Patent Grant
10981986
This application includes a Sequence Listing submitted electronically as a text file named 35264213—Teva Sequence Listing ST25.txt, created on Dec. 4, 2018, with a size of 1071 KB. The Sequence Listing is incorporated by reference herein.
The present invention relates to polypeptide constructs comprising mutated, attenuated polypeptide ligands attached to antibodies, wherein the antibodies direct the mutated ligands to cells that express on their surfaces the antigens to which said antibodies bind, as well as receptors for said ligands. The invention further relates to methods of treatment involving the use of these polypeptide constructs.
Numerous peptide and polypeptide ligands have been described to function by interacting with a receptor on a cell surface, and thereby stimulating, inhibiting, or otherwise modulating a biological response, usually involving signal transduction pathways inside the cell that bears the said receptor. Examples of such ligands include peptide and polypeptide hormones, cytokines, chemokines, growth factors, apoptosis-inducing factors and the like. Natural ligands can be either soluble or can be attached to the surface of another cell.
Due to the biological activity of such ligands, some have potential use as therapeutics. Several peptide or polypeptide ligands have been approved by regulatory agencies as therapeutic products, including, for example, human growth hormone, insulin, interferon (IFN)-α2b, IFNα2a, IFNβ, erythropoietin, G-CSF and GM-CSF. Many of these and other ligands have demonstrated potential in therapeutic applications, but have also exhibited toxicity when administered to human patients. One reason for toxicity is that most of these ligands trigger receptors on a variety of cells, including cells other than those that mediate the therapeutic effect. For example, when IFNα2b is used to treat multiple myeloma its utility resides, at least in part, in its binding to type I interferon receptors on the myeloma cells, which in turn triggers reduced proliferation and hence limits disease progression. Unfortunately, however, this IFN also binds to numerous other, normal cells within the body, triggering a variety of other cellular responses, some of which are harmful (e.g. flu-like symptoms, neutropenia, depression). A consequence of such “off target” activity of ligands is that many ligands are not suitable as drug candidates. In this context, “off target activity” refers to activity on the ligand's natural receptor, but on the surface of cells other than those that mediate therapeutically beneficial effects.
Even though some ligands, such as IFNα2b, are approved for the treatment of medical conditions, they are poorly tolerated due to their “off target” biological activity. The off-target activity and associated poor tolerability also mean that some of these peptide ligand-based drugs cannot be administered at sufficiently high dosages to produce optimal therapeutic effects on the target cells which mediate the therapeutic effect.
Similarly, it has been known since the mid-1980's that interferons, in particular IFNα, are able to increase apoptosis and decrease proliferation of certain cancer cells. These biological activities are mediated by type I interferon receptors on the surface of the cancer cells which, when stimulated, initiate various signal transduction pathways leading to reduced proliferation and/or the induction of terminal differentiation or apoptosis. IFNα has been approved by the FDA for the treatment of several cancers including melanoma, renal cell carcinoma, B cell lymphoma, multiple myeloma, chronic myelogenous leukemia (CML) and hairy cell leukemia. A “direct” effect of IFNα on the tumour cells is mediated by the IFNα binding directly to the type I IFN receptor on those cells and stimulating apoptosis, terminal differentiation or reduced proliferation. One “indirect” effect of IFNα on non-cancer cells is to stimulate the immune system, which may produce an additional anti-cancer effect by causing the immune system to reject the tumour.
Unfortunately, the type I interferon receptor is also present on most non-cancerous cells. Activation of this receptor on such cells by IFNα causes the expression of numerous pro-inflammatory cytokines and chemokines, leading to toxicity. Such toxicity prevents the dosing of IFNα to a subject at levels that exert the maximum anti-proliferative and pro-apoptotic activity on the cancer cells.
Ozzello et al. (Breast Cancer Research and Treatment 25:265-76, 1993) described covalently attaching human IFNα to a tumour-targeting antibody, thereby localizing the direct inhibitory activity of IFNα to the tumour as a way of reducing tumour growth rates, and demonstrated that such conjugates have anti-tumour activity in a xenograft model of a human cancer. The mechanism of the observed anti-cancer activity was attributed to a direct effect of IFNα on the cancer cells, since the human IFNα used in the experiments did not interact appreciably with the murine type I IFN receptor, which could have lead to an indirect anti-cancer effect. Because of this lack of binding of the human IFNα to the murine cells, however, the authors could not evaluate the toxicity of the antibody-IFNα conjugate relative to free IFNα. These authors used a chemical method to attach the IFNα to the antibody.
Alkan et al., (Journal of Interferon Research, volume 4, number 3, p. 355-63, 1984) demonstrated that attaching human IFNα to an antibody that binds to the Epstein-Barr virus (EBV) membrane antigen (MA) increased its antiproliferative activities towards cells that express the EBV-MA antigen. This increased potency was dependent on both antigen expression by the target cells and the binding specificity of the antibody. The cell line tested was the cancer cell line QIMR-WIL, a myeloblastic leukemia. The authors suggested that the attachment of IFNα to an antibody could be used as a treatment for cancer since it would reduce tumour growth. Alkan et al did not address the potential toxicity of these antibody-IFNα conjugates arising from their interactions with normal, antigen-negative cells.
It is also known that the linkage between an antibody and IFNα may be accomplished by making a fusion protein construct. For example, IDEC (WO01/97844) disclose a direct fusion of human IFNα to the C terminus of the heavy chain of an IgG targeting the tumour antigen CD20. Other groups have disclosed the use of various linkers between the C-terminus of an IgG heavy chain and the IFNα. For example, U.S. Pat. No. 7,456,257 discloses that the C-terminus of an antibody heavy chain constant region may be connected to IFNα via an intervening serine-glycine rich (S/G) linker of the sequence (GGGGS)n, where n may be 1, 2 or 3, and that there are no significant differences in the IFNα activity of the fusion protein construct regardless of linker length.
Morrison et al. (US2011/0104112 A1; and Xuan C, Steward K K, Timmerman J M, Morrison S L. Targeted delivery of interferon-α via fusion to anti-CD20 results in potent antitumor activity against B-cell lymphoma. Blood 2010; 115:2864-71) also disclose IFNα linked to the C-terminus of the heavy chain of a cancer-targeting IgG antibody, with an intervening S/G linker, and observed that the fusion of the IgG and linker to the IFNα reduced the activity of IFNα on cells that did not express the corresponding antigen on the cell surface. The decreased IFN activity of these fusion protein constructs was modest when compared to human non-fusion protein IFNα (free IFNα) acting on human cells, but appeared to be more significant for murine IFNα on murine cells. The decrease in the activity of human IFNα that results from fusing it to the C-terminus of an antibody, as observed by Morrison et al, and in U.S. Pat. No. 7,456,257 is modest and is generally considered to be a disadvantage since it reduces potency of the ligand. This disadvantage was pointed out, for example, by Rossi et al (Blood vol. 114, No. 18, pp 3864-71), who used an alternative strategy of attaching the IFNα to a tumor targeting antibody in such a way that no loss in IFNα activity was observed.
In general the prior art teaches to use a potent IFN and to target this IFN to cancer cells. While this approach results in an increase in activity of the IFN against cancer cells, it does not address the issue of activity of the IFN on normal “off-target” cells. In prior art examples referred to above, the human IFNα portion of the antibody-IFNα fusion protein maintained a high proportion of native IFNα activity when exposed to human cells that do not express the corresponding antigen on their cell surfaces. This activity may lead to toxicity arising from the activation of non-cancerous, normal (“off target”) cells by the IFNα portion of the fusion protein. Accordingly, there exists a need to decrease the “off-target” activity of ligand-based drugs, while retaining the “on-target”, therapeutic effect of such ligands. The maintenance of target-specific ligand activity and at the same time a reduction in non-target toxicity of ligand-based therapeutic agents would create a greater therapeutic concentration window for therapeutically useful ligands. It would for example be desirable to use human IFNα in a form such that its activity can be directed to the cancer cells while minimizing its effects on normal human cells. Ideally the type I interferon receptor on the cancer cells would be maximally stimulated, while the same receptor on non-cancerous cells would experience minimal stimulation. There is a need to target human IFNα to the cancer cells in such a way that it has dramatically more activity on the cancer cells, which display the antigen, than on the normal cells, which do not display the antigen. The same logic applies to other potentially therapeutic ligands, e.g. other cytokines, peptide and polypeptide hormones, chemokines, growth factors, apoptosis-inducing factors and the like.
The present inventors have found that when a peptide or polypeptide signaling ligand, having one or more mutations which substantially decrease the affinity of the ligand for its receptor, is linked to an antibody that targets the mutated ligand to target cells which display the antibody's corresponding antigen, the ligand's activity on target antigen-positive cells is maintained while the ligand's activity on non-target antigen-negative cells is substantially reduced. The net result is a ligand signaling molecule that has a much greater potency in activation of its receptors on antigen-positive target cells compared to antigen-negative non-target cells, which provides a means for reducing toxicity arising from off-target ligand activity.
Accordingly, a first aspect of the present invention provides a polypeptide construct comprising a peptide or polypeptide signaling ligand linked to an antibody or antigen binding portion thereof which binds to a cell surface-associated antigen, wherein the ligand comprises at least one amino acid substitution or deletion which reduces its potency on cells lacking expression of said antigen.
In a second aspect, the present invention provides a method of treating a tumour in a subject, comprising administering to the subject the polypeptide construct of the present invention.
In a third aspect, the present invention provides use of the polypeptide construct of the present invention in the treatment of cancer.
In a fourth aspect, the present invention provides a composition comprising the polypeptide construct of the present invention and a pharmaceutically acceptable carrier or diluent.
In a fifth aspect, the present invention provides method of reducing the potency of a peptide or polypeptide signaling ligand on an antigen negative cell which bears the ligand receptor whilst maintaining the potency of the ligand on an antigen positive cell which bears the ligand receptor to a greater extent when compared to the antigen negative cell, the method comprising modifying the ligand such that the ligand comprises at least one amino acid substitution or deletion which reduces its potency on the antigen negative cell and linking the modified ligand to an antibody or antigen-binding portion thereof, wherein the antibody or antigen binding portion thereof is specific for a cell surface-associated antigen on the antigen positive cell but not on the antigen negative cell.
Unlike the linking of a non-attenuated “native” or “wild-type” human ligand to an antibody or antigen-binding portion thereof, which typically results in from 1 to 15-fold higher potency of the ligand on antigen-positive compared to antigen-negative cells, the present invention demonstrates that the attachment of mutated, attenuated forms of the ligand to the same antibody is able to generate higher potency on antigen-positive cells compared to antigen negative cells.
In one embodiment the signaling ligand is IFNα or IFNβ and the polypeptide construct shows at least 10, at least 100, at least 1,000, at least 10,000 or at least 100,000-fold greater selectivity towards antigen positive cells over antigen negative cells compared to free, wild-type ligand using the “off-target” assay and the “on target (ARP)” or “on target (Daudi)” assays described herein.
The present invention also provides an antibody-attenuated ligand fusion proteins, wherein the attenuated ligand is IFNα or IFNβ and the wherein fusion protein construct, when injected into a mouse with an established human tumor, can eliminate the tumor.
The present invention also provides an antibody-attenuated ligand fusion proteins, wherein the attenuated ligand is IFNα or IFNβ and wherein the fusion protein construct, when injected into a mouse with an established human tumor with a volume of over 500 cubic millimeters, can eliminate the tumor.
The present invention also provides an antibody-attenuated ligand fusion proteins, wherein the attenuated ligand is IFNα or IFNβ and wherein the fusion protein construct, when injected as a single one-time treatment into a mouse with an established human tumor, can eliminate the tumor.
An antibody-attenuated ligand fusion proteins, wherein the attenuated ligand is IFNα or IFNβ and wherein the fusion protein construct can eliminate both established myeloma tumors and established lymphoma tumors in a mouse
In each of these cases it is preferred that cell surface-associated antigen is CD 38.
In one embodiment, the amino acid sequence of the signaling ligand comprising at least one amino acid substitution or deletion has greater than 90% or greater than 95%, or greater than 96%, or greater than 97%, or greater than 98% or greater than 99% sequence identity with the wild-type ligand amino acid sequence.
In one embodiment, the construct is a fusion protein.
In certain embodiments the signaling ligand is linked to the C-terminus of the heavy chain of the antibody or antigen binding portion thereof. In certain embodiments the signaling ligand is linked to the C-terminus of the light chain of the antibody or antigen binding portion thereof. In either of these embodiments, the ligand may be linked directly to the C-terminus of the heavy or light chain of the antibody or antigen binding portion thereof (ie without an intervening additional linker).
In one embodiment the cell surface associated antigen is selected from class I MHC or PD-1.
In certain embodiments, the cell surface-associated antigen is a myeloma associated antigen which is selected from the group consisting of CD38, HM1.24, CD56, CS1, CD138, CD74, IL-6R, Blys (BAFF), BCMA, HLA-SR, Kininogen, beta2 microglobulin, FGFR3, ICAM-1, matriptase, CD52, EGFR, GM2, alpha4-integrin, IFG-1R and KIR, and the ligand is an IFNα.
In one embodiment, the signaling ligand is selected from any one of IFNα2b, IFNβ, IL-4 or IL-6.
In certain embodiments in which the signaling ligand is an IFNα, the amino acid substitution or deletion may be at any one or more of amino acid positions R33, R144 or A145. In certain embodiments the signaling ligand is an IFNα and the substitution is selected from the group consisting of R144A (SEQ ID NO:30), R144S (SEQ ID NO:40), R144T (SEQ ID NO:41), R144Y (SEQ ID NO:43), R144I (SEQ ID NO:35), R144L (SEQ ID NO:37), A145D (SEQ ID NO:44), A145H (SEQ ID NO:47), A145Y (SEQ ID NO:58), A145K (SEQ ID NO:49), R33A+YNS (SEQ ID NO:65), R33A (SEQ ID NO:16) and R144A+YNS (SEQ ID NO:68).
In certain embodiments in which the signaling ligand is an IFNα and the cell surface associated antigen is CD38, the antibody is selected from any one of G003, G005, G024, MOR03077, MORO3079, MORO3080, MORO3100, 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, 38SB39, OKT10, X355/02, X910/12, X355/07, X913/15, R5D1, R5E8, R10A2, or an antigen binding portion thereof, or an antibody with greater than 95%, greater than 96%, greater than 97%, greater than 98% or at least 99% amino acid sequence identity with any one of R5D1, R5E8 or R10A2.
In certain embodiments in which the cell surface associated antigen is CD38, the signaling ligand of the polypeptide construct is an IFNα, the treatment is for a cancer in a subject selected from multiple myeloma, a leukemia or a lymphoma. In particular embodiments the subject is also treated with a retinoid, such as all-trans retinoic acid. In certain embodiments in which the cell surface associated antigen is CD38, the tumour or cancer may be selected from multiple myeloma, non-Hodgkin's lymphoma, chronic myelogenous leukemia, chronic lymphocytic leukemia or acute myelogenous leukemia.
In embodiments in which the ligand is linked to an antibody, the antibody may be an IgG4. In particular embodiments the IgG4 comprises an S228P amino acid substitution.
In certain embodiments in which the signaling ligand of the polypeptide construct is an IFNα, the antibody or antigen binding portion thereof may bind to a cell surface associated antigen on virally infected cells. In these embodiments the cell surface associated antigen may be selected from a virally encoded protein, phosphatidylserine or a phosphatidylserine-binding protein. In embodiments in which the cell surface associated antigen is phosphatidylserine or a phosphatidylserine-binding protein the construct may be used to treat Hepatitis C.
In certain embodiments in which the signaling ligand of the polypeptide construct is IFNα or IFNβ, the cell surface associated antigen is selected from CD20, CD38, CD138 or CS1. In certain embodiments in which the ligand is IFNα or IFNβ, the tumour or cancer may be selected from multiple myeloma, melanoma, renal cell carcinoma, chronic myelogenous leukemia or hairy cell leukemia.
In a particular embodiment the construct is G005-HC-L0-IFNα (A145D) IgG4.
In certain embodiments in which the signaling ligand of the polypeptide construct is an IFNβ, the cell surface associated antigen may be a T cell, a myeloid cell or an antigen presenting cell cell surface associated protein.
In certain embodiments in which the signaling ligand of the polypeptide construct is an IFNβ, the cell surface associated antigen may be selected from the group consisting of CD3, CD4, CD8, CD24, CD38, CD44, CD69, CD71, CD83, CD86, CD96, HLA-DR, PD-1, ICOS, CD33, CD115, CD11c, CD14, CD52 and PD-1. In these embodiments, the construct may be used to treat a disease characterized by excess inflammation, such as an autoimmune disease.
In certain embodiments in which the signaling ligand of the polypeptide construct is an IFNβ, the at least one amino acid substitution or deletion is selected from the group consisting of R35A, R35T, E42K, M62I, G78S, A141Y, A142T, E149K, R152H. In these embodiments, the IFNβ may also possess a C17S or C17A substitution.
In certain embodiments the signaling ligand of the polypeptide construct is an IFNγ. In these embodiments, the cell surface associated antigen may be a tumor-associated antigen. In other embodiments, the cell surface associated antigen may be selected from the group consisting of CD14, FSP1, FAP, PDGFR alpha and PDGFR beta. In these embodiments, the construct may be used to treat a disease characterized by excess fibrosis.
In certain embodiments in which the signaling ligand of the polypeptide construct is an IFNγ, the at least one amino acid substitution or deletion is selected from the group consisting of a deletion of residues A23 and D24, an S20I substitution, an A23V substitution, a D21K substitution and a D24A substitution.
In certain embodiments in which the signaling ligand of the polypeptide construct is an IL-4, the cell surface associated antigen is selected from the group consisting of CD3, CD4, CD24, CD38, CD44, CD69, CD71, CD96, PD-1, ICOS, CD52 and PD-1.
In certain embodiments in which the signaling ligand of the polypeptide construct is an IL-6, the cell surface associated antigen is selected from the group consisting of CD3, CD4, CD24, CD38, CD44, CD69, CD71, CD96, PD-1, ICOS, CD52 and PD-1.
In certain embodiments in which the signaling ligand of the polypeptide construct is an HGF, the cell surface associated antigen is selected from the group consisting of ASGR1, ASGR2, FSP1, RTI140/Ti-alpha, HTI56 and a VEGF receptor.
In certain embodiments in which the signaling ligand of the polypeptide construct is a IFNβ, the cell surface associated antigen is selected from the group consisting of CD3, CD4, CD8, CD24, CD38, CD44, CD69, CD71, CD83, CD86, CD96, HLA-DR, PD-1, ICOS, CD33, CD115, CD11c, CD14, CD52 and PD-1.
In certain embodiments in which the signaling ligand of the polypeptide construct is an erythropoietin, the cell surface associated antigen is selected from the group consisting of CD241 the product of the RCHE gene, CD117 (c-kit), CD71 (transferrin receptor), CD36 (thrombospondin receptor), CD34, CD45RO, CD45RA, CD115, CD168, CD235, CD236, CD237, CD238, CD239 and CD240.
In certain embodiments in which the signaling ligand of the polypeptide construct is an interleukin-10 and the cell surface associated antigen is selected from the group consisting of CD11c, CD33 or CD115, CD14, FSP1, FAP, or PDGFR (alpha or beta).
In a sixth aspect there is provided anti-CD38 antibodies with variable regions designated X910/12, X913/15, X355/02, X355/07, R5D1, R5E8, or R10A2, with sequences set out as follows:
From these sequences the person skilled in the field can readily identify the CDR sequences using known methods. As will be recognized by the skilled worker these CDR sequences can be used in differing framework sequences to those specified in the SEQ ID NO's specified above.
The constructs of the present invention are antibody-attenuated ligand constructs, which show an elevated antigen-specificity index with respect to activating signaling pathways due to the action of the attenuated ligand on a cell surface receptor. These constructs are based on the surprising discovery that, in the context of an antibody-ligand construct, the ligand portion can be mutated in such a way that the ligand activity on antigen-negative cells is dramatically attenuated, while the ligand activity on antigen-positive cells is only modestly, if at all, attenuated. Such constructs display one, two, three, four or five orders of magnitude greater potency on antigen-positive cells compared to antigen negative cells than does the free ligand. In one embodiment, the antibody-attenuated ligand construct retains at least 1%, at least 10%, at least 20%, at least 30%, at least 40% or at least 50% of the potency on antigen-positive cells as the non-attenuated free (i.e. not attached to an antibody) ligand. In addition, in one embodiment the antibody-attenuated ligand construct retains at least 30%, at least 50%, at least 75% or at least 90% of the maximal activity of the non-attenuated free (i.e. not attached to an antibody) ligand; in this context, “maximal activity” should be understood as meaning the amount of signaling activity (or downstream effect thereof) at the high, plateau portion of a dose-response curve, where further increases in the agent does not further increase the amount of response).
“Specificity” as used herein is not necessarily an absolute designation but often a relative term signifying the degree of selectivity of an antibody-ligand fusion protein construct for an antigen-positive cell compared to an antigen-negative cell. Thus for example, a construct may be said to have “100-fold specificity for antigen-positive cells compared to antigen-negative cells” and this would indicate that the construct has 100-fold higher potency on cells that express the antigen compared to cells that do not. In some cases, this degree of specificity of a construct comparing antigen-positive vs. antigen-negative cells may not be based on the absolute ratio of potency of the construct on antigen-positive vs. antigen-negative cells, but of the potency of the construct on each type of cell relative to the potency of the free, non attenuated ligand on the same same type of cell. This “ratio of ratio” approach for quantifying the degree of specificity of an antibody-ligand construct takes into consideration any inherent differences in the potency of a ligand on different cell types and is exemplified by the calculations of Antigen Specificity Index (ASI) in Table 25. Assays for determining potency of antibody-ligand fusion constructs are exemplified in the examples and include cell based assays for proliferation, apoptosis, phosphorylation of receptors and intracellular proteins, migration, differentiation (for example, differentiation of naïve CD4+ T cells into Th1, Th17, Th2 vs. Treg cells), increases or decreases in gene expression or gene product secretion into the media, etc.
Accordingly, in a first aspect the present invention provides a polypeptide construct comprising a peptide or polypeptide signaling ligand linked to an antibody or antigen binding portion thereof which binds to a cell surface-associated antigen wherein the ligand comprises at least one amino acid substitution or deletion which reduces its potency on cells lacking expression of said antigen.
In one embodiment the present invention provides a polypeptide construct comprising IFN linked to an antibody or antigen binding portion thereof which binds to a tumour associated antigen wherein the IFN comprises at least one amino acid substitution or deletion which reduces its potency on cells lacking expression of said antigen. Such a polypeptide will be capable of exerting with high potency the IFN's anti-proliferative activity on the antigen-positive tumor cells while exerting a much lower potency on the antigen-negative, non-tumour cells within the body.
In a second aspect the present invention provides a method of treating a tumour in a subject comprising administering to the subject the polypeptide construct of the present invention.
The term “antibody-ligand construct” as used herein refers to an antibody or antigen-binding fragment thereof covalently attached to a signaling ligand that has been attenuated by one or more substitutions or deletions that reduce the ligand's potency on cells that do not express the antigen corresponding to the antibody.
The term “antibody”, as used herein, broadly refers to any immunoglobulin (Ig) molecule comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art, non-limiting embodiments of which are discussed below.
In a full-length antibody, each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules 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.
The term “antigen binding domain” or “antigen binding portion” of an antibody, as used herein, refers to one or more fragments of an antibody or protein that retain 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 may also be bispecific, dual specific, or multi-specific formats, 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, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments in addition to a portion of the hinge region, linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a domain antibody (dAb) (Ward et al. 1989 Nature 341 544-6, Winter et al., PCT publication WO 90/05144 A1 herein incorporated by reference), which comprises a single variable domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. 1988 Science 242 423-6; Huston et al. 1988 Proc Natl Acad Sci USA 85 5879-83). Such 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 encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger, P., et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., 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 an embodiment the antibody binding portion is a Fab fragment.
The antibody described herein may be may be a humanized antibody. The term “humanized antibody” shall be understood to refer to a protein comprising a human-like variable region, which includes CDRs from an antibody from a non-human species (e.g., mouse or rat or non-human primate) grafted onto or inserted into FRs from a human antibody (this type of antibody is also referred to a “CDR-grafted antibody”). Humanized antibodies also include proteins in which one or more residues of the human protein are modified by one or more amino acid substitutions and/or one or more FR residues of the human protein are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found in neither the human antibody or in the non-human antibody. Any additional regions of the protein (e.g., Fc region) are generally human. Humanization can be performed using a method known in the art, e.g., U.S. Pat. Nos. 5,225,539, 6,054,297, 7,566,771 or 5,585,089. The term “humanized antibody” also encompasses a super-humanized antibody, e.g., as described in U.S. Pat. No. 7,732,578.
The antibody described herein may be human. The term “human antibody” as used herein refers to proteins having variable and, optionally, constant antibody regions found in humans, e.g. in the human germline or somatic cells or from libraries produced using such regions. The “human” antibodies can include amino acid residues not encoded by human sequences, e.g. mutations introduced by random or site directed mutations in vitro (in particular mutations which involve conservative substitutions or mutations in a small number of residues of the protein, e.g. in 1, 2, 3, 4 or 5 of the residues of the protein). These “human antibodies” do not necessarily need to be generated as a result of an immune response of a human, rather, they can be generated using recombinant means (e.g., screening a phage display library) and/or by a transgenic animal (e.g., a mouse) comprising nucleic acid encoding human antibody constant and/or variable regions and/or using guided selection (e.g., as described in or U.S. Pat. No. 5,565,332). This term also encompasses affinity matured forms of such antibodies. For the purposes of the present disclosure, a human protein will also be considered to include a protein comprising FRs from a human antibody or FRs comprising sequences from a consensus sequence of human FRs and in which one or more of the CDRs are random or semi-random, e.g., as described in U.S. Pat. No. 6,300,064 and/or U.S. Pat. No. 6,248,516.
The antibody portions of polypeptides of the present invention may be full length antibodies of any class, preferably IgG1, IgG2 or IgG4. The constant domains of such antibodies are preferably human. The variable regions of such antibodies may be of non-human origin or, preferably, be of human origin or be humanized. Antibody fragments may also be used in place of the full length antibodies.
The term “antibody” also includes engineered antibodies. As will be appreciated there are many variations of engineered antibodies (e.g. mouse monoclonal, chimeric, humanized and human monoclonal antibodies, single chain variable antibody fragments (scFv's), minibodies, aptamers, as well as bispecific antibodies and diabodies as described above).
Single variable region domains (termed dAbs) are, for example, disclosed in (Ward et al., Nature 341: 544-546, 1989; Hamers-Casterman et al., Nature 363: 446-448, 1993; Davies & Riechmann, FEBS Lett. 339: 285-290, 1994).
Minibodies are small versions of whole antibodies, which encode in a single chain the essential elements of a whole antibody. Suitably, the minibody is comprised of the VH and VL domains of a native antibody fused to the hinge region and CH3 domain of the immunoglobulin molecule as, for example, disclosed in U.S. Pat. No. 5,837,821.
In an alternate embodiment, the engineered antibody may comprise non-immunoglobulin derived, protein frameworks. For example, reference may be made to (Ku & Schutz, Proc. Natl. Acad. Sci. USA 92: 6552-6556, 1995) which discloses a four-helix bundle protein cytochrome b562 having two loops randomized to create CDRs, which have been selected for antigen binding.
There is a plethora of non-antibody recognition protein or protein domain scaffolds that may be utilised as the antigen binding domains in the constructs of this invention. These include scaffolds based on cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) (Evibody; U.S. Pat. No. 7,166,697); human transferrin (Trans-body); a three-helix bundle from the Z-domain of Protein A (Affibody); a 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 domains); lipocalins, FABP, Bilin-binding protein, Apoloproptein D (Anticalins); human α-crystallin or ubiquitin molecule (Affilin); trypsin inhibitor II (Microbody); α2p8 or Ankyrin repeat (repeat-motif proteins), Charybdotoxin (Scorpion toxins), Min-23, Cellulose binding domain (Knottins); Neocarzinostatin, CBM4-2 and Tendamistat.
Further, in addition to scaffolds provided for by antibody-derived domains or non-antibody folds as described above, there are naturally occurring ligand binding proteins or protein domains that may be utilised as the ligand binding domains in this invention. For example, protein domains that possess ligand binding properties include extracellular domains of receptors, PDZ modules of signalling proteins, such as Ras-binding protein AF-6, adhesion molecules, and enzymes.
The present invention further encompasses chemical analogues of amino acids in the subject antibodies. The use of chemical analogues of amino acids is useful inter alia to stabilize the molecules such as if required to be administered to a subject. The analogues of the amino acids contemplated herein include, but are not limited to, modifications of side chains, incorporation of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecule or their analogues.
Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH4; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH4.
The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.
The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivatisation, for example, to a corresponding amide.
Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.
Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.
Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids. A list of unnatural amino acid, contemplated herein is shown in Table 1.
Crosslinkers can be used, for example, to stabilize 3D conformations, using homo-bifunctional crosslinkers such as the bifunctional imido esters having (CH2)n spacer groups with n=1 to n=6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-bifunctional reagents which usually contain an amino-reactive moiety such as N-hydroxysuccinimide and another group specific-reactive moiety such as maleimido or dithio moiety (SH) or carbodiimide (COOH).
Using methods well known in the art to increase binding, by for example, affinity maturation, or to decrease immunogenicity by removing predicted MHC class II-binding motifs. The therapeutic utility of the antibodies described herein can be further enhanced by modulating their functional characteristics, such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), serum half-life, biodistribution and binding to Fc receptors or the combination of any of these. This modulation can be achieved by protein-engineering, glyco-engineering or chemical methods. Depending on the therapeutic application required, it could be advantageous to either increase or decrease any of these activities.
An example of glyco-engineering used the Potelligent® method as described in Shinkawa T. et al., 2003 (J Biol Chem 278: 3466-73).
Numerous methods for affinity maturation of antibodies are known in the art. Many of these are based on the general strategy of generating panels or libraries of variant proteins by mutagenesis followed by selection and/or screening for improved affinity. Mutagenesis is often performed at the DNA level, for example by error prone PCR (Thie, Voedisch et al. 2009), by gene shuffling (Kolkman and Stemmer 2001), by use of mutagenic chemicals or irradiation, by use of ‘mutator’ strains with error prone replication machinery (Greener 1996) or by somatic hypermutation approaches that harness natural affinity maturation machinery (Peled, Kuang et al. 2008). Mutagenesis can also be performed at the RNA level, for example by use of Qβ replicase (Kopsidas, Roberts et al. 2006). Library-based methods allowing screening for improved variant proteins can be based on various display technologies such as phage, yeast, ribosome, bacterial or mammalian cells, and are well known in the art (Benhar 2007). Affinity maturation can be achieved by more directed/predictive methods for example by site-directed mutagenesis or gene synthesis guided by findings from 3D protein modeling (see for example Queen, Schneider et al. 1989 or U.S. Pat. No. 6,180,370 or 5,225,539).
Methods of increasing ADCC have been described by Ferrara, Brunker et al. 2006; Li, Sethuraman et al. 2006; Stavenhagen, Gorlatov et al. 2007; Shields, Namenuk et al. 2001; Shinkawa, Nakamura et al. 2003; and WO 2008/006554.
Methods of increasing CDC have been described by Idusogie, Wong et al. 2001; Dall'Acqua, Cook et al. 2006; Michaelsen, Aase et al. 1990; Brekke, Bremnes et al. 1993; Tan, Shopes et al. 1990; and Norderhaug, Brekke et al. 1991.
References describing methods of increasing ADCC and CDC include Natsume, In et al. 2008. The disclosure of each of these references is included herein by cross reference.
A number of methods for modulating antibody serum half-life and biodistribution are based on modifying the interaction between antibody and the neonatal Fc receptor (FcRn), a receptor with a key role in protecting IgG from catabolism, and maintaining high serum antibody concentration. Dall'Acqua et al describe substitutions in the Fc region of IgG1 that enhance binding affinity to FcRn, thereby increasing serum half-life (Dall'Acqua, Woods et al. 2002) and further demonstrate enhanced bioavailability and modulation of ADCC activity with triple substitution of M252Y/S254T/T256E (Dall'Acqua, Kiener et al. 2006). See also U.S. Pat. Nos. 6,277,375; 6,821,505; 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). (Hinton, Xiong et al. 2006). See also U.S. Pat. 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). See also Shields et al 2001 and WO 2000/42072. Other examples of constant domain amino acid substitutions which modulate binding to Fc receptors and subsequent function mediated by these receptors, including FcRn binding and serum half-life, are described in U.S Pat. Application Nos 20090142340; 20090068175 and 20090092599.
The glycans linked to antibody molecules are known to influence interactions of antibody with Fc receptors and glycan receptors and thereby influence antibody activity, including serum half-life (Kaneko, Nimmerjahn et al. 2006; Jones, Papac et al. 2007; and Kanda, Yamada et al. 2007). Hence, certain glycoforms that modulate desired antibody activities can confer therapeutic advantage. Methods for generating engineered glycoforms are known in the art and include but are not limited to those described in U.S. Pat. Nos. 6,602,684; 7,326,681; 7,388,081 and WO 2008/006554.
Extension of half-life by addition of polyethylene glycol (PEG) has been widely used to extend the serum half-life of proteins, as reviewed, for example, by Fishburn 2008.
As will be recognised it is possible to make conservative amino acid substitutions within the sequences of the current invention. By “conservative substitution” is meant amino acids having similar properties. As used in this specification the following groups of amino acids are to be seen as 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.
The term “cell surface-associated antigen”, as used herein, broadly refers to any antigen expressed on surfaces of cells, including infectious or foreign cells or viruses.
In certain aspects of the present invention, the polypeptide constructs or compositions of the present invention may be used to treat patients with cancer. Cancers contemplated herein include: a group of diseases and disorders that are characterized by uncontrolled cellular growth (e.g. formation of tumor) without any differentiation of those cells into specialized and different cells. Such diseases and disorders include ABL1 protooncogene, AIDS related cancers, acoustic neuroma, acute lymphocytic leukaemia, acute myeloid leukaemia, adenocystic carcinoma, adrenocortical cancer, agnogenic myeloid metaplasia, alopecia, alveolar soft-part sarcoma, anal cancer, angiosarcoma, aplastic anaemia, astrocytoma, ataxia-telangiectasia, basal cell carcinoma (skin), bladder cancer, bone cancers, bowel cancer, brain stem glioma, brain and CNS tumors, breast cancer, CNS tumors, carcinoid tumors, cervical cancer, childhood brain tumors, childhood cancer, childhood leukaemia, childhood soft tissue sarcoma, chondrosarcoma, choriocarcinoma, chronic lymphocytic leukaemia, chronic myeloid leukaemia, colorectal cancers, cutaneous T-Cell lymphoma, dermatofibrosarcoma-protuberans, desmoplastic-small-round-cell-tumor, ductal carcinoma, endocrine cancers, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, extra-hepatic bile duct cancer, eye cancer, eye: melanoma, retinoblastoma, fallopian tube cancer, fanconi anemia, fibrosarcoma, gall bladder cancer, gastric cancer, gastrointestinal cancers, gastrointestinal-carcinoid-tumor, genitourinary cancers, germ cell tumors, gestational-trophoblastic-disease, glioma, gynaecological cancers, hematological malignancies, hairy cell leukaemia, head and neck cancer, hepatocellular cancer, hereditary breast cancer, histiocytosis, Hodgkin's disease, human papillomavirus, hydatidiform mole, hypercalcemia, hypopharynx cancer, intraocular melanoma, islet cell cancer, Kaposi's sarcoma, kidney cancer, Langerhan's-cell-histiocytosis, laryngeal cancer, leiomyosarcoma, leukemia, Li-Fraumeni syndrome, lip cancer, liposarcoma, liver cancer, lung cancer, lymphedema, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, male breast cancer, malignant-rhabdoid-tumor-of-kidney, medulloblastoma, melanoma, merkel cell cancer, mesothelioma, metastatic cancer, mouth cancer, multiple endocrine neoplasia, mycosis fungoides, myelodysplastic syndromes, multiple myeloma, myeloproliferative disorders, nasal cancer, nasopharyngeal cancer, nephroblastoma, neuroblastoma, neurofibromatosis, nijmegen breakage syndrome, non-melanoma skin cancer, non-small-cell-lung-cancer-(NSCLC), ocular cancers, oesophageal cancer, oral cavity cancer, oropharynx cancer, osteosarcoma, ostomy ovarian cancer, pancreas cancer, paranasal cancer, parathyroid cancer, parotid gland cancer, penile cancer, peripheral-neuroectodermal-tumors, pituitary cancer, polycythemia vera, prostate cancer, rare-cancers-and-associated-disorders, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, Rothmund-Thomson syndrome, salivary gland cancer, sarcoma, schwannoma, Sezary syndrome, skin cancer, small cell lung cancer (SCLC), small intestine cancer, soft tissue sarcoma, spinal cord tumors, squamous-cell-carcinoma-(skin), stomach cancer, synovial sarcoma, testicular cancer, thymus cancer, thyroid cancer, transitional-cell-cancer-(bladder), transitional-cell-cancer-(renal-pelvis-/-ureter), trophoblastic cancer, urethral cancer, urinary system cancer, uroplakins, uterine sarcoma, uterus cancer, vaginal cancer, vulva cancer, Waldenstrom's-macroglobulinemia and Wilms' tumor. In an embodiment the tumor is selected from a group of multiple myeloma or non-hodgkin's lymphoma.
As contemplated for the treatment of cancer, the antibody portions of the constructs of the present invention may bind to tumour-associated antigens, i.e., cell surface antigens that are selectively expressed by cancer cells or over-expressed in cancer cells relative to most normal cells. There are many tumour-associated antigens (TAAs) known in the art. Non-limiting examples of TAAs include enzyme tyrosinase; melanoma antigen GM2; alphafetoprotein (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 a number of 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).
A list of other TAAs is provided in US 2010/0297076, the disclosure of which is included herein by reference. Of particular interest are cell surface antigens associated with multiple myeloma cells, including but not limited to CD38, CD138, CS1, and HM1.24. In one embodiment an antigen for antibody-attenuated ligand constructs, for example, an antibody-attenuated interferon construct, is CD38.
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 on a significant proportion of normal bone marrow precursor cells (Malavasi, F., Hum. Immunol. 9:9, 1984). In lymphocytes, however, the expression appears to be dependent on the differentiation and activation state of the cell. Resting T and B cells are negative while immature and activated lymphocytes are predominantly positive for CD38 expression (Funaro, A., J. Immunol. 145:2390, 1990). Additional studies indicate mRNA expression in non-hemopoeitic organs such as pancreas, brain, spleen and liver (Koguma, T., Biochim. Biophys. Acta 1223:160, 1994.)
CD38 is a multifunctional ectoenzyme that is involved in transmembrane signaling and cell adhesion. It is also known as cyclic ADP ribose hydrolase because it can transform NAD+ and NADP+ into cADPR, ADPR and NAADP, depending on extracellular pH. These products induce Ca2+-mobilization inside the cell which can lead to tyrosine phosphorylation and activation of the cell. CD38 is also a receptor that can interact with a ligand, CD31. Activation of receptor via CD31 leads to intracellular events including Ca2+ mobilization, cell activation, proliferation, differentiation and migration (reviewed in Deaglio, S., Trends in Mol. Med. 14:210, 2008.)
CD38 is expressed at high levels on multiple myeloma cells, in most cases of T- and B-lineage acute lymphoblastic leukemias, some acute myelocytic leukemias, follicular center cell lymphomas and T lymphoblastic lymphomas. (Malavasi, F., J. Clin Lab Res. 22:73, 1992). More recently, CD38 expression has become a reliable prognostic marker in B-lineage chronic lymphoblastic leukemia (B-CLL) (Ibrahim, S., Blood. 98:181, 2001 and Durig, J., Leuk. Res. 25:927, 2002). Independent groups have demonstrated that B-CLL patients presenting with a CD38+ clone are characterized by an unfavorable clinical course with a more advance stage of disease, poor responsiveness to chemotherapy and shorter survival time (Morabito, F., Haematologica. 87:217, 2002). The consistent and enhanced expression of CD38 on lymphoid tumors makes this an attractive target for therapeutic antibody technologies.
Preferred antigens for the development of antibody-attenuated ligand fusion protein constructs which target cancer are antigens which show selective or greater expression on the cancer cells than on most other, non-transformed cells within the body. 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 Lewisx, lewisy and lewisxy carbohydrate antigens that can be displayed on proteins or glycolipids. Examples of protein antigens are HER-2/neu, human papillomavirus-E6 or -E7, MUC-1; KS 1/4 pan-carcinoma antigen (Perez and Walker, 1990, J. Immunol. 142:3662-3667; Bumal, 1988, Hybridoma 7(4):407-415); ovarian carcinoma 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 [U.S. Pat. No. 7,202,346],3A5 [U.S. Pat. No. 7,723,485]).NMB (U.S. Pat. No. 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's 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. 34:422-430); oncofetal antigens such as alpha-fetoprotein for liver cancer or bladder tumor oncofetal antigen (Hellstrom et al., 1985, Cancer. Res. 45:2210-2188); differentiation antigens such as human lung carcinoma antigen L6 or L20 (Hellstrom et al., 1986, Cancer Res. 46:3917-3923); antigens of fibrosarcoma; human leukemia T cell antigen-Gp37 (Bhattacharya-Chatterjee et al., 1988, J. Immunol. 141:1398-1403); tumor-specific transplantation type of cell-surface antigen (TSTA) such as virally-induced tumor antigens including T-antigen, DNA tumor virus and envelope antigens of RNA tumor viruses; neoglycoproteins, 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 (Feizi, 1985, Nature 314:53-57) such as I(Ma) found in gastric adenocarcinomas, SSEA-1 found in myeloid cells, VEP8, VEP9, Myl, VIM-D5, M18 and M39 found in breast epithelial cancers, D156-22 found in colorectal cancer, TRA-1-85 (blood group H), C14 found in colonic adenocarcinoma, F3 found in lung adenocarcinoma, AH6 found in gastric cancer, Y hapten found in embryonal carcinoma cells, TL5 (blood group A), E1 series (blood group B) antigens found in pancreatic cancer, FC10.2 found in embryonal carcinoma cells, gastric adenocarcinoma antigen, CO-514 (blood group Lea) found in adenocarcinoma, NS-10 found in adenocarcinomas, CO-43 (blood group Leb), G49 found in A431 cells, 19.9 found in colon cancer; gastric cancer mucins; R24 found in melanoma, MH2 (blood group ALeb/Ley) found in colonic adenocarcinoma, 4.2, D1.1, OFA-1, GM2, OFA-2 and M1:22:25:8 found in embryonal carcinoma cells and SSEA-3 and SSEA-4. HMW-MAA (SEQ ID NO:433), also known as melanoma chondroitin sulfate proteoglycan, is a membrane-bound protein of 2322 residues which is overexpressed on over 90% of the surgically removed benign nevi and melanoma lesions (Camploi, et. al, Crit Rev Immunol.; 24:267, 2004). Accordingly it may be a potential target cell surface associated antigen.
Other example cancer antigens for targeting with fusion protein constructs of the present invention include (exemplary cancers are shown in parentheses): CD5 (T-cell leukemia/lymphoma), CA15-3 (carcinomas), CA19-9 (carcinomas), L6 (carcinomas), CA 242 (colorectal), placental alkaline phosphatase (carcinomas), prostatic acid phosphatase (prostate), MAGE-1 (carcinomas), MAGE-2 (carcinomas), MAGE-3 (carcinomas), MAGE-4 (carcinomas), transferrin receptor (carcinomas), p97 (melanoma), MUC1 (breast cancer), MART1 (melanoma), CD20 (non Hodgkin's lymphoma), CD52 (leukemia), CD33 (leukemia), human chorionic gonadotropin (carcinoma), CD38 (multiple myeloma), CD21 (B-cell lymphoma), CD22 (lymphoma), CD25 (B-cell Lymphoma), CD37 (B-cell lymphoma), CD45 (acute myeloblastic leukemia), HLA-DR (B-cell lymphoma), IL-2 receptor (T-cell leukemia and lymphomas), CD40 (lymphoma), various mucins (carcinomas), P21 (carcinomas), MPG (melanoma), Ep-CAM (Epithelial Tumors), Folate-receptor alpha (Ovarian), A33 (Colorectal), G250 (renal), Ferritin (Hodgkin lymphoma), de2-7 EGFR (glioblastoma, breast, and lung), Fibroblast activation protein (epithelial) and tenascin metalloproteinases (glioblastoma). Some specific, 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. 62(13):3736-42) or MDX-0060 (U.S. Patent Publication No. 2004-0006215) and mAbs against the 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).
In certain embodiments, useful antibodies can bind to a receptor or a complex of receptors expressed on a target cell. The receptor or receptor complex can comprise an immunoglobulin gene superfamily member, a major histocompatibility protein, a cytokine receptor, a TNF receptor superfamily member, a chemokine receptor, an integrin, a lectin, a complement control protein, a growth factor receptor, a hormone receptor or a neuro-transmitter receptor. Non-limiting examples of appropriate 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, CD134/0X40, 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 lectin. Examples of antibodies to CEA are shown in Table 2.
Antibodies that bind the CD22 antigen expressed on human B cells include, for example, HD6, RFB4, UV22-2, To15, 4KB128 and a humanized anti-CD22 antibody (hLL2) (see, e.g., 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).
Antibodies to CD33 include, for example, HuM195 (see, e.g., Kossman et al. (1999) Clin. Cancer Res. 5: 2748-2755; U.S. Pat. No. 5,693,761) and CMA-676 (see, e.g., Sievers et al., (1999) Blood 93: 3678-3684).
Illustrative 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).
Illustrative 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.
Illustrative anti-HM1.24 antibodies include, but are not limited to a mouse monoclonal anti-HM1.24 and a humanized anti-HM1.24 IgG1kappa antibody (see, e.g., Ono et al. (1999) Mol. Immuno. 36: 387-395).
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 encoding a transmembrane receptor. Several antibodies have been developed against Her-2/neu, including trastuzumab (e.g., HERCEPTIN™; Fornier et al. (1999) Oncology (Huntingt) 13: 647-58), TAB-250 (Rosenblum et al. (1999) Clin. Cancer Res. 5: 865-874), BACH-250 (Id.), TA1 (Maier et al. (1991) Cancer Res. 51: 5361-5369), and the 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.
A number of antibodies have been developed that specifically bind HER2 and some are in clinical use. These include, for example, trastuzumab (e.g., HERCEPTIN™, Fornier et al. (1999) Oncology (Huntingt) 13: 647-658), TAB-250 (Rosenblum et al. (1999) Clin. Cancer Res. 5: 865-874), BACH-250 (Id.), TA1 (see, e.g., Maier et al. (1991) Cancer Res. 51: 5361-5369), and the antibodies described in U.S. Pat. Nos. 5,772,997; 5,770,195, and 5,677,171.
Other fully human anti-HER2/neu antibodies are well known to those of skill in the art. Such antibodies include, but are not limited to the 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, C6MH3-B46, C6MH3-B43, C6MH3-B41, C6MH3-B39, C6MH3-B34, C6MH3-B33, C6MH3-B31, C6MH3-B27, C6MH3-B25, C6MH3-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 U.S. Pat. Nos. 6,512,097 and 5,977,322, in PCT Publication 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 the like.
More generally, antibodies directed 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-EGF-R antibodies as described in U.S. Pat. Nos. 5,844,093 and 5,558,864, and in 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 and the like (see, e.g., U.S. Patent publications US 2006/0099205 A1 and US 2004/0071696 A1 which are incorporated herein by reference).
It may be advantageous for the cell surface-associated antigen to be expressed at sufficient levels on the target cell that a sufficently therapeutic amount of polypeptide construct is presented to ligand receptors on the target cell surface. Accordingly, in particular embodiments, the cell surface associated antigen is expressed at a density of greater than 12,600 copies per cell or greater than 15,000 copies per cell. Methods for determining copy number of a cell surface antigen are well known and readily available to a person of skill in the art, for example the method provided by Jilana (Am J Clin Pathol 118:560-566, 2002)
It may be advantageous for the cell surface-associated antigen to be expressed in a configuration on the cell surface such that the polypeptide construct is able to contact both the cell surface antigen and the ligand receptor on the target cell. Accordingly, in particular embodiments the cell surface associated antigen has an extracelluar domain having a molecular weight of less than 240 kD.
It may be advantageous for the antibody or antigen-binding portion thereof to bind to the cell surface associated antigen with sufficient affinity to facilitate ligand binding to the ligand receptor on the cell surface. Accordingly, in particular embodiments of the the present invention the polypeptide constructs exhibit an antigen binding affinity, as measured by EC50, of from 50 nM, from 25 nM, from 10 nM or from 5 nM to 0.1 pM.
As described in U.S. Pat. Nos. 6,512,097 and 5,977,322, other anti-EGFR family member antibodies can readily be produced by shuffling light and/or heavy chains followed by one or more rounds of affinity selection. Thus in certain embodiments, this invention contemplates the use of one, two, or three CDRs in the VL and/or VH region that are CDRs described in the above-identified antibodies and/or the above identified publications.
In various embodiments the targeting antibody or antigen binding portion thereof comprises an antibody or antigen binding portion thereof that specifically or preferentially binds CD20. Anti-CD20 antibodies are well known to those of skill and include, but are not limited to Rituximab, Ibritumomab, and Tositumomab, AME-133v (Applied Molecular Evolution), Ocrelizumab (Roche), Ofatumumab (Genmab), TRU-015 (Trubion) and IMMU-106 (Immunomedics).
WO 2010/105290 discloses an antibody designated “SC104” together with a range of humanised variants which bind an antigen expressed on a range of tumour cells.
In an embodiment, the antibody attachment and attenuating mutation in the ligand increases the antigen-specificity index (ASI) by greater than 10-fold, preferably greater than 50-fold, preferably greater than 100-fold, preferably greater than 1000-fold, or preferably greater than 10,000 fold. The antigen-specificity index (ASI) is defined herein as the fold increased potency in signaling activity of the polypeptide construct of the invention relative to the free non-mutated polypeptide ligand on target antigen-positive cells multiplied by the fold decreased potency in signaling activity relative to the free non-mutated polypeptide ligand on target antigen-negative cells. The term “potency” in this context may be quantitatively represented by the EC50 value, which is the mathematical midpoint of a dose-response curve, in which the dose refers to the concentration of ligand or antibody-ligand construct in an assay, and response refers to the quantitative response of the cells to the signaling activity of the ligand at a particular dose. Thus, for example, when a first compound is shown to possess an EC50 (expressed for example in Molar units) that is 10-fold lower than a second compound's EC50 on the same cells, typically when measured by the same method, the first compound is said to have a 10-fold higher potency. Conversely, when a first compound is shown to possess an EC50 that is 10-fold higher than a second compound's EC50 on the same cells, typically when measured by the same method, the first compound is said to have a 10-fold lower potency.
While the large majority of antibodies tested showed efficient targeting of attenuated IFNα the present inventors identified examples of two antigens where targeting attenuated IFNα to a target-expressing cell line did not exhibit an ASI that was appreciably greater than for the free, non-mutated ligand. The first example is demonstrated by the antigen CSPG4 (also known as HMW-MAA, high molecular weight melanoma-associated antigen). We tested two different anti-HMW-MAA-antibody-IFNα fusion protein constructs in on-target proliferation assays using A375 or CHL-1 cell lines. We did not see inhibitory activity with either cell line or antibody at the doses tested (EC50s>21 nM). The extracelluar domain of this antigen is exceptionally large (extracellular domain MW approx. 240 kD-450 kD depending on glycosylation). It is possible that certain antibody-IFN fusion protein constructs that bind to very large antigens may be sterically restricted from simultaneously interacting with the IFN receptors on the same cells. It is, however, possible that other antibodies that target other epitopes of this antigen may support the targeted IFN activity. Despite this possibility it is preferred that the antibody or antigen binding portion thereof of the polypeptide construct of the present invention binds an antigen wherein the extracellular domain thereof has a molecular weight of less than 240 kD.
A second example of an antibody-attenuated IFNα fusion protein construct that did not show potent activity was based on an antibody which binds to the myeloid antigen CD33. CD33 is expressed at a relatively low level on KG-1 cells, reported as 12,600 copies per cell (Sutherland, MAbs. 1(5): 481-490, 2009). Treatment of KG-1 cells with an anti-CD33 antibody-attenuated IFNα fusion protein construct failed to inhibit proliferation at all doses tested (IC50>76 nM). We believe that the relatively low copy number of this target may in some cases, depending on other factors such as epitope position, the receptor density of the IFN receptors, etc, limit the potency of the antibody-attenuated IFN fusion protein constructs. It is, however, possible that other antibodies that target other epitopes on this antigen may support the targeted IFN activity, or that other cells with low copy numbers of CD33 may nevertheless respond to such fusion protein constructs due to higher intrinsic IFN sensitivity, for example. Despite this possibility it is preferred that the antibody or antigen binding portion thereof of the polypeptide construct of the present invention binds an antigen wherein the antigen is present on the cell at a density of greater than 12,600 copies per cell, preferably greater than 15,000 copies per cell.
Another example of an antibody-attenuated fusion protein construct in which the antibody did not provide sufficient targeting to the cancer cells was an anti-GM2 ganglioside antibody attached to an attenuated IFNα. In this case, the antibody was to a carbohydrate epitope and, as typical of such antibodies, had a low affinity (EC50 for binding target cells was ˜50 nM by flow cytometry). Therefore, preferred embodiments of the present invention show high affinity binding to their antigens, with EC50s preferably below 50 nM, more preferably below 25 nM, still more preferably below 10 nM and ideally below 5 nM. In addition, preferred embodiments comprise antibodies that bind to protein and peptide epitopes rather than carbohydrate epitopes.
Multiple myeloma is of particular interest for certain embodiments of the present invention, namely fusion protein constructs comprising antibodies to multiple myeloma antigens and attenuated IFN peptides. Table 3 lists examples of multiple myeloma antigens and antibodies, with a reference to antibody sequences.
CD38 is of particular interest as an antibody target for fusion protein constructs of the present invention. Antibodies to CD38 include for example, AT13/5 (see, e.g., Ellis et al. (1995) J. Immunol. 155: 925-937), HB7, and the like. Table 4 discloses several known CD38 antibodies that may be used in this context:
The term “Signaling ligand” as used herein broadly includes any ligand involved in the activation of cell signaling pathways, including any molecule capable of activating or inhibiting cell surface receptors. The term should also be understood as including reference to molecules that can pass through the lipid bilayer of the cell membrane to activate cell signaling pathways within the cell. The term “polypeptide signaling ligand” as used herein refers to peptide and polypeptide sequences of length 6 amino acids through 1,000 amino acids in length, which bind to particular cell surface molecules (“receptors”) on certain cells and thereby transmit a signal or signals within those cells. Exemplary signaling ligands and polypeptide signaling ligands contemplated by the present invention include, but are not limited to cytokines, chemokines, growth factors, hormones, neurotransmitters, and apoptosis inducing factors.
Non-limiting examples of suitable cytokines include the interleukin's IL-1, IL-2 IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL 25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35 and their subfamiles; the interferon (IFN) subfamily including Interferon type I (IFN-α (IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, IFNA21), IFN-β (IFN-β1 (IFNB1) and IFN-β3 (IFNB3)), IFN-ω ((IFNW1), IFNWP2, IFNWP4, IFNWP5, IFNWP9, IFNWP15, IFNWP18, and IFNWP19 and IFNK), Interferon type II (IFN-γ) and Interferon type III (IFN-epsilon, -kappa, -omega, -delta, -tau, and -gamma) and interferon-like molecules (limitin, IL-28A, IL-28B, and IL-29; the IL-1 family including IL-1α, IL-10, the IL-1 Receptor antagonist (IL-1RA) and IL1F5, IL1F6, IL1F7, IL1F8, IL1F9 and IL1F10 and the IL-17 family including IL-17A, IL-17B, IL-17C, IL-17D, IL-17E (IL-25), and IL-17F. In an embodiment the peptide or polypeptide signaling ligand is selected from the group consisting of an IFN, IL-4 and IL-6. In an embodiment the peptide or polypeptide signaling ligand is selected from the group consisting of IFNα, IFNα2b, IFNβ1, IFNβ1b and IFNγ. Preferably the sequence of IFNα is selected from SEQ ID NOs 1 to 3, 80 to 90, 434 and 435.
Exemplary chemokines include, for example, RANTES, MCAF, MIP1-alpha, IP-10, monocyte chemoattractant protein-1 (MCP-1 or CCL2), interleukin-8 (IL-8), CXCL13, XCL1 (lymphotactin-α), XCL2 (lymphotactin-β) and fractalkine (CX3CL1).
Non-limiting examples of growth factors include, for example, Adrenomedullin (AM), Angiopoietin (Ang), Autocrine motility factor, Bone morphogenetic proteins (BMPs), Brain-derived neurotrophic factor (BDNF), Epidermal growth factor (EGF), Erythropoietin (EPO), Fibroblast growth factor (FGF), Glial cell line-derived neurotrophic factor (GDNF), Granulocyte colony-stimulating factor (G-CSF), Granulocyte macrophage colony-stimulating factor (GM-CSF), Growth differentiation factor-9 (GDF9), Hepatocyte growth factor (HGF), Hepatoma-derived growth factor (HDGF), Insulin-like growth factor (IGF), Migration-stimulating factor, Myostatin (GDF-8), Nerve growth factor (NGF) and other neurotrophins, Platelet-derived growth factor (PDGF), Thrombopoietin (TPO), Transforming growth factor alpha(TGF-α), Transforming growth factor beta(TGF-β), Tumor_necrosis_factor-alpha(TNF-α), Vascular endothelial growth factor (VEGF), placental growth factor (P1GF), IL-1-Cofactor for IL-3 and IL-6, IL-2-T-cell growth factor, IL-3, IL-4, IL-5, IL-6 and IL-7.
Exemplary apoptosis inducing factors include FasL and TRAIL.
Exemplary hormones include peptide hormones such as TRH and vasopressin, protein hormones such as insulin and growth hormone, glycoprotein hormones such as Luteinizing hormone, follicle-stimulating hormone and thyroid-stimulating hormone, Lipid and phospholipid-derived hormones such as steroid hormones e.g. testosterone and cortisol, Sterol hormones such as calcitriol, eicosanoids such as prostaglandins.
Non-limiting examples of suitable neurotransmitters include monoamines and other biogenic amines: dopamine (DA), norepinephrine (noradrenaline; NE, NA), epinephrine (adrenaline), histamine, serotonin (SE, 5-HT), somatostatin, substance P, opioid peptides and acetylcholine (ACh),
The linkage between the antibody and the ligand could be made via a simple peptide bond by creating a fusion protein between the ligand and the heavy or light chain, or both, of the antibody. The ligand could be attached at either the N- or C-terminus of either the heavy or the light chain of the antibody, with or without an intervening linker peptide sequence. In an embodiment the ligand is linked to the antibody or antigen binding portion thereof via a peptide bond. In one embodiment, the ligand is linked to the C-terminus of the heavy chain of a human, humanized or chimeric IgG1, IgG2 or IgG4, either directly or with an intervening linker of 1 to 20 amino acids in length.
The mutated polypeptide ligands may be attached to the antibody or antibody fragment by means of chemical conjugation, non-covalent protein-protein interactions, or by genetic fusion. Methods for conjugating the ligands described herein with antibodies may be readily accomplished by one of ordinary skill in the art. As will be readily ascertained, commonly used chemical coupling methods may be utilized to link ligands to antibodies via for example, free amino, carboxylic acid, or sulfhydryl groups. Ligands can also be linked to antibodies via Carbonyls (—CHO); these aldehyde groups can be created by oxidizing carbohydrate groups in glycoproteins.
Some commonly used cross-linking reagents include glutaraldehyde which links protein or peptide molecules to the N-terminal or aliphatic amine groups of peptides or polypeptides, carbodiimide (EDC) which attaches proteins or peptides to the C-terminus or side chain carboxyl groups of proteins or peptides, succinimide esters (e.g. MBS, SMCC) which conjugates free amino groups and thiols from Cys residues, benzidine (BDB) which links to Tyr residues, periodate which attaches to carbohydrate groups and isothiocyanate. The use of commercial chemical conjugation kits is contemplated.
In some embodiments, labels are attached via spacer arms of various lengths to reduce potential steric hindrance. For example, a chemical linker may be used between the ligand and the antibody. Exemplary linker sequences will be readily ascertained by those of skill in the art, and are likely to include linkers such as C6, C7 and C12 amino modifiers and linkers comprising thiol groups.
The antibody-ligand fusion protein constructs of the present invention have mutations or deletions in the ligand that render the ligands less active in stimulating their receptors on cells that lack cell surface expression of the antigen to which the antibody binds.
In one aspect of the present invention, the ligand is an interferon, examples of which are type I interferons (IFN-α (alpha), IFN-β (beta), IFN-κ (kappa), IFN-δ (delta), IFN-ε (epsilon), IFN-τ (tau), IFN-ω (omega), and IFN-ζ (zeta, also known as limitin), type II interferons (IFN-γ) or type III interferons (IFN-λ1, IFN-λ2 and IFN-λ3) (Pestka, Immunological Reviews 202(1):8-32, 2004).
Type I interferons all signal through the Type I interferon receptor, which is made of of IFNAR1 and IFNAR2. Signaling occurs when a type I IFN binds IFNAR1 and IFNAR2, thus bringing them together into a complex with the IFN. This initiates a cascade of intracellular events (the “signaling”) which leads, among other things, to changes in the expression of numerous interferon regulated genes. Details of the intracellular signaling events triggered by activation of the type I interferon receptor is described, for example, by Platanias, (Nature Reviews 5:375-86. 2005). Type I interferons include various interferon-alphas. Known human interferon-alphas are IFNα1b, α2α, α2β, α4b, α5, α6, α7, α8, α10, α1a/13, α14, α16, α17, ανδ α21, α2c and α4a. Some embodiments comprise IFNα2b, the sequence of which, SEQ ID NO:3, is shown in
Non-limiting examples of mutations in IFNα2b that can be used to reduce its potency are described in Tables 6 and 7, based on the sequence of human IFNα2b (SEQ ID NO:3):
These mutants have known reductions in binding to the type 1 interferon receptor IFNAR1 or IFNAR2, and/or have known reductions in IFNα potency based on cell-based assays.
The data in these tables was disclosed in the following references:
The abbreviation “YNS” is sometimes used herein to represent IFNα variants including the following mutation: H57Y, E58N and Q61S.
The present invention also contemplates combinations of the abovementioned mutations or deletions in IFNα.
The invention also contemplates the combination of the constructs of the present invention with other drugs and/or in addition to other treatment regimens or modalities such as radiation therapy or surgery. When the contructs of the present invention are used in combination with known therapeutic agents the combination may be administered either in sequence (either continuously or broken up by periods of no treatment) or concurrently or as an admixture. In the case of cancer, there are numerous known anticancer agents that may be used in this context. Treatment in combination is also contemplated to encompass the treatment with either the construct of the invention followed by a known treatment, or treatment with a known agent followed by treatment with the construct of the invention, for example, as maintenance therapy. For example, in the treatment of cancer it is contemplated that the constructs of the present invention may be administered in combination with an alkylating agent (such as mechlorethamine, cyclophosphamide, chlorambucil, ifosfamidecysplatin, or platinum-containing alkylating-like agents such as cysplatin, carboplatin and oxaliplatin), an antimetabolite (such as a purine or pyrimidine analogue or an antifolate agent, such as azathioprine and mercaptopurine), an anthracycline (such as Daunorubicin, Doxorubicin, Epirubicin Idarubicin, Valrubicin, Mitoxantrone, or anthracycline analog), a plant alkaloid (such as a vinca alkaloid or a taxane, such as Vincristine, Vinblastine, Vinorelbine, Vindesine, paclitaxel or Dosetaxel), a topoisomerase inhibitor (such as a type I or type II topoisomerase inhibitor), a Podophyllotoxin (such as etoposide or teniposide), or a tyrosine kinase inhibitor (such as imatinib mesylate, Nilotinib, or Dasatinib).
In the case of the treatment of multiple myeloma, it is contemplated that the constructs of the present invention may be administered in combination with current therapies, such as steroids such as dexamethasone, proteasome inhibitors (such as bortezomib or carfilzomib), immunomodulatory drugs (such as thalidomide, lenalidomide or pomalidomide), or induction chemotherapy followed by autologous haematopoietic stem cell transplantation, with or without other chemotherapeutic agents such as Melphalan hydrochloride or the chemotherapeutic agents listed above.
In the case of the treatment of Hodgkin's lymphoma, it is contemplated that the constructs of the present invention may be administered in combination with current therapeutic approaches, such as ABVD (Adriamycin (doxorubicin), bleomycin, vinblastine, and dacarbazine), or Stanford V (doxorubicin, bleomycin, vinblastine, vincristine, mechlorethamine, etoposide, prednisone), or BEACOPP (doxorubicin, bleomycin, vincristine, cyclophosphamide, procarbazine, etoposide, prednisone).
In the case of non-Hodgkin's lymphoma or other lymphomas, it is contemplated that the constructs of the present invention may be administered in combination current therapeutic approaches. Examples of drugs approved for non-Hodgkin lymphoma include Abitrexate (Methotrexate), Adriamycin PFS (Doxorubicin Hydrochloride), Adriamycin RDF (Doxorubicin Hydrochloride), Ambochlorin (Chlorambucil), Amboclorin (Chlorambucil), Arranon (Nelarabine), Bendamustine Hydrochloride, Bexxar (Tositumomab and Iodine I 131 Tositumomab), Blenoxane (Bleomycin), Bleomycin, Bortezomib, Chlorambucil, Clafen (Cyclophosphamide), Cyclophosphamide, Cytoxan (Cyclophosphamide), Denileukin Diftitox, DepoCyt (Liposomal Cytarabine), Doxorubicin Hydrochloride, DTIC-Dome (Dacarbazine), Folex (Methotrexate), Folex PFS (Methotrexate), Folotyn (Pralatrexate), Ibritumomab Tiuxetan, Istodax (Romidepsin), Leukeran (Chlorambucil), Linfolizin (Chlorambucil), Liposomal Cytarabine, Matulane (Procarbazine Hydrochloride), Methotrexate, Methotrexate LPF (Methotrexate), Mexate (Methotrexate), Mexate-AQ (Methotrexate), Mozobil (Plerixafor), Nelarabine, Neosar (Cyclophosphamide), Ontak (Denileukin Diftitox), Plerixafor, Pralatrexate, Rituxan (Rituximab), Rituximab, Romidepsin, Tositumomab and Iodine I 131 Tositumomab, Treanda (Bendamustine Hydrochloride), Velban (Vinblastine Sulfate), Velcade (Bortezomib), and Velsar (Vinblastine Sulfate), Vinblastine Sulfate, Vincasar PFS (Vincristine Sulfate), Vincristine Sulfate, Vorinostat, Zevalin (Ibritumomab Tiuxetan), Zolinza (Vorinostat). Examples of drug combinations used in treating non-Hodgkin lymphoma include CHOP (C=Cyclophosphamide, H=Doxorubicin Hydrochloride (Hydroxydaunomycin), O=Vincristine Sulfate (Oncovin), P=Prednisone); COPP (C=Cyclophosphamide, O=Vincristine Sulfate (Oncovin), P=Procarbazine Hydrochloride, P=Prednisone); CVP (C=Cyclophosphamide, V=Vincristine Sulfate, P=Prednisone); EPOCH (E=Etoposide, P=Prednisone, O=Vincristine Sulfate (Oncovin), C=Cyclophosphamide, H=Doxorubicin Hydrochloride (Hydroxydaunomycin)); ICE (I=Ifosfamide, C=Carboplatin, E=Etoposide) and R-CHOP (R=Rituximab, C=Cyclophosphamide, H=Doxorubicin Hydrochloride (Hydroxydaunomycin), O=Vincristine Sulfate (Oncovin), P=Prednisone.
Combination of retinoids with interferon-based fusion protein constructs is also contemplated. Retinoids are a family of molecules that play a major role in many biological functions including growth, vision, reproduction, epithelial cell differentiation and immune function (Meyskens, F. et al. Crit Rev Oncol Hematol 3:75, 1987, Herold, M. et al. Acta Dermatovener 74:29 1975). Early preclinical studies with the retinol all-trans retinoic acid or ATRA, either alone or in combination with other agents, demonstrated activity against acute promyelocytic leukemia (APL), myelodysplastic syndrome, chronic myelogenous leukemia (CML), mycosis fungoides and multiple myeloma (reviewed in Smith, M. J. Clin. Oncol. 10:839, 1992). These studies led to the approval of ATRA for the treatment of APL. Currently there are over 100 clinical trials evaluating the activity of ATRA in combination with other therapies for the treatment of hematological malignancies, kidney cancers, lung cancers, squamous cell carcinomas and more. Of particular interest and pertaining directly to this invention are the studies demonstrating enhanced efficacy of interferon-α treatment when combined with ATRA. This is described for mantle cell lymphoma (Col, J. et al. Cancer Res. 72:1825, 2012), renal cell carcinoma (Aass, N. et al. J. Clin. Oncol. 23:4172, 2005; Motzer, R. J. Clin. Oncol. 18:2972, 2000), CML, melanoma, myeloma and renal cell carcinoma (Kast, R. Cancer Biology and Therapy, 7:1515, 2008) and breast cancer (Recchia, F. et al. J. Interferon Cytokine Res. 15:605, 1995). We would therefor predict enhanced activity of our targeted attenuated IFNs when combined with therapeutic dosing of ATRA in the clinic. In addition, Mehta (Mol Cancer Ther 3(3):345-52, 2004) demonstrated that in vitro treatment of leukemia cells with retinoic acid induced expression of CD38 antigen. Thus, the enhanced efficacy of interferon plus the induced expression of the target CD38 would indicate a combination therapy of ATRA with our anti-CD38 antibody-attenuated IFNα in the treatment of IFN-sensitive cancers that express CD38 or may be induced by ATRA to express CD38. Example of such cancers are multiple myeloma, non-Hodgekin's lymphoma, CML and AML.
In addition, while the above constructs are based on IFNα2b, the mutations or deletions could also be made in the context of any of the other IFNαs or IFNβ. In another embodiment of the present invention, the type I IFN is an IFNβ. IFN-β is approved for the treatment of multiple sclerosis (MS). IFN-β could be attenuated by mutation or deletion and then attached to an antibody that targets cells involved in the pathogenesis of this disease. IFN-β is an effective drug in MS, but its use is associated with adverse events, including injection site inflammation, flu-like symptoms, leukocytopenia, liver dysfunction and depression, leading to discontinuation in a subset of patients. By directing IFN-β activity directly to pathogenic cells, these adverse events may be avoided.
Pathogenesis of MS is thought to be initiated and progressed by a number of events, including innate activation of dendritic and microglial cells through toll-like receptors, an imbalance between pro-inflammatory and anti-inflammatory/regulatory cytokines, differentiation of CD4+ T cells into Th1 and Th17 phenotypes, activation of Th1 cells by antigen presenting cells (APCs), reduction in the number of regulatory T (Treg) cells and migration of activated immune cells across the blood-brain barrier (BBB). The primary drivers of the clinical episodes of the disease are thought to be autoreactive, myelin-specific Th1 cells (reviewed in Gandhi, 2010 J Neuroimmunol 221:7; Boppana, 2011 Mt Sinai J Med 78:207; Loma, 2011 Curr Neuropharmacol 9:409).
In an embodiment of the invention, an attenuated version of IFN-β may be attached to an antibody targeting a cell surface marker specific for T cells, for the treatment of multiple sclerosis or other autoimmune indications where IFN-β may be effective. Direct effects of IFN-β on T cells include inhibition of proliferation (Rep, 1996 J Neuroimmunol 67:111), downregulation of the co-stimulatory molecule CD40L (Teleshova, 2000 Scand J Immunol. 51:312), decrease of metaloproteinase activity leading to reduced migration across the BBB (Stuve, 1996 Ann Neurol 40:853; Uhm, 1999 Ann Neurol 46:319), induction of apoptosis by upregulating intracellular CTLA-4 and cell surface Fas molecules (Hallal-Longo, 2007 J Interferon Cytokine Res 27:865), downregulation of anti-apoptotic proteins (Sharief, 2001 J Neuroimmunol. 120:199; Sharief, 2002 J Neuroimmunol. 129:224), and restoration of Treg function (De Andres, 2007 J Neuroimmunol 182:204; Korporal, 2008 Arch Neurol 65:1434; Sarasella, 2008 FASEB J 22:3500; Chen, 2012 J Neuroimmunol 242:39).
Therefore, in one aspect of the present invention, an attenuated IFN-β is attached to an anti-CD3 antibody that targets all T cells, which includes CD4+, CD8+, Treg, Th1, Th2 and Th17 cells. This comprehensive approach ensures full coverage of all T cells, as all of these cell types have reported roles in MS pathogenesis and are affected by IFN-β treatment (Dhib-Jalbut, 2010 Neurology 74:S17; Prinz, 2010 Trends Mol Med 16:379; Graber, 2010 Clin Neurol Neurosurg 112:58 and Loma, 2011 Curr Neuropharmacol 9:409). Examples of CD3 antibodies that may be incorporated into the fusion protein constructs of the present invention are listed in Table 8.
Alternatively, an attenuated IFN-β-anti-CD4 fusion protein construct presents a more restrictive approach, but would target autoreactive and regulatory T cells, including Th1 and Th17 cells and CD4+CD25+ Treg cells. In addition, subsets of dendritic cells (DCs) also express CD4 and direct therapeutic effects of IFN-β on DCs have been disclosed (Shinohara, 2008 Immunity 29:68; Dann, 2012 Nat Neurosci 15:98). Examples of CD4 antibodies that may be incorporated into the fusion protein constructs of the present invention are listed in Table 9.
A role for CD8+ T cells in MS has been reported (Friese, 2005 Brain 128:1747; Friese, 2009 Ann Neurol 66:132), as well as a direct effect of IFN-β on CD8+ T cells in MS patients (Zafranskaya, 2006 Immunol 121:29). Therefore, directing an attenuated IFN-β directly to CD8+ T cells with an anti-CD8 antibody may result in clinical benefits for MS patients. Examples of CD8 antibodies are shown in Table 10.
Markers of activated T cells, including, but not limited to CD25, CD38, CD44, CD69, CD71, CD83, CD86, CD96, HLA-DR, ICOS and PD-1, also represent attractive targets for this approach, since activated T cells are thought to be the main drivers of autoreactivity resulting in demyelination in MS (Gandhi, 2010 J Neuroimmunol 221:7; Boppana, 2011 Mt Sinai J Med 78:207; Loma, 2011 Curr Neuropharmacol 9:409). Antibodies targeting any of these antigens could be attached to an attenuated IFNβ. Examples antibodies that could be used in the present invention include the following: CD71 antibodies include BA120g (U.S. Pat. No. 7,736,647) and various antibodies mentioned in Wang et al (Di Yi Jun Yi Da Xue Xue Bao (Academic journal of the first medical college of PLA) 22(5):409-411, 2002). Examples of antibodies to CD83 include 20B08, 6G05, 20D04, 11G05, 14C12, 96G08 and 95F04 (U.S. Pat. No. 7,700,740). An example of an antibody to CD86 includes 1G10H6D10 (U.S. Pat. No. 6,071,519). HLA-DR antibodies include HD3, HD4, HD6, HD7, HD8 and HD10 (U.S. Pat. No. 7,262,278), DN1921 and DN1924 (US2005/0208048). One attractive target along these lines could be PD-1, which is expressed on recently activated T cells. Ideally, a non-antagonizing antibody could be used, such as the J110 antibody discussed in further detail below.
Examples of antibodies to ICOS include JMabs (U.S. Pat. No. 6,803,039) and JMab 136 (US2011/0243929).
Further of these examples of antibodies to these targets are shown in the Tables 11 and 12
In another embodiment of the invention, an attenuated version of IFN-β can be fused to an antibody targeting cell surface markers of myeloid cells, known to contribute to MS pathogenesis by driving T cell activation and differentiation. For example, the pan-myeloid markers CD33, CD115, or the dendritic cell marker CD11c may be targeted. A broad targeting approach may be preferred, for example, using antibodies against CD33 or CD115, since the exact contribution of each of the myeloid cell subsets to MS disease pathogenesis and response to IFN-β has been disputed (Prinz, 2008 Immunity 28:675; Shinohara, 2008 Immunity 29:68; Dann, 2012 Nat Neurosci 15:98). Antibodies to CD33 that could be used in the present invention include My9-6 (U.S. Pat. No. 7,557,189), any of 14 antibodies described in US patent application US2012/0082670, or the antibody known as huM195 (U.S. Pat. No. 5,693,761). Antibodies to CD115 that could be used include Ab1 and Ab 16 (U.S. Pat. No. 8,206,715) or CXIIG6 (US2011/0178278). An example of a CD11c antibody that could be used according to the present invention is mab 107 (U.S. Pat. No. 7,998,738, ATCC deposit number PTA-11614). The attenuated IFN-β could alternatively be directed to the CD14 antigen, present primarily on macrophages. Examples of CD14 antibodies are shown in Table 13.
In yet another embodiment, targeting CD52-expressing cells would deliver IFN-β to all lymphocytes and, in addition, to monocytes and peripheral dendritic cells (Buggins, 2002 Blood 100:1715; Ratzinger, 2003 Blood 101:1422), which are the key APCs responsible for proliferation and differentiation of autoreactive T cells in MS. This approach would direct the activity of IFN-β to the key cell types known to be directly affected by IFN-β and would facilitate its therapeutic activity in MS. Examples of CD52 antibodies that could be used according to the present invention include, but are not limited to DIVHv5/DIVKv2 (U.S. Pat. No. 7,910,104), any of the CD52 antibodies disclosed in (US2012/0100152) or CAMPATH.
Any of the above mentioned, antibody-targeted attenuated IFNβ fusion protein constructs may have therapeutic activity in the context of other inflammatory and autoimmune diseases beyond multiple sclerosis, due to their common underlying immunological etiologies.
Autoimmune diseases contemplated herein include inter alia alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease multiple sclerosis, autoimmune disease of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue syndrome (CFIDS), chronic inflammatory demyelinating, chronic inflammatory polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, crest syndrome, cold agglutinin disease, Crohn's disease, irritable bowel syndrome, inflammatory bowel disease, dermatitis herpetiformis, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia, glomerulonephritis, Grave's disease, Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA nephropathy, insulin dependent diabetes (Type I), lichen planus, lupus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, myocarditis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglancular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, pochitis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arrthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-man syndrome, systemic lupus erythematosus, Takayasu arteritis, temporal arteritis/giant cell arteritis, ulcerative colitis, uveitis, vasculitis and vitiligo. Of particular interest is Behcet's disease and chronic uveitic macular edema and other types of uveitis, since IFNα has been shown to render therapeutic benefit (Deuter, Dev Ophthalmol. 51:90-7. 2012)
Examples of inflammatory disease conditions contemplated by the present disclosure include but are not limited to those disease and disorders which result in a response of redness, swelling, pain, and a feeling of heat in certain areas that is meant to protect tissues affected by injury or disease. Inflammatory diseases which can be treated using the methods of the present disclosure, include, without being limited to, acne, angina, arthritis, aspiration pneumonia, disease, empyema, gastroenteritis, inflammation, intestinal flu, NEC, necrotizing enterocolitis, pelvic inflammatory disease, pharyngitis, PID, pleurisy, raw throat, redness, rubor, sore throat, stomach flu and urinary tract infections, chronic inflammatory demyelinating polyneuropathy, chronic inflammatory demyelinating polyradiculoneuropathy, chronic inflammatory demyelinating polyneuropathy, chronic inflammatory demyelinating polyradiculoneuropathy.
The sequence of human interferon-β1 is shown below: (SEQ ID NO: 191)
Using the numbering scheme above (residues 1-166), known mutations (at positions indicated by asterisks) in human IFNβ that reduce its activity include those listed in Table 14.
References:
In still another embodiment of the present invention, the IFN is IFN-λ (WO 2007/029041 A2), which may be used for any of the applications described more thoroughly for IFNα, or IFNβ.
Type I IFNs can have anti-cancer activity based on a direct stimulation of the type I IFN receptor on cancer cells. This has been shown for numerous types of cancer including multiple myeloma, melanoma, B cell lymphoma, non-small cell lung cancer, renal cell carcinoma, hairy cell leukemia, chronic myelogenous leukemia, ovarian cancer, fibrosarcoma, cervical cancer, bladder cancer, astrocytoma, pancreatic cancer, etc (Borden, Cancer Research 42:4948-53, 1982; Chawla-Sarkar, Clinical Cancer Research 7: 1821-31, 2001; Morgensen, Int J. Cancer 28:575-82, 1981; Otsuka, British Journal of Haematology 103:518-529, 1998; Lindner, J of Interferon and Cytokine Research 17:681-693, 1997; Caraglia, Cell Death and Differentiation 6:773-80, 1999; Ma, World J Gastroenterol 11(10):1521-8, 2005). One of skill in the art will recognize that the present invention has many aspects resulting from combining antibodies to tumor associated antigens with mutated type I interferons, and that the resulting fusion protein constructs may be used to reduce the proliferation of various interferon-sensitive cancers that express the corresponding tumor associated antigens. It will also be appreciated that type I interferons can be combined with other agents to further improve their effectiveness.
Type I interferons can also display anti-viral properties. IFNα2b, for example, has been FDA-approved for the treatment of chronic hepatitis C infections, and may have utility in treating other viral infections as well. Pegylated IFN-α is currently part of the standard of care regimen for hepatitis C, according to American and European guidelines, but results in side effects in over 80% of patients, often leading to discontinuation of treatment (Aman, 2012; Calvaruso, 2011). In one aspect of the present invention, a type I IFN with an attenuating mutation is attached to an antibody that binds to virally infected cells. The antigen to be recognized by the above referenced antibody could be a viral protein that is transiently expressed on the host cell surface, or it could be an endogenous host cell-produced antigen that is exposed on the cell surface to a greater extent after viral infection than before infection. Exemplary viral proteins that could serve as targets for the antibody portion include but are not limited to, Hepatitis C viral envelope glycoproteins, E1 and E2; Hepatitis B surface antigen (HBsAg); Herpes virus viral envelope glycoproteins B, C, D, E, G, H, I, J, K, L, M and UL32, and envelope protein UL49A; Human immune deficiency virus (HIV) Envelope proteins glycoprotein (gp) 120 and gp41; Adenoviruses knob domain of the fiber protein; Varicella-zoster virus envelope glycoproteins (gB, gC, gE, gH, gI, gK, gL); Epstein-barr virus viral glycoprotein gp350 and viral protein BMRF-2; Human cytomegalovirus UL16; Parvovirus B19 viral capsid proteins VP1-3; Human astrovirus structural proteins, e.g. VP26, VP29 and VP32; Noroviruses structural protein VP1 and capsid protein VP2; Poliovirus viral capsid proteins VP0, VP1, VP2, VP3 and VP4; Rhinovirus viral capsid proteins VP1, VP2, VP3 and VP4; and dengue virus virus particle proteins capsid (C), pre-membrane/membrane (prM/M) and envelope (E).
In one embodiment, IFN-α activity may be targeted with an antibody that binds, directly or indirectly via an intermediate protein such as annexin V or beta2-glycoprotein 1, to phosphatidylserine (PS), a phospholipid component of the inner leaflet of cellular membranes. Cells undergoing apoptosis, however, or cells infected with viruses, expose PS on the outer membrane, where it becomes accessible to antibodies. PS is exposed on the surface of cancer cells (Reidl, L. et al., J Immunol. 14:3623, 1991), the vascular endothelium in tumors (Ran, S. et al., Cancer Res. 62:6132. 2002; He, J. et al., Clin Cancer Res. 15:6871, 2009), and virus-infected cells (Soares, M. et al., Nat Med. 14:1357, 2008). An antibody indirectly (via beta2 glycoprotein 1) targeting PS, bavituximab, has been described. It mediates antibody-dependent cytotoxicity and is effective in a number of in vivo cancer models, including human breast and lymphoma xenografts and a rat glioblastoma model, as well as in viral disease models (Ran, S. et al., Clin Cancer Res; 11:1551, 2005; He, J. et al., Clin Cancer Res. 15:6871, 2009; Soares, M. et al., Nat Med. 14:1357, 2008). Currently, it is being developed as a therapeutic antibody for lung cancer treatment (DeRose, P. et al., Immunotherapy. 3:933, 2011; Gerber, D. et al., Clin Cancer Res. 17:6888, 2011). Alternative antibodies may be based on the variable regions from the anti-PS antibody 9D2 (Cancer Res Nov. 1, 2002 62; 6132). Yet another alternative for targeting PS would be to replace the antibody Fab portions with a natural PS-binding protein such annexin V or beta2-glycoprotein 1. An anti-PS antibody (or alternatively a direct or indirect PS binding protein) fused with an attenuated version of IFN-α would target IFN-α activity to PS expressing virus-infected cells without displaying the systemic safety issues related to IFN-α. Certain tumor cells, such as lung cancer cells, also express PS on their cell surfaces, so an antibody (or alternatively a direct or indirect PS binding protein) to PS, attached to an attenuated IFN, could also have use in the treatment of certain cancers.
It should be understood that antibody-targeted attenuated IFNλ could also be used in much the same way as IFNα for the targeting virally infected cells (S. V. Kotenko, G. Gallagher, V. V. Baurin et al., “IFN-λs mediate antiviral protection through a distinct class II cytokine receptor complex,” Nature Immunology, vol. 4, no. 1, pp. 69-77, 2003).
In one embodiment Type II IFNs, namely INFγ, may also be attenuated and attached to antibodies that direct them to specific cell types. IFNγ has anti-proliferative properties towards cancer cells (Kalvakolanu, Histol. Histopathol 15:523-37, 2000; Xu, Cancer Research 58:2832-7, 1998; Chawla-Sarkar, Apoptosis 8:237-49, 2003; Schiller, J Interferon Resarch 6:615-25, 1986). Sharifi has described how to make a fusion protein in which an IFNγ has been fused to the C-terminus of a tumor-targeting antibody (Sharifi, Hybridoma and Hybridomics 21(6):421-32, 2002). In this reference, Sharifi disclosed how to produce antibody-IFNγ fusion proteins in mammalian cells and showed that both the antibody and the IFN were functional. Alternatively, a single-chain dimer version of IFNγ, as described by Lander (J Mol Biol. 2000 May 26; 299(1):169-79) may be used in the fusion protein. In addition to IFNγ's anti-proliferative effect on the targeted tumor cells, it may also have another effect specifically on breast cancer cells: IFNγ has been shown to restore antiestrogen sensitivity to breast cancer cells (Mol Cancer Ther. 2010 May; 9(5): 1274-1285) and so an attenuated-IFNγ attached to a breast cancer antigen antibody may be therapeutically useful in combination with antiestrogen therapy. By attenuating IFNγ via mutation, a more cancer-selective form of IFNγ may be produced. Two attenuating mutations in IFNγ have been described by Waschutza (Eur J. Biochem. 256:303-9, 1998), namely des-(A23, D24), in which residues A23 and D24 are deleted, and des-(N25, G26), in which residues N25 and G26 are deleted. The des-(A23, D24) mutant has an ˜18-fold reduced affinity for the IFNγ receptor compared to wild type IFNγ, and had a ˜100-fold reduced antiviral activity compared to wild type IFNγ. The des-(N25, G26) variant had a ˜140-fold reduced affinity for the IFNγ receptor compared to wild type IFNγ, and had a ˜10-fold reduced antiviral activity compared to wild type IFNγ. Examples of fusion proteins comprising antibodies to tumor cell surface targets and attenuated mutants of IFNγ include the following: Rituximab may be used as fusion protein with one of these attenuated IFNγ using a 7 amino acid linker described by Sharifi to produce the fusion protein construct “Rituximab-HC-L7-IFNγ(Δ[A23,D24]) IgG1,” composed of SEQ ID NOS:378 (heavy chain) and 276 (light chain)). Such a fusion protein construct would be expected to have potent anti-proliferative activity against CD20 malignancies such as B cell lymphomas. Other attenuated mutants of IFNγ that may be appropriate for fusing to a cell-targeting antibody were described by Lundell (J Biol. Chem. 269(23):16159-62, 1994), namely S20I (˜50× reduced affinity), D21K (˜100× reduced affinity), A23Q (˜2,500-fold reduced binding), A23V (˜2,000-fold reduced binding) and D24A (˜4-fold reduced binding). These attenuated IFNγ may be used as fusions in combination with anti-CD38 antibodies, to generate the fusion protein construct “X355/02-HC-L7-IFNγ(S20I) IgG1” (composed of SEQ ID NOS:380 (heavy chain) and 226 (light chain)) or “R10A2-HC-L7-IFNγ(D21K) IgG1” (composed of SEQ ID NOS:382 (heavy chain) and 270 (light chain)). Other attenuating mutations in IFNγ that may be exploited for the current invention were described by Fish (Drug Des Deliv. 1988 February; 2(3):191-206.)
Targeted attenuated IFNγ may also be used to treat various indications characterized by pathological fibrosis, including kidney fibrosis, liver fibrosis and idiopathic pulmonary fibrosis (IPF). IPF is a chronic, progressive form of lung disease, characterized by fibrosis of unknown cause, occurring primarily in older adults. Despite the medical need, there has been little progress in the development of effective therapeutic strategies (O'Connell, 2011 Adv Ther 28:986). Pulmonary fibrosis can also be induced by exposure to drugs, particles, microorganisms or irradiation. The following relates to both IPF and lung fibrosis induced by known agents and potentially for treatment of fibrosis in other types of organs, including liver and kidney.
Fibroblasts play a key role in fibrotic diseases of the lung and their activation leads to collagen disposition, resulting in excessive scarring and destruction of the lung architecture. Yet there is little information on the origin of these pathogenic fibroblasts, though several precursor cell types have been proposed, including bone marrow progenitors, monocytes, circulating fibrocytes, and endogeneous cells, such as resident mesenchymal and epithelial cells (Stevens, 2008 Proc Am Thorac Soc 5:783; King, 2011 Lancet 378:1949).
CD14+ monocytes from peripheral blood are able to differentiate into fibrocytes, the precursors of fibroblasts, and this process is inhibited by interferon-γ (IFN-γ). A direct effect of IFN-γ on monocytes was demonstrated in in vitro differentiation studies, supporting the strategy of targeting an attenuated form of IFN-γ to CD14+ monocytes for the treatment of fibrotic disease (Shao, 2008 J Leukoc Biol 83:1323).
Experimental evidence exists that IFN-γ is capable of inhibiting proliferation and activation of fibroblasts (Rogliani, 2008 Ther Adv Respir Dis 2:75) and this fact has exploited successfully in preclinical models to reduce scaring and fibrosis. Clinical trials in IPF patients studying the benefit of subcutaneously administered IFN-γ failed to reach primary endpoints for survival benefits (O'Connell, 2011 Adv Ther 28:986; King, 2011). Current approaches focus on direct delivery of recombinant IFN-γ through inhalation of an aerosol form (Diaz, 2012 J Aerosol Med Pulm Drug Deliv 25:79), such that the lungs may achieve sufficient IFN-γ activity to produce benefit at an overall safe systemic dose.
Delivering IFN-γ activity directly to fibroblasts could be a powerful method to increase clinical response to this agent and at the same time reduce its side effects. Fusing attenuated IFN-γ to antibodies targeting fibroblast specific markers could facilitate this approach. There are several fibroblast cell surface molecules that are enriched in fibroblasts. These include, for example, fibroblast specific protein (FSP1; Strutz, 1995 J Cell Biol 130:393), fibroblast activation protein (FAP; Park, 1999 J Biol Chem 274:36505; Acharya, 2006 Hum Pathol 37:352), and platelet derived growth factor receptors (PDGFR-α and -β; Trojanowska, 2008 Rheumatology (Oxford) 47S5:2). Expression of these molecules is elevated in lung biopsies obtained from IPF patients and they have been directly implicated as drug targets in IPF or its pathogenesis (Lawson, 2005 Am J Respir Crit Care Med 171:899; Acharya, 2006 Hum Pathol 37:352; Abdollahi, 2005 J Exp Med 201:925). Examples of antibodies to FAP and the PDGF receptors are shown in Tables 15 and 16.
In a preclinical model of liver fibrosis, IFN-γ was delivered to hepatic stellate cells, the equivalent of fibroblasts and responsible for secreting collagen in liver fibrosis, through liposomes targeting PDGFR-β, thereby enhancing the anti-fibrotic effects of IFN-γ (Li, 2012 J Control Release 159:261). These data support the concept and the potential therapeutic benefit gained by delivering IFN-γ activity directly to fibroblasts in fibrotic diseases, including IPF and liver fibrosis, and validate PDGFR-β as a target for this approach.
The present invention also contemplates the attenuation and antibody-based targeting of type III IFNs, including IFNλ1 (IL29), IFNλ2 (IL28A), and IFNλ3 (IL28B) (S. V. Kotenko, G. Gallagher, V. V. Baurin et al., “IFN-λs mediate antiviral protection through a distinct class II cytokine receptor complex,” Nature Immunology, vol. 4, no. 1, pp. 69-77, 2003, P. Sheppard, W. Kindsvogel, W. Xu, et al., “IL-28, IL-29 and their class II cytokine receptor IL-28R,” Nature Immunology, vol. 4, no. 1, pp. 63-68, 2003). These IFNs act through receptors composed of the IFNλR1 chain (also known as IL28Rα) and the IL10R2 chain (shared with IL10, IL22, and IL26 receptor complexes [A. Lasfar, W. Abushahba, M. Balan, and K. A. Cohen-Solal, “Interferon lambda: a new sword in cancer immunotherapy,” Clinical and Developmental Immunology, vol. 2011, Article ID 349575, 11 pages, 2011]). IFNλRs are expressed on most cell types and mediate similar signalling pathways as the type I IFNs. The antiviral activity of λ IFNs has been demonstrated against several viruses including HBV and HCV (E. M. Coccia, M. Severa, E. Giacomini et al., “Viral infection and toll-like receptor agonists induce a differential expression of type I and A interferons in humans plasmacytoid and monocyte-derived dendritic cells,” European Journal of Immunology, vol. 34, no. 3, pp. 796-805, 2004; M. D. Robek, B. S. Boyd, and F. V. Chisari, “Lambda interferon inhibits hepatitis B and C virus replication,” Journal of Virology, vol. 79, no. 6, pp. 3851-3854, 2005; N. Ank, H. West, C. Bartholdy, K. Eriksson, A. R. Thomsen, and S. R. Paludan, “Lambda interferon (IFN-λ), a type III IFN, is induced by viruses and IFNs and displays potent antiviral activity against select virus infections in vivo,” Journal of Virology, vol. 80, no. 9, pp. 4501-4509, 2006; S. E. Doyle, H. Schreckhise, K. Khuu-Duong et al., “Interleukin-29 uses a type 1 interferon-like program to promote antiviral responses in human hepatocytes,” Journal of Hepatology, vol. 44, no. 4, pp. 896-906, 2006; T. Marcello, A. Grakoui, G. Barba-Spaeth et al., “Interferons α and λ inhibit hepatitis C virus replication with distinct signal transduction and gene regulation kinetics,” Gastroenterology, vol. 131, no. 6, pp. 1887-1898, 2006). Clinical studies with IFNλ for the treatment of hepatitis C have shown promise (E. L. Ramos, “Preclinical and clinical development of pegylated interferon-lambda 1 in chronic hepatitis C,” Journal of Interferon and Cytokine Research, vol. 30, no. 8, pp. 591-595, 2010). One aspect of the present invention is to target a mutated, attenuated for of an IFNλ towards virally infected cells, using for example the targeting antibodies describe above for the targeting of an attenuated form of IFNα. Mutated, attenuated forms of an IFNλ could also be used to target cancer cells, as described in more detail for IFNα, above.
Non-IFN ligands are also contemplated in the present invention and may also be attenuated by mutation and then targeted to specific cell types by antibodies or fragments thereof. The anti-inflammatory cytokine interleukin-10 (IL-10) plays a central role during innate and adaptive immune responses. IL-10 forms a homodimer and binds to the IL-10 receptor complex expressed on APCs, leading to reduced expression of MHC class II and reduced production of pro-inflammatory cytokines and chemokines, thereby inhibiting T cell development and differentiation. However, IL-10 has also been implicated in inducing the proliferation of several immune cells, including B cells (Hofmann, 2012 Clin Immunol 143:116).
Reduced expression of IL-10 is associated with a number of autoimmune disorders in humans and rodents, including psoriasis, inflammatory bowel disease and rheumatoid arthritis. Mice deficient in IL-10 develop chronic enterocolitis, which can be prevented by the administration of IL-10, but the clinical translation of these findings resulted in a number of failed trials in patients. One explanation of these failures is that the local IL-10 concentrations may be too low, even at maximum tolerable systemic administration (Herfarth, 2002 Gut 50:146). Another explanation may be the immunostimulatory effect of IL-10 on B cells and the resulting production of the pro-inflammatory IFN-γ, as was demonstrated in IL-10-treated Crohn's disease patients (Tilg, 2002 Gut 50:191).
Fusing attenuated IL-10 to an antibody specific for APCs, e.g. targeting dendritic cells through CD11c, or more broadly expressed myeloid markers, like CD33 or CD115, would decrease systemically active biologic activity and at the same time increase the targeted local active concentrations of IL-10. In addition, the demonstrated pro-inflammatory effect through B cells would be decreased or eliminated. The production of antibody-IL10 fusion proteins have been described previously (Schwager Arthritis Res Ther. 11(5): R142, 2009).
Evidence exists for an anti-fibrotic role of IL-10 in various models. A hallmark of fibrosis is the overproduction and deposition of collagen produced by fibroblasts, resulting in scarring tissue formation. IL-10 directly inhibits extracellular matrix synthesis by human fibroblasts (Reitamo, 1994 J Clin Invest 94:2489) and is anti-fibrotic in a rat hepatic fibrosis model through downregulation of TGF-β (Shi, 2006 World J Gastroenterol 12:2357; Zhang, 2007 Hepatogastroenterology 54:2092). Clinical use of IL-10 is hampered by its short half-life and a PEGylated version has shown promising pharmacokinetic improvements and efficacy in a preclinical model of fibrosis (Mattos, 2012 J Control Release 162:84). Targeting IL-10 activity through fusion with an antibody directing it to fibroblasts could result in therapeutic benefits in fibrotic diseases, including lung and liver fibrosis. Antibodies against fibroblast specific proteins such as fibroblast activation protein and platelet derived growth factor receptors, as described above in the description of IFN-γ-targeting, could deliver attenuated IL-10 directly to fibroblasts.
Recombinant erythropoietin (EPO) is a widely used and effective hormone for the treatment of anemia, often in cancer patients. It acts by signaling through the EPO receptor (EPOR), which is not only expressed by cells of the hematopoietic system, but also on non-hematopoietic cells, including cells from various tumor types. Many studies have examined the role of EPO and EPO-R stimulation in cancer models in vitro and in vivo, and a number of studies have demonstrated a stimulatory effect on tumor growth, either directly on cancer cells, or through increased angiogenesis in the tumors (reviewed in Jelkmann, 2008 Crit Rev Oncol Hematol 67:39). In several clinical trials, treatment with EPO has been associated with increased tumor growth and decreased survival, leading to the recommendation and black box warning to limit and monitor the exposure of EPO in cancer patients as much as clinically feasible (Farrell, 2004 The Oncologist 9:18; Jelkmann, 2008 Crit Rev Oncol Hematol 67:39; Elliott, 2012).
Erythropoiesis is a multi-step process, in which pluripotent stem cells undergo tightly controlled differentiation and proliferation steps. An intermediate cell type in this process, is the colony-forming-unit-erythroid (CFU-E) cell, which expresses high levels of EPOR, depends on EPO for survival and appears to be the main cell type in the differentiation process with this dependency (Elliott, 2008 Exp Hematol 36:1573).
Targeting EPO activity to CFU-E cells using specific markers would substantially reduce the effect of EPO on cancer and other non-hematopoietic cells, while maintaining the ability to drive erythrocyte formation and increase hemoglobin levels. Genome-wide analysis of CFU-E cells revealed several potential candidate cellular markers, including Rh-associated glycoproteins, e.g. CD241 and members of the Rh blood group system, e.g. the product of the RCHE gene (Terszowski, 2005 Blood 105:1937).
Additional example surface markers expressed on CFU-Es, and several other intermediates of erythropoiesis, include CD117 (c-kit), CD71 (transferrin receptor) and CD36 (thrombospondin receptor) (Elliott, 2012 Biologics 6:163), but these markers are overexpressed in certain cancer cells as well, as they are all involved in general growth and proliferation, and therefore represent less attractive targets for targeting EPO activity in cancer patients, but this approach may benefit patients with tumors not expressing these targets. CD117 antibodies include SR-1 (U.S. Pat. No. 7,915,391) and antibodies DSM ACC 2007, 2008 and 2009 (U.S. Pat. No. 5,545,533). Other antigens for targeting of an attenuated EPO include CD34, CD45RO, CD45RA, CD115, CD168, CD235, CD236, CD237, CD238, CD239 and CD240.
Fusing EPO activity to an antibody would also greatly increase the extent of the therapeutic activity. The half-life of recombinant EPO is about 5 hours in humans and this would likely be increased to weeks when attenuated EPO is fused to an antibody. This approach could benefit patients treated for anemia, who are dosed typically multiple times per week, often through intravenous injections. Importantly, it has been shown that the therapeutic response to EPO is primarily controlled by the length of time EPO concentrations are maintained, and not by the concentration levels (Elliott, 2008 Exp Hematol 36:1573).
Another example is transforming growth factor β (TGF-β) which is a critical factor in the regulation of T cell-mediated immune responses and the induction of immune tolerance. TGF-β knockout mice die from multifocal inflammation and autoimmune disorders, suggesting an immunosuppressive effect (Shull, 1992 Nature 359:693). However, TGF-β also has been shown to induce fibrotic disease through a prominent role in extracellular matrix regulation and by promoting fibroblast migration, proliferation and activation (Rosenbloom, 2010 Ann Intern Med 152:159; Wynn, 2011 J Exp Med 208:1339; King, 2011 Lancet 378:1949).
In the presence of TGF-β, CD4+CD25− naïve T cells can be converted into Treg cells, which can suppress antigen-specific T cell expansion in vivo and prevent allergic pathogenesis in a murine asthma model (Chen, 2003 J Exp Med 198:1875). Inflammatory responses also contribute to the transition of acute liver disease and perpetuation into chronic fibrosis and cirrhosis and TGF-β may help dampen these responses through its effect on Treg differentiation (Dooley, 2012 Cell Tissue Res 347:245). Similarly, TGF-β directed to naïve T cells in inflammatory bowel disease could lead to control and suppression of inflammation (Feagins, 2010 Inflamm Bowel Dis 16:1963).
Targeting TGF-β specifically to CD4+ T cells may leverage the anti-inflammatory potential of TGF-β, while minimizing its pro-fibrotic properties, and could provide a novel strategy to combat autoimmune disorders. Alternatively, TGF-β could be targeted soley to activated T cells using a T cell activation marker, as described above for the discussion of IFNβ targeting. One attractive target along these lines could be, for example, PD-1, which is expressed on recently activated CD4 T cells. Ideally, a non-antagonizing antibody could be used, such as the J110 antibody discussed in further detail below.
Another example is Interleukin-4 (IL-4) which is a cytokine that induces the differentiation of naïve CD4+ T cells into Th2 cells. Upon activation, Th2 cells produce more IL-4, and as a result, IL-4 is considered a main driver of Th2-mediated immune responses. The concept of a Th1/Th2 imbalance (favoring Th1) contributing to autoimmune and other inflammatory diseases was first postulated in the 1980s (reviewed in Kidd, 2003 Altern Med Rev 8:223), and indeed, a role of Th1/Th17 cells as drivers of disease in psoriasis (Ghoreschi, 2007 Clin Dermatol 25:574), certain types of inflammatory bowel disease, in particular Crohn's disease (Sanchez-Munoz, 2008 World J Gastroenterol 14:4280), or severe versus mild forms of asthma (Hansbro, 2011 Br J Pharmacol 163:81), has been documented.
In preclinical models of infectious diseases, deviation of the immune response away from Th1 to Th2 and activation of macrophages by IL-4 protected from immunopathology (Hunig, 2010 Med Microbiol Immunol 199:239), and IL-4 therapy of psoriasis patients resulted in an induction of Th2 differentiation and an improvement in clinical scores (Ghoreschi, 2003 Nat Med 9:40).
Diversion towards Th2 may provide a therapeutic benefit in certain types of diseases. Delivery of IL-4 to CD4+ T cells could accomplish this, or IL-4 activity could be targeted to macrophages to protect from immunopathology (Ghoreschi, 2007 Clin Dermatol 25:574; Hunig, 2010 Med Microbiol Immunol 199:239).
Attenuating mutations in IL-4 that may be exploited in the design of antibody-attenuated IL-4 fusion protein constructs of the present invention include those listed in Table 17.
The IL-4 mutants in this table, and their binding properties and biological activity, were described by Wang Y, Shen B and Sebald W. Proc. Natl. Acad. Sci. USA 1997 Mar. 4; 94(5): 1657-62.
In yet another example, Interleukin-6 (IL-6) may also be attenuated and targeted to specific cell types. A mechanism by which tumors can evade anti-tumor immunity is by recruiting Treg cells to the tumor microenvironment, resulting in tolerance at tumor sites. IL-6 is a cytokine involved in regulating the balance between Treg and Th17 cells and induces the development of Th17 cells, while it inhibits Treg differentiation (Kimura, 2010 Eur J Immunol 40:1830).
IL-6, by skewing the terminal differentiation of naïve CD4+ T cells towards the Th17 lineage, or reprogramming of Th17 cells, has the potential to reverse tumor-associated immune suppression by Treg cells in the context of cancer, thereby enabling the immune system to control the tumors.
This strategy has proven successful in a murine model of pancreatic cancer in which mice injected with tumor cells expressing IL-6 demonstrated a significant delay in tumor growth and enhanced survival, accompanied by an increase in Th17 cells in the tumor microenvironment, compared to mice bearing tumors not expressing IL-6 (Gnerlich, 2010 J Immunol 185:4063).
Adoptive transfer of T cells is an effective treatment for solid (Rosenberg, 2011 Clin Cancer Res 17:4550) and hematologic (Kochenderfer, 2012 Blood 119:2709) malignancies. Analysis of five different clinical trials in which adoptive T cell transfer was employed using a variety of preconditioning regimens revealed that the depth and duration of Treg depletion correlates with clinical response rate, highlighting the important role of residual Tregs controlling the anti-tumor response (Yao, 2012 Blood 119:5688). In mice, a direct link between surviving Tregs and efficacy of adoptive transfer therapy strongly supports these clinical observations (Baba, 2012 Blood 120:2417).
The importance of Tregs in controlling anti-tumor activity is further exemplified by a significant increase in the humoral response to peptide vaccination in glioblastoma patients after depletion of Tregs with the anti-IL-2 receptor antibody daclizumab (Sampson, 2012 PloS ONE 7:e31046).
Taken together, the published data strongly support a role for Tregs in inhibiting the immune response against tumors. By directing IL-6 activity to CD4+ cells in order to stimulate Th17 differentiation and decrease Treg formation, enhanced anti-tumor responses are expected. These may be achieved with or without accompanying vaccination strategies. Fusing attenuated IL-6 to an antibody against a T cell antigen (e.g. targeting CD4) or an activated T cell antigen (such as PD-1) would provide a comprehensive delivery directly to the target cells.
Attenuated mutants of IL-6 include those listed in Table 18.
These IL-6 mutants and their properties were described by Kalai M et. al. Blood. 1997 Feb. 15; 89(4):1319-1333
Another example is hepatocyte growth factor (HGF) discovered as a mitogen for hepatocytes (reviewed in Nakamura, 2010 Proc Jpn Acad Ser B Phys Biol Sci 86:588). Hepatocyte growth factor is a pleiotropic cytokine that regulates cell growth and motility, playing a central role in angiogenesis and tissue generation and repair in many organs.
HGF acts through its receptor, MET, which is expressed on epithelial and endothelial cells. Binding of HGF to MET results in a number of intracellular phosphorylation and signaling events, leading to a variety of biological responses including migration, proliferation and morphogenesis. Essential for embryogenesis, HGF's primary function in the adult is tissue repair (Nakamura, 2010 Proc Jpn Acad Ser B Phys Biol Sci 86:588).
HGF has been shown to alter the fate of epithelial cells and reduce epithelial-mesenchymal transition (EMT) through its intereference with TGF-β signaling, antagonizing the process of fibroblastogenesis (Shukla, 2009 Am J Respir Cell Mol Biol 40:643). After organ injury, TGF-β drives conversion of HGF-producing fibroblasts into collagen-producing myofibroblasts, while HGF in turn inhibits TGF-β production by myofibroblasts (Mizuno, 2004 Am J Physiol Renal Physiol 286:F134). Exogeneous HGF, or mimetics activating the MET receptor, act by restoring this imbalance imposed by tissue injury, and are therefore considered promising drug candidates for treating damaged tissues and fibrotic diseases (Nakamura, 2010 Proc Jpn Acad Ser B Phys Biol Sci 86:588).
Initially studied in models for liver damage and hepatitis (Roos, 1992 Endocrinology 131:2540; Ishiki, 1992 Hepatology 16:1227), HGF subsequently demonstrated therapeutic benefits in many additional damaged organs, including pulmonary, gastrointestinal, renal and cardiovascular models of injury and fibrosis (Nakamura, 2011 J Gastroenterol Hepatol 26:188).
In in vivo model systems of fibrosis, HGF prevents the progression of fibrotic changes and reduces collagen accumulation when administered prophylactically or therapeutically in murine lungs exposed to bleomycin (Yaekashiwa, 1997 Am J Respir Crit Care Med 156:1937; Mizuno, 2005 FASEB J 19:580), in an obstructive nephropathy model in mice (Yang, 2003 Am J Physiol Renal Physiol 284:F349) and in liver fibrosis models in rats (Matsuda, 1997 Hepatology 26:81); HGF also prevents fibrosis in cardiomyopathic hamsters (Nakamura, 2005 Am J Physiol Heart Circ Physiol 288:H2131).
Limitations of HGF as a therapeutic include its short half-life, which requires supra-physiological systemic concentrations to reach locally effective levels, and the role of its receptor, MET, in cancer. MET can activate oncogenic pathways in epithelial cells. Both of these limitations may be overcome by generation of an antibody-HGF fusion protein construct and targeting it to regenerating or fibrotic tissue. This strategy would produce a therapeutic with a much longer half-life directed primarily at the relevant cells types.
Clinical trials have investigated the therapeutic potential and regenerative activity of HGF, or HGF mimetics, in hepatic failure, chronic leg ulcers, limb ischemia, peripheral arterial disease, cardiovascular disease after myocardial infarction and neurological diseases (de Andrade 2009 Curr Opin Rheumatol 21:649; Nakamura, 2011 J Gastroenterol Hepatol 26:188; Madonna, 2012 Thromb Haemost 107:656).
Liver fibrosis, typically the result of chronic liver damage caused by infections or alcohol abuse, is, like fibrosis in other organs, characterized by excessive accumulation of extracellular matrix, including collagen produced by (myo) fibroblasts. Damaged hepatocytes release inflammatory cytokines and the resulting inflammatory milieu stimulates the transformation of hepatic stellate cells (HSC) into fibroblasts, producing collagen. The accumulation of extracellular matrix proteins results in scar tissue, which leads to liver cirrhosis (Bataller, 2005 J Clin Invest 115:209). Evidence exists for a direct effect of HGF on hepatocytes and HSC in vitro (Kwiecinski, 2012 PloS One 6:e24568; Namada, 2012 J Cell Physiol DOI 10.1002/jcp.24143). Targeting HGF specifically to hepatocytes or HSC may result in a therapeutic benefit in liver fibrosis patients, while eliminating the unwanted systemic effects of HGF.
Possible membrane proteins for hepatocytes include, for example, ASGR1, a subunit of the asialoglycoprotein, used as a target for liver specific drug delivery (Stockert, 1995 Physiol Rev 75:591), or alternatively the other subunit of this receptor, ASGR2. Fibroblast-specific protein (FSP1) expression is increased after liver injury and may be used to target fibroblasts or inflammatory macrophages in fibrotic liver tissue (Osterreicher, 2011 Proc Natl Acad Sci USA 108:308).
In lung fibrosis patients, the loss of pulmonary architecture is characterized by a loss of alveolar epithelial cells, the persistent proliferation of activated fibroblasts and the extensive alteration of the extracellular matrix (Panganiban, 2011 Acta Pharmacol Sin 32:12).
To treat lung fibrosis, HGF activity may be delivered to alveolar epithelial cells by attenuating it (by mutation) and attaching it to an antibody against a specific cell surface protein on these cells, such as RTI40/Tiα or HTI56 (McElroy, 2004 Eur Respir J 24:664).
Endothelial cell-specific markers, including VEGF receptors (Stuttfeld, 2009 IUBMB Life 61:915) may be used for targeting blood vessels for endothelial cell layer enhancement for a number of pathologic indications, including hindlimb ischemia. Examples of VEGF receptor antibodies are shown in Table 19.
Many other examples of signaling ligands are also known in the art and may, as described in the non-limiting exemplary embodiments above, be attenuated and attached to an antibody (or fragment thereof) that binds to an antigen on specific target cells, thereby allowing the ligand to generate its biological signal on those target cells to a much greater degree than it generates its signal on antigen-negative cells. Examples of ligands that have a direct negative effect on tumor proliferation include TNFα, TRAIL, Fas Ligand, IFNβ, IFNγ or IFNλ, which can be targeted to various tumor cell surface antigens as discussed above for INFα.
In many of the aspects of the present invention, specific mutations in various ligands are explicitly mentioned. There are, however, methods well known in the art for identifying other mutations in signalling ligands numerous methods for mutagenesis of proteins are known in the art. Such methods include random mutagenesis for example, exposing the protein to UV radiation or mutagenic chemicals and selecting mutants with desired characteristics. Random mutagenesis may also be done by using doped nucleotides in oligonucleotides synthesis, or conducting a PCR reaction in conditions that enhance misincorporation of nucleotide, thereby generating mutants. Another technique is site-directed mutagenesis which introduces specific changes to the DNA. One example of site directed mutagenesis is using mutagenic oligonucleotides in a primer extension reaction with DNA polymerase. This method allows for point mutation, or deletion or insertion of small stretches of DNA to be introduced at specific sites. The site-directed approach may be done systematically in such technique as alanine scanning mutagenesis whereby residues are systematically mutated to alanine and its effect on the peptide's activity is determined. Each of the amino acid residues of the peptide is analyzed in this manner to determine the important regions of the peptide.
Another example is combinatorial mutagenesis which allows the screening of a large number of mutants for a particular characteristic. In this technique, a few selected positions or a short stretch of DNA may be exhaustively modified to obtain a comprehensive library of mutant proteins. One approach of this technique is to excise a portion of DNA and replaced with a library of sequences containing all possible combinations at the desired mutation sites. The segment may be at an enzyme active site, or sequences that have structural significance or immunogenic property. A segment however may also be inserted randomly into the gene in order to assess the structural or functional significance of particular part of protein.
Methods of screening mutated ligands to determine potency includes assaying for the presence of a complex between the ligand and the target. One form of assay involves competitive binding assays. In such competitive binding assays, the target is typically labeled. Free target is separated from any putative complex and the amount of free (i.e. uncomplexed) label is a measure of the binding of the agent being tested to target molecule. One may also measure the amount of bound, rather than free, target. It is also possible to label the compound rather than the target and to measure the amount of compound binding to target in the presence and in the absence of the drug being tested.
One example of a cell free assay is a binding assay. Whilst not directly addressing function, the ability of a modulator to bind to a target molecule in a specific fashion is strong evidence of a related biological effect. For example, binding of a molecule to a target may, in and of itself, be inhibitory, due to steric, allosteric or charge-charge interactions. The target may be either free in solution, fixed to a support, expressed in or on the surface of a cell. Either the target or the compound may be labeled, thereby permitting determination of binding. Usually, the target will be the labeled species, decreasing the chance that the labeling will interfere with or enhance binding. Competitive binding formats can be performed in which one of the agents is labeled, and one may measure the amount of free label versus bound label to determine the effect on binding.
Depending on the assay, culture may be required. The cell is examined using any of a number of different physiologic assays. Alternatively, molecular analysis may be performed, for example, protein expression, mRNA expression (including differential display of whole cell or polyA RNA) and others. Non-limiting examples of in vitro biological assays that can be used to screen protein variants are shown in the Examples below and also include apoptosis assays, migration assays, invasion assays, caspase-activation assays, cytokine production assays and the like.
The present invention also provides compositions comprising the polypeptides of the present invention. These compositions can further comprise at least one of any suitable auxiliary, such as, but not limited to, diluent, binder, stabiliser, buffers, salts, lipophilic solvents, preservative, adjuvant or the like. Pharmaceutically acceptable auxiliaries are preferred. Non-limiting examples of, and methods of 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. Pharmaceutically acceptable carriers can be routinely selected that are suitable for the mode of administration, solubility and/or stability of the antibody composition as well known in the art or as described herein.
Pharmaceutical excipients and additives useful in the present composition include but are not limited to proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatised sugars such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination 1-99.99% by weight or volume. Exemplary protein excipients include serum albumin, such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acids which can also function in a buffering capacity include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. One preferred amino acid is histidine. A second preferred amino acid is arginine.
Carbohydrate excipients suitable for use in the invention include, for example, monosaccharides, such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), myoinositol and the like. Preferred carbohydrate excipients for use in the present invention are mannitol, trehalose, and raffinose.
Antibody compositions can also include a buffer or a pH adjusting agent; typically, the 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, or phosphate buffers. Preferred buffers for use in the present compositions are organic acid salts, such as citrate.
Additionally, the compositions of the invention can include polymeric excipients/additives, such as polyvinylpyrrolidones, ficolls (a polymeric sugar), dextrates (e.g., cyclodextrins, such as 2-hydroxypropyl-β-cyclodextrin), polyethylene glycols, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, surfactants (e.g., polysorbates such as “TWEEN® 20” and “TWEEN® 80”), lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol), and chelating agents (e.g., EDTA).
These and additional known pharmaceutical excipients and/or additives suitable for use in the antibody compositions according to the invention are known in the art, e.g., as listed in “Remington: The Science & Practice of Pharmacy”, 19 th ed., Williams & Williams, (1995), and in the “Physician's Desk Reference”, 52 nd ed., Medical Economics, Montvale, N.J. (1998), the disclosures of which are entirely incorporated herein by reference. Preferred carrier or excipient materials are carbohydrates (e.g., saccharides and alditols) and buffers (e.g., citrate) or polymeric agents.
Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
All publications mentioned in this specification are herein incorporated by reference. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia or elsewhere before the priority date of each claim of this application.
It must be noted that, as used in the subject specification, the singular forms “a”, “an” and “the” include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to “a” includes a single as well as two or more; reference to “an” includes a single as well as two or more; reference to “the” includes a single as well as two or more and so forth.
Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting.
Production of Antibody-IFNα Fusion Protein Constructs
Expression Vectors:
The DNA encoding the rituximab (Anderson et al., U.S. Pat. No. 5,843,439, Dec. 1, 1998) and palivizumab (Johnson, U.S. Pat. No. 5,824,307, Oct. 20, 1998) variable regions were generated from 18 (heavy chain) and 16 (light chain) DNA oligonucleotides, which were designed according to the published amino acid sequences, by PCR-based gene assembly. The DNA encoding the variable regions of the G005 anti-CD38 and nBT062 anti-CD138 monoclonal antibodies were drawn from the publications by De Weers et al. (U.S. Pat. No. 7,829,673) and by Daelken et al. (WO 2009/080832), respectively, and subjected to be synthesized by Integrated DNA Technology, Inc. (Coralville, Iowa) after the sequence modification to eliminate rare codons and unprefered restriction sites.
The DNA sequences encoding the variable regions of anti-human HLA (HB95), anti-human PD-1 (J110) and anti-yellow fever virus (2D12) monoclonal antibodies were determined after cloning from hybridoma W6/32 (ATCC HB-95, Barnstable et al. (1978), Cell 14:9-20), J110 (International Patent Organism Depositary FERM-8392, Iwai et al. (2002), Immunol. Lett, 83:215-220) and 2D12 (ATCC CRL-1689, Schlesinger et al. (1983), Virol. 125:8-17), respectively, using the SMART RACE cDNA Amplification kit (Clontech, Mountain View, Calif.) and Mouse Ig-Primer Sets (Novagen/EMD Chemicals, San Diego, Calif.). The sequence determination and sub-cloning of the newly isolated anti-CD38 antibodies is described in the following sections.
The DNA encoding human interferon-α2b (IFNα2b; amino acid sequence of SEQ ID NO:3) was isolated from genomic DNA of a HEK cell line by PCR. The sequences of human interferon-β1 (IFNβ1, SEQ ID NO:91), human interleukin-4 (IL-4, SEQ ID NO:119) and human interleukin-6 (IL-6, SEQ ID NO:123) were designed from the protein sequences such as NP_002167, NP_000580 and NP_000591, respectively, and synthesized by Integrated DNA Technology, Inc. (Coralville, Iowa) or GenScript USA Inc. (Piscataway, N.J.) using methods commonly known to those of skill in the art. Alterations of the cytokine sequences, for example the addition of linkers or point mutations, were introduced to the cytokine genes using overlap extension PCR techniques well known in the art.
The cytokine-endoding gene fragments were then cloned into the pTT5 expression vector (Durocher, Nucleic Acids Research volume 30, number 2, pages E1-9, 2002) containing either a human IgG1 heavy chain complete or partial constant region (such as Swissprot accession number PO1857), a human IgG4 heavy chain constant region (such as Swissprot accession number P01861 incorporating substitution S228P), human Ig kappa constant region (Swissprot accession number P01834) or human Ig lambda constant region (Swissprot accession number P0CG05) either as a naked Ig or as a cytokine gene fusion form using overlap extension PCR techniques and restriction sites according to cloning methods well known by those skilled in the art.
Production of IgG and IgG Interferon Fusion Protein Constructs:
DNA plasmids encoding the IgGs and IgG-cytokine fusion protein constructs were prepared using Plasmid Plus Maxi kit (Qiagen, Valencia, Calif.) and then transfected into HEK293-6E cells (CNRC, Montreal, Canada) grown in F17 synthetic medium supplemented with 0.1% Pluronic F-68, 4 mM L-glutamine (Invitrogen, Carlsbad, Calif.) using a commercially available transfection reagent and OptiMEM medium (Invitrogen, Carlsbad, Calif.). After allowing for expression for 6 days in an incubator supplied with 5% CO2 and gentle shaking, the culture media was isolated and subjected to IgG affinity purification using Protein G-agarose beads (GE Healthcare, Piscataway, N.J.). Purified IgG and IgG-cytokine fusion protein constructs were then concentrated and buffer-exchanged to phosphate buffered saline (PBS) pH 7.4 using Amicon Ultra centrifugal filter devices (Millipore, Billerica, Mass.), followed by protein concentration determination using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, Mass.).
Although different antibody-cytokine fusion protein constructs were expressed in the HEK system with differing yields, several of them, in particular several of those based on IFNα, were produce at at least 100 mg/L of media, showed high solubility and did not aggregate as determined by size exclusion chromatography.
The amino acid sequences of the antibodies and antibody-ligand construct fusion protein constructs are described below. For antibody-cytokine fusion protein constructs in which the cytokine was fused to the C-terminus of the heavy or light chain, the following naming convention was used:
Thus for example the construct “Rituximab-HC-L6-IFNα (A145G) IgG1” is the antibody rituximab, with IFNα2b (with the A145G point mutation), linked to the C-terminus of the IgG1 heavy chain, with an intervening linker L6.
The linkers used in the experiments were as follows:
Method for Measuring Antigen-Targeted Activity of Antibody-IFNα Fusion Protein Constructs
“On target (Daudi) assay”: This assay was used to quantify the anti-proliferative activity of IFNs and antibody-IFN fusion protein constructs on cells that display that antigen corresponding to the antibody to which the IFN is fused, and may be used as part of the assay for calculating the antigen-sensitivity index (ASI) defined herein. Daudi cells express both CD20 and CD38 as cell surface associated antigens. The viability of cells was measured using the reagent CellTiter-Glo®, Cat #G7570, from Promega (Madison, Wis.). This is a luminescence-based assay that determines the viability of cells in culture based on quantitation of ATP. The signal strength is proportional to the number of viable cells in a microtiter plate well. The details of the assay are as follows:
Daudi cells (obtained from ATCC, Manassas, Va.) were cultured in a T75 flask (TPP, Trasadingen, Switzerland, cat #90076) to a preferred density of between 0.5×105 and 0.8×105 viable cells/ml in RPMI 1640 (Mediatech, Inc., Manassas, Va., cat #10-040-CV) with 10% Fetal Bovine Serum (FBS; Hyclone, Logan, Utah cat #SH30070.03). Cells were harvested by centrifuging at 400 g for five minutes, decanting the supernatant, and resuspending the cell pellet in RPMI 1640+10% FBS. Cells were then counted andthe density was adjusted to 3.0×105 cells/ml in RPMI 1640+10% FBS. Then, 50 μl of the cell suspension was aliquoted into each well of a 96 well round bottom tissue culture plate (hereafter, “experimental plate”) (TPP, cat #92067). On a separate, sterile 96 well plate (hereafter, “dilution plate”; Costar, Corning, N.Y. cat #3879), test articles were serially diluted in duplicate in RPMI 1640+10% FBS. Then, 50 μl/well was transferred from the dilution plate to the experimental plate. The experimental plate was then incubated for four days at 37° C. with 5% CO2.
A mixture of the manufacturer-supplied assay buffer and assay substrate (hereafter, “CellTiterGlo reagent”, mixed according to the manufacturer's instructions) was added to the experimental plate at 100 μl/well. The plate was shaken for two minutes. Then, 100 μl/well was transferred from the experimental plate to a 96 well flat bottom white opaque plate (hereafter, “assay plate”; BD Biosciences, Franklin Lakes, N.J. cat #35 3296). The content of the assay plate was then allowed to stabilize in the dark for 15 minutes at room temperature. The plate was read on a Victor 3V Multilabel Counter (Perkin Elmer, Waltham, Mass., model #1420-041) on the luminometry channel and the luminescence was measured. Results are presented as “relative luminescence units (RLU)”.
Data was analyzed using Prism 5 (Graphpad, San Diego, Calif.) using non-linear regression and three parameter curve fit to determine the midpoint of the curve (EC50). For each test article, potency relative to free IFNα2b (or some other form of IFN with a known potency relative to IFNα2b) was calculated as a ratio of EC50s.
One of ordinary skill in the art will appreciate that there are many other commonly used assays for measuring cell viability that could also be used.
“On target (ARP) assay” (also sometimes referred to herein as a “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, Ark.). It is described in Hardin J. et al., (Interleukin-6 prevents dexamethasone-induced myeloma cell death. Blood; 84:3063, 1994). ARP-1 cells (CD38+) were used to test CD38 targeting antibody-IFN fusion protein constructs. Culture and assay conditions were the same as for Daudi-based assay outlined above, with the following exceptions: ARP-1 was cultured to a density of 4.0×105 to 6.0×105 cells/ml. ARP-1 concentration was adjusted to 1.0×104 cells/ml prior to assay.
Method for Measuring Non-Antigen-Targeted Activity of Antibody-IFNα Fusion Protein Constructs
“Off-target assay” (also sometimes referred to herein as the “not-targeted” assay): The iLite assay from PBL Interferon Source (Piscataway, N.J., Cat #51100), was performed largely as described by the manufacturer with the addition of a human IgG blocking step. The iLite cell line is described by the manufacturer as “a stable transfected cell line derived from a commercially available pro-monocytic human cell line characterized by the expression of MHC Class II antigens, in particular the human lymphocyte antigen (HLA-DR), on the cell surface.” The cell line contains a stably transfected luciferase gene, the expression of which is driven by an interferon-response element (IRE), which allows for interferon activity to be quantified based on luminescence output. The manufacturer-supplied iLite plate (hereafter “assay plate”) and diluent were removed from the −80° C. freezer and allowed to equilibrate to room temperature. Then, 50 μl of the diluent was added per well to the assay plate. The vial of manufacturer-supplied reporter cells was removed from the −80° C. freezer and thawed in a 37° C. water bath. Then, 25 μl aliquiots of cells were dispensed into each well of the assay plate. Next, 12.5 μl of 8 mg/ml human IgG that was diluted into RPMI 1640+10% FBS (Sigma Chemicals, St. Louis, Mo.; cat #14506) was added per well. The contents were mixed and incubated at 37° C. for 15 minutes. On a separate “dilution plate,” test articles were serially diluted in duplicate in RPMI 1640+10% FBS. Then, 12.5 μl of the test articles were transferred from the dilution plate to the assay plate. The assay plate was then incubated at 37° C. with 5% CO2 for 17 hours. The manufacturer-supplied assay buffer and substrate were removed from the −80° C. freezer and allowed to equilibrate to room temperature for two hours. The manufacturer-supplied assay buffer was added to the manufacturer-supplied substrate vial and mixed well according to the manufacturer's instructions to create the “luminescence solution.” Then, 100 μl of the luminescence solution was added to each well of the assay plate. The plate was shaken for 2 minutes. The plate was then incubated at room temperature for 5 minutes in the dark and finally read on a Victor 3V Multilabel Counter on a luminometry channel and the luminescence measured and presented as RLU. The data was analyzed with Graphpad Prism 5 as described for the “on-target (Daudi) assay,” above. To test anti-CD38 antibody-IFN fusion protein constructs in the iLte assay, the manufacturer-supplied diluent was supplenmented with 2 mg/ml human IgG and 0.5 mg/ml anti-CD38 antibody (same antibody clone being tested as an antibody-IFN fusion protein construct, to block any binding of the anti-CD38 antibody-IFN fusion protein constructs to the CD38 expressed on the iLite cells).
Results: Antigen-Specificity of Antibody-IFNα Fusion Protein Constructs
To determine whether the antibody-IFNα2b fusion protein constructs had enhanced activity relative to free IFNα2b on cells that do display the corresponding antigen on their cell surface, their effect on Daudi cells, which display the CD20 antigen of Rituximab, but which do not display the RSV F protein antigen corresponding to Palivizumab, was examined. The assay used in this case, described above as “Method for measuring antigen-targeted activity of antibody-IFNα fusion protein constructs” or simply the “on-target (Daudi) assay,” measured the effect of the test substances on the viability of Daudi cells. With these cells, the Rituximab-IFNα2b fusion protein construct (Rituximab-HC-L6-IFNα IgG1) was 3.25-fold (1.3/0.4=3.25) more potent than free IFNα2b (
The results presented in
The modest reduction in IFNα activity that occurred as a result of linking it to an antibody may not be sufficient to prevent the toxicity of the IFNα component of the construct in human subjects. Various mutations were therefore introduced into IFNα2b in order to reduce its activity and toxicity. For example, five different mutant versions of IFNα2b were generated and, in each case, linked to the C-terminus of the heavy chain of Rituximab via the six amino acid linker L6, which has the sequence SGGGGS (SEQ ID NO:132). These constructs were compared to the the Rituximab-wild type IFN fusion protein construct, Rituximab-HC-L6-IFNα IgG1 (as also used in the experiments shown in
Surprisingly, when the amount of interferon activity of these highly attenuated rituxumab-mutant IFNα2b fusion protein constructs was measured on antigen-positive cells (Daudi, CD20+), there was generally very little attenuation compared to the wild type IFNα2b version of the Rituximab-IFNα2b fusion protein construct (
Other Rituximab-IFNα2b constructs with mutations in the IFNα2b portion also showed surprisingly little reduced activity on antigen-positive cells (
To determine whether this dramatic difference in the ability of the mutations in the IFNα component of the fusion protein constructs to substantially reduce its activity on antigen-negative cells as compared to antigen-positive cells could be extended to other fusion protein constructs targeting other antigens, antibodies targeting the multiple myeloma antigen CD 38 (SEQ ID NO:131) were fused to both wild type and attenuated forms of IFNα and characterized. Some of these experiments were performed using the antibody G005 (De Weers et al. (U.S. Pat. No. 7,829,673)); the sequences of the heavy and light chains for this human antibody are shown as SEQ ID NOS:135 and 134, respectively.
In addition, several novel human and rat antibodies against CD38 were produced, as described below.
Development of Novel CD38 Antibodies
Formatting CD38 Constructs for Expression
The extracellular domains (ECD) of human and cynomolgus monkey CD38 proteins were each formatted to include a cleavable N-terminal leader sequence, an Avitag™, a poly-histidine tag and a thrombin cleavage site to yield proteins SEQ ID NO:127 and 128 respectively. These were back-translated into DNA sequences and synthesized de novo by assembly of synthetic oligonucleotides by methods known by those with skill in the art. Following gene synthesis, the genes were subcloned into vector pTT5 (Durocher, Nucleic Acids Research volume 30, number 2, pages E1-9, 2002) to yield constructs to produce soluble secreted forms of these proteins via transient expression in HEK293E cells (Durocher, supra).
Construction of Vectors for Antibody Expression
Heavy and light chain variable region sequences were subcloned into variants of the vector pTT5 containing either a human IgG1 heavy chain constant region (such as Swissprot accession number PO1857), a human IgG4 heavy chain constant region (such as Swissprot accession number P01861 incorporating substitution S228P), human kappa constant region (Swissprot accession number P01834) or human lambda region (Swissprot accession number POCG05) to yield full length antibody chains.
Transient Expression of Constructs in HEK293-6E Cells
HEK293-6E cells were cultured in complete cell growth media (1 L of F17 medium (Invitrogen™), 9 mL of Pluronic F68 (Invitrogen™), 2 mM Glutamine containing 20% (w/v) Tryptone NI (Organotechnie®) with Geneticin (50 mg/mL, Invitrogen™) at 50 μl/100 mL culture). The day before transfection, cells were harvested by centrifugation and resuspended in fresh media (without Geneticin). The next day DNA was mixed with a commercial transfection reagent and the DNA transfection mix added to the culture drop-wise. The culture was incubated overnight at 37° C. with 5% CO2 and 120 rpm without Geneticin. The next day, 12.5 mL of Tryptone was added along with 250 μl of Geneticin per 500 mL culture. The culture was incubated at 37° C., 5% CO2 and 120 rpm. After 7 days, the supernatant was harvested by centrifugation and was ready for purification.
Expression and Purification of Antibodies
Transient co-expression of heavy and light chains in HEK293-6E cells (as described above) generated antibodies that were subsequently purified by protein A chromatography. Briefly, supernatants derived from these transfections were adjusted to pH 7.4 before being loaded onto a HiTrap Protein A column (5 mL, GE Healthcare). The column was washed with 50 mL of 1×PBS (pH 7.4). Elution was performed using 0.1 M citric acid pH 2.5. The eluted antibody was desalted using Zeba Desalting columns (Pierce) into 1×PBS (pH 7.4). The antibodies were analyzed using SDS-PAGE. The concentration of the antibody was determined using the BCA assay kit (Pierce).
Purification of Histidine-Tagged Proteins from Tissue Culture Supernatants
Immobilized metal ion affinity chromatography (IMAC) was used to purify human and cynomolgous monkey CD38 extracellular domain (ED) proteins from tissue culture supernatants. Briefly, protein supernatants were diluted in binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 30 mM imidazole, pH 7.4) before being loaded onto a HisTrap™ FF column (1 mL, GE Healthcare). The column was washed with 5 mL of binding buffer (pH 7.4) and elution was performed using 20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH 7.4. The eluted proteins were desalted and buffer exchanged using Amicon Ultra-15 centrifugal filter unit with Ultracel-10 membrane (Millipore) into 1×PBS (pH 7.4). The absorbance at 280 nm (A280) of the protein was assessed using a Nanodrop spectrophotometer and readings corrected using the predicted extinction coefficients to determine protein concentrations.
Biotinylation of Antigens for Phage Display
The Avitag™ motifs of human and cynomolgus monkey CD38 EDs were biotinylated according to manufacturer's directions (Avidity LLC, Aurora, Colo.). Excess unconjugated biotin was removed from the biotinylated proteins by desalting into 1×PBS using a 7 KD molecular weight cut off (MWCO) Zeba spin column (Thermo Scientific, Logan, Utah) according to manufacturer's instructions. Successful biotinylation of CD38 ED proteins was confirmed using a combination of polyacrylamide gel electrophoresis and Western blotting. Western blots were probed using Streptavidin-HRP (BD Biosciences, San Diego, Calif.) and developed using TMB (Sigma-Aldrich, St. Louis, Mo.). For each antigen, monomeric biotinylated CD38 ED was detected.
Generation of Anti-CD38 Antibodies by Phage Display
FAbs that bind to both human and cynomolgus monkey CD38 EDs were isolated from a naïve phagemid library comprising approximately 2.5×1011 individual human FAb fragments. Methods of generating phage antibody fragment libraries are discussed in “Phage display: A Practical Approach” (Eds. Clackson and Lowman; Oxford University Press, Oxford, UK) and “Antibody Phage Display Methods and Protocols” (Eds. O'Brien and Aitken; Humana Press Inc, NJ 07512). Briefly, antibody heavy and light chain variable regions were amplified based on RNA from donor samples. Antibody heavy and light chain variable regions were then inserted into phagemid vectors to generate a library of antibody fragments fused to a phage coat protein. The antibody library used herein was a high diversity naïve phagemid library that expressed antibody fragments in the Fab format.
Anti-CD38 FAbs were isolated from the phage display library over the course of two panning ‘campaigns’ (i.e. discrete phage display experiments with different reagents or panning conditions). The general protocol followed the method outlined by Marks et al. (Marks, J. D. & Bradbury, A., 2004, Methods Mol Biol, 248, 161-76).
Each phage display campaign involved three rounds of panning. For each round, ˜2.5×1012 phage particles were blocked by mixing 1:1 with blocking buffer (4% skim milk in PBS, pH 7.4) and incubating for 1 hr at room temperature. The blocked phage library was then pre-depleted for any biotinylated protein tag motif binders used in panning through incubation for 45 mins with 50-200 pmols of an irrelevant antigen containing an identical biotinylated tag motif. Tag- and streptavidin-binders were captured by adding an excess (75-300 μL) of streptavidin-coated Dynabeads (Invitrogen), which were blocked as described for the library. The beads (including tag- and streptavidin-binders attached to them) were immobilized using a magnet and discarded.
Library panning was conducted by mixing the blocked and pre-depleted library with 50-200 pmols of biotinylated recombinant CD38 ED in a 2 mL microcentrifuge tube and rotating for 2 hrs at room temperature. Then, 100 μL of streptavidin-coupled Dynabeads (Invitrogen, Carlsbad, Calif.) were added and the mixture was incubated a further 15 minutes as described previously. Non-specifically bound phage were removed using a series of washes. Each wash involved pulling the bead complexes out of the solution onto the tube wall using a magnetic rack, aspirating the supernatant and then re-suspending the beads in fresh wash buffer. This was repeated multiple times with either PBS wash buffer (1×PBS with 0.5% skim milk) or PBS-T wash buffer (1×PBS supplemented with 0.05% TWEEN-20 [Sigma-Aldrich, St. Louis, Mo.] and 0.5% skim milk). Phage that remained bound after the washing process were eluted from the biotinylated-CD38 ED-bead complexes by incubation with either a twenty-fold excess of non-biotinylated CD38 ED for 1 hr at room temperature or 0.5 mL of 100 mM triethylamine (TEA) (Merck Chemicals, Darmstadt) for 20 mins at room temperature. TEA-eluted ‘output’ phage were neutralized by the addition of 0.25 mL of 1 M Tris-HCl pH 7.4 (Sigma-Aldrich, St. Louis, Mo.).
At the end of the first and second rounds of panning, the output phage were added to a 10 mL culture of exponentially growing TG1 E. coli (2× yeast-tryptone (2YT) growth media) and allowed to infect the cells during a 30 minute incubation at 37° C. without shaking, then with shaking at 250 rpm for 30 additional minutes. The phagemids encoding the phage display output were then rescued as phage particles following a standard protocol (Marks, J. D. & Bradbury, A., 2004, Methods Mol Biol, 248, 161-76). At the end of the third panning round, TG1 cells were infected with output phage and were plated on 2YT agar (supplemented with 2% glucose and 100 μg/mL carbenicillin) at a sufficient dilution to produce discrete E. coli colonies. These colonies were used to inoculate 1 mL liquid cultures to allow expression of FAb fragments for use in screening experiments.
ELISA-Based Screening of FAbs for CD38 Binding
Each individual E. coli colony was used to express a FAb that could be screened for CD38 ED-binding activity. Colonies were inoculated into 1 mL starter cultures (supplemented with 100 μg/mL carbenicillin and 2% glucose) in 96-well deep-well plates (Costar) and incubated overnight at 37° C. with shaking at 350 rpm (Innova R44 shaker; 1 inch orbit). These starter cultures were diluted 1:100 into a 1 mL expression culture (2YT supplemented with 100 μg/mL carbenicillin) and grown to an optical density (600 nm) of 0.5-0.8. FAb expression was induced by adding isopropyl-beta-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. Cultures were incubated at 25° C. for 16 hrs.
FAb samples were prepared by harvesting cells by centrifugation (2,000 g, 10 mins) and performing a lysozyme extraction. The cell pellet was resuspended in 200 μL of lysis buffer (160 μg/mL lysozyme, 10 μg/mL RNase A, 5 μg/mL DNase and complete protease inhibitors (Roche, Nutley, N.J.)) and shaken at 400 rpm for 30 minutes at 21° C. Following addition of a further 100 μl of lysis buffer, the reactions were incubated for a further 30 minutes as described previously. Clarified lysates were isolated following centrifugation at 3,000 g for 10 minutess and stored at 4° C. until required.
To screen by enzyme-linked immunosorbent assay (ELISA) for human CD38 ED-binders derived from the phage display biopanning, human CD38 extracellular domain (ED) (produced in HEK 293-6E cells and biotinylated as described above) was captured on streptavidin-coated ELISA plates (Nunc) at 1 μg/mL. Plates were then washed and individual FAb samples (prepared as described above) were added to individual wells on the ELISA plates. FAbs were allowed to bind the captured CD38 ED for an hour at room temperature and then washed three times with PBS-T (1×PBS supplemented with 0.1% Tween®20). FAbs that bound to CD38 ED were detected by incubation for 30 minutes at room temperature with an anti-V5-HRP conjugated antibody (Invitrogen, Carlsbad, Calif.) to detect the V5 tag fused to the C-terminus of the FAb heavy chain. Plates were washed to remove unbound antibody and the assay signal developed by incubation with 50 μL 3,3′,5,5′-Tetramethylbenzidine (Sigma-Adrich, St. Louis, Mo.) and quenching with 50 μL 1 M HCl. Assay signals were read at A450 nm using a microplate reader (BMG Labtech). Results were expressed as the raw A450 nm value, where any signal 2-fold greater than the average assay background was defined as ‘positive’.
In later assays FAb cross-reactivity with cynomolgus monkey CD38 ED was assessed by coating biotinylated cynomolgus monkey CD38 ED onto streptavidin coated ELISA plates and proceeding as described above. Plasmids encoding FAbs cross-reactive with both human and cynomolgus monkey CD38 ED were isolated and sequenced. Of approximately 1,000 FAbs screened for binding to human and cynomolgus monkey CD38 ED, six genetically unique FAbs were identified. Table 21 summarises the FAb sequence data obtained. The variable regions of some of these antibodies are shown in
All FAbs were converted into IgG1 format by cloning into the pTT5 vectors (described above), expressed in HEK293-6E cells and the resulting IgGs purified by protein A affinity chromatography as described above.
Assessing Binding of IgGs to Human CD38 Positive Cell Line RPMI-8226
The ability of the phage derived antibodies to bind the model human CD38 positive myeloma cell line RPMI-8226 (obtained from the Health Protection Agency Culture Collections, Porton Down, Salisbury, SP4 0JG, UK) in flow cytometry-based assays was tested. Briefly, viable RPMI-8226 cells (2×105, as judged by trypan blue exclusion) were incubated with each antibody or with a human IgG1 isotype control antibody preparation (Sigma-Aldrich, St. Louis, Mo.) at various concentrations in 100 μl of FACS buffer (PBS plus 1% fetal calf serum, FCS) in 96 well plates for 20 minutes on ice in the dark. Cells were washed twice with FACS buffer before incubation for 20 minutes in 100 μl of FACS buffer containing goat anti-human IgG (Fc-specific, conjugated to fluorescein isothiocyanate, FITC; Sigma-Aldrich, St. Louis, Mo.). After washing with FACS buffer, cells were resuspended in FACS buffer and analysed for antibody binding by flow cytometry on a FACS Canto (BD Biosciences, San Diego, Calif.) using EV, side scatter and FL-1 gating. Results are expressed as mean fluorescent intensity (MFI) plotted against protein concentration (
Generation of Anti-CD38 Antibodies by Genetic Immunization
Monoclonal antibodies against human CD38 ED were generated by genetic immunization with corresponding conventional protein immunization of rats. For genetic immunization, the DNA sequence of human CD38 ED is provided in SEQ ID NO:129. The corresponding conceptually translated protein sequence is given in SEQ ID NO:130. The DNA sequence of SEQ ID NO:129 was cloned into a plasmid for genetic immunization using restriction enzyme technology. Expression of the resulting plasmid allowed the secretion of soluble CD38 ED tagged by a c-myc epitope at the N- or C-terminus. The c-myc epitope was utilized to confirm expression of CD38 ED.
Rats were then immunized six times with the plasmid using a Helios gene gun (Bio-Rad, Germany) according to a published procedure (Kilpatrick et al., Hybridoma 17: 569-576, 1998). One week after the last application of the immunization plasmid, each rat was boosted by intradermal injection of untagged recombinant human CD38 ED. Untagged human CD38 ED for this purpose was produced by removing the protein tags from SEQ ID NO:127 by thrombin cleavage followed by purification over a size exclusion column.
Four days later, the rats were sacrificed and their lymphocytes fused with myeloma cells using polyethylene glycol (HybriMax™; Sigma-Aldrich, Germany), seeded at 100,000 cells per well in 96-well microtiter plates and grown in DMEM medium supplemented with 10% fetal bovine serum and HAT additive for hybridoma selection (Kilpatrick et al., 1998, supra).
Screening Hybridoma Supernatants for Human and Cynomolgus Monkey CD-38 Cross-Reactivity
Duplicate 100 μL samples of each hybridoma supernatant were coated onto separate wells of a maxisorp ELISA plate (Nunc Plasticware, Thermo Scientific, Rochester, N.Y. 14625, USA) through incubation at room temperature for an hour. Plates were washed three times in 1×PBS-T and subsequently blocked by addition of 2% BSA/1×PBS. Following incubation for 1 hour at room temperature, plates were washed as described previously. To one well of each rat antibody duplicate well was added 0.1 μg of biotinylated human CD38 in a final volume of 100 μL 1×PBS. To the second well of each rat antibody duplicate well was added 0.1 μg of biotinylated cynomolgus monkey CD38 ED in a final volume of 100 μL 1×PBS. Plate wells were washed as described previously prior to detection of bound biotinylated CD38 ED using a Streptavidin-HRP conjugate (BD Biosciences, San Diego, Calif.). Plates were washed as above to remove unbound Streptavidin-HRP conjugate and the assay signal developed by incubation with 50 μL 3,3′,5,5′-Tetramethylbenzidine (Sigma-Aldrich) and quenching with 50 μL 1 M HCl. Assay signals were read at A450 nm using a microplate reader (BMG Labtech, Cary, N.C.). Of the 15 hybridoma supernatants tested, all fifteen bound human CD38 ED and seven bound cynomolgus monkey CD38 ED (Table 22) as determined by ELISA. The cross-reactive antibodies are referred to as R5D1, R7F11, R5E8, R10A2, R10B10, R3A6 and R7H11.
Flow Cytometry Binding of Rat Antibodies to Human CD38 Positive Cell Line RPMI-8226
Viable RPMI-8226 cells (2×105, as judged by trypan blue exclusion) were incubated with 100 μL of rat hybridoma supernatant for 20 minutes on ice in the dark. Cells were washed twice with FACS buffer (1×PBS plus 1% FCS) before incubation for 20 minutes in 100 μl of FACS buffer containing anti-rat IgG-FITC conjugate (Sigma-Aldrich). After washing cells in FACS buffer, they were resuspended in FACS buffer and analysed for antibody-binding by flow cytometry on a FACS Canto (BD Biosciences, San Diego, Calif.) using EV, side scatter and FL-1 gating. Results were expressed as mean fluorescent intensity (MFI). Of the 15 rat antibodies exhibiting positive binding to human CD38 ED by ELISA, five showed weak or negligible binding to CD38 expressed on the human myeloma cell line RPMI-8226 by FACS (Table 22).
FACS Binding Background MFI Average was 153
Molecular Characterisation of Rat Antibodies
Six rat antibody hybridomas—R5D1, R7F11, R5E8, R10A2, R10B10 and R7H11—were selected for molecular characterization. RNA extraction from pelleted hybridoma cells of each clone was performed using TRI reagent (Sigma-Aldrich, St. Louis, Mo.) according to manufacturer's directions. The variable regions of each antibody were amplified using Rapid Amplification of cDNA Ends (RACE) reverse transcription polymerase chain reaction (RT-PCR) methodology according to manufacturer's directions (Clontech [Mountain View, Calif.] SMART RACE kit; Ambion Life Technologies [Foster City, Calif.] RLM-RACE kit). Gene-specific reverse PCR primers to amplify the rat heavy chain variable domains by 5′-RACE were designed to anneal to the available rat heavy chain constant region sequences. Similarly, gene specific reverse PCR primers to amplify the rat light chains were designed to anneal to the rat kappa chain constant region sequences, while further primers were designed to anneal to the rat lambda chain constant region sequences.
5′-RACE PCR was performed according to manufacturer's directions (Life Technologies; Clontech) using PfuUltrall polymerase (Agilent). Following 5′-RACE PCR, products were separated by agarose gel electrophoresis and bands of approximately the predicted size based on the location of the reverse primer in the constant region were excised from the gels. DNA was purified from agarose gel slices using a Qiaquick spin gel extraction kit (Qiagen) according to manufacturer's instructions. Insert DNA was cloned and propagated in E. coli using a StrataClone Blunt PCR Cloning Kit (Agilent, Santa Clara, Calif.) according to manufacturer's instructions. Single colonies from transformations were cultured and plasmid DNA prepared using a GenElute™ plasmid miniprep kit (Sigma-Aldrich, St. Louis, Mo.). DNA inserts were sequenced and antibody variable regions identified in the conceptually translated protein sequences.
Vectors were constructed using the rat antibody variable region sequences grafted onto human IgG1 constant sequences for the heavy chain variable region and, human kappa or lambda backbones (keeping the same light chain isotype as in the rat antibodies). The resulting variable region sequences of each clone are listed in Table 23. Subsequent co-expression of the corresponding heavy- and light chains in HEK293-6E cells, in the context of the pTT5 vectors, was followed by protein A purification of the resulting IgGs as described above.
Affinity of Anti-CD38 Antibodies for Human and Cynomolgus Monkey CD38
The binding affinities of a selection of the antibodies produced against human and cynomolgus monkey CD38 were measured. Briefly, using a Biacore T200, Protein A was immobilized onto Flow Cell (FC) 1 (FC1) and FC2 (or alternatively FC3 and FC4) of a CM5 research grade sensor chip using amine coupling, giving approximately 2000 RU. FC1 was used as a blank throughout the experiments. The experiments were run in HBS-P buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.005% v/v Surfactant P20). At a flow rate of 20 μl/min, 20 μl of 5 μg/mL of antibody was passed over FC2. Human CD38 ED or separately, cynomolgus monkey CD38 ED was passed over the surface of FC1 and FC2 at concentrations ranging from 25 nM to 200 nM. Regeneration of the surface was performed using 10 mM Glycine, pH 1.0. The FC1 sensorgram data was subtracted from FCS and the curves were fitted using a 1:1 Langmuir equation to generate the kd, ka and KD values. This data shows that cross-reactivity for human and cynomolgus monkey CD38 is maintained on conversion of the human phage-derived Fabs into human IgGs and rat antibodies into chimeric rat-human IgGs (Table 24).
Anti-CD38-Attenuated IFN Fusion Protein Constructs
To determine whether the surprising result obtained with an anti-CD20 antibody fused to an attenuated IFNα could be replicated with other antibodies, and in particular antibodies targeting an antigen unrelated to CD20, fusion protein constructs comprising the fully human IgG1:kappa anti-CD38 antibody G005 (composed of SEQ ID NOS:135 (heavy chain) and 134 (light chain)) and IFNα (SEQ ID NO:3), with or without various attenuating mutations was made.
In order to determine whether the ASI could be increased, as was observed for the anti-CD20-IFNα fusion protein constructs, several variants were constructed by attenuating the IFN portion of the anti-CD38-IFNα fusion protein construct by mutation. Numerous different attenuating mutations were made in the context of the G005 or other CD38 monoclonal antibodies. In addition, constructs of different IgG isotypes (IgG1 and IgG4) and linker lengths (L0, no linker; L6, 6 amino acid linker (SGGGGS, SEQ ID NO:132)) were made. The off-target assay and two types of on-target assays (using Daudi and ARP-1), both described in detail above, were run and the results are shown in
Table 26 characterizes the CD38 antibody G005, fused in various configurations via the C-terminus of the heavy chain to IFNα with the R144A attenuating mutation. Examples in this table are of IgG1 and IgG4 isotype and either have no linker between the antibody heavy chain and the IFN, L0, or have an intervening 6 amino acid linker, L6 (composed of SEQ ID NOS:138,140,152,146 (heavy chain) each combined with 134 (light chain)). In all cases, the fusion protein constructs had dramatically reduced potency on antigen-negative iLite cells (a reduction of from 8,300 to 100,000 fold compared to free IFNα), but substantially maintained the potency exhibited by free IFNα on CD38 positive cells (Daudi). The G005-HC-L0-IFNα (R144A) IgG4 construct (composed of SEQ ID NOS:152 (heavy chain) and 134 (light chain)), for example, has a 105-fold lower potency than free, wild type IFNα on antigen negative cells but its potency is reduced only 3.5-fold (2.7/0.77=3.5) vs. free, wild type IFNα on antigen positive cells (Table 26). This gives an Antigen Specificity Index (ASI) of 29,000 for this fusion protein construct.
Table 27 shows examples using another IFNα attenuating mutation, A145G, as a construct with the same G005 antibody in either IgG1 or IgG4 isotypes, with either no linker or the L6 linker (composed of SEQ ID NOS:142,144,148 (heavy chain) each combined with SEQ ID 134 (light chain)). The G005-HC-L6-IFNα (A145G) IgG4 construct (composed of SEQ ID NOS:148 (heavy chain) and 134 (light chain)), for example, showed an ASI of 20,000.
Table 28 shows examples in which the mutated IFNα is attached to the light chain rather than the heavy chain, with either no intervening linker or the L6 linker (composed of SEQ ID NOS:210 or 208 (light chain), respectively, each combined with SEQ ID NO:135 (heavy chain)). In both cases, the fusion protein constructs demonstrated a high ASI of 5,900 and 7,200, respectively.
Tables 29 and 30 demonstrate the ASI for the same fusion protein constructs but use an alternative cell line (ARP-1, a myeloma) for determining activity on CD38+ cells. Using this method, the ASIs for these fusion protein constructs ranged from 1,200-55,000.
Numerous other examples of mutated versions of IFNα, in the context of the G005-HC-L0-IFNα IgG4 fusion protein construct are set out in Table 25. The majority of these mutants (R144 mutated to A, S, E, G, H, N, Q, T, Y, I, L or V (composed of SEQ ID NOS:152,172,156,158,160,168,170,174,178,162,166,176 (heavy chain), respectively, each combined with SEQ ID NO:134 (light chain)) and A145 mutated to G, D, H, I, K, L, N, Q, R or Y (SEQ ID NOS:184,180,186,188,190,192,194,196,198,206 (heavy chain), respectively, each combined with SEQ ID NO:134 (light chain)) showed significant attenuation of IFNα activity compared to free wild type IFNα In this context, the A145V and A145S mutants did not show appreciable attenuation. Any of these point mutated, attenuated versions of IFNα could be used in the context of the present invention as antibody fusion protein constructs. Certain IFN variants may be preferred due to showing higher ASIs. Other considerations, such as expression level, immunogenicity, biophysical characteristics, etc., may also be considered in evaluating constructs for optimal utility. Of the numerous IFNα variants described in this document, those shown here to yield a high ASI in the context of an antibody-fusion protein construct include R144A, R144S, R144T, R144Y, R144I, R144L, R145G, R145D, R145H, R145Y (Table 25), R33A+YNS (as illustrated in the construct comprising SEQ ID NOS:286 (heavy chain) and 276 (light chain)), R33A (as illustrated in the construct comprising SEQ ID NOS:436 (heavy chain) and 276 (light chain)) and R144A+YNS (such as as illustrated in the construct comprising SEQ ID NOS:288 (heavy chain) and 276 (light chain)).
The mutation of A145D in the construct of SEQ ID NOS:180 (heavy chain) and 134 (light chain) compared to the A145E mutation in the construct of SEQ ID NO:182 (heavy chain) and 134 (light chain) produced unexpected results. Although both constructs have similar amino acid sequences, differing by only a single methylene group, they showed dramatically different effects on the non-targeted activity of the IFNα. The A145E mutation had minimal impact on the IFNα activity, however, the A145D mutation drastically reduced activity (by 20,000-fold) and resulted in a construct with high ASI (9,840).
Other examples of anti-CD38 antibody-attenuated IFNα fusion protein constructs are shown in Tables 31-33. In addition to the G005 antibody constructs, these tables show the on-target activity, the off-target activity, and the ASI for anti-CD38 antibody-attenuated IFNα fusion protein constructs based on certain novel antibodies. Fusion protein constructs of these antibodies with the IFNα A145D or R144A mutations include the following:
X910/12-HC-L0 IFNα (A145D) IgG4 composed of SEQ ID NOS:248 (heavy chain) and SEQ ID NO:242 (light chain);
X910/12-HC-L0 IFNα (R144A) IgG4 composed of SEQ ID NOS:246 (heavy chain) and SEQ ID NO:242 (light chain);
X913/15-HC-L0 IFNα (A145D) IgG4 composed of SEQ ID NOS:256 (heavy chain) and SEQ ID NO:250 (light chain);
X913/15-HC-L0 IFNα (R144A) IgG4 composed of SEQ ID NOS: 254 (heavy chain) and SEQ ID NO:250 (light chain);
X355/02-HC-L0 IFNα (A145D) IgG4 composed of SEQ ID NOS:232 (heavy chain) and SEQ ID NO:226 (light chain);
X355/02-HC-L0 IFNα (R144A) IgG4 composed of SEQ ID NOS:230 and SEQ ID NO:226 (light chain);
X355/07-HC-L0 IFNα (A145D) IgG4 composed of SEQ ID NOS:240 (heavy chain) and SEQ ID NO:234 (light chain);
X355/07-HC-L0 IFNα (R144A) IgG4 composed of SEQ ID NOS:238 and SEQ ID NO:234 (light chain);
R5D1-HC-L0 IFNα (A145D) IgG4 composed of SEQ ID NOS:262 (heavy chain) and 258 (light chain);
R5E8-HC-L0 IFNα (A145D) IgG4 composed of SEQ ID NOS:268 (heavy chain) and 264 (light chain); and
R10A2-HC-L0 IFNα (A145D) IgG4 composed of SEQ ID NOS:274 (heavy chain) and 270 (light chain).
These fusion protein constructs all showed high ASIs, ranging from 3,820 (X910/12-HC-L0-IFNα (R144A) IgG4) to 166,000 (X355/02-HC-L0-IFNα (A145D) IgG4).
The examples above demonstrate that mutated, attenuated forms of IFNα, attached to antibodies targeting CD20 (SEQ ID NO:430) or CD38 (SEQ ID NO:131), show orders of magnitude greater potency in IFN signaling on antigen-positive target cells than on antigen-negative off-target cells. The results below provide further examples using antibodies that target the attenuated IFNα to two other antigens: CD138 and class I MHC.
CD138 (SEQ ID NO:432), also called Syndecan-1, is a heparin sulfate proteoglycan that is thought to function as an adhesion molecule. It is expressed on most multiple myeloma cells (Dhodapkar, Blood; 91: 2679, 1998). Fusion protein constructs consisting of mutated, attenuated IFNα and the CD138-targeting antibody nBT062 (Ikeda, Clin Can Res., 15:4028, 2009; USPTO #20090175863, composed of SEQ ID NOS:330 (heavy chain) and 326 (light chain)) were generated. As shown in
Another antigen that has been described as a potential target for antibody therapy to treat cancer is the Class I MHC (see for example Stein, Leuk. Lymphhoma 52(2):273-84, 2011). In order to determine whether it was possible to apply the present invention in relation to this target, antibody W6/32 (Barnstable et al. (1978), Cell 14:9-20), was obtained by from ATCC (HB95). This antibody reacts with monomorphic determinants on human HLA A,B,C molecules. The antibody variable regions were cloned and sequenced using SMART RACE cDNA Amplification kit (Clontech, Mountain View, Calif.) and Mouse Ig-Primer Sets (Novagen/EMD Chemicals, San Diego, Calif.). The amino acid sequences of the heavy chain and light chain variable regions are shown as SEQ ID NOS:411 and 410, respectively. The chimeric version of HB95, with the murine variable regions and human IgG4 kappa constant regions, fused to IFNα with the A145D mutation (HB95-HC-L0-IFNα (A145D) IgG4, composed of SEQ ID NOS:316 (heavy chain) and 312 (light chain)) was expressed, and its activity was compared to an isotype control antibody fused in the same way to the same IFNα mutant (Isotype-HC-L0-IFNα (A145D) IgG4, where the isotype variable regions were derived from antibody 2D12). The “on-target (ARP-1)” assay was run as described above for the CD38-targeted antibodies (ARP-1 is class I MHC-positive). The results are shown in
Antiviral Activity of Targeted, Attenuated IFNα
The anti-viral activity of IFNα is well-known and recombinant IFNα is an FDA-approved treatment for hepatitis C viral infections. The effect of a host cell surface-targeted vs. non-targeted antibody-attenuated IFNα fusion protein construct on the cytopathic activity of the EMC virus on A549 cells, which are class I MHC-positive, was compared.
Methods:
IFN activity was measured using the cytopathic effect inhibition (CPE) assay as described Rubinstein (J. Virol. 37, 755-8, 1981). Briefly, 104 human adenocarcinoma A549 cells (ATCC, Manassas, Kans.) per well were incubated with test sample or IFN (human IFN-α2A) overnight. Cells were then challenged with EMC virus for 48-56 hours, followed by staining with crystal violet. A visual CPE determination was performed, followed by solubilization of the crystal violet and absorbance measurement at 570 nm. Nonlinear regression analysis was performed using a 4-parameter sigmoidal fit with variable slope (GraphPad Prism). One unit of IFNα activity is defined as the amount of interferon required to reduce the cytopathic effect by 50%. The units are determined with respect to the international reference standard for human IFNα2, provided by the National Institutes of Health (see Pestka, S. “Interferon Standards and General Abbreviations,” in Methods in Enzymology (S. Pestka, ed.), Academic Press, New York vol 119, pp. 14-23, 1986). The samples tested in this assay were IFNα (Intron A, inverted triangles), Anti-MHC class I targeted attenuated IFNα designated HB95-HC-L0-IFNα (R145D) IgG4 (closed squares), and istoype control (2D12)-attenuated IFNα (Isotype-HC-L0-IFNα (R145D) IgG4; triangles). Data is plotted as viability vs IFNα molar equivalents.
Results:
Results are shown in
Targeted, Attenuated IFNβ
IFNβ also has been shown in numerous publications (see above) to have anti-proliferative activity on various types of cancer cells. A fusion protein construct between an anti-CD38 antibody (G005) and IFNβ (SEQ ID NO:91), G005-HC-L0-IFNβ IgG4 (composed of SEQ ID NOS:212 (heavy chain) and 134 (light chain)) as well as an identical construct but carrying a single point mutation (R35A), known to reduce IFNβ potency (Runkel et al. J. Biol. Chem. 273:8003-8 (1998), composed of SEQ ID NOS:214 (heavy chain) and 134 (light chain)) was therefore made. In both constructs, the unpaired cysteine at position 17 of IFNβ was mutated to a serine in order to improve expression yields and product homogeneity.
Interleukin-4 (IL-4)
IL-4 is a helical bundle cytokine with multiple physiological activities, including the ability to bias T helper cell development towards Th2 and away from Th1. Since Th1 cells play a pathological role in certain autoimmune settings, it could be therapeutically advantageous to use IL-4 to influence T helper cell development away from Th1, i.e. to create a “Th1 diversion.” To avoid side effects related to IL-4's activity on other cell types, it would be advantageous to attenuate IL-4's activity by mutating it, and then attach it to an antibody that would direct it to activated (preferentially recently activated) helper T cells. The antibody chosen for this purpose was J110, a mouse anti-human PD-1 clone described by Iwai et. al. (Immunol Lett. 83:215-20, 2002). PD-1 (SEQ ID NO:431) is expressed on recently activated Th0 cells.
The J110 antibody (murine variable regions and human IgG1:kappa constant regions; the amino acid sequences of J110 heavy and light chain variable regions are shown as SEQ ID NOS:409 and 408, respectively) was fused to human IL-4 (SEQ ID NO:119), the latter being attached to the C-terminus of the heavy chains with an intervening six amino acid linker, L6 (SGGGGS, SEQ ID NO:132). In addition to this J110-HC-L6-IL4 IgG1 protein (composed of SEQ ID NOS:304 (heavy chain) and 300 (light chain)), a variant of this with a single substitution in the IL-4 component, J110-HC-L6-IL-4 (R88Q) IgG1 (composed of SEQ ID NOS:306 (heavy chain) and 300 (light chain)) was made. The R88Q mutation in IL-4 has been reported to reduce its potency in vitro (Kruse, EMBO Journal vol. 12 no. 13 pp. 5121-5129, 1993).
Methods:
The “off-target (HB-IL4) assay” was performed largely as described by the manufacturer of the HEK-Blue IL4/IL13 cell line. HEK-Blue™ IL-4/IL-13 Cells are specifically designed to monitor the activation of the STAT6 pathway, which is induced by IL-4. The cells were generated by introducing the human STAT6 gene into HEK293 cells to obtain a fully active STAT6 signaling pathway. The HEK-Blue™ IL-4/IL-13 Cells stably express a reporter gene, secreted embryonic alkaline phosphatase (SEAP), under the control of the IFNβ minimal promoter fused to four STAT6 binding sites. Activation of the STAT6 pathway in HEK-Blue™ IL-4/IL-13 cells induces the expression of the SEAP reporter gene. SEAP is then secreted into the media and can be quantitated using the colorimetric reagent QUANTI-Blue™. Briefly, HEK-Blue IL4/IL13 cells (Invivogen, San Diego Calif. cat #hkb-stat6) were thawed and cultured in DMEM media (Mediatech, Manassas Va., cat #10-013-CV)+10% FBS (Hyclone, Logan Utah, cat #SH30070.03) that had been heat inactivated (HI FBS). After one passage, 10 μg/ml blasticidin (Invivogen cat #ant-bl-1) and 100 micrograms/ml Zeocin (Invivogen cat #ant-zn-1) were added to the culture medium. After one more passage, cells were allowed to reach 60-80% confluence and then lifted with Cell Stripper (Mediatech, cat #25-056-C1). Cells were washed twice in DMEM+HI FBS and counted. Cells were adjusted to 2.8×105 viable cells/ml in DMEM+HI FBS and 180 μl was aliquoted per well into a flat bottom 96 well tissue culture plate (hereafter, the “experimental plate”). Then, 20 μl of IL-4 or fusion protein construct, diluted into DMEM+HI FBS, was added per well. The plate was incubated at 37° C. 5% CO2 for 16-24 hours. QUANTI-Blue (Invivogen, cat #rep-qbl) was prepared according to the manufacturer's directions. QUANTI-Blue (160 μl) was aliquoted into each well of a flat bottom plate (hereafter, the “assay plate”). Then, 40 μl supernatant per well from the experimental plate was transferred to assay plate. Assay plate was then incubated at 37° C. for 1-3 hours. Assay plate absorbance at 630 nm was read on a model 1420-41 Victor 3V Multilabel Counter from Perkin-Elmer. Data was analyzed using Graph Pad Prism.
The “on target (Th1 diversion) assay” was designed to monitor the percentage of CD4+ T cells that were of Th1 phenotype, as defined by their expression of IFN-γ. Th1 diversion is thereby quantified by a decrease in IFN-γ-positive CD4 T cells. The assay was performed as follows: “Loaded” Dynabeads (M450 Epoxy beads, Invitrogen Dynal, Oslo, Norway cat #140.11) were made as described by the manufacturer with 1.0 μg/107 beads anti-human CD3 epsilon antibody (R&D Systems, Minneapolis Minn., cat #MAB100), 1.0 μg/107 beads anti-human CD28 antibody (R&D Systems, cat #MAB342) and 3 μg/107 beads human IgG (R&D Systems, cat #1-001-A). PBMCs were obtained from the Stanford Blood Center; Palo Alto Calif. Naïve CD4+ T cells were purified from Leukocyte Reduction System (LRS) cones using the naïve CD4+ kit (Miltenyi Biotech cat #130-094-131) according to the manufacturer's directions. A total of 4.0×105 purified naïve CD4+ T-cells were aliquoted to each well of a 24 well tissue culture plate (hereafter, the “experimental plate”) in 1.3 ml in RPMI 1640 (Mediatech, cat #10-040-CV)+10% HI FBS+100 units/ml IL-2 (Peprotech, cat #200-02), hereafter referred to as Media-Q. Then, 4.0×105 “loaded” Dynabeads were added per well. IL-12 (Peprotech, cat #200-12) was diluted into Media-Q and 100 μl was added to appropriate wells, giving a final concentration of 10 ng/ml. Fusion protein constructs or IL-4 were diluted into Media-Q and 100 was added to the appropriate wells. Media-Q was added to appropriate wells to bring the total volume of each well to 1.5 ml. The experimental plate was incubated at 37° C. with 5% CO2 for five days. On the morning of the fifth day, Phorbol myristate acetate (PMA) was added to all wells at a final concentration of 50 ng/ml and ionomycin was also added to all wells at a final concentration of 1.0 Brefeldin A was added to a final concentration of 1.0 μg/ml of culture. The experimental plate was incubated at 37° C. with 5% CO2 for a minimum of four hours. Approximately ⅓ of the volume of each well of the experimental plate was then recovered and subjected to preparation for Intra-cellular Flow Cytometry according to the instructions supplied with the abovementioned Kit and utilizing the reagents supplied with the kit. The cells were stained for intra-cellular interferon-gamma with an anti human interferon-gamma antibody conjugated to AF647 (eBiosciences.com, cat #51-7319-42). The samples were analyzed by flow cytometry on a Becton Dickinson FACSort using Cell Quest software. Acquired samples were analyzed using FloJo Software and data were graphed using Graph Pad Prism software.
Results:
In the absence of antibody-based targeting, as measured by the “off-target (HB-IL4) assay,” IL4 showed an EC50 of 1.26 pM (
The “on target (Th1 diversion) assay” results are shown in
Interleukin-6 (IL-6)
IL-6 (SEQ ID NO:123) has numerous activities on different cell types and it may be advantageous to exploit some of these activities at the expense of others. For example, by targeting newly activated CD4+ T cells (via attachment to an anti-PD1 antibody, as in the example above with IL-4 targeting, for example), one may shift the T helper cell population away from the Treg pathway and in favor of the Th17 pathway. This could be advantageous to a cancer patient.
Methods:
The “IL-6 bioassay” was performed using the HEK-Blue™ IL-6 cells (Invivogen, cat #hkb-i16), an engineered reporter cell line that monitors the activation of the JAK-STAT pathway by IL-6. These cells were generated by introducing the human IL-6R gene into HEK293 cells. In addition, cells were further transfected with a reporter gene expressing SEAP under the control of the IFNβ minimal promoter fused to four STAT3 binding sites. In these cells, IL-6 stimulates the activation of STAT3 and leads to the secretion of SEAP. SEAP is then monitored when using the SEAP detection medium QUANTI-Blue™. The assay was run essentially according to the manufacturer's (Invivogen) instructions. Briefly, HEK-Blue IL6 cells were thawed and cultured in DMEM (Mediatech, Manassas Va., cat #10-013-CV)+10% FBS (Hyclone, Logan Utah, cat #SH30070.03) that had been heat inactivated (HI FBS). After one passage, 200 μg/ml HygroGold, (Invivogen cat #ant-hg-1) and 100 μg/ml Zeocin, (Invivogen cat #ant-zn-1) was added to the culture medium. After one more passage, cells were allowed to reach 60-80% confluence and then lifted with Cell Stripper (Mediatech, cat #25-056-C1). Cells were then washed twice in DMEM+HI FBS and counted. Cells were adjusted to 2.8×105 viable cells/ml in DMEM+HI FBS and 180 ul was aliquoted per well into a flat bottom 96 well tissue culture plate (hereafter, the “experimental plate”). Then, 20 μl of IL-6 or fusion protein construct, diluted into DMEM+HI FBS, was added per well. The plate was incubated at 37° C. with 5% CO2 for 16-24 hours. QUANTI-Blue (Invivogen, cat #rep-qbl), prepared according to the manufacturer's instructions, was then aliquoted (160 μl per well) into each well of a flat bottom plate (hereafter, the “assay plate”). Then, 40 μl supernatant per well from the experimental plate was transferred to the wells of the assay plate. The assay plate was incubated at 37° C. for 1-3 hours. Assay plate absorbance at 630 nm was read on a model 1420-41 Victor 3V Multilabel Counter from Perkin-Elmer. Data was analyzed using Graph Pad Prism.
In order to test whether IL-6 can be attenuated and targeted, such that a high Antigen Specificity Index (ASI) may be achieved, IL-6 carrying a 16-mer linker (L16, SGGGGSGGGGSGGGGS, SEQ ID NO:133) at the N-terminus was fused to an antibody targeting class I MHC, using the HB95 antibody (which binds to human class I MHC antigen, as described above) vs. an isotype control antibody, 2D12 (also described above). The non-targeting, isotype control fusion protein construct, 2D12-HC-L16-IL6 IgG1 (composed of SEQ ID NOS:360 (heavy chain) and 344 (light chain)), was compared to free IL-6 in the “IL-6 bioassay” described above (
In Vivo Studies of Antibody-Targeted Attenuated IFNα
To confirm that the antibody-attenuated ligand fusion protein constructs of the present invention were active in vivo several experiments, using constructs consisting of antibodies to CD38, which is expressed on the surface of multiple myeloma cells and attenuated versions of IFNα2b, were performed. In most studies this was compared to non-targeted control fusion protein constructs referred to below as “isotype control”. The variable regions for the isotype control antibodies were derived from the antibody 2D12 which was raised against the yellow fever virus (Shlesinger, Virology 125: 8-17, 1983).
In the first experiment, a xenograft model in which the multiple myeloma cell line NCI-H929 (ATCC CRL-9068, Gazdar, Blood 67: 1542-1549, 1986) is grown subcutaneously in immunocompromised (SCID) mice was used.
Methods:
Eight to 12 week old CB.17 SCID mice were injected subcutaneously in the flank with 1×107 NCI-H929 tumor cells in 50% Matrigel. When average tumor size reached 120-150 mm3, mice were grouped into 5 cohorts of 10 mice each and treatment began at time zero (T0). All treatments were given by intraperitoneal injection, (i.p.) twice weekly for 5 weeks (indicated by bar under graph). All compounds were dosed at 200 μg/dose (approximately 10 mg/kg) except for Interferon-α. IFNα2b (Intron A®, Schering Corp., Merck, Whitehouse Station, N.J.) was given at 2 million units/dose. Tumor volume was measured twice weekly by caliper measurement. Endpoint was tumor volume of 2,000 mm3.
Results:
Results are shown in
The anti-CD38-attenuated IFNα fusion protein construct (G005-HC-L6-IFNα (A145G) IgG1) was tested in a systemic multiple myeloma model based on the cell line MM1S (Crown Bioscience Inc., Santa Clara; Greenstein, Exp Hematol. April; 31(4):271-82, 2003).
Methods:
Six to 8 week old NOD-SCID mice were injected intravenously with 1×107MM1S tumor cells in 0.1 ml phosphate buffered saline (PBS) 24 hours after irradiation with 200 rad (60Co). Mice were grouped into 4 cohorts of 10 mice each at time zero and treatments began 7 days later. All treatments were given i.p. twice weekly for 9 weeks. All compounds were dosed at 200 μg/dose (approximately 10 mg/kg) except Interferon-α (given at 2 million units/dose). Body weights and overall health were monitored twice weekly and survival was the endpoint.
Results:
Results are shown in
An in vivo study using a third model of cancer, based on the Burkitt's lymphoma cell line Daudi (ATCC CCL-213, Klein, Cancer Res. 28: 1300-1310, 1968) was performed. Daudi cells are CD38+.
Methods:
Six to eight week old NOD-SCID mice were injected subcutaneously in the flank with 1×107 Daudi Burkitt's Lymphoma tumor cells in 50% Matrigel one day after irradiation with 200 rad (60Co). When mean tumor size reached 169 mm3 (Day 20), mice were grouped into 5 cohorts of 10 mice each and treatment began. All treatments were given i.p. twice weekly for 4 weeks. All compounds were dosed at 200 μg/dose (approximately 10 mg/kg) except Interferon-α, which was given at 2 million units (MIU)/dose. Tumor volume was measured twice weekly by caliper measurement. Endpoint was tumor volume of 2,000 mm3.
Results:
Results are shown in
aMean+/−SEM
bRatio of tumor size for treatment group divided by tumor size for vehicle group at day 37
cVs. vehicle control at day 37
This xenograft experiment shows that the CD38-targeted attenuated IFNα fusion protein constructs may be effective in treating lymphomas in addition to multiple myelomas.
The effect of different doses of the anti-CD38-attenuated IFNα fusion protein construct, were compared to the non-CD38-targeted fusion protein construct, on myeloma tumor growth. For these comparisons, the NCI-H929 s.c. multiple myeloma model was used.
Methods:
Eight to 12 week old CB.17 SCID mice were injected subcutaneously in the flank with 1×107 NCI-H929 tumor cells in 50% Matrigel. When mean tumor sizes reached 120-150 mm3, mice were grouped into 9 cohorts of 10 mice each and treatment began (time zero). All treatments were given i.p. twice weekly for 5 weeks. Two compounds, targeted anti-CD38-attenuated interferon (closed grey symbols) and non-targeted isotype control-interferon (open symbols), were compared in this study at different doses (see legend for doses). Tumor volume was measured twice weekly by caliper measurement. Endpoint was tumor volume of 2,000 mm3.
Results:
Results are shown in
The next example shows that antibodies of the present invention also include those of the IgG4 isotype.
Methods:
Eight to 12 week old CB.17 SCID mice were injected subcutaneously in the flank with 1×107 NCI-H929 tumor cells in 50% Matrigel. When average tumor size reached 120-150 mm3, mice were grouped into 5 cohorts of 10 mice each and treatment began (time zero). All treatments were given i.p. twice weekly for 5 weeks. All compounds were dosed at 70 μg/dose (approximately 3.5 mg/kg). Tumor volume was measured twice weekly by caliper measurement. Endpoint was 2,000 mm3.
Results:
Results are shown in
The next example extends the observation of in vivo efficacy of an antibody-targeted IFN to a second mutated form of IFNα in which A145 has been mutated to aspartic acid (D). In addition, the experiment below utilizes a different CD38 antibody, i.e. one based on the variable regions of human antibody clone X355/02; (SEQ ID NOS:391 (VH) and 390 (VX)). A third difference between this construct and the one presented in the preceding in vivo experiments is that the linker is removed (referred to as “L0”), i.e. the mutated IFNα is fused directly to the C terminus of the antibody heavy chain.
Methods:
Eight to 12 week old CB.17 SCID mice were injected subcutaneously in the flank with 1×107 NCI-H929 tumor cells in 50% Matrigel. When average tumor size reached 120-150 mm3, mice were grouped into 3 cohorts of 10 mice each and then treatment began (time zero). All treatments were given i.p. twice weekly for 5 weeks. All compounds were dosed at 60 μg/dose (approximately 3 mg/kg). Tumor volume was measured twice weekly by caliper measurement. Endpoint was tumor volume of 2,000 mm3.
Results:
Results are shown in
The next example shows that an anti-CD38 antibody-attenuated IFNα fusion protein construct is more effective than standard drugs used to treat multiple myeloma in the same xenograft model described above.
Methods:
Eight to 12 week old CB.17 SCID mice were injected subcutaneously in the flank with 1×107 NCI-H929 tumor cells in 50% Matrigel. When mean tumor sizes reached 120-150 mm3, mice were grouped into cohorts of 10 mice each and treatment began (time zero). Treatments were administered at doses and regimens described in legend. Tumor volume was measured twice weekly by caliper measurement. Endpoint was 2,000 mm3.
Results:
Results are shown in
The next example shows that an anti-CD38-attenuated IFN fusion protein construct can completely eliminate established human multiple myeloma tumors in a mouse model, even when the fusion protein construct is given as a single dose.
Methods:
Eight to 12 week old CB.17 SCID mice were injected subcutaneously in the flank with 1×107 NCI-H929 tumor cells in 50% Matrigel. When mean tumor sizes reached 120-150 mm3 mice were grouped into cohorts of 10 mice each and then treatment began (T0). Treatments with the anti-CD38 antibody-attenuated Interferon fusion protein construct were administered according to following regimens: single dose on day 0 (closed triangles), two doses (on day 0 and day 3; closed squares), 4 doses (on days 0, 3, 8, and 11; closed diamonds) and 6 doses (on days 0, 3, 8, 11, 15 and 18; closed black circles). One cohort received 6 doses of the isotype control-attenuated interferon fusion protein construct on days 0, 3, 8, 11, 15 and 18 (open squares). The vehicle treatment group is shown in grey filled circles. Tumor volume was measured twice weekly by caliper measurement. Endpoint was 2,000 mm3.
Results:
Results are shown in
The next example demonstrates that even very large tumors can be eliminated by treatment with an anti-CD38-attenuated IFN fusion protein construct.
Methods:
One cohort (n=9) from the immediately preceding study was not treated until the mean tumor volume reached 730 mm3. This cohort then received 6 doses of anti-CD38 targeted, attenuated interferon on days 12, 15, 19, 22, 26 and 29 (arrows).
Results:
Results are shown in
It was shown that in the in vitro experiments above (Table 27) that the G005-HC-L6-IFNα (A145G) IgG4 fusion protein construct has about 25,000-fold lower potency than free, wild-type IFNα2b under conditions where the antibody does not target the attenuated IFNα to the cells being tested (off target assay). The following experiments aimed to determine if the fusion protein construct also showed dramatic attenuation of IFN activity in an ex vivo assay of IFN activity that is relevant to the toxicity of IFNα. This effect of IFNα on hematopoiesis can be measured ex vivo by determining the effect of IFNα on the number of colony forming units derived from primary human bone marrow mononuclear cells. The IFNα vs. the antibody-attenuated IFNα fusion protein constructs were compared in terms of their effect on colony formation.
Methods:
Frozen normal human bone marrow mononuclear cells (AllCells, Inc., Emeryville, Calif.) from 3 donors were thawed in RPMI-1640 medium plus 10% fetal bovine serum (FBS) (complete medium) and washed with same medium two times. After washing, cells were kept in this medium at 1.75×106 cells/ml. Cell suspensions were diluted with MethoCult H4434 Classic medium (Stem Cell Technologies, Cat #04434) to a final cell concentration of 0.7×105 cells/ml. Cells were then mixed very well and 3 ml of this mixture was aliquoted into each tube.
Intron A (Schering Corp. Merck, NJ) and fusion protein constructs (G005-HC-L0-IFNα (145D) IgG4 and Isotype-HC-L0-IFNα (145D) IgG4) were diluted in tenfold serial dilutions in complete medium and 150 μl of each dilution was added to tubes containing the 3 ml of the bone marrow cells in the Methocult H4434 medium. Mixtures were plated at 1.1 ml per 35 mm tissue culture dish (Stem Cell Technologies, cat #27115). Plates were then incubated in a well-humidified incubator at 37° C. with 5% CO2 for two weeks. Colonies were counted on a microscope using a gridded scoring dish (Stem Cell Technologies, Cat #27500) and the number of colonies/plate was recorded. Percent colony recovery for a given test substance was calculated by dividing the number of colonies per plate by the number of colonies in the plates with no added test substance. A total of three human bone marrow MNC were tested using this method.
Results:
The results are shown in
Another activity of IFNα that can be measured ex vivo is the stimulation of cytokine and chemokine secretion. Normal human PBMCs were stimulated with various concentrations of IFNα vs the antibody-attenuated IFNα fusion protein construct Isotype-HC-L6-IFNα (A145G) IgG1 (based on the 2D12 antibody), and measured the resulting cytokine production.
Methods:
Normal human peripheral blood mononuclear cells (PBMC) from four normal donors were washed with Xvivo-15 medium (Lonza, Cat #04-418Q) and resuspended in the same medium at a cell density of 1×106cells/ml. The cells were then incubated with human IgG at 4 mg/ml and incubated at 37° C. for 30 min to block any nonspecific IgG binding. Without washing, 250 μl aliquots of cells were then added to wells of 24-well tissue culture treated plates. To these wells were then added 250 μl of free IFNα or an IgG-attenuated IFNα fusion protein construct (Isotype-HC-L6-IFNα (A145G) IgG1; isotype antibody is 2D12) at various concentrations. Plates were then incubated overnight at 37° C. in 5% CO2. The following day, the plates were spun down and 200 μl of supernatant was collected from each well. Supernatants were kept frozen until analysis using a Luminex cytokine assay.
Luminex Assay: Using the Premix 42-plex from Millipore (Cat #MPXHCYTO60KPMX42) we were able to measure the level of human cytokines produced by the PBMCs stimulated by the test substances. The culture supernatants were incubated with the pre-mixed polystyrene microbeads that were coated with anti-cytokine antibodies according to the manufacturer's instructions. After washing, the biotinylated detection antibody cocktail was introduced to the bead-captured analyte. Finally the reaction mixture was incubated with Streptavidin PE and the fluorescent intensity of PE was measured on the Luminex analyzer. Results were interpolated by the standard curve constructed based on the controls provided in the kit.
Results:
Results are shown in
Sequence Tables
| Number | Date | Country | Kind |
|---|---|---|---|
| 2011904502 | Oct 2011 | AU | national |
This application is a continuation of U.S. patent application Ser. No. 14/262,841, filed on Apr. 28, 2014, which is a continuation of International Patent Application No. PCT/AU2012/001323, filed on Oct. 29, 2012, which claims priority to Australian Patent Application No. 2011904502, filed on Oct. 28, 2011, the contents of each application are incorporated herein by reference in their entirety and for all purposes.
| Number | Name | Date | Kind |
|---|---|---|---|
| 4908431 | Colman et al. | Mar 1990 | A |
| 5055289 | Frincke | Oct 1991 | A |
| 5225539 | Winter | Jul 1993 | A |
| 5441734 | Reichert | Aug 1995 | A |
| 5545405 | Page | Aug 1996 | A |
| 5558864 | Bendig et al. | Sep 1996 | A |
| 5565332 | Hoogenboom et al. | Oct 1996 | A |
| 5585089 | Queen et al. | Dec 1996 | A |
| 5650150 | Gillies | Jul 1997 | A |
| 5677171 | Hudziak et al. | Oct 1997 | A |
| 5693761 | Queen et al. | Dec 1997 | A |
| 5770195 | Hudziak et al. | Jun 1998 | A |
| 5772997 | Hudziak et al. | Jun 1998 | A |
| 5795965 | Tsuchiya et al. | Aug 1998 | A |
| 5837821 | Wu | Nov 1998 | A |
| 5840299 | Bendig et al. | Nov 1998 | A |
| 5844093 | Kettleborough et al. | Dec 1998 | A |
| 5976531 | Mezes et al. | Nov 1999 | A |
| 5977322 | Marks et al. | Nov 1999 | A |
| 6054297 | Carter et al. | Apr 2000 | A |
| 6180370 | Queen et al. | Jan 2001 | B1 |
| 6217866 | Schlessinger et al. | Apr 2001 | B1 |
| 6248516 | Winter et al. | Jun 2001 | B1 |
| 6277375 | Ward | Aug 2001 | B1 |
| 6300064 | Knappik et al. | Oct 2001 | B1 |
| 6339070 | Emery | Jan 2002 | B1 |
| 6417337 | Anderson et al. | Jul 2002 | B1 |
| 6512097 | Marks et al. | Jan 2003 | B1 |
| 6569430 | Waldmann et al. | May 2003 | B1 |
| 6602684 | Umana et al. | Aug 2003 | B1 |
| 6800735 | Whitty | Oct 2004 | B2 |
| 6821505 | Ward | Nov 2004 | B2 |
| 6872392 | Nakamura et al. | Mar 2005 | B2 |
| 6872568 | Ni et al. | Mar 2005 | B1 |
| 6903203 | Copley et al. | Jun 2005 | B2 |
| 7083784 | Dall'Acqua et al. | Aug 2006 | B2 |
| 7091321 | Gillies | Aug 2006 | B2 |
| 7166697 | Galanis et al. | Jan 2007 | B1 |
| 7202346 | Payne et al. | Apr 2007 | B2 |
| 7217797 | Hinton et al. | May 2007 | B2 |
| 7252994 | Chuntharapai et al. | Aug 2007 | B2 |
| 7312318 | Hansen et al. | Dec 2007 | B2 |
| 7317089 | Kikly | Jan 2008 | B2 |
| 7326681 | Gerngross | Feb 2008 | B2 |
| 7355015 | Dickson et al. | Apr 2008 | B1 |
| 7371819 | Escary | May 2008 | B2 |
| 7388081 | Seki et al. | Jun 2008 | B2 |
| 7456257 | Jones et al. | Nov 2008 | B2 |
| 7521047 | Nagy et al. | Apr 2009 | B2 |
| 7566771 | Adair et al. | Jul 2009 | B1 |
| 7666422 | Siegall et al. | Feb 2010 | B2 |
| 7670595 | Gillies | Mar 2010 | B2 |
| 7700742 | Cohen et al. | Apr 2010 | B2 |
| 7709610 | Williams et al. | May 2010 | B2 |
| 7723485 | Junutula et al. | May 2010 | B2 |
| 7732572 | Cox, III | Jun 2010 | B2 |
| 7732578 | Foote | Jun 2010 | B2 |
| 7776330 | Yazaki et al. | Aug 2010 | B2 |
| 7790415 | Gillies | Sep 2010 | B2 |
| 7829673 | De et al. | Nov 2010 | B2 |
| 7919078 | Schreiber et al. | Apr 2011 | B2 |
| 7943744 | Frendeus et al. | May 2011 | B2 |
| 8039593 | Kuan et al. | Oct 2011 | B2 |
| 8088896 | Tesar et al. | Jan 2012 | B2 |
| 8119775 | Moretta et al. | Feb 2012 | B2 |
| 8124738 | Terret et al. | Feb 2012 | B2 |
| 8153765 | Park et al. | Apr 2012 | B2 |
| 8187601 | Weng et al. | May 2012 | B2 |
| 8980267 | Grewal et al. | Mar 2015 | B2 |
| 9139634 | Morrison | Sep 2015 | B2 |
| 9611322 | Wilson et al. | Apr 2017 | B2 |
| 9636334 | Pogue et al. | May 2017 | B2 |
| 9963515 | Clarke et al. | May 2018 | B2 |
| 20020142358 | Mikayama et al. | Oct 2002 | A1 |
| 20020164788 | Ellis et al. | Nov 2002 | A1 |
| 20020193569 | Hanna | Dec 2002 | A1 |
| 20030211100 | Bedian et al. | Nov 2003 | A1 |
| 20030211553 | Logtenberg et al. | Nov 2003 | A1 |
| 20040006215 | Keler et al. | Jan 2004 | A1 |
| 20040071696 | Adams et al. | Apr 2004 | A1 |
| 20060083736 | Law et al. | Apr 2006 | A1 |
| 20060099205 | Adams et al. | May 2006 | A1 |
| 20060269516 | Presta et al. | Nov 2006 | A1 |
| 20070098718 | Long et al. | May 2007 | A1 |
| 20070190068 | Hart | Aug 2007 | A1 |
| 20080166319 | Schreiber et al. | Jul 2008 | A1 |
| 20080166352 | Siu et al. | Jul 2008 | A1 |
| 20090006815 | Loubier | Jan 2009 | A1 |
| 20090068175 | Lazar et al. | Mar 2009 | A1 |
| 20090076249 | De et al. | Mar 2009 | A1 |
| 20090092599 | Lazar et al. | Apr 2009 | A1 |
| 20090123950 | Tesar | May 2009 | A1 |
| 20090142340 | Lazar et al. | Jun 2009 | A1 |
| 20090148449 | De Weers | Jun 2009 | A1 |
| 20090175863 | Kraus et al. | Jul 2009 | A1 |
| 20090202568 | Eriksson et al. | Aug 2009 | A1 |
| 20090304710 | Park et al. | Dec 2009 | A1 |
| 20100092489 | Van De Winkel et al. | Apr 2010 | A1 |
| 20100104557 | Bernett et al. | Apr 2010 | A1 |
| 20100172868 | Morrison et al. | Jul 2010 | A1 |
| 20100189689 | Chang et al. | Jul 2010 | A1 |
| 20100189729 | Hoerr et al. | Jul 2010 | A1 |
| 20100285004 | Tesar et al. | Nov 2010 | A1 |
| 20100297076 | Morrison | Nov 2010 | A1 |
| 20110104112 | Morrison et al. | May 2011 | A1 |
| 20120201827 | Elias et al. | Aug 2012 | A1 |
| 20130230517 | Grewal et al. | Sep 2013 | A1 |
| 20130302318 | Rojkjaer et al. | Nov 2013 | A1 |
| 20140248238 | Wilson et al. | Sep 2014 | A1 |
| 20150031395 | Wachter et al. | Jan 2015 | A1 |
| 20150203560 | Grewel | Jul 2015 | A1 |
| 20150313965 | Pogue et al. | Nov 2015 | A1 |
| 20150353485 | Hagen et al. | Dec 2015 | A1 |
| 20160068612 | Clarke et al. | Mar 2016 | A1 |
| 20160122410 | Behrens et al. | May 2016 | A1 |
| 20170202962 | Pogue et al. | Jul 2017 | A1 |
| 20170233449 | Wilson et al. | Aug 2017 | A1 |
| 20180305460 | Clarke et al. | Oct 2018 | A1 |
| Number | Date | Country |
|---|---|---|
| 101045156 | Oct 2007 | CN |
| 103118706 | May 2013 | CN |
| 0706799 | Apr 1996 | EP |
| 2905375 | Mar 2008 | FR |
| 2008-533977 | Aug 2008 | JP |
| 2009-501514 | Jan 2009 | JP |
| 2010-504363 | Feb 2010 | JP |
| 2010-540453 | Dec 2010 | JP |
| 2015-515453 | May 2015 | JP |
| 6184965 | Aug 2017 | JP |
| 9005144 | May 1990 | WO |
| 9700271 | Jan 1997 | WO |
| 199724137 | Jul 1997 | WO |
| 0042072 | Jul 2000 | WO |
| 200040265 | Jul 2000 | WO |
| 0197844 | Dec 2001 | WO |
| 200197844 | Dec 2001 | WO |
| 2004022593 | Mar 2004 | WO |
| 2004022747 | Mar 2004 | WO |
| 2005103083 | Nov 2005 | WO |
| 2006099875 | Sep 2006 | WO |
| 2006125640 | Nov 2006 | WO |
| 2007000769 | Jan 2007 | WO |
| 2007042309 | Apr 2007 | WO |
| 2008006554 | Jan 2008 | WO |
| 2008037257 | Apr 2008 | WO |
| 2008047242 | Apr 2008 | WO |
| 2008124086 | Oct 2008 | WO |
| 2008145139 | Dec 2008 | WO |
| 2009017823 | Feb 2009 | WO |
| 2009073975 | Jun 2009 | WO |
| 2010105290 | Sep 2010 | WO |
| 2011154453 | Dec 2011 | WO |
| 2012041800 | Apr 2012 | WO |
| 2012083370 | Jun 2012 | WO |
| 2012092612 | Jul 2012 | WO |
| 2013059885 | May 2013 | WO |
| 2013107791 | Jul 2013 | WO |
| 2013134138 | Sep 2013 | WO |
| 2014028502 | Feb 2014 | WO |
| 2014178820 | Nov 2014 | WO |
| Entry |
|---|
| Piehler et al. (2000, JBC, vol. 275 No. 51, pp. 40425-40433). (Year: 2000). |
| Yoshinaga et al., “Ig L-chain Shuffling for Affinity Maturation of Phage Lobrary-derived Human Anti-human MCP-1 Antibody Blocking its Chemotactic Activity”, J Biochem, 2008, 143, 593-601. |
| Ozzello, et al., “The use of natural interferon alpha conjugated to a monoclonal antibody anti mammary epithelial mucin (Mc5) for the treatment of human breast cancer xenograft”, Breast Cancer Research and Treatment, 1993, 25, 265-276. |
| Labrijn et al. “Therapeutic IgG4 antibodies engage in Fab-arm exchange with endogenous human IgG4 in vivo”, Nature Biotechnology, 2009, 27:8; 767-771. |
| Koguma, T., et al., “Cloning and characterization of cDNA encoding rat ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase (homologue to human CD38) from islets of Langerhans”, Biochim. Biophys. Acta., 1994, 160-162. |
| Al-Lazikani, B. et al., “Standard Conformations for the Canonical Structures of Immunoglobulins”, Journal of Molecular Biology, 1997, 273, 927-948. |
| Jorge Cortes et al: “Immune modulation of minimal residual disease in early chronic phase chronic myelogenous leukemia : A randomized trial of frontline high-dose imatinib mesylate with or without pegylated interferon alpha-2b and granulocyte-macrophage colony-stimulatin”, 1-15, Cancer., vol. 117, No. 3, Sep. 30, 2010, pp. 572-580. |
| Jorge Cortes et al: “Immune modulation of minimal residual disease in early chronic phase chronic myelogenous leukemia : A randomized trial of frontline high-dose imatinib mesylate with or without pegylated interferon alpha-2b and granulocyte-macrophage colony-stimulating factor”, 1-15, Cancer., vol. 117, No. 3, Sep. 30, 2010, pp. 572-580. |
| J. P. Laubach et al: “CD38-Targeted Immunochemotherapy in Refractory Multiple Myeloma: A New Horizon”, Clinical Cancer Research, vol. 21, No. 12, Apr. 15, 2015, pp. 2660-2662. |
| International Search Report dated Dec. 3, 2015. |
| International Search Report dated Nov. 23, 2015. |
| International Search Report and Written Opinion issued in related application PCT/IB2015/001600 dated Nov. 23, 2015. |
| International Search Report and Written Opinion issued in related application PCT/AU2015/050654 dated Dec. 3, 2015. |
| Ibrahim, et al., “CD38 Expression as an Important Prognostic Factor in B-Cell Chronic Lymphocytic Leukemia”, Blood, vol. 98, No. 1, Jul. 2001, pp. 181-186. |
| Hoon et al., Molecular Cloning of a Human Monoclonal Antibody Reactive to Ganlioside Gm3 Antigen on Human Cancers, Can. Res. vol. 53, Nov. 1993, pp. 5244-5250. |
| Honegger, et al., “Yet another numbering Scheme for Immunoglobulin Variable Domains: An Automatic Modeling and Analysis Tool”, J. Mol. Biology, 309, pp. 657-670, 2001. |
| Honegger, et al., “Yet another numbering scheme for immunoglobulin variable domains: An automatic modeling and analysis tool”, J. Mol. Biol., (2001) 309, 657-670. |
| Hellstroem et al. “Monoclonal Mouse Antibodies Raised Against Human Lung Carcinoma” Can. Res., vol. 46, Aug. 1986, pp. 3917-3923. |
| Hamers-Casteman, et al., Naturally Occurring Antibodies Devoid of Light Chains, Nature, vol. 363, Jun. 1993, pp. 446-448. |
| H. Ludwig et al: “Thaiidomide-dexamethasone compared with melphalan-prednisolone in elderly patients with multiple myeloma”, Blood, vol. 113, No. 15, Oct. 27, 2008, pp. 3435-3442. |
| Giudicelli, et al., “IMGT, the international ImMunoGeneTics database”, Nucleic Acids Research, 1997, vol. 25, No. 1, pp. 206-211. |
| Giudicelli, et al., “IMGT, The International Immunogene Tics database”, Nucleic Acids Research, vol. 25, No. 1, pp. 206-211, 1997. |
| Ghetie, et al., Anti-CD19 Inhibits the Growth of Human B-Cell Tumor Lines in Vitro and of Daudi Cells in SCIO Mice by Inducing Cell Cycle Arrest, Blood, vol. 83, No. 5, Mar. 1994, pp. 1329-1336. |
| Ghasriani et al., JBC, vol. 288, Jan. 4, 2013, No. 1, pp. 247-254. |
| Frey et al., Antibody-based Targeting of Interferon-Alpha to the Tumor Neovasculature: a Critical Evaluation; Royal Society of Chemistry, Integr. Biol., vol. 3, pp. 468-478, 2011. |
| Frederic Millot et al: “Results of a phase II trial testing interferon-alpha 2b and cytarabine in children and adolescents with chronic myelogenous leukemia”, Pediatric Blood and Cancer, vol. 47, No. 5, Jan. 1, 2006, pp. 555-559. |
| Edleman, et al., “The Covalent Stucture of an Entire yG Immunoglobulin Molecule”, Biochemisty, vol. 63, pp. 78-85, Mar. 1969. |
| Edelman, et al., “The Covalent Structure of an Entire yG Immunoglobulin Molecule”, Biochemistry, vol. 63, 1969, pp. 78-85. |
| de Weers, et al., “Daratumumab, a Novel Therapeutic Human CD38 Monoclonal Antibody, Induces Killing of Multiple Myeloma and other Hematological Tumors”, J. Immunol., 2011, 186:1840-1848. |
| De Weers, et al., Daratumumab a Novel Therapeutic Human CD38 Monoclonal Antibody, Induces Killing of Multiple Myeloma and other Hematological Tumors, J. Immunol, 186, pp. 1840-1848, 2011. |
| De Weers et al,, J. Immunol. 2011; 186:1840-48. |
| Chothia, et al., “Conformations of Immunoglobulin Hypervariable Regions”, Nature, vol. 342 pp. 877-883, Dec. 1989. |
| Chothia, et al., “Conformations of immunoglobulin hypervariable regions”, Nature, vol. 342, Dec. 1989, pp. 877-883. |
| Chothia, et al., “Canonical Structures for the Hypervariable Regions of Immunoglobulins”, J. Mol. Biol., (1987), No. 196, pp. 901-917. |
| Chothia et al., “Canonical Structures for the Hypervariable Regions of Immunoglobulins” J. Mol. Biol., No. 196, pp. 901-917, 1987. |
| CD38-Targeted Immunochemotherapy in Refractory Multiple Myeloma: A New Horizon, Jacob P. Laubach and Paul G. Richardson, 2660-2663. |
| Bork, et al., “Go hunting in sequence databases but watch out for traps”, Trends in Genetics, Oct. 1996, vol. 12, No. 10, pp. 425-427. |
| Bork, 2000, Genome Research 10:398-400. |
| Bork etal., 1996, Trends in Genetics 12:425-427. |
| Bonardi, et al. “Delivery of Saporin to Human B-Cell Lymphoma Using Bispecific Antibody: Targeting via DC22 byt not CD19, CD37 or Immunoglobulin Results in Efficient Kiling”, Can Res., vol. 53, Jul. 1991, pp. 3015-3021. |
| Ausiello, et al., “Functional topography of discrete domains of human CD38”, Tissue Antigens, 2000, 56:539-547. |
| Ausiello et al., Tissue Antigens, 2000; 56:539-47. |
| Ausiello et al., “Functional Topography of Discrete Domains of Human CD38”, Tissue Antigens, 56, pp. 539-547, 2000. |
| A. Aviles et al: “Randomized clinical trial of zoledronic acid in multiple myeloma patients undergoing high-dose chemotherapy and stem-cell transplantation”, Current Oncology, vol. 20, No. 1, Feb. 1, 2013, * the whole document *. |
| Yu et al., “Coexpression of Difference Antigenic Markers on Moieties that Bear CA 125 Determinants”, Can. Res., vol. 51, Jan. 1991, pp. 468-475. |
| Yokota et al., “Rapid Tumor Penetration of a Single-Chain Fv and Comparison with other Immunoglobulin Forms”, Can. Res., vol. 52, Jun. 1992, pp. 3402-3408. |
| Wells, “Additivity of Mutational Effects in Proteins”, Biochemistry, vol. 29, No. 37, Sep. 18, 1990, pp. 8509-8517. |
| Wells, “Additivity of Mutational Effects in Proteins”, Biochemistry, 1990, 29:8509-8517. |
| Wahl et al., “The Anti-CD30 Monoclonal Antibody SGN-30 Promotes Growth Arrest and DNA Fragmentation in Vitro and Afects Antbumor Activity in Models of Hodgkin's Disease”, Can. Res. vol. 62, Jul. 2002, pp. 3736-3742. |
| Vijayasaradhi et al., “The Melanoma Antigen gp75 is the Human Homologue of the Mouse b (Brown) Locus Gene Product”, J. Exp. Med., vol. 171, Apr. 1990, pp. 1375-1380. |
| Van Hof et al., “Biodistribution of Indium-Labeled Engineered Human Antibody CTMO1 in Ovarian Cancer Patients: Influence of Protein Dose” Can. Res., vol. 56, Nov. 1996, pp. 5179-5185. |
| Van Der Veer Michael S et al: “Towards effective immunotherapy of myeloma: enhanced elimination of myeloma cells by combination of lenalidomide with the human CD38 monoclonal antibody daratumumab”, Haematolo, Ferrata Storti Foundation, Italy, vol. 96, No. 2, Feb. 1, 2011 (Feb. 1, 2011), pp. 284-290. |
| Tse, et al., “CR011, a Fully Human Monoclonal Antibody-Auristatin E Conjugate, for the Treatment of Melanoma”, Clin. Cancer Res., 2006, 12(4):1373-1382. |
| Tse et al,, Clin Cancer Res, 2006; 12(4):1373-82. |
| Tse et al., “CR011, a Fully human Monoclonal Antibody-Auristatin E Conjugate, for theTreatment of Melanoma”, Clin. Cancer Res., 12(4), 1373-1382, 2006. |
| Trail et al., “Effect of Linker Variation on the Stability, Porency, and Efficacy of Carinoma—reactive BR64-Doxorubicin Immunoconjugates”, Can Res., vol. 56, Nov. 1996, pp. 5179-5185. |
| Tomoyuki, et al., “Engineering the variable region of therapeutic IgG antibodies”, MABS, Landes Bioscience, US, vol. 3, No. 3, May 1, 2011, p. 243-252. |
| Thalidomide-dexamethasone compared with melphalan-prednisolone in elderly patients with multiple myeloma, Heinz Ludwig, 1 Roman Hajek,2 Elena T6thova,3 Johannes Drach,4 Zdenek Adam,2 Boris Labar,5 Miklos Egyed,6, 3435-3442, 113/15. |
| Tailor et al., “Nucleotide Sequence of Human Prostatic Acid Phosphatase Determinded from a Full Length cDNA Clone” Nucleic Acids Research, vol. 18, No. 16, Jul. 1990, p. 4928. |
| Sievers et al., “Selective Abiation of Acute Myeloid Leukemia Using Antibody-Targeted Chemotherapy: a Phase I Study of Anti-CD33 Calichaemicin Immunoconjugate”, Blood, vol. 93, No. 11, Jun. 1999, pp. 3678-3784. |
| Shinkawa, et al., “The Absence of Fucose but Not the Presence of Galactose or Bisecting N-Acetylglucosamine of Human IgG1 Complex-type Oligosaccharides Shows the Critical Role of Enhancing Antibody-dependent Cellular Cytotoxicity”, J. Biological Chemistry, vol. 278, No. 5, Jan. 2003, pp. 3466-3473. |
| Sgouros et al., “Modeling and Dosimetry of Monoclonal Antibody M195 (Anti-CD33) in Acute Myelogenous Leukemia” J. Nuci. Med., vol. 34, No. 3, Mar. 1993, pp. 422-430. |
| Rossi et al., “Preclinical Studies on Targeted Delivery of Multiple IFNa2b to HLA-DR in Diverse Hemotologic Cancers” Lymphoid Neoplasia, Blood Journal, vol. 118, No. 7, Aug. 18, 2011. |
| Rosenblum et al, “Recombinant Immunotoxins Directed against c-erb-3/Her2/neu Oncogene Product: In Vitro Cytotoxicity, Pharmacokinetics, and in Vivo Efficacy Studies in Xenografl Models”, Clin. Can. Res., vol. 5, Apr. 1999, pp. 865-874. |
| Richardson, et al., “Monoclonal Antibodies in the Treatment of Multiple Myeloma”, British Journal of Haematology, 2011, 154:745-754. |
| Richardson et al,, Br. J. Haematol., 2011; 154:745-54. |
| Richardson et al., “Monoclonal Antibodies in the Treatment of Multiple myeloma”, British Journal of Hematology, 154, pp. 745-754, 2011. |
| Results of a Phase II Trial Testing Interferon-Alpha 2b and Cytarabine Children and Adolescents With Chronic Myelogenous Leukemia, Frederic Millot, MD,, 555-559. |
| Reff et al., Depletion of B Cells in Vivo by a Chimeric Mouse Human Monoclonal Antibody to CD20, Blood, vol. 83, No. 2, Jan. 1994, pp. 435-445. |
| Randomized clinical trial of zoledronic acid in multiple myeloma patients undergoing high-dose chemotherapy and stem-cell transplantation, A. Aviles MD,* N Neri MD/, e13-20, 20/1. |
| Pollack et al., Cancer Chemother Pharmacol 2007; 60: 423-35. |
| Pollack et al., Treatment parameters Modulating Regression of Human Melanoma Xenografls by an Antibody-drug conjugate (CR011-vcMMAE) targeting GPNMB, Cancer Chemother Pharmacol, 60, pp. 423-435, 2007. |
| Peterson et al., Effect of Multiple, Repeated Doses of Radioimmunotherapy on Target Antigen Expression (Breast MUC-1 Mucin) in Breast Carcinomas, Can. Res. vol. 57, Mar. 1997, pp. 1103-1108. |
| Pavlinkova et al., “Radioimmunotherapy of Human Colon Cancer Xenografls Using a Dimeric Single-Chain Fv Antibody Construct”, vol. 5, Sep. 1999, pp. 2613-2619. |
| Padlan, et al., “Identification of specificity-determining residues in antibodies”, FASEB Journal, vol. 9, Jan. 1995, pp. 133-139. |
| Padlan, et al., “Identification of Specificity-Determining Residues in Antibodies” FASEB Journal, vol. 9, pp. 133-139, Jan. 1999. |
| Ngo, et al., “Computational Complexity, Protein Structure Prediction, and the Levinthal Paradox”, The Protein Folding Problem and Tertiary Structure Prediction, 1994, pp. 492-495. |
| Mittelman et al, “Active Specific Immunotherapy in Patients with Melanoma”, J. Clin. Invest, vol. 86, Dec. 1990, pp. 2136-2144. |
| Mason, et al., “Value of Monoclonal Anti-CD22 (p135) Antibodies for the Detection of Normal and Neoplastic B Lymphoid Cells”, Blood, vol. 69, No. 3, Mar. 1987, pp. 836-840. |
| Maier et al., Requirements for the Internalization of Murine Monoclonal Antibody Directed Against the HER-2/neu Gene Product c-erB-2, Can. Res., vol. 51, Oct. 1991, pp. 5361-5369. |
| Isreli, et al. Molecular Cloning of a Complementary DNA Encoding a Prostate-specific Membrane Antigen, Can. Res. vol. 53, Jan. 15, 1993, pp. 227-230. |
| Igawa, et al., “Engineering the variable region of therapeutic IgG antibodies”, MASS, Landes Bioscience, US, vol. 3, No. 3, May 1, 2011, p. 243-252. |
| Igawa, et al., “Engineering the variable region of therapeutic IgG antibodies”, MABS, Landes Bioscience, US, vol. 3, No. 3, May 1, 2011, p. 243-252. |
| Lesinski, et al., “IFN-a Bortezomib Overcome BcI-2 and McI-1 Overexpression in Melanoma Cells by Stimulating the Extrinsic Pathway of Apoptosis”, Cancer Res., Oct. 2008; 68:(20), pp. 8351-8360. |
| Lesinski, et al., “IFN-a Bortezomib Overcome Bci-2 and Mci-1 Overexpression in Melanoma Cells by Stimulating the Extrinsic Pathway of Apoptosis”, Cancer Res., 68(20), pp. 8351-8360, 2008. |
| Laubach, et al., “Daratumumab granted breakthrough drug status”, Expert Opinion on Investigational Drugs, vol. 23, No. 4, Feb. 2014, pp. 445-452. |
| Laubach, et al., “Daratumumab granted breakthrough drug status”, Expert Opin. Investig. Drugs, 2014, 23(4):445-452. |
| Ku, et al., “Alternate protein frameworks for molecular recognition”, Proc. Natl. Acad. Sci. USA, vol. 92, Jul. 1995, pp. 6552-6556. |
| Ku, et al., “Alternate Protein Frameworks for Molecular Recognition”, Proc. Natl. Acad. Sci. USA, vol. 92, pp. 6552-6556, Jul. 1995. |
| Kossman et al., “A Phase I Trial of Humanized monoclonal Antibody HuM195 (anti-DC33) with Low-Dose Interleukin 2 in Acute Myelogenous Leukemia”, Clin. Can Res., vol. 5, Oct. 1999, pp. 2748-2755. |
| Koltchev, et al., “Synergy of Interferons and Bortezomib: Advantages of Combination Treatments in Facilitating Apoptosis in Multiple Myeloma Cells”, 2010, PBL InterferonSource. |
| Koltchev, et al., “Synergy of Interferons and Bortezomib: Advantages of Combination Treatments in Facilitating Apoptosis in Muliple Myeloma Cells”, PBL Interferon Source, 2010. |
| Kodama, et al., “Mutated SEA-D227 A-conjgated antibodies greatly enhance antitumor activity against MUC1-expressing bile duct carcinoma”, Cancer Immunology, Immnotherapy, vol. 50, No. 10, Dec. 2001, pp. 539-548. |
| “Peripheral blood CD38 expression predicts time to progression in B-cell chronic lymphocytic leukemia after first-line therapy with high-does chlorambucil”, Haematologica, vol. 87, No. 2, Feb. 2002, pp. 217-218. |
| Bonardi, et al., “Delivery of Saporin to Human B-Cell Lymphoma Using Bispecific Antibody: Targeting via DC22 byt not CD19, CD37, or Immunoglobulin Results in Efficient Killing”, Can. Res., vol. 53, Jul. 1991, pp. 3015-3021. |
| Bork, “Powers and Pitfalls in Sequence Analysis: The 70% Hurdle”, Genome Research, 2000, 10:398-400. |
| Bork, et al., “Go hunting in sequence databases but watch out for the traps”, Trends in Genetics, 1996, 12:425-427. |
| Francisco, et al., “Agonistic Properties and in Vivo Antitumor Activity of the Anti-CD40 Antibody SGN-14”, Can. Res., vol. 60, Jun. 15, 2000, pp. 3225-3231. |
| Ghetie, et al., “Anti-CD19 Inhibits the Growth of Human B-Cell Tumor Lines In Vitro and of Daudi Cells in SCID Mice by Inducing Cell Cycle Arrest”, Blood, vol. 83, No. 5, Mar. 1, 1994, pp. 1329-1336. |
| Hamers-Casterman, et al., “Naturally occurring antibodies devoid of light chains”, Nature, vol. 363, Jun. 3, 1993, pp. 446-448. |
| Hoon, et al., “Molecular Cloning of a Human Monoclonal Antibody Reactive to Ganglioside Gm3 Antigen on Human Cancers”, Can. Res. vol. 53, Nov. 1, 1993, pp. 5244-5250. |
| Ibrahim, et al., “CD38 expression as an important prognostic factor in B-cell chronic lymphocytic leukemia”, Blood, vol. 98, No. 1, Jul. 1, 2001, pp. 181-186. |
| International Search Report and Written Opinion from related application PCT/AU2012/001323 dated Mar. 13, 2013. |
| International Search Report and Written Opinion from related application PCT/US2013/038659 dated Feb. 12, 2014. |
| Israeli, et al., “Molecular Cloning of a Complementary DNA Encoding a Prostate-specific Membrane Antigen”, Can. Res., vol. 53, Jan. 15, 1993, pp. 227-230. |
| Maier, et al., “Requirements for the Internalization of a Murine Monoclonal Antibody Directed against the HER-2/neu Gene Product c-erB-2”, Can. Res., vol. 51, Oct. 1, 2991, pp. 5361-5369. |
| Pavlinkova, et al., “Radioimmunotherapy of Human Colon Cancer Xenografts Using a Dimeric Single-Chain Fv Antibody Construct”, vol. 5, Sep. 1999, pp. 2613-2619. |
| Peterson, et al., “Effect of Multiple, Repeated Doses of Radioimmunotherapy on Target Antigen Expression (Breast MUC-1 Mucin) in Breast Carcinomas”, Can. Res., vol. 57, Mar. 15, 1997, pp. 1103-1108. |
| Piehler, et al., “New structural and functional aspects of the Type 1 interferon-receptor interaction revealed by comprehensive mutational analysis of the binding interface”, vol. 275, No. 51, Dec. 2000, pp. 40425-40433. |
| Reff, et al., “Depletion of B Cells In Vivo by a Chimeric Mouse Human Monoclonal Antibody to CD20”, Blood, vol. 83, No. 2, Jan. 15, 1994, pp. 435-445. |
| Rosenblum, et al., “Recombinant Immunotoxins Directed against c-erb-3/HER2/neu Oncogene Product: In Vitro Cytotoxicity, Pharmacokinetics, and in Vivo Efficacy Studies in Xenograft Models”, Clin. Can. Res., vol. 5, Apr. 1999, pp. 865-874. |
| Sgouros, et al., “Modeling and Dosimetry of Monoclonal Antibody M195 (Anti-CD33) in Acute Myelogenous Leukemia”, J. Nucl. Med., vol. 34, No. 3, Mar. 1993, pp. 422-430. |
| Sievers, et al., “Selective Ablation of Acute Myeloid Leukemia Using Antibody-Targeted Chemotherapy: a Phase I Study of Anti-CD33 Calichaemicin Immunoconjugate”, Blood, vol. 93, No. 11, Jun. 1, 1999, pp. 3678-3784. |
| Tailor, et al., “Nucleotide sequence of human prostatic acid phosphatase determined from a full-length cDNA clone”, Nucleic Acids Research, vol. 18, No. 16, Jul. 11, 1990, p. 4928. |
| Trail, et al., “Effect of Linker Variation on the Stability, Porency, and Efficacy of Carcinoma-reactive BR64-Doxorubicin Immunoconjugates”, Can. Res., vol. 57, Jan. 1, 1997, pp. 100-105. |
| van Hof, et al., “Biodistribution of Indium-Labeled Engineered Human Antibody CTMO1 in Ovarian Cancer Patients: Influence of Protein Dose”, Can. Res., vol. 56, Nov. 15, 1996, pp. 5179-5185. |
| Wahl, et al., “The Anti-CD30 Monoclonal Antibody SGN-30 Promotes Growth Arrest and DNA Fragmentation in Vitro and Afects Antibumor Activity in Models of Hodgkin's Disease”, Can. Res., vol. 62, Jul. 1, 2002, pp. 3736-3742. |
| Yokota, et al., “Rapid Tumor Penetration of a Signle-Chain Fv and Comparison with other Immunoglobulin Forms”, Can. Res., vol. 52, Jun. 15, 1992, pp. 3402-3408. |
| Yu, et al., “Coexpression of Different Antigenic Markers on Moieties that Bear CA 125 Determinants”, Can. Res., vol. 51, Jan. 15, 1991, pp. 468-475. |
| Ellis, J.H. et al., J. Immunol., (1995), vol. 155, No. 2, pp. 925-937. |
| Huang, T.-H. et al., J. Immunol., (2007), vol. 179, pp. 6881-6888. |
| Kalie, E. et al., J. Biol. Chem., (2007), vol. 282, No. 15, pp. 11602-1161L. |
| Pan, M. et al., Biochemistry, (2008), vol. 47, pp. 12018-12027, abstract. |
| Stewart, A.G. et al., DNA, (1987), vol. 6, No. 2, pp. 119-128, abstract; 123, Table 1. |
| Thomas, C. et al., Cell, (Aug. 19, 2011), vol. 146, pp. 621-632. |
| Trzpis, M. et al., Am. J. Pathol., (2007), vol. 171, No. 2, pp. 386-395. |
| Norderhaug et al., “Chimeric mouse human IgG3 antibodies with an IgG4-like hinge region induce complement-mediated lysis more efficiently than IgG3 with normal hinge”, Eur. J. Immunol., 1991, 21, 2379-2384. |
| Ono et al., “The humanized anti-HM1.24 antibody effectively kills multiple myeloma cells by human effector cell-mediated cytotoxicity”, Mol. Immuno., 1999, 36, 387-395. |
| Otsuki et al., “Human Myeloma Cell Apoptosis induced by interferon-a”, Jul. 1998, 103, 518-529. |
| Peled et al., “The Biochemistry of Somatic Hypermutation”, Annu. Rev. Immunol., 2008, 26, 481-511. |
| Perez et al., “Isolation and Characterization of a cDNA encoding the KS1/4 epithelial carcinoma marker”, J. Immumnol., 1990, 142, 3662-3667. |
| Petkova et. al., “Enhanced half-life of genetically engineered human IgG1 antibodies in a humanized FcRn Mouse model: Potential Application in Humorally mediated autoimmune disease”, Immunol., 2006, 18(12), 1759-1769. |
| Poljak, “Production and Structure of Diabodies”, Structure, Dec. 1994, 2, 1121-1123. |
| Queen et al., “A Humanized antibody that binds to the interleukin 2 receptor”, PNAS, Dec. 1989, 86(24), 10029-10033. |
| Ragnhammar et al., “Effect of Monoclonal Antibody 17-1A and GM-CSF in patients with advanced colorectal carcinoma—long-lasting, complete remissions can be induced”, Int. J. Cancer, 1993, 53, 751-758. |
| Rossi et al., “CD20-Targeted Tetrameric Interferon-a, a novel and potent immunocytokine for the therapy of B-cell lymphomas”, Blood, Oct. 2009, vol. 114, No. 18, 3864-3871. |
| Saleh et al., “Generation of a human anti-idiotypic antibody that mimics the GD2 antigen”, J. Immunol., 1993, 151, 3390-3398. |
| Schier et al., “Isolation of High-Affinity Monomeric Human Anti-c-erbB-2 Single chain Fv Using Affinity-driven Selection”, J. Mol. Biol., 1996, 255, 28-43. |
| Schier et al., “Isolation of Picomolar Affinity Anti-c-erbB-2 Single-Chain Fv by Molecular Evolution of the Complementarity Determining Regions in the Center of the Antibody Binding Site”, J. Mol. Biol., 1996, 263, 551-567. |
| Shields et al., “High Resolution Mapping of the Binding Site on Human LgG1 for FcyRI, FcyRII, FcyRIII, and FcRn and Design of IgG1 Variants with Improved Binding to the FcyR”, J. Biol. Chem., Mar. 2001, 276(9), 6591-6604. |
| Shitara et al., “A mouse/human chimeric anti-(ganglioside GD3) antibody with enhanced antitumor activities”, Cancer Immunol. Immunother, 1993, 36, 373-380. |
| Stavenhagen et al., “Fc Optimization of Therapeutic Antibodies Enhances their ability to kill tumor cells in vitro and controls tumor expansion in vivo via low-affinity activating Fcy Receptors”, Cancer Res., 2007, 67, 8882-8890. |
| Tan et al., “Influence of the hinge region on complement activation, C1q binding, and segmental flexibility in chimeric human immunoglobulins”, PNAS, 1990, 87, 162-166. |
| Thie et al., “Affinity Maturation by Phage Display”, Methods, Mol. Biol., 2009, 525, 309-322. |
| Trail et al., “Cure of Xenografted Human Carcinomas by BR96-Doxorubicin Immunoconjugates”, Science, 1993, 261, 212-215. |
| Trauth et al., “Monoclonal Antibody-Mediated Tumor Regression by Induction of Apoptosis”, Science, Jul. 1989, 245, 301-304. |
| Wahl et al., Cancer Res., 2002, 62(13), 3736-3742. |
| Ward et al., “Binding Activities of a Repertoire of Single Immunoglobulin variable domains secreted from Escherichia coli”, Nature, Oct. 1989, 341, 544-546. |
| Xuan et al., “Targeted delivery of interferon-a via fusion to anti-CD20 results in potent antitumor activity against B-cell lymphoma”, Blood, 2010, 115, 2864-2871. |
| Alkan et al., “Antiviral and Antiproliferative Effects of Interferons Delivered via Monoclonal Antibodies”, Journal of Interferon Research, 1984, vol. 4, No. 3, p. 355-363. |
| Behr et al., “Low-Versus High-Dose Radioimmunotherapy with Humanized Anti-CD22 or Chimeric Anti-CD20 Antibodies in a Broad Spectrum of B Cell-associated Malignancies”, Clin. Cancer Res., Oct. 1999, 5, 3304s-3314s. |
| Benhar, “Design of Synthetic Antibody Libraries”, Expert Opin. Biol. Ther., May 2007, 7(5), 763-779. |
| Bhattacharya-Chatterjee et al., “Idiotype Vaccines against human T cell leukemia. II. Generation and Characterization of a Monoclonal idiotype cascade (Ab1, Ab2, and Ab3)”, J. Immunol., 1988, 141, 1398-1403. |
| Bird et al., “Single-Chain Antigen-Binding Proteins”, Science, Oct. 1988, 242, 423-426. |
| Brekke et al., “Human IgG3 Can Adopt the Disulfuide bond pattern characteristic for IgG1 without resembling it in complement mediated cell lysis”, Mol. Immuol., May 1993, 30, 1419-1425. |
| Bumal et al., “Characterization of the Human Tumor and Normal Tissue Reactivity of the KS1/4 Monoclonal Antibody”, Hybridoma, 1988, 7(4), 407-415. |
| Camploi et al., “Human High Molecular Weight-Melanoma-Associated Antigen (HMW-MAA): A Melanoma Cell Surface Chondroitin Sulfate Proteoglycan (MSCP) with Biological and Clinical Significance”, Crit. Rev. Immunol., 2004, 24(4), 267-296. |
| Crowder et al., Neoplasia, PML mediates IFN-a-induced apoptosis in myeloma by regulating TRAIL induction, 2005, pp. 1280-1287. |
| Dall'Acqua et al., “Increasing the Affinity of a Human IgG1 for the Neonatal Fc Receptor: Biological Consequences”, J. of Immunol., 2002, 169, 5171-5180. |
| Dall'Acqua et al., “Properties of Human IgG1s Engineered for Enhanced Binding to the Neonatal Fc Receptor (FcRn)”, J. Biol. Chem., Aug. 2006, 281(33), 23514-23524. |
| Davies & Riechmann, “Camelising Human Antibody Fragments: NMR Studies on VH Domains”, FEBS Letters, 1994, 339, 285-290. |
| Deaglio et al., “CD38 at the Junction between prognostic marker and therapeutic target”, Trends in Mol. Med., 2008, 14(5), 210-218. |
| Divgi et al., “Clinical Comparison of Radiolocalization of Two Monoclonal Antibodies (mAbs) Against the TAG-72 Antigen”, Nucl. Med. Biol., 1994, 21(1), 9-15. |
| During et al., “CD438 Expression is an Important Prognostic Marker in Chronic Lymphocytic Leukaemia”, Leuk. Res., 2002, 16, 30-35. |
| Estin et al., “Transfected Mouse Melanoma Lines That Express Various Levels of Human Melanoma-Associated Antigen p97”, J. Natl. Cancer Instit., Mar. 1989, 81(6), 445-448. |
| Feizi, “Demonstration by Monoclonal Antibodies that carbohydrate structures of glycoproteins and glycolipids are onco-developmental antigens”, Nature, Mar. 1985, 314(7), 53-57. |
| Ferrara et al., “Modulation of Therapeutic Antibody Effector Functions by Glycosylation Engineering: Influence of Golgi Enzyme Localization Domain and Co-Expression of Heterologous B1, 4-N-acetylgucosaminyltransferase III and Golgi a-mannosidase II”, Biotechnol. Bioeng., Apr. 2006, 93(5), 851-861. |
| Fishburn, “The Pharmacology of PEGylation: Balancing PD with PK to Generate Novel Therapeutics”, J. Pharm. Sci., 2008, 97, 4167-4183. |
| Foon et al., “Murine Anti-Idiotype (Id) Monoclonal Antibody (mAb) induces specific humoral responses to carcino-embryonic antigen (CEA) in Colorectal Cancer (CRC) Patients”, Proc. Am. Soc. Clin. Oncol., 1994, 13, 294. |
| Fornier et al., “Update on the Management of Advanced Breast Cancer”, Oncology, 1999, 13, 647-658. |
| Frankel et al., “Cell Surface Receptor-Targeted Therapy of Acute Myeloid Leukemia: A Review”, Cancer Biother. Radiopharm, 2000, 15(5), 459-477. |
| Funaro et al., “CD38 Functions Are Regulated Through an Internalization Step”, J. Immunol., 1998, 160, 2238-2247. |
| Gennaro, Ed., Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co., Easton, PA, 1990. |
| Harden et al., “Interleukin-6 Prevents Dexamethasone-induced myeloma cell death”, Blood, 1994, 84, 3063-3070. |
| Hellstrom et al., “Monoclonal Antibodies to Cell Surface Antigens Shared by Chemically Induced Mouse Bladder Carcinomas”, Cancer Res., 1985, 45, 2210-2188. |
| Henttu et al., “cDNA Coding for the Entire Human Prostate Specific Antigen Shows High Homologies to the Human Tissue Kallikrein Genes”, Biochem. Biophys. Res. Comm., 1989, 160(2), 903-910. |
| Herlyn et al., “Monoclonal Antibody Detection of a Circulating Tumor-Associates Antigen. I. Presence of Antigen in Sera of Patients with Colorectal, Gastric and Pancreatic Carcinoma”, J. Clin. Immunol., 1982, 2(2), 135-140. |
| Hilkens et al., “Cell Membrane-Associated mucins and their adhesion-modulating property”, Trends in Bio. Chem. Sci., Sep. 1992, 17, 359-363. |
| Hinton et al., “An Engineered Human IgG1 Antibody with Longer Serum Half-Life”, J. Immunol., 2006, 176, 346-356. |
| Hinton et al., “Engineered Human IgG Antibodies with Longer Serum Half-Lives in Primates”, J. Biol. Chem., Feb. 2004, 279(8), 6213-6216. |
| Holliger et al., “Diabodies: Small Bivalent and Bispecific Antibody Fragments”, PNAS USA, Jul. 1993, 90, 6444-6448. |
| Huston et al., “Protein Engineering of Antibody binding sites: Recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli”, PNAS USA, Aug. 1988, 85, 5879-5883. |
| Idusogie et al., “Engineered Antibodies with Increased Activity to Recruit Complement”, J. Immunol., 2001, 166, 2571-2575. |
| Jones et al., “Selective Clearance of Glycoforms of a Complex Glycoprotein pharmaceutical caused by terminal N-acetylglucosamine is similar in humans and cynomolgus monkeys”, Glycobiology, 2007, 17(5), 529-540. |
| Kanda et al., “Comparison of Biological Activity among nonfucosylated therapeutic IgG1 antibodies with three different N-linked Fc oligosaccharides: the high-mannose, hybrid, and complex types”, Glycobiology, 2006, 17(1), 104-118. |
| Kaneko et al., “Anti-Inflammatory Activity of Immunoglobulin G. Resulting from Fc Sialylation”, Science, Aug. 2006, 313, 670-673. |
| Kolkman et al., “Directed Evolution of Proteins by Exon Shuffling”, Nat. Biotechnol., May 2001, 19(5), 423-428. |
| Kopsidas et al., “In vitro Improvement of a Shark IgNAR antibody by QB replicase mutation and ribosome display mimics in vivo affinity maturation”, Immunol. Lett., Nov. 15, 2006, 107(2), 163-168. |
| Li et al., “Optimization of Humanized IgGs in Glycoengineered Pichia Pastoris”, Nat. Biotechnol., 2006, 24, 210-215. |
| Li et al., “The Epitope Specificity and Tissue Reactivity of Four Murine Monoclonal Anti-CD22 Antibodies”, Cell Immunol., 1989, 111, 85-99. |
| Liu et al., “Crystal Structure of Human CD38 Extracellular Domain”, Structure, 2005, 13, 1331-1339. |
| Livingston et al., “Improved Survival in Stage III Melanoma Patients with GM2 Antibodies: A Randomized Trail of Adjuvant Vaccination With GM2 Ganglioside” J. Clin. Oncol., 1994, 12, 1036-1044. |
| Malavasi et al., “CD38: A multi-lineage cell activation molecule with a split personality”, Intl. J. Clin. Lab. Res., 1992, 22, 73-80. |
| Malavasi, et al, Human Immunology, Characterization of a Murine Monoclonal Antibody Specific for Human Early Lymphohemopoietic Cells ,1984, 9, 9-20. |
| Matsui, et al., British Journal of Haematology, Anti-tumour activity of interferon-alpha in multiple myeloma: role of interleukin 6 and tumor cell differentiation 2003, 121, pp. 251-258. |
| Michaelsen et al., “Enhancement of Complement Activation and Cytolysis of Human IgG3 by Deletion of Hinge Exons”, Scand. J. Immunol, 1990, 32, 517-528. |
| Morabito, “Haematologica”, Feb. 2002, 87(2), 217-218. |
| Natali et al., “Immunohistochemical Detection of Antigen in Human Primary and Metastatic Melanomas by the Monoclonal Antibody 140.240 and Its Possible Prognostic Significance”, Cancer, 1987, 59, 55-63. |
| Natsume et al., “Engineered Antibodies of IgG1/IgG3 Mixed Isotype with Enhanced Cytotoxic Activities”, Cancer Research, May 2008, 68(10), 3863-3872. |
| Jiao, Y., et al. CD38: targeted therapy in multiple myeloma and therapeutic potential for solid cancers. Expert Opinion on Investigational Drugs., 2020, Sep. 2020, p. 1-14, published online ahead of print. |
| Mihara, K., et al. Activated T-cell-mediated immunotherapy with a chimeric receptor against CD38 in B-cell Non-Hodgkin lymphoma. J. Immunotherapy, 2009, 32:737-743. |
| Number | Date | Country | |
|---|---|---|---|
| 20170233449 A1 | Aug 2017 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14262841 | Apr 2014 | US |
| Child | 15447912 | US | |
| Parent | PCT/AU2012/001323 | Oct 2012 | US |
| Child | 14262841 | US |
