HK1158933B - Anti-igf antibodies - Google Patents

Description

anti-IGF antibodies

The present invention relates to the treatment of hyperproliferative diseases, in particular to the treatment of cancer.

Background

Insulin-like growth factor-1 (IGF-1; a 70 amino acid polypeptide) and insulin-like growth factor-2 (IGF-2; a 67 amino acid polypeptide) are 7.5kD soluble factors present in serum that strongly stimulate the growth of a variety of mammalian cells (reviewed in Pollack et al, 2004). Upon secretion into the bloodstream, IGFs form complexes with IGFBPs, preventing their proteolytic degradation in serum on their way to the target tissue and their binding to IGF receptors. IGF is also known to be secreted in the target tissue itself in an autocrine or paracrine fashion. This process is known to occur during normal fetal development, where IGF plays an important role in the growth of tissues, bones, and organs. It is also found in many cancer tissues where paracrine signaling between tumor cells and stromal cells or autocrine IGF production by tumor cells themselves is believed to occur (reviewed in LeRoith D, 2003).

IGF-1 and IGF-2 are capable of binding to the IGF-1 receptor (IGF-1R) expressed on many normal tissues, which is functionally a 460kD heterotetramer consisting of dimeric alpha-and beta-subunits with similar affinities (Rubin et al, 1995). IGF-2 also binds to the IGF-2 receptor, which is believed to prevent IGF-2 from binding to IGF-1R and signaling through IGF-1R. In this regard, IGF-2R has been demonstrated to be a tumor suppressor protein. IGF-1R is structurally similar to the insulin receptor that exists in two forms, IR-A and IR-B, except that the IR-A ectodomain lacks an alternatively spliced 12 amino acid exon. IR-B is the major IR isoform expressed in most normal adult tissues and functions to mediate the effects of insulin on metabolism. On the other hand, IR-A is known to be highly expressed in developing fetal tissues, not adult normal tissues. Recent studies have also shown that IR-A (but not IR-B) is highly expressed in certain cancers. Exon deletions in IR- A do not affect insulin binding but cause small conformational changes, resulting in much higher affinity for IGF-2 binding than for IR-B (frascA et al, 1999; Pandini et al, 2002). Since IR-A is expressed in cancer tissues and binds more readily to IGF-2, it may be as important as IGF1-R for IR-A to mediate the mitogenic effects of IGF-2 in cancer.

Binding of IGF to IGF-1R triggers a complex intracellular signaling cascade leading to activation of proteins that stimulate proliferation and survival (reviewed in Pollack et al, 2004).

Unlike the EGFR and Her2neu receptors, the absence of amplification of IGF1-R or IR-A receptors in cancer indicates that receptor activation is controlled by the presence of active ligands. A large body of scientific, epidemiological and clinical literature has shown the role of IGF in the formation, progression and metastasis of many different cancer types (reviewed in Jerome et al, 2003 and Pollack et al, 2004).

For example, in colorectal cancer, IGF-2mRNA and protein are expressed in clinical colorectal tumor samples more than in adjacent normal tissues (Freeer et al, 1999; Li et al, 2004). In patients with colorectal tumours, elevated IGF serum levels are also positively correlated with the proliferative cell index (Zhao et al, 2005). In addition, elevated circulating levels of IGF-2 are associated with an elevated risk of developing colorectal cancer and adenoma (Renehan et al, 2000a) and b); hassan et al, 2000). Parental loss of imprinting (LOI) of the IGF-2 gene, which results in epigenetic variations following high expression of IGF-2, is a heritable molecular trait that has recently been identified in patients with colorectal and other tumor types. IGF-2 imprinting loss has been shown to be associated with five-fold risk in colorectal tumors (Cui et al, 2003; Cruz-Correa et al, 2004) and adenomas (Woodson et al, 2004). Antibodies target the IGF-1R α -subunit, thereby blocking IGF binding and internalizing the receptor have been shown to delay the growth of xenograft colon cancer-derived cell lines, such as COLO205 (Burtrum et al, 2003).

High IGF levels are associated with poor prognosis in human lung adenocarcinoma (Takanami et al, 1996) and many cell lines derived from SCLC and NSCLC can express and secrete IGF (Quinn et al, 1996). Transgenic overexpression of IGF-2 induces spontaneous lung tumors in a murine model (Moorhead et al, 2003). In the case of hepatocellular carcinoma (HCC), human clinical samples and animal models of HCC express IGF mRNA and protein levels higher than corresponding normal tissues and this is associated with enhanced tumor growth (Wang et al, 2003; Ng et al, 1998). IGF-2 has also been shown to be a serological marker for HCC, with higher levels of IGF-2 in HCC patient serum compared to controls (Tsai et al, 2005).

The growth of many juvenile solid tumors, such as Ewing's sarcoma and rhabdomyosarcoma, appears to be dependent inter alia on the IGF signaling pathway (Scotlandi et al, 1996). LOI of the IGF-2 gene has been shown to be an early genetic event in the formation of embryonic rhabdomyosarcoma (Fukuzawa et al, 1999). In the case of type 1 EWS-FLI1 chimeric transcription factors expressed via chromosomal translocation leading to increased expression of target genes (including IGF ligand and IGF-1R) and decreased expression of IGFBP-3), autocrine IGF signaling is also thought to strongly influence ewing sarcoma growth. Antibodies and small molecule compounds targeting IGF-1R have been shown to reduce the growth of xenograft pediatric solid tumor-derived cell lines (Kolb et al, 2008; Manara et al, 2007).

The use of IGF ligand-specific antibodies has been shown to inhibit the growth of human prostate cancer cells in adult bone implanted in SCID mice (Goya et al, 2004). In addition, it has been demonstrated that the same IGF ligand antibody can block paracrine supply of IGF and inhibit liver metastasis in human colorectal cancer cells in a murine xenograft system (Miyamoto et al, 2005).

There is also substantial evidence that the IGF signaling system reduces the sensitivity of cancer to chemotherapeutic agents and radiation. One of the earliest findings in this regard was the demonstration that IGF-1R knockout mouse embryos are difficult to transform by viruses, oncogenes and overexpressed growth factor receptors (Sell et al, 1993; Sell et al, 1994) and that overexpression of IGF-1R protects cells from UV irradiation and gamma radiation induced apoptosis (Kulik et al, 1997). Furthermore, using hepatoma cell lines secreting large amounts of IGF-2, it was found that neutralization of IGF-2 in vitro can significantly enhance the response to chemotherapeutic agents, such as cisplatin and etoposide, especially at lower cytostatic doses, suggesting that IGF-2 can reduce sensitivity to chemotherapeutic agents (Lund et al, 2004). Consistent with these findings, antibodies targeting IGF-1R have been shown to enhance the sensitivity of tumor xenografts to the growth inhibitory effects of chemotherapeutic drugs and radiation (Goetsch et al, 2005).

Many antibodies have been reported to exhibit cross-reactive binding to human IGF-1 and human IGF-2. Antibody sm1.2 was raised against human IGF-1, showed 40% cross-reactivity with human IGF-2, and was shown to inhibit proliferation of the mouse fibroblast cell line BALB/c3T3 stimulated with 20ng/mL human IGF-1 (Russell et al, 1984). In a study designed to functionally localize IGF-1 epitopes by generating monoclonal antibodies to the entire IGF-1 protein and a portion of that protein, a number of antibodies that cross-react with IGF-2 have been identified (Manes et al, 1997). The percentage of cross-reactivity with IGF-2 ranged from 0 to 800%, and several antibodies were identified that reacted equally with IGF-1 and IGF-2. KM1486 is a rat monoclonal antibody that cross-reacts with human IGF-1 and IGF-2, and KM1486 has been shown to inhibit the growth of human prostate cancer cells in adult bone implanted in non-obese diabetic/Severe Complex immunodeficient mice (Goya et al, 2004). In addition, KM1486 has been shown to inhibit liver metastasis in human colorectal cancer (Miyamoto et al, 2005). KM1486 has also been described in WO 03/093317, JP 2003-310275, WO 2005/018671, WO 2005/028515 and WO 2005/027970.

For the treatment of human diseases, antibodies with fully human sequences are highly desirable to minimize the risk of human anti-antibody responses and neutralizing antibodies that rapidly eliminate the antibody administered in vivo and thus reduce the therapeutic effect. Thus (and given the role IGF-1 and IGF-2 dependent signaling play in cancer development and progression), there is a need to obtain fully human antibodies. WO 2007/070432 describes fully human antibodies that co-neutralize the mitogenic effects of two ligands.

It is an object of the present invention to provide alternative human anti-IGF antibodies with high affinity.

Another object of the present invention is to provide human anti-IGF antibodies with high affinity for IGF-1.

Another object of the present invention is to provide human anti-IGF antibodies with high affinity for IGF-1 and for IGF-2.

Another object of the invention is to provide human anti-IGF antibodies with appropriate relative affinity for IGF-1 and for IGF-2.

Another object of the present invention is to provide human anti-IGF antibodies with higher affinity for IGF-1 than for IGF-2.

It is another object of the present invention to provide human anti-IGF antibodies with high IGF-1 neutralizing potency.

Another object of the present invention is to provide human anti-IGF antibodies with high IGF-1 and IGF-2 neutralizing potency.

It is another object of the present invention to provide human anti-IGF antibodies with high solubility and stability.

Another object of the invention is to obtain antibodies that do not affect the binding of insulin to its receptor.

Pharmacodynamic biomarkers by drug activity may help in the clinical development of therapeutic agents. Clinical studies using antibodies targeting IGF-1R have demonstrated that an increase in total serum IGF-1 levels can be a useful pharmacodynamic marker for such agents (Pollack et al, 2007). The increase in total serum IGF-1 levels may be due to a feedback mechanism involving pituitary Growth Hormone (GH) secretion that promotes the release of IGF-1 and IGFBP from the liver. In fact, it has been demonstrated in humans that only about 1% of the total IGF-1 level is free or biologically active IGF-1 responsible for this feedback response (Chen et al, 2005).

It is therefore another object of the present invention to provide a therapy with biomarkers that allow pharmacological monitoring of the effectiveness of the therapy for the treatment of diseases whose development and/or progression is etiologically associated with IGF.

In the present experiments, it was demonstrated that total serum IGF-1 levels are elevated with the use of the anti-IGF antibodies of the present invention. Thus, total IGF-1 levels are useful pharmacodynamic markers of the effectiveness of anti-IGF antibody therapy. It is therefore highly advantageous that the antibodies of the invention cross-react with IGF of a suitable animal species, such as mouse or rat, so that the pharmacodynamic effects can be tested before clinical use.

"Total IGF-1 level" refers to the amount of IGF-1 added in plasma or serum, including the amount of IGF-1 bound to serum binding protein plus free (unbound) IGF-1.

Thus, in another aspect, the invention relates to a method of determining the effectiveness of an antibody molecule that binds to IGF-1 and IGF-2 for treating a patient with cancer. In the first step of the method, the total IGF-1 level is measured in a biological sample (e.g. serum or plasma) of the patient. The antibody molecule is then administered and, subsequently, after a period of time sufficient for the therapeutic antibody to exert its effect, the total IGF-1 level is again determined. The amount of increase in total IGF-1 level over that measured in the first step is indicative of the extent to which the patient is responsive to the anti-IGF antibody molecule. This method is preferably used to monitor therapy with the antibody of the invention.

Brief Description of Drawings

FIGS. 1A-1G show ELISA binding titrations of IgG1 antibodies, designated 60814, 60819 and 60833, against human IGF-1 (FIG. 1A), mouse IGF-1 (FIG. 1B), rat IGF-1 (FIG. 1C), human IGF-2 (FIG. 1D), mouse IGF-2 (FIG. 1E), rat IGF-2 (FIG. 1F) and human insulin (FIG. 1G).

FIG. 2 shows a typical titration of antibody 60833 to neutralize IGF-1R phosphorylation induced by IGF-1(20ng/mL) (FIG. 2A) and IGF-2(100ng/mL) (FIG. 2B) using a cell-based ELISA.

FIG. 3A shows A typical titration of antibody 60833 to neutralize IGF-2(100ng/mL) induced IR-A phosphorylation. FIG. 3B shows a typical titration of antibody 60833 to neutralize human serum (20%) induced IGF-1R phosphorylation. Both assays were performed using a cell-based ELISA.

FIGS. 4A-2D show the effect of antibodies 60814 and 60819 on MCF-7 (FIGS. 4A and 4B) and COLO205 (FIGS. 4C and 4D) cell proliferation stimulated by IGF-1 (FIGS. 4A and 4C) and IGF-2 (FIGS. 4B and 4D).

FIG. 5 shows the effect of antibodies 60819 and 60833 on the proliferation of the Ewing's sarcoma-derived cell line TC-71 in 10% growth medium.

FIG. 6 shows the effect of antibody 60819 on murine total serum IGF-1 levels 24 hours after single doses of 25mg/kg, 12.5mg/kg, 6.25mg/kg, 3.13mg/kg were administered; 0mg/kg represents the total serum IGF-1 level prior to antibody treatment;

FIG. 7 shows the effect of antibody 60819 on rat total plasma IGF-1 levels 24 hours after single doses of 30mg/kg, 100mg/kg, 200mg/kg administered by 10 minute intravenous infusion. 0mg/kg represents the total serum IGF-1 levels prior to antibody treatment.

FIG. 8 illustrates the effect of antibody 60819 and rapamycin (alone or in combination) on the proliferation of the Ewing's sarcoma-derived cell line SK-ES-1 in growth medium containing 10% FCS.

Figure 9 shows the effect of antibody 60819 and rapamycin (alone or in combination) on AKT phosphorylation and PTEN levels.

FIG. 10 illustrates the effect of antibody 60819 and erlotinib/derease (Tarceva), alone or in combination, on the proliferation of cell line A-549 derived from NSCLC in growth medium containing 10% FCS.

FIG. 11 shows the 3D structure of human IGF-1, with amino acids bound by antibody 60833 highlighted (dark gray). The linear amino acid sequence of human IGF-1 is shown below, with the amino acids that interact with antibody 60833 underlined.

FIG. 12 shows the amino acid and DNA sequences of the variable chains of antibodies 60814(A), 60819(B) and 60833 (C); the CDRs are in bold.

Summary of The Invention

In one aspect, the invention relates to an isolated human antibody molecule, which

a) Can combine human IGF-1 and IGF-2 to

i) Prevent binding of IGF-1 and IGF-2 to IGF-1 receptor, and

ii) inhibits IGF-1 receptor mediated signaling,

b) can combine mouse and rat IGF-1 and IGF-2,

c) does not bind human insulin;

wherein the antibody molecule is selected from the group comprising:

i) the heavy chain CDRs comprise SEQ ID NO: 1(CDR1), SEQ ID NO: 2(CDR2) and SEQ id no: 3(CDR3) and the light chain CDR comprises the amino acid sequence of SEQ ID NO: 4(CDR1), SEQ ID NO: 5(CDR2) and SEQ ID NO: 6(CDR 3);

ii) the heavy chain CDR comprises SEQ ID NO: 11(CDR1), SEQ ID NO: 12(CDR2) and seq id NO: 13(CDR3) and the light chain CDR comprises the amino acid sequence of SEQ id no: 14(CDR1), SEQ ID NO: 15(CDR2) and SEQ ID NO: 16(CDR 3);

iii) the heavy chain CDR comprises SEQ ID NO:21 (CDR1), SEQ ID NO:22 (CDR2) and SEQ ID NO:23 (CDR3) and the light chain CDR comprises the amino acid sequence of SEQ id no:24 (CDR1), SEQ ID NO:25 (CDR2) and SEQ ID NO:26 (CDR 3).

In another aspect, the invention relates to an anti-IGF antibody molecule, wherein the antibody molecule has a heavy chain variable region comprising SEQ ID NO: 1(CDR1), SEQ ID NO: 2(CDR2) and SEQ ID NO: 3(CDR3) and a light chain CDR comprising the amino acid sequence of SEQ ID NO: 4(CDR1), SEQ ID NO: 5(CDR2) and SEQ ID NO: 6(CDR 3).

In another aspect, the invention relates to an anti-IGF antibody molecule, wherein the antibody molecule has a heavy chain variable region comprising SEQ ID NO: 11(CDR1), SEQ ID NO: 12(CDR2) and SEQ ID NO: 13(CDR3) and a light chain CDR comprising the amino acid sequence of SEQ ID NO: 14(CDR1), SEQ ID NO: 15(CDR2) and SEQ ID NO: 16(CDR 3).

In another aspect, the invention relates to an anti-IGF antibody molecule, wherein the antibody molecule has a heavy chain variable region comprising SEQ ID NO:21 (CDR1), SEQ ID NO:22 (CDR2) and SEQ ID NO:23 (CDR3) and a light chain CDR having an amino acid sequence comprising SEQ ID NO:24 (CDR1), SEQ ID NO:25 (CDR2) and SEQ ID NO:26 (CDR3) in the amino acid sequence of seq id no.

In another aspect, the invention relates to an anti-IGF antibody molecule having a heavy chain and a light chain or CDR having amino acid sequences as depicted in FIGS. 12A-C.

In another aspect, the invention relates to an anti-IGF antibody molecule, wherein the antibody molecule binds a nonlinear epitope within IGF-1 comprising amino acid sequences LCGAELVDALQFVCGDR (SEQ ID NO: 41) and CCFRSCDLRRLEM (SEQ ID NO: 42) of human IGF-1(SEQ ID NO: 43). In a preferred embodiment, the antibody molecule contacts at least 8 amino acids within amino acid sequence LCGAELVDALQFVCGDR (SEQ ID NO: 41) and at least 10 amino acids within amino acid sequence CCFRSCDLRRLEM (SEQ ID NO: 42) of human IGF-1(SEQ ID NO: 43). In another preferred embodiment, such anti-IGF antibody molecules contact Leu (5), Cys (6), Glu (9), Leu (10), Asp (12), Ala (13), Phe (16), Val (17), Arg (21), Cys (47), Cys (48), Phe (49), Ser (51), Cys (52), Asp (53), Leu (54), Arg (55), Leu (57) and Glu (58) of human IGF-1(SEQ ID NO: 43), as determined by X-ray crystallography. A corresponding method is disclosed in example 9 herein. Preferably, the antibody molecule has an amino acid sequence comprising SEQ ID NO:21 (CDR1), SEQ ID NO:22 (CDR2) and SEQ ID NO:23 (CDR3) and a light chain CDR having an amino acid sequence comprising SEQ ID NO:24 (CDR1), SEQ ID NO:25 (CDR2) and SEQ id no:26 (CDR3) in the amino acid sequence of seq id no.

Antibody binding is defined as the interaction via non-covalent bonds that allow the antigen (or structurally similar protein or fragment thereof) to occupy the antibody binding site, i.e., the region of the immunoglobulin that binds to a determinant of the appropriate antigen (or structurally similar protein).

Affinity (i.e., the interaction between a single antigen binding site and a single epitope on an antibody) is determined by the binding constant K_A＝k_ass/k_dissOr dissociation constant K_D＝k_diss/k_assAnd (4) showing.

In one aspect, according to a), the antibody has a K in the range of 0.02nM to 20nM, e.g. 0.2nM to about 2nM_DThe affinity of values, for example, bind various IGF proteins with an affinity of 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0nM, as determined by surface plasmon resonance analysis. Based on this property, neutralization of IGF functional signaling is achieved.

In one aspect, according to c), the antibody does not bind human insulin at a concentration that is at least 100 times the minimum concentration required for binding to human IGF-1 or IGF-2.

On the other hand, the properties of the anti-IGF antibody molecule defined in c) can be characterized as follows: the affinity of the anti-IGF antibody molecule for IGF-1 and IGF-2 is at least 100 times and even more than 1000 times greater than its affinity for insulin, respectively. If weak binding cannot be completely ruled out, the anti-IGF antibody molecule does not bind insulin at therapeutic doses, even at very high doses (e.g., over 100 mg/kg).

In one embodiment, the antibody molecule of the invention does not affect the mitogenic properties of human insulin mediated by binding to the insulin receptor. (generally, mitogenic properties are defined as compounds that promote the initiation of cell division by a cell, thereby triggering mitosis, e.g., in the case of insulin, which promotes cell growth).

In another embodiment, the antibodies of the invention are capable of inhibiting IGF-2 signaling mediated viA the insulin receptor IR-A in addition to IGF signaling mediated viA the IGF-1 receptor.

The antibodies of the invention have surprisingly high neutralizing potency against IGF-1 and IGF-2. Furthermore, their potency and binding affinity for IGF-1 is unexpectedly higher than for IGF-2. They have high solubility and stability, they do not have unwanted glycosylation or hydrolysis motifs in the variable domain, and they have a long circulating half-life.

Detailed Description

Hereinafter, antibody molecules of the present invention that bind to human IGF-1 and IGF-2 are referred to as "anti-IGF antibody molecules".

The term "anti-IGF antibody molecule" encompasses human anti-IGF antibodies, anti-IGF antibody fragments, anti-IGF antibody-like molecules, and conjugates of any of the above antibody molecules. Antibodies within the meaning of the present invention include, but are not limited to, monoclonal antibodies, chimeric monoclonal antibodies and bispecific or multispecific antibodies. The term "antibody" encompasses intact immunoglobulins produced by lymphocytes and present, for example, in serum, monoclonal antibodies secreted by hybridoma cell lines, polypeptides produced by recombinant expression in host cells having the binding specificity of an immunoglobulin or monoclonal antibody, and molecules derived from such immunoglobulins, monoclonal antibodies or polypeptides that have been further processed while retaining their binding specificity.

In particular, the term "antibody molecule" preferably includes a fully human intact immunoglobulin comprising two heavy chains and two light chains.

In another aspect, the antibody molecule is an anti-IGF antibody fragment having an antigen binding region. To obtain antibody fragments (e.g., Fab fragments), digestion can be performed by conventional techniques (e.g., using papain or pepsin). Examples of papain digestion are described in WO 94/29348 and U.S. Pat. No. 4,342,566. Papain digestion of antibodies typically produces two identical antigen binding fragments (so-called Fab fragments), each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment produces F (ab') which has two antigen binding sites and still enables antigen cross-linking₂And (3) fragment. Antibody fragments may also be generated by molecular biological methods that produce corresponding encoding DNA fragments.

The Fab fragment also contains the constant domain of the light chain and the first constant domain of the heavy Chain (CH)₁). Fab' fragments differ from Fab fragments in that they are on the heavy chain CH₁The carboxy terminus of the domain contains other residues, including one or more cysteines from the antibody hinge region. Fab '-SH is referred to herein as Fab' in which the cysteine residues of the constant domains carry free thiol groups. F (ab')₂Antibody fragments are initially produced in the form of a Fab' fragment pair having hinge cysteines between them.

Antigen-binding antibody fragments or antibody-like molecules (including single chain antibodies and linear antibodies, as described in Zapata et al, 1995) may comprise variable regions on a single polypeptide alone or in combination with all or a portion of: the constant domains of the light chain, CH1, hinge region, CH2 and CH3 domains, for example the so-called "SMIP" ("Small Modular Immunopharmaceutical"), which is an antibody-like molecule that employs as its binding domain Fv a single polypeptide chain linked to a single-chain hinge and an effector domain lacking the constant domain CH1 (WO 02/056910). SMIP can be prepared in monomeric or dimeric form, but it does not exhibit the dimeric structure of a dimer of a conventional antibody. The invention also includes antigen binding fragments comprising any combination of constant domain regions of variable and light chains, VH1, CH1, hinge region, CH2, and CH3 domains.

Antibody fragments or antibody-like molecules may contain all or only a portion of the constant regions, as long as they exhibit specific binding to the relevant portion of the IGF-1/IGF-2 antigen. If effector functions such as complement fixation or antibody-dependent cytotoxicity are not required, the choice of constant region type and length will depend primarily on the desired pharmacological properties of the antibody protein. Antibody molecules are typically tetramers consisting of two light/heavy chain pairs, but may also be dimers, i.e. dimers consisting of one light/heavy chain pair, such as Fab or Fv fragments, or they may be monomeric single chain antibodies (scFv).

anti-IGF antibody-like molecules can also be single domain antibodies (e.g., so-called "nanobodies") that comprise an antigen binding site in a single Ig-like domain (described, for example, in WO03/050531 and Revets et al, 2005). Other examples of antibody-like molecules are immunoglobulin superfamily antibodies (IgSF; Srinivasan and Roeske, 2005) or molecules containing CDRs or CDR-grafts or "domain antibodies" (dAbs). dAB is a functional binding unit of an antibody, corresponding to the variable region of the human antibody heavy chain (VH) or light chain (VL). The domain antibody has a molecular weight of about 13kDa or less than one tenth of the size of the intact antibody. A series of large and fully functional fully human VH and VL dAb libraries have been developed. dABs can also be used for "dual targeting," i.e., a dAb can bind to another target in a molecule in addition to IGF-1/IGF-2. dAb libraries, selection and screening methods, dAb formats for dual targeting and extending serum half-life are described, for example, in U.S. Pat. No. 6,696,245, WO04/058821, WO 04/003019 and WO 03/002609.

In general, antibody fragments and antibody-like molecules are better expressed in bacterial, yeast and mammalian cell systems.

In a preferred embodiment, the antibody molecule of the invention as defined in i) above has a heavy chain variable region comprising SEQ ID NO: 8 and a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 10 (which sequence may contain additional gln at its C-terminus, this amino acid position may be considered to be the C-terminus of the variable region according to the Kabat numbering scheme, or, according to the sequence listing, it may represent the first amino acid of a constant light chain, see SEQ ID NO: 34).

The variable heavy chain comprises SEQ ID NO: 8 and the variable light chain comprises the amino acid sequence of SEQ id no: 10 preferably has an IgG1 constant heavy chain region. The antibody preferably has an Ig λ constant light chain region. The antibody is preferably the antibody designated 60814 having an amino acid sequence comprising SEQ ID NO:32 and a light chain constant region comprising the amino acid sequence of SEQ ID NO:34, or a light chain constant region of the amino acid sequence of seq id no. The complete amino acid sequence of the antibody designated 60814 is described in SEQ ID NO: 35 (heavy chain) and SEQ ID NO: 36 (light chain).

In another preferred embodiment, the antibody molecule of the invention as defined in ii) above has a heavy chain comprising SEQ ID NO: 18 and a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 20 (which sequence may contain additional gln at its C-terminus, this amino acid position may be considered to be the C-terminus of the variable region according to the Kabat numbering scheme, or, according to the sequence listing, it may represent the first amino acid of a constant light chain, see SEQ ID NO: 34).

The variable heavy chain comprises SEQ ID NO: 18 and the variable light chain comprises the amino acid sequence of SEQ id no: 20 preferably has an IgG1 constant heavy chain region. The antibody preferably has an Ig λ constant light chain region. The antibody is preferably the antibody designated 60819, which has an amino acid sequence comprising SEQ ID NO:32 and a light chain constant region comprising the amino acid sequence of SEQ ID NO:34, or a light chain constant region of the amino acid sequence of seq id no. The complete amino acid sequence of the antibody designated 60819 is depicted in SEQ ID NO: 37 (heavy chain) and seq id NO: 38 (light chain).

In another preferred embodiment, the antibody of the invention as defined in iii) above has a heavy chain comprising SEQ ID NO:28 and a variable heavy chain comprising the amino acid sequence of SEQ ID NO:30 (which sequence may contain additional gln at its C-terminus, this amino acid position may be considered to be the C-terminus of the variable region according to the Kabat numbering scheme, or, according to the sequence listing, it may represent the first amino acid of a constant light chain, see SEQ ID NO: 34).

The variable heavy chain comprises SEQ ID NO:28 and the variable light chain comprises the amino acid sequence of SEQ id no:30 preferably has an IgG1 constant heavy chain region. The antibody preferably has an Ig λ constant light chain region. The antibody is preferably the antibody designated 60833, which has an amino acid sequence comprising SEQ ID NO:32 and a light chain constant region comprising the amino acid sequence of SEQ ID NO:34, or a light chain constant region of the amino acid sequence of seq id no. The complete amino acid sequence of the antibody designated 60833 is depicted in SEQ ID NO:39 (heavy chain) and SEQ ID NO:40 (light chain).

Cross-reacting the antibodies of the invention with mouse and rat IGF-1 can allow for examination of their endocrine effects in these species, such as the effects on the growth hormone pathway. Cross-reacting with rat IGF is particularly advantageous since rat is an excellent animal model preferred for drug development to study toxicological effects.

The observed pharmacodynamic effects of antibodies on total IGF-1 levels are useful pharmacodynamic markers, probably due to removal of free IGF-1 leading to feedback regulation via the growth hormone pathway, leading to increased IGF-1 secretion by the liver. The use of this marker in animal species (allowing determination of dose/effect relationships early in drug development) helps prepare a phase I clinical study in which the pharmacodynamic response of patients to total IGF-1 levels is also monitored in addition to PK analysis.

The anti-IGF antibody molecule of the present invention may also be a variant of an antibody defined by the amino acid sequence shown in the sequence listing. Accordingly, the invention also comprises antibodies which are variants of such polypeptides having the features defined under a) to c) above. Functional variants of antibodies 60814, 60819, and 60833 will be prepared, tested, and used by those skilled in the art using conventional techniques. Variant antibodies with at least one position change in the CDR and/or framework are examples; variant antibodies in which single amino acid substitutions in the framework regions do not correspond to germline sequences are present; antibodies with conservative amino (acid) substitutions; antibodies encoded by DNA molecules which hybridize under stringent conditions to DNA molecules encoding the variable chains of the 60814, 60819 or 60833 antibodies described in the sequence listing; 60814. 60819 and 60833.

Variants may also be obtained by using the antibody of the invention as a starting point for optimization and diversifying one or more amino acid residues, preferably amino acid residues in one or more CDRs, and screening the resulting population of antibody variants for improved properties. Particularly preferred are one or more amino acid residues in the CDR3 of the diversified variable light chain, the CDR3 of the variable heavy chain, the CDR1 of the variable light chain and/or the CDR2 of the variable heavy chain. Diversification can be carried out by methods known in the art, such as the so-called TRIM technique mentioned in WO 2007/042309.

Given the nature of the individual amino acids, reasonable substitutions can be made to obtain antibody variants that retain the overall molecular structure of antibodies 60814, 60819, or 60833. Amino acid substitutions (i.e., "conservative substitutions") can be made, for example, based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the individual amino acids. Amino acid substitutions commonly performed (e.g., as described in WO 2007/042309) and methods of obtaining such modified antibodies are well known to those skilled in the art. Given the genetic code and recombinant and synthetic DNA techniques, DNA molecules encoding variant antibodies having one or more conservative amino acid exchanges can be routinely designed and readily obtained for each antibody.

Preferably, the antibody variant has at least 60%, more preferably at least 70% or 80%, still more preferably at least 90% and most preferably at least 95% sequence identity in the variable region. Preferably, the antibodies also have at least 80%, more preferably 90% and most preferably 95% sequence similarity in the variable region.

(the term "sequence identity" between two polypeptide sequences means the percentage of identical amino acids between the two sequences. "sequence similarity" means the percentage of identical amino acids or the percentage of amino acids that show conservative amino acid substitutions.)

In another embodiment, the anti-IGF antibody molecule of the invention is an "affinity matured" antibody.

An "affinity matured" anti-IGF antibody is an anti-IGF antibody derived from a parent anti-IGF antibody (e.g., 60814, 60819, or 60833) that has one or more mutations in one or more CDRs or in which one or more entire CDRs have been replaced, resulting in improved affinity for the antigen (as compared to the respective parent antibody). One procedure for generating these antibody mutants involves phage display (Hawkins et al, 1992 and Lowman et al, 1991). Briefly, multiple hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody mutants so produced are displayed from filamentous phage particles in a monovalent fashion as fusions to the gene III product of M13 encapsulated in each particle. The phage-displayed mutants are then screened for their biological activity (e.g., binding affinity), as disclosed herein.

Affinity matured antibodies can also be produced by methods described in the following references: for example Marks et al, 1992 (affinity maturation by shuffling of Variable Heavy (VH) and Variable Light (VL) domains); or Barbas et al, 1994; shier et al, 1995; yelton et al, 1995; jackson et al, 1995 and Hawkins et al, 1992 (random mutagenesis of CDR and/or framework residues). Preferably, the affinity matured antibody will have a very high affinity for the target antigen, e.g., a low picomolar concentration.

The invention also relates to DNA molecules encoding the anti-IGF antibody molecules of the invention. Such sequences include, but are not limited to, those DNA molecules encoding antibodies 60814, 60819 and 60833 as shown in the sequence listing: SEQ ID NOs: 7 and SEQ ID NO: 9; SEQ ID NOs: 17 and SEQ ID NO: 19; SEQ ID NOs: 27 and SEQ ID NO: 29.

SEQ ID NO: 9. SEQ ID NO: 19 and 29 may contain an additional Gln codon at its 3' end.

Thus, the present invention also relates to nucleic acid molecules that can hybridize to DNA molecules described in the sequence listing under high stringency binding and washing conditions (as defined in WO 2007/042309), wherein the nucleic acid molecules encode antibodies or functional fragments thereof having properties equivalent to or superior to those of antibodies 60814, 60819, or 60833. Preferably the molecules (from an mRNA perspective) are those having at least 75% or 80% (preferably at least 85%, more preferably at least 90% and most preferably at least 95%) homology or sequence identity to one of the DNA molecules described herein.

Another class of DNA variants falling within the scope of the present invention may be defined with reference to the polypeptide encoded thereby. These DNA molecules differ in their sequence from those described in the sequence listing (SEQ ID NO: 7, 17 and 27, or 9, 19, 29, respectively), but due to the degeneracy of the genetic code, these DNA molecules encode antibodies having the same amino acid sequence as antibodies 60814, 60819 or 60833, respectively. For example, as antibodies 60814, 60819, or 60833 are expressed in eukaryotic cells, the last nine nucleotides encoding the last three amino acids of the variable light chain can each be designed to match eukaryotic codon usage. If it is desired to express the antibody in E.coli, these sequences may be altered to match the E.coli codon usage.

Variants of the DNA molecules of the invention may be constructed in a number of different ways as described in WO 2007/042309.

In producing a recombinant anti-IGF antibody molecule of the invention, a DNA molecule (cDNA and/or genomic DNA) or fragment thereof encoding a full-length light chain (in the case of antibody 60814, the sequence comprises SEQ ID NO: 9 and SEQ ID NO: 33) and a heavy chain (in the case of antibody 60814, the sequence comprises SEQ ID NO: 7 and SEQ ID NO: 31) is inserted into an expression vector such that the sequences are operably linked to transcription and/or translation control sequences. In the case of antibody 60819, the sequences are SEQ ID NOs: 19 and SEQ ID NO: 33 and SEQ ID NO: 17 and SEQ ID NO: 31, their sequences; in the case of antibody 60833, the sequences are SEQ ID NOs: 29 and SEQ ID NO: 33 and SEQ ID NO:27 and SEQ ID NO: 31 to the sequence of the sequence.

In making the antibodies of the invention, one skilled in the art can select from a variety of expression systems known in the art (e.g., those reviewed by Kipriyanow and Le Gall, 2004).

In another aspect, the invention relates to an expression vector comprising a DNA molecule comprising a nucleotide sequence encoding a variable heavy chain and/or a variable light chain of an antibody molecule as described above. Preferably, such expression vectors contain a DNA molecule comprising the amino acid sequence of SEQ id no: 7 and/or SEQ ID NO: 9, or a nucleotide sequence comprising SEQ ID NO: 17 and/or SEQ id no: 19, or a sequence comprising SEQ ID NO:27 and/or SEQ ID NO:29, respectively, in sequence. Preferably, such expression vectors further comprise a DNA molecule encoding a constant heavy chain and/or a constant light chain, respectively, linked to a DNA molecule encoding a variable heavy chain and/or a variable light chain, respectively.

Expression vectors include plasmids, retroviruses, cosmids, EBV-derived episomes, and the like. The expression vector and expression control sequences are selected to be compatible with the host cell. The antibody light chain gene and the antibody heavy chain gene may be inserted into separate vectors. In certain embodiments, both DNA sequences are inserted into the same expression vector. Suitable vectors are those that encode functionally intact human CH (heavy chain constant region) or CL (light chain constant region) immunoglobulin sequences, wherein appropriate restriction sites are engineered so that any VH (heavy chain variable region) or VL (light chain variable region) sequence can be readily inserted and expressed as described above. In the case of antibodies having variable regions of 60814, 60819 and 60833, the constant chain is typically kappa or lambda for the antibody light chain and may be of (without limitation) any IgG isotype (IgG1, IgG2, IgG3, IgG4) or other immunoglobulin (including allelic variants) for the antibody heavy chain.

The recombinant expression vector may also encode a signal peptide that facilitates secretion of the antibody chain by the host cell. The DNA encoding the antibody chain may be cloned into a vector such that the signal peptide is linked in-frame to the amino terminus of the mature antibody chain DNA. The signal peptide may be an immunoglobulin signal peptide or a non-immunoglobulin heterologous peptide. Alternatively, the DNA sequence encoding the antibody chain may already contain a signal peptide sequence.

In addition to the antibody chain DNA sequences, the recombinant expression vectors contain regulatory sequences including promoters, enhancers, termination and polyadenylation signals, and other expression control elements that control the expression of the antibody chain in the host cell. Examples of promoter sequences, exemplified for expression in mammalian cells, are promoters and/or enhancers derived from CMV (such as the CMV simian virus 40(SV40) promoter/enhancer), adenoviruses (e.g. the adenovirus major late promoter (AdMLP)), polyoma (viruses) and strong mammalian promoters (such as the native immunoglobulin and actin promoters). Examples of polyadenylation signals are BGH polyA, SV40 late or early polyA; alternatively, 3' UTR such as immunoglobulin gene can be used.

Recombinant expression vectors may also contain sequences that regulate replication of the vector in a host cell (e.g., an origin of replication) and a selectable marker gene. Nucleic acid molecules encoding the anti-IGF antibody heavy chain or antigen-binding portion thereof and/or the anti-IGF antibody light chain or antigen-binding portion thereof, and vectors comprising such DNA molecules, can be introduced into host cells (e.g., bacterial cells or higher eukaryotic cells, such as mammalian cells) according to transfection methods well known in the art, including lipoplasmid-mediated transfection, polycation-mediated transfection, protoplast fusion, microinjection, calcium phosphate precipitation, electroporation, or transfer by viral vectors.

The DNA molecules encoding the heavy and light chains are preferably present in two vectors co-transfected into a host cell, preferably a mammalian cell.

In another aspect, the invention relates to a host cell, preferably a mammalian cell, carrying one or more expression vectors as described above.

Mammalian cell lines useful as expression hosts are well known in the art and include, inter alia, Chinese Hamster Ovary (CHO) cells, NSO, SP2/0 cells, HeLa cells, Baby Hamster Kidney (BHK) cells, monkey kidney Cells (COS), human cancer cells (e.g., Hep G2 and A-549 cells), 3T3 cells, or derivatives/progeny of any such cell line. Other mammalian cells can be used, including but not limited to human, mouse, rat, monkey, and rodent cell lines, or other eukaryotic cells, including but not limited to yeast, insect, and plant cells, or prokaryotic cells such as bacteria. The anti-IGF antibody molecules of the invention are produced by culturing the host cells for a time sufficient for the antibody molecule to be expressed in the host cells.

Thus, in another aspect, the invention relates to a method of producing an antibody molecule as described above, comprising transfecting a mammalian host cell with one or more vectors as described above, incubating the host cell, and recovering and purifying the antibody. In another embodiment, the invention relates to a method of producing an antibody as described above, comprising obtaining a mammalian host cell comprising one or more vectors as described above, and growing the host cell. In another embodiment, the method further comprises recovering and purifying the antibody.

The antibody molecule is preferably recovered from the culture medium as a secreted polypeptide or it may be recovered from the host cell lysate if, for example, there is no secretion signal upon expression. Antibody molecules must be purified in such a way that a substantially homogeneous antibody preparation is obtained using standard protein purification methods used for recombinant proteins and host cell proteins. For example, the most recent purification methods suitable for obtaining the anti-IGF antibody molecules of the invention comprise as a first step the removal of cells and/or particulate cell debris from the culture medium or lysate. The antibodies are then purified from contaminating soluble proteins, polypeptides and nucleic acids, for example, by fractionation on immunoaffinity or ion exchange columns, ethanol precipitation, reverse phase HPLC, Sephadex chromatography, chromatography on silica or cation exchange resins. In obtaining a preparation of anti-IGF antibody molecules, as a final step, the purified antibody molecules can be dried (e.g., lyophilized) as described below for therapeutic use.

In one embodiment, the anti-IGF antibody molecules of the invention can be purified by a series of prior art purification steps, including affinity chromatography (recombinant protein a), low pH viral inactivation, depth filtration (depthfiltration), cation exchange chromatography, anion exchange chromatography, nanofiltration, and 30kD ultrafiltration/diafiltration (Shukla et al, 2007).

In another aspect, the invention relates to an antibody molecule as described above for use in medicine.

In another aspect, the invention relates to a pharmaceutical composition comprising an anti-IGF antibody molecule, preferably a full antibody, according to the invention as active ingredient.

For use in therapy, anti-IGF antibody molecules are included in suitable pharmaceutical compositions for administration to animals or humans. Typical anti-IGF antibody molecule formulations can be prepared by mixing the anti-IGF antibody molecule with a physiologically acceptable carrier, excipient, or stabilizer in the form of a lyophilized or other dry formulation or an aqueous solution or an aqueous or non-aqueous suspension. The carrier, excipient, modulator or stabilizer is non-toxic at the dosages and concentrations employed. It includes buffer systems such as phosphates, citrates, acetates and other inorganic or organic acids and their salts; antioxidants, including ascorbic acid and methionine; preservatives, e.g. octadecyldimethylbenzylammonium chloride, chlorohexanediamine (hexamethonium chloride), benzalkonium chloride, benzethonium chloride, phenol, butanol or benzyl alcohol, alkyl parabens (such as methyl or propyl paraben), catechol, resorcinol, cyclohexanol, 3-pentanolAnd m-cresol; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone or polyethylene glycol (PEG); amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; mono-, di-, oligo-or polysaccharides and other carbohydrates including glucose, mannose, sucrose, trehalose, dextrins or dextrans; chelating agents, such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and/or ionic or nonionic surfactants, such as TWEEN^TM(Polysorbate), PLURONICS^TMOr fatty acid esters, fatty acid ethers or sugar esters. The antibody formulation may also contain an organic solvent such as ethanol or isopropanol. The excipient may also have a release-regulating or absorption-regulating function.

anti-IGF antibody molecules can also be dried (freeze-dried, spray-freeze-dried, dried by near-critical or supercritical gas, vacuum-dried, air-dried), precipitated or crystallized, or entrapped in microcapsules (prepared, for example, by coacervation techniques or interfacial polymerization using, for example, hydroxymethylcellulose or gelatin and poly (methylmethacrylate), respectively), in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), in macroemulsions, or precipitated or immobilized on carriers or surfaces, such as pcmc technology (protein coated microcrystals)). Such techniques are disclosed in Remington, 2005.

Formulations for in vivo administration must, of course, be sterile; sterilization may be accomplished by conventional techniques, such as filtration through sterile filtration membranes.

Increasing the concentration of anti-IGF antibodies can be used to achieve so-called High Concentration Liquid Formulations (HCLF); various methods of generating the HCLF have been described.

The anti-IGF antibody molecule may also be contained in a sustained release formulation. The formulations comprise a solid, semi-solid or liquid matrix of hydrophobic or hydrophilic polymers and may be shapedIn the form of an article (e.g., a film, a stick, or a microcapsule) and can be applied by an applicator device. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl methacrylate or sucrose acetate butyrate) or poly (vinyl alcohol)), polylactide (U.S. Pat. No.3,773,919), copolymers of L-glutamic acid and γ -ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic-glycolic acid copolymers (such as LUPRON DEPOT)^TM(injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate)) and poly-D- (-) -3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid are capable of releasing molecules for over 100 days, certain hydrogels release proteins in a shorter period of time. When the encapsulated antibody is left in the body for a long time, it may denature or aggregate due to exposure to moisture at 37 ℃, resulting in a loss of biological activity and possibly a change in immunogenicity. Depending on the mechanism involved, rational strategies can be devised to achieve stabilization. For example, if the aggregation mechanism is found to be intermolecular S-S bond formation via thio-disulfide interchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions (such as described in WO 89/011297), controlling moisture content, using appropriate additives, and forming specific polymer matrix compositions.

The formulations described in US 7,060,268 and US 6,991,790 may also be used in the anti-IGF antibody molecules of the present invention.

The IGF antibody molecule may also be incorporated into other forms of administration, such as dispersions, suspensions or liposomes, tablets, capsules, powders, sprays, transdermal or intradermal patches or creams with or without permeation enhancing devices, wafers, nasal, buccal or pulmonary formulations, or may be produced by implanted cells, or by the subject's own cells following gene therapy.

anti-IGF antibody molecules can also be derivatized with chemical groups such as polyethylene glycol (PEG), methyl or ethyl groups, or carbohydrate groups. Such groups may be useful for improving the biological characteristics of the antibody, such as increasing serum half-life or increasing tissue binding.

The preferred mode of administration is parenteral (intravenous, intramuscular, subcutaneous, intraperitoneal, intradermal) infusion or injection, but other modes of administration (such as inhalation, transdermal, intranasal, buccal, oral) may also be suitable.

In a preferred embodiment, the pharmaceutical composition of the invention contains an anti-IGF antibody (e.g., antibody 60814, 60819, or 60833) at a concentration of 10mg/ml and further comprises 25mM sodium citrate pH 6, 115mM NaCl, 0.02%(Polysorbitol 20).

In another embodiment, the pharmaceutical composition of the invention is an aqueous solution containing an anti-IGF antibody (e.g., antibody 60814, 60819, or 60833) at a concentration of 10mg/ml, and further comprising 25mM histidine HCl pH 6, 38.8g/L mannitol, 9.70g/L sucrose, and 0.02%(polysorbate 20).

For intravenous infusion, the pharmaceutical compositions of the present invention may be diluted with a physiological solution, for example, with 0.9% sodium chloride or G5 solution.

The pharmaceutical compositions may be freeze-dried and reconstituted with water for injection (WFI) prior to use.

For the prevention or treatment of disease, the appropriate dosage of antibody will depend on the following factors: the type of disease to be treated, the severity and course of the disease, the administration of the antibody for prophylactic or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody and the judgment of the attending physician. The antibody is preferably administered to the patient at once or over a series of treatments.

Depending on the type and severity of the disease, about 1 μ g/kg to 20mg/kg (e.g., 0.1mg/kg to 15mg/kg) of antibody is an initial candidate dose for administration to a patient, whether, for example, via one or more divided administrations or continuous infusion (1 hour infusion). Depending on the factors described above, a typical treatment schedule generally includes administration of the antibody at a dose in the range of about 0.1 μ g/kg to about 20mg/kg or more than 20mg/kg once a week to once every three weeks. For example, the weekly dose may be 5, 10, or 15 mg/kg. The course of the therapy is readily monitored by conventional techniques and assays.

A "therapeutically effective amount" of an antibody to be administered is the minimum amount required to prevent, ameliorate, or treat a disease or disorder.

The anti-IGF antibody molecules of the invention and pharmaceutical compositions containing them are useful for treating hyperproliferative disorders.

In certain embodiments, the hyperproliferative disorder is cancer.

Cancers are classified in two ways: the type of tissue from which the cancer originates (tissue type) and the primary site or location in the body where the cancer first occurs. The most common sites of cancer occurrence include skin, lung, breast/breast, prostate, colon and rectum, cervix and uterus.

The anti-IGF antibody molecules of the invention are useful for the treatment of various cancers, including (but not limited to) the following:

● AIDS-related cancers such as Kaposi's sarcoma;

● bone-related cancers, such as Ewing's family of tumors and osteosarcomas;

● brain-related cancers such as adult brain tumors, juvenile brain stem glioma, juvenile cerebellar astrocytoma, juvenile brain astrocytoma/malignant glioma, juvenile ependymoma, juvenile medulloblastoma, juvenile supratentorial primitive neuroectodermal tumors, juvenile optic and hypothalamic glioma, and other juvenile brain tumors;

● breast cancer;

● gastrointestinal/gastrointestinal related cancers such as anal cancer, extrahepatic bile duct cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), bile duct cancer, colon cancer, esophageal cancer, gallbladder cancer, adult primary liver cancer (hepatocellular carcinoma, hepatoblastoma), juvenile liver cancer, pancreatic cancer, rectal cancer, small intestine cancer, and gastric cancer;

● endocrine-related cancers such as adrenocortical carcinoma, gastrointestinal carcinoid tumor, islet cell carcinoma (endocrine pancreas), parathyroid carcinoma, pheochromocytoma, pituitary tumor, and thyroid cancer;

● eye-related cancers such as intraocular melanoma and retinoblastoma;

● genitourinary related cancers such as bladder cancer, renal (renal cell) cancer, penile cancer, prostate cancer, transitional cell renal pelvis and ureter cancer, testicular cancer, urethral cancer, Wilms' tumor and other juvenile stage renal tumors;

● germ cell related cancers such as juvenile extracranial germ cell tumor, extragonal germ cell tumor, ovarian germ cell tumor, and testicular cancer;

● gynecological cancers such as cervical cancer, endometrial cancer, gestational trophoblastic tumor (getstatinephospheric tumor), ovarian epithelial cancer, ovarian germ cell tumor, low malignant potential ovarian tumor (ovarial low malignant potential tumor), uterine sarcoma, vaginal cancer, and vulval cancer;

● head and neck related cancers such as hypopharynx cancer, larynx cancer, lip and oral cavity cancer, primary occult metastatic squamous neck cancer, nasopharyngeal carcinoma, oropharyngeal cancer, paranasal sinus and nasal cavity cancer, parathyroid cancer, and salivary gland cancer;

● hematologic/blood-related cancers such as leukemia, such as adult acute lymphoblastic leukemia, juvenile acute lymphoblastic leukemia, adult acute myeloid leukemia, juvenile acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, and hairy cell leukemia; and lymphomas such as AIDS-related lymphoma, cutaneous T-cell lymphoma, adult Hodgkin's lymphoma, juvenile Hodgkin's lymphoma, gestational Hodgkin's lymphoma, mycosis fungoides, adult non-Hodgkin's lymphomaLymphoma, juvenile non-Hodgkin's lymphoma, gestational non-Hodgkin's lymphoma, primary central nervous system lymphoma, Sezary's syndrome, cutaneous T-cell lymphoma and WaldenstromMacroglobulinemia and other hematologic/hematologic-related cancers, such as chronic myeloproliferative disorders, multiple myeloma/plasmacytoma, myelodysplastic syndrome, and myelodysplastic/myeloproliferative disorders;

● musculoskeletal-related cancers, such as Ewing's family of tumors, osteosarcomas, malignant fibrous histiocytomas of bone, juvenile rhabdomyosarcomas, adult soft tissue sarcomas, juvenile soft tissue sarcomas, and uterine sarcomas; angiosarcoma (hemangiosarcoma) and angiosarcoma (angiosarcoma);

● neural related cancers such as adult brain tumors, juvenile brain tumors, brain stem glioma, cerebellar astrocytoma, brain astrocytoma/malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, optic and hypothalamic glioma and other brain tumors such as neuroblastoma, pituitary tumors and primary central nervous system lymphoma;

● respiratory/thoracic related cancers such as non-small cell lung cancer, malignant mesothelioma, thymoma and thymus cancer;

● skin-related cancers such as cutaneous T-cell lymphoma, Kaposi's sarcoma, melanoma, Merkel cell carcinoma, and skin cancer;

● Small blue round cell tumor (small blue round cell tumor).

In particular, the anti-IGF antibody molecules of the invention and the pharmaceutical compositions containing them are useful for the treatment of the following cancers: cancers of the hematopoietic system including leukemia, lymphoma and myeloma; cancers of the gastrointestinal tract, including esophageal, gastric, colorectal, pancreatic, liver, and gallbladder and bile duct cancers; kidney, prostate and bladder cancer; gynecological cancers including breast, ovarian, cervical and endometrial cancers; skin and head and neck cancers, including malignant melanoma; pediatric cancers such as Wilms 'tumor, neuroblastoma, and Ewing's sarcoma; brain cancer such as glioblastoma; sarcomas, such as osteosarcoma, soft tissue sarcoma, rhabdomyosarcoma, angiosarcoma; lung cancer, mesothelioma, and thyroid cancer.

In a preferred aspect of the invention, the anti-IGF antibody molecules of the invention and the pharmaceutical compositions containing them are useful for the treatment of non-small cell lung cancer (NSCLC), in particular locally advanced or metastatic NSCLC (stage IIIB/IV). In this context, the anti-IGF antibody molecules of the invention may be combined with platinum-based chemotherapy, in particular platinum doublet therapy (platinum doublet therapy) of paclitaxel/carboplatin or gemcitabine/cisplatin.

In another preferred aspect of the invention, the anti-IGF antibody molecules of the invention and the pharmaceutical compositions containing them are useful for the treatment of hepatocellular carcinoma, particularly locally advanced or hepatocellular carcinoma (stage III/IV). In this context, the anti-IGF antibody molecules of the invention can be combined with Sorafenib (Sorafenib) (Strumberg d., 2005).

In another embodiment, the anti-IGF antibody molecules and pharmaceutical compositions containing them are useful for non-cancerous hyperproliferative disorders such as (but not limited to) psoriasis and post-angioplasty restenosis. In addition, according to recent observations (Reinberg, 2008): mutations in genes that reduce IGF-1 activity have a positive effect on longevity, and the antibodies of the invention have the potential to be useful for slowing aging and preventing age-related diseases when applied to adults in therapy.

Thus, in another aspect, the invention relates to the use of an antibody molecule as described above in the manufacture of a medicament for the treatment of a cancerous disease as described above.

In another aspect, the present invention relates to a pharmaceutical composition as described above for use in the treatment of a cancerous disease as described above.

In another aspect, the present invention relates to a method for treating a patient suffering from a cancerous disease as described above, comprising administering to the patient an effective amount of a pharmaceutical composition as described herein.

Depending on the condition to be treated, the anti-IGF antibody molecules of the invention can be used alone or in combination with one or more other therapeutic agents, in particular therapeutically active compounds selected from DNA damaging agents or compounds that inhibit angiogenesis, signal transduction pathways or mitotic checkpoints in cancer cells.

The other therapeutic agent may be administered simultaneously with (optionally as a component of the same pharmaceutical formulation) or before or after administration of the anti-IGF antibody molecule.

In certain embodiments, the additional therapeutic agent may be, but is not limited to, one or more inhibitors selected from the group consisting of: inhibitors of EGFR, VEGFR, HER2-neu, aurora A, aurora B, PLK, and PI3 kinase, FGFR, PDGFR, Raf, KSP, or PDK 1.

Other examples of other therapeutic agents are inhibitors of CDK, Akt, src/bcr-abl, cKit, cMet/HGF, c-Myc, Flt3, HSP90, hedgehog antagonists, JAK/STAT inhibitors, Mek, mTor, NF κ B, proteasome, Rho, wnt signaling inhibitors, or ubiquitination pathway inhibitors.

Examples of Aurora inhibitors are, but are not limited to, PHA-739358, AZD-1152, AT-9283, CYC-116, R-763, VX-667, MLN-8045, PF-3814735, SNS-314, VX-689, GSK-1070916, TTP-607, PHA-680626, MLN-8237 and ENMD-2076.

An example of a PLK inhibitor is GSK-461364.

Examples of RAF inhibitors are BAY-73-4506 (also a VEGFR inhibitor), PLX-4032, RAF-265 (also a VEGFR inhibitor), sorafenib (also a VEGFR inhibitor), XL-281, and Nevavar (also a VEGFR inhibitor).

Examples of KSP inhibitors are isbanus (ispinesib), ARRY-520, AZD-4877, CK-1122697, GSK-246053A, GSK-923295, MK-0731, SB-743921, LY-2523355 and EMD-534085.

Examples of src and/or bcr-abl inhibitors are dasatinib (dasatinib), AZD-0530, bosutinib (bosutinib), XL-228 (also IGF-1R inhibitors), nilotinib (nilotinib) (also PDGFR and cKit inhibitors), imatinib (imatinib) (also cKit inhibitors), NS-187, KX2-391, AP-2457 (also inhibitors of EGFR, FGFR, Tie2, Flt 3), KM-80 and LS-104 (also inhibitors of Flt3, Jak 2).

An example of a PDK1 inhibitor is AR-12.

An example of a Rho inhibitor is BA-210.

Examples of PI3 kinase inhibitors are PX-866, PX-867, BEZ-235 (also an mTor inhibitor), XL-147, XL-765 (also an mTor inhibitor), BGT-226, CDC-0941, GSK-1059615.

Examples of cMet or HGF inhibitors are XL-184 (also an inhibitor of VEGFR, cKit, Flt 3), PF-2341066, MK-2461, XL-880 (also an inhibitor of VEGFR), MGCD-265 (also an inhibitor of VEGFR, Ron, Tie 2), SU-11274, PHA-665752, AMG-102, AV-299, ARQ-197, MetMAb, CGEN-241, BMS-777607, JNJ-38877605, PF-4217903, SGX-126, CEP-17940, AMG-458, INCB-028060 and E-7050.

An example of a c-Myc inhibitor is CX-3543.

Examples of Flt3 inhibitors are AC-220 (also an inhibitor of cKit and PDGFR), KW-2449, LS-104 (also an inhibitor of bcr-abl and Jak 2), MC-2002, SB-1317, lestaurtinib (also an inhibitor of VEGFR, PDGFR, PKC), TG-101348 (also an inhibitor of JAK 2), XL-999 (also an inhibitor of cKit, FGFR, PDGFR and VEGFR), sunitinib (also an inhibitor of PDGFR, VEGFR and cKit), and tandutinib (also an inhibitor of PDGFR and cKit).

Examples of HSP90 inhibitors are tanespimycin (tanespimamycin), adriamycin (alvespimycin), IPI-504, STA-9090, MEDI-561, AUY-922, CNF-2024 and SNX-5422.

Examples of JAK/STAT inhibitors are CYT-997 (also interacting with tubulin), TG-101348 (also Flt3 inhibitors) and XL-019.

Examples of Mek inhibitors are ARRY-142886, AS-703026, PD-325901, AZD-8330, ARRY-704, RDEA-119, and XL-518.

Examples of mTor inhibitors are rapamycin (rapamycin), sirolimus (temsirolimus), de-sirolimus (also acting as a VEGF inhibitor), everolimus (also a VEGF inhibitor), XL-765 (also a PI3 kinase inhibitor) and BEZ-235 (also a PI3 kinase inhibitor).

Examples of Akt inhibitors are perifosine (perifosine), GSK-690693, RX-0201 and triciribine (triciribine).

Examples of cKit inhibitors are masitinib, OSI-930 (also acting as VEGFR inhibitor), AC-220 (also being an inhibitor of Flt3 and PDGFR), tandutinib (also being an inhibitor of Flt3 and PDGFR), axitinib (also being an inhibitor of VEGFR and PDGFR), sunitinib (also being an inhibitor of Flt3, PDGFR, VEGFR) and XL-820 (also acting as an inhibitor of VEGFR and PDGFR), imatinib (also being a bcr-abl inhibitor), nilotinib (nilotinib) (also being an inhibitor of bcr-abl and PDGFR).

Examples of hedgehog antagonists are IPI-609, CUR-61414, GDC-0449, IPI-926 and XL-139.

Examples of CDK inhibitors are Sericib (seliciclib), AT-7519, P-276, ZK-CDK (which also inhibits VEGFR2 and PDGFR), PD-332991, R-547, SNS-032, PHA-690509, PHA-848125, and SCH-727965.

Examples of proteasome inhibitors/inhibitors of the NF-. kappa.B pathway are bortezomib (bortezomib), carfilzomib (carfilzomib), NPI-0052, CEP-18770, MLN-2238, PR-047, PR-957, AVE-8680 and SPC-839.

An example of an inhibitor of the ubiquitination pathway is HBX-41108.

Examples of anti-angiogenic agents are inhibitors of FGFR, PDGFR and VEGF (R) and thalidomide, selected from (without limitation) BIBF 1120Bevacizumab (bevacizumab), motesanib (motesanib), CDP-791, SU-14813, tiratinib (telatinib), KRN-951, ZKCDK (also a CDK inhibitor), ABT-869, BMS-690514, RAF-265, IMC-KDR, IMC-18F1, IMiDs, thalidomide, CC-4047, lenalidomide, ENMD-0995, IMC-D11, Ki-23057, Borrenib (brivanib), Cediranib (cediranib), 1B3, CP-868596, IMC-3G3, R-1530 (also an Flt3 inhibitor), sunitinib (also an inhibitor of cKit and 3), Acitinib (also an inhibitor of cKittt), Lentatinib (also a 4) and a inhibitors of Kitavatib), Flt-24, Flt inhibitor of Abiranib (also a), Abiranib (also a-3), Ab-3, Abiranib (also a) and Flt inhibitors of Abiranib (also a) of Ab-3, and Ab-3, E-7080, CHIR-258, sorafenib tosylate (also a Raf inhibitor), vandetanib (vandetanib), CP-547632, OSI-930, AEE-788 (also inhibitors of EGFR and Her 2), BAY-57-9352 (also a Raf inhibitor), BAY-73-4506 (also a Raf inhibitor), XL-880 (also a cMet inhibitor), XL-647 (also inhibitors of EGFR and EphB 4), XL-820 (also a cKit inhibitor), nilotinib (also inhibitors of cKit and brc-abl), CYT-116, PTC-299, 119BMS-584622, CEP-81, dovitinib (dovitinib), CY-240BMS 1401, and ENMD-2976.

The other therapeutic agent may also be selected from EGFR inhibitors, which may be small molecule EGFR inhibitors or anti-EGFR antibodies. Examples of anti-EGFR antibodies are, without limitation, cetuximab (cetuximab), panitumumab (panitumumab), nimotuzumab (nimotuzumab), zalutumumab (zalutumumab); examples of small molecule EGFR inhibitors are gefitinib (gefitinib), erlotinib (erlotinib) and vandetanib (also VEGFR inhibitors). Another example of an EGFR modulator is EGF fusion toxin.

anti-IGF antibody molecules suitable for use with the present inventionThe other EGFR and/or Her2 inhibitor used in combination is BIBW 2992Lapatinib (lapatinib), trastuzumab (trastuzumab), pertuzumab (pertuzumab), XL-647, neratinib (neratinib), BMS-599626, ARRY-334543, AV-412, mAB-806, BMS-690514, JNJ-26483327, AEE-788 (also a VEGFR inhibitor), AZD-8931, ARRY-380, ARRY-333786, IMC-11F8, Zemab, TAK-285, AZD-4769.

Other agents that may be suitably treated in combination with the anti-IGF antibody molecules of the invention are tositumomab (tositumumab) and temetamicab (ibritumomab) (two radiolabeled anti-CD 20 antibodies), ovovatuzumab (ofatumumab), rituximab (rituximab), LY-2469298, orelizumab (ocrelizumab), TRU-015, PRO-131921, FBTA05, Vertuzumab (veltuzumab), R-7159(CD20 inhibitor), alemtuzumab (anti-CD 52 antibody), deoximab (deosomatomemab) (osteoclast differentiation factor ligand inhibitor), galiximab (galiximab) (CD 362 antagonist), zanolimumab (zanolimumab) (CD4 antagonist), SGN 4(CD 40 receptor modulator 829), Xlox-80135, 54 7/4 (Chikumab), ibritumomab (CD 085-49393), CD 085-EP-875 (CD 0893), rituximab (CD-9-4935), gamma-8723 (CD-3, gamma-3 (gamma-, Gemtuzumab ozolomide (exotuzumab) (a CD22 inhibitor), lumiximab (a CD23 inhibitor), TRU-016 (a CD37 inhibitor), MDX-1342, SAR-3419, MT-103 (a CD19 inhibitor) or mepiquat mab (mapatumumab), tegafuzumab (tigatuzumab), lexalimumab (lexatuzumab), Apomab, AMG-951, and AMG-655 (a TRAIL receptor modulator).

Other chemotherapeutic drugs that may be used in combination with the anti-IGF antibody molecules of the invention are selected from, but are not limited to, hormones, hormone analogs and anti-hormones (e.g., tamoxifen (tamoxifen), toremifene (toremifene), raloxifene (raloxifene), fulvestrant (fulvestrant), megestrol acetate (megestrol acetate), flutamide (flutamide), nilutamide (nilutamide), bicalutamide (bicalutamide), cyproterone acetate (cyproterone acetate), flupiride (astride), buterelin acetate (buserelin acetate), fludrocortisone (fludrocortisone), fluorometholone (fluoroxymesterone), medroxyprogesterone (medroxyprogesterone), octreotide (octreotide), zopentene (palentine), amethostatin (almetasone), amethostatin (e.g., omeprazole (omeprazole), amethone (e.g., omeprazole), amethone (e), amethostatin (e), ametholide (e.g., omeprazole (omeprazole), doxestride (e), amelione (e), ametholide (e (e.g., omeprazole (e), ametholide (e) inhibitors), ametholide (e), ametholide (e) or (e, e) may, e.g., a) may be, LHRH agonists and antagonists (e.g. goserelin acetate, leuprolide, abarelix, cetrorelix, deslorelin, histrelin, triptorelin), antimetabolites (e.g. antifolates such as methotrexate, pemetrexed), pyrimidine analogues such as 5-fluorouracil, capecitabine, decitabine, nerabine and gemcitabine), pyrimidine analogues such as mercaptopurine, thioguanine, trocaridine and anthramycin (e.g. epirubicin), and antibiotic analogues such as epirubicin, and epirubicin, mitomycin-C (mitomycin-C), bleomycin (bleomycin), dactinomycin (dactinomycin), plicamycin (plicamycin), mitoxantrone (mitoxantrone), pixantrone (pixantrone), streptozocin (streptozocin)), platinum derivatives (e.g., cisplatin (cissplatin), oxaliplatin (oxaliplatin), carboplatin (carboplatin), lobaplatin (lobaplatin), satraplatin (saraplatin)), alkylating agents (e.g., estramustine (estramustine), methyldichloroethylamine (mechlorethamine), melphalan (melphalan), chlorambucil (chlomethione), busulfan (busulosin), dacarbazine (badibazine), cyclophosphamide (cyclophosphatide), ifosfamide (ifosfamide), hydatidine (hydatidine), hydroxyl vincamine (vincamine), such as vincamine (vincamine), vincamine (such as alkaloids), vincamine (vincamine), vincamine (vincamine), such as), vincamine (vincamine), vincamine (vincamine), vinc, Vinorelbine (vinorelbine), vinflunine (vinflunine) and vincristine (vincristine); and taxanes such as paclitaxel (paclitaxel), docetaxel (docetaxel) and its formulations, larotaxel (larotaxel); semaxatriol (simotaxel) and epothilones (epothilones), such as ixabepilone (ixabepilone), patupilone (patupilone), ZK-EPO), topoisomerase inhibitors (e.g., epipodophyllotoxin (epidophyllotoxin), such as etoposide (etoposide) and fanpids (etophos), teniposide (teiposide), amsacrine (amsacrine), topotecan (topotecan), irinotecan (irinotecan)) and various chemotherapeutic agents (e.g., amifostine (amifostine), anagrelide (anagrelide), interferon alpha, procarbazine (procarbazine), mitotane (mitotane) and propamol (porrmer), bexarotene (bexarotene), and celecoxib (epothilones)).

In one aspect, the anti-IGF antibody molecules of the invention are used in combination with a platinum-based chemotherapy, e.g., in combination with a platinum doublet therapy of paclitaxel/carboplatin or gemcitabine/cisplatin. In one embodiment, such combination therapy may be repeated multiple times, e.g., 6 cycles (q3 weeks). This treatment may be followed by additional repeat treatments (e.g., 6 cycles of q3 weeks) with the anti-IGF antibody molecule alone. This regimen can be used, for example, to treat NSCLC. In another aspect, the anti-IGF antibody molecules of the invention are used in combination with sorafenib. In one embodiment, the anti-IGF antibody molecule can be administered repeatedly at 1-3 week intervals, e.g., 12 cycles, in combination with continuous administration of sorafenib. This protocol can be used, for example, to treat hepatocellular carcinoma.

The anti-IGF antibody molecules of the invention may also be combined with agents that target IGF-1R, e.g., when used at lower concentrations. Such agents include antibodies that bind IGF-1R (e.g., CP-751871, AMG-479, IMC-A12, MK-0646, AVE-1642, R-1507, BIIB-022, SCH-717454, rhuMab IGFR) and novel chemical entities that target the IGF1-R kinase domain (e.g., OSI-906 or BMS-554417, XL-228, BMS-754807).

The anti-IGF antibody molecules of the invention can also be used in combination with other therapies, including surgery, radiation therapy, endocrine therapy, biological response modifiers, hyperthermia and cryotherapy, and agents that attenuate any side effects, such as antiemetics, and in a preferred embodiment, diabetic agents, such as metformin (metformin).

The anti-IGF antibody molecules of the invention are also useful in the diagnosis of cancer, wherein elevated serum levels of IGF-1 and/or IGF-2 are correlated with the development or progression of the disease, e.g., by measuring elevated levels of IGF-2 due to loss of imprinting (LOI), a epigenetic variation affecting the insulin-like growth factor II gene (IGF 2). In certain embodiments, the antibody used for diagnostic applications (e.g., detection of IGF-1 in human tissue sections by immunohistological staining) is a chimeric antibody derived from a human antibody. In such antibodies, the constant region or a portion thereof has been replaced by the respective sequence of an antibody of another species (e.g., mouse). Using the chimeric antibody as a primary antibody, a secondary antibody (e.g., a goat antibody specifically reactive with the murine Fc portion) will specifically recognize the murine sequences of the chimeric antibody and will not bind to the Fc portion of other human immunoglobulin molecules present in human tissue samples. Thereby avoiding undesirable background staining.

The antibodies of the invention are also useful for controlling body weight and adipose tissue formation by blocking IGF-1 and IGF-2 mediated signal transduction. For this purpose, the antibodies of the invention may be administered alone or in combination with other anti-obesity drugs.

Materials and methods

Selection of high affinity fully human antibodies that bind IGF-1

Three rounds of panning were used to select specific Fab fragment clones from a human combinatorial antibody library (HuCAL Gold) (Knappik et al, 2000) that bind human IGF-1 with low nanomolar affinity, essentially as described by Rauchenberger et al, 2003. To identify Fab fragments with improved affinity for human IGF-1, a number of these "parent" Fab clones were subjected toExo-affinity maturation ", substantially as described by Nagy et al, 2002. The L-CDR3 (light chain CDR3) and H-CDR2 (heavy chain CDR2) sequences of each clone were expressed at about 10⁸The L-CDR3 and H-CDR2 boxes selected from HuCAL (Knappik et al, 2000) were diversified in place of the parental sequences. Phage were prepared from the resulting "mature libraries" and each library was solution panned against human IGF-1. To select the most avidity human IGF-1 binding agent, solution panning with reduced antigen and with and without human insulin blocking was performed under washing conditions of normal and elevated stringency according to methods known in the art. The panning products after three rounds of phage panning were subcloned into Fab expression vectors and the affinity of each Fab for human IGF-1 was determined by electrochemiluminescence-based equilibrium titration techniques developed by Bio Veris (Witney, Oxfordshire, UK), essentially as described by Haenel et al, 2005. Fab clones with optimal IGF-1 affinity were sequenced and then transformed into human IgG1 antibody as described by Krebs et al, 2001, which has sub-nanomolar affinity for human IGF-1 without any change in specificity compared to the parent antibody.

Cloning and recombinant expression of IgG1 antibodies

The variable heavy chain region (VH) and variable light chain region (VL) were excised from the Fab expression vector by restriction enzyme digestion and ligated into compatible restriction enzyme sites of pcdna3.1-based plasmids containing the human IgG1 heavy chain and human Ig λ light chain constant regions, respectively. EndoFree plasmid preparations (Qiagen) were prepared and the heavy and light chain plasmids were co-transfected into HEK293Freestyle cells (Invitrogen) at a concentration of 1mg/L of each plasmid according to the supplier's protocol. After 72 hours, supernatants were collected and IgG concentrations were determined by ELISA. The antibody was purified on a modified protein a column (GE Healthcare), eluted into citrate buffer and subsequently dialyzed in PBS to a concentration of 2.5 mg/ml. Alternatively, a CHO cell line stably integrated with an antibody expression plasmid is generated and used to produce antibodies.

Surface plasmon resonance analysis to determine affinity constants

a) Antibody capture method

The sensor chip was coated with about 1000RU of reference antibody in flow chamber 1 and about 1000RU of rabbit anti-human Fc- γ specific antibody in flow chamber 2 using the coupling reagents in the amine coupling kit. Using the surface preparation guide of Biacore 3000 software, 1000RU of target was set up at a flow rate of 5 μ l/min. The running buffer used was HBS-EP. Affinity measurements were performed using the following parameters: 20 μ l/min flow (HCB running buffer:); detecting the temperature at 25 ℃; fc1, Fc2 flow path; fc1 and Fc 2; anti-IGF-huMAb capture: 1. mu.g/ml solution for 3 minutes; IGF-Ag binding for 5 minutes; dissociation of IGF-Ag for 5 min; regeneration: the pulse was 30 seconds with 50mM HCl. IGF antigen was diluted to 500nM, 250nM, 125nM, 62.5nM and 31.3nM in running buffer (HCB) and different antigen dilutions were run separately in random order on Fc1 and Fc 2. During which a blank run using only running buffer was run. Prior to affinity analysis, blank run curves were subtracted from each binding curve. Data evaluation was performed using BIAevaluation software (version 4.1, Biacore, Freiburg, Germany). Kinetics of the dissociation and association phases were fitted separately. Fitting k separately_dissThe time range for the initial 200-300 seconds of the dissociation phase (the range in which the signal steadily decreases) is used. Fitting k separately_assIn value, an initial time frame of about 100 seconds (frame of signal stability enhancement) was used and the individual k's calculated using 1: 1 Langmuir (Langmuir) in combination with the model_dissThe value is obtained. Mean and standard deviation of the calculated kinetic data and corresponding dissociation constant (K)_D) And binding constant (K)_A)。

b) IGF coating method

When the sensor chip is coated with IGF ligand, the binding constants of IGF antibodies to IGF ligand are determined essentially as described above, except at 35.1pg/mm, respectively²And 38.5pg/mm²IGF-1 and IGF-2 coat the sensor chip. The antibody was then flowed through the chip at the following concentrations: 50nM, 25nM, 12.5nM, 6.25nM, 3.12 nM.

Measuring binding to human IGF, murine IGF, and rat IGF, and to human insulin in an immunoadsorption assay

The binding of fully human IgG1 antibody that binds IGF-1 with high affinity to human IGF-1 was also tested in a direct immunoadsorption assay (ELISA). The assay was performed by coating 96-well Maxisorb plates (100. mu.l/well) with human IGF-1(R & D Systems, No. 291-G1) at a concentration of 0.5. mu.g/mL overnight at 4 ℃. Coating buffer alone was used as a control for nonspecific binding. The wells were then washed once with wash buffer (1xTBS-T) and the residual binding sites were blocked with 200. mu.l blocking buffer on an orbital shaker at room temperature for 1 hour, followed by another round of washing. A series of three-fold dilutions of each test antibody in blocking buffer was prepared directly on the coated plate. Typical concentrations used were 50ng/mL, 16.6ng/mL, 5.6ng/mL, 1.8ng/mL, 0.6ng/mL, 0.2ng/mL and 0.07 ng/mL. Blocking buffer alone was used as a positive control. Plates were then incubated at room temperature for 2 hours with shaking. After three rounds of washing, 100 μ l of HRPO-conjugated anti-human IgG secondary reagent (Jackson ImmunoResearch Inc.) diluted in blocking buffer was added to each well. After incubation for 2 hours at room temperature with shaking, the plate was washed three times and the TMB substrate solution (equal amounts of solution a and B) was pipetted into all wells at 100 μ l/well. The plate was incubated at room temperature for 10-20 minutes with shaking, and then stopped by adding 100. mu.l of 1M phosphoric acid per well. The absorbance was measured at a wavelength of 450nm (reference wavelength 650 nm).

Fully human IGF-1 binding IgG1 antibodies were also tested for binding to mouse IGF-1(R & D Systems, number 791-MG), rat IGF-1(IBT, number RU100), human IGF-2(GroPep, number FM001), mouse IGF-2(R & D Systems, number 792-MG), rat IGF-2(IBT, number AAU100), and human insulin (Roche) as described above for human IGF-1, except that the human insulin concentration used for coating was 3 μ g/mL.

In vitro cell proliferation assay for determining neutralization potency

Cell lines derived from MCF-7 breast cancer (ATCC, HTB-22) and COLO205 colon cancer (ATCC # CCL-222) were seeded in 96-well plates at a cell density of 1000 cells per well in serum-free RPMI medium. 10ng/mL IGF-1 or IGF-2 was added in the presence or absence of humanized isotype control antibodies or antibodies 60814, 60819 and 60833 that do not bind IGF-1 or IGF-2 at concentrations of 12ng/mL, 37ng/mL, 111ng/mL, 333ng/mL, 1000ng/mL and 3000 ng/mL. Cells were cultured for 5 days, followed by relative cell numbers in each well using the CellTiter-Glo luminescent cell viability assay (Promega). The luminosity (LU ═ light units) was recorded using XFluor GENios Pro 4.

Growth assays for cell lines derived from Ewing's sarcoma

The Ewing's sarcoma derived cell lines TC-71(ATCC # ACC516) and SK-ES-1(ATCC # HTB86) were seeded at a density of 1000 cells per well in 96-well plates in DMEM medium containing 1 XNEAA, 1 Xsodium pyruvate, 1 Xglutamax and 10% Fetal Calf Serum (FCS) and incubated overnight at 37 ℃ and in a humidified atmosphere of 5% CO 2. The next day test antibody, humanized isotype control antibody that does not bind to IGF-1 or IGF-2 (humanized IgG1 antibody targeting CD44-v 6), rapamycin, or serial dilutions of rapamycin in combination with test antibody were added to the cells. Typical concentrations used were 30. mu.g/ml, 10. mu.g/ml, 3.3. mu.g/ml, 1.1. mu.g/ml, 0.37. mu.g/ml and 0.12. mu.g/ml (or 100nM, 10nM, 1nM, 0.1nM, 0.01nM, 0.001nM rapamycin and test antibody for the combined study) and each dilution was performed in triplicate wells. The cells were then incubated with the antibody for 120 hours, after which the relative cell number in each well was determined using the CellTiter-Glo luminescent cell viability assay (Promega). The Luminosity (LU) was recorded using XFluor GENios Pro 4 and the mean of triplicate wells was taken for data analysis and fitted by iterative calculations using a sigmoidal curve analysis program (GraphPad Prism) with variable Hill slope (Hill slope).

Western blot analysis of phosphorylated AKT and PTEN levels

SK-ES-1 cells were seeded in 6-well plates in medium containing 10% fetal bovine serum and, after overnight incubation, treated with 100nM of an isotype control antibody that does not bind IGF-1 or IGF-2 (humanized IgG1 antibody targeting CD44-v 6), 100nM 60819, 100nM rapamycin or 100nM 60819 in combination with 100nM rapamycin. After 24 hours, the cells were lysed and the cell lysate was frozen after determination of the protein concentration by the Bradford assay. Western blot analysis was performed by applying 30 μ g of protein lysate to SDS PAGE gels (BioRad) and blotting the gels on a Citerian gel blot sandwich. Western blots were incubated overnight with rabbit anti-beta actin (control) antibody, rabbit anti-PTEN antibody (CellSignaling #9559), or rabbit anti-phosphorylated pAKT antibody (Cell Signaling #4060) (diluted 1: 5000 (anti-beta actin), 1: 1000 (anti-PTEN), or 1: 2000 (anti-phosphorylated AKT) in 1% milk powder). After washing in TBS, anti-rabbit IgG HRPO conjugated secondary antibody (Amersham) was applied for 1 hour and after re-washing in TBS, antibody reactivity was detected by ECL and captured on hyperfilm (Amersham).

In vitro combination of an anti-IGF antibody and an EGFR inhibitor in a cell line derived from NSCLC

A cell line A-549 derived from NSCLC (ATCC # CCL-185) was seeded at a density of 1000 cells per well in RPMI 1640 medium containing 2mM L-glutamine and 10% fetal bovine serum in 96-well plates at 37 ℃ and 5% CO₂Incubated overnight in a humidified atmosphere. The next day serial dilutions of test IGF antibody, erlotinib/delustering, or a combination of test IGF antibody and erlotinib were added to the cells. Typical concentrations of test IGF antibodies used were 30000ng/mL, 10000ng/mL, 3333ng/mL, 1111ng/mL, 370ng/mL, 123ng/mL, 41ng/mL, 14ng/mL, and typical concentrations of erlotinib used were 20000nM, 6667nM, 2222nM, 741nM, 247nM, 82nM, 27nM, 9nM, and each dilution was performed in triplicate wells. The cells were then incubated for 120 hours, after which the relative cell number in each well was determined using the CellTiter-Glo luminescent cell viability assay (Promega). The luminosity (LU ═ luminescence units) was recorded using XFluor GENios Pro 4 and the mean of triplicate wells was taken for data analysis and fitted by iterative calculations using a sigmoidal curve analysis program (Graph PadPrism) with variable hill slope.

Determining the Effect on Total IGF-1 levels in murine serum and Total IGF-1 levels in rat serum

Test IGF antibodies were administered intravenously in single doses (bolus injections) of 25mg/kg, 12.5mg/kg, 6.25mg/kg, and 3.13mg/kg in 6-8 week old female athymic NMRI nude mice (n ═ 5). 30mg/kg, 100mg/kg, 200mg/kg of antibody 60819 was administered intravenously in a single 10 minute dose to 6-8 week old male and female Wistar Han rats (n ═ 4 males, 4 females). Blood samples were taken before antibody treatment and 24 hours after administration, sera collected and murine or rat total IGF-1 levels determined using an octenia rat/mouse total IGF-1 immunocytoassay. Assays were performed according to manufacturer's instructions, absorbance was measured at 450nm and evaluated using SoftMax Pro software. Serum total IGF-1 concentration (ng/mL) was determined using a standard curve. Statistical analysis was performed using GraphPad Prism software.

Cell-based IGF-1R phosphorylation ELISA

Mouse fibroblast cell lines recombinantly expressing human IGF-1R or human IR-A were maintained in DMEM supplemented with 10% heat-inactivated FCS, 1mM sodium pyruvate (sodium pyruvate), 0.075% sodium bicarbonate, MEM NEAA, and 0.3. mu.g/ml puromycin at 37 ℃ and 5% CO₂In a humidified incubator. Cells were detached with trypsin/EDTA, resuspended in growth medium, and diluted to 100,000 cells/mL. mu.L (10,000 cells) were seeded into wells of sterile 96-well plates and incubated at 37 ℃ and 5% CO₂Incubated overnight in a humidified incubator. Cells were then starved with 100 μ L/well assay medium (DMEM supplemented with 0.5% heat-inactivated FCS, 1mM sodium pyruvate, 0.075% sodium bicarbonate, and MEM NEAA) and incubated overnight as above. A series of test antibody concentrations prepared in assay medium were added to the cells and all samples were prepared in triplicate to determine the standard deviation for each assay condition. An IGF-1R antibody, α IR-3(Calbiochem, No. GR11L), was also tested in these experiments. IGF-1(20ng/mL final concentration), IGF-2(100ng/mL final concentration), or human serum (20% final concentration) was then added and the plates were incubated in a humidified incubator for 30 minutes. Cells were fixed by replacing the growth medium with 4% formaldehyde in PBS for 20 minutes at room temperature. Wash with 300. mu.L/well wash buffer (PBS containing 0.1% Triton X-100) for 5 minutes with shakingAfter two rounds, cells were quenched with 100. mu.L/well of 1.2 wt% hydrogen peroxide in wash buffer for 30 minutes at room temperature. The cells were washed again with 300. mu.L/well wash buffer and blocked with 100. mu.L/well blocking buffer (wash buffer containing 5% BSA) for 60 min at room temperature with shaking. Blocking buffer was removed and 50. mu.l/well of phosphorylated IGF-I receptor beta (tyr 1135/1136)/insulin receptor beta (tyr1150/1151) primary antibody (Cell Signaling, No.3024) diluted 1: 1000 in blocking buffer was added. Plates were incubated overnight at 4 ℃ in a shaker, then washed three times as above, and horseradish peroxidase conjugated anti-rabbit IgG goat immunoglobulin (Dako, No. P0448) diluted 1: 500 in blocking buffer was added at 50. mu.L/well. After incubation for 60 minutes at room temperature with shaking, the wells were washed twice with wash buffer as above and once with 300 μ L PBS. 100 μ L/well TMB substrate solution (Bender MedSystems, No. BMS406.1000) was added to the wells and incubated for 10 minutes in shaking, after which the reaction was stopped by adding 100 μ L/well 1M phosphoric acid and the absorbance was read using a spectrometer (OD 450nm, OD 650nm as reference). Determination of IGF-1R or IR-A phosphorylation inhibitory IC by graphical analysis₅₀The value is obtained.

Fab-IGF-1 cocrystallization and Structure determination

The monoclonal antibody was prepared in a buffer of 100mM sodium phosphate (Na-phosphate) (pH 7.0) followed by papain digestion. Papain (SigmaAldrich, P #3125) was activated in digestion buffer (phosphate buffer containing 10mM cysteine hydrochloride, 4mM EDTA, pH 7.0) following the manufacturer's instructions. The IgG antibody was mixed with activated papain (ratio enzyme: IgG 1: 100) and the reaction was incubated overnight at 37 ℃ on a rotary shaker. Digestion was stopped by adding iodoacetamide (iodacetamid) to a final concentration of 30 mM. To separate the Fab fragments from the Fc fragment, Fc cleavage products and intact Mab, the digestion mixture was loaded onto a protein a MabSelect column equilibrated with phosphate buffer. The column was washed with 5 column volumes of PBS and the Fab fragments were collected in the flow-through and wash fractions. The Fc fragment and intact Mab were eluted from the column with 100mM citrate buffer (pH 3.0), followed by size exclusion chromatography of the Fab fragment using a HiLoad Superdex 75 column. The column was run with 20mM triethanolamine, 130mM NaCl, pH 8.0 at 0.5 mL/min. The protein concentration of the Fab fragment was determined by measuring the absorbance at 280 nm. The quality of the Fab fragments was analyzed by Western blotting and ELISA.

Fab-IGF-1 complexes were generated by adding a 2-fold molar excess of recombinant IGF-1 (Gropep; Receptor Grade) to the purified Fab, followed by incubation at 4 ℃ overnight on a rotary shaker. Concentration of the complex to (15mg/mL) and removal of unbound IGF-1 were performed using an Amicon-Ultra device. Crystallization of the Fab-IGF-1 complex is performed using various techniques, such as hanging drop (hanging drop), sitting drop (sitting drop), and seed sowing (seed). In one embodiment, the crystals are precipitated by contacting the solution with a stock solution that reduces the solubility of the protein due to the presence of a precipitating agent, i.e., an agent that induces precipitation. Screening of the various conditions resulted in a suitable buffer system which was operated by the addition of precipitants and additives. The concentration of the precipitating agent is preferably between 5-50% w/v. The pH of the buffer is preferably from about 3 to about 6. The protein concentration in the solution is preferably supersaturated to allow precipitation. The temperature during crystallization is preferably between 4 and 25 ℃.

The three-dimensional structure of the Fab: IGF-1 complex, defined in atomic coordinates, is obtained from the X-ray diffraction pattern of the crystal and its derived electron density map. The diffraction of the crystal is better than that of the crystalResolution. The crystal preferably has space group P3221 (number 154) and aboutCell size of (a); and γ is 120 °. The method for determining three-dimensional structures is molecular replacement, which involves the use of closely related molecules or the structure of a receptor ligand complex. Model building and refinement proceeds in several iterative steps to final R-factors (R and R)_{Free form}) 21 and 23%, respectively.

Determination of pharmacokinetic parameters of rat

Wistar rats were given five intravenous bolus administrations of 18mg/kg, 52mg/kg, and 248mg/kg antibody every 72 hours. At each time point, blood samples were taken and the human antibody concentration in plasma was determined by sandwich ELISA. This allowed calculation of the mean pharmacokinetic parameters of the antibodies on the first day of dosing and calculation of the half-life (t (n) ═ 1008 hours) after the last day of dosing.

Example 1: selection of high affinity antibodies that bind IGF-1

To identify Fab fragments with improved affinity for human IGF-1, multiple "parent" Fab clones identified as binding IGF-1 with low nanomolar affinity were subjected to "in vitro affinity maturation" in which the L-CDR3 and H-CDR2 sequences of each clone were diversified by substituting the parent sequences with a library of new L-CDR3 and H-CDR2 sequences, respectively. The resulting "mature library" was subjected to solution panning against human IGF-1 and the clone with the best affinity was selected for transformation into IgG1 antibody and further tested. The three antibodies with the best affinity for human IGF-1 have affinities of 180pM, 190pM and 130pM, respectively (K)_D) 60814, 60819, and 60833 (shown in table 1), as determined by an electrochemiluminescence-based equilibrium titration method.

Table 1: summary of IGF-1 binding

Antibodies	Affinity (pM)
		60814	180
60819	190
		60833	130

Antibodies were also tested for their binding to human, murine, and rat IGF-1 and IGF-2, as well as human insulin, in an immunoadsorption assay. This demonstrated that 60814, 60819, and 60833 showed comparable cross-reactive binding to mouse and rat IGF-1, as well as human, murine, and rat IGF-2, but were not reactive to human insulin (50 ng/ml at the highest concentrations tested) (fig. 1A-1G).

The affinity constants for antibody binding to human, mouse, and rat IGF-1 and IGF-2 were also determined by surface plasmon resonance (Biacore) analysis. The method involves capturing the antibody on a sensor and flowing IGF antigen over the captured antibody, thus overcoming any affinity effects that may occur when the IGF antigen is coated onto the sensor and the antibody is added. The affinity constants obtained for antibody 60833 using this method are shown in Table 2, where KD measurements for human IGF-1 and human IGF-2 were found to be 0.07nM and 0.9nM, respectively.

Table 2: affinity constants of antibody 60833 for human, mouse, and rat IGF-1 and IGF-2 were determined by surface plasmon resonance (antibody capture method)

Antigens	Kon[M-1s-1]	Koff[s-1]	KD[nM]
				Human IGF-1	4.74x 106	3.01x 10-4	0.07
Mouse IGF-1	1.00x 106	3.23x 10-4	0.33
				Rat IGF-1	3.81x 106	2.53x 10-4	0.07
Human IGF-2	3.97x 106	3.53x 10-3	0.913
				Mouse IGF-2	8.68x 105	1.1x 10-2	13.4
Rat IGF-2	2.56x 106	6.13x 10-3	2.41

Example 2: inhibition of IGF signaling

The first signaling event that occurs upon binding of IGF-1R is IGF-1R phosphorylation. Inhibition of IGF-induced IGF-1R phosphorylation by antibody 60833 was measured using a cell-based ELISA assay.

60833 were tested for their potency and effectiveness in neutralizing recombinant bioactive IGF-1 and IGF-2 induced phosphorylation of IGF-1R up to 15. mu.g/mL (100 nM). As shown in Table 3 and, for example, FIG. 2, 60833 potently and effectively inhibited IGF-1 (FIG. 2A) and IGF-2 (FIG. 2B) induced signaling. In the same assay, mab IR3 targeting IGF-1R was much less potent and effective in IGF-1-induced signaling and demonstrated a very weak effect on IGF-2-induced signaling.

A similar cell-based IR-A phosphorylation ELISA was used to demonstrate that 60833 also inhibited IGF-2 signaling by IR-A. As shown in Table 4 and, for example, FIG. 3A, 60833 potently and effectively inhibited IGF-2-induced IR-A phosphorylation. In contrast, IR3 which was not able to bind IR-A showed no inhibitory effect.

The level of IGF bioactivity in human serum or plasma samples can also be measured using a cell-based ELISA for IGF-1R phosphorylation. This was used to determine 60833 potency and effectiveness in neutralizing human serum IGF bioactivity (up to 15 μ g/mL (100 nM)). As shown in table 3 and, for example, fig. 3B, 60833 potently and effectively inhibited IGF bioactivity in human serum.

Table 3: 60833 Effect on IGF-1R phosphorylation

Table 4: 60833 Effect on IR-A phosphorylation

Example 3: effect on IGF-1 and IGF-2 induced cell proliferation

The effect of antibodies 60814, 60819 and 60833 on IGF-1 and IGF-2 induced proliferation of MCF-7 (from breast cancer) and COLO205 (from colon cancer) cell lines was determined. Examples of the effects of antibodies 60814 and 60819 are shown in FIGS. 4A-D. All three antibodies showed dose-dependent inhibition of IGF-1 (FIGS. 4A and 4C) and IGF-2 (FIGS. 4B and 4D) induced cell proliferation of MCF-7 (FIGS. 4A and 4B) and COLO205 (FIGS. 4C and 4D). The concentration of each antibody required to inhibit IGF-1 or IGF-2-induced cell line proliferation by 50% is shown in Table 5.

Table 5: inhibition of IGF-1 and IGF-2 induced proliferation of MCF-7 and COLO205 cancer cell lines

Example 4: effect on proliferation of cell lines derived from Ewing's sarcoma

The effect of antibodies 60819 and 60833 on the proliferation of the ewing's sarcoma-derived cell line TC-71 grown in medium containing 10% FCS is shown in fig. 5. 60819 and 60833 showed dose-dependent inhibition of TC-71 cell proliferation relative to humanized IgG1 isotype control antibodies that did not bind IGF-1 or IGF-2.

Example 5: effect on murine and rat Total IGF-1 levels

Neutralization of active IGF-1 with IGF-targeting antibodies is expected to lead to feedback via internal secretion of the GH pathway, resulting in elevated serum total IGF-1 levels. Antibodies 60814, 60819 and 60833 cross-react with mouse and rat IGF-1, allowing measurement of any pharmacodynamic effect on total IGF-1 levels in the serum of these species. As shown in fig. 6 and 7, administration of antibody 60819 to mice (fig. 6) and rats (fig. 7) resulted in a dose-dependent increase in total IGF-1 levels in murine and rat sera 24 hours after administration. This represents a useful pharmacodynamic marker of the activity of these antibodies, which can be tested in humans during clinical development.

Example 6: effect of IGF ligand-targeting antibodies in combination with rapamycin on cell line proliferation and intracellular signaling from Ewing's sarcoma

The effect of antibody 60819 and the mTOR inhibitor rapamycin (alone or in combination) on proliferation of the Ewing's sarcoma-derived cell line SK-ES-1 is shown in FIG. 8. There was a dose-dependent inhibition of proliferation of antibody 60819 with rapamycin alone, and both single agents achieved approximately 60% inhibition of proliferation at 100 nM. The equivalent dose combination of antibody 60819 and rapamycin demonstrated an additive inhibitory effect on cell proliferation, with about 95% inhibition when combined at a dose of 100 nM.

IGF-induced cell proliferation is mediated by a series of intracellular protein phosphorylation events. One protein whose phosphorylation is enhanced by IGF stimulation is AKT. FIG. 9 shows the effect of antibody 60819 and rapamycin (alone or in combination) on AKT phosphorylation in SK-ES-1 cells 24 hours after treatment with 100nM dose. The 100nM antibody 60819 inhibited AKT phosphorylation compared to proliferation of untreated cells showing AKT phosphorylation. In contrast, 100nM rapamycin treatment caused higher levels of phosphorylated AKT than controls, which is thought to be due to compensatory feedback mechanisms following mTOR inhibition. However, phosphorylation of AKT was inhibited when 100nM rapamycin was combined with 100nM antibody 60819. This suggests that compensatory feedback leading to AKT phosphorylation after rapamycin treatment is due to elevated IGF ligands and that these ligands can be inhibited by antibody 60819. Figure 9 also shows that antibody 60819 and rapamycin (alone or in combination) did not affect the total content of PTEN.

Example 7: effect of IGF ligand-targeting antibodies in combination with EGFR inhibitors on proliferation of NSCLC-derived cell lines

The effect of antibody 60819 and the EGFR inhibitor erlotinib/deluxe (alone or in combination) on the proliferation of cell line a-549 derived from NSCLC is shown in figure 10. In this model, antibody 60819 alone had only a minor effect on cell proliferation, but rather was relieved of the dose-dependent effect of exhibiting about 60% inhibition of cell proliferation at the maximum tested dose (20 μ M). However, when antibody 60819 was combined with bradysia, inhibition of cell proliferation was more intense and more effective, indicating a synergistic effect.

Example 8: pharmacokinetic profiles in Wistar rats

Mean pharmacokinetic parameters of antibody 60833 in Wistar rats on day one at 18mg/kg, 52mg/kg and 248mg/kg are shown in Table 6. The terminal half-life after the last day of dosing (t (n) ═ 1008 hours) was calculated, and the mean terminal half-life for all three dose levels was 221 hours (9.2 days).

Table 6: mean pharmacokinetic parameters of day one antibody 60833 on WISTAR rats

Phi, the last day after administration and t (n) 1008hr

Example 9: Fab-IGF-1 cocrystallization and structural assays to identify antibody binding sites on IGF-1

To determine with certainty the residues on human IGF-1 that interact with IGF antibodies, Fab and IGF-1 were co-crystallized and better thanThe resolution of (a) determines the structure of the interaction. Residues on IGF-1 that were contacted by antibody (Fab)60833 are shown in Table 7. Throughout, 19 residues on IGF-1 contact 15 CDR residues on 60833. Of these 19 IGF-1 residues, 17 were identical in human IGF-2 when the amino acid sequences of human IGF-1 and IGF-2 were aligned (listed in Table 7). FIG. 11 shows the 3D structure of IGF-1 with amino acids bound by 60833 highlighted and the linear amino acid sequence of human IGF-1 with the interacting amino acids underlined.

Table 7: residues of human IGF-1 that contact residue 60833FAB

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Claims (37)

1. An isolated human antibody molecule which

a) Combining human IGF-1 and IGF-2 to

i) Prevent binding of IGF-1 and IGF-2 to IGF-1 receptor, and

ii) inhibits IGF-1 receptor mediated signaling,

b) binding mouse and rat IGF-1 and IGF-2,

c) does not bind human insulin;

wherein the antibody molecule is

An antibody molecule having heavy chain CDR1 shown in the amino acid sequence of SEQ ID NO. 21, heavy chain CDR2 shown in the amino acid sequence of SEQ ID NO. 22 and heavy chain CDR3 shown in the amino acid sequence of SEQ ID NO. 23 and having light chain CDR1 shown in the amino acid sequence of SEQ ID NO. 24, light chain CDR2 shown in the amino acid sequence of SEQ ID NO. 25 and light chain CDR3 shown in the amino acid sequence of SEQ ID NO. 26.

2. The antibody molecule of claim 1 having the variable heavy chain as set forth in the amino acid sequence of SEQ ID NO. 28.

3. The antibody molecule of claim 1, which has a variable light chain as set forth in the amino acid sequence of SEQ ID NO. 30.

4. The antibody molecule of claim 1, which has a variable heavy chain as set forth in the amino acid sequence of SEQ ID NO. 28 and a variable light chain as set forth in the amino acid sequence of SEQ ID NO. 30.

5. The antibody molecule of any one of claims 1 to 4 comprising a heavy chain constant region selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgM, IgA and IgE constant regions.

6. The antibody molecule of claim 5, wherein the heavy chain constant region is the IgG1 heavy chain constant region represented by the amino acid sequence of SEQ ID NO. 32.

7. An antibody molecule according to any one of claims 1 to 4 wherein the light chain constant region is that of Ig λ.

8. The antibody molecule of claim 7, wherein the light chain constant region is represented by the amino acid sequence of SEQ ID NO. 34.

9. An antibody molecule according to any one of claims 2 to 4 having

a) The heavy chain of the amino acid sequence of SEQ ID NO:39, and

b) 40 in the sequence of SEQ ID NO.

10. The antibody molecule of any one of claims 1 to 4 which is a Fab fragment, a F (ab')2 fragment, or a single chain Fv fragment.

11. A DNA molecule comprising a nucleotide sequence encoding the variable heavy chain of an antibody molecule according to any one of claims 1 to 10 and a nucleotide sequence encoding the variable light chain of an antibody molecule according to any one of claims 1 to 10.

12. The DNA molecule of claim 11, the nucleotide sequence encoding the variable heavy chain of the antibody molecule of any one of claims 1 to 10 being represented by the nucleotide sequence of SEQ ID No. 27, encoding the variable heavy chain of an antibody as defined in claim 2 or 4.

13. A DNA molecule according to claim 11, wherein the nucleotide sequence encoding the variable light chain of an antibody molecule according to any one of claims 1 to 10 is represented by the nucleotide sequence of SEQ ID NO. 29, which encodes the variable light chain of an antibody as defined in claim 3 or 4.

14. An expression vector comprising a DNA molecule consisting of nucleotide sequences encoding the variable heavy and variable light chains of the antibody molecule of any one of claims 1 to 10.

15. The expression vector of claim 14, comprising a DNA molecule represented by the sequences of SEQ ID NO. 27 and SEQ ID NO. 29.

16. The expression vector of claim 14 or 15, further comprising a DNA molecule encoding a constant heavy chain and/or a constant light chain, respectively, linked to DNA molecules encoding a variable heavy chain and a variable light chain, respectively.

17. A host cell carrying one or more expression vectors according to claim 14, 15 or 16.

18. The host cell of claim 17 which is a mammalian cell.

19. A method of producing an antibody according to any one of claims 1 to 10, comprising transfecting a mammalian host cell with one or more vectors according to claim 14, 15 or 16, incubating the host cell, and recovering and purifying the antibody.

20. A method of producing an antibody according to any one of claims 1 to 10, comprising obtaining a mammalian host cell comprising one or more vectors according to claim 14, 15 or 16, and growing the host cell.

21. The method of claim 20, further comprising recovering and purifying the antibody.

22. An antibody molecule according to any one of claims 1 to 10 for use in medicine.

23. Use of an antibody molecule according to any one of claims 1 to 10 for the preparation of a medicament for the treatment of a cancerous disease selected from the group consisting of: colorectal cancer, breast cancer, and ewing's sarcoma.

24. The use as in claim 23, wherein the medicament is used in combination with: platinum-based chemotherapy, sorafenib, or a compound selected from the group of inhibitors of EGFR, VEGF, HER2-neu, AuroraB, Plk1, PI3 kinase or mTor.

25. The use as in claim 24, wherein the medicament is used in combination with: platinum doublet therapy of paclitaxel/carboplatin or gemcitabine/cisplatin.

26. A pharmaceutical composition comprising an antibody molecule according to any one of claims 1 to 10 and a pharmaceutically acceptable carrier.

27. The pharmaceutical composition of claim 26, further comprising one or more additional therapeutic agents selected from the group consisting of:

a) a DNA damaging agent which is capable of damaging DNA,

b) therapeutically active compounds that inhibit the signal transduction pathway or mitotic checkpoint in cancer cells,

c) a Chinese medicinal composition for treating diabetes.

28. The pharmaceutical composition of claim 27, wherein the one or more compounds b) are selected from the group of inhibitors of EGFR, VEGF, HER2-neu, AuroraB, Plk1, PI3 kinase or mTor.

29. The pharmaceutical composition according to any one of claims 26 to 28 for use in the treatment of a cancerous disease selected from the group consisting of: colorectal cancer, breast cancer, and ewing's sarcoma.

30. The pharmaceutical composition of claim 29, further used in combination with: platinum-based chemotherapy, sorafenib, or a compound selected from the group of inhibitors of EGFR, VEGF, HER2-neu, AuroraB, Plk1, PI3 kinase or mTor.

31. The pharmaceutical composition of claim 30, wherein the medicament is used in combination with: platinum doublet therapy of paclitaxel/carboplatin or gemcitabine/cisplatin.

32. Use of an antibody molecule according to any one of claims 1 to 10 for the preparation of a medicament for a method of inhibiting binding of IGF-1 and IGF-2 to an IGF-1 receptor in a mammalian cell, the method comprising administering an antibody molecule according to any one of claims 1 to 10 to the cell, thereby inhibiting signaling mediated by the IGF-1 receptor and proliferation and anti-apoptosis mediated by IGF-1 and IGF-2.

33. Use of an antibody molecule according to any one of claims 1 to 10 for the preparation of A medicament for A method of inhibiting IGF-2 binding to insulin receptor IR- A in A mammalian cell, which method comprises administering an antibody molecule according to any one of claims 1 to 10 to the cell, thereby inhibiting signaling mediated by insulin receptor IR- A and thereby inhibiting IGF-2 mediated proliferation and anti-apoptosis.

34. The use of claim 32 or 33, wherein the administration is performed in vitro.

35. An in vitro method of inhibiting binding of IGF-1 and IGF-2 to IGF-1 receptors in a mammalian cell, comprising administering to the cell an antibody molecule of any one of claims 1 to 10, thereby inhibiting signaling mediated by IGF-1 receptors and proliferation and anti-apoptosis mediated by IGF-1 and IGF-2.

36. An in vitro method of inhibiting IGF-2 binding to insulin receptor IR- A in A mammalian cell, comprising administering to the cell an antibody molecule of any one of claims 1 to 10, thereby inhibiting signaling mediated by insulin receptor IR- A and thereby inhibiting IGF-2 mediated proliferation and anti-apoptosis.

37. Use of an antibody molecule according to any one of claims 1 to 10 for the preparation of a medicament for a method of monitoring the effectiveness of an antibody molecule that binds IGF-1 and IGF-2 for the treatment of a patient with cancer, wherein the method comprises:

(a) measuring the total IGF-1 level in a biological sample of the patient,

(b) administering to the patient an antibody molecule according to any one of claims 1 to 10,

(c) measuring the total IGF-1 level in the patient's biological sample, wherein an amount of increase in the total IGF-1 level over the total IGF-1 level measured in step (a) is indicative of the extent to which the patient is responsive to treatment with the anti-IGF antibody molecule.