ES2359176T3 - PROTEINS OF UNION TO THE HEPATOCITE GROWTH FACTOR (HGF). - Google Patents

Abstract

Isolated binding protein that binds to human hepatocyte growth factor (HGF), comprising: (a) a variable region of an immunoglobulin heavy chain comprising the structure CDRH1-CDRH2-CDRH3, in which (i) CDRH1 comprises an amino acid sequence SEQ ID No. 15, (ii) CDRH2 comprises an amino acid sequence selected from the group consisting of SEQ ID No. 204 and SEQ ID No. 205, and (iii) CDRH3 comprises an amino acid sequence of SEQ ID No. 17 and (b) a variable region of an immunoglobulin light chain comprising the structure CDRL1-CDRL2-CDRL3, in which (i) CDRL1 comprises an amino acid sequence SEQ ID NO: 18, (ii) CDRL2 comprises an amino acid sequence selected from the group consisting of SEQ ID No. 19 and SEQ ID No. 206, and (iii) CDRL3 comprises an amino acid sequence SEQ ID No. 20, in which the variable region of the immunoglobulin heavy chain and the variable region of the chain light immunoglobulin a jointly define a single binding site for human HGF binding.

Description

RELATED APPLICATIONS [0001] This application claims the benefit and priority over the Requests U.S. Provisionals No. 60 / 810,714, filed June 2, 2006, and 60 / 860,509, filed on November 21, 2006.

FIELD OF THE INVENTION [0002] The field of the invention is molecular biology, immunology and oncology. More particularly, the field are binding proteins based on antibodies that bind to human hepatocyte growth factor (HGF).

BACKGROUND [0003] Hepatocyte Growth Factor (HGF), also known as dispersion factor (SF), is a multifunctional heterodimeric protein produced predominantly by mesenchymal cells, and is an effector of cells that express the Met tyrosine kinase receptor (Bottaro et al. (1991) SCIENCE 251: 802-804, Rubin et al. (1993) BIOCHIM. BIOPHYS ACT 1155: 357-371). He Human Met receptor is also known as "c-Met". Mature HGF contains two polypeptide chains, chain � and chain �. Published studies suggest that it is the � chain that contains the c-Met receptor binding domain of the HGF. [0004] When it joins its related receptor, the HGF mediates several activities cell phones. The HGF-Met signaling pathway plays a role in the liver regeneration, wound healing, neuronal regeneration, angiogenesis and malignant tumors. See, for example, Cao et al. (2001) PROC. NATL. ACAD SCI. USA 98: 7443-7448, Burges et al. (2006) CANCER RES. 66: 1721-1729 and U.S. Patent Nos. 5,997,868 and 5,707,624. The researchers have been developing several HGF modulators, including antibodies, to treat various disorders that involve HGF activity, by For example, certain HGF-sensitive cancers. See, for example, the Publication of International Application No. WO 2005/017107. [0005] The basic structure common to all antibodies is shown schematically in Figure 1. Antibodies are multimeric proteins that They contain four polypeptide chains. Two of the polypeptide chains are they are called heavy chains or H, and two of the polypeptide chains are called light or L. Heavy and light immunoglobulin chains are

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connected by an intercatenary disulfide bond. The immunoglobulin heavy chains are connected by several intercatenary disulfide bonds. A light chain is composed of a variable region (VL in Figure 1) and a constant region (CL in Figure 1), while the heavy chain is composed of a variable region (VH in Figure 1) and at least three constant regions (CH1, CH2 and CH3 in Figure 1). The variable regions determine the specificity of the antibody and the constant regions have other functions. [0006] Structural and amino acid information indicates that each variable region comprises three hypervariable regions (also known as complementarity determining regions or CDRs) flanked by four relatively conserved framework regions ("framework regions") or FR. The three CDRs, called CDR1, CDR2 and CDR3 are responsible for the specificity of individual antibody binding. When antibodies are to be used as diagnostic and therapeutic agents, it is typically desirable to create antibodies that have the highest specificity and binding affinity towards the target molecule. It is believed that differences in the variable regions can have a great effect on the specificity and affinity of the antibody. [0007] In WO 2005/017107 A, antibodies against the human HGF protein are described. US Patent No. 5,707,624 describes the use of anti-HGF antibodies in the treatment of Kaposi's sarcoma. Similarly, US Patent No. 5,997,868 describes the treatment of a tumor by administering an anti-HGF antibody to the patient to be treated to block the ability of endogenous HGF to promote angiogenesis in the tumor. More recently, the researchers proposed that antibodies that bind to the � chain of HGF may have potential as therapeutic agents in patients with HGF-dependent tumors (Burgess (2006), above). [0008] However, there is still a need for additional HGF modulators that can be used as therapeutic and diagnostic agents.

SUMMARY DESCRIPTION OF THE INVENTION [0009] The invention is based, in part, on the discovery of a family of binding proteins that specifically bind HGF, in particular, human HGF. Binding proteins are antibody based to the extent that they contain antigen binding sites (i.e., HGF) based on the CDRs of a family of antibodies that specifically bind HGF. CDRs confer the binding specificity of HGF binding proteins. Binding proteins can be used as diagnostic and therapeutic agents. When used as a therapeutic agent, binding proteins are engineered (for example, they are humanized) to reduce or eliminate the risk of inducing an immune response against the binding protein when administered to the recipient (e.g., a being human). [0010] Binding proteins neutralize the activity of HGF and, therefore, can be used as a therapeutic agent. In certain embodiments, the binding proteins prevent the binding of HGF to its related receptor, c-Met, thereby neutralizing HGF activity. Because HGF has been implicated in the growth and proliferation of cancer cells, binding proteins can be used to inhibit the proliferation of cancer cells. In addition, when administered to a mammal, binding proteins can inhibit or reduce the growth of a tumor in the mammal. [0011] The invention provides an isolated binding protein that binds to human hepatocyte growth factor (HGF), which comprises:

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(to)

an immunoglobulin heavy chain variable region comprising the structure CDRH1-CDRH2-CDRH3, in which

(i)

 CDRH1 comprises the amino acid sequence SEQ ID NO: 15,

(ii)

 CDRH2 comprises an amino acid sequence selected from the group consisting of SEQ ID No. 204 and SEQ ID No. 205 and

(iii) the CDRH3 comprises the amino acid sequence SEQ ID NO: 17 and

(b)

an immunoglobulin light chain variable region comprising the structure CDRL1-CDRL2-CDRL3, in which

(i)

 CDRL1 comprises the amino acid sequence SEQ ID NO: 18,

(ii)

 CDRL2 comprises an amino acid sequence selected from the group consisting of SEQ ID No. 19 and SEQ ID No. 206 and

(iii)

 CDRL3 comprises the amino acid sequence SEQ ID NO: 20, in which the immunoglobulin heavy chain variable region and the immunoglobulin light chain variable region together define a single binding site for binding to human HGF. [0012] The invention further provides an isolated nucleic acid comprising a nucleotide sequence encoding an immunoglobulin light chain variable region comprising the structure CDRL1-CDRL2-CDRL3, in which

(i)

 CDRL1 comprises the amino acid sequence SEQ ID NO: 18,

(ii)

 CDRL2 comprises the amino acid sequence SEQ ID No. 206 and

(iii)

 CDRL3 comprises the amino acid sequence SEQ ID NO: 20 and an expression vector comprising said nucleic acid. [0013] The invention further provides an isolated nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain variable region comprising the structure CDRH1-CDRH2-CDRH3, in which

(i)

 CDRH1 comprises the amino acid sequence SEQ ID NO: 15,

(ii)

 CDRH2 comprises an amino acid sequence selected from the group consisting of SEQ ID No. 204 and SEQ ID No. 205 and

(iii)

 CDRH3 comprises the amino acid sequence SEQ ID NO: 17 and an expression vector comprising said nucleic acid. [0014] An expression vector is also provided comprising a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin light chain variable region comprising the structure CDRL1CDRL2-CDRL3, in which

(i)

 CDRL1 comprises the amino acid sequence SEQ ID NO: 18,

(ii)

 CDRL2 comprises the amino acid sequence SEQ ID No. 206 and

(iii)

 CDRL3 comprises the amino acid sequence SEQ ID NO: 20 and a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain variable region comprising the structure CDRH1-CDRH2-CDRH3, in which

(i)

 CDRH1 comprises the amino acid sequence SEQ ID NO: 15,

(ii)

 CDRH2 comprises an amino acid sequence selected from the group consisting of SEQ ID No. 204 and SEQ ID No. 205 and

(iii)

 CDRH3 comprises the amino acid sequence SEQ ID No. 17. [0015] The invention also provides a host cell comprising at least one expression vector encoding a light variable region of immunoglobulin comprising the structure CDRL1-CDRL2-CDRL3, in the that

(i)

 CDRL1 comprises the amino acid sequence SEQ ID NO: 18,

(ii)

 CDRL2 comprises the amino acid sequence SEQ ID No. 206 and

(iii)

 CDRL3 comprises the amino acid sequence SEQ ID No. 20 [0016] A host cell is also provided that comprises at least one expression vector encoding an immunoglobulin heavy chain variable region comprising the structure CDRH1-CDRH2-CDRH3, in which that

(i)

 CDRH1 comprises the amino acid sequence SEQ ID NO: 15,

(ii)

 CDRH2 comprises an amino acid sequence selected from the group consisting of SEQ ID No. 204 and SEQ ID No. 205 and

(iii)

 CDRH3 comprises the amino acid sequence SEQ ID No. 17. [0017] The invention further provides a host cell comprising at least one expression vector encoding a light variable region of immunoglobulin comprising the structure CDRL1-CDRL2-CDRL3, in the that

(i)

 CDRL1 comprises the amino acid sequence SEQ ID NO: 18,

(ii)

 CDRL2 comprises the amino acid sequence SEQ ID No. 206 and

(iii)

 CDRL3 comprises the amino acid sequence SEQ ID No. 20 and an immunoglobulin heavy chain variable region comprising the structure CDRH1-CDRH2-CDRH3, in which

(i)

 CDRH1 comprises the amino acid sequence SEQ ID NO: 15,

(ii)

 CDRH2 comprises an amino acid sequence selected from the group consisting of SEQ ID No. 204 and SEQ ID No. 205 and

(iii)

 the CDRH3 comprises the amino acid sequence SEQ ID No. 17. [0018] In another aspect, the invention provides a method of producing a polypeptide comprising a heavy chain variable region of immunoglobulin, or a method of producing a polypeptide comprising a variable region of immunoglobulin light chain, the method comprising:

(i)

 culturing the host cell of the invention under conditions for the host cell to express the polypeptide comprising the immunoglobulin heavy chain variable region or the polypeptide comprising the immunoglobulin light chain variable region, respectively; Y

(ii)

 purifying the polypeptide comprising the immunoglobulin heavy chain variable region or the polypeptide comprising the immunoglobulin light chain variable region, respectively.

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[0019] In further embodiments, the isolated binding protein that binds to human hepatocyte growth factor (HGF) comprises an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region selected from the group consisting of:

(to)

 an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID No. 193, and an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID No. 183;

(b)

 an immunoglobulin light chain variable region comprising

the amino acid sequence of SEQ ID No. 193, and an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID No. 189;

(C)

 an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID No. 199, and an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID No. 183; Y

(d)

 an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID No. 199, and an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID No. 189.

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[0020] In a preferred embodiment, the binding protein comprises an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID No. 199, and an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 189. [0021] In further embodiments, the isolated binding protein that binds to human hepatocyte growth factor (HGF) comprises an immunoglobulin light chain sequence and an immunoglobulin heavy chain sequence selected from the group consisting of:

(to)

 an immunoglobulin light chain sequence comprising the amino acid sequence of SEQ ID No. 197, and an immunoglobulin heavy chain sequence comprising the amino acid sequence of SEQ ID No. 187;

(b)

 an immunoglobulin light chain sequence comprising the amino acid sequence of SEQ ID No. 197, and an immunoglobulin heavy chain sequence comprising the amino acid sequence of SEQ ID No. 191;

(C)

 an immunoglobulin light chain sequence comprising the amino acid sequence of SEQ ID No. 201, and an immunoglobulin heavy chain sequence comprising the amino acid sequence of SEQ ID No. 187; Y

(d)

 an immunoglobulin light chain sequence comprising the amino acid sequence of SEQ ID No. 201, and an immunoglobulin heavy chain sequence comprising the amino acid sequence of SEQ ID No. 191.

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[0022] In a preferred embodiment, the binding protein comprises an immunoglobulin light chain comprising the amino acid sequence of SEQ ID No. 201, and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID No. 191. [0023] The binding protein of the invention is also provided for use in therapy. In certain embodiments, the binding protein of the invention is for use in inhibiting or reducing the proliferation of a tumor cell or for use in inhibiting or reducing the growth of a tumor in a mammal. [0024] In another aspect, the invention provides a method of producing a binding protein that binds to human hepatocyte growth factor (HGF), which is an intact antibody, such as a monoclonal antibody, or a binding fragment. to antigen thereof, the method comprising:

(i) culturing the host cell of the invention under conditions for the host cell to express a polypeptide comprising the immunoglobulin heavy chain variable region and a polypeptide comprising the immunoglobulin light chain variable region; Y

(ii) purify the antibody or antigen-binding fragment of the antibody. [0025] These and other aspects and advantages of the invention will be apparent upon consideration of the following figures, detailed description and claims.

DESCRIPTION OF THE FIGURES [0026] The invention can be more fully understood with respect to the following drawings. [0027] Figure 1 is a schematic representation of a typical antibody. [0028] Figure 2 is a schematic diagram showing the amino acid sequence defining the full immunoglobulin heavy chain variable region of the antibodies indicated as 1A3, 1D3, 1F3, 2B8, 2F8, 3A12, 3B6 and 3D11. The amino acid sequences for each antibody are aligned with each other and the regions that define the signal peptide, CDR1, CDR2 and CDR3 are identified in boxes. Unframed sequences represent FR sequences. [0029] Figure 3 is a schematic diagram showing the sequences of CDR1, CDR2 and CDR3 for each of the immunoglobulin heavy chain variable region sequences presented in Figure 2. [0030] Figure 4 is a schematic diagram which shows the amino acid sequence that defines the complete immunoglobulin light chain variable region of antibodies 1A3, 1D3, 1F3, 2B8, 2F8, 3A12, 3B6 and 3D11. The amino acid sequences for each antibody are aligned with each other and the regions that define the signal peptide, CDR1, CDR2 and CDR3 are identified in boxes. Unframed sequences represent FR sequences. [0031] Figure 5 is a schematic diagram showing the sequences of CDR1, CDR2 and CDR3 for each of the immunoglobulin light chain variable region sequences presented in Figure 4. [0032] Figure 6 is a graph that summarizes the results of an experiment to measure the tumor inhibitory activity of anti-HGF antibodies 1D3, 1F3 1A3 and 2B8 in a U87MG xenograft model. The rhombuses correspond to PBS; the triangles correspond to the anti-HGF 1A3 antibody; the X correspond to the anti-HGF 1D3 antibody; the squares correspond to the anti-HGF 1F3 antibody and the circles correspond to the anti-HGF 2B8 antibody. [0033] Figure 7 is a graph summarizing the results of an experiment to measure the inhibitory activity of anti-HGF antibody tumors 1D3, 1F3, 1A3 and 2B8 in a U118 xenograft model. The rhombuses correspond to IgG; the squares correspond to anti-HGF 1F3 antibody, the triangles correspond to anti-HGF 1D3 antibody; the X correspond to anti-HGF 1A3 antibody; and the circles correspond to anti-HGF 2B8 antibody. [0034] Figure 8 is a table summarizing surface plasmon resonance data on the affinity of antigen binding and the interaction kinetics between human HGF and chimeric, chimeric / humanized or humanized 2B8 antibodies. The table lists the Kappa light chain and IgG1 heavy chain pairs tested. The antibodies with the typical deviations (DT) listed were analyzed in three independent experiments. [0035] Figure 9 is a bar graph summarizing experimental data indicating that Hu2B8 binds to a mutually exclusive epitope for murine monoclonal antibody 2B8. The humanized chimeric 2B8 was captured on an anti-human Fc microplate. Then, HGF joined humanized or chimeric 2B8. The ability of mouse 2B8 or control antibody (goat polyclonal anti-HGF antibody) to bind to the captured HGF was measured. Both humanized 2B8 and chimeric 2B8 antibodies prevent the binding of murine 2B8 to HGF. The white bars correspond to the chimeric 2B8 antibody; the gray bars correspond to the humanized Hu2B8 antibody (kappa variable region Kv1-39.1 and heavy chain variable region Hv5-51.1); the black bars correspond to the humanized Hu2B8 antibody (kappa variable region Kv3-15.1 and heavy chain variable region Hv5-51.1).

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DETAILED DESCRIPTION OF THE INVENTION [0036] The invention is based, in part, on the discovery of a family of binding proteins that specifically bind to, and neutralize the activity of HGF, in particular, human HGF. Binding proteins can be used in a variety of therapeutic and diagnostic applications. Binding proteins are based on the antigen binding sites of a monoclonal antibody that has been selected for its ability to bind to and neutralize HGF activity. In particular, binding proteins contain immunoglobulin variable region CDR sequences that together define a binding site for HGF. [0037] In view of the neutralizing activity of these antibodies, they are particularly useful in modulating the growth and / or proliferation of HGF-sensitive cells, for example, cancer cells. When used as a therapeutic agent. Binding proteins can be engineered to minimize or eliminate the risk of inducing an immune response against binding proteins when administered to the recipient. Furthermore, depending on the particular application, it is contemplated that the binding proteins can be conjugated with other moieties, for example, detectable markers, for example, radiolabels and effector molecules, for example, other therapeutic products based on proteins and small molecules. Each of these features and aspects of the invention is discussed in more detail below.

I - Binding proteins that bind to HGF [0038] In one aspect, the invention provides an isolated binding protein that binds to human HGF. The binding protein comprises (i) an immunoglobulin light chain variable region comprising the CDRL1-CDRL2CDRL3 structure and (ii) an immunoglobulin heavy chain variable region comprising three complementarity determining regions (CDR), in which the immunoglobulin light chain variable region and immunoglobulin heavy chain variable region together define a single binding site to bind to human HGF. As described herein, the CDRL1 comprises the amino acid sequence X1 X2 Ser X4 X5 X6 X7 X8 X9 X10 X11 X12 X13 X14 X15, wherein amino acid X1 is Arg, Lys or Ser, X2 is Ala or Thr, X4 is Glu, Gln or Ser, X5 is Asn, Asp or Ser, X6 is Ile or Val, X7 is Asp, Lys, Ser, Val or Tyr, X8 is a peptide bond

image9

or Tyr, X9 is a peptide bond or Asp, X10 is a peptide bond or Gly, X11 is a peptide bond or Asn, X12 is a peptide bond, Ile or Ser, X13 is Asn or Tyr, X14 is Ile, Leu, Met or Val, X15 is Ala, Asn, His or Ser. CDRL2 comprises the amino acid sequence X16 X17 X18 X19 X20 X21 X22, in which amino acid X16 is Ala, Asp, Arg, Gly or Val, X17 is Ala, Thr or Val, X18 is Asn, Ser or Thr, X19 is Arg, Asn, Lys or His, X20 is Leu or Arg, X21 is Ala, Asn, Glu, Val or Pro, X22 is Asp, Ser or Thr. The CDRL3 comprises the amino acid sequence X23 X24 X25 X26 X27 X28 Pro X30 Thr, in which amino acid X23 is Leu, Gly or Gln, X24 is His or Gln, X25 is Phe, Ser, Trp

or Tyr, X26 is Asp, Ile, ser, Trp or Tyr, X27 is Gly, Glu, Asn or Ser, X28 is Asp, Asn, Phe, Thr or Tyr, X30 is Leu, Phe, Pro or Tyr. [0039] In another aspect, the invention provides an isolated binding protein that binds to human HGF, comprising (i) an immunoglobulin heavy chain variable region comprising the structure CDRH1-CDRH2-CDRH3 and (ii) a region immunoglobulin light chain variable comprising three complementarity determining regions (CDR), wherein the immunoglobulin heavy chain variable region and the immunoglobulin light chain variable region together define a single binding site for HGF binding human. As described herein, the CDRH1 comprises the amino acid sequence X1 Tyr X3 X4 X5, wherein amino acid X1 is Asp, Asn, Ser or Thr, X3 is Phe, Ser, Trp or Tyr, X4 is Ile, Leu or Met, X5 is Asn, His or Ser. CDRH2 comprises the amino acid sequence X6 Ile X8 X9 X10 X11 Gly X13 X14 X15 Tyr X17 X18 X19 X20 X21 X22, wherein amino acid X6 is Lys, Gln, Glu, Val or Tyr, X8 is Asn, Gly, Ser, Trp or Tyr, X9 is Ala, Pro or Ser, X10 is Gly or Thr, X11 is a peptide bond, Asp, Asn, Gly or Ser, X13 is Asp, Asn, His or Ser, X14 is Ser or Thr, X15 is Asn or Tyr, X17 is Asn or Pro, X18 is Ala, Asp, Gly, Gln, Glu, Pro or Ser, X19 is Asn, Lys, Met or Ser, X20 is Leu, Phe or Val, X21 is Lys, Met or Gln, X22 is Asp, Gly or Ser. The CDRH3 comprises the amino acid sequence X23 X24 X25 X26 X27 X28 X29 X30 X31 X32 X33 X34 Tyr, wherein amino acid X23 is Arg, Asn, Gln or Glu, X24 is Gly, Leu, Arg or Tyr, X25 is a peptide bond, Asp or Gly, X26 is a peptide bond or Gly, X27 is a peptide bond or Tyr, X28 is a peptide bond, Leu or Tyr, X29 is a peptide bond, Gly, Leu, Arg or Val, X30 is a peptide bond, Asp, Gly or Glu, X31 is a peptide bond, Asn, Arg, Ser or Tyr, X32 is a peptide bond, Ala, Gly,

image10

Ile or Tyr, X33 is Met or Phe, X34 is Ala or Asp. [0040] It is understood that the binding protein may comprise the sequences of both the immunoglobulin light chain and the immunoglobulin heavy chain, or fragments thereof, indicated above. In addition, it is understood that the binding protein can be an intact antibody or an antigen binding fragment thereof, or a biosynthetic antibody site. [0041] In certain embodiments, the CDR sequences of the immunoglobulin light chain and the immunoglobulin heavy chain are interposed between framework regions (FR). [0042] In certain other embodiments, the CDR sequences of the immunoglobulin light chain and the immunoglobulin heavy chain are interposed between human or humanized framework regions. [0043] In one embodiment, the binding protein comprises an immunoglobulin light chain variable region comprising a CDRL1 comprising the sequence of SEQ ID No. 18 (2B8), a CDRL2 comprising the sequence of SEQ ID No. 19 (2B8) or SEQ ID No. 206 (LRMR2B8LC) and a CDRL3 comprising the sequence of SEQ ID No. 20 (2B8). [0044] In each of the above embodiments, the sequences of CDRL1, CDRL2 and CDRL3 are preferably interposed between FR of human or humanized immunoglobulins. It is understood that the binding protein can be an intact antibody, an antigen binding fragment thereof or a biosynthetic antibody site. [0045] In another embodiment, the binding protein comprises an immunoglobulin heavy chain variable region comprising: a CDRH1 comprising the sequence of SEQ ID NO: 15 (2B8); a CDRH2 comprising the sequence of SEQ ID No. 204 (LR2B8HC) or SEQ ID No. 205 (LRMR2B8HC); and a CDRH3 comprising the sequence of SEQ ID No. 17 (2B8). [0046] In each of the above embodiments, the sequences of CDRH1, CDRH2 and CDRH3 are preferably interposed between FR of human or humanized immunoglobulins. It is understood that the binding protein can be an intact antibody, an antigen binding fragment thereof or a biosynthetic antibody site. [0047] In another aspect, the invention provides an isolated binding protein that binds to human HGF, comprising (i) an immunoglobulin light chain variable region selected from the group consisting of SEQ ID No. 193 (variable region of light chain LR2B8LC) and SEQ ID No. 199 (variable region light chain LRMR2B8LC); and (ii) an immunoglobulin heavy chain variable region selected from the group consisting of SEQ ID No. 183 (heavy chain variable region LR2B8HC) and SEQ ID No. 189 (heavy chain variable region LRMR2B8HC). The binding protein can be an intact antibody, an antigen binding fragment thereof or a biosynthetic antibody site. [0048] In another aspect, the invention provides an isolated binding protein that binds to human HGF, comprising (i) an immunoglobulin light chain selected from the group consisting of SEQ ID No. 197 (LR2B8LC + Kappa constant (allotype) Km (3) (allele 1)) and SEQ ID No. 201 (LRMR2B8LC + Kappa constant (allotype Km (3) (allele 1)); and (ii) an immunoglobulin heavy chain selected from the group consisting of SEQ ID No. 187 (LR2B8HC + IgG1 constant (allotype G1m (3) (allele 1)) and SEQ ID No. 191 (LRMR2B8HC + constant of IgG1 (allotype G1m (3) (allele 1)). The binding protein may be a intact antibody, an antigen binding fragment thereof or a biosynthetic antibody site. [0049] An isolated binding protein that binds to reduced human HGF is also described herein. The binding protein comprises (i) a immunoglobulin light chain variable region comprising three CDRs and (ii) a p chain variable region immunoglobulin assay comprising three CDRs. CDRs typically interpose between FR. The CDRs of the immunoglobulin light chain and the immunoglobulin heavy chain together define a binding site that binds to the reduced human HGF, for example, the � chain of the reduced HGF. Reduced HGF refers to HGF treated with an amount of reducing agent, for example, dithiothreitol (DTT), 2-mercaptoethanol or glutathione, sufficient to reduce the disulfide bond between the � chain and the � chain. Exemplary concentrations include, for example, 100 mM DTT and 5% 2-mercaptoethanol. [0050] A binding protein comprising an immunoglobulin light chain variable region comprising at least one CDR selected from the group consisting of CDRL1, CDRL2 and CDRL3 is also described. Optionally, the binding protein comprises two CDRs, for example, CDRL1 and CDRL2, or CDRL1 and CDRL3, or CDRL1 and CDRL3. Optionally, the binding protein comprises all three CDRs, that is, CDRL1, CDRL2 and CDRL3. The CDRL1 described herein comprises the amino acid sequence X1 X2 Ser X4 X5 X6 X7 X8 X9 X10 X11 X12 X13 X14 X15, wherein amino acid X1 is Arg or Lys, X2 is Ala or Thr, X4 is Glu or Gln , X5 is Asn, Ser or Asp, X6 is Ile or Val, X7 is Tyr, Asp or Lys, X8 is a peptide bond or Tyr, X9 is a peptide bond or Asp, X10 is a peptide bond or Gly, X11 is a peptide bond or Asn, X12 is a peptide bond or Ser, X13 is Asn or Tyr, X14 is Ile or Leu, X15 is Ala, Asn or Ser. CDRL2 comprises the amino acid sequence X16 X17 X18 X19 Leu X21 X22, in the that amino acid X16 is Ala, Asp, Val or Arg, X17 is Ala or Val, X18 is Asn, Ser or Thr, X19 is Arg, Asn or His, X21 is Ala, Glu, Val or Pro, X22 is Asp or Ser CDRL3 comprises the amino acid sequence X23 X24 X25 X26 X27 X28 Pro X30 Thr, in which amino acid X23 is Leu or Gln, X24 is His or Gln, X25 is Phe, Ser or Tyr, X26 is Asp, Ile or Trp , X27 is Gly or Glu, X28 is Asp, Phe or Thr, X30 is Phe, Pro or Tyr. [0051] A binding protein comprising an immunoglobulin heavy chain variable region comprising at least one CDR selected from the group consisting of CDRH1, CDRH2 and CDRH3 is also described. Optionally, the binding protein comprises two CDRs, for example, CDRH1 and CDRH2, or CDRH1 and CDRH3, or CDRH1 and CDRH3. Optionally, the binding protein comprises all three CDRs, that is, CDRH1, CDRH2 and CDRH3. The CDRH1 described herein comprises the amino acid sequence X1 Tyr X3 X4 X5, wherein amino acid X1 is Asp, Asn, Ser or Thr, X3 is Phe, Trp or Tyr, X4 is Ile or Met, X5 is Asn , His or Ser. The CDRH2 comprises the amino acid sequence X6 Ile X8 X9 Gly X11 Gly X13 X14 X15 Tyr X17 X18 X19 X20 Lys X22, wherein amino acid X6 is Lys, Gln or Tyr, X8 is Gly, Ser or Tyr , X9 is Pro or Ser, X11 is Asp, Gly or Ser, X13 is Asp or Ser, X14 is Ser or Thr, X15 is Asn or Tyr, X17 is Asn or Pro, X18 is Ala, Asp, Gly or Glu, X19 is Asn, Met or Ser, X20 is Phe or Val, X22 is Asp or Gly. The CDRH3 comprises the amino acid sequence X23 X24 X25 X26 X27 X28 X29 X30 X31 X32 X33 Asp Tyr, wherein amino acid X23 is Arg or Gln, X24 is Gly or Leu, X25 is Asp, Gly or a peptide bond, X26 is Gly or a peptide bond, X27 is a peptide bond or Tyr, X28 is Leu, a peptide bond or Tyr, X29 is a Gly, Arg or Leu, X30 is Asp, Gly or Glu, X31 is a Tyr, Arg or Asn, X32 is Ala, Gly or Tyr, X33 is Met or Phe. [0052] It is understood that the binding protein may comprise both immunoglobulin heavy chain and immunoglobulin light chain sequences, or fragments thereof, indicated above. In addition, it is understood that the binding protein can be an intact antibody or an antigen binding fragment thereof, or a biosynthetic antibody site.

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[0053] It is understood that each of the binding proteins discussed above may be an intact antibody, for example, a monoclonal antibody. Alternatively, the binding protein may be an antigen binding fragment of an antibody, or it may be a biosynthetic antibody binding site. Antibody fragments include Fab, Fab ’, (Fab’) 2 or Fv fragments. Those skilled in the art are aware of methods for preparing said antibody fragments. Several biosynthetic antibody binding sites are known in the art, for example, single Fv molecules or sFv described, for example, in US Patent No. 5,476,786. Other biosynthetic antibody binding sites include bispecific or bifunctional binding proteins, for example, bispecific or bifunctional antibodies, which are antibodies or antibody fragments that bind to at least two different antigens. For example, bispecific binding proteins can bind HGF, for example, human HGF, and another antigen of interest. Methods for generating bispecific antibodies are known in the art and include, for example, by hybridoma fusion or by binding of Fab ’fragments. See, for example, Songsivilai et al. (1990) CLIN. EXP. IMMUNOL 79: 315-325; Kostelny et al. (1992)

J. IMMUNOL. 148: 1547-1553. [0054] The binding proteins of the invention may bind to hHGF containing a substitution of cysteine to arginine at position 561 or a substitution of glycine to glutamate at position 555. [0055] In another aspect, the invention provides a protein of isolated junction that binds to human HGF with a kd of 4.0 x 10-5 s-1 or less, 3.0 x 10-5 s-1 or less or 2.0 x 10-5 s-1 or lower Isolated binding proteins can bind to human HGF with a kd of 5.0 x 10-5 s-1 to 0.5 x 10-5 s-1, or 4.0 x 10-5 s-1 to 1 , 0 x 10-5 s-1, or 3.0 x 10-5 s-1 to 1.5 x 10-5 s-1. In another aspect, the invention provides an isolated binding protein that binds to human HGF with a KD of 100 pM or less, or 20 pM or less, or 10 pM or less or 5 pM or less. Isolated binding proteins can bind to human HGF with a KD of 100 pM at 5 pM, or 20 pM at 5 pM, or 15 pM at 10 pM, or 20 pM at 10 pM or 15 pM at 5 pM . Unless otherwise specified, KD values are determined by the methods, and under the conditions, described in Example 6. [0056] In another aspect, the invention provides an isolated binding protein that binds to human HGF. , in which the antibody binds to human HGF with a KD lower than 37 ° C than at 25 ° C. The binding protein that binds optionally binds to human HGF with a KD of less than 5 pM at 37 ° C. [0057] In other aspects and embodiments, binding proteins can inhibit hHGF binding to c-Met. For example, binding proteins may have an IC50 (50% concentration of maximum inhibition) of at least about 4.0, 4.5, 5.0, 5.5, 6.0, 6.5 and 7 , 0 nM when tested using the protocol described in Example 7 (a). In certain other embodiments, binding proteins can neutralize the incorporation of HGF BrdU into 4 MBr-5 cells (ATCC, Catalog No. CCL208) using the method described in Example 7 (b). [0058] Binding proteins have an IC50 of 50 nM or less, preferably 45, 40, 35, 30, 25, 20, 15, 10, 5, 1, 0.5 nM or less, when tested using the protocol described in Example 7 (b). In certain other embodiments, binding proteins can be used to inhibit HGF-stimulated c-Met phosphorylation in PC-3 cells (ATCC, Manassus, VA Catalog No. CRL-1435) using the assay described in Example 9. Proteins Binding inhibit phosphorylation of c-Met stimulated by HGF (1.25 nM) in PC-3 cells with an IC50 of 2 nM or less (Table 8), using the assay described in Example 9.

image14

II - Production of binding proteins [0059] The binding proteins of the invention can be produced in various ways using strategies known in the art. For example, DNA molecules that encode light chain variable regions and heavy chain variable regions can be chemically synthesized using a commercial synthesizer and the sequence information provided herein. Said synthetic DNA molecules can be ligated with other appropriate nucleotide sequences including, for example, constant region coding sequences and expression control sequences, to produce conventional gene expression constructs that encode the desired binding proteins. The production of defined gene constructs is included within the usual specialty in the art. Alternatively, the sequences provided herein may be cloned from hybridomas by hybridization techniques or conventional PCR techniques, using synthetic nucleic acid probes whose sequences are based on the sequence information provided herein or on the information. of prior art sequence regarding genes encoding the light and heavy chains of murine antibodies in hybridoma cells. The production and use of said probes is included in the specialty in the art. [0060] Nucleic acids encoding the desired binding proteins can be introduced (ligated) into expression vectors that can be introduced into a host cell by means of conventional transfection or transformation techniques known in the art. Exemplary host cells include, for example, E. coli cells, Chinese hamster ovary (CHO) cells, HeLa cells, kidney hamster breeding cells (BHK), monkey kidney cells (COS), carcinoma cells Human hepatocellular (for example, Hep G2) and myeloma cells that do not otherwise produce immunoglobulin protein. Transfected host cells can be cultured under conditions that allow host cells to express the genes of interest, for example, the genes encoding the heavy or light chain variable regions of immunoglobulin. The resulting expression products can be collected using methods known in the art. [0061] The particular expression and purification conditions will vary depending on which expression system is used. For example, if the gene is to be expressed in E. coli, it is first cloned into an expression vector. This is achieved by placing the genetically engineered gene downstream of a suitable bacterial promoter, for example, Trp or Tac, and a signal sequence, for example a sequence encoding fragment B of protein A (FB). The resulting expressed fusion protein typically accumulates in inclusion or refractory bodies in the cytoplasm of the cells and can be collected after cell breakdown by French press or sonication. The refractory bodies are then solubilized and the expressed proteins are folded back and cleaved by the methods already established for many other recombinant proteins. [0062] If the genetically engineered gene is to be expressed in eukaryotic host cells, for example, myeloma cells or CHO cells, it is first inserted into an expression vector containing a suitable eukaryotic promoter, a secretion signal, enhancers of immunoglobulin and various introns. This expression vector may optionally contain sequences encoding all or part of a constant region, allowing all or part of a heavy or light chain to be expressed. The gene construct can be used to transfect myeloma cells or CHO cells using established transfection protocols. Said transfected cells can express VL or VH fragments, VL-VH heterodimers, VH-VL or VL-VH single chain polypeptides, complete light or heavy immunoglobulin chains, or portions thereof, each of which can be linked to a domain protein that has another function (for example, cytotoxicity).

image15

image16

III - Modifications in binding proteins [0063] It is understood that binding proteins can be modified to optimize performance depending on the desired use of binding proteins. For example, when the binding protein is being used as a therapeutic agent, the binding protein can be modified to reduce its immunogenicity in the desired recipient. Alternatively, or in addition, the binding protein can be fused or coupled to another protein or peptide, for example, a growth factor, cytokine or cytotoxin. Such modifications can be achieved using routine gene manipulation techniques known in the art. [0064] Various methods for reducing the antigenicity of antibodies and antibody fragments are known in the art. These techniques can be used to reduce or eliminate the antigenicity of the binding proteins of the invention. For example, when the binding proteins are to be administered to a human being, the binding proteins are preferably engineered to reduce their antigenicity in humans. This procedure is often called humanization. Preferably, the humanized binding proteins have the same or substantially the same affinity for the antigen as the original non-humanized binding protein from which they were obtained. [0065] In a well-known humanization strategy, chimeric proteins are created in which the immunoglobulin constant regions of antibodies of one species, for example, mouse, are replaced with immunoglobulin constant regions of a second different species, for example, a human being. In this example, the resulting antibody is a mouse-human chimera in which the sequences of the human constant region, in principle, are less immunogenic than homologous murine sequences. This type of genetic engineering modification of antibodies is described, for example, in Morrison, et al (1984) PROC. NAT. ACAD SCI. 81: 6851-6855, Neuberger et al. (1984) NATURE

312: 604-608; United States Patents No. 6,893,625 (Robinson); 5,500,362 (Robinson); and 4,816,567 (Cabilly). [0066] In another strategy, known as CDR grafting, the CDRs of the heavy and light chain variable regions of an antibody of interest are grafted into the framework regions (FR) of another species. For example, murine CDRs can be grafted into human FR sequences. In some embodiments, the CDRs of the heavy and light chain variable regions of an anti-HGF antibody are grafted into human FR or human consensus FR. To create human consensus FR, FR of several human light chain or heavy chain amino acid sequences are aligned to identify a consensus amino acid sequence. CDR grafting is described, for example, in US Pat. Nos.

image17

(2)

Aplicaciones de diagnóstico [0090] Cuando las proteínas de unión se usan para fines de diagnóstico, in vitro o in vivo, las proteínas de unión típicamente se marcan directa o indirectamente con un resto detectable. El resto detectable puede ser cualquier resto que sea capaz de producir, directa o indirectamente una señal detectable. Por ejemplo, el resto detectable puede ser un radioisótopo, tal como 3Hidrógeno (3H), 14Carbono (14C), 32Fósforo (32P), 35Azufre (35S) o 125Yodo (l25I); un compuesto fluorescente o quimioluminiscente, tal como isotiocianato de fluoresceína, rodamina o luciferina; una enzima, tal como fosfatasa alcalina, beta-galactosidasa o peroxidasa de rábano picante; una sonda de espín, tal como un marcador de espín; o una partícula coloreada, por ejemplo una partícula de látex u oro. Se entiende que la proteína de unión puede conjugarse con el resto detectable usando varias estrategias conocidas en la técnica, por ejemplo, como se describe en Hunter et al. (1962) NATURE 144: 945; David et al. (1974) BIOCHEMISTRY 13: 1014; Pain et al. (1981)

J.

IMMUNOL. METH. 40:219; y Nygren (1982) J. HISTOCHEM. AND CYTOCHEM.

30:407. Los marcadores pueden detectarse, por ejemplo, visualmente o con la ayuda de un espectrofotómetro u otro detector. [0091] Las proteínas de unión pueden emplearse en una amplia variedad de técnicas de inmunoensayo disponibles en la técnica. Los inmunoensayos ejemplares incluyen, por ejemplo, inmunoensayos tipo sándwich, inmunoensayos competitivos, procedimientos inmunohistoquímicos. [0092] En un inmunoensayo tipo sándwich, se usan dos anticuerpos que se unen a un analito o antígeno de interés, por ejemplo, uno inmovilizado sobre un soporte sólido y uno libre en solución y marcado con un resto detectable. Cuando una muestra que contiene el antígeno se introduce en este sistema, el antígeno se une tanto al anticuerpo inmovilizado como al anticuerpo marcado para formar un complejo inmune tipo “sándwich” en la superficie del soporte. La proteína que forma un complejo se detecta eliminando por lavado los componentes de muestra no unidos y el exceso de anticuerpo marcado, y midiendo la cantidad de anticuerpo marcado que forma un complejo con proteína en la superficie del soporte. Como alternativa, el anticuerpo libre en solución puede detectarse por un tercer anticuerpo marcado con un resto detectable que se une al anticuerpo libre. Puede encontrarse una revisión detallada del diseño, la teoría y los protocolos del ensayo inmunológico en numerosos textos, incluyendo Butt, ed., (1984) PRACTICAL IMMUNOLOGY, Marcel Dekker, Nueva York; Harlow et al eds. (1988) ANTIBODIES, A LABORATORY APPROACH, Cold Spring Harbor Laboratory; y Diamandis et al., eds. (1996) IMMUNOASSAY, Academic Press, Boston. [0093] Se contempla que las proteínas de unión marcadas sean útiles como agentes de formación de imágenes in vivo, por lo que las proteínas de unión pueden dirigir a los agentes de formación de imágenes a tejidos particulares de interés en el destinatario. Un resto detectable remotamente preferido para formación de imágenes in vivo incluye el átomo radiactivo Tecnecio-99m (99mTc), un emisor gamma con una semivida de aproximadamente seis horas. Los restos no radiactivos que también son útiles en la formación de imágenes in vivo incluyen marcadores de espín de nitróxido así como iones de metales de transición y lantánidos, induciendo todos ellos la relajación de protones in situ. Además de la formación de inmunoimágenes, los restos radiactivos que forman complejos pueden usarse en protocolos de radioinmunoterapia convencionales para destruir las células diana. Los nucleótidos preferidos para radioinmunoterapia de alta dosis incluyen los átomos radiactivos 90Itrio (90Yt), 131Yodo (131I) e 111Indio (111ln). La proteína de unión puede marcarse con 1311, 111In y 99mTC usando procedimientos de acoplamiento conocidos en la técnica de formación de imágenes. De forma similar, son bien conocidos en la técnica de formación de imágenes procedimientos para preparar y administrar el agente de formación de imágenes, así como capturar y procesar imágenes, y por tanto no se analizan en detalle en la presente memoria. De forma similar, se conocen bien en la técnica métodos para realizar inmunoterapias basadas en anticuerpos. Véase, por ejemplo, la Patente de Estados Unidos Nº 5.534.254. [0094] Durante toda la descripción, en la que se describen composiciones que tienen, que incluyen o que comprenden componentes específicos, se contempla que las composiciones también consistan esencialmente en, o consistan en los componentes enumerados. De forma similar, cuando se describe que los procedimientos tienen, o incluyen o comprenden etapas de procedimiento específicas, los procedimientos también consisten esencialmente en, o consisten en las etapas de procesamiento enumeradas. Excepto cuando se indique otra cosa, el orden de las etapas o el orden para realizar determinadas acciones es irrelevante siempre que la invención continúe siendo realizable. Además, a menos que se indique otra cosa, dos o más etapas o acciones pueden realizarse simultáneamente. imagen24 imagen25
EJEMPLOS [0095] Los Ejemplos siguientes analizan la producción y caracterización de varios anticuerpos monoclonales anti-hHGF. Se incluyen como referencia secuencias que no están dentro del alcance de las reivindicaciones.
Ejemplo 1 - Producción de anticuerpos monoclonales anti-hHGF [0096] Este Ejemplo describe la producción de varios anticuerpos monoclonales anti-hHGF. [0097] Se realizaron inmunizaciones, fusiones y exploraciones primarias en MBS Inc. (Portland, ME), siguiendo el protocolo de Sitios Múltiples de Inmunización Repetitiva (RIMMS). Se inmunizaron cinco ratones AJ y cinco ratones Balb/c con HGF humano recombinante (R&D Systems, Minneapolis, MN; Nº Catálogo. 294HGN-025). Se escogieron dos ratones con sueros que presentaban la mayor actividad anti-HGF por Ensayo Inmunoabsorbente Ligado a Enzimas (ELISA) para una fusión posterior. Se recogieron los bazos y ganglios linfáticos de los ratones apropiados. Después se recogieron las células B y se fusionaron con una línea de mieloma. Los productos de fusión se diluyeron en serie en una o más placas hasta casi la clonalidad. Los sobrenadantes de las fusiones resultantes se exploraron para determinar su unión a hHGF mediante ELISA. Los sobrenadantes que se identificó que contenían anticuerpos contra HGF se caracterizaron adicionalmente por ensayo funcional in vitro como se analiza en los ejemplos siguientes. Se seleccionó un panel de hibridomas y los hibridomas se subclonaron y se expandieron. Los anticuerpos monoclonales se purificaron después por cromatografía de afinidad en resina de Proteína A/G en condiciones convencionales.
Ejemplo 2 - Análisis de secuencia de anticuerpos monoclonales anti-hHGF [0098] Este Ejemplo describe el análisis de secuencia y de isotipo de los imagen26
anticuerpos monoclonales anti-hHGF producidos en el Ejemplo 1.
a. Determinación de isotipos de anticuerpos monoclonales murinos de HGF [0099] El isotipo de cadena pesada y tipo de cadena ligera de cada anticuerpo monoclonal se determinó usando el kit de isotipado de anticuerpos monoclonales de ratón IsoStrip de acuerdo con las instrucciones del fabricante (Roche Applied Science). [0100] Se determinó que todos los anticuerpos contenían una cadena ligera de inmunoglobulina Kappa y una cadena pesada de inmunoglobulina IgG1.
b. Determinación de secuencias de nucleótidos que codifican las regiones
variables de cadena ligera y pesada de inmunoglobulina [0101] Se extrajo el ARN total de cada línea celular de hibridoma monoclonal usando el kit de minipreparaciones RNeasy de acuerdo con las instrucciones del fabricante (Qiagen Venlo, Países Bajos). Se generó ADNc de primera cadena de longitud completa usando el kit de amplificación de ADNc por RACE BD SMART� de acuerdo con las instrucciones del fabricante (Clontech) usando los cebadores oligonucleotídicos BD SMART II A (5’ aagcagtggtatcaacgcagagtacgcggg 3’) (SEC ID Nº 85) y el cebador CDS de RACE-5' (5’ tttttttttttttttttttttttttvn 3’, donde v = a, g ó c y n = a, g, c ó t) (SEC ID Nº 86) con el fin de RACE 5’ (Amplificación Rápida de Extremos de ADNc). [0102] Las regiones variables de las cadenas de inmunoglobulina Kappa y pesada (IgG1) se amplificaron por PCR (Reacción en Cadena de la Polimerasa) usando el sistema de PCR de Alta Fidelidad Expand (Roche Applied Science) de acuerdo con las instrucciones del fabricante. Se amplificaron las regiones variables de la cadena pesada con la mezcla de cebadores oligonucleotídicos 5’ Mezcla de Cebadores Universales A (mezcla de 5’ ctaatacgactcactatagggcaagcagtggtatcaacgcagagt 3’ (SEC ID Nº 87) y 5’ ctaatacgactcactatagggc 3’ (SEC ID Nº 88)) y un cebador específico de la Región Constante de IgG1 3’, 5’ tatgcaaggcttacaaccaca 3’ (SEC ID Nº 89) o 5’ gccagtggatagacagatgggggtgtcg 3’ (SEC ID Nº 90). Se amplificaron las regiones variables de la cadena kappa con la mezcla de cebadores oligonucleotídicos 5’ Mezcla de Cebadores Universales A y un cebador específico de la Región Constante Kappa 3’, 5’ ctcattcctgttgaagctcttgacaat 3’ (SEC ID Nº 91) o 5’ cgactgaggcacctccagatgtt 3’ (SEC ID Nº 92). [0103] Los productos de PCR individuales se fraccionaron mediante electroforesis en gel de agarosa y se purificaron usando el kit de Purificación en Gel Qiaquick de acuerdo con las instrucciones del fabricante (Qiagen). Los productos de PCR se clonaron posteriormente en el plásmido pCR2.1 TOPO usando el kit de clonación basado en topoisomerasa TOPO TA Cloning�Kit (con vector pCR�2.1-TOPO�) de acuerdo con las instrucciones del fabricante (Invitrogen, Carlsbad, CA) y se usaron para transformar bacterias DH5 usando técnicas de transformación convencionales. El ADN plasmídico aislado de clones bacterianos transformados se secuenció usando los cebadores T7 (5’ TAATACGACTCACTATAGGG 3’) (SEC ID Nº 93), M13 Directo (5’ GTAAAACGACGGCCAGT 3’) (SEC ID Nº 94) y M13 Inverso (5’ CAGGAAACAGCTATGACC 3’) (SEC ID Nº 95) por Agencourt Bioscience usando métodos de secuenciación de ADN didesoxi convencionales para identificar la secuencia de las secuencias de la región variable. Las secuencias se analizaron usando el software Vector NTI (Invitrogen, Carlsbad, CA) y el servidor web IMGT/V-Quest (http://imgt.cines.fr/textes/vquest) para identificar y confirmar secuencias de la región variable. imagen27
c. Determinación de secuencias de nucleótidos que codifican secuencias de la región constante de cadena ligera y pesada de inmunoglobulina para las cadenas de IgG1 y Kappa de 1A3, 1D3, 1F3 y 2B8
[0104] Se amplificaron por PCR ADNc de longitud completa para las cadenas de IgG1 de 1A3, 1D3 y 1F3 a partir del ADNc creado anteriormente usando el cebador directo 5’ ggggacaagtttgtacaaaaaagcaggctgccaccatgaactttgggctcagattgattttcc 3’ (subrayado el codón de inicio) (SEC ID Nº 96) y el cebador inverso 5’ ggggaccactttgtacaagaaagctgggttcatttaccaggagagtgggagagg 3’ (subrayado el codón de terminación) (SEC ID Nº 97). Se amplificó el ADNc de longitud completa para la cadena de IgG1 de 2B8 a partir del ADNc creado anteriormente usando el cebador directo 5’ ggggacaagtttgtacaaaaaagcaggctgccaccatgggatggagctatatcatcctcttt 3’ (subrayado el codón de inicio) (SEC ID Nº 98) y el cebador inverso 5’ ggggaccactttgtacaagaaagctgggttcatttaccaggagagtgggagag 3’ (subrayado el codón de terminación) (SEC ID Nº 99). [0105] El ADNc de longitud completa para la cadena kappa de 2B8 se amplificó usando el cebador directo 5’ ggggacaagtttgtacaaaaaagcaggctgccaccatggaatcacagactctggtcttcata 3’ (subrayado el codón de inicio) (SEC ID Nº 100) y el cebador inverso 5’ ggggaccactttgtacaagaaagctgggtctaacactcattcctgttgaagctc 3’ (subrayado el codón de terminación) (SEC ID Nº 101). Los fragmentos de PCR se subclonaron en pDONR221 (Invitrogen, Carlsbad, CA) por reacción de recombinación de Gateway BP (Invitrogen, Carlsbad, CA) y se secuenciaron por Agencourt Bioscience usando métodos de secuenciación de ADN didesoxi convencionales para identificar la secuencia de la región constante y confirmar adicionalmente secuencias de la región variable. imagen28
d. Análisis de secuencia [0106] Se identificaron las Regiones Variables (texto normal) usando el software del servidor web IMGT/V-QUEST (http://imgt.cines.fr/textes/vquest/). Se predijeron las secuencias de péptido señal basándose en la identificación del codón de inicio en fase de lectura (ATG) que estaba cadena arriba de la Región Variable identificada. Se identificaron las secuencias de Péptido Señal y están subrayadas a continuación. [0107] El último nucleótido de cada región variable es la primera base del siguiente codón generado por la unión de región variable/constante. Este nucleótido se incluye en la región variable porque es parte de ese exón. Las secuencias de aminoácidos de las regiones constantes enumeradas a continuación incluyen la traducción de este codón de unión. [0108] Para crear las secuencias de anticuerpo de cadena kappa o pesada completas, las secuencias de la región variable indicadas a continuación se combinan con sus secuencias de región constante respectivas (las secuencias señal están subrayadas).
[0109] (1) Región variable de la cadena pesada de 1A3 (SEC ID Nº 1) imagen29
[0110] (2) Región variable de la cadena ligera kappa de 1A3 (SEC ID Nº 3) imagen30 imagen29
[0111] (3) Región variable de la cadena pesada de 2B8 (SEC ID Nº 11) imagen31
[0112] (4) Región variable de la cadena ligera kappa de 2B8 (SEC ID Nº 13) imagen29 imagen32
[0113] (5) Región variable de la cadena pesada de 2F8 (SEC ID Nº 21) imagen33
[0115] (7) Región variable de la cadena pesada de 3B6 (SEC ID Nº 31) imagen29 imagen34
[0116] (8) Región variable de la cadena ligera kappa de 3B6 (2 codones de inicio ATG posibles (en mayúsculas)) (SEC ID Nº 33) imagen29
[0117] (9) Región variable de la cadena pesada de 3D11 (SEC ID Nº 41) imagen29
[0118] (10) Región variable de la cadena ligera kappa de 3D11 (SEC ID Nº 43) imagen29 imagen35
[0119] (11) Región variable de la cadena pesada de 1D3 (SEC ID Nº 51) imagen36
[0121] (13) Región variable de la cadena pesada de 1F3 (SEC ID Nº 61) imagen29
10 [0122] (14) Región variable de la cadena ligera kappa de 1F3 (SEC ID Nº 63) imagen29 imagen37 imagen29
[0123] (15) Región variable de la cadena pesada de 3A12 (SEC ID Nº 71) imagen29
[0124] (16) Región variable de la cadena ligera kappa de 3A12 (SEC ID Nº 73) imagen29
[0125] (17) Región constante de la cadena pesada de IgG1 de ratón de referencia 10 (J00453) (SEC ID Nº 81) imagen29 imagen38 imagen29
[0126] (18) Región constante de la cadena pesada de IgG1 de ratón determinada para 1A3, 1D3, 1F3 y 2B8 (obtenida de ratones de cepa AJ) (SEC ID Nº 82) imagen31 imagen39
[0127] (19) Región constante de la cadena ligera kappa de ratón de referencia (V00807) y región constante de la cadena ligera kappa de ratón determinada para 1D3, 1F3 y 2B8 (obtenida de ratones de cepa AJ) (SEC ID Nº 83) imagen29
[0128] (20) Región constante de la cadena ligera kappa de ratón determinada para 1A3 que contiene un nucleótido alterado en comparación con 1D3, 1F3 y 2B8 (subrayado) (SEC ID Nº 84) imagen29
10 [0129] Cada una de las secuencias de aminoácidos que definen las regiones variables de cadena pesada de inmunoglobulina para los anticuerpos producidos en el Ejemplo 1 se exponen en la Figura 2. Cada una de las secuencias se alinean entre sí y las secuencias que definen el péptido señal, la CDR1, la CDR2 y la CDR3
15 se identifican mediante recuadros. La Figura 3 muestra un alineamiento de las secuencias de CDR1, CDR2 y CDR3 separadas para cada uno de los anticuerpos. [0130] Cada una de las secuencias de aminoácidos que definen las regiones variables de cadena ligera de inmunoglobulina para cada uno de los anticuerpos producidos en el Ejemplo 1 se exponen en la Figura 4. Cada una de las secuencias
20 se alinean entre sí y las secuencias que definen el péptido señal, la CDR1, la CDR2 y la CDR3 se identifican mediante recuadros. La Figura 5 muestra un alineamiento de las secuencias de CDR1, CDR2 y CDR3 separadas para cada uno de los anticuerpos. [0131] Por comodidad, la Tabla 1 proporciona una gráfica de concordancia que muestra la correspondencia entre las secuencias de anticuerpos analizadas en este Ejemplo con las presentadas en la Lista de Secuencias. imagen40

SEC ID Nº

Proteína o Ácido Nucleico

1

Región variable de la cadena pesada de 1A3 - ácido nucleico

2

Región variable de la cadena pesada de 1A3 - proteína

3

Región variable de la cadena ligera (kappa) de 1A3 - ácido nucleico

4

Región variable de la cadena ligera (kappa) de 1A3 - proteína

5

CDR1 de la cadena pesada de 1A3

6

CDR2 de la cadena pesada de 1A3

7

CDR3 de la cadena pesada de 1A3

8

CDR1 de la cadena ligera (kappa) de 1A3

9

CDR2 de la cadena ligera (kappa) de 1A3

10

CDR3 de la cadena ligera (kappa) de 1A3

11

Región variable de la cadena pesada de 2B8 - ácido nucleico

12

Región variable de la cadena pesada de 2B8 - proteína

13

Región variable de la cadena ligera (kappa) de 2B8 - ácido nucleico

14

Región variable de la cadena ligera (kappa) de 2B8 - proteína

15

CDR1 de la cadena pesada de 2B8

16

CDR2 de la cadena pesada de 2B8

17

CDR3 de la cadena pesada de 2B8

18

CDR1 de la cadena ligera (kappa) de 2B8

19

CDR2 de la cadena ligera (kappa) de 2B8

20

CDR3 de la cadena ligera (kappa) de 2B8

21

Región variable de la cadena pesada de 2F8 - ácido nucleico

22

Región variable de la cadena pesada de 2F8 - proteína

23

Región variable de la cadena ligera (kappa) de 2F8 - ácido nucleico

24

Región variable de la cadena ligera (kappa) de 2F8 - proteína

25

CDR1 de la cadena pesada de 2F8

26

CDR2 de la cadena pesada de 2F8

27

CDR3 de la cadena pesada de 2F8

28

CDR1 de la cadena ligera (kappa) de 2F8

29

CDR2 de la cadena ligera (kappa) de 2F8

30

CDR3 de la cadena ligera (kappa) de 2F8

imagen41

SEC ID Nº

Proteína o Ácido Nucleico

31

Región variable de la cadena pesada de 3B6 - ácido nucleico

32

Región variable de la cadena pesada de 3B6 - proteína

33

Región variable de la cadena ligera (kappa) de 3B6 - ácido nucleico

34

Región variable de la cadena ligera (kappa) de 3B6 - proteína

35

CDR1 de la cadena pesada de 3B6

36

CDR2 de la cadena pesada de 3B6

37

CDR3 de la cadena pesada de 3B6

38

CDR1 de la cadena ligera (kappa) de 3B6

39

CDR2 de la cadena ligera (kappa) de 3B6

40

CDR3 de la cadena ligera (kappa) de 3B6

41

Región variable de la cadena pesada de 3D11 - ácido nucleico

42

Región variable de la cadena pesada de 3D11 - proteína

43

Región variable de la cadena ligera (kappa) de 3D11 - ácido nucleico

44

Región variable de la cadena ligera (kappa) de 3D11 - proteína

45

CDR1 de la cadena pesada de 3D11

46

CDR2 de la cadena pesada de 3D11

47

CDR3 de la cadena pesada de 3D11

48

CDR1 de la cadena ligera (kappa) de 3D11

49

CDR2 de la cadena ligera (kappa) de 3D11

50

CDR3 de la cadena ligera (kappa) de 3D11

51

Región variable de la cadena pesada de 1D3 - ácido nucleico

52

Región variable de la cadena pesada de 1D3 - proteína

53

Región variable de la cadena ligera (kappa) de 1D3 - ácido nucleico

54

Región variable de la cadena ligera (kappa) de 1D3 - proteína

55

CDR1 de la cadena pesada de 1D3

56

CDR2 de la cadena pesada de 1D3

57

CDR3 de la cadena pesada de 1D3

58

CDR1 de la cadena ligera (kappa) de 1D3

59

CDR2 de la cadena ligera (kappa) de 1D3

60

CDR3 de la cadena ligera (kappa) de 1D3

61

Región variable de la cadena pesada de 1F3 - ácido nucleico

62

Región variable de la cadena pesada de 1F3 - proteína

63

Región variable de la cadena ligera (kappa) de 1F3 - ácido nucleico

imagen42

SEC ID Nº

Proteína o Ácido Nucleico

64

Región variable de la cadena ligera (kappa) de 1F3 - proteína

65

CDR1 de la cadena pesada de 1F3

66

CDR2 de la cadena pesada de 1F3

67

CDR3 de la cadena pesada de 1F3

68

CDR1 de la cadena ligera (kappa) de 1F3

69

CDR2 de la cadena ligera (kappa) de 1F3

70

CDR3 de la cadena ligera (kappa) de 1F3

71

Región variable de la cadena pesada de 3A12 - ácido nucleico

72

Región variable de la cadena pesada de 3A12 - proteína

73

Región variable de la cadena ligera (kappa) de 3A12 - ácido nucleico

74

Región variable de la cadena ligera (kappa) de 3A12 - proteína

75

CDR1 de la cadena pesada de 3A12

76

CDR2 de la cadena pesada de 3A12

77

CDR3 de la cadena pesada de 3A12

78

CDR1 de la cadena ligera (kappa) de 3A12

79

CDR2 de la cadena ligera (kappa) de 3A12

80

CDR3 de la cadena ligera (kappa) de 3A12

[0132] También por comodidad, las secuencias siguientes representan las secuencias de cadena ligera y de cadena pesada de longitud completa contempladas o reales (es decir, que contienen secuencias de región tanto
5 constante como variable) para cada uno de los anticuerpos descritos en este Ejemplo. Se señala que las regiones constantes de los anticuerpos murinos 2F8, 3A12, 3B6 y 3D11 no se secuenciaron pero se supone que tienen las mismas secuencias de región constante que los anticuerpos 1D3, 1F3 y 2B8 que se secuenciaron, ya que todas proceden de ratones de cepa AJ. Se aprecia sin
10 embargo que las secuencias de región variable descritas en la presente memoria pueden ligarse con cada una de varias otras secuencias de región constante conocidas por los expertos en la materia para producir cadenas ligeras y pesadas de inmunoglobulina de longitud completa activas. imagen43
[0133] (1) Secuencia de ácido nucleico que codifica la secuencia de la cadena pesada de 1A3 de longitud completa (región variable de la cadena pesada de 1A3 y región constante de IgG1) (secuencia señal subrayada) (SEC ID Nº 122) imagen29
[0134] (2) Secuencia proteica que define la secuencia de la cadena pesada de 1A3 de longitud completa (región variable de la cadena pesada de 1A3 y región constante de IgG1) (sin secuencia señal) (SEC ID Nº 123) imagen29
[0135] (3) Secuencia de ácido nucleico que codifica la secuencia de la cadena ligera de 1A3 de longitud completa (región constante y región variable kappa de 1A3) (secuencia señal subrayada) (SEC ID Nº 124) [0136] (4) Secuencia proteica que define la secuencia de la cadena ligera de 1A3 de longitud completa (región constante y región variable kappa de 1A3) (sin secuencia señal) (SEC ID Nº 125) imagen29 imagen44 imagen29 imagen29
[0137] (5) Secuencia de ácido nucleico que codifica la secuencia de la cadena pesada de 2B8 de longitud completa (región variable de la cadena pesada de 2B8 y 10 región constante de IgG1) (secuencia señal subrayada) (SEC ID Nº 126) imagen29
[0138] (6) Secuencia proteica que define la secuencia de la cadena pesada de 2B8 de longitud completa (región variable de la cadena pesada de 2B8 y región
15 constante de IgG1) (sin secuencia señal) (SEC ID Nº 127) [0139] (7) Secuencia de ácido nucleico que codifica la secuencia de la cadena ligera de 2B8 de longitud completa (región constante y región variable kappa de 2B8) (secuencia señal subrayada) (SEC ID Nº 128) imagen29 imagen45 imagen29 imagen29
[0140] (8) Secuencia proteica que define la secuencia de la cadena ligera de 2B8 de longitud completa (región constante y región variable kappa de 2B8) (sin
10 secuencia señal) (SEC ID Nº 129) imagen29
[0141] (9) Secuencia de ácido nucleico que codifica la secuencia de la cadena pesada de 2F8 de longitud completa (región variable de la cadena pesada de 2F8 y 15 región constante de IgG1) (secuencia señal subrayada) (SEC ID Nº 130) imagen29 imagen46 imagen29
[0142] (10) Secuencia proteica que define la secuencia de la cadena pesada de 2F8 de longitud completa (región variable de la cadena pesada de 2F8 y región constante de IgG1) (sin secuencia señal) (SEC ID Nº 131) imagen29
[0143] (11) Secuencia de ácido nucleico que codifica la secuencia de la cadena ligera de 2F8 de longitud completa (región constante y región variable kappa de 10 2F8) (secuencia señal subrayada) (SEC ID Nº 132) imagen29
[0144] (12) Secuencia proteica que define la secuencia de la cadena ligera de 2F8 de longitud completa (región constante y región variable kappa de 2F8) (sin
15 secuencia señal) (SEC ID Nº 133) imagen29
[0145] (13) Secuencia de ácido nucleico que codifica la secuencia de la cadena pesada de 3B6 de longitud completa (región variable de la cadena pesada de 3B6 y 20 región constante de IgG1) (secuencia señal subrayada) (SEC ID Nº 134) imagen29 imagen29
[0146] (14) Secuencia proteica que define la secuencia de la cadena pesada de 3B6 de longitud completa (región variable de la cadena pesada de 3B6 y región constante de IgG1) (sin secuencia señal) (SEC ID Nº 135) imagen29
[0147] (15) Secuencia de ácido nucleico que codifica la secuencia de la cadena ligera de 3B6 de longitud completa (región constante y región variable kappa de 10 3B6) (secuencia señal subrayada) (SEC ID Nº 136) imagen29 imagen47
[0148] (16) Secuencia proteica que define la secuencia de la cadena ligera de 3B6 de longitud completa (región constante y región variable kappa de 3B6) (sin secuencia señal) (SEC ID Nº 137) imagen29
[0149] (17) Secuencia de ácido nucleico que codifica la secuencia de la cadena pesada de 3D11 de longitud completa (región variable de la cadena pesada de 3D11 y región constante de IgG1) (secuencia señal subrayada) (SEC ID Nº 138) imagen29
[0150] (18) Secuencia proteica que define la secuencia de la cadena pesada de 3D11 de longitud completa (región variable de la cadena pesada de 3D11 y región constante de IgG1) (sin secuencia señal) (SEC ID Nº 139) [0151] (19) Secuencia de ácido nucleico que codifica la secuencia de la cadena ligera de 3D11 de longitud completa (región constante y región variable kappa de 3D11) (secuencia señal subrayada) (SEC ID Nº 140) imagen29 imagen48 imagen29
[0152] (20) Secuencia proteica que define la secuencia de la cadena ligera de 3D11 de longitud completa (región constante y región variable kappa de 3D11) (sin secuencia señal) (SEC ID Nº 141) imagen29
[0153] (21) Secuencia de ácido nucleico que codifica la secuencia de la cadena pesada de 1D3 de longitud completa (región variable de la cadena pesada de 1D3 y región constante de IgG1) (secuencia señal subrayada) (SEC ID Nº 142) [0154] (22) Secuencia proteica que define la secuencia de la cadena pesada de 1D3 de longitud completa (región variable de la cadena pesada de 1D3 y región constante de IgG1) (sin secuencia señal) (SEC ID Nº 143) imagen29 imagen49 imagen29
[0155] (23) Secuencia de ácido nucleico que codifica la secuencia de la cadena ligera de 1D3 de longitud completa (región constante y región variable kappa de 1D3) (secuencia señal subrayada) (SEC ID Nº 144) imagen29
[0156] (24) Secuencia proteica que define la secuencia de la cadena ligera de 1D3 de longitud completa (región constante y región variable kappa de 1D3) (sin secuencia señal) (SEC ID Nº 145) imagen29
[0157] (25) Secuencia de ácido nucleico que codifica la secuencia de la cadena pesada de 1F3 de longitud completa (región variable de la cadena pesada de 1F3 y región constante de IgG1) (secuencia señal subrayada) (SEC ID Nº 146) [0158] (26) Secuencia proteica que define la secuencia de la cadena pesada de 1F3 de longitud completa (región variable de la cadena pesada de 1F3 y región constante de IgG1) (sin secuencia señal) (SEC ID Nº 147) imagen29 imagen50 imagen29 imagen29
[0159] (27) Secuencia de ácido nucleico que codifica la secuencia de la cadena ligera de 1F3 de longitud completa (región constante y región variable kappa de 10 1F3) (secuencia señal subrayada) (SEC ID Nº 148) imagen29
[0160] (28) Secuencia proteica que define la secuencia de la cadena ligera de 1F3 de longitud completa (región constante y región variable kappa de 1F3) (sin
15 secuencia señal) (SEC ID Nº 149) imagen29
[0161] (29) Secuencia de ácido nucleico que codifica la secuencia de la cadena pesada de 3A12 de longitud completa (región variable de la cadena pesada de 3A12 y región constante de IgG1) (secuencia señal subrayada) (SEC ID Nº 150) imagen29
[0162] (30) Secuencia proteica que define la secuencia de la cadena pesada de 3A12 de longitud completa (región variable de la cadena pesada de 3A12 y región constante de IgG1) (sin secuencia señal) (SEC ID Nº 151) imagen29
[0163] (31) Secuencia de ácido nucleico que codifica la secuencia de la cadena ligera de 3A12 de longitud completa (región constante y región variable kappa de 3A12) (secuencia señal subrayada) (SEC ID Nº 152) [0164] (32) Secuencia proteica que define la secuencia de la cadena ligera de 3A12 de longitud completa (región constante y región variable kappa de 3A12) (sin secuencia señal) (SEC ID Nº 153) imagen51 imagen29
5 [0165] Por comodidad, la Tabla 2 proporciona una gráfica de concordancia que muestra la correspondencia entre las secuencias de longitud completa de los anticuerpos analizados en este Ejemplo con las presentadas en la Lista de Secuencias.
10 TABLA 2

SEC ID Nº

Proteína o Ácido Nucleico

122

Variable pesada de 1A3 + constante de IgG1 - ácido nucleico

123

Variable pesada de 1A3 + constante de IgG1 - proteína

124

Variable ligera de 1A3 + constante - ácido nucleico

125

Variable ligera de 1A3 + constante - proteína

126

Variable pesada de 2B8 + constante de IgG1 - ácido nucleico

127

Variable pesada de 2B8 + constante de IgG1 - proteína

128

Variable ligera de 2B8 + constante - ácido nucleico

129

Variable ligera de 2B8 + constante - proteína

130

Variable pesada de 2F8 + constante de IgG1 - ácido nucleico

131

Variable pesada de 2F8 + constante de IgG1 - proteína

132

Variable ligera de 2F8 + constante - ácido nucleico

133

Variable ligera de 2F8 + constante - proteína

134

Variable pesada de 3B6 + constante de IgG1 - ácido nucleico

135

Variable pesada de 3B6 + constante de IgG1 - proteína

136

Variable ligera de 3B6 + constante - ácido nucleico

137

Variable ligera de 3B6 + constante - proteína

138

Variable pesada de 3D11 + constante de IgG1 - ácido nucleico

139

Variable pesada de 3D11 + constante de IgG1 - proteína

140

Variable ligera de 3D11 + constante - ácido nucleico

141

Variable ligera de 3D11 + constante - proteína

142

Variable pesada de 1D3 + constante de IgG1 - ácido nucleico

143

Variable pesada de 1D3 + constante de IgG1 - proteína

imagen52

144

Variable ligera de 1D3 + constante - ácido nucleico

145

Variable ligera de 1D3 + constante - proteína

146

Variable pesada de 1F3 + constante de IgG1 - ácido nucleico

147

Variable pesada de 1F3 + constante de IgG1 - proteína

148

Variable ligera de 1F3 + constante - ácido nucleico

149

Variable ligera de 1F3 + constante - proteína

150

Variable pesada de 3A12 + constante de IgG1 - ácido nucleico

151

Variable pesada de 3A12 + constante de IgG1 - proteína

152

Variable ligera de 3A12 + constante - ácido nucleico

153

Variable ligera de 3A12 + constante - proteína

Ejemplo 3 - Producción de diversas proteínas hHGF recombinantes [0166] Este Ejemplo describe la clonación y expresión de varias proteínas recombinantes usadas para caracterizar los anticuerpos creados en el Ejemplo 1 y
5 en el Ejemplo 14. En particular, este ejemplo describe la clonación y expresión de proteína hHGF recombinante, una proteína hHGF recombinante que contiene una sustitución de glicina a glutamato en la posición 555 (G555E), una proteína hHGF recombinante que contiene una sustitución de cisteína a arginina en la posición 561 (C561R), una proteína HGF quimérica de ratón-humano-ratón (mhm) recombinante
10 que contiene la secuencia de HGF V495-L585 humana dispuesta dentro de la secuencia de HGF de ratón, una proteína de HGF quimérica mhm recombinante que contiene la secuencia de HGF I499-R566 humana dispuesta dentro de la secuencia de HGF de ratón, y una proteína HGF quimérica mhm recombinante que contiene la secuencia de HGF W507-L585 humana dispuesta dentro de la
15 secuencia de HGF de ratón. [0167] Las construcciones de expresión siguientes se generaron usando técnicas moleculares convencionales, y las secuencias de ADNc resultantes se confirmaron mediante secuenciación de ADN.
20 a. hHGF-Fc [0168] En una primera ronda de PCR, se generaron dos fragmentos de PCR solapantes introduciendo un sitio Not I y que codificaban un marcador 6xHis entre hHGF y hlgFc. Los fragmentos de PCR solapantes sirvieron como molde en una segunda ronda para amplificar el hHGF-his-IgFc. El fragmento resultante se digirió
25 mediante Nhel y BamHI y se clonó en pcDNA5/FRT (Invitrogen, Nº 35-3014). imagen53
Después, el hHGF se amplificó a partir del clon ID de Invitrogen: IOH29794 (ADNc de HGF humano). Se descubrió que la secuencia correspondía a la secuencia depositada en el NCBI bajo el número de acceso NM_000601.4.

(1)

Cebador 5’ hHGF NheI [0169] ACTGGCTAGCATGTGGGTGACCAAACTCCT (SEC ID Nº 102)

(2)

Cebador 3’ hHGF Notl marcador His [0170] GTGATGGTGATGGTGATGGCGGCCGCATGACTGTGGTACCTTATATG (SEC ID Nº 103)

(3)

Cebador 5’ HisIgFc [0171] ACTGGCGGCCGCCATCACCATCACCATCAC (SEC ID Nº 104)

(4)

Cebador 3’ IgFc BamHI [0172] ACTGGGATCCTCACTATTTACCCGGGGACAG (SEC ID Nº 105)

b.

hHGF-Fc G555E y hHGF-Fc C561R [0173] Se generaron los mutantes de hHGF-Fc G555E y C561R por mutagénesis dirigida usando el kit de mutagénesis dirigida QuikChange II XL (Stratagene) de acuerdo con las instrucciones del fabricante.

(1)

Cebador con sentido hHGF-Fc (G555E) [0174] CATGATGTCCACGAAAGAGGAGATGAG (SEC ID Nº 106)

(2)

Cebador antisentido hHGF-Fc (G555E) [0175] CTCATCTCCTCTTTCGTGGACATCATG (SEC ID Nº 107)

(3)

Cebador con sentido hHGF-Fc (C561R) [0176] GGAAGAGGAGATGAGAAACGCAAACAGGTTCTCAATG (SEC ID Nº 108)

(4)

Cebador antisentido hHGF-Fc (C561R) [0177] CATTGAGAACCTGTTTGCGTTTCTCATCTCCTCTTCC (SEC ID Nº 109)

c.

Fc quimera de ratón-humano-ratón [0178] La construcción de IgFc quimera de ratón-humano- ratón contiene la cadena alfa de mHGF-hHGF, los aminoácidos de la cadena � VaI 495-Leu 585 del HGF humano y la cadena beta C-terminal de mHGF, seguida de marcador 6xHis y de IgG-Fc. [0179] El ADNc de HGF humano que codifica los aminoácidos V495-L585 se amplificó a partir del clon ID de Invitrogen: IOH29794 (ADNc de HGF humano). La secuencia corresponde a la secuencia depositada en el NCBI bajo el número de acceso NM_000601.4. Se amplificaron las secuencias de HGF de ratón mediante RT-PCR a partir de ARN total de hígado de ratón (Clontech, Nº 636603) usando el kit de RT-PCR de una etapa Super Script de Invitrogen (Nº 10928-034) de acuerdo con las instrucciones del fabricante. La secuencia de ADNc de mHGF corresponde a la secuencia depositada en el NCBI bajo el número de acceso D10213.1. [0180] Se generaron tres fragmentos, denominados Fragmentos 1, 2 y 3, usando cebadores de PCR solapantes, y se hibridaron en rondas consecutivas de amplificación por PCR. El producto final se escindió con Nhel y Notl y se clonó en pcDNA5/FRT IgGFc.

(1)

Cebadores del Fragmento 1 para la cadena alfa de mHGF 5’Nhel [0181] 5’ ATCGGCTAGCATGATGTGGGGGACCAAAC (SEC ID Nº 110) [0182] 3’ GAATCCCATTTACAACCCGCAGTTGTTTTGTTTTGG (SEC ID Nº 111)

(2)

Cebadores del Fragmento 2 para los aminoácidos de la cadena beta de hHGF V495-L585 [0183] 5’ CCAAAACAAAACAACTGCGGGTTGTAAATGGGATTC (SEC ID Nº 112) [0184] 3’ CAGGATTGCAGGTCGAGCAAGCTTCATTAAAACCAGATCT (SEC ID Nº 113)

(3)

Cebador del Fragmento 3 para el extremo C-terminal de la cadena beta de mHGF 3’Notl [0185] 5’ AGATCTGGTTTTAATGAAGCTTGCTCGACCTGCAATCCTG (SEC ID Nº 114) [0186] 3’GTAATTTTGACATACAAGTTGTGCGGCCGCCATCACCATCACCATCAC (SEC ID Nº 115)

d.

Construcción de hHGF y quimera mhm [0187] Los vectores que codifican el hHGF y la quimera mhm (V495-L585), pcDNA5/FRT hHGF y pcDNA5/FRT-quimera mhm (V495-L585), sin Fc-marcador se generaron por mutagénesis dirigida. Se introdujo un codón de terminación 3’ del marcador 6xHis usando el kit de mutagénesis dirigida QuikChange II XL (Stratagene) de acuerdo con las instrucciones del fabricante. El cebador de mutagénesis incluía el Cebador 1: CATCACCATCACCATCACTAAGCGGGTCTGGTGCCACG (SEC ID Nº 116) y el Cebador 2: CGTGGCACCAGACCCGCTTAGTGATGGTGATGGTGATG (SEC ID Nº 117). [0188] Además, se crearon dos quimeras mhm adicionales a partir de la construcción pcDNA5/FRT-mhm (V495-L585) por mutagénesis dirigida usando el kit de mutagénesis dirigida QuikChange II XL (Stratagene) de acuerdo con las instrucciones del fabricante. Una construcción de mhm contenía la región de I499R556 de hHGF dispuesta entre secuencias murinas. La otra construcción mhm contenía la región de W507-L585 de hHGF dispuesta entre las secuencias murinas. [0189] Para la quimera mhm (I499-R556), las siguientes mutaciones puntuales se realizaron en orden en la construcción de molde pcDNA5/FRT-quimera mhm (V495L585): D558E, C561R, V564I, V567I y M583L, usando las secuencias oligonucleotídicas apropiadas. Para la quimera mhm (W507-L585), las mutaciones puntuales siguientes se introdujeron en una etapa en la construcción de molde pcDNA5/FRT-quimera mhm (V495-L585): Q502R, N504T e I505V, usando las secuencias oligonucleotídicas apropiadas. [0190] La secuencia de nucleótidos resultante de la proteína hHGF-Fc se expone como SEC ID Nº 118, incluyendo la secuencia señal (nucleótidos 1-93) y el prodominio (nucleótidos 94-162). La secuencia de aminoácidos de la proteína hHGF-Fc se expone como SEC ID Nº 119. [0191] La secuencia de nucleótidos resultante que codifica la proteína quimérica mhm (V495-L585)-Fc se expone en la SEC ID Nº 120, incluyendo la secuencia señal (nucleótidos 1-96) y el prodominio (nucleótidos 97-165). La secuencia de aminoácidos de la proteína quimérica mhm (V495-L585)-Fc se expone en la SEC ID Nº 121. [0192] La secuencia de nucleótidos resultante que codifica, y la secuencia proteica que define, la construcción mhm (V495-L585) se expone en las SEC ID Nº 211 y 212, respectivamente. La secuencia de ácido nucleico expuesta en la SEC ID Nº

imagen54 imagen55 imagen56
211 incluye la secuencia de señal (nucleótidos 1-96) y el prodominio (nucleótidos 97-165), y la secuencia proteica expuesta en la SEC ID Nº 212 incluye la secuencia de la proteína activa (sin la secuencia señal o el prodominio). La secuencia de nucleótidos resultante que codifica, y la secuencia proteica que define, la construcción mhm (I499-R556) se exponen en las SEC ID Nº 213 y 214, respectivamente. La secuencia de ácido nucleico expuesta en la SEC ID Nº 213 incluye la secuencia señal (nucleótidos 1-96) y el prodominio (nucleótidos 97-165), y la secuencia proteica expuesta en la SEC ID Nº 214 incluye la secuencia de la proteína activa (sin la secuencia señal o el prodominio). La secuencia de nucleótidos resultante que codifica, y la secuencia proteica que define, la mhm (W507-L585) se exponen en las SEC ID Nº 215 y 216, respectivamente. La secuencia de ácido nucleico expuesta en la SEC ID Nº 215 incluye la secuencia señal (nucleótidos 1-96) y el prodominio (nucleótidos 97-165), y la secuencia proteica expuesta en la SEC ID Nº 216 incluye la secuencia de la proteína activa (sin la secuencia señal o el prodominio).
e. Expresión de proteína
(1) Cultivo celular. [0193] Se cultivaron células CHO FlpIn (Invitrogen, Nº de Catálogo R758-07) en medios F12K (ATCC, Nº de Catálogo 30-2004), FCS al 10% (Invitrogen, Nº de Catálogo 10438026), penicilina (10000 unidades/ml)/estreptomicina (10.000 �g/ml) al 1 % (Invitrogen, Nº de Catálogo 15140-122) a 37ºC, CO2 al 5%, zeocina 100
�g/ml (Invitrogen, Nº de catálogo R250-01).

(2)

Generación de Líneas Celulares CHO FIpIn Estables [0194] Se transfectaron células hospedadoras CHO FlpIn con una proporción 9:1 de ADN plasmídico de expresión pOG44:pcDNA5/FRT usando lipofectamina 2000 de acuerdo con las instrucciones del fabricante (Invitrogen, Nº de Catálogo 11668027). Como controles, se transfectaron células con pcDNA5/vector FRT/pOG44 vacío y plásmido pOG44 (Invitrogen, Nº de Catálogo 35-3018) solamente. Veinticuatro horas después de la transfección, las células se dividieron y, después de cuarenta y ocho horas, se añadió higromicina B 0,5 mg/ml (Sigma, Nº de Catálogo H0654-SPEC) a las células. La selección policlonal de células estables se realizó en F12K, FCS al 10%, penicilina/estreptomicina al 1%, higromicina B 0,5 mg/ml.

(3)

Expresión de proteína en líneas celulares CHO FlpIn estables [0195] Se sembraron aproximadamente 2 x 106 células en placas de 15 cm y se cultivaron en F12K (ATCC, Nº de Catálogo 30-2004)/DMEM con alto contenido en glucosa (Invitrogen, Nº de Catálogo 11995065) 1:1, FCS con nivel ultra reducido de IgG al 5% (Invitrogen, Nº 16250-78) a 37ºC, CO2 al 5% durante 5-6 días. Se recogieron los sobrenadantes y las proteínas resultantes se analizaron mediante ELISA y mediante resonancia de plasmón superficial.

imagen57
Ejemplo 4 - Características de unión de anticuerpos monoclonales anti-hHGF [0196] Los anticuerpos monoclonales producidos en el Ejemplo 1 estaban caracterizados por su capacidad para unirse a hHGF y a ciertas de las proteínas HGF recombinantes producidas en el Ejemplo 3. [0197] Los anticuerpos se analizaron por resonancia de plasmón superficial usando un instrumento BIAcore T100 para evaluar su capacidad para unirse a HGF y a ciertas de las proteínas de fusión analizadas en el Ejemplo 3. Cada anticuerpo se inmovilizó en una microplaca detectora CM5 de dextrano carboximetilado (BIAcore, Nº de Catálogo BR-1006-68) por acoplamiento con amina (BIAcore, Nº de Catálogo BR-1000-50) usando un protocolo de acoplamiento convencional de acuerdo con las instrucciones del fabricante. [0198] Se realizaron análisis a 25ºC usando PBS (GIBCO, Nº de Catálogo 14040133) que contenía tensioactivo P20 al 0,05% (BIAcore, Nº de Catálogo R-1000-54), BSA 2 mg/ml (EMD, Nº de Catálogo 2930) y sal sódica de CM-dextrano 10 mg/ml (Fluka, Nº de Catálogo 86524) como tampón de procesamiento. El sobrenadante que contenía diferentes proteínas de fusión de HGF o el sobrenadante de células transfectadas con vector vacío se inyectó sobre cada anticuerpo a un caudal de 30
�l/min durante 3 minutos. La unión resultante se determinó como unidades de resonancia (UR) sobre el nivel basal 30 segundos después finalizar la inyección. Se comparó la unión a HGF humano (R&D Systems, Nº de Catálogo 294-HGN-025) diluido en tampón de procesamiento. Se controló la unión inespecífica por comparación de la unión a una superficie de control en la que se inmovilizó IgG de ratón (Rockland, Nº de Catálogo 010-0102) usando el mismo procedimiento de acoplamiento con amina. [0199] Los resultados se resumen en la Tabla 3. imagen58
TABLA 3

Anticuerpo

rhHGF (R&D Systems) mHGF (R&D Systems) Quimera mhm (V495-L585) HGF Humano G555E C561R

1A3

Sí No No Sí Sí Sí

1D3

Sí No Sí Sí Sí Sí

1F3

Sí Sí Sí Sí Sí Sí

2B8

Sí No Sí Sí Sí Sí

2F8

Sí Sí No Sí Sí Sí

3A12

Sí No No Sí Sí Sí

3B6

Sí No No Sí Sí Sí

3D11

Sí No No Sí Sí Sí

[0200] Los resultados de la Tabla 3 demuestran que cada uno de los anticuerpos se une a rHGF y a HGF humano purificado. Además, todos los anticuerpos se unen
5 a hHGF que contiene las mutaciones puntuales G555E y C561R. En general, todos los anticuerpos excepto 1F3 y 2F8 no se unían a HGF murino, demostrando que los anticuerpos 1A3, 1D3, 2B8, 3A12, 3B6 y 3D11 se unen específicamente a HGF humano. Los anticuerpos 1D3, 1F3 y 2B8 se unen a la quimera de ratón-humanoratón, mientras que los anticuerpos restantes no lo hacían. Los resultados sugieren
10 que los anticuerpos 1D3 y 2B8 al menos en parte se unen a los restos 495-585 de HGF humano. Los anticuerpos 1A3, 3A12, 3B6 y 3D11 parecen unirse a porciones de hHGF humano distintos de los restos 495-585. En la actualidad, no se sabe claramente por qué 2F8 no se une a la quimera mhm, ya que parece unirse tanto a hHGF como a mHGF.
15 Ejemplo 5 - Capacidad de los anticuerpos monoclonales anti-hHGF para unirse a HGF reducido y no reducido [0201] En este Ejemplo, los anticuerpos monoclonales anti-hHGF producidos en el Ejemplo 1 se analizaron por su capacidad para unirse a HGF reducido y no
20 reducido. [0202] La reactividad de los sueros anti-HGF con el hHGF recombinante se evaluó por inmunotransferencia. Ocho �g de proteína hHGF recombinante en tampón de procesamiento NuPAGE MOPS SDS (Invitrogen) con o sin tampón reductor de muestras NuPAGE (Invitrogen) se fraccionaron en un gel de pocillos Bis-Tris 1,0 mm X2D al 4-12% (Invitrogen, Carlsbad, CA). Las proteínas fraccionadas se transfirieron después sobre una membrana de nitrocelulosa usando procedimientos convencionales. Las membranas de nitrocelulosa se bloquearon con solución de leche desnatada en polvo al 5% en solución salina tamponada con Tris con Tweenimagen59
5 20� al 0,1% (TBST), y después se montaron en un aparato Mini Protean II Multi-Screen (BioRad) para un bloqueo adicional. [0203] Las membranas resultantes se sondaron con los anticuerpos purificados en un aparato Multi-Screen. Los anticuerpos purificados se diluyeron hasta 5 �g/ml en tampón de bloqueo. Después se retiró la membrana de nitrocelulosa del aparato y
10 se incubó con anticuerpos anti-IgG de ratón marcados con peroxidasa de rábano picante. Los resultados se resumen en la Tabla 4, en la que los números reflejan el grado de unión, representando – la mínima (escasa o ninguna unión) y representando 3 + la mayor unión. TABLA 4

Anticuerpo

Reducido (exposición: 3-5 min) No Reducido (exposición 20 s)

1A3

2+ 2+

1D3

2+ 2+

1F3

2+ 2+

2B8

– 1+

2F8

2+ 2+

3A12

– 2+

3B6

3+ 2+

3D11

– 3+

15 [0204] Los datos en la Tabla 4 demuestran que todos los anticuerpos se unen a rhHGF no reducido. Por el contrario, los anticuerpos monoclonales 1A3, 1D3, 1F3, 2F8, 3B6 se unían a rhHGF reducido, pero los anticuerpos 2B8, 3A12 y 3D11 no se unían a rhHGF reducido.
20
Ejemplo 6 - Afinidades de unión [0205] Las afinidades de unión y la cinética de la interacción de cada uno de los anticuerpos producidos en el Ejemplo 1 contra hHGF se midieron por resonancia de plasmón superficial.
25 [0206] Se inmovilizaron inmunoglobulinas de conejo anti-ratón (BIAcore, Nº de Catálogo BR-1005-14) sobre microplacas detectoras CM5 de dextrano carboximetilado (BIAcore, Nº de Catálogo BR-1006-68) por acoplamiento con amina (BIAcore, Nº de Catálogo BR-1000-50) usando un protocolo de acoplamiento convencional de acuerdo con las instrucciones del fabricante. Los análisis se realizaron a 25ºC usando PBS (GIBCO, Nº de Catálogo 14040-133) que contenía imagen60
5 tensioactivo P20 al 0,05% (BIAcore, Nº de Catálogo BR-1000-54), BSA 2 mg/ml (EMD, Nº de Catálogo 2930) y sal sódica de CM-dextrano 10 mg/ml (Fluka, Nº de Catálogo 86524) como tampón de procesamiento. [0207] Los anticuerpos se capturaron en una celda de flujo individual a un caudal de 10 �l/min. El tiempo de inyección era variable para cada anticuerpo para producir
10 aproximadamente 20 UR de anticuerpo capturado para cada ciclo. Tampón o HGF (R&D Systems, Nº de Catálogo 294-HGN-025) diluido en tampón de procesamiento se inyectó secuencialmente sobre una superficie de referencia (sin anticuerpo capturado) y la superficie activa (anticuerpo a ensayar) durante 2 minutos a 60
�l/min. La fase de disociación se controló durante 15 ó 90 minutos dependiendo de
15 la concentración. Después, la superficie se regeneró con glicina-HCl 10 mM, pH 1,7 (BIAcore, Nº de Catálogo BR-1003-54) inyectado durante 3 minutos a un caudal de 60 �l/min antes de que se iniciase otro ciclo. Las concentraciones de HGF ensayadas eran de 0,46 nM a 7,5 nM. [0208] Los parámetros cinéticos se determinaron usando la función cinética del
20 software BIAevaluation con sustracción de referencia. Los parámetros cinéticos para cada anticuerpo, ka (constante de velocidad de asociación), kd (constante de velocidad de disociación) y KD (constante de disociación en equilibrio) se resumen en la Tabla 5.
TABLA 5

Anticuerpo

ka (1/Ms) ET (ka) kd (1/s) ET (kd) KD (pM) DT

1A3

1,7 x 106 7,3 x 104 5,2 x 10-5 8,4 x 10-7 30,1 5,6

1D3

1,7 x 106 3,1 x 104 8,2 x 10-5 1,7 x 10-6 54,2 27,4

1F3

1,5 x 106 5,0 x 104 2,6 x 10-5 6,6 x 10-7 18,1 8,2

2B8

1,6 x 106 2,9 x 104 2,1 x 10-5 1,4 x 10-7 13,5 4,4

3A12

1,6 x 106 3,7 x 104 1,6 x 10-4 1,6 x 10-6 103,0 10,4

3B6

2,0 x 106 6,5 x 104 3,9 x 10-5 3,2 x 10-7 17,0 3,4

[0209] Los datos de la Tabla 5 demuestran que los anticuerpos se unen a hHGF con una KD de aproximadamente 100 pM o menos, aproximadamente 50 pM o menos, o 20 pM o menos. imagen61
Ejemplo 7 - Actividad de neutralización de anticuerpos anti-hHGF [0210] En este Ejemplo, los anticuerpos producidos en el Ejemplo 1 se caracterizaron por su capacidad para (a) inhibir la unión de hHGF a c-Met y (b) inhibir la incorporación de BrdU estimulada por HGF en células 4MBr-5.
a. Ensayo de inhibición de la unión de HGF-Met (Ensayo de neutralización) [0211] Los anticuerpos se ensayaron mediante ELISA por su capacidad para inhibir la unión de hHGF a c-Met. [0212] En concreto, se recubrieron placas de ensayo DELFIA de 96 pocillos Wallac (Wallac Inc., Nº de Catálogo AAAND-0001) con 100 �l de HGF 6,25 �g/ml (R&D Systems, Nº de Catálogo 294-HGN-025) en tampón de recubrimiento de carbonato (Na2CO3 15 mM y NaHCO3 34 mM, pH 9,0) durante 16 horas a 4ºC. Después, las placas se bloquearon con 200 �l de leche desnatada en polvo al 5% en PBS durante 1 hora a temperatura ambiente. Los anticuerpos se prepararon en una placa separada añadiendo concentraciones crecientes de los anticuerpos en investigación (0,033-667 nM, dilución en serie de 3 veces) a c-Met 2 nM (R&D Systems, Nº de Catálogo 358-MT/CF) en leche desnatada en polvo al 5% en PBS. Se transfirieron 100 �l de muestra por pocillo a la placa de ensayo y se incubaron durante una noche a 4ºC. Las placas de ensayo se lavaron después 3 veces con PBS-Tween 20 al 0,1 % y se incubaron durante 2 horas a temperatura ambiente con 100 �l/pocillo de anticuerpo anti-c-Met humano biotinilado (R&D Systems, Nº de Catálogo BAF358) preparado en leche desnatada en polvo al 5% en PBS. [0213] Las placas resultantes se lavaron después tres veces con PBS-Tween 20 al 0,1% y se incubaron durante 1 hora a temperatura ambiente con estreptavidina marcada con Eu (Wallac, Nº de Catálogo 1244-360) diluida 1:1000 en tampón de ensayo DELFIA (Wallac, Nº de Catálogo 4002-0010). Las placas resultantes se lavaron 3 veces con solución de lavado DELFIA (Wallac, Nº de Catálogo 40100010) y se incubaron con solución de potenciación DELFIA 100 �l/pocillo (Wallac Nº 4001-0010) durante 15 minutos a temperatura ambiente con agitación. [0214] Las placas se leyeron en un instrumento de Victor3V (Perkin Elmer) usando el método de europio. Se calcularon los valores de CI50 y se resumen en la Tabla 6. imagen62
TABLA 6

Anticuerpo

CI50 (nM) DT

1A3

5,65 0,91

1D3

4,43 2,27

1F3

6,57 0,28

2B8

5,57 1,19

2F8

5,36 0,88

3A12

5,26 2,11

3B6

– –

3D11

5,66 2,75

[0215] Los resultados demuestran que todos los anticuerpos (es decir, 1D3, 1A3, 2B8, 3A12, 1F3, 3D11 y 2F8) distintos de 3B6 neutralizan eficazmente la unión de 5 HGF a c-Met.
b. Neutralización de la incorporación de BrdU estimulada por HGF en células 4MBr-5 [0216] Se dispensaron diez �l de hHGF 12,5 nM en pocillos individuales de una
10 placa de microtitulación de cultivo de tejidos de 96 pocillos (Costar Nº de Catálogo 3903). Se añadieron diez �l de anticuerpos diluidos en serie a las concentraciones de 6667, 2222, 740, 247, 82, 27, 9,1, 3,0, 1,0, 0,33 nM a cada pocillo. La mezcla de anticuerpos de HGF se incubó después a temperatura ambiente durante 30 minutos. Células epiteliales bronquiales de mono 4MBr-5 (ATCC, CCL208)
15 cultivadas en F-12K (ATCC, 30-2004), FBS al 15% (Gibco 10438-026), EGF 30 ng/ml (Sigma E9644), penicilina/estreptomicina al 1% (PS, Gibco Nº de Catálogo 15140-122) se disociaron con tripsina (Gibco Nº de Catálogo 25200-056), se resuspendieron en medios de ensayo (F-12K, FBS al 2,5%, PS al 1%) a 75.000 células/ml y se dispensaron 80 �l de la suspensión celular en la mezcla de
20 anticuerpos de HGF. [0217] Las células resultantes se incubaron a 37ºC, CO2 al 5%. Cuarenta y ocho horas después, se añadieron 10 �l de BrdU 100 �M (Roche Nº de Catálogo 1669915). Setenta y dos horas después, los medios se eliminaron, las placas se secaron con un secador de pelo y se procesaron con el ELISA de BrdU de acuerdo
25 con las instrucciones del fabricante (Roche Nº de Catálogo 1669915). [0218] La señal luminiscente se cuantificó mediante un lector de placas Synergy HT (Bio-Tek). Los datos se ajustaron a una respuesta a la dosis sigmoidea con pendiente variable con la ecuación y = parte inferior + (parte superior – parte inferior)/(1 + 10^(log(CE50 – x)*pendiente)) en GraphPad Prism (GraphPad Software). Cada experimento se repitió al menos 3 veces por duplicado y los valores promedios de la CE50 se presentan en la Tabla 7. imagen63
TABLA 7

Anticuerpo

CI50 (nM)

1A3

4,69

1D3

4,99

1F3

1,94

2B8

1,41

2F8

19,24

3A12

30,30

3B6

36,08

3D11

51,12

[0219] [0220] Los resultados de la Tabla 7 demuestran que todos los anticuerpos, 1A3, 1D3, 1F3, 2B8, 2F8, 3A12, 3B6 y 3D11 inhiben la proliferación inducida por HGF en
10 células 4MBr-5.
Ejemplo 8 - Actividad anti-dispersión de anticuerpos anti-hHGF [0221] Este Ejemplo describe una caracterización de los anticuerpos producidos en el Ejemplo 1 para determinar su capacidad para inhibir la actividad de dispersión
15 inducida por HGF. El HGF induce la “dispersión” (motilidad) de grupos en células MDCK (ATCC, Manassas, VA, Nº Catálogo CCL-34). [0222] Se sembraron células MDCK en placas de cultivo tisular Costar de 96 pocillos (Corning Incorporated, Corning, NY Nº de Catálogo 3595) a una densidad de 4 x 103 células por pocillo en 80 �l de MEM (ATCC, Manassas, VA, Nº de
20 Catálogo 30-2003) que contenía suero bovino fetal al 10% (Invitrogen Nº de Catálogo 10438026) y penicilina-estreptomicina al 1% (Invitrogen Nº de Catálogo 15140122). Cada uno de los anticuerpos a investigar se diluyó hasta 6,667 nM en MEM que contenía suero bovino fetal al 10% y penicilina-estreptomicina al 1%. Cada una de las diluciones de anticuerpo diferentes, así como el MEM que contenía
25 suero bovino fetal al 10% y penicilina-estreptomicina al 1% sin anticuerpo, se combinó después por separado con un volumen equivalente de MEM que contenía suero bovino fetal al 10% y penicilina-estreptomicina al 1% y HGF 100 ng/ml (R&D Systems Nº de Catálogo 294-HGN-025). Las diluciones de anticuerpo/HGF se incubaron durante 30 minutos a 25ºC. Se añadieron veinte �l de cada dilución de anticuerpo/HGF por separado a pocillos individuales, produciendo una imagen64
5 concentración final de anticuerpo de 666,7 nM y una concentración final de HGF de 10 ng/ml. Las células MDCK se incubaron después durante 24 horas a 37ºC con CO2 al 5%. [0223] Después de 24 horas de incubación, las células MDCK se lavaron cuidadosamente una vez con 100 �l por pocillo de PBS helado (Invitrogen Nº de
10 Catálogo 14190144) y se fijaron con 100 �l por pocillo de metanol helado con balanceo durante 10 minutos a 25ºC. Después, las placas se lavaron cuidadosamente una vez con agua destilada. Se añadió a cada pocillo un volumen de 100 �l de solución de cristal violeta, que consistía en cristal violeta al 0,5% (Sigma, St. Louis, MO, Nº de Catálogo C3886) y etanol al 50% en agua destilada, y
15 las células se incubaron durante 20 minutos a 25ºC con balanceo. [0224] Después de la tinción con solución de cristal violeta, las células se lavaron cuidadosamente tres veces con agua destilada. Después, se añadió PBS a cada pocillo para evitar que las muestras se secaran. Se tomaron imágenes de las células usando el microscopio Leica DMIRB (Leica Microsystems GmbH, Wetzler,
20 Alemania), la cámara DC500 (Leica Microsystems GmbH, Wetzler, Alemania) y el software MagnaFire 2.1C (Optronics, Goleta, CA) y las muestras se clasificaron por el nivel de dispersión. Los resultados se resumen en la Tabla 8. TABLA 8

Inhibición de la dispersión de células MDCK inducida por HGF

Anticuerpo

Ensayo 1 Ensayo 2

1A3

++ +

1D3

++ ++

1F3

+ +

2B8

+++ +++

2F8

+ +

3A12

– –/+

3B6

++ ++

3D11

– –

– Sin inhibición +++ Inhibición muy fuerte, casi completa imagen65
++ Inhibición fuerte
+ Inhibición detectable [0225] Los resultados de la Tabla 8 demuestran que el anticuerpo 2B8 inhibía la dispersión inducida por HGF más que otros anticuerpos. Los anticuerpos 1D3 y 3B6 mostraban un nivel intermedio de inhibición; el anticuerpo 1A3 mostraba un nivel de bajo a intermedio de inhibición; los anticuerpos 1F3 y 2F8 mostraban un nivel bajo de inhibición; y los anticuerpos 3A12 y 3D11 proporcionaban una inhibición escasa
o no detectable.
Ejemplo 9 - Inhibición de la fosforilación de c-Met estimulada por HGF [0226] Este ejemplo describe una caracterización de los anticuerpos producidos en el Ejemplo 1 por su capacidad para inhibir la fosforilación de c-Met estimulada por HGF en células PC-3. El HGF induce la fosforilación de Met en células PC-3 (ATCC Nº CRL-1435). [0227] Se sembraron células PC-3 en pocillos individuales de placas de cultivo de tejidos Costar de 96 pocillos (Corning Nº de Catálogo 3595) a una densidad de 4,5 x 104 células por pocillo en 100 �l de F-12K (ATCC, Manassas, VA, Nº de Catálogo 30-2004) que contenía suero bovino fetal al 10% (Invitrogen Nº de Catálogo 10438026) y penicilina-estreptomicina al 1% (Invitrogen Nº de Catálogo 15140122). Después de 24 horas a 37ºC con CO2 al 5% se retiraron los medios y las células se aclararon una vez con F-12K sin suero que contenía penicilina-estreptomicina al 1%. Después, las células se incubaron durante 24 horas en 100 �l de F-12K sin suero que contenía penicilina-estreptomicina al 1%. [0228] Las siguientes 10 diluciones diferentes de cada uno de los anticuerpos que se estaban investigando se prepararon en F-12K sin suero que contenía penicilinaestreptomicina al 1%: 6667 nM, 2222 nM, 741 nM, 247 nM, 82,3 nM, 27,4 nM, 9,1 nM, 3,0 nM, 1,0 nM y 0,3 nM. Cada dilución de anticuerpo y F-12K sin suero que contenía penicilina-estreptomicina al 1% sin anticuerpo se combinó por separado con un volumen equivalente de F-12K sin suero que contenía penicilinaestreptomicina al 1% y HGF 500 ng/ml (R&D Systems Nº de Catálogo 294-HGN025). Estas diluciones de anticuerpo/HGF se incubaron durante 30 minutos a 25ºC. Esto dio como resultado una concentración final de HGF 1,25 nM. [0229] Después, las células PC-3 se aclararon una vez con F-12K sin suero que contenía penicilina-estreptomicina al 1%. A continuación, se añadieron 70 �l de F12K sin suero que contenía penicilina-estreptomicina al 1% a las células, seguido de Na3VO4 10 mM (Sigma Nº de Catálogo S6508) en F-12K sin suero que contenía penicilina-estreptomicina al 1%. Después, las células se incubaron durante 60 minutos a 37ºC con CO2 al 5%. Después de esta incubación, se añadieron 20 �l de cada dilución de anticuerpo/HGF por separado a pocillos separados, produciendo una concentración final de HGF de 50 ng/ml y las concentraciones finales siguientes de cada anticuerpo: 666,7 nM, 222,2 nM, 74,1 nM, 24,7 nM, 8,23 nM, 2,74 nM, 0,91 nM, 0,30 nM, 0,10 nM, 0,03 nM. Las células se incubaron después durante 10 minutos a 37ºC con CO2 al 5%, punto después del cual se retiró la mezcla de medios/anticuerpo/HGF y las placas se pusieron en hielo. Después, las células se aclararon una vez con 100 �l por pocillo de PBS helado (Invitrogen Nº de Catálogo 14190144) que contenía Na3VO4 1 mM. Después, las células se incubaron durante 30 minutos a 4ºC en 100 �l por pocillo de tampón de lisis helado que consistía en OmniPur Triton X-100 al 1% (MERCK KGaA, Darmstadt, Alemania, Nº de Catálogo 9410), Tris-HCl 50 mM pH 8,0, NaCl 100 mM, Na3VO4 0,3 mM, cóctel inhibidor de proteasas 1x (Sigma Nº de Catálogo P8340) y cóctel inhibidor de fosfatasa 2 1x (Sigma Nº de Catálogo Nº 5726). [0230] Se diluyó anticuerpo anti-HGF-R (c-met) humano biotinilado (R&D Systems Nº de Catálogo BAF358) a una concentración de 2 �l/ml en tampón de ensayo DELFIA (PerkinElmer, Turku, Finlandia, Nº de Catálogo 4002-0010) que contenía albúmina de suero bovino al 1% (Sigma Nº de Catálogo A2153), y se añadieron 50 imagen66
�l de esta dilución por pocillo de placas de microtitulación con estreptavidina amarillas (PerkinElmer Nº de Catálogo AAAND-0005). Después, las placas se incubaron con anticuerpo durante 30 minutos a 25ºC con balanceo. Después de la incubación, las placas se lavaron con solución de lavado DELFIA (PerkinElmer Nº de Catálogo 4010-0010) y se añadieron 80 �l de cada uno de los diferentes lisados de células PC-3 por separado a pocillos individuales de las placas de microtitulación con estreptavidina lavadas. [0231] Las placas de microtitulación con estreptavidina que contenían los lisados de células PC-3 se incubaron durante 60 minutos a 25ºC con agitación, y después se lavaron con solución de lavado DELFIA. 100 �l de anticuerpo DELFIA Eu-N1 PTyr-100 600 ng/ml (PerkinElmer Nº de Catálogo AD0159) diluido en tampón de ensayo DELFIA que contenía albúmina de suero bovino al 1% se añadieron a cada pocillo de las placas de microtitulación con estreptavidina lavadas, previamente incubadas con lisados de células PC-3. Las placas se incubaron durante 60 minutos a 25ºC con balanceo. Las placas se lavaron un tiempo final con solución de lavado DELFIA. Después, se añadieron 200 �l de solución de potenciación DELFIA (PerkinElmer Nº de Catálogo 4001-0010) a cada pocillo de las placas de microtitulación con estreptavidina lavadas, y las placas se incubaron en la oscuridad durante 5 minutos a 25º C con agitación. imagen67
5 [0232] Después, se midió la señal usando el protocolo de europio en el lector Victor3V (PerkinElmer). Los valores de CE50 se calcularon usando Prism 4 para Windows (GraphPad Software, Inc., San Diego, CA) y la ecuación de respuesta a la dosis sigmoidea. [0233] Los resultados resumidos como CE50 en nM se representan en la Tabla 9.
10 TABLA 9

Anticuerpo

Promedio de dos ensayos Desviación típica

1A3

0,684 0,242

1D3

0,984 0,129

1F3

1,19 1,01

2B8

0,287 0,104

2F8

1,39 2,12

3A12

2,00 0,553

3B6

1,01 1,11

3D11

2,28 N/A

[0234] Los datos de la Tabla 9 demuestran que los ocho anticuerpos son potentes inhibidores de la fosforilación de c-Met inducida por HGF en células PC-3.
15 Ejemplo 10 - Inhibición de tumores en el modelo de xenoinjerto U87MG [0235] La capacidad de anticuerpos monoclonales murinos de la invención para inhibir el crecimiento de tumores se ensayó en un modelo de xenoinjerto U87MG. Se expandieron células U87MG (ATCC) en cultivo a 37ºC en una atmósfera que contenía CO2 al 5% y aire al 95%, usando un medio que comprendía medio de
20 Eagle modificado por Dulbecco (DMEM) con suero bovino fetal al 10%, penicilina 100 unidades/ml y estreptomicina 100 �g/ml. Las células se subcultivaron y se mantuvieron por desprendimiento de las células de la pared de la placa de cultivo usando tripsina-EDTA. [0236] Se recogieron células casi confluentes por tratamiento con tripsina, y
25 después se inyectaron 5 x 106 células en Matrigel al 50% (BD Biosciences; Nº de Catálogo 356237) subcutáneamente en el área dorsal superior entre las escápulas de ratones ICR SCID hembra de 7 semanas de edad (Taconic Labs). Los diámetros largo (L) y corto (W) (mm) de los tumores se midieron con un calibrador. Se calculó el volumen tumoral (vol.) como: volumen (mm3) = L x W2 / 2. Cuando los tumores crecieron hasta aproximadamente 200 mm3, los ratones que portaban tumores se imagen68
5 aleatorizaron en 5 grupos de 10 ratones cada uno. Un grupo recibió PBS. Cada uno de los otros 4 grupos recibió uno de los anticuerpos 1A3, 1D3, 1F3 ó 2B8. Todos los anticuerpos se dosificaron a 1 mg/kg de peso corporal dos veces por semana por inyección intraperitoneal de 5 dosis. Los volúmenes tumorales y los pesos corporales de los ratones se registraron dos veces por semana. Se analizó la
10 inhibición del crecimiento tumoral usando la prueba t de Student. Los resultados se resumen en la Figura 6 y en la Tabla 10. Tabla 10

Porcentaje de Inhibición

2B8 frente a PBS

93% p = 0,001

1A3 frente a PBS

73% p = 0,0075

1D3 frente a PBS

51% p = 0,075

1F3 frente a PBS

60% p = 0,027

[0237] Se consiguió una regresión parcial en el grupo tratado con 2B8 (Figura 6).
15 Se observó una inhibición del crecimiento estadísticamente significativa en los grupos tratados con 1A3 y tratados con 1F3 (Tabla 10). Había una inhibición del crecimiento tumoral del 51% para 1D3 con un valor p de 0,075. No se observaron pérdidas de peso corporal significativas.
20 Ejemplo 11 - Inhibición de tumores en el modelo de xenoinjerto U118 [0238] La capacidad de los anticuerpos 1A3, 1D3, 1F3 y 2B8 para inhibir el crecimiento de tumores se ensayó en un modelo de xenoinjerto U118. Se expandieron células U118 (ATCC) como se describe en el Ejemplo 10 (anterior) con respecto a las células U87MG.
25 [0239] Se establecieron tumores subcutáneos como se ha descrito en el Ejemplo 10 anterior, excepto porque los ratones usados eran ratones atímicos NCr hembra de 7 semanas de edad (Taconic) y el tratamiento comenzó cuando los tumores crecieron hasta aproximadamente 80 mm3. Como en el modelo U87MG, todos los anticuerpos se dosificaron a 1 mg/kg de peso corporal dos veces por semana
30 mediante inyecciones intraperitoneales durante 4 dosis. Los volúmenes tumorales y los pesos corporales de los ratones se registraron dos veces por semana. Se analizó la inhibición del crecimiento tumoral usando la prueba t de Student. Los resultados se resumen en la Figura 7 y en la Tabla 11. imagen69
Tabla 11

Porcentaje de Inhibición

2B8 frente a IgG

75% p = 0,007

1A3 frente a IgG

57% p = 0,01

1D3 frente a IgG

47% p = 0,12

1F3 frente a IgG

30% p = 0,39

5 [0240] Se observó una inhibición del crecimiento tumoral estadísticamente significativa en los grupos tratados con 2B8 y 1A3 (Figura 7). Había una inhibición del crecimiento tumoral modesta en los grupos con 1F3 y 1D3 con valores p inferiores a 0,05, que se definieron como estadísticamente significativos en este
10 estudio (Tabla 11). No se observaron pérdidas de peso corporal significativas.
Ejemplo 12: Humanización de anticuerpos monoclonales murinos [0241] Este ejemplo describe la humanización del anticuerpo 2B8 murino junto con una caracterización de los anticuerpos humanizados resultantes. Las regiones
15 variables ligera y pesada del 2B8 murino se “humanizaron” mediante dos métodos.
A. Procedimiento de humanización 1 [0242] En el primer método, se diseñaron tres regiones variables de cadena pesada humanizadas y dos regiones variables de cadena ligera kappa
20 humanizadas basándose en el método de “superhumanización” descrito en Hwang et al. (2005) METHODS 36:35-42; Tan et al. (2002) J. IMMUNOL. 169:1119-1125; Patente de Estados Unidos Nº 6.881.557. [0243] Se determinó la clase estructural canónica de Chotia para cada CDR de 2B8 de ratón basándose en la longitud de la CDR y en la composición de aminoácidos.
25 Se identificaron regiones variables de línea germinal humana que consistían en regiones variables pesadas y ligeras de la misma clase estructural canónica de Chotia basándose en alelos de referencia de la región variable de línea germinal humana conocidos descritos en el sitio web del International Immunogenetics Information System (IMGT) (disponible en la red global mundial (world wide web) en
30 imgt.cines.fr y biochem.unizh.ch/antibody/Sequences/index.html). Estas regiones variables de línea germinal humana de la misma clase estructural se compararon con regiones variables de 2B8 murino calculando el porcentaje de identidad o similitud entre restos aminoacídicos de CDR. Aquellas regiones variables de línea germinal humana con la mayor identidad y/o similitud con restos de CDR de 2B8 de ratón se seleccionaron para el injerto de CDR. Los restos flanqueantes de las regiones variables de línea germinal humana se conservaron, mientras que los restos de CDR de 2B8 de ratón se usaron para sustituir los restos de la región variable de línea germinal humana correspondientes que eran diferentes entre CDR de 2B8 de ratón y CDR de línea germinal humana. La región J humana que era la más similar a la región J de ratón de 2B8 se añadió después al extremo carboxilo terminal de la región variable “superhumanizada”. Después, se añadió una secuencia señal al extremo amino-terminal de las regiones variables “superhumanizadas”, y estas secuencias de aminoácidos se convirtieron en secuencias de ácido nucleico. [0244] La secuencia de ácido nucleico de la región variable completa se construyó usando métodos de PCR de síntesis de genes (Young et al. (2004). NUCL. ACIDS RES 32:e59) y se clonó en un vector de expresión de mamífero (basado en pcDNA3.2 DEST (Invitrogen)) que contenía regiones Kappa (alotipo Km(3) (alelo 2)) imagen70
o de IgG1 (alotipo G1m(17,1)) constantes humanas (cadena abajo de las regiones variables) usando técnicas de biología molecular convencionales. Los cuatro anticuerpos IgG1 de cadena pesada (2B8 quimérico y 3 cadenas pesadas humanizadas (Hu2B8 Hv1-f.1, Hu2B8 Hv5-a.1, Hu2B8 Hv5-51.1) se expresaron en las combinaciones posibles con los 3 anticuerpos de cadena kappa (2B8 quimérico y 2 cadenas ligeras humanizadas (Hu2B8 Kv1-39.1 y Hu2B8 Kv3-15.1), creando 12 proteínas de anticuerpo diferentes. La unión de los anticuerpos quiméricos, quiméricos/humanizados y humanizados a HGF humano se midió después como se describe a continuación, y los resultados se resumen en la Figura 8. Cada una de las posibles combinaciones de regiones variables de cadena ligera de inmunoglobulina y cadena pesada de inmunoglobulina se expone a continuación en la Tabla 12 A.
Tabla 12A

Región Variable de Cadena Pesada

Región Variable de Cadena Ligera

2B8 quimérico (SEC ID Nº: 12)

2B8 quimérico (SEC ID Nº: 14)

2B8 quimérico (SEC ID Nº: 12)

Hu2B8 Kv1-39.1 (SEC ID Nº: 173)

2B8 quimérico (SEC ID Nº: 12)

Hu2B8 Kv3-15.1 (SEC ID Nº: 179)

imagen71

Región Variable de Cadena Pesada

Región Variable de Cadena Ligera

Hu2B8 Hv1-f.1 (SEC ID Nº: 159)

2B8 quimérico (SEC ID Nº: 14)

Hu2B8 Hv1-f.1 (SEC ID Nº: 159)

Hu2B8 Kv1-39.1 (SEC ID Nº: 173)

Hu2B8 Hv1-f.1 (SEC ID Nº: 159)

Hu2B8 Kv3-15.1 (SEC ID Nº: 179)

Hu2B8 Hv5-a.1 (SEC ID Nº: 165)

2B8 quimérico (SEC ID Nº: 14)

Hu2B8 Hv5-a.1 (SEC ID Nº: 165)

Hu2B8 Kv1-39.1 (SEC ID Nº: 173)

Hu2B8 Hv5-a.1 (SEC ID Nº: 165)

Hu2B8 Kv3-15.1 (SEC ID Nº: 179)

Hu2B8 Hv5-51.1 (SEC ID Nº: 169)

2B8 quimérico (SEC ID Nº: 14)

Hu2B8 Hv5-51.1 (SEC ID Nº: 169)

Hu2B8 Kv1-39.1 (SEC ID Nº: 173)

Hu2B8 Hv5-51.1 (SEC ID Nº: 169)

Hu2B8 Kv3-15.1 (SEC ID Nº: 179)

[0245] Cada una de las posibles combinaciones de cadenas pesadas de inmunoglobulina y cadenas ligeras de inmunoglobulina se expone a continuación en la Tabla 12B.
Tabla 12B

Cadena Pesada de Inmunoglobulina

Cadena Ligera de Inmunoglobulina

IgG1 de 2B8 quimérica (SEC ID Nº: 155)

Kappa de 2B8 quimérica (Km(3)) (SEC ID Nº: 157)

IgG1 de 2B8 quimérica (SEC ID Nº: 155)

Hu2B8 Kv1-39.1 + Constante Kappa (alotipo Km(3)) (alelo 2) (SEC ID Nº: 177)

IgG1 de 2B8 quimérica (SEC ID Nº: 155)

Hu2B8 Kv3-15.1 + Constante Kappa (alotipo Km(3)) (alelo 2) (SEC ID Nº: 181)

Hu2B8 Hv1-f.1 + Constante de IgG1 (alotipo G1M(17,1)) (SEC ID Nº: 163)

Kappa de 2B8 quimérica (Km(3)) (SEC ID Nº: 157)

Hu2B8 Hv1-f.1 + Constante de IgG1 (alotipo G1M(17,1)) (SEC ID Nº: 163)

Hu2B8 Kv1-39.1 + Constante Kappa (alotipo Km(3)) (alelo 2) (SEC ID Nº: 177)

Hu2B8 Hv1-f.1 + Constante de IgG1 (alotipo G1M(17,1)) (SEC ID Nº: 163)

Hu2B8 Kv3-15.1 + Constante Kappa (alotipo Km(3)) (alelo 2) (SEC ID Nº: 181)

Hu2B8 Hv5-a.1 + Constante de IgG1 (alotipo G1M(17,1)) (SEC ID Nº: 167)

Kappa de 2B8 quimérica (Km(3)) (SEC ID Nº: 157)

Hu2B8 Hv5-a.1 + Constante de IgG1 (alotipo G1M(17,1)) (SEC ID Nº: 167)

Hu2B8 Kv1-39.1 + Constante Kappa (alotipo Km(3)) (alelo 2) (SEC ID Nº: 177)

Hu2B8 Hv5-a.1 + Constante de IgG1 (alotipo G1M(17,1)) (SEC ID Nº: 167)

Hu2B8 Kv3-15.1 + Constante Kappa (alotipo Km(3)) (alelo 2) (SEC ID Nº: 181)

imagen72

Cadena Pesada de Inmunoglobulina

Cadena Ligera de Inmunoglobulina

Hu2B8 Hv5-51.1 + Constante de IgG1 (alotipo G1M(17,1)) (SEC ID Nº: 171)

Kappa de 2B8 quimérica (Km(3)) (SEC ID Nº: 157)

Hu2B8 Hv5-51.1 + Constante de IgG1 (alotipo G1M(17,1)) (SEC ID Nº: 171)

Hu2B8 Kv1-39.1 + Constante Kappa (alotipo Km(3)) (alelo 2) (SEC ID Nº: 177)

Hu2B8 Hv5-51.1 + Constante de IgG1 (alotipo G1M(17,1)) (SEC ID Nº: 171)

Hu2B8 Kv3-15.1 + Constante Kappa (alotipo Km(3)) (alelo 2) (SEC ID Nº: 181)

[0246] Dos de las construcciones de anticuerpos posibles que contienen las cadenas ligeras y pesadas de inmunoglobulina de longitud completa que contienen regiones variables humanizadas se indican a continuación:
5 sh2B8-9 (G1m(17,1)) = hu2B8 Hv5-51.1 (+ región constante de IgG1 (alotipo G1m(17,1)) (SEC ID Nº 171) más hu2B8 Kv 1-39.1 (+ región constante kappa (alotipo Km(3) (alelo 2))) (SEC ID Nº 177)
sh2B8-12 (G1m(17,1)) = hu2B8 Hv5-51.1 (+ región constante de IgG1
10 (alotipo G1m(17,1)) (SEC ID Nº 171) más hu2B8 Kv 3-15.1 (+ región constante kappa (alotipo Km(3) (alelo 2))) (SEC ID Nº 181) [0247] Las secuencias de ácido nucleico que codifican, y las secuencias proteicas que definen cada uno de los anticuerpos humanizados se resumen a continuación.
15 En esta sección, el último nucleótido de cada región variable es la primera base del siguiente codón generado por la unión de la región variable/constante. Este nucleótido se incluye en la Región Variable porque es parte de ese exón. Las secuencias de aminoácidos de las Regiones Constantes enumeradas a continuación incluyen la traducción de este codón de unión. imagen73
[0248] Secuencia de ácido nucleico que codifica la cadena pesada de 2B8 quimérica de longitud completa (región variable de ratón y región constante de IgG1 humana) (alotipo G1m(17,1)) (secuencia señal subrayada) (SEC ID Nº 154) imagen29
[0249] (2) Secuencia proteica que define la cadena pesada de 2B8 quimérica de longitud completa (IgG1 de 2B8 quimérica (alotipo G1m(17,1)) (sin secuencia señal) (SEC ID Nº 155) imagen29
[0250] (3) Secuencia de ácido nucleico que codifica la cadena ligera de 2B8 quimérica de longitud completa (región variable de ratón y región constante humana) (kappa de 2B8 quimérica (Km(3))) (secuencia señal subrayada) (SEC ID Nº 156) [0251] (4) Secuencia proteica que define la cadena ligera de 2B8 quimérica de longitud completa (kappa de 2B8 quimérica (Km(3))) (sin secuencia señal) (SEC ID Nº 157) imagen74 imagen75 imagen29 imagen29
[0252] (5) Secuencia de ácido nucleico que codifica la región variable de la cadena pesada de Hu2B8 Hv1-f.1 humanizada (secuencia señal subrayada) (SEC ID Nº
10 158) imagen29
[0253] (6) Secuencia proteica que define la región variable de la cadena pesada de imagen76 imagen77
[0254] (7) Secuencia de ácido nucleico que codifica la región constante de la cadena pesada de IgG1 humana (alotipo G1m(17,1)) (SEC ID Nº 160) imagen29 imagen78
[0255] (8) Secuencia proteica que define la región constante de la cadena pesada de IgG1 humana (alotipo G1m(17,1)) (SEC ID Nº 161). El primer aminoácido procede de la traducción del último nucleótido de la región variable y los dos nucleótidos del comienzo de la secuencia de la Cadena Pesada de IgG1. imagen31
[0256] (9) Secuencia de ácido nucleico que codifica la región variable de la cadena pesada de Hu2B8 Hv1-f.1 humanizada de longitud completa y la región constante de la cadena pesada de IgG1 humana (alotipo G1m(17,1) (secuencia señal
10 subrayada) (SEC ID Nº 162) [0257] (10) Secuencia proteica que define la región variable de la cadena pesada de Hu2B8 Hv1-f.1 humanizada de longitud completa y la región constante de la cadena pesada de IgG1 humana (alotipo G1m(17,1) (sin secuencia señal) (SEC ID Nº 163) imagen29 imagen79 imagen31
[0258] (11) Secuencia de ácido nucleico que codifica la región variable de la cadena pesada de Hu2B8 Hv5a.1 humanizada (secuencia señal subrayada) (SEC ID Nº 164) imagen80
[0259] (12) Secuencia proteica que define la región variable de la cadena pesada de Hu2B8 Hv5a.1 humanizada (sin secuencia señal) (SEC ID Nº 165) imagen74 imagen81
[0260] (13) Secuencia de ácido nucleico que codifica la región variable de la cadena pesada de Hu2B8 Hv5a.1 humanizada de longitud completa y la región constante de la cadena pesada de IgG1 humana (alotipo G1m(17,1) (secuencia señal subrayada) (SEC ID Nº 166) imagen31
[0261] (14) Secuencia proteica que define la región variable de la cadena pesada de Hu2B8 Hv5a.1 humanizada de longitud completa y la región constante de la cadena pesada de IgG1 humana (alotipo G1m(17,1) (sin secuencia señal) (SEC ID
10 Nº 167) [0262] (15) Secuencia de ácido nucleico que codifica la región variable de la cadena pesada de Hu2B8 Hv5-51.1 humanizada (secuencia señal subrayada) (SEC ID Nº 168) imagen29 imagen82 imagen29
[0263] (16) Secuencia proteica que define la secuencia variable de la cadena pesada de Hu2B8 Hv5-51.1 humanizada (sin secuencia señal) (SEC ID Nº 169) imagen29
10 [0264] (17) Secuencia de ácido nucleico que codifica la región variable de la cadena pesada de Hu2B8 Hv5-51.1 humanizada de longitud completa y la región constante de la cadena pesada de IgG1 humana (alotipo G1m(17,1) (secuencia señal subrayada) (SEC ID Nº 170) [0265] (18) Secuencia proteica que define la región variable de la cadena pesada de Hu2B8 Hv5-51.1 humanizada de longitud completa y la región constante de la cadena pesada de IgG1 humana (alotipo G1m(17,1) (sin secuencia señal) (SEC ID Nº 171) imagen74 imagen83 imagen31
[0266] (19) Secuencia de ácido nucleico que codifica la región variable de la cadena kappa de Hu2B8 Kv1-39.1 humanizada (secuencia señal subrayada) (SEC ID Nº 172). Se muestran en mayúsculas dos posibles ATG de inicio. imagen80
[0267] (20) Secuencia proteica que define la región variable de la cadena kappa de Hu2B8 Kv1-39.1 humanizada (sin secuencia señal) (SEC ID Nº 173) imagen29
[0268] (21) Secuencia de ácido nucleico que codifica la región constante de la cadena kappa humana (alotipo Km(3)) (alelo 2) (SEC ID Nº 174) imagen84
[0269] (22) Secuencia proteica que define la región constante de la cadena kappa humana (alotipo Km(3)) (alelo 2) (SEC ID Nº 175). El primer aminoácido procede de la traducción del último nucleótido de la región variable y los dos nucleótidos del comienzo de la secuencia de la Cadena Ligera Kappa. imagen31
[0270] (23) Secuencia de ácido nucleico que codifica la región variable de la cadena ligera de Hu2B8 Kv1-39.1 humanizada de longitud completa y la región constante de la cadena kappa humana (alotipo Km(3)) (alelo 2) (secuencia señal
10 subrayada) (SEC ID Nº 176) imagen29
[0271] (24) Secuencia proteica que define la región variable de la cadena ligera de Hu2B8 Kv1-39.1 humanizada de longitud completa y la región constante de la
15 cadena kappa humana (alotipo Km(3)) (alelo 1) (SEC ID Nº 177) imagen29
[0272] (25) Secuencia de ácido nucleico que codifica la región variable de la cadena ligera de Hu2B8 Kv3-15.1 humanizada (secuencia señal subrayada) (SEC 20 ID Nº 178) imagen29 imagen85 imagen29
[0273] (26) Secuencia proteica que define la región variable de la cadena ligera de Hu2B8 Kv3-15.1 humanizada (sin secuencia señal) (SEC ID Nº 179) imagen31
[0274] (27) Ácido nucleico que codifica la región variable de la cadena ligera de Hu2B8 Kv3-15.1 humanizada de longitud completa y la región constante de la cadena kappa humana (alotipo Km(3)) (alelo 2) (secuencia señal subrayada) (SEC
10 ID Nº 180) imagen29
[0275] (28) Secuencia proteica que define la región variable de la cadena ligera de Hu2B8 Kv3-15.1 humanizada y la región constante de la cadena kappa humana 15 (alotipo Km(3)) (alelo 2) (sin secuencia señal) (SEC ID Nº 181) imagen29
[0276] Por comodidad, la Tabla 13 proporciona una gráfica de concordancia que muestra la correspondencia entre las secuencias de longitud completa y de los 20 anticuerpos analizados en esta sección con las presentadas en la Lista de Secuencias. imagen86
TABLA 13

SEC ID Nº

Proteína o Ácido Nucleico

154

IgG1 de 2B8 quimérica (G1m(17,1)) - ácido nucleico

155

IgG1 de 2B8 quimérica (G1m(17,1)) - proteína

156

Kappa de 2B8 quimérica (Km(3)) - ácido nucleico

157

Kappa de 2B8 quimérica (Km(3)) - proteína

158

Región variable de la cadena pesada de Hu2B8 Hv1f.1 - ácido nucleico

159

Región variable de la cadena pesada de Hu2B8 Hv1f.1- proteína

160

Región constante de la cadena pesada de IgG1 humana (alotipo G1m(17,1)) - ácido nucleico

161

Región constante de la cadena pesada de IgG1 humana (alotipo G1m(17,1)) - proteína

162

Hu2B8 Hv1f.1 + constante de IgG1 (alotipo G1m(17,1)) - ácido nucleico

163

Hu2B8 Hv1f.1 + constante de IgG1 (alotipo G1m(17,1)) - proteína

164

Región variable de la cadena pesada de Hu2B8 Hv5a.1- ácido nucleico

165

Región variable de la cadena pesada de Hu2B8 Hv5a.1- proteína

166

Hu2B8 Hv5a.1 + constante de IgG1 (alotipo G1m(17,1)) - ácido nucleico

167

Hu2B8 Hv5a.1 + constante de IgG1 (alotipo G1m(17,1)) - proteína

168

Región variable de la cadena pesada de Hu2B8 Hv5-51.1 - ácido nucleico

169

Región variable de la cadena pesada de Hu2B8 Hv5-51.1 - proteína

170

Hu2B8 Hv5-51.1 + constante de IgG1 (alotipo G1m(17,1)) - ácido nucleico

171

Hu2B8 Hv5-51.1 + constante de IgG1 (alotipo G1m(17,1)) - proteína

172

Región variable de la cadena kappa de Hu2B8 Hv1-39.1 - ácido nucleico

173

Región variable de la cadena kappa de Hu2B8 Hv1-39.1 - proteína

174

Región constante de la cadena kappa humana (alotipo Km(3)) (alelo 2) - ácido nucleico

imagen87

SEC ID Nº

Proteína o Ácido Nucleico

175

Región constante de la cadena kappa humana (alotipo Km(3)) (alelo 2) - proteína

176

Hu2B8 Hv1-39.1 + constante kappa (alotipo Km(3)) (alelo 2) - ácido nucleico

177

Hu2B8 Hv1-39.1 + constante kappa (alotipo Km(3)) (alelo 2) - proteína

178

Región variable de la cadena kappa de Hu2B8 Hv3-15.1 - ácido nucleico

179

Región variable de la cadena kappa de Hu2B8 Hv3-15.1 - proteína

180

Hu2B8 Hv3-15.1 + constante kappa (alotipo Km(3)) (alelo 2) - ácido nucleico

181

Hu2B8 Hv3-15.1 + constante kappa (alotipo Km(3)) (alelo 2) - proteína

B. Procedimiento de humanización 2 [0277] El segundo método de humanización empleado para reducir la inmunogenicidad del anticuerpo 2B8 de ratón se basa en el método descrito en
5 Studnicka et al. (1994) PROTEIN ENG. 7: 805-814. Se identificaron las regiones variables de línea germinal humana kappa y pesada más idénticas (a nivel de aminoácido) a las de 2B8 de ratón. Los restos que diferían entre ratón y humano se convirtieron en la secuencia humana dependiendo de la probabilidad de riesgo de que dicho cambio afectase a la unión o a la inmunogenicidad. Los residuos de bajo
10 riesgo (es decir, restos que cuando se cambiasen probablemente no afectarían a la unión a antígeno y que también reducirían la inmunogenicidad potencial) se cambiaron al aminoácido humano en la región variable pesada (creando LR2B8HC) y la región variable kappa (creando LR2B8LC). Además, los de bajo riesgo y riesgo medio (es decir, restos que cuando se cambiasen es algo probable que tuviesen un
15 efecto sobre los restos de unión a antígeno y que también redujesen la inmunogenicidad potencial) se cambiaron al aminoácido humano en la región variable pesada (creando LRMR2B8HC) y la región variable kappa (creando LRMR2B8LC). La región constante de la cadena pesada de IgG1 humana (alotipo G1m(3) (alelo1)) se añadió al extremo carboxilo terminal de las dos regiones
20 variables pesadas modificadas por ingeniería genética humanas, y la región constante kappa humana (alotipo Km(3) (alelo 1)) se añadió al extremo carboxilo terminal de dos regiones variables ligeras modificadas por ingeniería genética humanas, creando de este modo cuatro cadenas de anticuerpo modificadas por ingeniería genética humanas. Las secuencias de ácido nucleico de la región variable se sintetizaron primero por métodos de síntesis de genes y después se imagen88
5 añadieron a secuencias de la región constante humana. Estos anticuerpos modificados por ingeniería genética humanos se clonaron en vectores de expresión de proteínas de mamíferos y la proteína se expresó en las cuatro combinaciones posibles de cadena pesada más cadena ligera. La unión de los anticuerpos quiméricos, quiméricos/humanizados o humanizados al HGF humano se midió
10 usando técnicas convencionales que se describen a continuación. [0278] Las secuencias de ácido nucleico que codifican, y las secuencias proteicas que definen cada uno de los anticuerpos humanizados se resumen a continuación. En esta sección, el último nucleótido de cada región variable es la primera base del siguiente codón generado por la unión de región variable/constante. Este nucleótido
15 se incluye en la Región Variable porque es parte de ese exón. Las secuencias de aminoácidos de las Regiones Constantes enumeradas a continuación incluyen la traducción de este codón de unión.
[0279] (1) Secuencia de ácido nucleico que codifica la región variable de la cadena 20 pesada de LR2B8HC humanizada (secuencia señal subrayada) (SEC ID Nº 182) imagen29
[0280] (2) Secuencia proteica que define la región variable de la cadena pesada de LR2B8HC humanizada (sin secuencia señal) (SEC ID Nº 183) imagen89 imagen90
[0281] (3) Secuencia de ácido nucleico que codifica la región constante de la cadena pesada de IgG1 humana (alotipo G1m(3)) (alelo1) (SEC ID Nº 184) imagen29
[0282] (4) Secuencia proteica que define la región constante de la cadena pesada de IgG1 humana (alotipo G1m(3)) (alelo1 ó 2) (SEC ID Nº 185). El primer aminoácido procede de la traducción del último nucleótido de la región variable y los [0283] (5) Secuencia de ácido nucleico que codifica la región variable de la cadena pesada de LR2B8HC humanizada de cadena pesada de longitud completa y la región constante de la cadena pesada de IgG1 humana (alotipo G1m(3)) (alelo1) (secuencia señal subrayada) (SEC ID Nº 186) imagen91 imagen92 imagen31
[0284] (6) Secuencia proteica que define la región variable de la cadena pesada de LR2B8HC humanizada de cadena pesada de longitud completa y la región constante de la cadena pesada de IgG1 humana (alotipo G1m(3)) (alelo1) (sin
10 secuencia señal) (SEC ID Nº 187) [0285] (7) Secuencia de ácido nucleico que codifica la región variable de la cadena pesada de LRMR2B8HC humanizada (secuencia señal subrayada) (SEC ID Nº 188) imagen29 imagen93 imagen29
[0286] (8) Secuencia proteica que define la región variable de la cadena pesada de LRMR2B8HC humanizada (sin secuencia señal) (SEC ID Nº 189) imagen29
10 [0287] (9) Secuencia de ácido nucleico que codifica la región variable de la cadena pesada de LRMR2B8HC humanizada de cadena pesada de longitud completa y la región constante de la cadena pesada de IgG1 humana (alotipo G1m(3)) (alelo1) (secuencia señal subrayada) (SEC ID Nº 190) [0288] (10) Secuencia proteica que define la región variable de la cadena pesada de LRMR2B8HC humanizada de cadena pesada de longitud completa y la región constante de la cadena pesada de IgG1 humana (alotipo G1m(3)) (alelo1) (sin secuencia señal) (SEC ID Nº 191) imagen29 imagen94 imagen31
[0289] (11) Secuencia de ácido nucleico que codifica la región variable de la cadena ligera de LR2B8LC humanizada (secuencia señal subrayada) (SEC ID Nº 192) imagen80
[0290] (12) Secuencia proteica que define la región variable de la cadena ligera de LR2B8LC humanizada (sin secuencia señal) (SEC ID Nº 193) imagen29
[0291] (13) Secuencia de ácido nucleico que codifica la región constante de la cadena kappa humana (alotipo Km(3)) (alelo1) (SEC ID Nº 194) imagen29 imagen95
[0292] (14) Secuencia proteica que define la región constante de la cadena kappa humana (alotipo Km(3)) (alelo1) (SEC ID Nº 195). El primer aminoácido procede de la traducción del último nucleótido de la región variable y los dos nucleótidos del comienzo de la secuencia de la Cadena Ligera Kappa. imagen31
[0293] (15) Secuencia de ácido nucleico que codifica la región variable de la cadena ligera de LR2B8LC humanizada de longitud completa y la región constante de la cadena kappa humana (alotipo Km(3)) (alelo1) (SEC ID Nº 196) imagen80
[0294] (16) Secuencia proteica que codifica la región variable de la cadena ligera de LR2B8LC humanizada de longitud completa y la región constante de la cadena kappa humana (alotipo Km(3)) (alelo1) (SEC ID Nº 197) imagen74
[0295] (17) Secuencia de ácido nucleico que codifica la región variable de la cadena ligera de LRMR2B8LC humanizada (secuencia señal subrayada) (SEC ID Nº 198) [0296] (18) Secuencia proteica que define la región variable de la cadena ligera de LRMR2B8LC humanizada (sin secuencia señal) (SEC ID Nº 199) imagen96 imagen29
[0297] (19) Secuencia de ácido nucleico que codifica la región variable de la cadena ligera de LRMR2B8LC humanizada de longitud completa y la región constante de la cadena kappa humana (alotipo Km(3)) (alelo1) (secuencia señal subrayada) (SEC ID Nº 200) imagen29
[0298] (20) Secuencia proteica que define la región variable de la cadena ligera de LRMR2B8LC humanizada de longitud completa y la región constante de la cadena kappa humana (alotipo Km(3)) (alelo1) (SEC ID Nº 201) imagen29
15 [0299] Por comodidad, la Tabla 14 proporciona una gráfica de concordancia que muestra la correspondencia entre las secuencias de longitud completa y de los anticuerpos analizados en esta sección con las presentadas en la Lista de Secuencias.
20 TABLA 14

SEC ID Nº

Proteína o Ácido Nucleico

182

Región variable de la cadena pesada de LR2B8HC - ácido nucleico

183

Región variable de la cadena pesada de LR2B8HC - proteína

184

Región constante de la cadena pesada de IgG1 humana (alotipo G1m(3)) (alelo1) - ácido nucleico

imagen97

SEC ID Nº

Proteína o Ácido Nucleico

185

Región constante de la cadena pesada de IgG1 humana (alotipo G1m(3)) (alelo1) - proteína

186

LR2B8HC + constante de IgG1 (alotipo G1m(3)) (alelo1) - ácido nucleico

187

LR2B8HC + constante de IgG1 (alotipo G1m(3)) (alelo1) - proteína

188

Región variable de la cadena pesada de LRMR2B8HC - ácido nucleico

189

Región variable de la cadena pesada de LRMR2B8HC - proteína

190

LRMR2B8HC + constante de IgG1 (alotipo G1m(3)) (alelo1) - ácido nucleico

191

LRMR2B8HC + constante de IgG1 (alotipo G1m(3)) (alelo1) - proteína

192

Región variable de la cadena ligera de LR2B8LC - ácido nucleico

193

Región variable de la cadena ligera de LR2B8LC - proteína

194

Región constante de la cadena kappa humana (alotipo Km(3)) (alelo1) - ácido nucleico

195

Región constante de la cadena kappa humana (alotipo Km(3)) (alelo1) - proteína

196

LR2B8LC + constante de kappa (alotipo Km(3)) (alelo1) - ácido nucleico

197

LR2B8LC + constante de kappa (alotipo Km(3)) (alelo1) - proteína

198

Región variable de la cadena ligera de LRMR2B8LC - ácido nucleico

199

Región variable de la cadena ligera de LRMR2B8LC - proteína

200

LRMR2B8LC + constante de kappa (alotipo Km(3)) (alelo1) - ácido nucleico

201

LRMR2B8LC + constante de kappa (alotipo Km(3)) (alelo1) proteína

[0300] La Tabla 15 resume las secuencias de CDR de la cadena pesada (definición de Kabat) de los anticuerpos 2B8 humanizados preparados por el procedimiento de humanización 1 y por el procedimiento de humanización 2 descritos anteriormente en la presente memoria en este Ejemplo. imagen98
TABLA 15

Anticuerpo

CDR1 CDR2 CDR3 Región variable de cadena pesada de longitud completa

Pesada de 2B8 murino

TYWMH (SEC ID Nº 15) EINPTNGHTNYNEKFKS (SEC ID Nº 16) NYVGSIFDY (SEC ID Nº 17) SEC ID Nº 12

Hu2B8 Hv1f.1

TYWMH (SEC ID Nº 15) EINPTNGHTNYNEKFQG (SEC ID Nº 202) NYVGSIFDY (SEC ID Nº 17) SEC ID Nº 159

Hu2B8 Hv5a.1

TYWMH (SEC ID Nº 15) EINPTNGHTNYNPSFQG (SEC ID Nº 203) NYVGSIFDY (SEC ID Nº 17) SEC ID Nº 165

Hu2B8 Hv5-51.1

TYWMH (SEC ID Nº 15) EINPTNGHTNYNPSFQG (SEC ID Nº 203) NYVGSIFDY (SEC ID Nº 17) SEC ID Nº 169

LR2B8HC

TYWMH (SEC ID Nº 15) EINPTNGHTNYNEKFKG (SEC ID Nº 204) NYVGSIFDY (SEC ID Nº 17) SEC ID Nº 183

LRMR2B8HC

TYWMH (SEC ID Nº 15) EINPTNGHTNYNQKFQG (SEC ID Nº 205) NYVGSIFDY (SEC ID Nº 17) SEC ID Nº 189

[0301] La Tabla 16 resume las secuencias de CDR de la cadena ligera (definición de Kabat) de los anticuerpos 2B8 humanizados preparados por el procedimiento de humanización 1 y por el procedimiento de humanización 2 descritos anteriormente en la presente memoria en este Ejemplo.
TABLA 16

Anticuerpo

CDR1 CDR2 CDR3 Región variable de cadena ligera de longitud completa

Ligera de

KASENVVSYVS GASNRNT GQSYNYPYT SEC ID Nº 14

2B8 murino

(SEC ID Nº 18) (SEC ID Nº 19) (SEC ID Nº 20)

Hu2B8 Kv1

KASENVVSYVS GASNRNT GQSYNYPYT SEC ID Nº 173

39.1

(SEC ID Nº 18) (SEC ID Nº 19) (SEC ID Nº 20)

Hu2B8 Kv3

KASENVVSYVS GASNRNT GQSYNYPYT SEC ID Nº 179

15.1

(SEC ID Nº 18) (SEC ID Nº 19) (SEC ID Nº 20)

LR2B8LC

KASENVVSYVS GASNRNT GQSYNYPYT SEC ID Nº 193

(SEC ID Nº 18)

(SEC ID Nº 19) (SEC ID Nº 20)

LRMR2B8L

KASENVVSYVS GASNRES GQSYNYPYT SEC ID Nº 199

C

(SEC ID Nº 18) (SEC ID Nº 206) (SEC ID Nº 20)

imagen99
C. Afinidad de unión de anticuerpos 2B8 humanizados [0302] Se evaluó la afinidad de unión a antígeno y la cinética de interacción mediante tecnología de resonancia de plasmón superficial usando un instrumento BIAcore T100. Se inmovilizaron inmunoglobulinas de ratón anti-humano (Jackson ImmunoResearch Labs, 209-005-098) sobre microplacas detectoras CM4 de dextrano carboximetilado (BIAcore, Nº de Catálogo BR-1005-34) mediante acoplamiento con amina (BIAcore, Nº de Catálogo BR-1000-50) usando un protocolo de acoplamiento convencional de acuerdo con las recomendaciones del fabricante. Los análisis se realizaron a 25ºC usando PBS (GIBCO, Nº de Catálogo 14040-133) que contenía tensioactivo P20 al 0,05% (BIAcore, Nº de Catálogo BR1000-54), BSA 2 mg/ml (EMD, Nº de Catálogo 2930) y sal sódica de CM-Dextrano 10 mg/ml (Fluka, Nº de Catálogo 86524) como tampón de procesamiento. [0303] Los anticuerpos se capturaron en celdas de flujo individuales a un caudal de 10 �l/min. El tiempo de inyección era variable para cada anticuerpo para producir aproximadamente 20 UR de anticuerpo capturado para cada ciclo. Tampón o HGF (R&D Systems, Nº de Catálogo 294-HGN-025) diluido en tampón de procesamiento se inyectó secuencialmente sobre una superficie de referencia (sin anticuerpo capturado) y la superficie activa (anticuerpo a ensayar) durante dos minutos a 60

�l/min.

La fase de disociación se controló durante 15 ó 90 minutos dependiendo de la concentración. Después, la superficie se regeneró con glicina-HCl 10mM, pH 2,0 (BIAcore, Nº de Catálogo BR-1003-55) inyectado durante 3 minutos a un caudal de 60 �l/min antes de que se iniciase otro ciclo. Las concentraciones de HGF ensayadas eran de 1,88, 3,75 y 7,5 nM. La determinación de los parámetros cinéticos se consiguió usando la función cinética del software BIAevaluation con sustracción de referencia. Los parámetros cinéticos para cada anticuerpo, ka (constante de velocidad de asociación), kd (constante de velocidad de disociación) y KD (constante de disociación en equilibrio) se resumen en la Figura 8. [0304] Los resultados resumidos en la Figura 8 muestran que ciertas combinaciones de cadenas pesadas (Hu2B8 Hv5a.1, Hu2B8 Hv5-51.1 o Hu2B8 Hv1-f.1) y cadenas ligeras (Hu2B8 Kv1-39.1 o Hu2B8 Kv3-15.1) superhumanizadas conservan una afinidad de unión (KD) a HGF similar a la de 2B8 quimérico (regiones variables de ratón con regiones constantes humanas) y 2B8 (Tabla 5).

D.

Ensayo de unión mutuamente excluyente [0305] Se evaluó la unión mutuamente excluyente a HGF por tecnología de

imagen100
resonancia de plasmón superficial usando un instrumento BIAcore T100. Se inmovilizaron inmunoglobulinas de ratón anti-humano (Jackson ImmunoResearch Labs, 209-005-098) sobre microplacas detectoras CM5 de dextrano carboximetilado (BIAcore, Nº de Catálogo BR-1006-68) por acoplamiento con amina (BIAcore, Nº de Catálogo BR-1000-50) usando un protocolo de acoplamiento convencional de acuerdo con las recomendaciones del fabricante. Los análisis se realizaron a 25ºC usando PBS (GIBCO, Nº de Catálogo 14040-133) que contenía tensioactivo P20 al 0,05% (BIAcore, Nº BR-1000-54), BSA 2 mg/ml (EMD, Nº de Catálogo 2930) y sal sódica de CM-dextrano 10 mg/ml (Fluka, Nº de Catálogo 86524) como tampón de procesamiento. [0306] Los anticuerpos humanizados se capturaron en una celda de flujo individual a un caudal de 30 �l/min. El tiempo de inyección era variable para cada anticuerpo para producir aproximadamente 150 UR de anticuerpo capturado para cada ciclo. El HGF (R&D Systems, Nº de Catálogo 294-HGN-025) diluido en tampón de procesamiento a una concentración final de 7,5 �g/ml se inyectó durante 90 s a 30
�l/min sobre los anticuerpos humanizados capturados. La unión de HGF se controló antes de una inyección posterior de anticuerpo 2B8 de ratón o anticuerpo policlonal de cabra anti-HGF (R&D Systems, AF294) durante 3 min a 30 �l/min. Después, la superficie se regeneró con glicina-HCl 10 mM, pH 2,0 (BIAcore, Nº de Catálogo BR1003-55) inyectado durante 3 min a un caudal de 60 �l/min antes de que se ensayase otro anticuerpo. Los resultados se resumen en la Figura 9. [0307] Los resultados resumidos en la Figura 9 muestran que tanto los anticuerpos 2B8 humanizados como los anticuerpos 2B8 quiméricos impiden la unión de 2B8 murino a HGF. Estos resultados demuestran que los anticuerpos humanizados se unen todavía al mismo epítopo de HGF que el anticuerpo 2B8 original.
Ejemplo 13 - Producción de variantes de 2B8 humanizadas
a. Anticuerpos HUMAN ENGINEERED� [0308] Cadenas pesadas (LR2B8HC y LRMR2B8HC, respectivamente) y cadenas ligeras (LR2B8LC y LRMR2B8LC, respectivamente) Human Engineered de riesgo bajo más moderado y de bajo riesgo optimizadas para codones y expresión se clonaron en fase de lectura en vectores de expresión transitoria de anticuerpos XOMA, que contienen módulos de regiones constantes Gamma-1 y Kappa humanas. Las cuatro variantes 2B8 Human Engineered se produjeron por transfección transitoria en células HEK293E. Se produjeron los cuatro anticuerpos imagen101
siguientes: HE2B8-1 = LR2B8HC (+ región constante de IgG1 (alotipo G1m(3) (alelo 1)) (SEC ID Nº 187) más LR2B8LC (+ región constante Kappa (alotipo Km(3) (alelo 1))) (SEC ID Nº 197) HE2B8-2 = LR2B8HC (+ región constante de IgG1 (alotipo G1m(3) (alelo 1)) (SEC ID Nº 187) más LRMR2B8LC (+ región constante Kappa (alotipo Km(3) (alelo 1))) (SEC ID Nº 201) HE2B8-3 = LRMR2B8HC (+ región constante de IgG1 (alotipo G1m(3) (alelo 1)) (SEC ID Nº 191) más LR2B8LC (+ región constante Kappa (alotipo Km(3) (alelo 1))) (SEC ID Nº 197) HE2B8-4 = LRMR2B8HC (+ región constante de IgG1 (alotipo G1m(3) (alelo 1)) (SEC ID Nº 191) más LRMR2B8LC (+ región constante Kappa (alotipo Km(3) (alelo 1))) (SEC ID Nº 201)
[0309] Las cadenas ligeras y pesadas se usaron para cotransfectar células HEK293E adaptadas a suspensión de XOMA en medios IS293 (Irvine Scientific, Irvine, CA) usando matraces con agitación de 2 litros. Después de 24 horas en los matraces con agitación, se centrifugaron 200 ml de células transfectadas, se resuspendieron en 40 ml de medio recién preparado y se transfirieron a matraces Integra (Wilson Wolf Manufacturing Inc., MN) para su producción. Después de la incubación durante siete días, las suspensiones celulares se retiraron de los matraces Integra, se centrifugaron y se conservaron los sobrenadantes de cultivo. Los anticuerpos en los sobrenadantes de cultivo se purificaron sobre columnas Spin de proteína A (Pro-Chem), se dializaron contra PBS, se concentraron y se esterilizaron por filtración.
b. Anticuerpos SUPERHUMANIZED� [0310] Se clonó ADNc de Hu2B8_Hv5-51.1 de longitud completa + dominio constante de IgG1 humana (alotipo G1m(3)) en pEE6.4 (Lonza Biologics, Berkshire, Reino Unido) usando sitios de restricción HindIII y EcoRI. El ADNc de la región variable Hu2B8_Kv1-39.1 de longitud completa + dominio constante Kappa humano y el ADNc de la región variable Hu2B8_Kv3-15.1 de longitud completa + dominio constante Kappa humano se clonaron cada uno en pEE14.4 (Lonza Biologics) usando sitios de restricción HindIII y EcoRI. El ADNc de promotor de hCMV-MIE + Hu2B8_Hv5-51.1 de longitud completa + dominio constante de IgG1 humana (alotipo G1m(3)) + fragmento poli A de SV40 (en pEE6.4) se eliminó por digestión con NotI/SalI y se insertó en el vector pEE14.4 de cadena Kappa por medio de sitios NotI/SalI, generando de este modo 2 vectores de expresión diferentes, expresando cada uno simultáneamente cadena pesada y ligera para generar los imagen102
anticuerpos siguientes: 5 sh2B8-9 (G1m(3)) =
sh2B8-12 (G1m(3)) = 10
hu2B8 Hv5-51.1 (+ región constante de IgG1 (alotipo G1m(3)) (alelo 2)) (SEC ID Nº 210) más hu2B8 Kv 1-39.1 (+ región constante kappa (alotipo Km(3) (alelo 2))) (SEC ID Nº 177) hu2B8 Hv5-51.1 (+ región constante de IgG1 (alotipo G1m(3)) (alelo 2)) (SEC ID Nº 210) más hu2B8 Kv 3-15.1 (+ región constante kappa (alotipo Km(3) (alelo 2))) (SEC ID Nº 181)
[0311] Las secuencias de ácido nucleico que codifican, y las secuencias proteicas que definen la región constante pesada de IgG1 humana, alotipo G1m(3) (alelo 2), y
15 cada una de las secuencias de cadena pesada de longitud completa se exponen a continuación. Las secuencias de cadena ligera eran las mismas que se describen en el Ejemplo 12. [0312] (1) Secuencia de ácido nucleico que codifica la región constante de la cadena pesada de IgG1 humana (alotipo G1m(3)) (alelo 2) (SEC ID Nº 207). imagen103 imagen104 imagen29
[0313] (2) Secuencia proteica que define la región constante de la cadena pesada de IgG1 humana (alotipo G1m(3)) (alelo 1 ó 2) (SEC ID Nº 208). El primer aminoácido procede de la traducción del último nucleótido de la región variable y los dos nucleótidos del comienzo de la secuencia de la cadena pesada de IgG1. imagen29
[0314] (3) Secuencia de ácido nucleico que codifica la cadena de longitud completa
10 que contiene la región variable de la cadena pesada Hu2B8 Hv5-51.1 humanizada y la región constante de la cadena pesada de IgG1 humana, alotipo G1m(3) (alelo 2) (secuencia señal subrayada) (SEC ID Nº 209) [0315] (4) Secuencia proteica que define la cadena pesada de longitud completa que contiene Hu2B8 Hv5-51.1 humanizado y la región constante de la cadena pesada de IgG1 humana, alotipo G1m(3) (alelo 2) (sin secuencia señal) (SEC ID Nº 210) imagen29 imagen29 imagen29
[0316] Cada vector de expresión doble se usó para transfectar células 293T para
10 su expresión transitoria, usando DMEM con suero bovino fetal al 10%. Cuarenta y ocho horas después de la transfección, las células se lavaron con y después se sustituyeron con medio sin suero, IS GRO� (Irvine Scientific, Santa Ana, CA) que contenía L-glutamina 4 mM. Se recogió el sobrenadante diariamente y se sustituyó con medios recién preparados durante 10 días. Los sobrenadantes de cultivo se
15 centrifugaron, se filtraron (0,45 �m) y se concentraron 10-100 veces. Los anticuerpos se purificaron sobre resina ProSep vA (Millipore), se dializaron contra PBS, se concentraron y se esterilizaron por filtración.
Ejemplo 14 - Características de unión de variantes de 2B8 humanizadas
20 [0317] Los anticuerpos humanizados producidos en el Ejemplo 13 se caracterizaron por su capacidad para unirse a hHGF y a las proteínas HGF recombinantes producidas en el Ejemplo 3. [0318] Los anticuerpos se analizaron por resonancia de plasmón superficial usando un instrumento BIAcore T100 para evaluar su capacidad para unirse a hHGF y las
25 proteínas de fusión analizadas en el Ejemplo 3. Cada anticuerpo se inmovilizó en una microplaca detectora CM5 de dextrano carboximetilado (BIAcore, Nº de Catálogo BR-1006-68) por acoplamiento con amina (BIAcore, Nº de Catálogo BR1000-50) usando un protocolo de acoplamiento convencional de acuerdo con las instrucciones del fabricante. [0319] Los análisis se realizaron a 25ºC usando PBS (GIBCO, Nº de Catálogo imagen105
5 14040-133) que contenía tensioactivo P20 al 0,05% (BIAcore, Nº de Catálogo R1000-54), BSA 2 mg/ml (EMD, Nº de Catálogo 2930) y sal sódica de CM-dextrano 10 mg/ml (Fluka, Nº de Catálogo 86524) como tampón de procesamiento. El sobrenadante que contenía diferentes proteínas de fusión de HGF o el sobrenadante de células transfectadas con vector vacío se inyectó sobre cada
10 anticuerpo a un caudal de 30 �l/min durante 3 minutos. La unión resultante se determinó como unidades de resonancia (UR) sobre el nivel basal 30 segundos después finalizar la inyección. Se comparó la unión a HGF humano (R&D Systems, Nº de Catálogo 294-HGN-025) diluido en tampón de procesamiento. Se controló la unión inespecífica por comparación de la unión a una superficie de control. Los
15 resultados se resumen en la Tabla 17.
TABLA 17

Anticuerpo

rhHGF (R&D Systems) mHGF (R&D Systems) Quimera MHM (495585) Quimera MHM (507585) Quimera MHM (499556)

2B8

Sí No Sí Sí Sí

HE2B8-1

Sí No Sí Sí Sí

HE2B8-2

Sí No Sí Sí Sí

HE2B8-3

Sí No Sí Sí Sí

HE2B8-4

Sí No Sí Sí Sí

sh2B8-9 (G1m(3))

Sí No Sí Sí Sí

sh2B8-12 (G1m(3))

Sí No Sí Sí Sí

[0320] Los resultados de la Tabla 17 demuestran que cada uno de los anticuerpos basados en 2B8 humanizados se une a rhHGF y las tres quimeras de ratón
20 humano-ratón.
Ejemplo 15 - Afinidades de unión de variantes de 2B8 humanizadas [0321] Las afinidades de unión y las cinéticas de interacción de los anticuerpos enumerados en la Tabla 15 se midieron por resonancia de plasmón superficial. [0322] Se inmovilizaron inmunoglobulinas de ratón anti-humano (Jackson Labs, Nº de Catálogo 209-005) sobre microplacas detectoras CM5 de dextrano carboximetilado (BIAcore, Nº de Catálogo BR-1006-68) por acoplamiento con amina imagen106
5 (BIAcore, Nº de Catálogo BR-1000-50) usando un protocolo de acoplamiento convencional de acuerdo con las instrucciones del fabricante. Los análisis se realizaron a 25ºC usando PBS (GIBCO, Nº de Catálogo 14040-133) que contenía tensioactivo P20 al 0,05% (BIAcore, Nº de Catálogo BR-1000-54) y BSA 2 mg/ml (EMD, Nº de Catálogo 2930).
10 [0323] Los anticuerpos se capturaron en una celda de flujo individual a un caudal de 10 �l/min. El tiempo de inyección era variable para cada anticuerpo para producir aproximadamente 20 UR de anticuerpo capturado para cada ciclo. Tampón o HGF (R&D Systems, Nº de Catálogo 294-HGN-025) diluido en tampón de procesamiento se inyectó secuencialmente sobre una superficie de referencia (sin anticuerpo
15 capturado) y la superficie activa (anticuerpo a ensayar) durante 2 minutos a 60
�l/min. La fase de disociación se controló durante 15 ó 90 minutos dependiendo de la concentración. Después, la superficie se regeneró con glicina-HCl 10 mM, pH 2,2 (BIAcore, Nº de Catálogo BR-1003-54) inyectado durante 3 minutos a un caudal de 60 �l/min antes de que se iniciase otro ciclo. Las concentraciones de HGF
20 ensayadas eran de 0,46 nM a 7,5 nM. [0324] Se determinaron los parámetros cinéticos usando la función cinética del software BIAevaluation� con sustracción de referencia. Los parámetros cinéticos para cada anticuerpo, ka (constante de velocidad de asociación), kd (constante de velocidad de disociación) y KD (constante de disociación en equilibrio) se resumen
25 en la Tabla 18.
TABLA 18

Anticuerpo

ka (1/Ms) kd (1/s) KD (pM) DT

2B8

1,4 x 106 1,0 x 10-5 7,3

HE2B8-1

2,2 x 106 1,4 x 10-5 7,1 5,2

HE2B8-2

1,8 x 106 9,6 x 10-6 5,2 2,7

HE2B8-3

2,0 x 106 4,1 x 10-6 2,0 1,1

HE2B8-4

1,7 x 106 1,1 x 10-5 6,5 1,3

sh2B8-9 (G1m(17,1)

2,0 x 106 1,7 x 10-5 8,1 5,3

sh2B8-12 (G1m(17,1)

1,9 x 106 2,3 x 10-5 12 0,4

imagen107
[0325] Estos datos muestran que los anticuerpos humanizados tienen velocidades de asociación rápidas (ka), velocidades de disociación muy lentas (kd) y afinidades muy elevadas (KD). En particular, los anticuerpos tienen afinidades que varían de 2,0-12 pM.
Ejemplo 16 - Comparación de las afinidades de unión a 25ºC y 37ºC [0326] Las afinidades de unión y las cinéticas de interacción de los anticuerpos HE2B8-4, sh2B8-9, sh2B8-12 y 2B8 murino se midieron por resonancia de plasmón superficial en condiciones diferentes. [0327] Se inmovilizaron inmunoglobulinas de ratón anti-humano (Jackson Labs, Nº de Catálogo 209-005) o inmunoglobulinas de conejo anti-ratón (BIAcore, Nº de Catálogo BR-1005-14) sobre microplacas detectoras CM4 de dextrano carboximetilado (BIAcore, Nº de Catálogo BR-1006-68) por acoplamiento con amina (BIAcore, Nº de Catálogo BR-1000-50) usando un protocolo de acoplamiento convencional de acuerdo con las instrucciones del fabricante. En el caso de mediciones a 25ºC para sh2b8-9 y sh2B8-12, se usó una microplaca detectora CM5 (BIAcore, Nº de Catálogo BR-1006-68). Los análisis se realizaron a 25ºC y 37ºC usando PBS (GIBCO, Nº de Catálogo 14040-133) que contenía tensioactivo P20 al 0,05% (BIAcore, Nº de Catálogo BR-1000-54) y BSA a 2 mg/ml (EMD, Nº de Catálogo 2930) como tampón de procesamiento. [0328] Los anticuerpos se capturaron en una celda de flujo individual a un caudal de 10 �l/min. El tiempo de inyección era variable para cada anticuerpo para producir aproximadamente 20 UR de anticuerpo capturado para cada ciclo. Tampón o HGF (R&D Systems, Nº de Catálogo 294-HGN-025) diluido en tampón de procesamiento se inyectó secuencialmente sobre una superficie de referencia (sin anticuerpo capturado) y la superficie activa (anticuerpo a ensayar) durante 2 minutos a 60
�l/min. La fase de disociación se controló durante 15 ó 90 minutos dependiendo de la concentración. La superficie de las microplacas detectoras con inmunoglobulinas de ratón anti-humano se regeneró después con glicina-HCl 10 mM, pH 2,2 (BIAcore, Nº de Catálogo BR-1003-54) inyectado durante 3 minutos a un caudal de 60 �l/min antes de que se iniciase otro ciclo. La superficie de las microplacas detectoras con inmunoglobulinas de conejo anti-ratón se regeneró con glicina-HCl 10 mM, pH 1,7 (BIAcore, Nº de Catálogo BR-1003-54) inyectado durante 3 minutos a un caudal de 60 �l/min antes de que se iniciase otro ciclo. Las concentraciones de HGF ensayadas eran de 0,46 nM a 7,5 nM. imagen108
[0329] Se determinaron los parámetros cinéticos usando la función cinética del software BIAevaluation con sustracción de referencia. Los parámetros cinéticos para cada anticuerpo, ka (constante de velocidad de asociación), kd (constante de velocidad de disociación) y KD (constante de disociación en equilibrio) se resumen a continuación en la Tabla 19.
TABLA 19

Anticuerpo

Temp. (ºC) ka (1/MS) kd (1/s) KD(pM)

2B8

25 1,6 x 106 2,1 x 10-5 13,5

2B8

37 2,8 x 106 1,3 x 10-5 4,5

HE2B8-4

25 2,0 x 106 1,2 x 10-5 5,6

HE2B8-4

37 3,1 x 106 1,0 x 10-5 3,3

sh2B8-9 (G1m(17,1))

25 2,0 x 106 1,7 x 10-5 8,1

sh2B8-9 (G1m(3))

37 2,5 x 106 1,4 x 10-5 5,8

sh2B8-12 (G1m(17,1))

25 1,9 x 106 2.,3 x 10-5 12,0

sh2B8-12 (G1m(3))

37 2,4 x 106 1,0 x 10-5 4,8

[0330] Como se esperaba, las constantes de velocidad de asociación aumentaban con un aumento en la temperatura. Sorprendentemente, las constantes de
10 disociación no cambiaban significativamente con un aumento en la temperatura correspondiente. Por consiguiente, las constantes de disociación en equilibrio (KD) globales eran de aproximadamente 1,4 a 3 veces menores (mayor afinidad) a temperatura fisiológica (37ºC).
15 Ejemplo 17 - Actividad de neutralización de variantes de 2B8 humanizadas [0331] Los anticuerpos descritos en el Ejemplo 14 se caracterizaron por su capacidad para (a) inhibir la unión de hHGF a c-Met y (b) inhibir la incorporación de BrdU estimulada por HGF en células 4MBr-5. [0332] Se realizó un ensayo de inhibición de la unión de HGF-Met (Ensayo de
20 neutralización) como se describe a continuación. Los anticuerpos se ensayaron mediante ELISA para determinar su capacidad para inhibir la unión de hHGF a c-Met. En concreto, se recubrieron placas de ensayo DELFIA de 96 pocillos Wallac (Wallac Inc., Nº de Catálogo AAAND-0001) con 100 �l de HGF 6,25 �g/ml (R&D Systems, Nº de Catálogo 294-HGN-025) en tampón de recubrimiento de carbonato (Na2CO3 15 mM y NaHCO3 34 mM, pH 9,0) durante 16 horas a 4ºC. Después, las placas se bloquearon con 200 �l de leche desnatada en polvo al 5% en PBS imagen109
5 durante 1 hora a temperatura ambiente. Los anticuerpos se prepararon en una placa separada por adición de concentraciones crecientes de los anticuerpos en investigación (0,033-250 nM, dilución en serie de 2 veces) a c-Met biotinilado 2 nM en leche desnatada en polvo al 5% en PBS. Se biotiniló el c-Met (R&D Systems, Nº de Catálogo 358-MT/CF) de acuerdo con las instrucciones del fabricante a una
10 proporción de biotina respecto a c-Met de 10:1 (Pierce, Nº de Catálogo 21335). Se transfirieron 100 �l de muestra por pocillo a la placa de ensayo y se incubaron durante 2 horas a temperatura ambiente. Las placas resultantes se lavaron tres veces con PBS-Tween 20 al 0,1% y se incubaron durante 1 hora a temperatura ambiente con estreptavidina marcada con Eu (Wallac, Nº de Catálogo 1244-360)
15 diluida 1:1000 en tampón de ensayo DELFIA (Wallac, Nº de Catálogo 4002-0010). Las placas resultantes se lavaron 3 veces con solución de lavado DELFIA (Wallac, Nº de Catálogo 4010-0010) y se incubaron con solución de potenciación DELFIA 100 �l/pocillo (Wallac, Nº 4001-0010) durante 15 minutos a temperatura ambiente con agitación. Las placas se leyeron en un instrumento Victor3V (Perkin Elmer)
20 usando el método de europio. Los valores de CI50 se calcularon usando Prism. [0333] Los valores de CI50 obtenidos se muestran en la Tabla 20. TABLA 20

Anticuerpo

CI50 (nM) DT

2B8

9,2 1,2

HE2B8-1

6,0 1,2

HE2B8-2

5,7 1,1

HE2B8-3

5,9 1,1

HE2B8-4

6,5 1,2

sh2B8-9 (G1m(3))

4,2 -

sh2B8-12 (G1m(3)

6,8 -

[0334] Estos resultados de la Tabla 20 demuestran que los anticuerpos
25 humanizados ensayados neutralizan eficazmente la unión de HGF a c-Met. [0335] Los anticuerpos de la Tabla 17 se ensayaron también en el ensayo de proliferación celular descrito en el Ejemplo 7(b). Los resultados se resumen a imagen110
continuación en la Tabla 21.
TABLA 21

Anticuerpo

CI50 (nM) DT

2B8

0,86 0,35

HE2B8-1

0,47 0,15

HE2B8-2

0,66 0,13

HE2B8-3

0,55 0,28

HE2B8-4

0,58 0,26

sh2B8-9 (G1m(3))

0,52 0,11

sh2B8-12 (G1m(3))

0,81 0,22

[0336] Los resultados de la Tabla 21 demuestran que todos los anticuerpos 5 humanizados ensayados inhiben la proliferación inducida por HGF de células 4MBr-5.
Ejemplo 18 - Actividad anti-dispersión de variantes de 2B8 humanizadas [0337] Los anticuerpos de la Tabla 17 se ensayaron en el ensayo de anti-dispersión 10 descrito en el Ejemplo 8. Los resultados se resumen a continuación en la Tabla 22.
TABLA 22

Inhibición de la dispersión de células MDCK inducida por HGF

Anticuerpo

Ensayo 1 Ensayo 2

2B8

++ ++

HE2B8-1

++ ++

HE2B8-2

++ ++

HE2B8-3

++ ++

HE2B8-4

++ ++

sh2B8-9 (G1m(3))

++ ++

sh2B8-12 (G1m(3))

++ ++

– Sin Inhibición +++ Inhibición muy fuerte, casi completa ++ Inhibición fuerte
+ Inhibición detectable [0338] Los resultados de la Tabla 22 demuestran que todos los anticuerpos humanizados ensayados inhibían la dispersión inducida por HGF en la misma medida que el anticuerpo monoclonal murino 2B8. imagen111
Ejemplo 19 - Inhibición de la fosforilación de c-Met estimulada por HGF [0339] Los anticuerpos de la Tabla 17 se ensayaron en el ensayo de fosforilación de c-Met descrito en el Ejemplo 9. Los resultados se resumen a continuación en la Tabla 23.
TABLA 23

Anticuerpo

Promedio de dos ensayos Desviación típica

2B8

0,91 0,02

he2B8-1

0,80 0,04

he2B8-2

0,88 0,15

he2B8-3

0,79 0,05

he2B8-4

0,75 0,14

sh2B8-9 (G1m(3))

0,93 0,03

sh2B8-12 (G1m(3))

0,81 0,07

[0340] Los resultados de la Tabla 23 demuestran que todos los anticuerpos 10 humanizados ensayados eran potentes inhibidores de la fosforilación de c-Met inducida por HGF en células PC-3.
Ejemplo 20 - Inhibición de tumores en el modelo de xenoinjerto U87MG [0341] La capacidad de los anticuerpos monoclonales humanizados de la invención
15 para inhibir el crecimiento de tumores se ensayó en un modelo de xenoinjerto U87MG. Las células U87MG (ATCC) se expandieron en cultivo a 37ºC en una atmósfera que contenía CO2 al 5% y aire al 95%, usando un medio que comprendía medio de Eagle modificado por Dulbecco (DMEM) con suero bovino fetal al 10%, penicilina 100 unidades/ml y estreptomicina 100 �g/ml. Las células se subcultivaron
20 y se mantuvieron por desprendimiento de las células de la pared de la placa de cultivo usando tripsina-EDTA. [0342] Se recogieron células casi confluentes por tratamiento con tripsina y después se inyectaron 5 x 106 células en Matrigel al 50% (BD Biosciences; nº de catálogo 356237) subcutáneamente en el área dorsal superior entre las escápulas
25 de ratones ICR SCID hembra de 7 semanas de edad (Taconic Labs). Los diámetros largo (L) y corto (W) (mm) de los tumores se midieron con un calibrador. Se calculó el volumen tumoral (vol.) como: volumen (mm3) = L x W2 / 2. Cuando los tumores crecieron hasta aproximadamente 200 mm³, los ratones que portaban tumores se aleatorizaron en 5 grupos de 10 ratones cada uno. Un grupo recibió PBS y un grupo recibió control de IgG humana. Cada uno de los otros 4 grupos recibió uno de los anticuerpos humanizados (HE2B8-1, HE2B8-2, HE2B8-3 y HE2B8-4). Todos los imagen112
5 anticuerpos se dosificaron a 0,25 mg/kg de peso corporal dos veces por semana, por inyección intraperitoneal de 5 dosis. Se registraron los volúmenes tumorales y los pesos corporales de los ratones dos veces por semana. La inhibición del crecimiento tumoral se analizó usando la prueba t de Student. [0343] Los anticuerpos humanizados ensayados eran activos in vivo. Había una
10 inhibición del crecimiento tumoral del 57% para HE2B8-1 con un valor p de 0,02, una inhibición del crecimiento tumoral del 61% para HE2B8-2 con un valor p de 0,02, una inhibición del crecimiento tumoral del 85% para HE2B8-3 con un valor p de 0,0004 y una inhibición del crecimiento tumoral del 74% para HE2B8-4 con un valor p de 0,001. No se observaron pérdidas de peso corporal significativas. 7,022,500 (Queen); 6,982,321 (Winter); 6,180,370 (Queen); 6,054,297 (Carter);
5,693,762 (Queen); 5,859,205 (Adair); 5,693,761 (Queen); 5,565,332 (Hoogenboom); 5,585,089 (Queen); 5,530,101 (Queen); Jones et al (1986) NATURE 321: 522-525; Riechmann et al. (1988) NATURE 332: 323-327; Verhoeyen et al. (1988) SCIENCE 239: 1534-1536; and Winter (1998) FEBS LETT
430: 92-94. [0067] In a strategy called "superhumanization," antibodies are created in which human immunogenicity is reduced or eliminated by an alternative form of grafting. In superhumanization, human FR sequences are selected from a set of human germline genes based on the structural similarity of human CDRs with those of the mouse antibody to be humanized. This strategy is described, for example, in US Patent No. 6,881,557 (Foote) and in Tan et al (2002) J. IMMUNOL 169: 1119-1125. [0068] Other strategies to reduce immunogenicity include techniques known as "reconformation,""hyper-polymerization," or "coating / surface coating" to produce humanized antibodies. See, for example, Vaswami et al. (1998) ANNALS OF ALLERGY, ASTHMA, & IMMUNOL. 81: 105; Roguska et al. (1996) PROT. ENGINEER 9: 895-904; and U.S. Patent No. 6,072,035 (Hardman). In the coating / surface coating strategy, the surface accessible amino acid residues in the murine antibody are replaced by amino acid residues that are more frequently found in the same positions in a human antibody. This type of surface coating of antibodies is described, for example, in US Patent No. 5,639,641 (Pedersen). [0069] An exemplary strategy for converting a mouse antibody into a form suitable for medical use in humans is known as ACTIVMAB� technology (Vaccinex, Inc., Rochester, NY), which implies that a vaccinia virus-based vector expresses antibodies. in mammalian cells. It is said that high levels of combinatorial diversity of heavy and light chains of immunoglobulin are produced. See, for example, United States Patents No. image18
6,706,477 (Zauderer); 6,800,442 (Zauderer); and 6,872,518 (Zauderer). [0070] Another exemplary strategy for converting a mouse antibody into a form suitable for use in humans is the technology commercially practiced by KaloBios Pharmaceuticals, Inc. (Palo Alto, CA). This technology involves the use of a proprietary human "acceptor" library to produce an "epitope-centered" library for antibody selection. [0071] Another exemplary strategy to modify a mouse antibody in a manner suitable for medical use in humans is the HUMAN ENGINEERING� (HE�) technology, which is commercially implemented by XOMA (United States) LLC. See, for example, International Application Publication No. WO 93/11794 and United States Patents No. 5,766,886; 5,770,196; 5,821,123; and 5,869,619. [0072] Any suitable strategy, including any of the above strategies, can be used to reduce or eliminate human immunogenicity of a binding protein of interest. [0073] In addition, it is possible to create fully human antibodies in mice. In this strategy, human antibodies are prepared using a transgenic mouse in which the mouse antibody producing genes have been replaced by a substantial part of the human antibody producing genes. Such mice produce human immunoglobulin instead of murine immunoglobulin molecules. See, for example, WO 98/24893 (Jacobovitz et al.) And Mendez et al. (1997) NATURE GENETICS 15: 146-156. Fully human anti-HGF monoclonal antibodies can be produced using the following strategy. Transgenic mice that contain human immunoglobulin genes are immunized with the antigen of interest, for example, HGF. The lymphatic cells of the mice are then obtained from the mice, which are then fused with a myeloid-like cell line to prepare immortal hybridoma cell lines. Hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce specific antibodies against HGF. [0074] The binding proteins of the invention can be conjugated with other molecules depending on their desired use. For example, if the binding protein is to be used as a therapeutic product, then the binding protein can be conjugated with another agent, for example, an effector molecule that modulates or otherwise promotes the therapy. To the extent that the effector is a non-protein based agent, for example, a small molecular drug, a radiolabel or toxin, then the agent can be chemically coupled to the binding protein using conventional in vitro coupling chemicals. If, on the other hand, the effector molecule is a protein or peptide, for example, an enzyme, receptor, toxin, growth factor, cytokine or other immunomodulator, then the binding protein can be chemically coupled to the effector using in vitro coupling chemicals or it can be coupled to the effector as a fusion protein. Fusion proteins can be constructed and expressed using techniques similar to those discussed in section II. image19
IV - Use of binding proteins [0075] The binding proteins described herein can be used as a diagnostic agent or as a therapeutic agent.
(1) Therapeutic Applications [0076] Because the binding proteins of the invention neutralize the activity of HGF, they can be used in various therapeutic applications. For example, certain binding proteins of the invention are useful in the prevention or treatment of hyperproliferative diseases or disorders, for example, various forms of cancer. [0077] Binding proteins can be used to inhibit or reduce the proliferation of tumor cells. In such a strategy, the tumor cells are exposed to a therapeutically effective amount of the binding protein to inhibit or reduce the proliferation of the tumor cell. In certain embodiments, the binding proteins inhibit the proliferation of tumor cells by at least 50%, 60%, 70%, 80%, 90%, 95% or 100%. [0078] In certain embodiments, the binding protein is used to inhibit or reduce the proliferation of a tumor cell, in which the binding protein reduces the ability of hHGF to bind to c-Met. The binding protein can be used to inhibit or reduce the proliferation of a tumor cell even when the binding protein binds to hHGF but does not substantially inhibit the binding of hHGF to c-Met, as shown by the 3B6 antibody in Tables 5 and 6. image20
[0079] In addition, the binding protein can be used to inhibit or slow the growth or development of tumors in a mammal. In said method, an effective amount of the mammalian binding protein is administered to inhibit or slow tumor growth in the mammal. Accordingly, binding proteins can be used to treat tumors, for example, in a mammal. The method comprises administering to the mammal a therapeutically effective amount of the binding protein. The binding protein can be administered alone or in combination with another pharmaceutically active molecule, to treat the tumor. [0080] It is contemplated that the binding proteins of the invention can be used in the treatment of a variety of HGF-sensitive disorders including, for example, HGF-sensitive tumor cells in lung cancer, breast cancer, colon cancer, cancer of prostate, ovarian cancer, head and neck cancer, ovarian cancer, multiple myeloma, liver cancer, gastric cancer, esophageal cancer, renal cancer, nasopharyngeal cancer, pancreatic cancer, mesothelioma, melanoma and glioblastoma. [0081] As used herein, the terms "treat", "treating" and "treatment" refer to the treatment of a pathological state in a mammal, particularly in a human being, and include: (a) preventing that the pathology appears in a mammal, in particular, when said mammal is predisposed to the pathology but has not yet been diagnosed as suffering from it; (b) inhibit the pathology, that is, stop its development; and / or (c) alleviate the pathology, that is, cause the regression of the pathology. [0082] Generally, a therapeutically effective amount of active component will be in the range of about 0.1 mg / kg to about 100 mg / kg, optionally from about 1 mg / kg to about 100 mg / kg, optionally about 1 mg / kg to 10 mg / kg. The amount administered will depend on variables such as the type and degree of the disease or indication to be treated, the general health status of the particular patient, the relative biological efficacy of the administered binding protein, the formulation of the binding protein, the presence and types of excipients in the formulation and route of administration. The initial dosage administered can be increased beyond the upper level to quickly reach the desired blood level or tissue level,
or the initial dosage may be less than optimal and the daily dosage may be progressively increased during the course of the treatment depending on the particular situation. The human dosage can be optimized, for example, in a study of scale-up of the conventional Phase I dose designed to run from 0.5 mg / kg to 20 mg / kg. The dosage frequency may vary depending on factors such as the route of administration, the amount of dosage and the pathological condition to be treated. They are exemplary dosing frequencies once a day, once a week and once every two weeks. A preferred route of administration is parenteral, for example, intravenous infusion. The formulation of drugs based on monoclonal antibodies is included in the specialty in the art. In some embodiments of the invention, the binding protein, for example, monoclonal antibody, is lyophilized and reconstituted in buffered saline at the time of administration. [0083] Binding proteins can be administered alone or in combination with other pharmaceutically active ingredients. The other active ingredients, for example, immunomodulators, can be administered together with the binding protein or can be administered before or after the binding protein. [0084] Formulations containing the binding proteins for therapeutic use typically include the binding proteins combined with a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" refers to buffers, vehicles and excipients that are, within the scope of good medical judgment, suitable for use in contact with the tissues of humans and animals without toxicity, irritation, allergic response or other excessive problem or complication, in proportion to a reasonable benefit / risk ratio. The vehicle or vehicles should be "acceptable" in the sense of being compatible with the other ingredients of the formulations and not harmful to the recipient. Pharmaceutically acceptable carriers, in this regard, are intended to include any and all buffers, solvents, dispersion media, coatings, isotonic and absorption retarding agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art. [0085] The formulations may conveniently be presented in a unit dosage form and may be prepared by any suitable method, including any of the methods well known in the pharmaceutical art. A pharmaceutical composition of the invention should be formulated to be compatible with its desired route of administration. Examples of routes of administration include parenteral administration or non-parenteral administration, for example, intravenous, intradermal, inhalation, transdermal (topical), transmucosal and rectal administration. Solutions useful for oral or parenteral administration can be prepared by any of the methods well known in the pharmaceutical art described, for example, in Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990). [0086] Other formulations suitable for oral administration may be in the form of: discrete units such as injectables, capsules, gelatin capsules, sachets, tablets, troches or dragees, each containing a predetermined amount of the binding protein; a powder or granular composition; a solution or suspension in an aqueous liquid or non-aqueous liquid; or an oil-in-water emulsion or a water-in-oil emulsion. [0087] Formulations suitable for parenteral administration include, for example, the following components: a sterile diluent such as water for injection, saline solution, nonvolatile oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparation can be included in ampoules, disposable syringes or multi-dose vials made of glass or plastic. [0088] In general, compositions suitable for injectable use include aqueous solutions (water soluble) or dispersions and powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable vehicles include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). It should be stable under the conditions of manufacture and storage and should be preserved from the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol) and suitable mixtures thereof. [0089] Pharmaceutical formulations are preferably sterile. Sterilization can be achieved, for example, by filtration through sterile filtration membranes. When the composition is lyophilized, sterilization can be performed using this method before or after lyophilization and reconstitution. Once the pharmaceutical composition has been formulated, it can be stored, for example, in vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. image21 image22 image23

(2)

Diagnostic applications [0090] When binding proteins are used for diagnostic purposes, in vitro or in vivo, binding proteins are typically labeled directly or indirectly with a detectable moiety. The detectable moiety can be any moiety that is capable of directly or indirectly producing a detectable signal. For example, the detectable moiety can be a radioisotope, such as 3 Hydrogen (3H), 14 Carbon (14C), 32 Phosphorus (32P), 35 Sulfur (35S) or 125 Iodo (l25I); a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine or luciferin; an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase; a spin probe, such as a spin marker; or a colored particle, for example a latex or gold particle. It is understood that the binding protein can be conjugated to the detectable moiety using several strategies known in the art, for example, as described in Hunter et al. (1962) NATURE 144: 945; David et al. (1974) BIOCHEMISTRY 13: 1014; Pain et al. (1981)

J.

IMMUNOL METH 40: 219; and Nygren (1982) J. HISTOCHEM. AND CYTOCHEM.

30: 407 Markers can be detected, for example, visually or with the help of a spectrophotometer or other detector. [0091] Binding proteins can be employed in a wide variety of immunoassay techniques available in the art. Exemplary immunoassays include, for example, sandwich immunoassays, competitive immunoassays, immunohistochemical procedures. [0092] In a sandwich immunoassay, two antibodies are used that bind to an analyte or antigen of interest, for example, one immobilized on a solid support and one free in solution and labeled with a detectable moiety. When a sample containing the antigen is introduced into this system, the antigen binds both the immobilized antibody and the labeled antibody to form a "sandwich" immune complex on the surface of the support. The protein that forms a complex is detected by washing away the unbound sample components and the excess of labeled antibody, and measuring the amount of labeled antibody that forms a complex with protein on the surface of the support. Alternatively, the free antibody in solution can be detected by a third antibody labeled with a detectable moiety that binds to the free antibody. A detailed review of the design, theory and protocols of the immunological assay can be found in numerous texts, including Butt, ed., (1984) PRACTICAL IMMUNOLOGY, Marcel Dekker, New York; Harlow et al eds. (1988) ANTIBODIES, A LABORATORY APPROACH, Cold Spring Harbor Laboratory; and Diamandis et al., eds. (1996) IMMUNOASSAY, Academic Press, Boston. [0093] It is contemplated that labeled binding proteins are useful as in vivo imaging agents, whereby binding proteins can direct imaging agents to particular tissues of interest in the recipient. A remotely preferred detectable moiety for in vivo imaging includes the radioactive atom Technetium-99m (99mTc), a gamma emitter with a half-life of approximately six hours. Non-radioactive moieties that are also useful in in vivo imaging include spin markers of nitroxide as well as transition metal ions and lanthanides, all inducing proton relaxation in situ. In addition to the formation of immunoimaging, radioactive residues that form complexes can be used in conventional radioimmunotherapy protocols to destroy target cells. Preferred nucleotides for high dose radioimmunotherapy include radioactive atoms 90Itrium (90Yt), 131Iodo (131I) and 111Indium (111ln). The binding protein can be labeled with 1311, 111In and 99mTC using coupling procedures known in the art of imaging. Similarly, methods for preparing and administering the imaging agent, as well as capturing and processing images, are well known in the art of imaging, and therefore are not discussed in detail herein. Similarly, methods for performing antibody-based immunotherapies are well known in the art. See, for example, U.S. Patent No. 5,534,254. [0094] Throughout the description, in which compositions are described which have, which include or comprise specific components, it is contemplated that the compositions also consist essentially of, or consist of, the enumerated components. Similarly, when it is described that the procedures have, or include or comprise specific process steps, the procedures also consist essentially of, or consist of the processing steps listed. Except where otherwise indicated, the order of the stages or the order to perform certain actions is irrelevant as long as the invention continues to be realizable. In addition, unless otherwise indicated, two or more stages or actions may be performed simultaneously. image24 image25
EXAMPLES [0095] The following Examples analyze the production and characterization of several anti-hHGF monoclonal antibodies. Sequences that are not within the scope of the claims are included as reference.
Example 1 - Production of anti-hHGF monoclonal antibodies [0096] This Example describes the production of various anti-hHGF monoclonal antibodies. [0097] Immunizations, fusions, and primary scans were performed at MBS Inc. (Portland, ME), following the Multiple Repeat Immunization Sites (RIMMS) protocol. Five AJ mice and five Balb / c mice were immunized with recombinant human HGF (R&D Systems, Minneapolis, MN; Catalog No. 294HGN-025). Two mice were chosen with sera that had the highest anti-HGF activity by Enzyme-linked Immunoabsorbent Assay (ELISA) for subsequent fusion. Spleens and lymph nodes were collected from the appropriate mice. Then B cells were collected and fused with a myeloma line. The fusion products were serially diluted in one or more plates until almost clonal. The supernatants of the resulting fusions were screened to determine their hHGF binding by ELISA. Supernatants that were identified to contain antibodies against HGF were further characterized by functional in vitro assay as analyzed in the following examples. A panel of hybridomas was selected and the hybridomas were subcloned and expanded. The monoclonal antibodies were then purified by affinity chromatography on Protein A / G resin under conventional conditions.
Example 2 - Sequence analysis of anti-hHGF monoclonal antibodies [0098] This Example describes the sequence and isotype analysis of image26
anti-hHGF monoclonal antibodies produced in Example 1.
to. Determination of murine monoclonal antibody isotypes of HGF [0099] The heavy chain and light chain type isotype of each monoclonal antibody was determined using the IsoStrip mouse monoclonal antibody isotyping kit according to the manufacturer's instructions (Roche Applied Science ). [0100] All antibodies were determined to contain a Kappa immunoglobulin light chain and an IgG1 immunoglobulin heavy chain.
b. Determination of nucleotide sequences encoding regions
immunoglobulin light and heavy chain variables [0101] Total RNA was extracted from each monoclonal hybridoma cell line using the RNeasy mini-preparation kit according to the manufacturer's instructions (Qiagen Venlo, The Netherlands). Full-length first-chain cDNA was generated using the RACE BD SMART cDNA amplification kit according to the manufacturer's instructions (Clontech) using BD SMART II A (5 'aagcagtggtatcaacgcagagtacgcggg 3') oligonucleotide primers (SEQ ID NO. 85) and the RACE-5 'CDS primer (5' tttttttttttttttttttttttttvn 3 ', where v = a, g or cyn = a, g, c or t) (SEQ ID No. 86) for the purpose of RACE 5' (Amplification Rapid cDNA Ends). [0102] The variable regions of the Kappa and heavy immunoglobulin (IgG1) chains were amplified by PCR (Polymerase Chain Reaction) using the Expand Fidelity PCR System (Roche Applied Science) according to the manufacturer's instructions . The variable regions of the heavy chain were amplified with the mixture of oligonucleotide primers 5 'Universal Primer Mix A (mixture of 5' ctaatacgactcactatagggcaagcagtggtatcaacgcagagt 3 '(SEQ ID No. 87) and 5' ctaatacgactcactatagggc 3 'and SEQ ID No. 88) (SEQ ID No. 88) a specific primer of the 3 ', 5' tatgcaaggcttacaaccaca 3 'IgG1 Constant Region (SEQ ID No. 89) or 5' gccagtggatagacagatgggggtgtcg 3 '(SEQ ID No. 90). Variable regions of the kappa chain were amplified with the mixture of oligonucleotide primers 5 'Mix of Universal Primers A and a specific primer of the Kappa Constant Region 3', 5 'ctcattcctgttgaagctcttgacaat 3' (SEQ ID No. 91) or 5 'cgactgaggcacctccagatgtt 3 '(SEQ ID No. 92). [0103] Individual PCR products were fractionated by agarose gel electrophoresis and purified using the Qiaquick Gel Purification kit according to the manufacturer's instructions (Qiagen). PCR products were subsequently cloned into plasmid pCR2.1 TOPO using the TOPO TA Cloning�Kit topoisomerase-based cloning kit (with pCR�2.1-TOPO� vector) according to the manufacturer's instructions (Invitrogen, Carlsbad, CA ) and were used to transform DH5 bacteria using conventional transformation techniques. Plasmid DNA isolated from transformed bacterial clones was sequenced using primers T7 (5 'TAATACGACTCACTATAGGG 3') (SEQ ID No. 93), M13 Direct (5 'GTAAAACGACGGCCAGT 3') (SEQ ID No. 94) and M13 Inverse (5 'CAGGAAACAGCTATGACC 3 ') (SEQ ID No. 95) by Agencourt Bioscience using conventional dideoxy DNA sequencing methods to identify the sequence of the variable region sequences. The sequences were analyzed using Vector NTI software (Invitrogen, Carlsbad, CA) and the IMGT / V-Quest web server (http://imgt.cines.fr/textes/vquest) to identify and confirm sequences from the variable region. image27
C. Determination of nucleotide sequences encoding sequences of the immunoglobulin light and heavy chain constant region for the IgG1 and Kappa chains of 1A3, 1D3, 1F3 and 2B8
[0104] Full length cDNAs for the IgG1 chains of 1A3, 1D3 and 1F3 were amplified from the cDNA previously created using the direct primer 5 'ggggacaagtttgtacaaaaaagcaggctgccaccatgaactttgggctcagattgattttcc 3' (underline start codon No. 96) (SEC 96 start codon) and the reverse primer 5 'ggggaccactttgtacaagaaagctgggttcatttaccaggagagtgggagagg 3' (underline the termination codon) (SEQ ID No. 97). the full length cDNA was amplified for chain IgG1 2B8 from the cDNA created above using the forward primer 5'ggggacaagtttgtacaaaaaagcaggctgccaccatgggatggagctatatcatcctcttt 3 '(underlined the start codon) (SEQ ID NO: 98) and reverse primer 5'ggggaccactttgtacaagaaagctgggttcatttaccaggagagtgggagag 3 '(underline the termination codon) (SEQ ID No. 99). [0105] The full length cDNA for the kappa chain of 2B8 was amplified using the forward primer 5'ggggacaagtttgtacaaaaaagcaggctgccaccatggaatcacagactctggtcttcata 3 '(underlined the start codon) (SEQ ID NO: 100) and reverse primer 5'ggggaccactttgtacaagaaagctgggtctaacactcattcctgttgaagctc 3' (underlined termination codon) (SEQ ID No. 101). The PCR fragments were subcloned into pDONR221 (Invitrogen, Carlsbad, CA) by recombination reaction of Gateway BP (Invitrogen, Carlsbad, CA) and sequenced by Agencourt Bioscience using conventional dideoxy DNA sequencing methods to identify the region sequence constant and further confirm sequences of the variable region. image28
d. Sequence analysis [0106] Variable Regions (normal text) were identified using the IMGT / V-QUEST web server software (http://imgt.cines.fr/textes/vquest/). The signal peptide sequences were predicted based on the identification of the start-reading codon (ATG) that was upstream of the identified Variable Region. Signal Peptide sequences were identified and are underlined below. [0107] The last nucleotide of each variable region is the first base of the next codon generated by the variable / constant region junction. This nucleotide is included in the variable region because it is part of that exon. The amino acid sequences of the constant regions listed below include the translation of this binding codon. [0108] To create the complete kappa or heavy chain antibody sequences, the variable region sequences indicated below are combined with their respective constant region sequences (the signal sequences are underlined).
[0109] (1) Heavy chain variable region of 1A3 (SEQ ID No. 1) image29
[0110] (2) Variable region of the kappa light chain of 1A3 (SEQ ID No. 3) image30 image29
[0111] (3) 2B8 heavy chain variable region (SEQ ID No. 11) image31
[0112] (4) Kappa light chain variable region of 2B8 (SEQ ID NO: 13) image29 image32
[0113] (5) Heavy chain variable region of 2F8 (SEQ ID No. 21) image33
[0115] (7) 3B6 heavy chain variable region (SEQ ID No. 31) image29 image34
[0116] (8) Variable region of the 3B6 kappa light chain (2 possible ATG start codons (in uppercase)) (SEQ ID No. 33) image29
[0117] (9) Variable region of the 3D11 heavy chain (SEQ ID No. 41) image29
[0118] (10) Variable region of the kappa light chain of 3D11 (SEQ ID No. 43) image29 image35
[0119] (11) Heavy chain variable region of 1D3 (SEQ ID No. 51) image36
[0121] (13) Heavy chain variable region of 1F3 (SEQ ID No. 61) image29
10 [0122] (14) Kappa light chain variable region of 1F3 (SEQ ID No. 63) image29 image37 image29
[0123] (15) Heavy chain variable region of 3A12 (SEQ ID No. 71) image29
[0124] (16) Variable region of the 3A12 kappa light chain (SEQ ID No. 73) image29
[0125] (17) Constant region of the mouse IgG1 heavy chain of reference 10 (J00453) (SEQ ID No. 81) image29 image38 image29
[0126] (18) Constant region of the mouse IgG1 heavy chain determined for 1A3, 1D3, 1F3 and 2B8 (obtained from mice of strain AJ) (SEQ ID No. 82) image31 image39
[0127] (19) Constant region of the reference mouse kappa light chain (V00807) and constant region of the mouse kappa light chain determined for 1D3, 1F3 and 2B8 (obtained from AJ strain mice) (SEQ ID No. 83 ) image29
[0128] (20) Constant region of the mouse kappa light chain determined for 1A3 containing an altered nucleotide compared to 1D3, 1F3 and 2B8 (underlined) (SEQ ID No. 84) image29
[0129] Each of the amino acid sequences that define the immunoglobulin heavy chain variable regions for the antibodies produced in Example 1 are set forth in Figure 2. Each of the sequences are aligned with each other and the sequences they define. the signal peptide, CDR1, CDR2 and CDR3
15 are identified by boxes. Figure 3 shows an alignment of the separate CDR1, CDR2 and CDR3 sequences for each of the antibodies. [0130] Each of the amino acid sequences that define the immunoglobulin light chain variable regions for each of the antibodies produced in Example 1 are set forth in Figure 4. Each of the sequences
20 align with each other and the sequences that define the signal peptide, CDR1, CDR2 and CDR3 are identified by boxes. Figure 5 shows an alignment of the separate CDR1, CDR2 and CDR3 sequences for each of the antibodies. [0131] For convenience, Table 1 provides a concordance graph showing the correspondence between the antibody sequences analyzed in this Example with those presented in the Sequence List. image40

SEQ ID NO.

Protein or Nucleic Acid

one

Variable region of the heavy chain of 1A3 - nucleic acid

2

1A3 heavy chain variable region - protein

3

Variable light chain region (kappa) of 1A3 - nucleic acid

4

Variable light chain region (kappa) of 1A3 - protein

5

1R3 heavy chain CDR1

6

1A3 heavy chain CDR2

7

1A3 heavy chain CDR3

8

CDR1 of the light chain (kappa) of 1A3

9

CDR2 of the light chain (kappa) of 1A3

10

CDR3 of the light chain (kappa) of 1A3

eleven

Variable region of the 2B8 heavy chain - nucleic acid

12

Variable region of the 2B8 heavy chain - protein

13

Variable light chain region (kappa) of 2B8 - nucleic acid

14

Variable light chain region (kappa) of 2B8 - protein

fifteen

2B8 heavy chain CDR1

16

2B8 heavy chain CDR2

17

2R8 heavy chain CDR3

18

CDR1 of the light chain (kappa) of 2B8

19

CDR2 of the light chain (kappa) of 2B8

twenty

CDR3 of the light chain (kappa) of 2B8

twenty-one

Variable region of the 2F8 heavy chain - nucleic acid

22

Variable region of the 2F8 heavy chain - protein

2. 3

Variable light chain region (kappa) of 2F8 - nucleic acid

24

Variable light chain region (kappa) of 2F8 - protein

25

2R8 heavy chain CDR1

26

2R8 heavy chain CDR2

27

2R8 heavy chain CDR3

28

CDR1 of the light chain (kappa) of 2F8

29

CDR2 of the light chain (kappa) of 2F8

30

CDR3 of the light chain (kappa) of 2F8

image41

SEQ ID NO.

Protein or Nucleic Acid

31

3B6 heavy chain variable region - nucleic acid

32

3B6 heavy chain variable region - protein

33

Variable light chain region (kappa) of 3B6 - nucleic acid

3. 4

Variable light chain region (kappa) of 3B6 - protein

35

3R6 heavy chain CDR1

36

3R6 heavy chain CDR2

37

3R6 heavy chain CDR3

38

CDR1 of the light chain (kappa) of 3B6

39

CDR2 of the light chain (kappa) of 3B6

40

CDR3 of the light chain (kappa) of 3B6

41

Variable region of the 3D11 heavy chain - nucleic acid

42

Variable region of the 3D11 heavy chain - protein

43

Variable light chain region (kappa) of 3D11 - nucleic acid

44

Variable region of the light chain (kappa) of 3D11 - protein

Four. Five

3D11 Heavy Chain CDR1

46

3D11 CDR2 heavy chain

47

3D11 CDR3 heavy chain

48

3D11 light chain (kappa) CDR1

49

3D11 light chain CDR2 (kappa)

fifty

3D11 light chain CDR3 (kappa)

51

1D3 heavy chain variable region - nucleic acid

52

1D3 heavy chain variable region - protein

53

Variable light chain region (kappa) of 1D3 - nucleic acid

54

Variable light chain region (kappa) of 1D3 - protein

55

1R3 heavy chain CDR1

56

1D3 heavy chain CDR2

57

1D3 heavy chain CDR3

58

CDR1 of the light chain (kappa) of 1D3

59

CDR2 of the light chain (kappa) of 1D3

60

CDR3 of the light chain (kappa) of 1D3

61

1F3 heavy chain variable region - nucleic acid

62

1F3 heavy chain variable region - protein

63

Variable light chain region (kappa) of 1F3 - nucleic acid

image42

SEQ ID NO.

Protein or Nucleic Acid

64

Variable light chain region (kappa) of 1F3 - protein

65

1R3 heavy chain CDR1

66

1R3 heavy chain CDR2

67

1R3 heavy chain CDR3

68

CDR1 of the light chain (kappa) of 1F3

69

CDR2 of the light chain (kappa) of 1F3

70

CDR3 of the light chain (kappa) of 1F3

71

3A12 heavy chain variable region - nucleic acid

72

3A12 heavy chain variable region - protein

73

Variable light chain region (kappa) of 3A12 - nucleic acid

74

Variable region of the light chain (kappa) of 3A12 - protein

75

3R12 heavy chain CDR1

76

3A12 heavy chain CDR2

77

3A12 heavy chain CDR3

78

CDR1 of the light chain (kappa) of 3A12

79

CDR2 of the light chain (kappa) of 3A12

80

CDR3 of the light chain (kappa) of 3A12

[0132] Also for convenience, the following sequences represent the contemplated or actual full-length light chain and heavy chain sequences (ie, they contain both region sequences
5 constant as variable) for each of the antibodies described in this Example. It is noted that the constant regions of murine antibodies 2F8, 3A12, 3B6 and 3D11 were not sequenced but it is assumed that they have the same constant region sequences as antibodies 1D3, 1F3 and 2B8 that were sequenced, since they all come from mice of strain AJ. It is appreciated without
However, the variable region sequences described herein can be linked to each of several other constant region sequences known to those skilled in the art to produce active full length immunoglobulin light and heavy chains. image43
[0133] (1) Nucleic acid sequence encoding the full length 1A3 heavy chain sequence (1A3 heavy chain variable region and IgG1 constant region) (underlined signal sequence) (SEQ ID No. 122) image29
[0134] (2) Protein sequence defining the sequence of the full length 1A3 heavy chain (variable region of the heavy chain of 1A3 and constant region of IgG1) (without signal sequence) (SEQ ID No. 123) image29
[0135] (3) Nucleic acid sequence encoding the full length 1A3 light chain sequence (constant region and kappa variable region of 1A3) (underlined signal sequence) (SEQ ID No. 124) [0136] (4) Protein sequence defining the sequence of the full-length 1A3 light chain (constant region and kappa variable region of 1A3) (without signal sequence) (SEQ ID No. 125) image29 image44 image29 image29
[0137] (5) Nucleic acid sequence encoding the full length 2B8 heavy chain sequence (variable region of the 2B8 heavy chain and 10 constant region of IgG1) (underlined signal sequence) (SEQ ID No. 126) image29
[0138] (6) Protein sequence defining the sequence of the full length 2B8 heavy chain (variable region of the 2B8 heavy chain and region
15 IgG1 constant) (without signal sequence) (SEQ ID No. 127) [0139] (7) Nucleic acid sequence encoding the full-length 2B8 light chain sequence (constant region and kappa variable region of 2B8) ( signal sequence underlined) (SEQ ID No. 128) image29 image45 image29 image29
[0140] (8) Protein sequence defining the sequence of the full-length 2B8 light chain (constant region and kappa variable region of 2B8) (without
10 signal sequence) (SEQ ID No. 129) image29
[0141] (9) Nucleic acid sequence encoding the full-length 2F8 heavy chain sequence (variable region of the 2F8 heavy chain and 15 constant region of IgG1) (underlined signal sequence) (SEQ ID No. 130) image29 image46 image29
[0142] (10) Protein sequence defining the sequence of the full-length 2F8 heavy chain (variable region of the 2F8 heavy chain and constant region of IgG1) (without signal sequence) (SEQ ID No. 131) image29
[0143] (11) Nucleic acid sequence encoding the full-length 2F8 light chain sequence (constant region and kappa variable region of 10 2F8) (underlined signal sequence) (SEQ ID NO: 132) image29
[0144] (12) Protein sequence defining the sequence of the full-length 2F8 light chain (constant region and kappa variable region of 2F8) (without
15 signal sequence) (SEQ ID No. 133) image29
[0145] (13) Nucleic acid sequence encoding the full length 3B6 heavy chain sequence (variable region of the 3B6 heavy chain and 20 constant region of IgG1) (underlined signal sequence) (SEQ ID No. 134) image29 image29
[0146] (14) Protein sequence defining the sequence of the full-length 3B6 heavy chain (variable region of the 3B6 heavy chain and IgG1 constant region) (without signal sequence) (SEQ ID No. 135) image29
[0147] (15) Nucleic acid sequence encoding the full-length 3B6 light chain sequence (constant region and kappa variable region of 10 3B6) (underlined signal sequence) (SEQ ID No. 136) image29 image47
[0148] (16) Protein sequence defining the sequence of the full-length 3B6 light chain (constant region and kappa variable region of 3B6) (without signal sequence) (SEQ ID No. 137) image29
[0149] (17) Nucleic acid sequence encoding the full-length 3D11 heavy chain sequence (3D11 heavy chain variable region and IgG1 constant region) (underlined signal sequence) (SEQ ID NO: 138) image29
[0150] (18) Protein sequence defining the sequence of the full-length 3D11 heavy chain (variable region of the 3D11 heavy chain and constant region of IgG1) (without signal sequence) (SEQ ID No. 139) [0151] (19) Nucleic acid sequence encoding the full-length 3D11 light chain sequence (constant region and kappa variable region of 3D11) (underlined signal sequence) (SEQ ID No. 140) image29 image48 image29
[0152] (20) Protein sequence defining the sequence of the full-length 3D11 light chain (constant region and kappa variable region of 3D11) (without signal sequence) (SEQ ID No. 141) image29
[0153] (21) Nucleic acid sequence encoding the full-length 1D3 heavy chain sequence (1D3 heavy chain variable region and IgG1 constant region) (underlined signal sequence) (SEQ ID No. 142) [ 0154] (22) Protein sequence defining the sequence of the full length 1D3 heavy chain (variable region of the 1D3 heavy chain and constant region of IgG1) (without signal sequence) (SEQ ID No. 143) image29 image49 image29
[0155] (23) Nucleic acid sequence encoding the full length 1D3 light chain sequence (constant region and 1D3 kappa variable region) (underlined signal sequence) (SEQ ID No. 144) image29
[0156] (24) Protein sequence defining the sequence of the full-length 1D3 light chain (constant region and kappa variable region of 1D3) (without signal sequence) (SEQ ID No. 145) image29
[0157] (25) Nucleic acid sequence encoding the full-length 1F3 heavy chain sequence (1F3 heavy chain variable region and IgG1 constant region) (underlined signal sequence) (SEQ ID No. 146) [ 0158] (26) Protein sequence defining the sequence of the full-length 1F3 heavy chain (variable region of the 1F3 heavy chain and constant region of IgG1) (without signal sequence) (SEQ ID No. 147) image29 image50 image29 image29
[0159] (27) Nucleic acid sequence encoding the full-length 1F3 light chain sequence (constant region and kappa variable region of 10 1F3) (underlined signal sequence) (SEQ ID No. 148) image29
[0160] (28) Protein sequence defining the sequence of the full-length 1F3 light chain (constant region and kappa variable region of 1F3) (without
15 signal sequence) (SEQ ID No. 149) image29
[0161] (29) Nucleic acid sequence encoding the full-length 3A12 heavy chain sequence (3A12 heavy chain variable region and IgG1 constant region) (underlined signal sequence) (SEQ ID No. 150) image29
[0162] (30) Protein sequence defining the sequence of the full-length 3A12 heavy chain (variable region of the 3A12 heavy chain and IgG1 constant region) (without signal sequence) (SEQ ID No. 151) image29
[0163] (31) Nucleic acid sequence encoding the full-length 3A12 light chain sequence (constant region and kappa variable region of 3A12) (underlined signal sequence) (SEQ ID No. 152) [0164] (32) Protein sequence defining the sequence of the full-length 3A12 light chain (constant region and 3A12 kappa variable region) (without signal sequence) (SEQ ID No. 153) image51 image29
[0165] For convenience, Table 2 provides a concordance graph showing the correspondence between the full length sequences of the antibodies analyzed in this Example with those presented in the Sequence List.
10 TABLE 2

SEQ ID NO.

Protein or Nucleic Acid

122

Heavy variable of 1A3 + IgG1 constant - nucleic acid

123

Heavy variable of 1A3 + IgG1 constant - protein

124

Light variable of 1A3 + constant - nucleic acid

125

Light variable of 1A3 + constant - protein

126

2B8 heavy variable + IgG1 constant - nucleic acid

127

Heavy variable of 2B8 + IgG1 constant - protein

128

Light variable of 2B8 + constant - nucleic acid

129

Light variable of 2B8 + constant - protein

130

Heavy variable of 2F8 + IgG1 constant - nucleic acid

131

Heavy variable of 2F8 + IgG1 constant - protein

132

Light variable of 2F8 + constant - nucleic acid

133

Light variable of 2F8 + constant - protein

134

Heavy variable of 3B6 + IgG1 constant - nucleic acid

135

Heavy variable of 3B6 + IgG1 constant - protein

136

Light variable of 3B6 + constant - nucleic acid

137

Light variable of 3B6 + constant - protein

138

Heavy variable of 3D11 + IgG1 constant - nucleic acid

139

Heavy variable of 3D11 + IgG1 constant - protein

140

Light variable of 3D11 + constant - nucleic acid

141

Light variable of 3D11 + constant - protein

142

1D3 heavy variable + IgG1 constant - nucleic acid

143

1D3 heavy variable + IgG1 constant - protein

image52

144

Light variable of 1D3 + constant - nucleic acid

145

Light variable of 1D3 + constant - protein

146

1F3 heavy variable + IgG1 constant - nucleic acid

147

1F3 heavy variable + IgG1 constant - protein

148

Light variable of 1F3 + constant - nucleic acid

149

Light variable of 1F3 + constant - protein

150

Heavy variable of 3A12 + IgG1 constant - nucleic acid

151

Heavy variable of 3A12 + IgG1 constant - protein

152

Light variable of 3A12 + constant - nucleic acid

153

Light variable of 3A12 + constant - protein

Example 3 - Production of various recombinant hHGF proteins [0166] This Example describes the cloning and expression of several recombinant proteins used to characterize the antibodies created in Example 1 and
5 in Example 14. In particular, this example describes the cloning and expression of recombinant hHGF protein, a recombinant hHGF protein that contains a glycine to glutamate substitution at position 555 (G555E), a recombinant hHGF protein that contains a substitution of arginine cysteine at position 561 (C561R), a recombinant mouse-human-mouse (mhm) chimeric HGF protein
10 containing the human HGF V495-L585 sequence disposed within the mouse HGF sequence, a recombinant mhm chimeric HGF protein containing the human HGF sequence I499-R566 arranged within the mouse HGF sequence, and a Recombinant mhm chimeric HGF protein containing the human HGF sequence W507-L585 arranged within the
15 mouse HGF sequence. [0167] The following expression constructs were generated using conventional molecular techniques, and the resulting cDNA sequences were confirmed by DNA sequencing.
20 a. hHGF-Fc [0168] In a first round of PCR, two overlapping PCR fragments were generated by introducing a Not I site and encoding a 6xHis marker between hHGF and hlgFc. The overlapping PCR fragments served as a template in a second round to amplify hHGF-his-IgFc. The resulting fragment was digested
25 by Nhel and BamHI and was cloned into pcDNA5 / FRT (Invitrogen, No. 35-3014). image53
Then, hHGF was amplified from clone Invitrogen ID: IOH29794 (human HGF cDNA). The sequence was found to correspond to the sequence deposited in the NCBI under the accession number NM_000601.4.

(one)

Primer 5 'hHGF NheI [0169] ACTGGCTAGCATGTGGGTGACCAAACTCCT (SEQ ID No. 102)

(2)

Primer 3 'hHGF Notl His marker [0170] GTGATGGTGATGGTGATGGCGGCCGCATGACTGTGGTACCTTATATG (SEQ ID No. 103)

(3)

Primer 5 'HisIgFc [0171] ACTGGCGGCCGCCATCACCATCACCATCAC (SEQ ID NO. 104)

(4)

Primer 3 'IgFc BamHI [0172] ACTGGGATCCTCACTATTTACCCGGGGACAG (SEQ ID No. 105)

b.

hHGF-Fc G555E and hHGF-Fc C561R [0173] The hHGF-Fc G555E and C561R mutants were generated by directed mutagenesis using the QuikChange II XL directed mutagenesis kit (Stratagene) according to the manufacturer's instructions.

(one)

HHGF-Fc sense primer (G555E) [0174] CATGATGTCCACGAAAGAGGAGATGAG (SEQ ID NO. 106)

(2)

HHGF-Fc antisense primer (G555E) [0175] CTCATCTCCTCTTTCGTGGACATCATG (SEQ ID No. 107)

(3)

HHGF-Fc sense primer (C561R) [0176] GGAAGAGGAGATGAGAAACGCAAACAGGTTCTCAATG (SEQ ID NO. 108)

(4)

HHGF-Fc antisense primer (C561R) [0177] CATTGAGAACCTGTTTGCGTTTCTCATCTCCTCTTCC (SEQ ID No. 109)

C.

Fc mouse-human-mouse chimera [0178] The construction of mouse-human-mouse chimera IgFc contains the alpha chain of mHGF-hHGF, the amino acids of the � VaI 495-Leu 585 chain of human HGF and the beta C chain -terminal of mHGF, followed by 6xHis marker and IgG-Fc. [0179] The human HGF cDNA encoding amino acids V495-L585 was amplified from the Invitrogen ID clone: IOH29794 (human HGF cDNA). The sequence corresponds to the sequence deposited in the NCBI under the accession number NM_000601.4. Mouse HGF sequences were amplified by RT-PCR from total mouse liver RNA (Clontech, No. 636603) using the Invitrogen Super Script one-step RT-PCR kit (No. 10928-034) according to Manufacturer's instructions. The mHGF cDNA sequence corresponds to the sequence deposited in the NCBI under accession number D10213.1. [0180] Three fragments, called Fragments 1, 2 and 3, were generated using overlapping PCR primers, and hybridized in consecutive rounds of PCR amplification. The final product was cleaved with Nhel and Notl and cloned into pcDNA5 / FRT IgGFc.

(one)

Fragment 1 primers for the mHGF alpha chain 5'Nhel [0181] 5 'ATCGGCTAGCATGATGTGGGGGACCAAAC (SEQ ID No. 110) [0182] 3' GAATCCCATTTACAACCCGCAGTTGTTTTGTTTTGG (SEQ ID No. 111)

(2)

Fragment 2 primers for the amino acids of the hHGF beta chain V495-L585 [0183] 5 'CCAAAACAAAACAACTGCGGGTTGTAAATGGGATTC (SEQ ID No. 112) [0184] 3' CAGGATTGCAGGTCGAGCAAGCTTCATTAA SECACAGAT 113CAGAT

(3)

Fragment 3 primer for the C-terminal end of the mHGF beta chain 3'Notl [0185] 5 'AGATCTGGTTTTAATGAAGCTTGCTCGACCTGCAATCCTG (SEQ ID No. 114) [0186] 3'GTAATTTTGACATACAAGTTGTGCGCATCACCACCACCATC SECAC # 115

d.

Construction of hHGF and chimera mhm [0187] Vectors encoding hHGF and chimera mhm (V495-L585), pcDNA5 / FRT hHGF and pcDNA5 / FRT-chimera mhm (V495-L585), without Fc-marker were generated by mutagenesis directed. A 3 'termination codon of the 6xHis marker was introduced using the QuikChange II XL directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. The mutagenesis primer included Primer 1: CATCACCATCACCATCACTAAGCGGGTCTGGTGCCACG (SEQ ID No. 116) and Primer 2: CGTGGCACCAGACCCGCTTAGTGATGGTGATGGTGATG (SEQ ID No. 117). [0188] In addition, two additional mhm chimeras were created from the pcDNA5 / FRT-mhm (V495-L585) construct by site-directed mutagenesis using the QuikChange II XL-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. An mhm construct contained the I499R556 region of hHGF arranged between murine sequences. The other mhm construct contained the W507-L585 region of hHGF arranged between murine sequences. [0189] For the mhm chimera (I499-R556), the following point mutations were made in order in the construction of pcDNA5 / FRT-chimera mhm mold (V495L585): D558E, C561R, V564I, V567I and M583L, using oligonucleotide sequences appropriate. For the mhm chimera (W507-L585), the following point mutations were introduced at a stage in the construction of pcDNA5 / FRT-chimera mhm mold (V495-L585): Q502R, N504T and I505V, using the appropriate oligonucleotide sequences. [0190] The nucleotide sequence resulting from the hHGF-Fc protein is set forth as SEQ ID No. 118, including the signal sequence (nucleotides 1-93) and the dominance (nucleotides 94-162). The amino acid sequence of the hHGF-Fc protein is set forth as SEQ ID No. 119. [0191] The resulting nucleotide sequence encoding the chimeric protein mhm (V495-L585) -Fc is set forth in SEQ ID No. 120, including the signal sequence (nucleotides 1-96) and the dominance (nucleotides 97-165). The amino acid sequence of the chimeric protein mhm (V495-L585) -Fc is set forth in SEQ ID No. 121. [0192] The resulting nucleotide sequence that encodes, and the protein sequence that defines, the mhm construct (V495-L585 ) is set out in SEQ ID NOS 211 and 212, respectively. The nucleic acid sequence set forth in SEQ ID NO.

image54 image55 image56
211 includes the signal sequence (nucleotides 1-96) and the dominance (nucleotides 97-165), and the protein sequence set forth in SEQ ID No. 212 includes the active protein sequence (without the signal sequence or the dominance). The resulting nucleotide sequence that encodes, and the protein sequence that defines, the mhm construct (I499-R556) are set forth in SEQ ID Nos. 213 and 214, respectively. The nucleic acid sequence set forth in SEQ ID No. 213 includes the signal sequence (nucleotides 1-96) and the domain (nucleotides 97-165), and the protein sequence set forth in SEQ ID No. 214 includes the active protein sequence. (without the signal sequence or the dominance). The resulting nucleotide sequence that encodes, and the protein sequence that defines, the mhm (W507-L585) are set forth in SEQ ID Nos. 215 and 216, respectively. The nucleic acid sequence set forth in SEQ ID No. 215 includes the signal sequence (nucleotides 1-96) and the domain (nucleotides 97-165), and the protein sequence set forth in SEQ ID No. 216 includes the active protein sequence. (without the signal sequence or the dominance).
and. Protein expression
(1) Cell culture. [0193] FlpIn CHO cells (Invitrogen, Catalog No. R758-07) were grown in F12K media (ATCC, Catalog No. 30-2004), 10% FCS (Invitrogen, Catalog No. 10438026), penicillin (10,000 units / ml) / streptomycin (10,000 �g / ml) 1% (Invitrogen, Catalog No. 15140-122) at 37 ° C, 5% CO2, zeocin 100
�g / ml (Invitrogen, Catalog No. R250-01).

(2)

Generation of Stable CHO FIpIn Cell Lines [0194] CHO FlpIn host cells were transfected with a 9: 1 ratio of pOG44 expression plasmid DNA: pcDNA5 / FRT using lipofectamine 2000 according to the manufacturer's instructions (Invitrogen, Catalog No. 11668027) . As controls, cells were transfected with empty pcDNA5 / empty FRT / pOG44 vector and plasmid pOG44 (Invitrogen, Catalog No. 35-3018) only. Twenty-four hours after transfection, the cells were divided and, after forty-eight hours, 0.5 mg / ml hygromycin B (Sigma, Catalog No. H0654-SPEC) was added to the cells. Polyclonal stable cell selection was performed in F12K, 10% FCS, 1% penicillin / streptomycin, 0.5 mg / ml hygromycin B.

(3)

Protein expression in stable CHp FlpIn cell lines [0195] Approximately 2 x 106 cells were seeded in 15 cm plates and cultured in F12K (ATCC, Catalog No. 30-2004) / DMEM with high glucose content (Invitrogen, No. Catalog 11995065) 1: 1, FCS with ultra-low level of 5% IgG (Invitrogen, No. 16250-78) at 37 ° C, 5% CO2 for 5-6 days. Supernatants were collected and the resulting proteins were analyzed by ELISA and by surface plasmon resonance.

image57
Example 4 - Binding characteristics of anti-hHGF monoclonal antibodies [0196] The monoclonal antibodies produced in Example 1 were characterized by their ability to bind hHGF and certain of the recombinant HGF proteins produced in Example 3. [0197] Antibodies They were analyzed by surface plasmon resonance using a BIAcore T100 instrument to assess their ability to bind HGF and certain of the fusion proteins analyzed in Example 3. Each antibody was immobilized on a carboxymethylated dextran CM5 detector microplate (BIAcore, No. of Catalog BR-1006-68) by amine coupling (BIAcore, Catalog No. BR-1000-50) using a conventional coupling protocol according to the manufacturer's instructions. [0198] Analyzes were performed at 25 ° C using PBS (GIBCO, Catalog No. 14040133) containing 0.05% P20 surfactant (BIAcore, Catalog No. R-1000-54), BSA 2 mg / ml (EMD, No. Catalog 2930) and 10 mg / ml CM-dextran sodium salt (Fluka, Catalog No. 86524) as a processing buffer. The supernatant containing different HGF fusion proteins or the supernatant of cells transfected with an empty vector was injected onto each antibody at a flow rate of 30
He / min for 3 minutes. The resulting binding was determined as resonance units (UR) above baseline 30 seconds after the end of the injection. Binding to human HGF (R&D Systems, Catalog No. 294-HGN-025) diluted in processing buffer was compared. Non-specific binding was controlled by comparison of binding to a control surface on which mouse IgG (Rockland, Catalog No. 010-0102) was immobilized using the same amine coupling procedure. [0199] The results are summarized in Table 3. image58
TABLE 3

Antibody

rhHGF (R&D Systems) mHGF (R&D Systems) Chimera mhm (V495-L585) Human HGF G555E C561R

1A3

Yes No No Yes Yes Yes

1D3

Yes No Yes Yes Yes Yes

1F3

Yes yes yes yes yes yes

2B8

Yes No Yes Yes Yes Yes

2F8

Yes Yes No Yes Yes Yes

3A12

Yes No No Yes Yes Yes

3B6

Yes No No Yes Yes Yes

3D11

Yes No No Yes Yes Yes

[0200] The results in Table 3 demonstrate that each antibody binds to rHGF and purified human HGF. In addition, all antibodies bind
5 to hHGF containing the point mutations G555E and C561R. In general, all antibodies except 1F3 and 2F8 did not bind to murine HGF, demonstrating that antibodies 1A3, 1D3, 2B8, 3A12, 3B6 and 3D11 specifically bind human HGF. The 1D3, 1F3 and 2B8 antibodies bind to the mouse-humanoid chimera, while the remaining antibodies did not. The results suggest
10 that antibodies 1D3 and 2B8 at least in part bind to residues 495-585 of human HGF. Antibodies 1A3, 3A12, 3B6 and 3D11 appear to bind to portions of human hHGF other than residues 495-585. At present, it is not clear why 2F8 does not bind to the mhm chimera, as it seems to bind both hHGF and mHGF.
Example 5 - Ability of anti-hHGF monoclonal antibodies to bind to reduced and non-reduced HGF [0201] In this Example, the anti-hHGF monoclonal antibodies produced in Example 1 were analyzed for their ability to bind to reduced and non-HGF
20 reduced. [0202] The reactivity of the anti-HGF sera with the recombinant hHGF was evaluated by immunoblot. Eight �g of recombinant hHGF protein in NuPAGE MOPS SDS (Invitrogen) processing buffer with or without NuPAGE sample reducing buffer (Invitrogen) was fractionated in a 4-12% Bis-Tris 1.0 mm well gel (Invitrogen) , Carlsbad, CA). The fractionated proteins were then transferred onto a nitrocellulose membrane using conventional procedures. Nitrocellulose membranes were blocked with 5% skim milk powder solution in Tris buffered saline with Tween image59
5 20� at 0.1% (TBST), and then mounted on a Mini Protean II Multi-Screen (BioRad) device for additional blocking. [0203] The resulting membranes were probed with the purified antibodies in a Multi-Screen apparatus. The purified antibodies were diluted to 5 g / ml in blocking buffer. Then the nitrocellulose membrane was removed from the apparatus and
10 was incubated with anti-mouse IgG antibodies labeled with horseradish peroxidase. The results are summarized in Table 4, in which the numbers reflect the degree of union, representing - the minimum (little or no union) and representing 3 + the largest union. TABLE 4

Antibody

Reduced (exposure: 3-5 min) Not Reduced (exposure 20 s)

1A3

2+ 2+

1D3

2+ 2+

1F3

2+ 2+

2B8

- 1+

2F8

2+ 2+

3A12

- 2+

3B6

3+ 2+

3D11

- 3+

[0204] The data in Table 4 demonstrate that all antibodies bind to non-reduced rhHGF. In contrast, monoclonal antibodies 1A3, 1D3, 1F3, 2F8, 3B6 bound to reduced rhHGF, but antibodies 2B8, 3A12 and 3D11 did not bind to reduced rhHGF.
twenty
Example 6 - Binding affinities [0205] Binding affinities and the kinetics of the interaction of each of the antibodies produced in Example 1 against hHGF were measured by surface plasmon resonance.
[0206] Anti-mouse rabbit immunoglobulins (BIAcore, Catalog No. BR-1005-14) were immobilized on carboxymethylated dextran CM5 microplates (BIAcore, Catalog No. BR-1006-68) by amine coupling (BIAcore, Catalog No. BR-1000-50) using a conventional coupling protocol according to the manufacturer's instructions. The analyzes were performed at 25 ° C using PBS (GIBCO, Catalog No. 14040-133) containing image60
5 0.05% P20 surfactant (BIAcore, Catalog No. BR-1000-54), BSA 2 mg / ml (EMD, Catalog No. 2930) and 10 mg / ml CM-dextran sodium salt (Fluka, No. Catalog 86524) as processing buffer. [0207] Antibodies were captured in an individual flow cell at a flow rate of 10 µl / min. The injection time was variable for each antibody to produce
About 10 UR of captured antibody for each cycle. Buffer or HGF (R&D Systems, Catalog No. 294-HGN-025) diluted in processing buffer was sequentially injected onto a reference surface (without captured antibody) and the active surface (antibody to be tested) for 2 minutes at 60
He / min The dissociation phase was monitored for 15 or 90 minutes depending on
15 concentration. Then, the surface was regenerated with 10 mM glycine-HCl, pH 1.7 (BIAcore, Catalog No. BR-1003-54) injected for 3 minutes at a flow rate of 60 µl / min before another cycle was started. The concentrations of HGF tested were 0.46 nM to 7.5 nM. [0208] Kinetic parameters were determined using the kinetic function of the
20 BIAevaluation software with reference subtraction. The kinetic parameters for each antibody, ka (association rate constant), kd (dissociation rate constant) and KD (equilibrium dissociation constant) are summarized in Table 5.
TABLE 5

Antibody

ka (1 / Ms) ET (ka) kd (1 / s) ET (kd) KD (pM) DT

1A3

1.7 x 106 7.3 x 104 5.2 x 10-5 8.4 x 10-7 30.1 5.6

1D3

1.7 x 106 3.1 x 104 8.2 x 10-5 1.7 x 10-6 54.2 27.4

1F3

1.5 x 106 5.0 x 104 2.6 x 10-5 6.6 x 10-7 18.1 8.2

2B8

1.6 x 106 2.9 x 104 2.1 x 10-5 1.4 x 10-7 13.5 4.4

3A12

1.6 x 106 3.7 x 104 1.6 x 10-4 1.6 x 10-6 103.0 10.4

3B6

2.0 x 106 6.5 x 104 3.9 x 10-5 3.2 x 10-7 17.0 3.4

[0209] The data in Table 5 demonstrate that antibodies bind to hHGF with a KD of about 100 pM or less, about 50 pM or less, or 20 pM or less. image61
Example 7 - Anti-hHGF antibody neutralization activity [0210] In this Example, the antibodies produced in Example 1 were characterized by their ability to (a) inhibit hHGF binding to c-Met and (b) inhibit incorporation of BrdU stimulated by HGF in 4MBr-5 cells.
to. HGF-Met Binding Inhibition Assay (Neutralization Assay) [0211] Antibodies were tested by ELISA for their ability to inhibit hHGF binding to c-Met. [0212] Specifically, Wallac 96-well DELFIA test plates (Wallac Inc., Catalog No. AAAND-0001) were coated with 100 μl of HGF 6.25 μg / ml (R&D Systems, Catalog No. 294- HGN-025) in carbonate coating buffer (15 mM Na2CO3 and 34 mM NaHCO3, pH 9.0) for 16 hours at 4 ° C. The plates were then blocked with 200 µl of 5% skimmed milk powder in PBS for 1 hour at room temperature. The antibodies were prepared on a separate plate by adding increasing concentrations of the antibodies under investigation (0.033-667 nM, 3-fold serial dilution) to c-Met 2 nM (R&D Systems, Catalog No. 358-MT / CF) in milk 5% skimmed powder in PBS. 100 µl of sample was transferred per well to the assay plate and incubated overnight at 4 ° C. The test plates were then washed 3 times with 0.1% PBS-Tween 20 and incubated for 2 hours at room temperature with 100 µl / well of biotinylated human anti-c-Met antibody (R&D Systems, Catalog No. BAF358) prepared in 5% skimmed milk powder in PBS. [0213] The resulting plates were then washed three times with 0.1% PBS-Tween 20 and incubated for 1 hour at room temperature with Eu-labeled streptavidin (Wallac, Catalog No. 1244-360) diluted 1: 1000 in DELFIA assay buffer (Wallac, Catalog No. 4002-0010). The resulting plates were washed 3 times with DELFIA wash solution (Wallac, Catalog No. 40100010) and incubated with 100 µl / well DELFIA enhancement solution (Wallac No. 4001-0010) for 15 minutes at room temperature with stirring. [0214] The plates were read on a Victor3V instrument (Perkin Elmer) using the europium method. IC50 values were calculated and summarized in Table 6. image62
TABLE 6

Antibody

IC50 (nM) DT

1A3

5.65 0.91

1D3

4.43 2.27

1F3

6.57 0.28

2B8

5.57 1.19

2F8

5.36 0.88

3A12

5.26 2.11

3B6

- -

3D11

5.66 2.75

[0215] The results demonstrate that all antibodies (ie, 1D3, 1A3, 2B8, 3A12, 1F3, 3D11 and 2F8) other than 3B6 effectively neutralize the binding of 5 HGF to c-Met.
b. Neutralization of HGF-stimulated BrdU incorporation in 4MBr-5 cells [0216] Ten μl of 12.5 nM hHGF were dispensed into individual wells of one
10 96-well tissue culture microtiter plate (Costar Catalog No. 3903). Ten μl of serially diluted antibodies were added at the concentrations of 6667, 2222, 740, 247, 82, 27, 9.1, 3.0, 1.0, 0.33 nM to each well. The HGF antibody mixture was then incubated at room temperature for 30 minutes. Monkey bronchial epithelial cells 4MBr-5 (ATCC, CCL208)
15 grown in F-12K (ATCC, 30-2004), 15% FBS (Gibco 10438-026), 30 ng / ml EGF (Sigma E9644), 1% penicillin / streptomycin (PS, Gibco Catalog No. 15140- 122) were dissociated with trypsin (Gibco Catalog No. 25200-056), resuspended in assay media (F-12K, 2.5% FBS, 1% PS) at 75,000 cells / ml and 80 µl were dispensed of the cell suspension in the mixture of
20 HGF antibodies. [0217] The resulting cells were incubated at 37 ° C, 5% CO2. Forty-eight hours later, 10 ll of 100 �M BrdU (Roche Catalog No. 1669915) was added. Seventy-two hours later, the media was removed, the plates were dried with a hair dryer and processed with the BrdU ELISA according to
25 with the manufacturer's instructions (Roche Catalog No. 1669915). [0218] The luminescent signal was quantified using a Synergy HT (Bio-Tek) plate reader. The data were adjusted to a sigmoid dose response with variable slope with the equation y = lower part + (upper part - lower part) / (1 + 10 ^ (log (EC50 - x) * pending)) in GraphPad Prism ( GraphPad Software). Each experiment was repeated at least 3 times in duplicate and the average EC50 values are presented in Table 7. image63
TABLE 7

Antibody

IC50 (nM)

1A3

4.69

1D3

4.99

1F3

1.94

2B8

1.41

2F8

19.24

3A12

30.30

3B6

36.08

3D11

51.12

[0219] [0220] The results in Table 7 demonstrate that all antibodies, 1A3, 1D3, 1F3, 2B8, 2F8, 3A12, 3B6 and 3D11 inhibit HGF-induced proliferation in
10 4MBr-5 cells.
Example 8 - Anti-dispersion activity of anti-hHGF antibodies [0221] This Example describes a characterization of the antibodies produced in Example 1 to determine their ability to inhibit dispersion activity
15 induced by HGF. HGF induces the "dispersion" (motility) of groups in MDCK cells (ATCC, Manassas, VA, Catalog No. CCL-34). [0222] MDCK cells were seeded in 96-well Costar tissue culture plates (Corning Incorporated, Corning, NY Catalog No. 3595) at a density of 4 x 103 cells per well in 80 µl of MEM (ATCC, Manassas, VA , Nº of
20 Catalog 30-2003) containing 10% fetal bovine serum (Invitrogen Catalog No. 10438026) and 1% penicillin-streptomycin (Invitrogen Catalog No. 15140122). Each of the antibodies to be investigated was diluted to 6,667 nM in MEM containing 10% fetal bovine serum and 1% penicillin-streptomycin. Each of the different antibody dilutions, as well as the MEM containing
25 10% fetal bovine serum and 1% penicillin-streptomycin without antibody, was then combined separately with an equivalent volume of MEM containing 10% fetal bovine serum and 1% penicillin-streptomycin and 100 ng / ml HGF ( R&D Systems Catalog No. 294-HGN-025). The antibody / HGF dilutions were incubated for 30 minutes at 25 ° C. Twenty of each antibody / HGF dilution was added separately to individual wells, producing a image64
5 final antibody concentration of 666.7 nM and a final HGF concentration of 10 ng / ml. The MDCK cells were then incubated for 24 hours at 37 ° C with 5% CO2. [0223] After 24 hours of incubation, MDCK cells were carefully washed once with 100 µl per well of ice cold PBS (Invitrogen No.
10 Catalog 14190144) and were fixed with 100 per well of frozen methanol with rolling for 10 minutes at 25 ° C. Then, the plates were carefully washed once with distilled water. A volume of 100 µl of violet crystal solution was added to each well, consisting of 0.5% violet crystal (Sigma, St. Louis, MO, Catalog No. C3886) and 50% ethanol in distilled water, Y
The cells were incubated for 20 minutes at 25 ° C with rocking. [0224] After staining with violet crystal solution, the cells were carefully washed three times with distilled water. Then, PBS was added to each well to prevent the samples from drying out. Images of the cells were taken using the Leica DMIRB microscope (Leica Microsystems GmbH, Wetzler,
20 Germany), the DC500 camera (Leica Microsystems GmbH, Wetzler, Germany) and the MagnaFire 2.1C software (Optronics, Goleta, CA) and the samples were classified by the level of dispersion. The results are summarized in Table 8. TABLE 8

Inhibition of HGF-induced MDCK cell dispersion

Antibody

Trial 1 Trial 2

1A3

++ +

1D3

++ ++

1F3

+ +

2B8

+++ +++

2F8

+ +

3A12

- - / +

3B6

++ ++

3D11

- -

- Without inhibition +++ Very strong inhibition, almost complete image65
++ Strong inhibition
+ Detectable inhibition [0225] The results in Table 8 demonstrate that the 2B8 antibody inhibited HGF-induced dispersion more than other antibodies. The 1D3 and 3B6 antibodies showed an intermediate level of inhibition; antibody 1A3 showed a low to intermediate level of inhibition; 1F3 and 2F8 antibodies showed a low level of inhibition; and 3A12 and 3D11 antibodies provided poor inhibition
or not detectable.
Example 9 - Inhibition of HGF stimulated c-Met phosphorylation [0226] This example describes a characterization of the antibodies produced in Example 1 for their ability to inhibit HGF stimulated c-Met phosphorylation in PC-3 cells. HGF induces Met phosphorylation in PC-3 cells (ATCC No. CRL-1435). [0227] PC-3 cells were seeded in individual wells of 96-well Costar tissue culture plates (Corning Catalog No. 3595) at a density of 4.5 x 104 cells per well in 100 µl of F-12K ( ATCC, Manassas, VA, Catalog No. 30-2004) containing 10% fetal bovine serum (Invitrogen Catalog No. 10438026) and penicillin-streptomycin 1% (Invitrogen Catalog No. 15140122). After 24 hours at 37 ° C with 5% CO2 the media was removed and the cells rinsed once with serum-free F-12K containing 1% penicillin-streptomycin. The cells were then incubated for 24 hours in 100 µl of serum-free F-12K containing 1% penicillin-streptomycin. [0228] The following 10 different dilutions of each of the antibodies being investigated were prepared in serum-free F-12K containing 1% penicillin esterptomycin: 6667 nM, 2222 nM, 741 nM, 247 nM, 82.3 nM, 27.4 nM, 9.1 nM, 3.0 nM, 1.0 nM and 0.3 nM. Each dilution of serum-free antibody and F-12K containing 1% penicillin-streptomycin without antibody was combined separately with an equivalent volume of serum-free F-12K containing 1% penicillin-esterptomycin and 500 ng / ml HGF (R&D Systems No. from Catalog 294-HGN025). These antibody / HGF dilutions were incubated for 30 minutes at 25 ° C. This resulted in a final concentration of 1.25 nM HGF. [0229] Next, PC-3 cells were rinsed once with serum-free F-12K containing 1% penicillin-streptomycin. Next, 70 µl of serum-free F12K containing 1% penicillin-streptomycin was added to the cells, followed by 10 mM Na3VO4 (Sigma Catalog No. S6508) in F-12K without serum containing 1% penicillin-streptomycin . Then, the cells were incubated for 60 minutes at 37 ° C with 5% CO2. After this incubation, 20 µl of each antibody / HGF dilution was added separately to separate wells, yielding a final HGF concentration of 50 ng / ml and the following final concentrations of each antibody: 666.7 nM, 222, 2 nM, 74.1 nM, 24.7 nM, 8.23 nM, 2.74 nM, 0.91 nM, 0.30 nM, 0.10 nM, 0.03 nM. The cells were then incubated for 10 minutes at 37 ° C with 5% CO2, at which point the mixture of media / antibody / HGF was removed and the plates were placed on ice. The cells were then rinsed once with 100 µl per well of ice cold PBS (Invitrogen Catalog No. 14190144) containing 1 mM Na3VO4. The cells were then incubated for 30 minutes at 4 ° C in 100 µl per well of ice lysis buffer consisting of 1% OmniPur Triton X-100 (MERCK KGaA, Darmstadt, Germany, Catalog No. 9410), Tris-HCl 50 mM pH 8.0, 100 mM NaCl, 0.3 mM Na3VO4, 1x protease inhibitor cocktail (Sigma Catalog No. P8340) and 2x phosphatase inhibitor cocktail 2 (Sigma Catalog No. 5726). [0230] Biotinylated human anti-HGF-R (c-met) antibody (R&D Systems Catalog No. BAF358) was diluted to a concentration of 2 l / ml in DELFIA assay buffer (PerkinElmer, Turku, Finland, Catalog No. 4002-0010) containing 1% bovine serum albumin (Sigma Catalog No. A2153), and 50 were added image66
This dilution per well of yellow streptavidin microtiter plates (PerkinElmer Catalog No. AAAND-0005). Then, the plates were incubated with antibody for 30 minutes at 25 ° C with rocking. After incubation, the plates were washed with DELFIA wash solution (PerkinElmer Catalog No. 4010-0010) and 80 µl of each of the different PC-3 cell lysates were added separately to individual wells of the plates. microtiter with streptavidin washes. [0231] Streptavidin microtiter plates containing the lysates of PC-3 cells were incubated for 60 minutes at 25 ° C with shaking, and then washed with DELFIA wash solution. 100 µl of DELFIA Eu-N1 PTyr-100 600 ng / ml antibody (PerkinElmer Catalog No. AD0159) diluted in DELFIA assay buffer containing 1% bovine serum albumin was added to each well of streptavidin microtiter plates washed, previously incubated with lysates of PC-3 cells. Plates were incubated for 60 minutes at 25 ° C with rocking. The plates were washed a final time with DELFIA wash solution. Then, 200 µl of DELFIA enhancement solution (PerkinElmer Catalog No. 4001-0010) was added to each well of the washed streptavidin microtiter plates, and the plates were incubated in the dark for 5 minutes at 25 ° C with stirring. image67
5 [0232] Next, the signal was measured using the europium protocol in the Victor3V (PerkinElmer) reader. The EC50 values were calculated using Prism 4 for Windows (GraphPad Software, Inc., San Diego, CA) and the equation of sigmoid dose response. [0233] The results summarized as EC50 in nM are shown in Table 9.
10 TABLE 9

Antibody

Average of two tests Standard deviation

1A3

0.684 0.222

1D3

0.984 0.129

1F3

1.19 1.01

2B8

0.287 0.104

2F8

1.39 2.12

3A12

2.00 0.553

3B6

1.01 1.11

3D11

2.28 N / A

[0234] The data in Table 9 demonstrate that all eight antibodies are potent inhibitors of HGF-induced c-Met phosphorylation in PC-3 cells.
Example 10 - Inhibition of tumors in the U87MG xenograft model [0235] The ability of murine monoclonal antibodies of the invention to inhibit tumor growth was tested in a U87MG xenograft model. U87MG cells (ATCC) were grown in culture at 37 ° C in an atmosphere containing 5% CO2 and 95% air, using a medium comprising medium of
20 Eagle modified by Dulbecco (DMEM) with 10% fetal bovine serum, 100 units / ml penicillin and 100 g / ml streptomycin. Cells were subcultured and maintained by detachment of cells from the culture plate wall using trypsin-EDTA. [0236] Almost confluent cells were collected by trypsin treatment, and
25 5 x 106 cells were then injected into 50% Matrigel (BD Biosciences; Catalog No. 356237) subcutaneously in the upper dorsal area between the scapulae of 7-week-old female ICR SCID mice (Taconic Labs). The long (L) and short (W) (mm) diameters of the tumors were measured with a caliper. Tumor volume (vol.) Was calculated as: volume (mm3) = L x W2 / 2. When tumors grew to approximately 200 mm3, mice carrying tumors were image68
5 randomized into 5 groups of 10 mice each. One group received PBS. Each of the other 4 groups received one of the 1A3, 1D3, 1F3 or 2B8 antibodies. All antibodies were dosed at 1 mg / kg body weight twice a week by intraperitoneal injection of 5 doses. Tumor volumes and body weights of the mice were recorded twice a week. The
10 tumor growth inhibition using Student's t-test. The results are summarized in Figure 6 and Table 10. Table 10

Inhibition Percentage

2B8 vs. PBS

93% p = 0.001

1A3 vs. PBS

73% p = 0.0075

1D3 vs. PBS

51% p = 0.075

1F3 vs. PBS

60% p = 0.027

[0237] A partial regression was achieved in the group treated with 2B8 (Figure 6).
15 Statistically significant growth inhibition was observed in the groups treated with 1A3 and treated with 1F3 (Table 10). There was a 51% tumor growth inhibition for 1D3 with a p-value of 0.075. No significant body weight losses were observed.
Example 11 - Inhibition of tumors in the U118 xenograft model [0238] The ability of antibodies 1A3, 1D3, 1F3 and 2B8 to inhibit tumor growth was tested in a U118 xenograft model. U118 cells (ATCC) were expanded as described in Example 10 (above) with respect to U87MG cells.
[0239] Subcutaneous tumors were established as described in Example 10 above, except that the mice used were 7 weeks old female NCr athymic mice (Taconic) and treatment began when the tumors grew to approximately 80 mm 3. As in the U87MG model, all antibodies were dosed at 1 mg / kg body weight twice a week
30 by intraperitoneal injections for 4 doses. Tumor volumes and body weights of the mice were recorded twice a week. Tumor growth inhibition was analyzed using Student's t-test. The results are summarized in Figure 7 and Table 11. image69
Table 11

Inhibition Percentage

2B8 vs. IgG

75% p = 0.007

1A3 vs. IgG

57% p = 0.01

1D3 vs. IgG

47% p = 0.12

1F3 vs. IgG

30% p = 0.39

[0240] Statistically significant tumor growth inhibition was observed in the groups treated with 2B8 and 1A3 (Figure 7). There was a modest tumor growth inhibition in the groups with 1F3 and 1D3 with p values lower than 0.05, which were defined as statistically significant in this
10 study (Table 11). No significant body weight losses were observed.
Example 12: Humanization of murine monoclonal antibodies [0241] This example describes the humanization of the murine 2B8 antibody together with a characterization of the resulting humanized antibodies. The regions
15 light and heavy variables of murine 2B8 were "humanized" by two methods.
A. Humanization procedure 1 [0242] In the first method, three humanized heavy chain variable regions and two kappa light chain variable regions were designed
20 humanized based on the "superhumanization" method described in Hwang et al. (2005) METHODS 36: 35-42; Tan et al. (2002) J. IMMUNOL. 169: 1119-1125; U.S. Patent No. 6,881,557. [0243] The canonical structural class of Chotia was determined for each mouse 2B8 CDR based on the length of the CDR and the amino acid composition.
25 Variable regions of human germline were identified that consisted of heavy and light variable regions of the same canonical structural class of Chotia based on reference alleles of the known human germline variable region described on the International Immunogenetics Information System website ( IMGT) (available on the world wide web) at
30 imgt.cines.fr and biochem.unizh.ch/antibody/Sequences/index.html). These human germline variable regions of the same structural class were compared with murine 2B8 variable regions calculating the percentage of identity or similarity between CDR amino acid residues. Those human germline variable regions with the highest identity and / or similarity with mouse 2R8 CDR residues were selected for CDR grafting. The flanking residues of the human germline variable regions were preserved, while the mouse 2R8 CDR residues were used to replace the corresponding human germline variable region residues that were different between mouse 2B8 CDR and CDR of human germ line. The human J region that was the most similar to the 2B8 mouse J region was then added to the carboxyl terminus of the "superhumanized" variable region. Then, a signal sequence was added to the amino-terminal end of the "superhumanized" variable regions, and these amino acid sequences were converted to nucleic acid sequences. [0244] The nucleic acid sequence of the entire variable region was constructed using gene synthesis PCR methods (Young et al. (2004). NUCL. ACIDS RES 32: e59) and cloned into a mammalian expression vector (based on pcDNA3.2 DEST (Invitrogen)) containing Kappa regions (allotype Km (3) (allele 2)) image70
or of IgG1 (allotype G1m (17,1)) human constants (downstream of the variable regions) using conventional molecular biology techniques. The four heavy chain IgG1 antibodies (2B8 chimeric and 3 humanized heavy chains (Hu2B8 Hv1-f.1, Hu2B8 Hv5-a.1, Hu2B8 Hv5-51.1) were expressed in the possible combinations with the 3 kappa chain antibodies (2B8 chimeric and 2 humanized light chains (Hu2B8 Kv1-39.1 and Hu2B8 Kv3-15.1), creating 12 different antibody proteins.The binding of chimeric, chimeric / humanized and humanized antibodies to human HGF was then measured as described below, and The results are summarized in Figure 8. Each of the possible combinations of immunoglobulin light chain and immunoglobulin heavy chain variable regions is set forth below in Table 12 A.
Table 12A

Variable Heavy Chain Region

Variable Light Chain Region

2B8 chimeric (SEQ ID NO: 12)

2B8 chimeric (SEQ ID NO: 14)

2B8 chimeric (SEQ ID NO: 12)

Hu2B8 Kv1-39.1 (SEQ ID NO: 173)

2B8 chimeric (SEQ ID NO: 12)

Hu2B8 Kv3-15.1 (SEQ ID NO: 179)

image71

Variable Heavy Chain Region

Variable Light Chain Region

Hu2B8 Hv1-f.1 (SEQ ID NO: 159)

2B8 chimeric (SEQ ID NO: 14)

Hu2B8 Hv1-f.1 (SEQ ID NO: 159)

Hu2B8 Kv1-39.1 (SEQ ID NO: 173)

Hu2B8 Hv1-f.1 (SEQ ID NO: 159)

Hu2B8 Kv3-15.1 (SEQ ID NO: 179)

Hu2B8 Hv5-a.1 (SEQ ID NO: 165)

2B8 chimeric (SEQ ID NO: 14)

Hu2B8 Hv5-a.1 (SEQ ID NO: 165)

Hu2B8 Kv1-39.1 (SEQ ID NO: 173)

Hu2B8 Hv5-a.1 (SEQ ID NO: 165)

Hu2B8 Kv3-15.1 (SEQ ID NO: 179)

Hu2B8 Hv5-51.1 (SEQ ID NO: 169)

2B8 chimeric (SEQ ID NO: 14)

Hu2B8 Hv5-51.1 (SEQ ID NO: 169)

Hu2B8 Kv1-39.1 (SEQ ID NO: 173)

Hu2B8 Hv5-51.1 (SEQ ID NO: 169)

Hu2B8 Kv3-15.1 (SEQ ID NO: 179)

[0245] Each of the possible combinations of immunoglobulin heavy chains and immunoglobulin light chains is set forth below in Table 12B.
Table 12B

Immunoglobulin Heavy Chain

Immunoglobulin Light Chain

Chimeric 2B8 IgG1 (SEQ ID NO: 155)

Chimeric 2B8 Kappa (Km (3)) (SEQ ID NO: 157)

Chimeric 2B8 IgG1 (SEQ ID NO: 155)

Hu2B8 Kv1-39.1 + Kappa constant (allotype Km (3)) (allele 2) (SEQ ID NO: 177)

Chimeric 2B8 IgG1 (SEQ ID NO: 155)

Hu2B8 Kv3-15.1 + Kappa constant (allotype Km (3)) (allele 2) (SEQ ID NO: 181)

Hu2B8 Hv1-f.1 + IgG1 constant (allotype G1M (17,1)) (SEQ ID NO: 163)

Chimeric 2B8 Kappa (Km (3)) (SEQ ID NO: 157)

Hu2B8 Hv1-f.1 + IgG1 constant (allotype G1M (17,1)) (SEQ ID NO: 163)

Hu2B8 Kv1-39.1 + Kappa constant (allotype Km (3)) (allele 2) (SEQ ID NO: 177)

Hu2B8 Hv1-f.1 + IgG1 constant (allotype G1M (17,1)) (SEQ ID NO: 163)

Hu2B8 Kv3-15.1 + Kappa constant (allotype Km (3)) (allele 2) (SEQ ID NO: 181)

Hu2B8 Hv5-a.1 + IgG1 constant (allotype G1M (17,1)) (SEQ ID NO: 167)

Chimeric 2B8 Kappa (Km (3)) (SEQ ID NO: 157)

Hu2B8 Hv5-a.1 + IgG1 constant (allotype G1M (17,1)) (SEQ ID NO: 167)

Hu2B8 Kv1-39.1 + Kappa constant (allotype Km (3)) (allele 2) (SEQ ID NO: 177)

Hu2B8 Hv5-a.1 + IgG1 constant (allotype G1M (17,1)) (SEQ ID NO: 167)

Hu2B8 Kv3-15.1 + Kappa constant (allotype Km (3)) (allele 2) (SEQ ID NO: 181)

image72

Immunoglobulin Heavy Chain

Immunoglobulin Light Chain

Hu2B8 Hv5-51.1 + IgG1 constant (allotype G1M (17,1)) (SEQ ID NO: 171)

Chimeric 2B8 Kappa (Km (3)) (SEQ ID NO: 157)

Hu2B8 Hv5-51.1 + IgG1 constant (allotype G1M (17,1)) (SEQ ID NO: 171)

Hu2B8 Kv1-39.1 + Kappa constant (allotype Km (3)) (allele 2) (SEQ ID NO: 177)

Hu2B8 Hv5-51.1 + IgG1 constant (allotype G1M (17,1)) (SEQ ID NO: 171)

Hu2B8 Kv3-15.1 + Kappa constant (allotype Km (3)) (allele 2) (SEQ ID NO: 181)

[0246] Two of the possible antibody constructs that contain the full-length immunoglobulin light and heavy chains containing humanized variable regions are indicated below:
5 sh2B8-9 (G1m (17.1)) = hu2B8 Hv5-51.1 (+ constant region of IgG1 (allotype G1m (17.1)) (SEQ ID No. 171) plus hu2B8 Kv 1-39.1 (+ constant region kappa ( allotype Km (3) (allele 2))) (SEQ ID No. 177)
sh2B8-12 (G1m (17.1)) = hu2B8 Hv5-51.1 (+ constant region of IgG1
10 (allotype G1m (17,1)) (SEQ ID No. 171) plus hu2B8 Kv 3-15.1 (+ constant region kappa (allotype Km (3) (allele 2))) (SEQ ID No. 181) [0247] The sequences of nucleic acid they encode, and the protein sequences that define each of the humanized antibodies are summarized below.
15 In this section, the last nucleotide of each variable region is the first base of the next codon generated by the union of the variable / constant region. This nucleotide is included in the Variable Region because it is part of that exon. The amino acid sequences of the Constant Regions listed below include the translation of this binding codon. image73
[0248] Nucleic acid sequence encoding the full length chimeric 2B8 heavy chain (mouse variable region and human IgG1 constant region) (G1m allotype (17.1)) (underlined signal sequence) (SEQ ID No. 154) image29
[0249] (2) Protein sequence defining the full length chimeric 2B8 heavy chain (chimeric 2B8 IgG1 (G1m allotype (17.1)) (no signal sequence) (SEQ ID No. 155) image29
[0250] (3) Nucleic acid sequence encoding the full-length chimeric 2B8 light chain (mouse variable region and human constant region) (chimeric 2B8 kappa (Km (3))) (underlined signal sequence) (SEC ID No. 156) [0251] (4) Protein sequence defining the full-length chimeric 2B8 light chain (chimeric 2B8 kappa (Km (3))) (without signal sequence) (SEQ ID No. 157) image74 image75 image29 image29
[0252] (5) Nucleic acid sequence encoding the human Hu2B8 Hv1-f.1 heavy chain variable region (underlined signal sequence) (SEQ ID NO.
10 158) image29
[0253] (6) Protein sequence defining the variable region of the heavy chain of image76 image77
[0254] (7) Nucleic acid sequence encoding the constant region of the human IgG1 heavy chain (allotype G1m (17.1)) (SEQ ID No. 160) image29 image78
[0255] (8) Protein sequence defining the constant region of the human IgG1 heavy chain (allotype G1m (17.1)) (SEQ ID No. 161). The first amino acid comes from the translation of the last nucleotide of the variable region and the two nucleotides of the beginning of the IgG1 Heavy Chain sequence. image31
[0256] (9) Nucleic acid sequence encoding the full-length humanized Hu2B8 Hv1-f.1 heavy chain variable region and the human IgG1 heavy chain constant region (allotype G1m (17,1)) ( signal sequence
10 underlined) (SEQ ID No. 162) [0257] (10) Protein sequence defining the heavy-chain human Hu2B8 Hv1-f.1 heavy chain variable region and the human IgG1 heavy chain constant region (allotype G1m (17.1) (without signal sequence) (SEQ ID No. 163) image29 image79 image31
[0258] (11) Nucleic acid sequence encoding the heavy chain variable region of humanized Hu2B8 Hv5a.1 (underlined signal sequence) (SEQ ID NO: 164) image80
[0259] (12) Protein sequence defining the variable region of the humanized Hu2B8 Hv5a.1 heavy chain (without signal sequence) (SEQ ID No. 165) image74 image81
[0260] (13) Nucleic acid sequence encoding the full-length humanized Hu2B8 Hv5a.1 heavy chain variable region and the human IgG1 heavy chain constant region (G1m allotype (17.1) (signal sequence underlined) (SEQ ID No. 166) image31
[0261] (14) Protein sequence defining the variable region of the full-length humanized Hu2B8 Hv5a.1 heavy chain and the constant region of the human IgG1 heavy chain (allotype G1m (17.1) (no signal sequence) (SEQ ID
10 No. 167) [0262] (15) Nucleic acid sequence encoding the human Hu2B8 heavy chain variable region Hv5-51.1 (underlined signal sequence) (SEQ ID No. 168) image29 image82 image29
[0263] (16) Protein sequence that defines the human Hu2B8 Hv5-51.1 heavy chain variable sequence (without signal sequence) (SEQ ID No. 169) image29
10 [0264] (17) Nucleic acid sequence encoding the full-length humanized Hu2B8 Hv5-51.1 heavy chain variable region and the human IgG1 heavy chain constant region (G1m allotype (17.1) (sequence underlined signal) (SEQ ID No. 170) [0265] (18) Protein sequence defining the heavy-chain Hu2B8 Hv5-51.1 heavy chain variable region and the human IgG1 heavy chain constant region (G1m allotype ( 17.1) (without signal sequence) (SEQ ID No. 171) image74 image83 image31
[0266] (19) Nucleic acid sequence encoding the variable region of the humanized Hu2B8 Kv1-39.1 kappa chain (underlined signal sequence) (SEQ ID No. 172). Two possible starting ATGs are shown in uppercase. image80
[0267] (20) Protein sequence defining the variable region of the humanized Hu2B8 Kv1-39.1 kappa chain (without signal sequence) (SEQ ID No. 173) image29
[0268] (21) Nucleic acid sequence encoding the constant region of the human kappa chain (allotype Km (3)) (allele 2) (SEQ ID No. 174) image84
[0269] (22) Protein sequence that defines the constant region of the human kappa chain (allotype Km (3)) (allele 2) (SEQ ID No. 175). The first amino acid comes from the translation of the last nucleotide of the variable region and the two nucleotides of the beginning of the Kappa Light Chain sequence. image31
[0270] (23) Nucleic acid sequence encoding the variable region of the full-length humanized Hu2B8 Kv1-39.1 light chain and the constant region of the human kappa chain (allotype Km (3)) (allele 2) (sequence signal
10 underlined) (SEQ ID No. 176) image29
[0271] (24) Protein sequence defining the variable region of the full-length humanized Hu2B8 Kv1-39.1 light chain and the constant region of the
15 human kappa chain (allotype Km (3)) (allele 1) (SEQ ID No. 177) image29
[0272] (25) Nucleic acid sequence encoding the variable region of the humanized Hu2B8 Kv3-15.1 light chain (underlined signal sequence) (SEC 20 ID No. 178) image29 image85 image29
[0273] (26) Protein sequence defining the variable region of the humanized Hu2B8 Kv3-15.1 light chain (without signal sequence) (SEQ ID No. 179) image31
[0274] (27) Nucleic acid encoding the variable region of the full-length humanized Hu2B8 Kv3-15.1 light chain and the constant region of the human kappa chain (allotype Km (3)) (allele 2) (signal sequence underlined ) (SEC
10 ID No. 180) image29
[0275] (28) Protein sequence defining the variable region of the humanized Hu2B8 Kv3-15.1 light chain and the constant region of the human kappa chain 15 (allotype Km (3)) (allele 2) (without signal sequence) ( SEQ ID No. 181) image29
[0276] For convenience, Table 13 provides a concordance graph showing the correspondence between the full length sequences and the 20 antibodies analyzed in this section with those presented in the Sequence List. image86
TABLE 13

SEQ ID NO.

Protein or Nucleic Acid

154

Chimeric 2B8 IgG1 (G1m (17.1)) - nucleic acid

155

Chimeric 2B8 IgG1 (G1m (17.1)) - protein

156

Kappa of 2B8 chimeric (Km (3)) - nucleic acid

157

Kappa of 2B8 chimeric (Km (3)) - protein

158

Variable region of the heavy chain of Hu2B8 Hv1f.1 - nucleic acid

159

Variable region of the Hu2B8 Hv1f.1-protein heavy chain

160

Constant region of the human IgG1 heavy chain (allotype G1m (17,1)) - nucleic acid

161

Constant region of the human IgG1 heavy chain (allotype G1m (17,1)) - protein

162

Hu2B8 Hv1f.1 + IgG1 constant (allotype G1m (17,1)) - nucleic acid

163

Hu2B8 Hv1f.1 + IgG1 constant (allotype G1m (17,1)) - protein

164

Variable region of the heavy chain of Hu2B8 Hv5a.1- nucleic acid

165

Variable region of the heavy chain of Hu2B8 Hv5a.1- protein

166

Hu2B8 Hv5a.1 + IgG1 constant (allotype G1m (17,1)) - nucleic acid

167

Hu2B8 Hv5a.1 + IgG1 constant (allotype G1m (17,1)) - protein

168

Variable region of the Hu2B8 Hv5-51.1 heavy chain - nucleic acid

169

Variable region of the Hu2B8 Hv5-51.1 heavy chain - protein

170

Hu2B8 Hv5-51.1 + IgG1 constant (allotype G1m (17,1)) - nucleic acid

171

Hu2B8 Hv5-51.1 + IgG1 constant (allotype G1m (17,1)) - protein

172

Variable region of the kappa chain of Hu2B8 Hv1-39.1 - nucleic acid

173

Variable region of the kappa chain of Hu2B8 Hv1-39.1 - protein

174

Constant region of the human kappa chain (allotype Km (3)) (allele 2) - nucleic acid

image87

SEQ ID NO.

Protein or Nucleic Acid

175

Constant region of the human kappa chain (allotype Km (3)) (allele 2) - protein

176

Hu2B8 Hv1-39.1 + kappa constant (allotype Km (3)) (allele 2) - nucleic acid

177

Hu2B8 Hv1-39.1 + kappa constant (allotype Km (3)) (allele 2) - protein

178

Variable region of the kappa chain of Hu2B8 Hv3-15.1 - nucleic acid

179

Variable region of the kappa chain of Hu2B8 Hv3-15.1 - protein

180

Hu2B8 Hv3-15.1 + kappa constant (allotype Km (3)) (allele 2) - nucleic acid

181

Hu2B8 Hv3-15.1 + kappa constant (allotype Km (3)) (allele 2) - protein

B. Humanization procedure 2 [0277] The second humanization method used to reduce the immunogenicity of mouse 2B8 antibody is based on the method described in
5 Studnicka et al. (1994) PROTEIN ENG. 7: 805-814. The most identical kappa and heavy human germline variable regions (at the amino acid level) to those of mouse 2B8 were identified. The debris that differed between mouse and human became the human sequence depending on the likelihood of such a change affecting the binding or immunogenicity. Low waste
10 risk (i.e. residues that when changed would probably not affect antigen binding and also reduce potential immunogenicity) were changed to the human amino acid in the heavy variable region (creating LR2B8HC) and the kappa variable region (creating LR2B8LC) . In addition, those of low risk and medium risk (that is, remains that when changed were likely to have a
The effect on the antigen-binding residues and also reducing the potential immunogenicity) was changed to the human amino acid in the heavy variable region (creating LRMR2B8HC) and the kappa variable region (creating LRMR2B8LC). The constant region of the human IgG1 heavy chain (allotype G1m (3) (allele1)) was added to the carboxyl terminus of the two regions
20 heavy variables modified by human genetic engineering, and the human kappa constant region (allotype Km (3) (allele 1)) were added to the terminal carboxyl end of two light variable regions modified by human genetic engineering, thus creating four chains of Antibodies modified by human genetic engineering. The nucleic acid sequences of the variable region were first synthesized by gene synthesis methods and then image88
5 added to sequences of the human constant region. These human genetically engineered antibodies were cloned into mammalian protein expression vectors and the protein was expressed in the four possible heavy chain plus light chain combinations. The binding of chimeric, chimeric / humanized or humanized antibodies to human HGF was measured
10 using conventional techniques described below. [0278] The nucleic acid sequences that encode, and the protein sequences that define each of the humanized antibodies are summarized below. In this section, the last nucleotide of each variable region is the first base of the next codon generated by the variable / constant region junction. This nucleotide
15 is included in the Variable Region because it is part of that exon. The amino acid sequences of the Constant Regions listed below include the translation of this binding codon.
[0279] (1) Nucleic acid sequence encoding the heavy chain variable region of humanized LR2B8HC (underlined signal sequence) (SEQ ID No. 182) image29
[0280] (2) Protein sequence that defines the heavy chain variable region of humanized LR2B8HC (without signal sequence) (SEQ ID No. 183) image89 image90
[0281] (3) Nucleic acid sequence encoding the constant region of the human IgG1 heavy chain (allotype G1m (3)) (allele1) (SEQ ID No. 184) image29
[0282] (4) Protein sequence defining the constant region of the human IgG1 heavy chain (allotype G1m (3)) (allele1 or 2) (SEQ ID No. 185). The first amino acid comes from the translation of the last nucleotide of the variable region and the [0283] (5) Nucleic acid sequence encoding the heavy chain variable region of humanized full-length heavy chain LR2B8HC and the constant region of the heavy chain of human IgG1 (allotype G1m (3)) (allele1) (underlined signal sequence) (SEQ ID No. 186) image91 image92 image31
[0284] (6) Protein sequence defining the heavy chain variable region of the full-length humanized heavy chain LR2B8HC and the constant region of the human IgG1 heavy chain (allotype G1m (3)) (allele1) (without
10 signal sequence) (SEQ ID No. 187) [0285] (7) Nucleic acid sequence encoding the heavy chain variable region of humanized LRMR2B8HC (underlined signal sequence) (SEQ ID No. 188) image29 image93 image29
[0286] (8) Protein sequence defining the heavy chain variable region of humanized LRMR2B8HC (without signal sequence) (SEQ ID NO. 189) image29
[0287] (9) Nucleic acid sequence encoding the heavy chain variable region of the full-length humanized heavy chain LRMR2B8HC and the constant region of the human IgG1 heavy chain (allotype G1m (3)) (allele1) (underlined signal sequence) (SEQ ID No. 190) [0288] (10) Protein sequence defining the heavy chain variable region of the full-length heavy chain humanized LRMR2B8HC and the constant region of the human IgG1 heavy chain (allotype G1m (3)) (allele1) (without signal sequence) (SEQ ID No. 191) image29 image94 image31
[0289] (11) Nucleic acid sequence encoding the variable region of the humanized LR2B8LC light chain (underlined signal sequence) (SEQ ID No. 192) image80
[0290] (12) Protein sequence defining the variable region of the humanized LR2B8LC light chain (without signal sequence) (SEQ ID No. 193) image29
[0291] (13) Nucleic acid sequence encoding the constant region of the human kappa chain (allotype Km (3)) (allele1) (SEQ ID No. 194) image29 image95
[0292] (14) Protein sequence defining the constant region of the human kappa chain (allotype Km (3)) (allele1) (SEQ ID No. 195). The first amino acid comes from the translation of the last nucleotide of the variable region and the two nucleotides of the beginning of the Kappa Light Chain sequence. image31
[0293] (15) Nucleic acid sequence encoding the variable region of the full-length humanized LR2B8LC light chain and the constant region of the human kappa chain (allotype Km (3)) (allele1) (SEQ ID No. 196) image80
[0294] (16) Protein sequence encoding the variable region of the full-length humanized LR2B8LC light chain and the constant region of the human kappa chain (allotype Km (3)) (allele1) (SEQ ID No. 197) image74
[0295] (17) Nucleic acid sequence encoding the light chain variable region of humanized LRMR2B8LC (underlined signal sequence) (SEQ ID No. 198) [0296] (18) Protein sequence defining the light chain variable region of humanized LRMR2B8LC (without signal sequence) (SEQ ID No. 199) image96 image29
[0297] (19) Nucleic acid sequence encoding the variable region of the full-length humanized LRMR2B8LC light chain and the constant region of the human kappa chain (allotype Km (3)) (allele1) (underlined signal sequence) ( SEQ ID No. 200) image29
[0298] (20) Protein sequence defining the variable region of the full-length humanized LRMR2B8LC light chain and the constant region of the human kappa chain (allotype Km (3)) (allele1) (SEQ ID No. 201) image29
[0299] For convenience, Table 14 provides a concordance graph showing the correspondence between the full length sequences and the antibodies analyzed in this section with those presented in the Sequence List.
20 TABLE 14

SEQ ID NO.

Protein or Nucleic Acid

182

Variable region of the heavy chain of LR2B8HC - nucleic acid

183

LR2B8HC heavy chain variable region - protein

184

Constant region of the human IgG1 heavy chain (allotype G1m (3)) (allele1) - nucleic acid

image97

SEQ ID NO.

Protein or Nucleic Acid

185

Constant region of the human IgG1 heavy chain (allotype G1m (3)) (allele1) - protein

186

LR2B8HC + IgG1 constant (allotype G1m (3)) (allele1) - nucleic acid

187

LR2B8HC + IgG1 constant (allotype G1m (3)) (allele1) - protein

188

Variable region of the heavy chain of LRMR2B8HC - nucleic acid

189

LRMR2B8HC heavy chain variable region - protein

190

LRMR2B8HC + IgG1 constant (allotype G1m (3)) (allele1) - nucleic acid

191

LRMR2B8HC + IgG1 constant (allotype G1m (3)) (allele1) - protein

192

Variable region of the light chain of LR2B8LC - nucleic acid

193

LR2B8LC light chain variable region - protein

194

Constant region of the human kappa chain (allotype Km (3)) (allele1) - nucleic acid

195

Constant region of the human kappa chain (allotype Km (3)) (allele1) - protein

196

LR2B8LC + kappa constant (allotype Km (3)) (allele1) - nucleic acid

197

LR2B8LC + kappa constant (allotype Km (3)) (allele1) - protein

198

Variable region of the light chain of LRMR2B8LC - nucleic acid

199

LRMR2B8LC light chain variable region - protein

200

LRMR2B8LC + kappa constant (allotype Km (3)) (allele1) - nucleic acid

201

LRMR2B8LC + kappa constant (allotype Km (3)) (allele1) protein

[0300] Table 15 summarizes the heavy chain CDR sequences (Kabat definition) of humanized 2B8 antibodies prepared by the humanization procedure 1 and by the humanization procedure 2 described hereinbefore in this Example. image98
TABLE 15

Antibody

CDR1 CDR2 CDR3 Full-length heavy chain variable region

Heavy 2B8 murine

TYWMH (SEQ ID No. 15) EINPTNGHTNYNEKFKS (SEQ ID No. 16) NYVGSIFDY (SEQ ID No. 17) SEQ ID No. 12

Hu2B8 Hv1f.1

TYWMH (SEQ ID No. 15) EINPTNGHTNYNEKFQG (SEQ ID No. 202) NYVGSIFDY (SEQ ID No. 17) SEQ ID No. 159

Hu2B8 Hv5a.1

TYWMH (SEQ ID No. 15) EINPTNGHTNYNPSFQG (SEQ ID No. 203) NYVGSIFDY (SEQ ID No. 17) SEQ ID No. 165

Hu2B8 Hv5-51.1

TYWMH (SEQ ID No. 15) EINPTNGHTNYNPSFQG (SEQ ID No. 203) NYVGSIFDY (SEQ ID No. 17) SEQ ID No. 169

LR2B8HC

TYWMH (SEQ ID No. 15) EINPTNGHTNYNEKFKG (SEQ ID No. 204) NYVGSIFDY (SEQ ID No. 17) SEQ ID No. 183

LRMR2B8HC

TYWMH (SEQ ID No. 15) EINPTNGHTNYNQKFQG (SEQ ID No. 205) NYVGSIFDY (SEQ ID No. 17) SEQ ID No. 189

[0301] Table 16 summarizes the CDR sequences of the light chain (Kabat definition) of humanized 2B8 antibodies prepared by the humanization procedure 1 and by the humanization procedure 2 described hereinbefore in this Example.
TABLE 16

Antibody

CDR1 CDR2 CDR3 Full-length light chain variable region

Light of

KASENVVSYVS GASNRNT GQSYNYPYT SEC ID No. 14

2B8 murine

(SEQ ID No. 18) (SEQ ID No. 19) (SEQ ID No. 20)

Hu2B8 Kv1

KASENVVSYVS GASNRNT GQSYNYPYT SEC ID No. 173

39.1

(SEQ ID No. 18) (SEQ ID No. 19) (SEQ ID No. 20)

Hu2B8 Kv3

KASENVVSYVS GASNRNT GQSYNYPYT SEC ID No. 179

15.1

(SEQ ID No. 18) (SEQ ID No. 19) (SEQ ID No. 20)

LR2B8LC

KASENVVSYVS GASNRNT GQSYNYPYT SEC ID No. 193

(SEQ ID No. 18)

(SEQ ID No. 19) (SEQ ID No. 20)

LRMR2B8L

KASENVVSYVS GASNRES GQSYNYPYT SEC ID No. 199

C

(SEQ ID No. 18) (SEQ ID No. 206) (SEQ ID No. 20)

image99
C. Binding affinity of humanized 2B8 antibodies [0302] Antigen binding affinity and interaction kinetics were evaluated by surface plasmon resonance technology using a BIAcore T100 instrument. Anti-human mouse immunoglobulins (Jackson ImmunoResearch Labs, 209-005-098) were immobilized on carboxymethylated dextran CM4 detection microplates (BIAcore, Catalog No. BR-1005-34) by amine coupling (BIAcore, Catalog No. BR- 1000-50) using a conventional coupling protocol according to the manufacturer's recommendations. The analyzes were performed at 25 ° C using PBS (GIBCO, Catalog No. 14040-133) containing 0.05% P20 surfactant (BIAcore, Catalog No. BR1000-54), BSA 2 mg / ml (EMD, Catalog No. 2930 ) and 10 mg / ml CM-Dextran sodium salt (Fluka, Catalog No. 86524) as a processing buffer. [0303] Antibodies were captured in individual flow cells at a flow rate of 10 µl / min. The injection time was variable for each antibody to produce approximately 20 UR of captured antibody for each cycle. Buffer or HGF (R&D Systems, Catalog No. 294-HGN-025) diluted in processing buffer was sequentially injected onto a reference surface (without captured antibody) and the active surface (antibody to be tested) for two minutes at 60

He / min

The dissociation phase was monitored for 15 or 90 minutes depending on the concentration. Then, the surface was regenerated with 10mM glycine-HCl, pH 2.0 (BIAcore, Catalog No. BR-1003-55) injected for 3 minutes at a flow rate of 60 µl / min before another cycle was started. The concentrations of HGF tested were 1.88, 3.75 and 7.5 nM. The determination of the kinetic parameters was achieved using the kinetic function of the BIAevaluation software with reference subtraction. The kinetic parameters for each antibody, ka (association rate constant), kd (dissociation rate constant) and KD (equilibrium dissociation constant) are summarized in Figure 8. [0304] The results summarized in Figure 8 show that certain combinations of heavy chains (Hu2B8 Hv5a.1, Hu2B8 Hv5-51.1 or Hu2B8 Hv1-f.1) and light chains (Hu2B8 Kv1-39.1 or Hu2B8 Kv3-15.1) retain a binding affinity (KD) to HGF similar to that of chimeric 2B8 (mouse variable regions with human constant regions) and 2B8 (Table 5).

D.

Mutually exclusive binding assay [0305] Mutually exclusive binding to HGF was evaluated by technology

image100
Superficial plasmon resonance using a BIAcore T100 instrument. Anti-human mouse immunoglobulins (Jackson ImmunoResearch Labs, 209-005-098) were immobilized on carboxymethylated dextran CM5 microplates (BIAcore, Catalog No. BR-1006-68) by amine coupling (BIAcore, Catalog No. BR- 1000-50) using a conventional coupling protocol according to the manufacturer's recommendations. The analyzes were performed at 25 ° C using PBS (GIBCO, Catalog No. 14040-133) containing 0.05% P20 surfactant (BIAcore, No. BR-1000-54), BSA 2 mg / ml (EMD, Catalog No. 2930 ) and 10 mg / ml CM-dextran sodium salt (Fluka, Catalog No. 86524) as a processing buffer. [0306] Humanized antibodies were captured in an individual flow cell at a flow rate of 30 µl / min. The injection time was variable for each antibody to produce approximately 150 UR of captured antibody for each cycle. HGF (R&D Systems, Catalog No. 294-HGN-025) diluted in processing buffer to a final concentration of 7.5 g / ml was injected for 90 s at 30
He / min on captured humanized antibodies. HGF binding was monitored before a subsequent injection of mouse 2B8 antibody or goat anti-HGF polyclonal antibody (R&D Systems, AF294) for 3 min at 30 µl / min. Then, the surface was regenerated with 10 mM glycine-HCl, pH 2.0 (BIAcore, Catalog No. BR1003-55) injected for 3 min at a flow rate of 60 µl / min before another antibody was tested. The results are summarized in Figure 9. [0307] The results summarized in Figure 9 show that both humanized 2B8 antibodies and chimeric 2B8 antibodies prevent the binding of murine 2B8 to HGF. These results demonstrate that humanized antibodies still bind to the same HGF epitope as the original 2B8 antibody.
Example 13 - Production of humanized 2B8 variants
to. HUMAN ENGINEERED� Antibodies [0308] Heavy chains (LR2B8HC and LRMR2B8HC, respectively) and light chains (LR2B8LC and LRMR2B8LC, respectively). of transient expression of XOMA antibodies, which contain modules of human Gamma-1 and Kappa constant regions. The four 2B8 Human Engineered variants were produced by transient transfection in HEK293E cells. The four antibodies were produced image101
following: HE2B8-1 = LR2B8HC (+ constant region of IgG1 (allotype G1m (3) (allele 1)) (SEQ ID No. 187) plus LR2B8LC (+ constant region Kappa (allotype Km (3) (allele 1)))) ( SEQ ID No. 197) HE2B8-2 = LR2B8HC (+ constant region of IgG1 (allotype G1m (3) (allele 1)) (SEQ ID No. 187) plus LRMR2B8LC (+ constant region Kappa (allotype Km (3) (allele 1) )) (SEQ ID No. 201) HE2B8-3 = LRMR2B8HC (+ constant region of IgG1 (allotype G1m (3) (allele 1)) (SEQ ID No. 191) plus LR2B8LC (+ constant region Kappa (allotype Km (3) ( allele 1))) (SEQ ID No. 197) HE2B8-4 = LRMR2B8HC (+ constant region of IgG1 (allotype G1m (3) (allele 1)) (SEQ ID No. 191) plus LRMR2B8LC (+ constant region Kappa (allotype Km ( 3) (allele 1))) (SEQ ID No. 201)
[0309] Light and heavy chains were used to co-transfect HEK293E cells adapted to XOMA suspension in IS293 media (Irvine Scientific, Irvine, CA) using 2 liter shake flasks. After 24 hours in the flasks with shaking, 200 ml of transfected cells were centrifuged, resuspended in 40 ml of fresh medium and transferred to Integra flasks (Wilson Wolf Manufacturing Inc., MN) for production. After incubation for seven days, cell suspensions were removed from Integra flasks, centrifuged and culture supernatants were preserved. The antibodies in the culture supernatants were purified on Spin columns of protein A (Pro-Chem), dialyzed against PBS, concentrated and sterilized by filtration.
b. SUPERHUMANIZED Antibodies [0310] Full-length Hu2B8_Hv5-51.1 cDNA + human IgG1 constant domain (allotype G1m (3)) was cloned into pEE6.4 (Lonza Biologics, Berkshire, UK) using HindIII and EcoRI restriction sites. The cDNA of the Hu2B8_Kv1-39.1 full-length variable region + human Kappa constant domain and the full-length Hu2B8_Kv3-15.1 variable region cDNA + human Kappa constant domain were each cloned into pEE14.4 (Lonza Biologics) using sites of HindIII and EcoRI restriction. The hCMV-MIE + Hu2B8_Hv5-51.1 full-length promoter cDNA + human IgG1 constant domain (allotype G1m (3)) + SV40 poly A fragment (in pEE6.4) was removed by digestion with NotI / SalI and inserted into the vector pEE14.4 of the Kappa chain by means of NotI / SalI sites, thereby generating 2 different expression vectors, each expressing simultaneously heavy and light chain to generate the image102
following antibodies: 5 sh2B8-9 (G1m (3)) =
sh2B8-12 (G1m (3)) = 10
hu2B8 Hv5-51.1 (+ constant region of IgG1 (allotype G1m (3)) (allele 2)) (SEQ ID No. 210) plus hu2B8 Kv 1-39.1 (+ constant region kappa (allotype Km (3) (allele 2)) ) (SEQ ID No. 177) hu2B8 Hv5-51.1 (+ constant region of IgG1 (allotype G1m (3)) (allele 2)) (SEQ ID No. 210) plus hu2B8 Kv 3-15.1 (+ constant region kappa (allotype Km ( 3) (allele 2))) (SEQ ID No. 181)
[0311] The nucleic acid sequences encoding, and the protein sequences defining the heavy constant region of human IgG1, allotype G1m (3) (allele 2), and
Each of the full-length heavy chain sequences are set forth below. The light chain sequences were the same as described in Example 12. [0312] (1) Nucleic acid sequence encoding the constant region of the human IgG1 heavy chain (allotype G1m (3)) (allele 2) ( SEQ ID No. 207). image103 image104 image29
[0313] (2) Protein sequence defining the constant region of the human IgG1 heavy chain (allotype G1m (3)) (allele 1 or 2) (SEQ ID No. 208). The first amino acid comes from the translation of the last nucleotide of the variable region and the two nucleotides of the beginning of the IgG1 heavy chain sequence. image29
[0314] (3) Nucleic acid sequence encoding the full length chain
10 containing the variable region of the humanized Hu2B8 Hv5-51.1 heavy chain and the constant region of the human IgG1 heavy chain, allotype G1m (3) (allele 2) (underlined signal sequence) (SEQ ID No. 209) [0315] (4) Protein sequence defining the full length heavy chain containing humanized Hu2B8 Hv5-51.1 and the constant region of the human IgG1 heavy chain, allotype G1m (3) (allele 2) (without signal sequence) (SEQ ID NO. 210) image29 image29 image29
[0316] Each double expression vector was used to transfect 293T cells to
10 its transient expression, using DMEM with 10% fetal bovine serum. Forty-eight hours after transfection, the cells were washed with and then replaced with serum-free medium, IS GRO (Irvine Scientific, Santa Ana, CA) containing 4 mM L-glutamine. The supernatant was collected daily and replaced with freshly prepared media for 10 days. The culture supernatants are
15 centrifuged, filtered (0.45 µm) and concentrated 10-100 times. The antibodies were purified on ProSep vA resin (Millipore), dialyzed against PBS, concentrated and sterilized by filtration.
Example 14 - Binding characteristics of humanized 2B8 variants
[0317] The humanized antibodies produced in Example 13 were characterized by their ability to bind hHGF and the recombinant HGF proteins produced in Example 3. [0318] The antibodies were analyzed by surface plasmon resonance using a BIAcore T100 instrument for evaluate your ability to join hHGF and the
25 fusion proteins analyzed in Example 3. Each antibody was immobilized on a carboxymethylated dextran CM5 detector microplate (BIAcore, Catalog No. BR-1006-68) by amine coupling (BIAcore, Catalog No. BR1000-50) using a Conventional coupling protocol according to the manufacturer's instructions. [0319] The analyzes were performed at 25 ° C using PBS (GIBCO, Catalog No. image105
5 14040-133) containing 0.05% P20 surfactant (BIAcore, Catalog No. R1000-54), BSA 2 mg / ml (EMD, Catalog No. 2930) and 10 mg / ml CM-dextran sodium salt ( Fluka, Catalog No. 86524) as processing buffer. The supernatant containing different HGF fusion proteins or the supernatant of cells transfected with an empty vector was injected onto each
10 antibody at a flow rate of 30 l / min for 3 minutes. The resulting binding was determined as resonance units (UR) above baseline 30 seconds after the end of the injection. Binding to human HGF (R&D Systems, Catalog No. 294-HGN-025) diluted in processing buffer was compared. Nonspecific binding was controlled by comparison of binding to a control surface. The
15 results are summarized in Table 17.
TABLE 17

Antibody

rhHGF (R&D Systems) mHGF (R&D Systems) Chimera MHM (495585) Chimera MHM (507585) Chimera MHM (499556)

2B8

Yes No Yes Yes Yes

HE2B8-1

Yes No Yes Yes Yes

HE2B8-2

Yes No Yes Yes Yes

HE2B8-3

Yes No Yes Yes Yes

HE2B8-4

Yes No Yes Yes Yes

sh2B8-9 (G1m (3))

Yes No Yes Yes Yes

sh2B8-12 (G1m (3))

Yes No Yes Yes Yes

[0320] The results in Table 17 demonstrate that each of the humanized 2B8-based antibodies binds to rhHGF and the three mouse chimeras
20 human-mouse.
Example 15 - Binding affinities of humanized 2B8 variants [0321] Binding affinities and interaction kinetics of the antibodies listed in Table 15 were measured by surface plasmon resonance. [0322] Anti-human mouse immunoglobulins (Jackson Labs, Catalog No. 209-005) were immobilized on carboxymethylated dextran CM5 detection microplates (BIAcore, Catalog No. BR-1006-68) by amine coupling image106
5 (BIAcore, Catalog No. BR-1000-50) using a conventional coupling protocol according to the manufacturer's instructions. The analyzes were performed at 25 ° C using PBS (GIBCO, Catalog No. 14040-133) containing 0.05% P20 surfactant (BIAcore, Catalog No. BR-1000-54) and BSA 2 mg / ml (EMD, No. Catalog 2930).
10 [0323] The antibodies were captured in an individual flow cell at a flow rate of 10 µl / min. The injection time was variable for each antibody to produce approximately 20 UR of captured antibody for each cycle. Buffer or HGF (R&D Systems, Catalog No. 294-HGN-025) diluted in processing buffer was sequentially injected onto a reference surface (without antibody
15 captured) and the active surface (antibody to be tested) for 2 minutes at 60
He / min The dissociation phase was monitored for 15 or 90 minutes depending on the concentration. Then, the surface was regenerated with 10 mM glycine-HCl, pH 2.2 (BIAcore, Catalog No. BR-1003-54) injected for 3 minutes at a flow rate of 60 µl / min before another cycle was started. HGF concentrations
20 tested were 0.46 nM to 7.5 nM. [0324] Kinetic parameters were determined using the kinetic function of the BIAevaluation software with reference subtraction. The kinetic parameters for each antibody, ka (association rate constant), kd (dissociation rate constant) and KD (equilibrium dissociation constant) are summarized
25 in Table 18.
TABLE 18

Antibody

ka (1 / Ms) kd (1 / s) KD (pM) DT

2B8

1.4 x 106 1.0 x 10-5 7.3

HE2B8-1

2.2 x 106 1.4 x 10-5 7.1 5.2

HE2B8-2

1.8 x 106 9.6 x 10-6 5.2 2.7

HE2B8-3

2.0 x 106 4.1 x 10-6 2.0 1.1

HE2B8-4

1.7 x 106 1.1 x 10-5 6.5 1.3

sh2B8-9 (G1m (17.1)

2.0 x 106 1.7 x 10-5 8.1 5.3

sh2B8-12 (G1m (17.1)

1.9 x 106 2.3 x 10-5 12 0.4

image107
[0325] These data show that humanized antibodies have rapid association rates (ka), very slow dissociation rates (kd) and very high affinities (KD). In particular, antibodies have affinities that vary from 2.0-12 pM.
Example 16 - Comparison of binding affinities at 25 ° C and 37 ° C [0326] The binding affinities and interaction kinetics of murine HE2B8-4, sh2B8-9, sh2B8-12 and 2B8 antibodies were measured by surface plasmon resonance in different conditions. [0327] Anti-human mouse immunoglobulins (Jackson Labs, Catalog No. 209-005) or rabbit anti-mouse immunoglobulins (BIAcore, Catalog No. BR-1005-14) were immobilized on carboxymethylated dextran CM4 detection microplates (BIAcore , Catalog No. BR-1006-68) by amine coupling (BIAcore, Catalog No. BR-1000-50) using a conventional coupling protocol according to the manufacturer's instructions. In the case of measurements at 25 ° C for sh2b8-9 and sh2B8-12, a CM5 detector microplate was used (BIAcore, Catalog No. BR-1006-68). The analyzes were performed at 25 ° C and 37 ° C using PBS (GIBCO, Catalog No. 14040-133) containing 0.05% P20 surfactant (BIAcore, Catalog No. BR-1000-54) and BSA at 2 mg / ml (EMD , Catalog No. 2930) as processing buffer. [0328] The antibodies were captured in an individual flow cell at a flow rate of 10 µl / min. The injection time was variable for each antibody to produce approximately 20 UR of captured antibody for each cycle. Buffer or HGF (R&D Systems, Catalog No. 294-HGN-025) diluted in processing buffer was sequentially injected onto a reference surface (without captured antibody) and the active surface (antibody to be tested) for 2 minutes at 60
He / min The dissociation phase was monitored for 15 or 90 minutes depending on the concentration. The surface of the detection microplates with anti-human mouse immunoglobulins was then regenerated with 10 mM glycine-HCl, pH 2.2 (BIAcore, Catalog No. BR-1003-54) injected for 3 minutes at a flow rate of 60 µl / min before another cycle started. The surface of the detection microplates with rabbit anti-mouse immunoglobulins was regenerated with 10 mM glycine-HCl, pH 1.7 (BIAcore, Catalog No. BR-1003-54) injected for 3 minutes at a flow rate of 60 μl / min before another cycle began. The concentrations of HGF tested were 0.46 nM to 7.5 nM. image108
[0329] Kinetic parameters were determined using the kinetic function of the BIAevaluation software with reference subtraction. The kinetic parameters for each antibody, ka (association rate constant), kd (dissociation rate constant) and KD (equilibrium dissociation constant) are summarized below in Table 19.
TABLE 19

Antibody

Temp. (ºC) ka (1 / MS) kd (1 / s) KD (pM)

2B8

25 1.6 x 106 2.1 x 10-5 13.5

2B8

37 2.8 x 106 1.3 x 10-5 4.5

HE2B8-4

25 2.0 x 106 1.2 x 10-5 5.6

HE2B8-4

37 3.1 x 106 1.0 x 10-5 3.3

sh2B8-9 (G1m (17.1))

25 2.0 x 106 1.7 x 10-5 8.1

sh2B8-9 (G1m (3))

37 2.5 x 106 1.4 x 10-5 5.8

sh2B8-12 (G1m (17.1))

25 1.9 x 106 2., 3 x 10-5 12.0

sh2B8-12 (G1m (3))

37 2.4 x 106 1.0 x 10-5 4.8

[0330] As expected, association rate constants increased with an increase in temperature. Surprisingly, the constants of
10 dissociation did not change significantly with an increase in the corresponding temperature. Therefore, global equilibrium dissociation constants (KD) were approximately 1.4 to 3 times lower (higher affinity) at physiological temperature (37 ° C).
Example 17 - Neutralization activity of humanized 2B8 variants [0331] The antibodies described in Example 14 were characterized by their ability to (a) inhibit hHGF binding to c-Met and (b) inhibit the incorporation of stimulated BrdU by HGF in 4MBr-5 cells. [0332] An HGF-Met binding inhibition assay was performed (Test of
20 neutralization) as described below. The antibodies were tested by ELISA to determine their ability to inhibit hHGF binding to c-Met. Specifically, Wallac 96-well DELFIA test plates (Wallac Inc., Catalog No. AAAND-0001) were coated with 100 μl of HGF 6.25 μg / ml (R&D Systems, Catalog No. 294-HGN-025 ) in carbonate coating buffer (15 mM Na2CO3 and 34 mM NaHCO3, pH 9.0) for 16 hours at 4 ° C. Then, the plates were blocked with 200 µl of 5% skimmed milk powder in PBS image109
5 for 1 hour at room temperature. The antibodies were prepared on a separate plate by adding increasing concentrations of the antibodies under investigation (0.033-250 nM, 2-fold serial dilution) to 2 nM biotinylated c-Met in 5% skimmed milk powder in PBS. The c-Met (R&D Systems, Catalog No. 358-MT / CF) was biotinylated according to the manufacturer's instructions to a
10 ratio of biotin to c-Met of 10: 1 (Pierce, Catalog No. 21335). 100 µl of sample per well was transferred to the assay plate and incubated for 2 hours at room temperature. The resulting plates were washed three times with 0.1% PBS-Tween 20 and incubated for 1 hour at room temperature with Eu-labeled streptavidin (Wallac, Catalog No. 1244-360)
15 diluted 1: 1000 in DELFIA assay buffer (Wallac, Catalog No. 4002-0010). The resulting plates were washed 3 times with DELFIA wash solution (Wallac, Catalog No. 4010-0010) and incubated with 100 µl / well DELFIA enhancement solution (Wallac, No. 4001-0010) for 15 minutes at room temperature with agitation. The plates were read on a Victor3V instrument (Perkin Elmer)
20 using the europium method. IC50 values were calculated using Prism. [0333] The IC50 values obtained are shown in Table 20. TABLE 20

Antibody

IC50 (nM) DT

2B8

9.2 1.2

HE2B8-1

6.0 1.2

HE2B8-2

5.7 1.1

HE2B8-3

5.9 1.1

HE2B8-4

6.5 1.2

sh2B8-9 (G1m (3))

4.2 -

sh2B8-12 (G1m (3)

6.8 -

[0334] These results from Table 20 demonstrate that antibodies
Humanized assays effectively neutralize the binding of HGF to c-Met. [0335] The antibodies in Table 17 were also tested in the cell proliferation assay described in Example 7 (b). The results are summarized to image110
continued in Table 21.
TABLE 21

Antibody

IC50 (nM) DT

2B8

0.86 0.35

HE2B8-1

0.47 0.15

HE2B8-2

0.66 0.13

HE2B8-3

0.55 0.28

HE2B8-4

0.58 0.26

sh2B8-9 (G1m (3))

0.52 0.11

sh2B8-12 (G1m (3))

0.81 0.22

[0336] The results in Table 21 demonstrate that all humanized antibodies tested inhibit HGF-induced proliferation of 4MBr-5 cells.
Example 18 - Anti-dispersion activity of humanized 2B8 variants [0337] The antibodies of Table 17 were tested in the anti-dispersion assay 10 described in Example 8. The results are summarized below in Table 22.
TABLE 22

Inhibition of HGF-induced MDCK cell dispersion

Antibody

Trial 1 Trial 2

2B8

++ ++

HE2B8-1

++ ++

HE2B8-2

++ ++

HE2B8-3

++ ++

HE2B8-4

++ ++

sh2B8-9 (G1m (3))

++ ++

sh2B8-12 (G1m (3))

++ ++

- Without Inhibition +++ Very strong inhibition, almost complete ++ Strong inhibition
+ Detectable inhibition [0338] The results in Table 22 demonstrate that all humanized antibodies tested inhibited HGF-induced dispersion to the same extent as murine monoclonal antibody 2B8. image111
Example 19 - Inhibition of HGF-stimulated c-Met phosphorylation [0339] The antibodies in Table 17 were tested in the c-Met phosphorylation assay described in Example 9. The results are summarized in Table 23 below. .
TABLE 23

Antibody

Average of two tests Standard deviation

2B8

0.91 0.02

he2B8-1

0.80 0.04

he2B8-2

0.88 0.15

he2B8-3

0.79 0.05

he2B8-4

0.75 0.14

sh2B8-9 (G1m (3))

0.93 0.03

sh2B8-12 (G1m (3))

0.81 0.07

[0340] The results in Table 23 demonstrate that all humanized antibodies tested were potent inhibitors of HGF-induced c-Met phosphorylation in PC-3 cells.
Example 20 - Inhibition of tumors in the U87MG xenograft model [0341] The ability of the humanized monoclonal antibodies of the invention
15 to inhibit the growth of tumors was tested in a U87MG xenograft model. U87MG (ATCC) cells were grown in culture at 37 ° C in an atmosphere containing 5% CO2 and 95% air, using a medium comprising Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum, penicillin 100 units / ml and streptomycin 100 �g / ml. The cells were subcultured
20 and were maintained by detachment of the cells from the culture plate wall using trypsin-EDTA. [0342] Almost confluent cells were collected by trypsin treatment and then 5 x 106 cells were injected into 50% Matrigel (BD Biosciences; catalog no. 356237) subcutaneously in the upper dorsal area between the scapulae
25 of 7-week-old female ICR SCID mice (Taconic Labs). The long (L) and short (W) (mm) diameters of the tumors were measured with a caliper. Tumor volume (vol.) Was calculated as: volume (mm3) = L x W2 / 2. When the tumors grew to approximately 200 mm³, the mice carrying tumors were randomized into 5 groups of 10 mice each. One group received PBS and one group received control of human IgG. Each of the other 4 groups received one of the humanized antibodies (HE2B8-1, HE2B8-2, HE2B8-3 and HE2B8-4). All the image112
5 antibodies were dosed at 0.25 mg / kg body weight twice a week, by intraperitoneal injection of 5 doses. Tumor volumes and body weights of the mice were recorded twice a week. Tumor growth inhibition was analyzed using Student's t-test. [0343] The humanized antibodies tested were active in vivo. There was a
10 tumor growth inhibition of 57% for HE2B8-1 with a p-value of 0.02, a tumor growth inhibition of 61% for HE2B8-2 with a p-value of 0.02, a tumor growth inhibition of 85% for HE2B8-3 with a p-value of 0.0004 and a tumor growth inhibition of 74% for HE2B8-4 with a p-value of 0.001. No significant body weight losses were observed.

LISTA DE SECUENCIAS <110> Winston, William M.

Wright, Kirk S.

Han, May

Breault, Lyne

Lin, Jie

Etemad-Gilbertson, Bijan

Knuehl, Christine

Gyuris, Jeno

<120> Proteínas de Unión al Factor de Crecimiento de Hepatocitos (HGF)

<130> AVO-001B

<150> 60/810.714

<151> 02-06-2006

<150> 60/860.509

<151> 21-11-2006

<160> 216

<170> PatentIn Versión 3.3

<210> 1

<211> 424

<212> ADN

<213> Secuencia artificial

<220>

<223> Región variable de la cadena pesada sintética de 1A3

<400> 1

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<220>
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<220>
<223> Secuencia proteica que define la región variable de la cadena pesada LR2B8HC humanizada de cadena pesada de longitud completa sintética y la región constante de la cadena pesada de IgG1 humana (alotipo G1m(3)) (alelo 1) (sin secuencia señal)
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[0344] A subsequent study was performed as previously described in female NCR athymic mice (Taconic Labs) carrying subcutaneous U87MG tumors inoculated on the flank. Each group (10 mice each) received one of the following treatments at 0.5 mg / kg: PBS vehicle control, huIgG control, HE2B8-4 or sh2B8-9. The treatment was administered intraperitoneally twice.
20 per week for a minimum of 5 weeks. Each treatment group demonstrated a similar tumor regression with tumor growth inhibition of 113% for sh2B8-9 and 115% for HE2B8-4, and a minimum tumor growth delay of 30 days. Both treatments were well tolerated without significant body weight losses. image113

LIST OF SEQUENCES <110> Winston, William M.

Wright, Kirk S.

Han, May

Breault, Lyne

Lin, Jie

Etemad-Gilbertson, Bijan

Knuehl, Christine

Gyuris, Jeno

<120> Hepatocyte Growth Factor Binding Proteins (HGF)

<130> AVO-001B

<150> 60 / 810,714

<151> 06-02-2006

<150> 60 / 860.509

<151> 11-21-2006

<160> 216

<170> PatentIn Version 3.3

<210> 1

<211> 424

<212> DNA

<213> Artificial sequence

<220>

<223> Variable region of the synthetic heavy chain of 1A3

<400> 1

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<213> Artificial sequence
<220>
<223> Protein sequence defining the sequence of the synthetic full length 2F8 light chain (constant region and kappa variable region of 2F8) (without signal sequence)
<400> 133 image214 image215 image216
<212> PRT
<213> Artificial sequence
<220>
<223> Protein sequence defining the sequence of the synthetic full-length 3B6 heavy chain (variable region of the 3B6 heavy chain and constant region of IgG1) (without signal sequence)
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<213> Artificial sequence
<220>
<223> Protein sequence defining the variable region of the humanized LR2B8HC heavy chain synthetic full-length heavy chain and the constant region of the human IgG1 heavy chain (allotype G1m (3)) (allele 1) (without signal sequence)
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Claims (20)

1. Isolated binding protein that binds to human hepatocyte growth factor (HGF), which comprises:

(to)

a variable region of an immunoglobulin heavy chain comprising the structure CDRH1-CDRH2-CDRH3, in which

(i)

 CDRH1 comprises an amino acid sequence SEQ ID NO: 15,

(ii)

 CDRH2 comprises an amino acid sequence selected from the group consisting of SEQ ID No. 204 and SEQ ID No. 205, and

(iii) CDRH3 comprises an amino acid sequence of SEQ ID NO: 17 and

(b)

a variable region of an immunoglobulin light chain comprising the structure CDRL1-CDRL2-CDRL3, in which

(i)

 CDRL1 comprises an amino acid sequence SEQ ID NO: 18,

(ii)

 CDRL2 comprises an amino acid sequence selected from the group consisting of SEQ ID No. 19 and SEQ ID No. 206, and

(iii) CDRL3 comprises an amino acid sequence SEQ ID No. 20, in which the variable region of the immunoglobulin heavy chain and the variable region of the immunoglobulin light chain together define a single binding site for human HGF binding. 2. Binding protein of claim 1, wherein the variable region of the immunoglobulin heavy chain comprises the structure CDRH1-CDRH2-CDRH3, wherein

(i)

 CDRH1 comprises the amino acid sequence SEQ ID NO: 15,

(ii)

 CDRH2 comprises the amino acid sequence SEQ ID No. 205 and

(iii) CDRH3 comprises the amino acid sequence SEQ ID NO: 17, and the variable region of the immunoglobulin light chain comprises the structure CDRL1-CDRL2-CDRL3, in which

(i)

 CDRL1 comprises the amino acid sequence SEQ ID NO: 18

(ii)

 CDRL2 comprises the amino acid sequence SEQ ID No. 206, and

(iii) CDRL3 comprises the amino acid sequence SEQ ID NO: 20.

3.

Binding protein of claim 1 or claim 2, wherein the complementarity determining regions (CDRs) are interposed between human or humanized immunoglobulin framework regions.

Four.

Binding protein of any of the preceding claims, wherein the binding protein is an antibody or an antigen binding fragment thereof.

5.

Binding protein of claim 4, wherein the antibody is a monoclonal antibody.

6.

Isolated nucleic acid comprising a nucleotide sequence encoding a variable region of an immunoglobulin light chain of any of claims 2-5.

7.

Isolated nucleic acid comprising a nucleotide sequence encoding a variable region of an immunoglobulin heavy chain of any of claims 1-5.

8.

Expression vector comprising the nucleic acid sequence of claim 6 and / or claim 7.

9.

Host cell comprising at least one expression vector encoding a variable region of an immunoglobulin heavy and / or light chain of any of claims 2-5.

10.

Method of producing a polypeptide comprising a variable region of an immunoglobulin heavy chain, the process comprising:

(i)

 culturing the host cell of claim 9 under conditions for the host cell to express the polypeptide comprising the variable region of the immunoglobulin heavy chain; Y

(ii)

 purifying the polypeptide comprising the variable region of the immunoglobulin heavy chain.

eleven.

Method of producing a polypeptide comprising a variable region of an immunoglobulin light chain, the process comprising:

(i)

 culturing the host cell of claim 9 under conditions for the host cell to express the polypeptide comprising the variable region of the immunoglobulin light chain; Y

(ii)

 purifying the polypeptide comprising the variable region of the immunoglobulin light chain.

12.

Isolated binding protein of any of claims 1-5, wherein the binding protein binds to human hepatocyte growth factor with a kd of 4.0 x 10-5 s-1 or less.

13.

Isolated binding protein of any of claims 1-5, wherein the binding protein binds to human hepatocyte growth factor with a KD of 20 pM or less.

14.

Binding protein of claim 1, which binds to human hepatocyte growth factor (HGF), comprising a variable region of an immunoglobulin light chain and a variable region of an immunoglobulin heavy chain selected from the group consisting of:

image 1 image2

(to)

a variable region of an immunoglobulin light chain comprising the amino acid sequence of SEQ ID No. 193 and a variable region of an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID No. 183;

(b)

a variable region of an immunoglobulin light chain comprising the amino acid sequence of SEQ ID No. 193 and a variable region of an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID No. 189;

(C)

a variable region of an immunoglobulin light chain comprising the amino acid sequence of SEQ ID No. 199, and a variable region of an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID No. 183; Y

(d)

a variable region of an immunoglobulin light chain comprising the amino acid sequence of SEQ ID No. 199 and a variable region of an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID No. 189.

image3

fifteen.

Binding protein of claim 14, wherein the variable region of the immunoglobulin light chain comprises the amino acid sequence of SEQ ID No. 199, and the variable region of the immunoglobulin heavy chain comprises the amino acid sequence of SEQ ID No. 189.

16.

Binding protein of claim 1, which binds to human hepatocyte growth factor (HGF), comprising a sequence of an immunoglobulin light chain and a sequence of an immunoglobulin heavy chain selected from the group consisting of:

(to)

a sequence of an immunoglobulin light chain comprising the amino acid sequence of SEQ ID No. 197 and a sequence of an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID No. 187;

(b)

a sequence of an immunoglobulin light chain comprising the amino acid sequence of SEQ ID No. 197 and a sequence of an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID No. 191;

(C)

a sequence of an immunoglobulin light chain comprising the amino acid sequence of SEQ ID No. 201 and a sequence of an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID No. 187; Y

(d)

a sequence of an immunoglobulin light chain comprising the amino acid sequence of SEQ ID No. 201 and a sequence of an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID No. 191.

17.

Binding protein of claim 16, wherein the immunoglobulin light chain comprises the amino acid sequence of SEQ ID No. 201 and the immunoglobulin heavy chain comprises the amino acid sequence of SEQ ID No. 191.

18.

Binding protein of any of claims 1-5 or 14-17 for use in therapy.

19.

Binding protein of any of claims 1-5 or 14-17 for use in inhibiting or reducing the proliferation of a tumor cell.

twenty.

Binding protein of any of claims 1-5 or 14-17 for use in inhibiting or reducing tumor growth in a mammal.

image4 21. Method of producing a binding protein that binds to human hepatocyte growth factor (HGF), which is an intact antibody, such as a monoclonal antibody, or an antigen binding fragment thereof, the method comprising : (i) growing the host cell of claim 9 under conditions 10 allowing the host cell to express a polypeptide comprising the variable region of an immunoglobulin heavy chain and a polypeptide comprising the variable region of an immunoglobulin light chain; Y (ii) purify the antibody or antigen-binding fragment of the antibody. fifteen