, CA) was transfected into cells grown in Freestyle TM CHO cells (Lonza) were grown in CHO expression medium (Invitrogen, Carlsbad, CA). After 6 days of expression in an incubator supplied with 10% CO2 and shaking at 120 rpm, the culture medium was separated and the protein A Mab Select SuRe was used. TMAffinity purification was performed using agarose beads (GE Healthcare, Piscataway, NJ). The purified protein construct was buffer-exchanged to 0.2 M arginine.HCl, 25 mM citric acid, 71.5 mM sodium hydroxide, pH 6.0 using a PD Midi-Trap G-25 column (GE Healthcare, Piscataway, NJ) or a HiPrep 26 / 10 desalting column (HiTrap Desalting HiPrep 26 / 10 Desalting). The purified protein construct was then concentrated using a 50 kDa Amicon Ultra centrifugal filter device (Millipore, Billerica, MA), and protein concentration was determined by reading the absorbance at 280 nm.

[0195] Methods for measuring the antigen targeting activity of antibody-IFNα2b fusion protein constructs

[0196] "On target (Daudi) assay": This assay is used to quantify the antiproliferative activity of IFNα2b and antibody-IFNα2b fusion protein constructs on cells displaying antigens targeted by antibodies fused to IFN receptors and IFNα2b. Daudi cells express CD20 and CD38 as cell surface associated antigens, as well as cell surface IFN receptors. The reagent CellTiter- Cat# G7570 measures the viability of Daudi cells. This is a luminescence-based assay that determines the viability of cells in culture based on ATP quantification. The signal intensity is proportional to the number of viable cells in the microtiter plate well. The assay details are as follows:

[0197] Daudi cells (obtained from ATCC, Manassas, VA) were cultured in T75 flasks (TPP, Trasadingen, Switzerland, cat#90076) to a preferred density of 0.5 × 10 cells / mL in RPMI 1640 (Mediatech, Inc., Manassas, VA, cat#10-040-CV) with 10% fetal bovine serum (FBS; Hyclone, Logan, UT cat#SH30070.03). 5 to 0.8×10 5 Cells were collected by centrifugation at 400 g for 5 minutes, the supernatant was decanted, and the cell pellet was resuspended in RPMI 1640 + 10% FBS. The cells were then counted and the density was adjusted to 3.0 × 10 cells / ml in RPMI 1640 + 10% FBS. 5cells / ml. Then, 50 μl of the cell suspension was aliquoted into each well of a 96-well round-bottom tissue culture plate (hereinafter referred to as the "test plate") (TPP, cat#92067). The test article was serially diluted in duplicate in RPMI 1640 + 10% FBS in a separate sterile 96-well plate (hereinafter referred to as the "dilution plate"; Costar, Corning, NY cat#3879). 50 μl / well was then transferred from the dilution plate to the test plate. The test plate was then incubated at 37°C, 5% CO2 for 4 days.

[0198] The mixture of assay buffer and assay substrate provided by the manufacturer (hereinafter referred to as "CellTiterGlo reagent", mixed according to the manufacturer's instructions) is added to the experimental plate with 100 μl / well. The plate is shaken for two minutes. Then, 100 μl / well is transferred from the experimental plate to a 96-well flat-bottomed white opaque plate (hereinafter referred to as "assay plate"; BD Biosciences, Franklin Lakes, NJ cat#35 3296). The contents of the assay plate are then stabilized at room temperature in the dark for 15 minutes. The plate is read on the luminescence measurement channel on a Victor 3V Multilabel Counter (Perkin Elmer, Waltham, MA, model 1420-041), and luminescence is measured. The results are expressed as "relative luminescence units (RLU)".

[0199] Data were analyzed using Prism 5 (Graphpad, San Diego, CA) using nonlinear regression and three-parameter curve fitting to determine the midpoint of the curve (EC50). For each test article, potency relative to free IFNα2b (or some other form of IFN with known potency relative to IFNα2b) was calculated as the ratio of the EC50.

[0200] Those of ordinary skill in the art will appreciate that many other commonly used assays for measuring cell viability can also be used.

[0201] "On-target (ARP) assay" (sometimes referred to herein as "targeted assay"): The multiple myeloma cell line ARP-1 was a gift from Bart Barlogie MD, PhD, Director of the Myeloma Institute at the University of Arkansas Medical Center (Little Rock, AK). This was described in Hardin J. et al. (Interleukin-6 prevents dexamethasone-induced myeloma cell death, Blood; 84:3063, 1994). ARP-1 cells (CD38 +) CD38 targeting antibody-IFN fusion protein constructs were tested. Culture and assay conditions were the same as those outlined above for the Daudi-based assay with the following exceptions: ARP-1 was cultured to 4.0×10 5 to 6.0×10 5 The ARP-1 concentration was adjusted to 1.0×10 cells / ml before the assay. 4 cells / ml.

[0202] Example 1

[0203] Isoelectric point of IFNα2b fusion protein attenuated by anti-CD38 antibody

[0204] Various transiently transfected cells expressing anti-CD38 antibody-attenuated IFNα2b fusion constructs (Table 2) were harvested and purified using Mab Select Sure Protein A columns. Samples were desalted to 200 mM arginine, 25 mM histidine pH 6.5 using HiLoad Superdex 200 columns.

[0205] Table 2: Table of constructs

[0206]

[0207]

[0208]

[0209] Isoelectric focusing gels are used to determine the isoelectric point (pi) of fusion polypeptides and to detect subtle changes in proteins due to post-translational modifications such as phosphorylation and glycosylation.

[0210] Prefabricated IEF gel is arranged in the gel tank to ensure the tight seal between the gel and the buffer. Then 200mL 1x cathode buffer is poured into the inner chamber to ensure that no buffer enters the outer chamber. Then 500mL 1x anode buffer is poured into the outer chamber and fills 3 / 4 of the groove. After sample and ladder are loaded on the gel, electrophoresis is then performed at 100 volts for 1 hour, at 200 volts for 1 hour, and at 500 volts for 1 / 2 hour. After gel electrophoresis is completed, the gel is taken out and fixed in the TCA solution in the glass container for 30 minutes. Immediately use deionized water washing gel 3 times then. The gel is dyed in SimplyBlueSafeStain (Invitrogen Life Technologies) for a full hour and placed in water overnight to decolorize. Use a scanner to scan the final image.

[0211] The O-linked glycosylation site of the IFNα2b portion of the antibody-attenuated interferon fusion construct was removed by replacing interferon threonine 106 (T106) with alanine (shown as T106A), serine (T106S), valine (T106V), glycine (T106G), or glutamic acid (T106E) or by deleting T106 (shown as ΔT106). The effect of these changes on the pI and heterogeneity of the fusion constructs was investigated by comparing constructs with and without O-linked glycosylation on IEF gels.

[0212] In each case, deletion of T106 or replacement of T106 with alanine, serine, valine, glycine, or glutamate reduced the amount of charged species observed on the IEF gel, as evidenced by a decrease in the number of bands in direct comparison with unmodified T106, thus reducing the heterogeneity of the fusion construct ( Figure 9 The reduced heterogeneity of molecules incorporating T106S is consistent with the removal of O-linked glycosylation at residue 106 of IFNα2b, as evidenced by the reduction in charged species on the IEF gel.

[0213] Removal of O-linked glycosylation sites in the attenuated IFN portion of the antibody fusion construct resulted in an increased pI relative to the O-linked glycosylated protein. This trend was consistent regardless of whether the antibody was IgG4, IgG1, IgG1AA (IgG1 L235A, G237A, a reduced effector function version of IgG1), IgG2, or IgG2SS (IgG2 (A330S, P331S)) using the same antibody front end as A10.21. Figure 10 ).

[0214] YTE substitutions (M252Y, S254T, T256E) have been shown to confer increased affinity for FcRn, presumably increasing the half-life of the antibody. Further experiments examined whether substitutions in other parts of the antibody IFN fusion construct would affect the reduced heterogeneity caused by T106 deletion or substitution. The heterogeneity of the glycosylated A10.21 anti-CD38-attenuated IFNα2b fusion construct (YTE, T106T) and the non-glycosylated IFN fusion construct with the YTE substitution (YTE, T106A) was assessed on an IEF gel. Removal of glycosylation from the IFNα2b component of the fusion construct with the YTE mutation reduced heterogeneity ( Figure 11 ).

[0215] Attenuation of IFNα2b is achieved by replacing key amino acid residues responsible for binding to the IFN receptor. A10.21IgG4(S228P) IFN constructs with various attenuating amino acid substitutions in IFNα2b were evaluated, as well as the number of charged species with O-linked glycosylation (T106T) or without O-linked glycosylation (T106A) of the IFNα2b component. The individual amino acid residues arginine-33, arginine-144, and alanine-145 of IFNα2b were replaced one at a time with alanine for residue 33 (R33A), isoleucine for residue 144 (R144I), or lysine, glycine, or glutamine for residue 145 (A145K, A145G, A145Q). When directly compared, the deglycosylated IFN fusion constructs were consistently more heterogeneous than their glycosylated counterparts ( Figure 12 ).

[0216] The reduction in heterogeneity shown was independent of the antibody portion of the construct. Removal of the O-linked glycosylation site in the attenuated IFNα2b portion of an antibody (IgG4 (S228P))-attenuated IFN fusion construct specific for HLA, CD138, and CD38 (directed to different epitopes on CD38 for antibody A10.21-antibody A02.12) also resulted in a decrease in heterogeneity detected by IEF. Figure 13 ).

[0217] Example 2

[0218] Anti-proliferative activity of interferon α2b fusion protein attenuated by antibodies

[0219] The anti-proliferative effect of IFNα2b is composed of direct and indirect activity. Direct activity occurs by inhibiting the growth of cancer cells by cell cycle arrest (Matsui et al., 2003), by apoptosis or differentiation (Matsui et al. 2003) of death receptor-dependent (Crowder et al. 2005) and independent (Otsuki et al. 1998) cell pathways. Luminescent cell viability assay was used to measure the target-specific direct cytotoxicity of the interferon fusion protein weakened by the antibody against target-positive cell lines.

[0220] Anti-CD38-IFN leader sequence sample

[0221] Constructs (Table 3) were stably cloned or transiently transfected and harvested and purified using Mab Select SureProtein A columns. Samples were desalted to 200 mM arginine, 25 mM histidine pH 6.5 using HiLoad Superdex 200 columns.

[0222] Table 3: Table of constructs

[0223]

[0224]

[0225]

[0226] Commercially available IFNα2b

[0227] Will A (commercially available bacterially produced IFNα2b from Schering-Plough) was used as a positive control.

[0228] Antiproliferative activity measurement

[0229] Antiproliferative activity was measured using the "Daudi cell proliferation assay" and "ARP-1 cell proliferation assay" described above. Antiproliferative activity of antibody-attenuated IFNα2b fusion proteins was measured using the ARP-1 cell proliferation assay with an additional cell line, NCI-H929. In some experiments, a dedicated The plates were read using a 96-well microplate luminometer and the CellTiter- The 2.0 reagent replaced the original CellTiter-Glo, neither of which affected the results. The ARP-1 / NCI-H929 or Daudi cell proliferation assay was used to quantify the antiproliferative activity of IFN and antibody-attenuated IFNα2b fusion protein constructs against CD38-displaying cells. Daudi, ARP1, and NCI-H929 cells express CD38 as a cell surface antigen. Assay details are as follows.

[0230] In the ARP-1 / NCI-H929 cell proliferation assay, the reagent CellTiter- Cell viability was measured using the ELISA kit 2.0 (Cat# G9242). This is a luminescence-based assay that determines the viability of cells in culture based on the quantification of ATP. The signal intensity is proportional to the number of viable cells in the microtiter plate wells. Cells (NCI-H929 and ARP-1 from ATCC, Manassas, VA, a gift from Bart Barlogie MD, PhD, Director of the Myeloma Institute at the University of Arkansas Medical Center, Little Rock, AK) were cultured in T75 flasks (TPP, Trasadingen, Switzerland, cat# 90076) to a preferred density of 0.5 × 10 in RPMI 1640 (Mediatech, Inc., Manassas, VA, cat# 10-040-CV) with 10% fetal bovine serum (FBS; Hyclone, Logan, UT cat# SH30070.03). 5 to 0.8×10 5 Cells were collected by centrifugation at 400 × g for 5 minutes, the supernatant was decanted, and the cell pellet was resuspended in RPMI 1640 + 10% FBS. The cells were then counted and the density was adjusted to 3.0 × 10 cells / mL in RPMI 1640 + 10% FBS. 5 cells / mL. 50 μL of cell suspension was inoculated into each well of a 96-well round-bottom tissue culture plate (hereinafter referred to as the "experimental plate") (TPP, cat#92067). The cells were incubated at 4°C for 1 hour before the addition of the test compound. The test article was serially diluted in duplicate in RPMI1640+10% FBS on a separate sterile 96-well plate (hereinafter referred to as the "dilution plate"; Costar, Corning, NY cat#3879). 50 μL / well was transferred from the dilution plate to the experimental plate. The experimental plate was then incubated at 37°C, 5% CO2 for 4 days. The "CellTiter- Reagent 2.0" was added to the assay plate at 100 μL / well. The plate was shaken for two minutes. 100 μL / well was transferred from the assay plate to a 96-well flat-bottom white opaque plate (hereinafter referred to as "assay plate"; BD Biosciences, Franklin 5 Lakes, NJ cat#35 3296). The contents of the assay plate were then allowed to stabilize at room temperature in the dark for 15 minutes. The plate was read on a 96-well microplate luminometer. The results were expressed as "relative luminescence units" (RLU).

[0231] Data were analyzed using Prism 5 (Graphpad, San Diego, CA) and IC50s were determined using nonlinear regression and three-parameter curve fitting.

[0232] The O-linked glycosylation sites of anti-CD38 antibody attenuated interferon fusion constructs were removed by replacing threonine 106 (T106) with alanine (T106A), serine (T106S), valine (T106V), glycine (T106G), or glutamic acid (T106E) or deleting T106 (shown as ΔT106). The effect on cell proliferation was investigated by comparing A10.21 anti-CD38 antibody fused to attenuated IFNα2b with and without O-linked glycosylation. Removal of the O-linked glycosylation sites in the attenuated IFN portion of the antibody fusion construct resulted in increased anti-proliferative activity, as demonstrated by ARP1 ( Figure 1 A) and NCI-H929 cells ( Figure 1 B) shows a lower IC50 (nM) relative to the corresponding O-linked glycosylated fusion proteins, namely A10.21IgG4(S228P))IFN(A145D, T106T) and A10.21IgG1IFN(A145D, T106T). This trend is consistent regardless of the antibody isotype, IgG4 or IgG1.

[0233] Substitution of threonine 106 (T106) for alanine (T106A), serine (T106S), valine (T106V), glycine (T106G), or glutamic acid (T106E) resulted in increased antiproliferative activity as indicated by lower IC50 (nM) relative to O-linked glycosylated fusion proteins. All non-glycosylated constructs showed higher "on-target" potency ( Figure 14 ).

[0234] The effect of removing the O-linked glycosylation site from the weakened IFNα2b component on anti-proliferative activity was examined in a series of anti-CD38 antibody-weakened IFNα2b fusion proteins. The variants (T106A, T106G, T106N, T106F, T106R, T106D, T106E, T106Q, T106H, T106I, T106L, T106K, T106M, T106F, T106P, T106S, T106V, T106Y and T106W) of the anti-CD38 antibody-weakened IFNα2b fusion protein R10A2IgG4(S228P)IFN(A145D) with different amino acid substitutions at T106 to remove the O-linked glycosylation site on the IFN component were examined (Table 4), and the results are shown in Figure 2.

[0235] While the level of potency varies among the different substitutions at the T106 position, all substitutions that result in removal of O-linked glycosylation are expected to increase potency relative to the corresponding O-linked glycosylated protein ( FIG. 2 ).

[0236] Table 4: R10A2 anti-CD38 antibody-attenuated IFN fusion construct variants with various amino acid substitutions at T106 to remove O-linked glycosylation.

[0237]

[0238] In a similar manner, the relative changes in potency between two different anti-CD38 antibody interferon fusion proteins (A02.12 and A10.21) that bind to different epitopes on CD38, with or without O-linked glycosylation (A02.12 IgG4 (S228P) IFN (A145D, T106T), A02.12 IgG4 (S228P) IFN (A145D, T106A), A10.21 IgG4 (S228P) IFN (A145D, T106T), and A10.21 IgG4 (S228P) IFN (A145D, T106A). Removal of the O-linked glycosylation site in the attenuated IFN portion of the antibody (IgG4 (S228P)) fusion constructs with different specificities for anti-CD38 resulted in an increase in anti-proliferative activity, regardless of the target of the antibody portion. Figure 15 ).

[0239] Attenuation of IFNα2b was achieved by various attenuating substitutions. The antiproliferative activity of A10.21IgG4(S228P) IFN constructs with various attenuating substitutions in IFNα2b with (T106T) and without O-linked glycosylation (T106A) was also evaluated (Table 5). Removal of the O-linked glycosylation site in the IFNα2b component of the constructs with various attenuating substitutions was more potent than their glycosylated counterparts ( Figure 16 ).

[0240] Table 5: A10.21 IgG4 (S228P) IFN constructs with various attenuating substitutions in IFNα2b with (T106T) and without O-linked glycosylation (T106A).

[0241]

[0242]

[0243] To investigate whether other attenuated IFNα2b fusion antibodies / proteins with different binding targets modulate anti-proliferative activity as a result of removal of O-linked glycosylation, two constructs (anti-CD138 antibody and anti-HLA antibody) were generated and fused to IFN(A145D) with or without O-linked glycosylation (Table 6) and tested for their anti-proliferative activity.

[0244] Removal of the O-linked glycosylation sites in the attenuated IFN portion of an antibody (IgG4(S228P)) fusion construct with specificity against HLA and CD138 resulted in increased antiproliferative activity, regardless of the target of the antibody portion of the construct. This was demonstrated in an antibody fusion construct directed against HLA and CD138 ( Figure 17 ).

[0245] Table 6: Constructs of various IFN fusion proteins - anti-CD138 antibodies and anti-HLA antibodies fused to IFN (A145D) with or without O-linked glycosylation

[0246]

[0247] Effect of Removal of O-Linked Glycosylation from Fusion Proteins Containing Substitutions in the Fc Region to Extend Half-Life or Reduce Effector Function on Antiproliferative Activity

[0248] YTE substitutions have been shown to confer increased affinity for FcRn, presumably increasing the half-life of the antibody. IFN fusion antibodies containing YTE substitutions were tested for antiproliferative activity in the presence and absence of O-linked glycosylation. The following variants were made from IFN fusion proteins attenuated with the A10.21 anti-CD38 antibody (Table 7).

[0249] The introduction of the YTE substitution to A10.21 IgG4 (S228P) (A145D, T106A) did not affect the increase in potency caused by the attenuated removal of O-linked glycosylation of IFN ( Figure 18 ).

[0250] L235A and G237A in the Fc part of IgG1 variant replace and A330S and P331S in the Fc part of IgG2 variant replace and cause effector function to reduce.In the presence and absence of the glycosylation that O-is connected, the anti-proliferative activity of the IFN fusion containing L235A and G237A replacement IgG1 antibody and the IFN fusion containing A330S and P331S replacement of weakened test.These variants are made (table 7) by the IFN fusion protein that A10.21 anti-CD38 antibody weakens.Removing the glycosylation site that O-is connected in the weakening IFN part of antibody fusion construct causes effectiveness to increase, and no matter the isotype of antibody in antibody construct is IgG1 or IgG1AA (IgG1 (L235A, G237A)), IgG2 or IgG2SS (IgG2 (A330S, P331S)) Figure 19 ).

[0251] Table 7: A10.21 fusion proteins composed of substitutions in the Fc region that extend half-life or reduce effector function

[0252]

[0253]

[0254] Removal of the O-linked glycosylation site from the attenuated interferon portion of the anti-CD38 antibody interferon fusion protein by amino acid substitution or deletion of T106 resulted in increased antiproliferative activity (1.3-12 fold) on target CD38, CD138, or HLA-positive cells.

[0255] Example 3

[0256] Antibody-attenuated on-target and off-target activities of IFNα2b fusion proteins

[0257] iLite using luciferase to generate bioluminescence TM The reporter gene assay is used to quantitatively measure the biological activity (IU / ml) of human interferon alpha (IFNα2b). The cells used in this assay express CD38 and are used to measure the "on-target" activity of the anti-CD38-attenuated IFNα2b fusion protein. When CD38 is blocked with an anti-CD38 antibody that recognizes the same epitope, these cells can also be used to measure "off-target" activity. These assays can be used to determine the selectivity index (SI), which is the selective activity of the anti-CD38 IFN fusion protein against CD38+ target cells and against cells where CD38 is blocked (simulating CD38). - The SI is a measure of the inactivity of an agent against a target cell (i.e., CD38). The larger the SI, the more selective the agent is for the target, while a number close to 1 indicates no selectivity for either target or non-target. Intron A is used as a positive control because it is active against cells expressing the interferon alpha receptor IFNAR1 / 2, but has no selectivity for other cell surface-expressed antigens (i.e., CD38), with an SI of approximately 1.

[0258] Antibody-attenuated IFN constructs

[0259] The sequences of the constructs used are listed in the Sequence Listing and in Table 8 below.

[0260] Table 8

[0261]

[0262]

[0263]

[0264] A was used as a positive control.

[0265] On-target / off-target activity measurement

[0266] Measure on-target and off-target activity using the same method as the "iLite reporter assay" described above.

[0267] The iLite reporter gene assay (PBL Interferon Source, Piscataway, NJ, Cat# 51100) was performed primarily as described by the manufacturer with the addition of a human IgG blocking step. The manufacturer describes the iLite cell line as "a stably transfected cell line derived from a commercially available pro-monocyte human cell line characterized by the expression of MHC class II antigens, specifically human lymphocyte antigens (HLADR) on the cell surface." The cell line expresses CD38 and contains a stably transfected luciferase gene, whose expression is driven by an interferon response element (IRE), which allows quantification of interferon activity based on luminescent output.

[0268] The iLite plates (hereinafter referred to as assay plates) and diluent provided by the manufacturer were taken out of the -80°C freezer and equilibrated to room temperature. 50 μL of diluent was added to each well of the assay plate. The vial of reporter cells provided by the manufacturer was taken out of the -80°C freezer and thawed in a 37°C water bath. 25 μL aliquots of cells were distributed to each well of the assay plate. Next, 12.5 μL of 8 mg / mL human IgG diluted in RPMI 1640+10% FBS (Sigma Chemicals, St. Louis, MO; cat# I4506) was added to each well. The contents were mixed and incubated at 37°C for 15 minutes. On a separate "dilution plate", the test article was serially diluted in RPMI 1640+10% FBS in duplicate. Then, 12.5 μL of the test article was transferred from the dilution plate to the assay plate. The assay plate was then incubated at 37°C, 5% CO2 for 17 hours. Remove the manufacturer's assay buffer and substrate from the -80°C freezer and allow them to equilibrate to room temperature for 2 hours. Add the manufacturer's assay buffer to the manufacturer's substrate vial and mix thoroughly according to the manufacturer's instructions to create a "luminescent solution." Then, add 100 μL of the luminescent solution to each well of the assay plate. Shake the plate for 2 minutes. The plate is then incubated in the dark at room temperature for 5 minutes and read on a Victor 3V Multilabel counter on the photometric channel. Luminescence is measured and expressed as RLU.

[0269] To test the off-target activity of the anti-CD38 antibody-IFN fusion protein construct in the iLite assay, the manufacturer's provided diluent was supplemented with 0.25 mg / mL anti-CD38 antibody (an antibody that recognizes the same epitope on CD38 as the antibody-IFN fusion protein construct being tested to prevent any binding of the anti-CD38 antibody-IFN fusion protein construct to CD38 expressed on iLite cells). This blocking phase was followed by treatment with either anti-CD38 antibody-IFN fusion protein or IFNα2b.

[0270] Prism 5 (Graphpad, San Diego, CA) analysis of data was used, and the midpoint (EC50) of the curve was determined using nonlinear regression and three-parameter curve fitting. Selectivity index (SI) was calculated by EC50 (off-target activity) / EC50 (on-target activity). Selectivity index (SI) is a measure of how selectively active and inactive the anti-CD38 IFN construct is for cells expressing CD38 and for cells without CD38. The larger the number, the more selective it is to the target, and the number close to 1 indicates no selectivity for the target. Intron A is used as a positive control, and its SI is approximately 1.

[0271] Removal of the O-linked glycosylation site of the interferon fusion construct attenuated by anti-CD38 antibodies by replacing threonine 106 (T106) with alanine (shown as T106A). This activity was investigated by comparing A10.21 and A10.43 anti-CD38 antibodies fused to IFNα2b with and without O-linked glycosylation.

[0272] Removal of the O-linked glycosylation site (shown as T106A) in the attenuated IFN portion of the antibody fusion construct resulted in a slight increase in on-target activity relative to the O-linked glycosylated protein ( Figure 3 and Table 9) and slight to no increase in off-target activity. O-linked glycosylated and non-glycosylated proteins showed high selectivity for CD38+ cells, while Intron A showed no selectivity.

[0273] Table 9: EC50 and SI of IFNα2b fusion proteins A10.21 and A10.43 attenuated by anti-CD38 antibodies with and without O-linked glycosylation

[0274]

[0275] The effect of removing O-linked glycosylation sites from attenuated IFN fusion proteins on on-target / off-target activities can also be examined in a similar manner.

[0276] An iLite™ reporter gene on-target and off-target activity assay was performed to demonstrate the selective activity of anti-CD38 attenuated IFN fusion proteins on target CD38+ cells, as well as limited activity on cells when blocking CD38 with an anti-CD38 antibody (recognizing the same epitope) mimicked the expected activity on target cells.

[0277] Removal of the O-linked glycosylation site in the attenuated IFN portion of the antibody fusion construct (via T106A substitution) resulted in a slight increase in on-target activity relative to the O-linked glycosylated protein and a slight to no increase in off-target activity. Both O-linked glycosylated and non-glycosylated anti-CD38 antibody attenuated interferon fusion proteins showed high selectivity for CD38+ cells, while Intron A showed no selectivity.

[0278] In order to further examine the regulation of the selectivity index of the result of the removal of the glycosylation site of O-connection in the weakening IFN part of the antibody fusion construct, in a series of constructs, check on-target activity.The variation examined is based on A10.21IgG4 (S228P) IFN (A145D, T106T) construct.These comprise that the disappearance (Δ T106), T106 of threonine 106 are replaced with serine (T106S), YTE replaces (YTE T106T and YTE T106A), by being replaced with the IFN of glutamine (A145Q) from alanine 145, weakening and changing antibody isotype is IgG1 (IgG1T106T and IgG1T106A).

[0279] Most constructs showed selectivity indices >1, with the magnitude of selectivity for targeting CD38+ cells varying depending on the anti-CD38 IFN fusion construct ( Figure 20 The highest selectivity was observed with A10.21 IgG4(S228P) IFN(A145D, T106A).

[0280] Example 4

[0281] Anti-CD38 antibody-attenuates off-target activity of IFNα2b fusion protein

[0282] By monitoring the activation of the ISGF3 pathway, HEK-Blue TM IFN-α / β cells allow the detection of biologically active human type I IFNs. HEK-Blue is stimulated with human IFN-α TM IFN-α / β cells activate the JAK / STAT / ISGF3 pathway, which subsequently induces the production of SEAP (a reporter gene under the control of the IFN-α / β-inducible ISG54 promoter). The level of SEAP in the supernatant can be measured using QUANTI-Blue TMEasy Assay. Use this reporter gene assay to evaluate the effects of removing O-linked glycosylation from the interferon-attenuating portion of an antibody-interferon fusion protein.

[0283] Generation of antibody / Fc fusion protein constructs

[0284] Transiently transfected cells expressing interferon fusion proteins were harvested and purified using Mab Select SureProtein A columns. Samples were desalted to 200 mM arginine, 25 mM histidine, pH 6.5 using a HiLoad Superdex 200 column.

[0285] Table 10

[0286]

[0287]

[0288]

[0289]

[0290] A was used as a positive control.

[0291] Measurement of off-target activity by HEK-Blue assay

[0292] The off-target activity of antibody-IFN fusions was measured using the same method as in the "HEK-Blue off-target assay," except for the seeded cells, diluted antibodies, and supernatant volumes used. The details of the assay are as follows.

[0293] HEK-Blue off-target assay was used to analyze the TM The ability of antibody-IFN fusion constructs to bind to interferon-α / β receptors (IFNARs) was quantified using the HEK-Blue IFN-α / β cell line (InvivoGen, San Diego, CA). The "off-target (HB-IFN) assay" was performed primarily as described by the manufacturer of the HEK-Blue IFN-α / β cell line. TM IFN-α / β cells are specifically designed to monitor the activation of the JAK-STAT pathway induced by type I IFN. The cells are generated by introducing human STAT2 and IRF9 genes into HEK293 cells to obtain a fully active type I IFN signaling pathway. TMIFN-α / β cells stably express the reporter gene secreted embryonic alkaline phosphatase (SEAP) under the control of the ISG54 promoter. ISG54 is a well-known ISG activated by type I IFN through an ISRE-dependent mechanism. Upon stimulation with IFN-α or IFNβ, HEK-Blue TM IFN-α / β cells activate the JAK-STAT pathway and subsequently express the SEAP reporter gene. SEAP is secreted into the culture medium and can be detected using the colorimetric reagent QUANTI-Blue. TM Quantitative.

[0294] Briefly, HEK-Blue IFN-α / β cells (Invivogen, San Diego CA cat#hkb-ifnab) were thawed and cultured in DMEM medium (Mediatech, Manassas VA, cat#10-013-CV) + 10% heat-inactivated FBS (Hyclone, Logan UT, cat#SH30070.03) (HI FBS). When the cells reached 60-80% confluence, they were lifted using CellStripper (Mediatech, cat#25-056-Cl). The cells were washed twice in DMEM+HI FBS and counted. The cells were adjusted to 2.77×10 5 Viable cells / mL were plated and inoculated into a flat-bottomed 96-well tissue culture plate (hereinafter referred to as the "assay plate") at 180 μL per well. 20 μL of IFNα2b or fusion protein construct diluted in DMEM+HI FBS was then added to each well. The plate was incubated at 37°C, 5% CO2 for 16-24 hours. QUANTI-Blue (Invivogen, cat# rep-qb1) was prepared according to the manufacturer's instructions. QUANTI-Blue (180 μL) was inoculated into each well of a flat-bottomed plate (hereinafter referred to as the "assay plate"). 20 μL of supernatant from each well of the experimental plate was then transferred to the assay plate. The assay plate was then incubated at 37°C for 1-3 hours. The assay plate absorbance at 620 nm was read on a SpectraMaxPlus 384 microplate reader from Molecular Devices. Data were analyzed using Graph Pad Prism.

[0295] The effect of the presence or absence of O-linked glycosylation in IFNα2b on attenuating the off-target activity of an IFN-fusion anti-CD38 antibody was assessed.

[0296] The O-linked glycosylation site of the anti-CD38 antibody with attenuated interferon fusion construct was removed by replacing threonine 106 (T106) with alanine (shown as T106A) or deleting T106 (shown as ΔT106). The impact on off-target activity was investigated by comparing the X10.21 anti-CD38 antibody with and without O-linked glycosylation fused to attenuated IFNα2b.

[0297] Removal of the O-linked glycosylation sites in the attenuated IFN portion of the antibody fusion construct resulted in a slight increase in off-target activity relative to the O-linked glycosylated proteins, A10.21IgG4IFN(145D,T106T) and A10.21IgG1IFN(145D,T106T). Figure 5 This trend was consistent regardless of isotype ( Figure 5 ). However, IFNα2b(A145D) is so attenuated that even at the highest dose used, the level of SEAP induction in this assay is limited. Therefore, the EC50 is only an approximation.

[0298] The off-target activity of antibodies fused to attenuated IFN (R10A2IgG4(S228P)IFN(A145D)) with different amino acid substitutions to remove O-linked glycosylation was examined. All possible amino acid substitutions were tested and the constructs are listed in Table 11.

[0299] Table 11

[0300]

[0301] Each of these substitutions has been shown to exhibit similar or slightly less off-target activity (increased EC) relative to the O-glycosylated IFN fusion antibody R10A2IgG4(S228P)IFN(A145D, T106T). 50 ). However, IFNα2b(A145D) was so attenuated that even at the highest dose used, the level of SEAP induction in this assay was limited. 50 Only approximations.

[0302] The off-target activity of another anti-CD38 antibody attenuated IFNα2b construct and the effect of removal of O-linked glycosylation by T106A substitution were investigated. Compared to A10.21, the heavy chain of A10.43 has 7 amino acid changes, while the two constructs have the same light chain sequence (see sequence listing). The results showed that removal of the O-linked glycosylation site by T106A substitution slightly increased the off-target activity of A10.43. However, IFNα2b (A145D) was so attenuated that the level of SEAP induction in this assay was limited even at the highest dose used. Therefore, EC 50 Only approximate.

[0303] Removal of O-linked glycosylation sites from the attenuated IFN portion of the antibody-interferon fusion construct resulted in slight to no increase in off-target activity and a slight increase in on-target activity relative to the O-linked glycosylated protein. Both O-linked glycosylated and non-glycosylated anti-CD38 antibody-attenuated interferon fusion proteins showed high selectivity for CD38+ cells, while Intron A showed no selectivity.

[0304] Example 5

[0305] Evaluation of Human Neonatal FC Receptor (FCRN) Binding to Anti-CD38-Attenuated IFN

[0306] The polyhistidine-tagged FcRn was fixed to the designated active flow cell of the CM5 sensor chip in the Biacore T200 enhanced instrument using an amine coupling protocol, while the reference flow cell was blank-fixed. A pulse of polyhistidine-tagged FcRn was injected onto the active surface to ensure that the solution was pre-concentrated onto the flow cell. The surface was then washed with 50mM NaOH. Both the reference surface and the active surface were activated with a mixture of 50:50 EDC / NHS for 7 minutes. This was followed by a series of pulses of 2 μg / mL polyhistidine-tagged FcRn in 10mM sodium acetate pH 5.0 only on the active surface. Once the target of 150RU was reached, both surfaces were deactivated for 7 minutes with 1M ethanolamine pH 8.5. This protocol resulted in approximately 150RU of polyhistidine-tagged FcRn being fixed on the active surface. Each active surface was used for one run.

[0307] The test antibodies were desalted in PBS-P running buffer (DPBS, pH 7.4 and 0.005% Tween-20, adjusted to pH 6.0 with HCl at 25° C.) using a Zeba Spin desalting column, 7K MWCO, 0.5 mL (Pierce Product No. 89882). After desalting, the concentration of each sample was adjusted to 1 mg / mL.

[0308] On the day of the assay, the 1 mg / mL solution was further diluted in running buffer to prepare the highest concentration, followed by a 1:2 dilution to generate a concentration series. Test samples were passed over the surface at a flow rate of 50 μL / min. The association phase was 100 seconds, while the dissociation phase was 300 seconds for all concentrations tested. A 60-second injection of 100 mM Tris, 50 mM NaCl, pH 8.0 was used to remove test antibody regeneration activity and the reference surface. Binding constants were determined at 25°C.

[0309] A two-state response model was used to evaluate antibody polyhistidine FcRn binding interactions, in which R max The RI (refractive index) parameter was set to local. All data were reference subtracted twice: first, the signal in the reference cell (blank fixation) resulting from antibody binding to the dextran matrix was subtracted, and second, the signal of 0 nM antibody on the active surface was subtracted.

[0310] Binding constants were determined for at least two separate runs for each antibody. Prolia Lot 1035726 was tested as a positive control for each run along with the anti-CD38 / IFN samples. a1 、k d1 、k a2 、k d2 and K D The average value of .

[0311] The average kinetics and affinity values ​​for FcRn binding were measured for four anti-CD38 / IFNs (A10.21 IgG4 (S228P) IFN (A145D, T106A), A10.21 IgG4 (S228P) IFN (A145D, T106T), A10.43 IgG4 (S228P) IFN (A145D, T106A), A10.43 IgG4 (S228P) IFN (A145D, T106T). In this sample set, clone A10.21 IgG4 (S228P) IFN (A145D, T106A) showed the highest affinity for FcRn.

[0312] Example 6

[0313] Efficacy of anti-CD38 antibodies fused to attenuated interferon α2B with and without O-linked glycosylation in the mouse NCI-H929 multiple myeloma model

[0314] Drugs and treatment

[0315] Table 12

[0316]

[0317]

[0318] #-Control group

[0319] program:

[0320] ·Use 1x10 dissolved in 50% Matrigel 7 H929 tumor cells were placed sc in the flank of CR female CB.17 SCID mice.

[0321] The cell injection volume was 0.2 mL per mouse.

[0322] Age of mice at the start date: 8 to 12 weeks.

[0323] When the tumor reaches 170-350mm 3 The average size of is used for pair matching and processing begins.

[0324] Dosing volume = 0.2 mL / mouse. No adjustment for body weight was performed.

[0325] · Weight: qd x 5 then biwk to the end

[0326] Caliper measurement: biwk to end

[0327] Endpoint TGD. Animals should be monitored individually. The endpoint of the experiment is a tumor volume of 2000 mm 3 Or 60 days, whichever comes first. Responders may be followed for longer periods. When endpoint is reached, animals will be euthanized according to SOP #687.

[0328] The results are shown in Figure 8 Roughly comparable efficacy was observed with and without O-linked glycosylation, with the deglycosylated form being slightly more effective in preventing tumor growth.

[0329] Example 7

[0330] When evaluating the binding of human neonatal Fc receptor (FcRn) to anti-CD38 antibodies with and without O-linked glycosylation attenuating IFN fusion, the protein without O-linked glycosylation showed the highest affinity for FcRn. This effect could be evaluated in cynomolgus monkeys and humanized FcRn mice.

[0331] Cynomolgus monkey studies

[0332] Monkey study design - comparison of + / - O-glyc

[0333] 3 mg / kg as a single intravenous infusion over 1 hour via an indwelling catheter

[0334] –A10.21IgG4(S228P)IFN(145D,T106A)(n=4)

[0335] –A10.21IgG4(S228P)IFN(145D,T106T)(n=4)

[0336] –A10.21IgG4(S228P)IFN(145D,T106A)(n=4)

[0337] –A10.21IgG4(S228P)IFN(145D,T106T)(n=4)

[0338] Comparison of PK and PD (biological effect, i.e. serum neopterin levels)

[0339] PK: All monkeys, ≤1 ml, 11 time points (pre-dose, 0 min (immediately after the end of infusion), 2, 6, 12, 24, 48, 96, 120, 168, and 240 h post-infusion). Samples (80) were analyzed by ELISA.

[0340] TK Modeling: WinNonlin Table Assembly (non-compartmental analysis)

[0341] Clinical Pathology: Hematology and Blood Chemistry - All monkeys, 3 occasions (pretreatment, 24 hours post-dose, and day 8)

[0342] Serum neopterin: All monkeys, approximately 0.5 ml, 3 times at 5 time points (before administration, 12, 24, 96, and 168 hours after administration)

[0343] Pharmacokinetic study of humanized FcRN mice

[0344] 1. On day 1, 32 B6.Cg-Fcgr tm1Dcr Tg(CAG-FCGRT)276Dcr / DcrJ (JAX stock#004919) female mice were injected IP with 1 mg / kg of:

[0345] –A10.21IgG4(S228P)IFN(145D,T106A)(n=8)

[0346] –A10.21IgG4(S228P)IFN(145D,T106T)(n=8)

[0347] –A10.43IgG4(S228P)IFN(145D,T106A)(n=8)

[0348] –A10.43IgG4(S228P)IFN(145D,T106T)(n=8)

[0349] 2. Measure body weight 3 days before administration, on the day of administration, and then weekly.

[0350] 3. Perform cage-side observations daily and clinical observations weekly.

[0351] 4. Pharmacokinetics Blood collection: Mice were bled 3 days before administration and 1, 12, 24, 48, and 72 hours, and 5, 7, 10, and 14 days after administration (25 μl). Mice were divided into two groups (4 mice / group / group).

[0352] 5. All mice were sacrificed on day 14. Terminal cardiac puncture was performed to collect blood.

[0353] 6. Collect blood into lithium heparin tubes and centrifuge at 10,000 rpm for 2 minutes at 4°C.

[0354] 7. Plasma samples were diluted 1:10 in PBS and frozen, then analyzed by binding ELISA for A10.21 IgG4 IFN(145D)A106, A10.21 IgG4 IFN(145D)T106, A10.43 IgG4 IFN(145D)A106, or A10.43 IgG4 IFN(145D)T106.

Claims

1. A fusion polypeptide comprising a first and a second domain, wherein the first domain comprises a polypeptide ligand that binds to a cell surface-associated antigen, and the second domain consists of deglycosylated IFNα2b, wherein the deglycosylated IFNα2b consists of the following sequence: (i) SEQ ID NO: 1, wherein the residue at position 106 is selected from the group consisting of A, D, E, F, G, H, I, K, L, M, P, Q, R, S, V, and Y, and the residue at position 145 is mutated to D, or (ii) SEQ ID NO: 2, wherein the residue at position 144 is mutated to D, or (iii) SEQ ID NO: 1, wherein the residue at position 106 is A, and the residue at position 33 is mutated to A or the residue at position 144 is mutated to I or the residue at position 145 is mutated to K, G or Q.

2. The fusion polypeptide of claim 1, wherein the sequence of the deglycosylated IFNα2b is SEQ ID NO: 1, wherein the residue at position 106 is A.

3. The fusion polypeptide of claim 1 or claim 2, wherein the cell surface associated antigen is selected from CD38, CD138, RANK ligand, HM1.24, CD56, CS1, CD20, CD74, IL-6R, BAFF, BCMA, HLA-SR, HLA-DR, kininogen, β2 microglobulin, FGFR3, ICAM-1, matriptase, CD52, EGFR, GM2, α4 integrin, IFG-1R, KIR, CD3, CD4, CD8, CD24, CD44, CD69, CD71, CD79, CD8 3, CD86, CD96, HLA, PD-1, ICOS, CD33, CD115, CD11c, CD19, CD52, CD14, FSP1, FAP, PDGFRα, PDGFRβ, ASGR1, ASGR2, FSP1, RTI140 / Ti-α, HTI56, VEGF receptor, CD241, CD117, CD71, CD36, CD34, CD45RO, CD45RA, CD115, CD168, CD235, CD236, CD237, CD238, CD239, and CD240.

4. The fusion polypeptide of any one of claims 1 to 3, wherein the polypeptide ligand is an antibody or an antigen-binding portion thereof.

5. The fusion polypeptide of any one of claims 1 to 4, wherein the polypeptide ligand is an antibody that binds CD38.

6. The fusion polypeptide of claim 5, wherein the V H The sequence is selected from any one of SEQ ID Nos: 48-56 and 58, and wherein the V L The sequence is selected from any one of SEQ ID Nos: 81, 82 and 84.

7. The fusion polypeptide of any one of claims 1 to 6, wherein the first domain is linked to the second domain via a peptide bond.

8. The fusion polypeptide of any one of claims 1 to 6, wherein the first domain is directly linked to the second domain via a peptide bond.

9. The fusion polypeptide of any one of claims 1 to 6, wherein the C-terminus of the first domain is linked to the N-terminus of the second domain.

10. A fusion polypeptide comprising: (i) V of SEQ ID NO: 81 L Sequence and any one of SEQ ID NOs: 31, 61-70, 72-74, 83 and 87; (ii) V of SEQ ID NO: 82 L sequence and the sequence of SEQ ID NO: 75; or (iii) the sequence of SEQ ID NO: 84 L sequence and the sequence of SEQ ID NO:

77.

11. V comprising SEQ ID NO: 81 L A fusion polypeptide of the sequence of SEQ ID NO:

87.

12. A composition comprising the fusion polypeptide of any one of claims 1 to 11 and a pharmaceutically acceptable carrier.

13. Use of the fusion polypeptide of any one of claims 1 to 11 or the composition of claim 12 in the preparation of a medicament for treating a tumor in a subject, wherein the first domain of the fusion polypeptide binds to a tumor cell, and wherein the tumor is selected from multiple myeloma or non-Hodgkin's lymphoma.

14. One or more isolated polynucleotides encoding the fusion polypeptide of any one of claims 1 to 11.

15. A vector comprising one or more isolated polynucleotides of claim 14.

16. A transformed cell comprising the vector of claim 15.

17. A method for producing a polypeptide ligand-attenuated IFNα2b fusion polypeptide in a mammalian cell, wherein the polypeptide ligand-attenuated IFNα2b fusion polypeptide has reduced heterogeneity and / or enhanced FcRn binding and / or enhanced target selectivity, the method comprising culturing a recombinant mammalian cell comprising one or more polynucleotides encoding the polypeptide ligand-attenuated IFNα2b fusion polypeptide, wherein the attenuated IFNα2b consists of the sequence: (i) SEQ ID NO: 1, wherein the residue at position 106 is selected from the group consisting of A, D, E, F, G, H, I, K, L, M, P, Q, R, S, V, and Y, and the residue at position 145 is mutated to D, or (ii) SEQ ID NO: 2, wherein the residue at position 144 is mutated to D, or (iii) SEQ ID NO: 1, wherein the residue at position 106 is A, and the residue at position 33 is mutated to A or the residue at position 144 is mutated to I or the residue at position 145 is mutated to K, G or Q.

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