HK1167412B

HK1167412B - Humanized anti-cdcp1 antibodies

Info

Publication number: HK1167412B
Application number: HK12108145.3A
Authority: HK
Inventors: Johannes Auer; Birgit Bossenmaier; Guy Georges; Alexander Lifke; Ekkehard Moessner; Gerhard Niederfellner
Original assignee: 罗切格利卡特公司
Priority date: 2009-08-28
Filing date: 2010-08-26
Publication date: 2015-03-27

Description

Humanized anti-CDCP 1 antibodies

The present invention relates to humanized antibodies against human CDCP1 (anti-CDCP 1 antibodies), methods of their production, pharmaceutical compositions containing the antibodies, and uses thereof.

Background

Human CDCP1 ((protein 1 containing the CUB domain, B345, CD318, SIMA135, TRASK; SEQ ID NO: 29 and variant having the mutation R525Q (i.e., arginine (R) replaced by glutamine (Q) at amino acid position 525 of SEQ ID NO: 29)) and/or the mutation G709D (i.e., glycine (G) replaced by aspartic acid (D) at amino acid position 709 of SEQ ID NO: 29)) is a transmembrane protein containing three extracellular CUB domains, it was found that this protein is overexpressed in breast, colon and lung cancers, the expression level of which correlates with the metastatic capacity of cancer cells (Uekita, T. et al, am. J. Pathol.172 (1729) 1739.) it has been shown to be tyrosine phosphorylated in cancer cell lines (WO 2002/004508; Scherl-Mostageer, M. et al, Oncogene 20(2001) 4402-8; Hooper J. oncogene.2008. 2003. Pergene et al.;. 2003-1794), s., e, et al, FEBS lett.581(2007) 1137-42; brown, t, a, et al, j.biol.chem.279(2004) 14772-; ota, T, et al, nat. Genet.36(2004) 40-45). Alternatively spliced transcript variants encoding unique isoforms have been reported.

WO 2002/004508 mentions CDCP1 as tumor associated antigen B345. WO 2004/074481 mentions that CDCP1 is the glycoprotein antigen SIMA135 expressed in metastatic tumor cells. WO2005/042102 mentions CDCP1 as a protein involved in ovarian cancer. WO 2007/005502 relates to methods and compositions targeting CDCP1 for the treatment of disease.

US 2004/0053343 (and Conze, t. et al, ann.n.y.acad.sci.996(2003)222-6 and Buehring, h.j. et al, Stem Cells 22(2004)334-43) relates to CDCP1 antibodies for identifying certain Stem cell populations.

Summary of The Invention

One aspect of the invention is an antibody that specifically binds to human CDCP1, comprising the heavy chain variable domain (VH) of the CUB4 antibody (accession number DSM ACC2551) SEQ ID NO:1 and light chain variable domain (VL) SEQ ID NO:2,

the antibody is characterized in that: a) is humanized and b) comprises in the VH sequence:

lysine (K) at position 57 instead of threonine (T), and valine (V) at position 60 instead of proline (P) (all positions are numbered according to Kabat).

Another aspect of the invention is an antibody that specifically binds to human CDCP1, comprising the heavy chain variable domain (VH) of the CUB4 antibody (accession number DSM ACC2551) SEQ ID NO:1 and light chain variable domain (VL) SEQ ID NO:2,

the antibody is characterized in that: a) is humanized and b) comprises in the VL sequence:

leucine (L) at position 33 instead of valine (V), and tryptophan (W) at position 47 (all positions are numbered according to Kabat).

the antibody is characterized in that: a) is a humanized form of the human-derived,

and b) comprises in the VH sequence:

lysine (K) at position 57 in place of threonine (T), and valine (V) at position 60 in place of proline (P);

and in said VL sequence:

Preferably, the humanized antibody according to the invention is characterized in that the heavy chain variable domain (VH) is seq id NO: 3.

preferably, the humanized antibody according to the invention is characterized in that the light chain variable domain (VL) is SEQ id no: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17. or SEQ ID NO: 18.

preferably, the humanized antibody according to the invention is characterized in that:

the heavy chain variable domain (VH) is SEQ ID NO:3,

and is

The light chain variable domain (VL) is SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18. SEQ ID NO: 19. SEQ ID NO: 20. SEQ ID NO: 21. SEQ ID NO: 23. or SEQ ID NO: 24.

the heavy chain variable domain (VH) is SEQ ID NO:3,

and is

The light chain variable domain (VL) is SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17. or SEQ ID NO: 18.

preferably, the humanized antibody according to the invention is characterized in that: the antibody is of the subclass human IgG 1.

Preferably, the humanized antibody according to the invention is characterized in that: the antibody is glycosylated with a sugar chain at Asn297, wherein the amount of fucose within the sugar chain is 65% or less.

Yet another embodiment of the invention is a pharmaceutical composition comprising a humanized antibody according to the invention.

Yet another embodiment of the invention is said pharmaceutical composition for the treatment of cancer comprising a humanized antibody according to the invention.

The invention further comprises a humanized antibody according to the invention for use in the treatment of cancer.

The invention further comprises the use of an antibody according to the invention for the manufacture of a medicament for the treatment of cancer.

The invention provides nucleic acids encoding the humanized antibodies according to the invention. The invention further provides expression vectors containing a nucleic acid according to the invention and capable of expressing said nucleic acid in a prokaryotic or eukaryotic host cell and host cells containing such vectors for the recombinant production of antibodies according to the invention.

The invention further comprises a prokaryotic or eukaryotic host cell comprising a vector according to the invention.

The invention further comprises a method for producing a recombinant humanized antibody according to the invention, characterized in that a nucleic acid according to the invention is expressed in a prokaryotic or eukaryotic host cell and the antibody is recovered from the cell or cell culture supernatant. The invention further includes antibodies obtained by such recombinant methods.

The invention further provides a method for treating a patient suffering from cancer comprising administering to a patient diagnosed with (and therefore in need of) such a disease an effective amount of an antibody according to the invention. Preferably, the antibody is administered in a pharmaceutical composition.

It has now surprisingly been found that a particular humanized version of the CDCP1 antibody CUB4 according to the invention shows improved CDCP1 binding properties compared to other humanized versions derived from humanization known in the prior art. This is due to specific amino acid changes in the CDRH2, and/or in the CDRL1 and in the light chain framework. Surprisingly, the specific humanized version of the CDCP1 antibody CUB4 according to the invention showed improved tumor growth inhibition in vivo compared to chimeric and mouse CUB4 antibodies.

Detailed Description

The CUB4 antibody refers to the deposited antibody from DE 10242146(EP 1396501, US 7,541,030) with deposit number DSM ACC2551(DSMZ) having the heavy chain variable domain (VH) SEQ ID NO:1 and light chain variable domain (VL) SEQ ID NO: 2. the CUB4 antibody specifically binds to human CDCP 1. (deposit No. DSM ACC2551(DSMZ) by Eberhard-Karls-university Tubingen,tubingen, Geisswag 372076 Tubingen).

As used herein, the term "humanized" means a polypeptide based on a polypeptide having the VH SEQ id no:1 and VL SEQ ID NO:2, wherein (after chimerization with human constant regions) the VH and VL are humanized by grafting murine CDRs into the framework regions of human antibodies (see, e.g., Riechmann, l. et al, Nature 332(1988)323-61; WO 90/07861; and U.S. Pat. No.5,225,539). The heavy and light chain variable framework regions may be derived from the same or different human antibody sequences. The human antibody sequence may be that of a naturally occurring human antibody. The human heavy and light chain variable framework regions are listed, for example, in Lefranc, M.P., Current Protocols in Immunology (2000) -appendix 1PA.1P.1-A.1P.37, and via IMGT, the International ImmunoGeneTiCs information System(http:// imgt. circuits. fr) or accessible via http:// vbase. mrc-cpe. cam. ac. uk.

In addition, the humanized antibody according to the present invention has

a) Specific mutations in the CDRH2 of the VH (mutations T57K and P60V), and/or

b) A specific mutation in the CDRL1 of VL (mutation V33L) and a specific mutation in the framework region of VL (back mutation at position 47 from the human VL framework amino acid to the mouse amino acid W).

Surprisingly, such mutations in the humanized CUB4 antibody resulted in improved binding properties (compared to a humanized CUB4 antibody without such modifications). Furthermore, such modifications in the CDRs and/or framework result in humanized antibodies according to the invention with improved in vivo tumor growth inhibition (compared to the chimeric and mouse parent antibodies).

the antibody is characterized in that: is humanized and comprises in the VH sequence:

lysine (K) at position 57 (in CDRH 2) replacing threonine (T), and valine (V) at position 60 (in CDRH 2) replacing proline (P) (all positions are numbered according to Kabat). This means that seq id NO:1 contains mutations T57K and P60V in the CDRH2 of the VH.

the antibody is characterized in that: is humanized and comprises in the VL sequence:

leucine (L) at position 33 (in CDRL 1) replacing valine (V), and tryptophan (W) at position 47 (replacing an amino acid from the human VL framework region) (all positions numbered according to Kabat). This means that SEQ ID NO:2 comprises the mutation V33L in CDRL1 and a back mutation from human to mouse amino acid W at position 47 in framework region VL.

the antibody is characterized in that: is a humanized form of the human-derived,

and in said VH sequence:

lysine (K) 57 (in CDRH 2) replacing threonine (T), and valine (V) 60 (in CDRH 2) replacing proline (P);

and in said VL sequence:

leucine (L) at position 33 instead of valine (V), and tryptophan (W) at position 47 (all positions are numbered according to Kabat). This means that SEQ ID NO:1 comprises the mutations T57K and P60V in the CDRH2 of the VH and SEQ ID NO:2 mutation V33L in CDRL1 comprising VL and a back mutation at position 47 in the VL framework region from human to mouse amino acid W.

leucine (L) at position 33 (in CDRL 1) replacing valine (V), and tryptophan (W) at position 47 (replacing an amino acid from the human VL framework region);

and further characterized by: (further) comprising in said VL sequence:

methionine (M) at position 21 (replacing an amino acid from the human VL framework region) (all positions are numbered according to Kabat).

and in said VH sequence:

and in said VL sequence:

leucine (L) at position 33 and tryptophan (W) at position 47 in place of valine (V) (in place of an amino acid from a human VL framework region);

and further characterized by: (further) comprising in said VL sequence:

methionine (M) at position 21 (replacing an amino acid from a human VL framework region); glycine (G) or arginine (R) at position 24 (in CDRL 1) instead of serine (S), and alanine (a) at position 25 (in CDRL 1) instead of valine (V) (all positions numbered according to Kabat).

and in said VH sequence:

and in said VL sequence:

and further characterized by: (further) comprising in said VL sequence:

arginine (R) at position 24 (in CDRL 1) instead of serine (S), and alanine (a) at position 25 (in CDRL 1) instead of valine (V) (all positions are numbered according to Kabat).

and in said VH sequence:

and in said VL sequence:

and further characterized by: (further) comprising in said VL sequence:

In one embodiment of the invention, the humanized antibody according to the invention is characterized in that:

the heavy chain variable domain (VH) is SEQ ID NO: 3.

in another embodiment of the invention, the humanized antibody according to the invention is characterized in that: the light chain variable domain (VL) is SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17. or SEQ ID NO: 18.

in another embodiment of the invention, the humanized antibody according to the invention is characterized in that:

the heavy chain variable domain (VH) is SEQ ID NO:3,

and is

the heavy chain variable domain (VH) is SEQ ID NO:3,

and is

the heavy chain variable domain (VH) is SEQ ID NO:3,

and is

The light chain variable domain (VL) is SEQ ID NO: 14.

the heavy chain variable domain (VH) is SEQ ID NO:3,

and is

The light chain variable domain (VL) is SEQ ID NO: 15.

the heavy chain variable domain (VH) is SEQ ID NO:3,

and is

The light chain variable domain (VL) is SEQ ID NO: 16.

the heavy chain variable domain (VH) is SEQ ID NO:3,

and is

The light chain variable domain (VL) is SEQ ID NO: 17.

the heavy chain variable domain (VH) is SEQ ID NO:3,

and is

The light chain variable domain (VL) is SEQ ID NO: 18.

the heavy chain variable domain (VH) is SEQ ID NO:3,

and is

The light chain variable domain (VL) is SEQ ID NO: 23.

in another embodiment of the invention, the humanized antibody according to the invention is characterized in that: the antibody is of the subclass human IgG 1.

In another embodiment of the invention, the humanized antibody according to the invention is characterized in that: the antibody is glycosylated with a sugar chain at Asn297, wherein the amount of fucose within the sugar chain is 65% or less.

A preferred embodiment of the humanized antibody according to the invention is characterized in that: one of the following combinations of humanized heavy chain variable domain VH and humanized light chain variable domain VL, as shown in table 1 (see examples below).

Table 1: preferred combinations of humanized heavy chain variable domain VH and humanized light chain variable domain VL.

Humanized CUB4 antibody example number	VL(SEQ ID NO：)	VL(SEQ ID NO：)
			80	hHC4-H(SEQ ID NO：3)	hLC-M(SEQ ID NO：14)
69	hHC4-H(SEQ ID NO：3)	hLC-L2(SEQ ID NO：15)
			47	hHC4-H(SEQ ID NO：3)	hLC-K(SEQ ID NO：16)
58	hHC4-H(SEQ ID NO：3)	hLC-L(SEQ ID NO：17)
			36	hHC4-H(SEQ ID NO：3)	hLC-J(SEQ ID NO：18)
102	hHC4-H(SEQ ID NO：3)	hLC-b(SEQ ID NO：19)
			113	hHC4-H(SEQ ID NO：3)	hLC-c(SEQ ID NO：20)
91	hHC4-H(SEQ ID NO：3)	hLC-a(SEQ ID NO：21)
			124	hHC4-H(SEQ ID NO：3)	hLC-d(SEQ ID NO：22)
135	hHC4-H(SEQ ID NO：3)	hLC-e(SEQ ID NO：23)
			146	hHC4-H(SEQ ID NO：3)	hLC-f(SEQ ID NO：24)

Thus, one embodiment of the present invention is an antibody specifically binding to human CDCP1, characterized by comprising

Heavy chain variable domain (VH) SEQ ID NO: 3.

one embodiment of the present invention is an antibody specifically binding to human CDCP1, characterized by comprising

Heavy chain variable domain (VH) SEQ ID NO:3

And

light chain variable domain (VL) SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18. SEQ ID NO: 19. SEQ ID NO: 20. SEQ ID NO: 21. SEQ ID NO: 22. SEQ ID NO: 23. or SEQ ID NO: 24.

Heavy chain variable domain (VH) SEQ ID NO:3

And

light chain variable domain (VL) SEQ ID NO: 14.

Heavy chain variable domain (VH) SEQ ID NO:3

And

light chain variable domain (VL) SEQ ID NO: 15.

Heavy chain variable domain (VH) SEQ ID NO:3

And

light chain variable domain (VL) SEQ ID NO: 16.

Heavy chain variable domain (VH) SEQ ID NO:3

And

light chain variable domain (VL) SEQ ID NO: 17.

Heavy chain variable domain (VH) SEQ ID NO:3

And

light chain variable domain (VL) SEQ ID NO: 18.

Heavy chain variable domain (VH) SEQ ID NO:3

And

light chain variable domain (VL) SEQ ID NO: 19.

Heavy chain variable domain (VH) SEQ ID NO:3

And

light chain variable domain (VL) SEQ ID NO: 20.

Heavy chain variable domain (VH) SEQ ID NO:3

And

light chain variable domain (VL) SEQ ID NO: 21.

Heavy chain variable domain (VH) SEQ ID NO:3

And

light chain variable domain (VL) SEQ ID NO: 22.

Heavy chain variable domain (VH) SEQ ID NO:3

And

light chain variable domain (VL) SEQ ID NO: 23.

Heavy chain variable domain (VH) SEQ ID NO:3

And

light chain variable domain (VL) SEQ ID NO: 24.

a further preferred embodiment of the humanized antibody according to the invention is characterized in that: one of the following combinations of humanized heavy chain variable domain VH and humanized light chain variable domain VL, as shown in table 2. Such combinations include the following example numbers of humans, as shown in table 2.

Table 2: other preferred combinations of humanized heavy chain variable domain VH and humanized light chain variable domain VL.

Humanized CUB4 antibody example number	VL(SEQ ID NO：)	VL(SEQ ID NO：)
			80	hHC4-H(SEQ ID NO：3)	hLC-M(SEQ ID NO：14)
69	hHC4-H(SEQ ID NO：3)	hLC-L2(SEQ ID NO：15)
			47	hHC4-H(SEQ ID NO：3)	hLC-K(SEQ ID NO：16)
58	hHC4-H(SEQ ID NO：3)	hLC-L(SEQ ID NO：17)
			36	hHC4-H(SEQ ID NO：3)	hLC-J(SEQ ID NO：18)

Thus, one embodiment of the invention is an antibody that specifically binds to human CDCP1, characterized by comprising:

heavy chain variable domain (VH) SEQ ID NO: 3.

one embodiment of the present invention is an antibody that specifically binds to human CDCP1, characterized by comprising:

light chain variable domain (VL) SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17. or SEQ ID NO: 18.

heavy chain variable domain (VH) SEQ ID NO:3

And

unless otherwise specified, the terms "Kabat numbering" or "numbering according to Kabat" or "EU index" are defined as using the EU index as in Kabat et al (Sequences of Proteins of immunologica interest, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) for residue numbering in, e.g., IgG antibodies.

As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to a preparation of antibody molecules of a single amino acid composition.

The term "chimeric antibody" refers to a monoclonal antibody comprising at least a portion of a mouse-derived variable, i.e., binding, region and constant regions derived from different sources or species, typically prepared by recombinant DNA techniques. Chimeric antibodies comprising mouse variable regions and human constant regions are particularly preferred. Such mouse/human chimeric antibodies are the product of an expressed immunoglobulin gene comprising a DNA segment encoding a mouse immunoglobulin variable region and a DNA segment encoding a human immunoglobulin constant region. Other forms of "chimeric antibodies" encompassed by the present invention are those in which the class or subclass has been modified or altered from that of the original antibody. Such "chimeric" antibodies are also known as "class switch antibodies". Methods for generating chimeric antibodies involve conventional recombinant DNA and gene transfection techniques now known in the art (see, e.g., Morrison, S.L. et al, Proc. Natl. Acad Sci. USA 81(1984) 6851-6855; US 5,202,238 and US 5,204,244).

Human CDCP1 ((protein 1 containing the CUB domain, B345, CD318, SIMA135, TRASK; SEQ ID NO: 29 and variant with mutation R525Q (i.e., arginine (R) replaced by glutamine (Q) at amino acid position 525 of SEQ ID NO: 29)) and/or mutation G709D (i.e., glycine (G) replaced by aspartic acid (D) at amino acid position 709 of SEQ ID NO: 29)) is a transmembrane protein containing three extracellular CUB domains this protein was found to be overexpressed in breast, colon and lung cancers (Uekita, T.et al, am.J.Pathol.172(2008) 1729) whose expression level correlates with the metastatic capacity of cancer cells it has been shown to be tyrosine phosphorylated in cancer cell lines (WO 2002/004508; Scherl-Mostager, M. et al, Oncogene 20(2001) 4402-8; Hooper, J.17322, D. 2003. oncogene.22; Pergene 1783), s.e. et al, FEBS lett.581(2007) 1137-42; brown, T.A., et al, J.biol.chem.279(2004) 14772-14783; ota, T, et al, nat. Genet.36(2004) 40-45). Alternatively spliced transcript variants encoding unique isoforms have been reported.

As used herein, "specifically binds to human CDCP 1" refers to an antibody that specifically binds to human CDCP1 antigen. The binding affinity was 1.0x10^-8mol/l or less (e.g., 1.0x 10)^-8mol/l to 1.0x10^-13mol/l), preferably 5.0x10^-9mol/l or less (e.g., 5.0x 10)^-9mol/l to 1.0x10^-13mol/l) of KD values. Using standard binding assays, such as surface plasmon resonance techniquesTo determine binding affinity.

The term "epitope" means a protein determinant in human CDCP1 that is capable of specifically binding to an antibody. Epitopes are usually composed of chemically active surface aggregates of molecules such as amino acids or sugar side chains, and usually epitopes have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished by the loss of binding to the former, but not the latter, in the presence of denaturing solvents.

As used herein, "variable domain" (light chain variable domain (VL), heavy chain variable domain (VH)) means each of a pair of light and heavy chain domains that are directly involved in binding of an antibody to an antigen. The light and heavy chain variable domains have the same general structure, and each domain comprises four Framework (FR) regions whose sequences are widely conserved, connected by three "hypervariable regions" (or complementarity determining regions, CDRs). The framework regions adopt a β -sheet conformation, and the CDRs can form loops connecting the β -sheet structure. The CDRs in each chain retain their three-dimensional structure through the framework regions and form together with the CDRs from the other chain an antigen binding site. The heavy and light chain CDR3 regions of the antibody play a particularly important role in the binding specificity/affinity of the antibody according to the invention and thus provide further objects of the invention.

The "framework" or "FR" regions are those variable domain regions that differ from the hypervariable region residues as defined herein. Thus, the light and heavy chain variable domains of the antibody comprise domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the N to the C terminus. CDR and FR regions are defined according to the standard definition of Kabat et al, Sequences of proteins of Immunological Interest, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991) and/or those residues from "hypervariable loops".

As used herein, the term "antigen-binding portion of an antibody" refers to the amino acid residues of an antibody that are responsible for antigen binding. The antigen-binding portion of an antibody comprises amino acid residues from a "complementarity determining region" or "CDR". The term "antigen-binding portion" of an antibody of the invention contains 6 Complementarity Determining Regions (CDRs) that contribute to the affinity of the binding site for the antigen to varying degrees. There are three heavy chain variable domain CDRs (CDRH1, CDRH2 and CDRH3) and three light chain variable domain CDRs (CDRL1, CDRL2 and CDRL 3). The term "CDRH 1" means the CDR1 region of the heavy chain variable region calculated according to Kabat. CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 refer to the respective regions from the heavy (H) or light (L) chain. The extent of the CDRs and Framework Regions (FRs) is determined by comparison with a compiled database of amino acid sequences in which those regions have been defined according to the inter-sequence variability according to Kabat et al (supra).

As used herein, the term "nucleic acid" or "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, but preferably is double-stranded DNA.

As used within this application, the term "amino acid" means the group of naturally occurring carboxy alpha-amino acids, including alanine (three letter code: ala, one letter code: a), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

The antibodies according to the invention are characterized in that the constant regions are of human origin and preferably of the human IgG1 subclass. The constant regions include the heavy and light chain constant regions of an antibody. The heavy chain constant region comprises, in the N-terminal to C-terminal direction, an antibody heavy chain constant domain 1(CH1), an antibody Hinge Region (HR), an antibody heavy chain constant domain 2(CH2), and an antibody heavy chain constant domain 3(CH3), and optionally an antibody heavy chain constant domain 4(CH4) in the case of IgE subclass antibodies. The light chain constant region comprises an antibody light chain constant domain (CL). The antibody light chain constant domain (CL) may be kappa (kappa) or lambda (lambda). Such invariant chains are well known in the art and are described, for example, by Kabat, e., a. (see, e.g., Johnson, g., and Wu, t., Nucleic Acids res.28(2000) 214-. For example, a useful IgG1 subclass human heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 26. for example, a useful human light chain constant region comprises the amino acid sequence of a kappa light chain constant region of SEQ ID NO: 27; another useful human light chain constant region comprises the amino acid sequence of a lambda light chain constant region SEQ ID NO: 28.

the "Fc portion" of an antibody is not directly involved in binding of the antibody to an antigen, but exhibits various effector functions. The "Fc portion of an antibody" is a term well known to the skilled artisan and is defined based on the cleavage of the antibody by papain. Antibodies or immunoglobulins are classified according to the amino acid sequence of their heavy chain constant region: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, and IgG4, IgA1, and IgA 2. Depending on the heavy chain constant region, different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

The Fc portion of antibodies is directly involved in ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity) based on complement activation, C1q binding and Fc receptor binding. Complement activation (CDC) is initiated by the binding of complement factor C1q to the Fc portion of most IgG antibody subclasses. Although the effect of antibodies on the complement system depends on certain conditions, binding to C1q is caused by a defined binding site in the Fc portion. Such binding sites are known in the art and are described, for example, by Boackle, R.J., et al, Nature 282(1979) 742-; lukas, T.J., et al, J.Immunol.127(1981) 2555-2560; brunhouse, r. and Cebra, j.j., mol. immunol.16(1979) 907-; burton, D.R. et al, Nature 288(1980) 338-344; thommesen, J.E., et al, mol.Immunol.37(2000) 995-1004; idusogene, E.E., et al, J.Immunol.164(2000) 4178-; hezareh, M. et al, J.virology 75(2001) 12161-12168; morgan, A. et al, Immunology 86(1995) 319-324; EP 0307434 describes. Such binding sites are for example L234, L235, D270, N297, E318, K320, K322, P331 and P329 (numbering according to the EU index of Kabat, e.a., see below). Antibodies of subclasses IgG1, IgG2 and IgG3 generally show complement activation and binding to C1q and C3, whereas IgG4 does not activate the complement system and does not bind to C1q and C3.

The antibodies according to the invention comprise an Fc part derived from human origin, and preferably all other parts of the human constant region. As used herein, the term "Fc portion derived from human origin" means an Fc portion of a human antibody that is of the IgG1, IgG2, IgG3, or IgG4 subclasses, preferably an Fc portion from the human IgG1 subclass, a mutated Fc portion from the human IgG1 subclass (preferably having a mutation in the aspect of L234A + L235A), an Fc portion from the human IgG4 subclass, or an Fc portion from the mutated Fc portion of the human IgG4 subclass (preferably having a mutation in the aspect of S228P). Most preferred are polypeptides having the sequence of SEQ ID NO: 26 or 31, human IgG1 subclass, human IgG1 subclass with mutations L234A and L235A, human IgG having the amino acid sequence of SEQ ID NO: the human IgG4 subclass of 32, or the human heavy chain constant region of the human IgG4 subclass with the mutation S228P.

The term "antibody-dependent cellular cytotoxicity (ADCC)" refers to the lysis of human target cells by an antibody according to the invention in the presence of effector cells. ADCC is preferably measured by treating a preparation of CDCP 1-expressing cells with an antibody according to the invention in the presence of effector cells, such as freshly isolated PBMCs or purified effector cells from buffy coats, such as monocytes or Natural Killer (NK) cells or permanently growing NK cell lines.

The term "Complement Dependent Cytotoxicity (CDC)" means a process initiated by the binding of complement factor C1q to the Fc portion of most IgG antibody subclasses. Binding of C1q to antibodies is caused by defined protein-protein interactions at the so-called binding site. Such Fc moiety binding sites are known in the art (see above). Such Fc moiety binding sites are for example characterized by amino acids L234, L235, D270, N297, E318, K320, K322, P331, and P329 (numbering according to EU index of Kabat). Antibodies of subclasses IgG1, IgG2, and IgG3 are generally shown to include complement activation with C1q and C3 binding, whereas IgG4 does not activate the complement system and does not bind C1q and/or C3.

The cell-mediated effector functions of monoclonal antibodies can be enhanced by engineering their oligosaccharide components as described in Umana, P.et al, Nature Biotechnol.17(1999)176-180, and U.S. Pat. No.6,602,684. IgG 1-type antibodies (the most commonly used therapeutic antibodies) are glycoproteins with a conserved N-linked glycosylation site at Asn297 in each CH2 domain. Two complex biantennary oligosaccharides attached to Asn297 are buried between CH2 domains, which form extensive contacts with the polypeptide backbone, and their presence is critical for antibody-mediated effector functions such as antibody-dependent cellular cytotoxicity (ADCC) (Lifely, m., r. et al, Glycobiology 5(1995) 813-822; Jefferis, r. et al, immunol. rev.163(1998) 59-76; Wright, a. and Morrison, s., l., Trends biotechnol.15(1997) 26-32). Umana, P.et al, Nature Biotechnol.17(1999)176-180 and WO99/54342 show that overexpression of β (1, 4) -N-acetylglucosaminyltransferase III ("GnTIII") (a glycosyltransferase that catalyzes the formation of bisected oligosaccharides) in Chinese Hamster Ovary (CHO) cells significantly increases the ADCC activity of the antibody in vitro. Modification of Asn297 carbohydrate composition or elimination thereof also affects binding to Fc γ R and C1q (Umana, P. et al, Nature Biotechnol.17(1999) 176-180; Davies, J. et al, Biotechnol.Bioeng.74(2001) 288-294; Mimura, Y. et al, J.biol.chem.276(2001) 45539-45547; Radaev, S. et al, J.biol.chem.276(2001) 16478-16483; Shields, R.L. et al, J.biol.chem.276(2001) 6591-6604; Shields, R.L. et al, J.biol.chem.277(2002) 26740; Simmons, L.C. et al, J.immunool.Methol.263 (2002) 147-133).

For example in WO 2005/044859, WO 2004/065540, WO2007/031875, Umana, P.et al, Nature Biotechnol.17(1999)176-180, WO 99/154342, WO 2005/018572, WO 2006/116260, WO 2006/114700, WO 2004/065540, WO 2005/011735, WO2005/027966, WO 1997/028267, US 2006/0134709, US 2005/0054048, US2005/0152894, WO 2003/035835 and WO 2000/061739, or for example in Niwa, R.et al, J.Immunol.methods 306(2005) 151-160; shinkawa, T, et al, J.biol.chem.278(2003) 3466-; methods for enhancing cell-mediated effector function of monoclonal antibodies are reported in WO 03/055993 and US 2005/0249722.

Thus, in one embodiment of the invention, an antibody according to the invention is glycosylated with a sugar chain at Asn297 (if it comprises an Fc part of the subclass IgG1 or IgG 3), wherein the amount of fucose within said sugar chain is 65% or less (numbering according to Kabat). In another embodiment, the amount of fucose within said sugar chain is between 5% and 65%, preferably between 20% and 40%. In an alternative embodiment, the amount of fucose is 0% of the oligosaccharides at Asn297 in the Fc region. "Asn 297" according to the invention means the amino acid asparagine located at about position 297 in the Fc region. Asn297 may also be located some amino acids (usually no more than ± 3 amino acids) upstream or downstream of position 297, i.e. between positions 294 and 300, based on minor sequence variations of the antibody. In one embodiment, the IgG subclass of the glycosylated antibody according to the invention is that of human IgG1 subclass or IgG3 subclass. In yet another embodiment, the amount of N-glycolylneuraminic acid (NGNA) is 1% or less and/or the amount of N-terminal alpha-1, 3-galactose is 1% or less within the sugar chain. Preferably, the sugar chain shows the characteristics of an N-linked glycan attached to Asn297 of an antibody recombinantly expressed in CHO cells.

The term "sugar chain shows the characteristics of an N-linked glycan attached to Asn297 of an antibody recombinantly expressed in CHO cells" means that the sugar chain at Asn297 of an antibody according to the invention has the same structure and sugar residue sequence (except for fucose residues) as the sugar chain of the same antibody expressed in unmodified CHO cells, e.g. as those reported in WO 2006/103100.

As used within this application, the term "NGNA" means the sugar residue N-glycolylneuraminic acid.

Glycosylation of human IgG1 or IgG3 occurred at Asn297 as a core fucosylated biantennary complex oligosaccharide terminated by up to two Gal residues. Kabat, E., A. et al, supra and Brueggemann, M. et al, J.Exp.Med.166(1987) 1351-; love, T.W. et al, Methods enzymol.178(1989) 515-. Depending on the amount of terminal Gal residues, these structures are designated G0, G1 (. alpha. -1, 6-or. alpha. -1, 3-), or G2 glycan residues (Raju, T., S., Bioprocess int.1(2003) 44-53). CHO-type glycosylation of the Fc part of antibodies is described, for example, by Router, F.H., Glycoconjugate J.14(1997) 201-207. Antibodies recombinantly expressed in CHO host cells that are not sugar modified typically undergo fucosylation at Asn297 in an amount of at least 85%. The modified oligosaccharides of the antibody may be hybrid or complex. Preferably, the bisected, reduced/nonfucosylated oligosaccharides are hybrid. In another embodiment, the bisected, reduced/nonfucosylated oligosaccharides are complex.

According to the present invention, "amount of fucose" means the amount of said sugar within the sugar chain at Asn297, measured by MALDI-TOF mass spectrometry and calculated as an average value, relative to the sum of all sugar structures attached to Asn297 (e.g. complex, hybrid and high mannose structures) (see e.g. WO 2008/077546). The relative amount of fucose is the percentage of fucose-containing structures relative to all sugar structures identified in the N-glycosidase F-treated sample (e.g. complexed, heterozygous and oligo and high mannose structures, respectively) by MALDI-TOF.

Preferably, the antibodies according to the invention are generated by recombinant means. Such methods are generally known in the art and include protein expression in prokaryotic and eukaryotic cells, followed by isolation of the antibody polypeptide, and usually purification to a pharmaceutically acceptable purity. For protein expression, nucleic acids encoding the light and heavy chains or fragments thereof are inserted into the expression vector by standard methods. Expression is carried out in suitable prokaryotic or eukaryotic host cells, such as CHO cells, NS0 cells, SP2/0 cells, HEK293 cells, COS cells, yeast, or E.coli cells, and the antibody is recovered from the cells (supernatant or lysed cells).

Recombinant production of antibodies is well known in the art and is described, for example, in review articles Makrides, S., C., Protein Expr. Purif.17(1999) 183-202; geisse, S.et al, protein Expr. Purif.8(1996) 271-282; kaufman, R.J., mol.Biotechnol.16(2000) 151-160; werner, R.G., Drug Res.48(1998) 870-.

The antibody may be present in the whole cell, in a cell lysate, or in a partially purified or substantially pure form. Purification to eliminate other cellular components or other contaminants, such as other cellular nucleic acids or proteins, is carried out by standard techniques, including alkali/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other techniques well known in the art (see Ausubel, F. et al (eds.) Current protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987)).

For example, Barnes, L.M., et al, Cytotechnology 32(2000)109- & 123; and Barnes, L.M., et al, Biotech.Bioeng.73(2001)261-270 describe expression in NS0 cells. Transient expression is described, for example, by Durocher, Y., et al, Nucl. acids. Res.30(2002) E9. Orlandi, R.et al, Proc.Natl.Acad.Sci.USA 86(1989) 3833-3837; carter, P.et al, Proc. Natl.Acad.Sci.USA 89(1992) 4285-; and Norderhaug, l, et al, j.immunol.methods 204(1997)77-87, describe the cloning of variable domains. A preferred transient expression system (HEK 293) is described by Schlaeger, E.J. and Christensen, K., in Cytotechnology 30(1999)71-83 and Schlaeger, E.J., in J.Immunol. methods 194(1996) 191-199.

Suitable control sequences for prokaryotes include, for example, promoters, optionally operator sequences, and ribosome binding sites. Eukaryotic cells are known to utilize promoters, enhancers, and polyadenylation signals.

A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or operably linked to a coding sequence if the ribosome binding site is positioned so as to facilitate translation. In general, "operably linked" means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers need not be contiguous. Ligation is achieved by ligation at convenient restriction sites. If such sites are not present, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

Monoclonal antibodies are suitably isolated from the culture broth by conventional immunoglobulin purification procedures, such as, for example, protein a-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. DNA and RNA encoding the monoclonal antibodies are readily isolated and sequenced using conventional procedures. Hybridoma cells can serve as a source of such DNA and RNA. Once isolated, the DNA may be inserted into an expression vector, which is then transfected into a host cell such as an HEK293 cell, CHO cell, or myeloma cell that does not otherwise produce immunoglobulin protein, to obtain synthesis of the recombinant monoclonal antibody in the host cell.

As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny. As such, the words "transformant" and "transformed cell" include the primary subject cell and cultures derived therefrom, regardless of the number of deliveries. It is also understood that all progeny may not be exactly identical in DNA content due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where a unique name is intended, it will be clear from the context.

As used herein, the term "transformation" refers to the process of transferring a vector/nucleic acid into a host cell. If cells without an intractable cell wall barrier are used as host cells, transfection is carried out, for example, by calcium phosphate precipitation, as described by Graham, F., L. and van der Eb, Virology 52(1973) 456-467. However, other methods for introducing DNA into cells may also be used, such as by nuclear injection or by protoplast fusion. If prokaryotic cells or cells containing rigid cell wall structures are used, one method of transfection is, for example, calcium treatment with calcium chloride, as described by Cohen, S.N. et al, PNAS.69(1972) 2110-2114.

As used herein, "expression" refers to the process of transcribing a nucleic acid into mRNA and/or the subsequent translation of the transcribed mRNA (also called transcript) into a peptide, polypeptide, or protein. The transcripts and the encoded polypeptides are collectively referred to as gene products. If the polynucleotide is derived from genomic DNA, expression in the eukaryotic cell may include splicing of mRNA.

"vector" refers to a nucleic acid molecule, particularly self-replicating, that transfers an inserted nucleic acid molecule into and/or between host cells. The term includes vectors that function primarily in the insertion of DNA or RNA into a cell (e.g., chromosomal integration), replicating vectors that function primarily in the replication of DNA or RNA, and expression vectors that function in the transcription and/or translation of DNA or RNA. Also included are vectors that provide more than one function, as described.

An "expression vector" refers to a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. An "expression system" generally refers to a suitable host cell containing an expression vector that can function to produce a desired expression product.

One aspect of the invention is a pharmaceutical composition comprising an antibody according to the invention. Another aspect of the invention is the use of an antibody according to the invention for the manufacture of a pharmaceutical composition. Yet another aspect of the invention is a method for manufacturing a pharmaceutical composition comprising an antibody according to the invention. In another aspect, the invention provides a composition, e.g., a pharmaceutical composition, comprising an antibody according to the invention formulated with a pharmaceutically acceptable carrier.

Furthermore, such specific humanized versions of the CDCP1 antibody CUB4 have proven to be particularly useful for the treatment of cancer, compared to, for example, other anti-CDCP 1 antibodies.

Accordingly, one aspect of the present invention is the pharmaceutical composition for use in the treatment of cancer.

Another aspect of the invention is a humanized antibody according to the invention for use in the treatment of cancer.

Another aspect of the invention is the use of a humanized antibody according to the invention for the manufacture of a medicament for the treatment of cancer.

Another aspect of the invention is a method of treating a patient suffering from cancer by administering to said patient in need of such treatment a humanized antibody according to the invention.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coating materials, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g. by injection or infusion).

The compositions of the present invention may be administered by a variety of methods known in the art. As the skilled artisan will appreciate, the route and/or pattern of administration will vary with the desired result. In order to administer a compound of the invention by certain routes of administration, it may be necessary to coat the compound with a material or to co-administer the compound with a material to prevent its inactivation. For example, the compounds can be administered to a subject in a suitable carrier, such as a liposome or diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Pharmaceutical carriers include sterile aqueous solutions or dispersions and sterile powders for the in situ preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art.

As used herein, the phrase "parenteral administration" means modes of administration other than enteral and topical administration, typically by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

As used herein, the term "cancer" may be, for example, lung cancer, non-small cell lung (NSCL) cancer, bronchioloalveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer (stomach cancer), stomach cancer (gastrotic cancer), colon cancer, breast cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulval cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney or ureter cancer, renal cell cancer, renal pelvis cancer, mesothelioma, hepatocellular carcinoma, biliary cancer (biliary cancer), Central Nervous System (CNS) neoplasms, spinal axis tumors, brain stem glioma, Glioblastoma multiforme (glioblastomas multiforme), astrocytomas, schwanoma (schwanoma), ependymomas, medulloblastomas, meningiomas, squamous cell carcinomas, pituitary adenomas, lymphomas, lymphocytic leukemias, including refractory forms of any of the foregoing, or combinations of one or more of the foregoing. Preferably, such cancer is breast, ovarian, cervical, lung or prostate cancer, and more preferably lung cancer. Preferably, such cancers are further characterized by CDCP1 expression or overexpression, more preferably, CDCP1 overexpression.

These compositions may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. Prevention of the presence of microorganisms can be ensured by sterilization procedures, see above, and by the inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to incorporate isotonic agents, such as sugars, sodium chloride, and the like into the composition. In addition, delayed absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

Regardless of the route of administration chosen, the compounds of the invention (which may be used in a suitable hydrated form) and/or the pharmaceutical compositions of the invention are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

The actual dosage level of the active ingredient in the pharmaceutical compositions of the invention can be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response, composition, and mode of administration for a particular patient, yet is non-toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular composition of the invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

The composition must be sterile and fluid to the extent that the composition is deliverable by syringe. In addition to water, the carrier is preferably an isotonic buffered saline solution.

Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it is preferred to include isotonic agents (e.g., sugars, polyols such as mannitol or sorbitol, and sodium chloride) in the composition.

The following examples, sequence listing and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is to be understood that modifications may be made to the procedures set forth without departing from the spirit of the invention.

Brief Description of Drawings

FIG. 1 VH Domain amino acid sequence of the mouse (mVH-CUB4) antibody (CDRH1, CDRH2 and CDRH3 are labeled-bold letters) and VH Domain amino acid sequence of a different humanized CUB4 anti-CDCP 1 antibody (specific modifications according to the invention are labeled-bold letters)

FIG. 2 VL domain amino acid sequences of the mouse (mVL-CUB4) antibody (CDRH1, CDRH2 and CDRH3 are marked in bold letters) and of a different humanized CUB4 anti-CDCP 1 antibody (specific modifications according to the invention are marked in bold letters)

Figure 3 relative binding ratios of different humanized anti-CDCP 1 antibodies CUB 4. The binding ratios of combinations of different humanized VH and VL domains are shown relative to the chimeric CUB4((chHC4 ═ mouse VH and VL and human IgG1 constant region) antibody.

Figure 4 in vitro ADCC of Glycoengineered (GE) humanized CUB4 antibody No.69GE with an amount of fucose of 65% or less compared to ADCC of chimeric Glycoengineered (GE) CUB4 and wild type (wt ═ non-glycoengineered chimeric CUB4 antibody and negative human IgG control.

FIGS. 5a and 5b in vivo tumor growth inhibition in human lung carcinoma H322M xenografts by humanized CUB4 antibodies No.69 and No.135, chimeric CUB4 antibody, and mouse CUB4 antibody.

Examples

Example 1

Antigen-specific ELISA

The soluble CDCP1 extracellular domain (CDCP1-ECD) (SEQ ID NO: 30) fused to Streptavidin Binding Protein (SBP) was captured on streptavidin plates. To define the optimal binding of the antibody to SBP-CDCP1-ECD, 384-well polystyrene plates (NUNC, streptavidin-coated) delivered by MicroCoat, Bernarid, Germany (ID-No.1734776-001) have been coated with pure and stepwise diluted HEK293 supernatant (in BSA/IMDM buffer: 100mg/ml BSA fraction V, Roche 10735078001, dissolved in Iscove's modified Dulbeccos medium). Using a calibration curve of the mouse CUB4 antibody, the optimal dilution factor of HEK293 supernatant was identified relative to the streptavidin binding performance of the microtiter plates. For standard coatings, HEK293 supernatant containing SBP-CDCP1-ECD (between 1: 15 and 1: 40) was diluted and incubated overnight (25. mu.l per well) at 2-8 ℃. An intensive wash of the microtiter plate was necessary to remove the remaining unbound SBP-CDCP 1-ECD.

The humanized CUB4 antibody and/or the reference antibody (chimeric (chHC4) CUB4 antibody comprising human constant regions and mouse VH and VL of SEQ ID NOS: 1 and 2) were tested undiluted or using 12-step dilution. 12, 5. mu.l of each sample per well were incubated for 90 minutes at room temperature. After intense washing with PBS-T (0.1% Tween20 in PBS), 25. mu.l of either of the goat anti-human IgG antibodies coupled to HRP against the human antibody (Jackson ImmunoResearch, code No.: 109. 036. sup. 098, diluted 1: 10000) was added and incubated for 1 hour. After intensive washing, antibody binding was detected with ABTS tablets (Roche Diagnostics GmbH, catalog No.: 1112422). Absorbance at 405nm/492nm was measured using a standard photometer.

In fig. 3, the binding ratios of different combinations of humanized VH and VL are shown relative to the chimeric (chHC4) CUB4 antibody. The results show that surprisingly, the specific modified humanized antibodies as follows lead to significantly improved binding properties compared to the humanized CUB4 antibody without such specific modifications:

a) specific mutations in the CDRH2 of VH (mutations T57K and P60V). (see VH domain: hHC4-H (SEQ ID NO: 3)),

and/or

b) A specific mutation in CDRL1 of VL (mutation V33L) and a mutation in the VL framework region from human to mouse amino acid W at position 47; (see VL domain: hLC4-M (SEQ ID NO: 14), hLC4-L2(SEQ ID NO: 15), hLC4-K (SEQ ID NO: 16), hLC4-L (SEQ ID NO: 17), hLC4-J (SEQ ID NO: 18)).

Example 2

anti-CDCP 1 antibody to human CDCP1 fragment comprising the extracellular domain (ECD) SEQ ID No.: characterization of binding of 30 (extracellular domain ECD comprising human CDCP 1):

for affinity measurements, 30. mu.g/ml anti-mouse Fc γ antibody (from goat, Jackson Immuno research JIR 115-005-. After conjugation, different anti-CDCP 1 antibodies were injected at 25 ℃ at a flow rate of 5. mu.L/min, followed by injection of a dilution series (0nM to 1000nM) of CDCP1ECD at 30. mu.L/min. As a binding experiment of the running buffer, PBS/0.1% BSA. Then, the chip was regenerated with a 60 second pulse of 10mM glycine-hydrochloric acid, pH 2.0 solution.

Calculation of thermodynamic parameters (K) Using Langmuir (Langmuir) 1: 1 binding model_DBinding constant) and kinetic parameters (k)_onRate, k_offRate) is calculated.

Table 3: exemplary binding parameters of humanized antibodies according to the invention

Example 3

Glycoengineered humanized CUB4 antibody (humanized CUB4 antibody)GE) Preparation of

To be compared to the amino acid sequence SEQ ID NO:3 and SEQ ID NO:15 (antibody 69) the entire antibody heavy and light chain DNA sequences corresponding to were subcloned into mammalian expression vectors (one for the light chain and one for the heavy chain) under the control of the MPSV promoter and upstream of the synthetic poly a site, each vector carrying the EBV OriP sequence.

Antibodies were generated by co-transfecting HEK293-EBNA cells with mammalian antibody heavy and light chain expression vectors using calcium phosphate transfection methods. Exponentially growing HEK293-EBNA cells were transfected by the calcium phosphate method. For the production of unmodified antibodies, cells were transfected with antibody heavy and light chain expression vectors alone in a 1: 1 ratio. For glycoengineered antibody production, cells were CO-transfected with four plasmids (two for antibody expression, one for fusion GnTIII polypeptide expression (GnT-III expression vector), and one for mannosidase II expression (Golgi mannosidase II expression vector) at a ratio of 4: 1: 1.cells were cultured in T flasks as adherent monolayer cultures using DMEM medium supplemented with 10% FCS and transfected at between 50 and 80% confluence. for transfection of T150 flasks 1500 ten thousand cells were seeded 24 hours later, transfected in 25ml DMEM medium supplemented with FCS (at final 10% V/V) and cells were placed overnight in an incubator at 37 ℃ in a 5% CO2 atmosphere. for each T150 flask to be transfected, 94 μ g total plasmid vector DNA, equally separated by mixing between light and heavy chain expression vectors, To a final volume of 469. mu.l of water and 469. mu.l of 1M CaCl2 solution a solution of DNA, CaCl2 and water was prepared. To this solution 938. mu.l of 50mM HEPES, 280mM NaCl, 1.5mM Na2HPO4 solution (pH 7.05) was added, mixed immediately for 10 seconds, and left upright at room temperature for 20 seconds. The suspension was diluted with 10ml of DMEM supplemented with 2% FCS and added to T150, replacing the existing culture medium. Then, an additional 13ml of transfection medium was added. Cells were incubated at 37 ℃ for approximately 17 to 20 hours with 5% CO2, and then the medium was replaced with 25ml DMEM, 10% FCS. Conditioned media were harvested 7 days post-transfection by centrifugation at 210Xg for 15 minutes, the solution was sterile filtered (0.22 μm filter) and sodium azide was added to a final concentration of 0.01% w/v and maintained at 4 ℃.

Secreted antibody glycoengineered humanized CUB4 antibody No.69 (humanized CUB4 antibody No.69GE) was purified by protein a affinity chromatography, followed by cation exchange chromatography and a final size exclusion chromatography step on a Superdex 200 column (Amersham Pharmacia) which exchanged the buffer for 25mM potassium phosphate, 125mM sodium chloride, 100mM glycine solution (pH 6.7), and collected pure monomeric IgG1 antibody. Antibody concentration was estimated from absorbance at 280nm using a spectrophotometer. Oligosaccharides attached to the Fc region of the antibody were analyzed by MALDI/TOF-MS (as described, for example, in WO 2008/077546). The oligosaccharides were enzymatically released from the antibodies by PNG enzyme F digestion, wherein the antibodies were immobilized on PVDF membranes or in solution. The resulting digestion solution containing the released oligosaccharides is either directly ready for MALDI/TOF-MS analysis or further digested with EndoH glycosidase, after which the sample is prepared for MALDI/TOF-MS analysis,

in yet another experiment, the amount of fucose within the sugar chains at antibody 69 and 135. Asn297 of the glycoengineered humanized CUB4 antibody (humanized CUB4 antibody GE) was analyzed to be 50-10% by cotransfection with four plasmids, two for antibody expression, one for fusion GnTIII polypeptide expression (GnT-III expression vector), and one for mannosidase II expression (Golgi mannosidase II expression vector), at a ratio of 4: 1 in CHO cells replacing HEK293-EBNA cells.

Example 4

In vitro ADCC of humanized CUB4 antibodies

Target cells PC-3(DSMZ # ACC 465, prostatic adenocarcinoma, cultured in Ham's F12 nutrient mix +2mM L-alanyl-L-glutamine + 10% FCS) and H322M (non-small cell lung carcinoma, cultured in RPMI1640+2mM L-alanyl-L-glutamine + 10% FCS) were harvested in exponential growth phase in the presence of trypsin/EDTA (Gibco # 25300-054). After washing steps and examination of cell number and viability, the required aliquots were labeled with calcein (Invitrogen # C3100 MP; 1 vial was resuspended in 50. mu.l DMSO for 5 million (Mio) cells in 5ml of medium) in a cell culture incubator for 30 minutes at 37 ℃. Then, the cells were washed three times with AIM-V medium, the cell number and viability were checked, and the cell number was adjusted to 30 ten thousand/ml.

Meanwhile, PBMCs were prepared as effector cells by density gradient centrifugation (Histopaque-1077, Sigma # H8889) according to the manufacturer's protocol (washing step 1x at 400g and 2x at 350g, 10 min each). Cell number and viability were examined and cell number was adjusted to 1500 ten thousand/ml.

100 μ l of calcein-stained target cells were dispensed in a round bottom 96-well plate, and 50 μ l of diluted antibody and 50 μ l of effector cells were added. In some experiments, target cells were incubated with 10mg/ml RedimoneNF liquids (ZLB ehring) were mixed.

Spontaneous lysis (which was determined by co-culturing target cells and effector cells in the absence of antibody) and maximal lysis (which was determined by lysis of target cells with 1% Triton X-100 only) served as controls. The plates were incubated at 37 ℃ for 4 hours in a humidified cell incubator.

Killing of target cells was assessed by measuring LDH release from injured cells using a cytotoxicity detection kit (LDH detection kit, Roche # 1644793) according to the manufacturer's instructions. Briefly, 100 μ l of supernatant from each well was mixed with 100 μ l of substrate from the kit in a clear flat-bottomed 96-well plate. The Vmax value of the color reaction of the substrate was determined in an ELISA reader at 490nm for at least 10 minutes. The percentage of specific antibody-mediated killing was calculated as follows: ((A-SR)/(MR-SR) x100, where A is the mean Vmax at the particular antibody concentration, SR is the mean Vmax released spontaneously, and MR is the mean Vmax released maximally.

As an additional readout, calcein retention of intact target cells was assessed by lysing the remaining target cells in borate buffer (5mM sodium borate + 0.1% Triton) and measuring calcein fluorescence in a fluorescence plate reader.

Figure 4 shows in vitro ADCC of Glycoengineered (GE) humanized CUB4 antibody No.69GE with an amount of fucose of 65% or less compared to ADCC of chimeric Glycoengineered (GE) CUB4 and wild type (wt ═ non-glycoengineered chimeric CUB4 antibody and negative human IgG control.

Example 5

Stimulation of CDCP1 phosphorylation in DU-145 cells

2x10 every 6 holes⁵Du-145 cells were cultured overnight in DMEM (Paa catalog number E15-0011), 2 mML-glutamine (Sigma catalog number G7513, 2mM sodium pyruvate, 10% FCS (PAA catalog number E15-0011.) cells were incubated with 20. mu.g/mL of various humanized CUB4 antibodies for 10 minutes, cells were lysed with freshly prepared ice-cold RIPA lysis buffer (RIPA-buffer 1% NP40, 1% DOC, 0, 1% SDS, 150mM NaCl, 10mM Tris/HCl, pH 7,4, 1mMPMSF in ethanol, 10. mu.g/mL aprotinin, 0,4mM orthovanadate) after 10 minutes on ice, the cell lysate was centrifuged at 10000rpm for 10 minutes, the lysate was separated on SDS-PAGE by standard protocol and transferred to nitrocellulose by Western blot to nitrocellulose-phosphotyrosine antibody (4G10) or anti-phosphodensitometric antibody detection by Western blot GS 1 (Biorad scanning 800) The intensity of phosphorylated CDCP1 was determined.

Table 4: percent (%) stimulation of humanized CUB4 antibody (relative to chimeric CUB4)

Humanized CUB4 antibody No.	Percent (%) stimulation
		Chimeric CUB4	100％
80	133％
		69	112％

47	96％
		135	72％

All humanized CUB4 antibodies No.80, No.69, 47 and 135 were shown to stimulate CDCP1 phosphorylation in DU-145 cells.

Example 6

In vivo tumor suppression of humanized CUB4 antibodies

A) Study name: CDCP1_ PZ _ H322M _007

An in vivo study was performed to compare the efficacy of the chimeric anti-CDCP 1 antibody CUB4 and a humanized version of the CUB4 antibody in the NCI-H322M non-small cell lung cancer model.

H322M non-small cell lung cancer cells were obtained from the NCI collection. Tumor cell lines were routinely cultured in RPMI1640 medium supplemented with 10% fetal bovine serum and 2mM L-glutamine at 37 ℃ in a water-saturated atmosphere at 5% CO 2. Passage 4 was used for cell transplantation.

Human non-small cell lung cancer cellsLine H322M was inoculated subcutaneously with Matrigel (5X 10)⁶Individual cells) into the right flank of the mouse.

Animal treatment was started 19 days after cell transplantation on the day of randomization. The antibody was administered at i.p.q7d at days 19, 26, 33, 40 and 47 at the indicated dose of 25 mg/kg. The corresponding vehicle was also administered on the same day. The volume administered was 10 ml/kg.

Humanized CUB4 antibody No.69 is based on SEQ ID NO:3 and SEQ ID NO:15 VH and VL.

Humanized CUB4 antibody No.135 is based on SEQ ID NO:3 and SEQ ID NO:23 VH and VL.

Group (2):

treatment group 1: media

Treatment group 2: chimeric CUB4(25mg/kg i.p);

treatment group 3: humanized CUB4 antibody No.69(25mg/kg i.p);

treatment group 4: humanized CUB4 antibody No.135(25mg/kg i.p);

in fig. 5a, the in vivo tumor growth inhibition of humanized CUB4 antibodies nos. 69 and 135 and chimeric CUB4 antibody in human lung cancer H322M xenografts is shown, with the in vivo tumor growth inhibition of both humanized CUB4 antibodies nos. 69 and 135 being significantly improved compared to the chimeric CUB4 antibody.

B) Study name: CDCP1_ PZ _ H322M _004

An in vivo study was performed to compare the efficacy of the murine anti-CDCP 1 antibody CUB4 with the chimeric anti-CDCP 1 antibody CUB4 (mouse VH and VL and human IgG1 constant regions) in the NCI-H322M non-small cell lung cancer model.

The human non-small cell lung cancer cell line H322M was inoculated subcutaneously with Matrigel (5X 10)⁶Individual cells) into the right flank of the mouse.

Animal treatment was started on the day of randomization 17 days after cell transplantation. The antibody was administered at the indicated dose of 10mg/kg i.p.q.7d until day 59 of study termination. The corresponding vehicle was also administered on the same day. The volume administered was 10 ml/kg.

Group (2):

treatment group 1: media

Treatment group 4: mouse CUB4(10mg/kg i.p.)

Treatment group 5: chimeric CUB4(10mg/kg i.p.)

In fig. 5b, in vivo tumor growth inhibition in human lung carcinoma H322M xenografts was shown for the mouse CUB4 antibody and the chimeric CUB4 antibody, with the in vivo tumor growth inhibition of the chimeric CUB4 antibody being improved compared to the mouse CUB4 antibody.

Claims

1. An antibody that specifically binds to human CDCP1, comprising:

heavy chain variable domain (VH) SEQ ID NO 3, and

light chain variable domain (VL) SEQ ID NO 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24.

2. The antibody according to claim 1, characterized in that:

the heavy chain variable domain (VH) is SEQ ID NO 3,

and is

The light chain variable domain (VL) is SEQ ID NO 14.

3. The antibody according to claim 1, characterized in that:

the heavy chain variable domain (VH) is SEQ ID NO 3,

and is

The light chain variable domain (VL) is SEQ ID NO 15.

4. The antibody according to claim 1, characterized in that:

the heavy chain variable domain (VH) is SEQ ID NO 3,

and is

The light chain variable domain (VL) is SEQ ID NO 23.

5. The antibody according to any one of claims 1 to 4, characterized in that said antibody is of the subclass human IgG 1.

6. Antibody according to any one of claims 1 to 5, characterized in that the antibody is glycosylated with a sugar chain at Asn297 wherein the amount of fucose within said sugar chain is 65% or lower.

7. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 6.

8. The antibody according to any one of claims 1 to 6 for use in the treatment of cancer.

9. Use of an antibody according to any one of claims 1 to 6 for the manufacture of a medicament for the treatment of cancer.

10. Nucleic acid encoding an antibody according to any one of claims 1 to 6.

11. An expression vector containing a nucleic acid according to claim 10, which is capable of expressing said nucleic acid in a prokaryotic or eukaryotic host cell.

12. A prokaryotic or eukaryotic host cell comprising a vector according to claim 11.

13. A method for the production of an antibody according to any one of claims 1 to 6, characterized in that a nucleic acid according to claim 10 is expressed in a prokaryotic or eukaryotic host cell and the antibody is recovered from the cell or cell culture supernatant.

14. An antibody obtained by the method of claim 13.