IL267022B2 - Monoclonal antibodies aganist her2 - Google Patents

Monoclonal antibodies aganist her2

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IL267022B2
IL267022B2 IL267022A IL26702219A IL267022B2 IL 267022 B2 IL267022 B2 IL 267022B2 IL 267022 A IL267022 A IL 267022A IL 26702219 A IL26702219 A IL 26702219A IL 267022 B2 IL267022 B2 IL 267022B2
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antibody
region
seq
her2
sequence
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IL267022A
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IL267022A (en
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Genmab As
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Priority claimed from PCT/EP2011/056388 external-priority patent/WO2011131746A2/en
Priority claimed from PCT/EP2011/058779 external-priority patent/WO2011147986A1/en
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Publication of IL267022A publication Critical patent/IL267022A/en
Publication of IL267022B2 publication Critical patent/IL267022B2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/526—CH3 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/528—CH4 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Description

MONOCLONAL ANTIBODIES AGAINST HER2 The present application is a divisional patent application of Israel Patent Application No. 245275, which in turn is a divisional patent application of Israel Patent Application No. 223162.
FIELD OF THE INVENTION The present invention relates to monoclonal antibodies directed to human epidermal growth factor receptor 2 (HER2) and to uses of such antibodies, in particular their use in the treatment of cancer.
BACKGROUND OF THE INVENTION HER2 is a 185-kDa cell surface receptor tyrosine kinase and member of the epidermal growth factor receptor (EGFR) family that comprises four distinct receptors: EGFR/ErbB-1, HER2/ErbB-2, HER3/ErbB-3, and HER4/ErbB-4. Both homo- and heterodimers are formed by the four members of the EGFR family, with HER2 being the preferred and most potent dimerization partner for other ErbB receptors (Graus-Porta et al., Embo J 1997; 16:1647-1655; Tao et al., J Cell Sci 2008;121:3207-3217). HER2 can be activated by overexpression or by heterodimerization with other ErbBs that can be activated by ligand binding (Riese and Stern, Bioessays 1998; 20:41-48). For HER2, no ligand has been identified. HER2 activation leads to receptor phosphorylation, which triggers a cascade of downstream signals through multiple signaling pathways, such as MAPK, phosphoinositol 3-kinase/AKT, JAK/STAT and PKC, which ultimately results in the regulation of multiple cellular functions, such as growth, survival and differentiation (Huang et al., Expert Opin Biol Ther 2009;9:97-110).
Much of the attention on HER2 in tumors has been focused on its role in breast cancer, in which HER2 overexpression is reported in approximately 20% of the cases and is correlated with poor prognosis (Reese et al., Stem Cells 1997;15:1-8; Andrechek et al., Proc Natl Acad Sci U S A 2000;97:3444-3449; and Slamon et al., Science 1987;235:177-182). Besides breast cancer, HER2 expression has also been associated with other human carcinoma types, including prostate cancer, non-small cell lung cancer, bladder cancer, ovarian cancer, gastric cancer, colon cancer, esophageal cancer and squamous cell carcinoma of the head & neck (Garcia de Palazzo et al., Int J Biol Markers 1993;8:233-239; Ross et al., Oncologist 2003;8:307- 325; Osman et al., J Urol 2005;174:2174-2177; Kapitanovic et al., Gastroenterology 1997;112:1103-1113; Turken et al., Neoplasma 2003;50:257-261; and Oshima et al., Int J Biol Markers 2001;16:250-254).
Trastuzumab (Herceptin®) is a recombinant, humanized monoclonal antibody directed against domain IV of the HER2 protein, thereby blocking ligand-independent HER2 homodimerization, and to a lesser extend heterodimerization of HER2 with other family members in cells with high HER2 overexpression (Cho et al., Nature 2003;421:756-760 and PCT/EP2G11/058779 WO 2011/147986 Wehrman et a/., Proc Nat( Acad Sci U S A 2006;103:19063-19068). In cells with modest HER2 expressing levels, trastuzumab was found to inhibit the formation of HER2/EGFR heterodimers (Wehrman et al.f (2006), supra; Schmitz et al,r Exp Cell Res 2009:315:659־ 670). Trastuzumab mediates antibody-dependent cellular cytotoxicity (ADCC) and prevents ectodomain shedding, which would otherwise result in the formation of a truncated constitutively active protein in HER2 overexpressing cells. Also inhibition of both in vitro and in vivo proliferation of tumor ceils expressing high levels of HER2 has been reported for trastuzumab (reviewed in Nahta and Esteva, Oncogene 2007;26:3637-3643). Herceptin® has been approved both for first-line and adjuvant treatment of HER2 overexpressing metastatic breast cancer, either in combination with chemotherapy, or as a single agent following one or more chemotherapy regimens. Trastuzumab has been found to be effective only in 20-50% of HER2 overexpressing breast tumor patients and many of the initial responders show relapse after a few months (Dinh et a/., Clin Adv Hematol Oncol 2007;5:707-717).Pertuzumab (Omnitarg™) is another humanized monoclonal antibody. It is directed against domain II of the HER2 protein, resulting in inhibition of iigand-induced heterodimerization (i.e., HER2 dimerizing with another member of the ErbB family to which a ligand has bound); a mechanism reported to not strictly require high HER2 expression levels (Franklin et a/., Cancer Ceil 2004;5:317-328.). Although pertuzumab also mediates ADCC, the main mechanism of action of pertuzumab relies on its dimerization blockade (Hughes et a!., Mol Cancer Ther 2009;8:1885-1892). Moreover, pertuzumab was found to enhance EGFR internalization and downregulatron by inhibiting the formation of EGFR/HERheterodimers, which otherwise tethers EGFR at the plasma membrane (Hughes et al.f 2009, supra). This correlates with the observation that EGFR homodimers internalize more efficient than EGFR/HER2 dimers (Pedersen et a/., Mol Cancer Res 2009;7:275-284. The complementary mechanisms of action of pertuzumab and trastuzumab reportedly results in enhanced anti-tumor effects and efficacy when combined in patients who progressed during prior trastuzumab therapy (Baselga et al., 3 Clin Oncol 2010;28:1138-1144), and a phase III trial to evaluate this antibody combination together with Docetaxel in previously untreated HER2־positive metastatic breast cancer is underway.An alternative approach to improve targeted antibody therapy is by delivering cytotoxic cells or drugs specifically to the antigen-expressing cancer cells. For example, the so-called trifunctional antibodies are bispecific antibodies, targeting with one arm the antigen on the tumor cell and with the other arm for instance CD3 on T cells. Upon binding, a complex of T cells, tumor ceils and effector cells that bind Fc is formed, leading to killing of the tumor cells (Muller and Kontermann, BioDrugs 2010;24:89-98.). Ertumaxomab is one PCT/EP2011/058779 WO 2011/147986 such trifunctional antibody against HER2, which induces cytotoxicity in cell lines with low HER2 expression and which is in Phase II clinical development in metastatic breast cancer (Jones et at., Lancet Oncol 2009;10:1179-1187 and Kiewe et at., Clin Cancer Res 2006;12:3085-3091).A HER2 antibody drug conjugate (ADC) is currently in clinical development. T-DMconsists of trastuzumab conjugated to the fungal toxin maytansine. In Phase II trials, responses in a heavily pretreated patient cohort including prior trastuzumab and/or lapatinib therapy were reported Burris et al, 2011, 3 Clin Oncol 29: 398-405 and Lewis Phillips et at.f Cancer Res 2008;68:9280-9290). Preliminary data from a Phase II trial determining efficacy and safety of T-DM1 versus trastuzumab plus docetaxel in her2-positive metastatic breast cancer patients with no rior chemotherapy for metastatic disease were reported (Perez et al, Abstract BA3, European Society for Medical Oncology meeting 2010). A Phase III trial to evaluate T-DM1 efficacy and safety versus capecitabine + lapatinib in patients with HER2- positive locally advanced or metastatic breast cancer who received prior trastuzumab therapy is ongoing.While many factors are involved in selecting a suitable antibody for HER2 targeted therapy, it is typically an advantage for an ADC approach if the HER2-antibody complex efficiently internalizes upon antibody binding. Studies on murine HER2 antibodies have shown that certain combinations of antibodies instigate HER2 endocytosis (Ben-Kasus et at., PIMAS 2009;106:3294-9). Human HER2 antibodies F5 and Cl have been reported to internalize relatively rapidly on their own and to bind the same epitope (WO 99/55367 and WO 2006/116107). As compared to EGFR, however, internalization of HER2 is impaired. Indeed, EGFR homodimers internalize much more efficiently than HER2 homodimers (Dinh et at., Clin Adv Hematol Oncol 2007;5:707-717). EGFR, and also HER3, can increase endocytosis of HER2 by the formation of EGFR/HER2 and HER3/HER2 heterodimers, respectively (Baulida et at., 3 Biol Chem 1996;271:5251-5257; Pedersen NM, et at., Mol Cancer Res 2009;7:275-84).The complex mechanisms regulating the function of HER2 warrant further research on new and optimized therapeutic strategies against this proto-oncogene. Accordingly, there remains a need for effective and safe products for treating HER2־related diseases, such as cancer.
SUMMARY OF THE I NVENTI ON It is an object of the present invention to provide novel highly specific and effective monoclonal HER2 antibodies for medical use. The antibodies of the invention exhibit HERbinding characteristics that differ from antibodies described in the art. Particularly, althoughmost of the antibodies apparently bind to HER2 segments overlapping with those bound by3 PCT/EJP2011/058779 WO 2011/147986 trastuzumab, pertuzumab or F5/C1 as shown in a cross-blocking HER2 binding assay, the novel antibodies are characterized by a higher efficiency in killing HER2־expressing tumor cells in an ADC assay, improved internalization and/or other advantages over the known antibodies.In preferred embodiments, the antibodies of the invention are fully human, bind to novel epitopes, and/or have favorable properties for therapeutic use in human patients. Exemplary properties include, but are not limited to, favorable binding characteristics to cancer cells expressing human HER2 at high or low levels, specific binding rhesus epithelial cells expressing a HER2 ortholog, efficient internalization upon binding to HER2, high capacity for killing cancer cells expressing high- or low-levels of HER2 when administered as an ADC, promoting proliferation of HER2-expressing cells less than F5, a neutral or inhibitory effect on the proliferation of HER2־expressing cancer cells, and provide for effective ADCC-mediated killing of HER2-expressing cells, as well as any combination of the foregoing properties.These and other aspects of the invention are described in further detail below.
BRI EF DESCRI PTI ON OF THE DRAW! NGS Figure t : Alignment of HuMab heavy chain variable region (VH) sequences with germline (reference) sequences (A-K). In each VH sequence, the amino acids that differ from those of the germtine (reference) at specific positions are highlighted. Consensus VH sequences are shown, where UX״ indicates positions at which alternative amino acids (selected from those aligned at each position) are possible. The CDR1, CDR2, and CDR3 sequences are underlined in each VH sequence. The consensus CDR sequences are further defined in Table 4.Figure 2: Alignment of HuMab light chain variable region (Vt) sequences with germline (reference) sequences (panels A-B). In each VL sequence, the amino adds that differ from those of the germline (reference) at specific positions are highlighted. In Figure 2A, ail VL sequences derived from the same V-segment (IgKVl-12-01), but the closest !-segment differed between antibodies. Consensus VL sequences are shown, where ,‘X״ indicates positions at which alternative amino acids (selected from those aligned at the indicated position) are possible. The CDR1, CDR2, and CDR3 sequences are underlined in each VL sequence. The consensus CDR sequences are further defined in Table 4.Figure 3: Binding curves of HER2 antibodies to (A, B) high (AU565) and (C, D) low (A431) HER2 expressing cel! lines, determined as described in Example 12. Data shown are mean fluorescence intensities (MFI) of one representative experiment for each cell line. The EC5o values indicate the apparent affinities.
PCT/EP2011/058779 WO 2011/147986 Figure 4: Binding of HER2 antibodies to HER2 expressed on monkey Rhesus epithelial cells. Data shown are mean fluorescence intensities (MFI) of one experiment, described in Example 13.Figure 5: Chromium-release (ADCC) assay of HER2 antibodies, showing PBMC-mediated lysis of 51Cr-labeled SK-BR-3 cells after incubation with HER2 antibody. Values depicted are the mean maximum percentages 51Cr-release ± the standard deviation from one representative in vitro ADCC experiment with SK-BR-3 cells. See Example 15 for details. Figure 6: Effect of HER2 antibodies on the proliferation of AU565 cells, as compared to untreated cells (set to 100%). Data shown are percentages proliferation of AU565 cells compared to untreated cells measured in three independent experiments ± the standard deviation. * Significant (P<0.05). See Example 16 for details.Figure 7: Percentage of viable MCF7 cells stimulated with Heregulin-J3l and treated with the indicated HER2 antibodies, relative to cells stimulated with Heregulin-pi only. As a control, the percentage proliferation of unstimulated cells is shown (none). Data was obtained from three independent experiments ± the stdev. * Significant inhibition of Heregulin-pl-induced proliferation (P<0.05). See Example 17 for details.Figure 8: ADC assay, showing killing of AU565 cells (A, B) or A431 cells (C, D) via anti- kappa-ETA'-conjugated HER2 antibodies. (A, B) Data shown are fluorescence intensities (FI) of one representative experiment with AU565 cells treated with non-conjugated and anti- kappa-ETA'-conjugated HER2 antibodies. (C, D) Data shown are mean fluorescence intensities (MFI) of one representative experiment with A431 cells treated with non- conjugated and anti-kappa-ETA'-conjugated HER2 antibodies. See Example 18 for details. Figure 9: Killing of A431 cells induced by anti-kappa-ETA׳ pre-incubated HER2 x HERbispecific antibodies. The viability of A431 cells after 3 days incubation with HERantibodies, pre-incubated with anti-kappa-ETA'. Cell viability was quantified using Aiamarblue. Data shown are fluorescence intensities (FI) of one experiment with A431 ceils treated with anti-kappa-ETA'-conjugated HER2 antibodies and HER2 x HER2 bispecific antibodies. Staurosporin was used as positive control, whereas an isotype control antibody was used as negative control.Figure 10: HER2 x HER2 bispecific molecules induced downmoduiation of HER2 receptor. Relative percentage of HER2 expression levels in AU565 cell lysates after 3 days incubation with 10 pg/mL mAb. The amount of HER2 was quantified using a HER2-specific capture ELISA and depicted as percentage inhibition compared to untreated ceils. Data shown is the mean of two experiments plus standard deviation, except for combinations of monospecific IgGl antibodies which were tested once.
PGT/EP2011/058779 WO 2011/147986 Figure 11 : Colocalization analysis of HER2 x HER2 bispecific antibodies (FITC) with lysosomal marker LAMP1 (Cy5). FITC pixel intensity overlapping with Cy5 for various monospecific HER2 antibodies and HER2 x HER2 bispecific antibodies (Figure 11(A)) FITC pixel intensity in LAMP1/Cy5 positive pixels of three different images is plotted for each antibody tested. Monospecifics show lower FITC pixel intensities in the LAMP1/Cy5 positive pixels compared to bispecifics. Figure 11(B) represents the mean value of FITC pixel intensity per LAMP1/Cy5 positive pixel calculated from the three different images. Together these results indicate that after internalization higher levels of bispecific antibodies, compared to monospecifics antibodies, localize to Lampl/Cy5 positive vesicles. Figure 12:Inhibition of proliferation by HER-2 mono and bispecific antibodies. AU565 cells were seeded in the presence of 10 pg/mL HER2 antibody or HER2 x HER2 bispecific antibody in serum-free cell culture medium. After three days, the amount of viable cells was quantified with Alamarblue and cell viability was presented as a percentage relative to untreated ceils. An isotype control antibody was used as negative control. Data shown are percentage viable AU565 cells compared to untreated cells measured in five-fold ± the standard deviation. * indicates only one data point was depicted. Figure13; T cell mediated cytotoxicity of AU565 cells by HER2 x CD3 bispecific antibodies as well as by N297Q mutants of HER2 x CD3 bispecific antibodies (bispecific indicated as Duo in the figure). Figure 14:Antibody induced downmodulation of HER2. Relative percentage of HERexpressed in AU565 cell lysate after 3 days incubation with 10 pg/mL antibody. The amount of HER2 was quantified using a HER2-specific capture ELISA and plotted as a percentage relative to untreated cells. Data shown are mean of three experiments ± standard deviation. Figure 15: Colocalization analysis of HER2 antibodies (FITC) with lysosomal marker LAMP(Cy5). FITC pixel intensity overlapping with Cy5 for various monospecific HER2 antibodies. FITC pixel intensity in LAMP1/Cy5 positive pixels of three different images is plotted for each antibody. Group 3 antibodies 098 and 153 show higher FITC pixel intensities in the LAMP1/Cy5 positive compartments compared to antibodies 025 and pertuzumab from Group and 169 and Herceptin from Group 1. Figure 16:HER2 antibody binding to CHO-S cells transfected with different HER2 ECD construct analyzed by means of flow cytometry. Hu-HER2 = fully human HER2, Hu-HER2- ch(I) CR1 = hu-HER2 with chicken domain I, Hu-HER2-ch(II) = hu־HER2 with chicken domain IIf hu-HER2-ch(III) = hu־HER2 with chicken domain III and Hu-HER2־cb(IV) = hu- HER2 with chicken domain IV. Data shown are mean fluorescence intensities (MFI) of one representative antibody, TH1014-153. See example 25 for details.
PCT/EP2011/058779 WO 2011/147986 Figure 17: In vivo effect of HER2־HuMab$ in the NCI-N87 human gastric carcinoma xenograft model in female CB.17 severe combined immunodeficiency (SCID) mice. Data shown are mean tumorsize ± S.E.M. per group (n =10 ־ mice per group) (A) and survival (B). See example 29 for details.Figure 18: In vivo effect of HER2 HuMabs in BT-474 breast tumor xenografts in Balb/C nude mice. Data shown are mean tumorsi2e ± S.E.M. per group (n = 8 mice per group) (A) and survival (B). See example 30 for details.
DETAI LED DESCRI PTI ON OF THE I NVENTI ON DefinitionsThe term "HER2" (also known as ErbB-2, NEU, HER2־f and CD34Q), when used herein, refers to human epidermal growth factor receptor 2 (SwissProt P04626) and includes any variants, isoforms and species homologs of HER2 which are naturally expressed by cells, including tumor cells, or are expressed on cells transfected with the HER2 gene. Species homologs include rhesus monkey HER2 (macaca mulatta; Genbank accession No. 61:109114897).The term "immunoglobulin" refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light (L) low molecular weight chains and one pair of heavy (H) chains, all four inter-connected by disulfide bonds. The structure of immunoglobulins has been well characterized. See for instance Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). Briefly, each heavy chain typically is comprised of a heavy chain variable region (abbreviated herein as VH or VH) and a heavy chain constant region. The heavy chain constant region typically is comprised of three domains, CH1, CH2, and CH3. Each light chain typically is comprised of a light chain variable region (abbreviated herein as VL or VL) and a light chain constant region. The light chain constant region typically is comprised of one domain, CL. The VH and VL regions may be further subdivided into regions of hypervariability (or hypervariabie regions which may be hypervariabie in sequence and/or form of structurally defined loops), also termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk 1. Mol. Biol. 196, 901-917 (1987)). Unless otherwise stated or contradicted by context, CDR sequences herein are identified according to IMGT rules (Brochet X., Nucl Acids Res. 2008;36:W5G3-508 and Lefranc MR., Nucleic Acids Research 1999;27:209-212; see also internet http address imgt.cines.fr/IMGT_vquest/vquest?Iivret=0&Option=humanIg.However, the numbering of amino acid residues in an antibody sequence can also be7 PCT/EP2011/058779 WO 2011/147986 performed by the method described in Kabat et a!Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991) (phrases such as ''variable domain residue numbering as in Kabat״, "Kabat position" or "according to Kabat" herein refer to this numbering system). Particularly, for numbering of amino acids in the constant region, the EU index numbering system according to Kabat et al, supra, can be used. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a "standard" Kabat numbered sequence.The term "antibody" (Ab) in the context of the present invention refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, ora derivative of either thereof, which has the ability to specifically bind to an antigen under typical physiological conditions with a half life of significant periods of time, such as at least about minutes, at least about 45 minutes, at least about one hour, at least about two hours, at least about four hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 or more days, etc., or any other relevant functionally-defined period (such as a time sufficient to induce, promote, enhance, and/or modulate a physiological response associated with antibody binding to the antigen and/or time sufficient for the antibody to recruit an effector activity). The variable regions of the heavy and light chains of the immunoglobulin molecule contain a binding domain that interacts with an antigen. The constant regions of the antibodies (Abs) may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as Clq, the first component in the classical pathway of complement activation. A HERantibody may also be a bispecific antibody, diabody, or similar molecule (see for instance PNAS USA 90(14), 6444-8 (1993) for a description of diabodies). Indeed, bispedfic antibodies, diabodies, and the like, provided by the present invention may bind any suitable target in addition to a portion of HER2. As indicated above, the term antibody herein, unless otherwise stated or clearly contradicted by context, includes fragments of an antibody that are antigen-binding fragments, i.e., retain the ability to specifically bind to the antigen. It has been shown that the antigen-binding function of an antibody may be performed by fragments of a full-length antibody. Examples of antigen-binding fragments encompassed within the term "antibody" include (i) a Fab׳ or Fab fragment, a monovalent fragment consisting of the VL/ vH, Cl and CH1 domains, or a monovalent antibody as described in WO20Q7059782 (Genmab); (ii) F(ab’)2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting essentially of the VH and CH1 domains; (iv) a Fv fragment consisting essentially of the VL PCT/EP2011/058779 WO 2011/147986 and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward ef a/., Nature 341. 544-546 (1989)), which consists essentially of a VH domain and also called domain antibodies (Holt et ai; Trends Biotechnol. 2003 Nov: 21(11484-901: ו: (vi) cam el id or nanobodies (Revets et al; Expert Opin Biol Ther. 2005 Jan;5(l): 111-24) and (vii) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they may be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the Vt and VH regions pair to form monovalent molecules (known as single chain antibodies or single chain Fv (scFv), see for instance Bird et ai, Science 212,423-426 (1988) and Huston et ai., PNAS USA £5, 5879-5883 (1988)). Such single chain antibodies are encompassed within the term antibody unless otherwise noted or clearly indicated by context. Although such fragments are generally included within the meaning of antibody, they collectively and each independently are unique features of the present Invention, exhibiting different biological properties and utility. These and other useful antibody fragments in the context of the present invention, as well as bispecific formats of such fragments, are discussed further herein. It also should be understood that the term antibody, unless specified otherwise, also includes polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like polypeptides, such as chimeric antibodies and humanized antibodies, and antibody fragments retaining the ability to specifically bind to the antigen (antigen-binding fragments) provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques. An antibody as generated can possess any isotype.As used herein, "isotype" refers to the immunoglobulin class (for instance IgGl,IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) that is encoded by heavy chain constant region genes.The term "monovalent antibody" means in the context of the present invention that an antibody molecule is capable of binding a single molecule of the antigen, and thus is not able of antigen crosslinking.An "antibody deficient in effector function" or an "effector-function-deficient antibody״ refers to an antibody which has a significantly reduced or no ability to activate one or more effector mechanisms, such as complement activation or Fc receptor binding. Thus, effector-function deficient antibodies have significantly reduced or no ability to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement- dependent cytotoxicity (CDC). An example of such an antibody is IgG4.A "HER2 antibody״ or "anti־HER2 antibody" is an antibody as described above, which binds specifically to the antigen HER2.
PCT/EP2011/058779 WO 2011/147986 The term "human antibody", as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences.The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site- specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.As used herein, a human antibody is "derived from" a particular germline sequence if the antibody is obtained from a system using human immunoglobulin sequences, for instance by immunizing a transgenic mouse carrying human immunoglobulin genes or by screening a human immunoglobulin gene library, and wherein the selected human antibody is at least 90%, such as at least 95%, for instance at least 96%, such as at least 97%, for instance at least 98%, or such as at least 99% identical in amino add sequence to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, outside the heavy chain CDR3, a human antibody derived from a particular human germline sequence will display no more than 20 amino acid differences, e.g. no more than 10 amino acid differences, such as no more than 9, 8, 7, 6 or 5, for instance no more than 4, 3, 2, or amino acid difference from the amino add sequence encoded by the germline immunoglobulin gene.In a preferred embodiment, the antibody of the invention is isolated. An "isolated antibody," as used herein, is intended to refer to an antibody which is substantially free of other antibodies having different antigenic specificities (for instance an isolated antibody that specifically binds to HER2 is substantially free of antibodies that specifically bind antigens other than HER2). An isolated antibody that specifically binds to an epitope, isoform or variant of HER2 may, however, have cross-reactivity to other related antigens, for instance from other species (such as HER2 species homologs). Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals. In one embodiment of the present invention, two or more "isolated" monoclonal antibodies having different antigen-binding specificities are combined in a well-defined composition.When used herein in the context of two or more antibodies, the term "competes with" or "cross-competes with" indicates that the two or more antibodies compete for binding to HER2, e.g. compete for HER2 binding in the assay described in Example 14. An antibody "blocks״ or "cross-blocks" one or more other antibodies from binding to HER2 if the antibody competes with the one or more other antibodies 25% or more, with 25%-74% representing "partial black" and 75%-100% representing "full block", preferably as PCT/EP2011/058779 WO 2011/147986 determined using the assay of Example 14. For some pairs of antibodies, competition or blocking in the assay of the Examples is only observed when one antibody is coated סח the plate and the other is used to compete, and not vice versa. Unless otherwise defined or negated by context, the terms "competes with״, "cross-competes with", "blocks" or "cross- blocks״ when used herein is also intended to cover such pairs of antibodies.The term "epitope" means a protein determinant capable of specific binding to an antibody. Epitopes usually consist of surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. Conformational and nonconformationaI epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. The epitope may comprise amino acid residues directly involved in the binding (also called immunodominant component of the epitope) and other amino acid residues, which are not directly involved in the binding, such as amino acid residues which are effectively blocked or covered by the specifically antigen binding peptide (in other words, the amino acid residue is within the footprint of the specifically antigen binding peptide).The term "monoclonal antibody" as used herein refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Accordingly, the term "human monoclonal antibody" refers to antibodies displaying a single binding specificity which have variable and constant regions derived from human germline immunoglobulin sequences.The human monoclonal antibodies may be generated by a hybridoma which includes a B cell obtained from a transgenic or transchramosomal nonhuman animal, such as a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene, fused to an Immortalized ceil.As used herein, the term "binding" in the context of the binding of an antibody to a predetermined antigen or epitope typically is a binding with an affinity corresponding to a KD of about 10'7 M or less, such as about 10'8 M or less, such as about 10'9 M or less, about 10'10 M or less, or about 10"11 M or even less when determined by for instance surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using the antigen as the ligand and the antibody as the analyte, and binds to the predetermined antigen with an affinity corresponding to a KD that is at least ten-fold lower, such as at least 100 fold lower, for instance at least 1,000 fold lower, such as at least 10,000 fold lower, for instance at least 100,000 fold lower than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen. The amount with which the affinity is lower is dependent on the KD of the antibody, so that when the KD of PCT/EP2011/058779 WO 2011/147986 the antibody is very low (that is, the antibody is highly specific), then the amount with which the affinity for the antigen is lower than the affinity for a non-specific antigen may be at least 10,000 fold.The term "kd" (sec1־), as used herein, refers to the dissociation rate constant of a particular antibody-antigen interaction. Said value is also referred to as the value.The term "ka״ (M1־־ x sec‘1), as used herein, refers to the association rate constant of a particular antibody-antigen interaction.The term "KD" (M), as used herein, refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.The term "KA" (N1־), as used herein, refers to the association equilibrium constant of a particular antibody-antigen interaction and is obtained by dividing the ka by the kd.As used herein, the term "inhibits proliferation" (e.g. referring to cells, such as tumor cells) is intended to include any substantial decrease in the cell proliferation when contacted with a HER2 antibody as compared to the proliferation of the same cells not in contact with a HER2 antibody, e.g., the inhibition of proliferation of a cell culture by at least about 10%, at least about 20% or at least about 30%, or at least as much as a reference antibody such as trastuzumab, e.g., as determined by an assay in the Examples, e.g. example 16,As used herein, the term "promotes proliferation״ (e.g. referring to cells, such as tumor cells) is intended to include any substantial increase in the cell proliferation when contacted with a HER2 antibody as compared to the proliferation of the same cells not in contact with a HER2 antibody, e.g., the promotion of proliferation of a cell culture by at least about 10%, at least about 20% or at least about 30%, or at least as much as a reference antibody as F5, e.g., as determined by an assay in the Examples.As used herein, the term "internalization", when used in the context of a HERantibody includes any mechanism by which the antibody is internalized into a HER2- expressing cell from the cell-surface and/or from surrounding medium, e.g., via endocytosis. The internalization of an antibody can be evaluated using a direct assay measuring the amount of internalized antibody (such as, e.g., the fab-CypHer5E assay described in Example 18), or an indirect assay where the effect of an internalized antibody- toxin conjugate is measured (such as, e.g., the anti-kappa-ETA'assay of Example 17).The present invention also provides antibodies comprising functional variants of the VL region, VH region, or one or more CDRs of the antibodies of the examples. A functional variant of a Vu VH, or CDR used in the context of a HER2 antibody still allows the antibody to retain at least a substantial proportion (at least about 50%, 60%, 70%, 80%, 90%, 95% or more) of the affinity/avidity and/or the specificity/selectivity of the parent antibody and PCT/EP2011/058779 WO 2011/147986 in some cases such a HER2 antibody may be associated with greater affinity, selectivity and/or specificity than the parent antibody.Such functional variants typically retain significant sequence identity to the parent antibody. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = # of identical positions/total # of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The percent identity between two nucleotide or amino acid sequences may e.g. be determined using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci 4, 11-17 (1988) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences may be determined using the Needleman and Wunsch, J. Mol. Biol. 48, 444-453 (1970) algorithm.Exemplary variants include those which differ from a parent antibody VH and/or VL sequence shown in Figures 1 and 2 at one or more "variant" amino acid positions, denoted "X״ in the corresponding consensus sequence. Preferred variants are those in which the new amino add is selected from those at the corresponding position in one of the aligned sequences in Figure 1 or 2 (for details on CDR sequence variants, see Table 4).Alternatively or additionally, the sequence of VH, VL or CDR variants may differ from the sequence of the VH, VL or CDR of the parent antibody sequences mainly by conservative substitutions; for instance at least 10, such as at least 9, 8, 7, 6, 5, 4, 3, 2 or 1 of the substitutions in the variant are conservative amino acid residue replacements.In the context of the present invention, conservative substitutions may be defined by substitutions within the classes of amino acids reflected in the following table: Amino acid residue classes for conservative substitutions Acidic Residues Asp (D) and Glu (E)Basic Residues Lys (K), Arg (R), and His (H)Hydrophilic Uncharged Residues Ser (S), Thr (T), Asn (N), andGin (Q)Aliphatic Uncharged Residues Gly (G), Ala (A), Val (V), Leu (L),and lie (I)Non-polar Uncharged Residues Cys (C), Met (M), and Pro (P)Aromatic Residues Phe (F), Tyr (Y), and Trp (W) PCT/EP2011/058779 WO 2011/147986 The term ״recombinant host cell" (or simply "host cell״),, as used herein, is intended to refer to a cell into which an expression vector has been introduced, e.g, an expression vector encoding an antibody of the invention. Recombinant host cells include, for example, transfectomas, such as CHO cells, HEK293 cells, NS/0 cells, and lymphocytic cells.The term "transgenic non-human animal" refers to a non-human animal having a genome comprising one or more human heavy and/or light chain transgenes or transchromosomes (either integrated or non-integrated into the animal's natural genomic DIMA) and which is capable of expressing fully human antibodies. For example, a transgenic mouse can have a human light chain transgene and either a human heavy chain transgene or human heavy chain transchromosome, such that the mouse produces human HERantibodies when immunized with HER2 antigen and/or cells expressing HER2. The human heavy chain transgene may be integrated into the chromosomal DNA of the mouse, as is the case for transgenic mice, for instance HuMAb mice, such as HC07, HC012, or HC0mice, or the human heavy chain transgene may be maintained extrachromosomally, as is the case for transchromosomai KM mice as described in WO02/43478. Similar mice, having a larger human Ab gene repertoire, include HC07 and HC020 (see e.g. W02009097006). Such transgenic and transchromosomai mice (collectively referred to herein as "transgenic mice") are capable of producing multiple isotypes of human monoclonal antibodies to a given antigen (such as IgG, IgA, IgM, IgD and/or IgE) by undergoing V-D-3 recombination and isotype switching. Transgenic, nonhuman animal can also be used for production of antibodies against a specific antigen by introducing genes encoding such specific antibody, for example by operatively linking the genes to a gene which is expressed in the miik of the animal,"Treatment" refers to the administration of an effective amount of a therapeutically active compound of the present invention with the purpose of easing, ameliorating, arresting or eradicating (curing) symptoms or disease states.An "effective amount" or "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of a HER2 antibody may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the HERantibody to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects.An "anti-idiotypic" antibody is an antibody which recognizes unique determinants generally associated with the antigen-binding site of an antibody.
PCT/EP2011/058779 WO 2011/147986 Further aspects and embodiments of the inventionAs described above, in a first aspect, the invention relates to a monoclonal antibody which binds HER2.Monoclonal antibodies of the present invention may be produced, e.g., by the hybridoma method first described by Kohler et at., Nature 256, 495 (1975), or may be produced by recombinant DNA methods. Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in, for example, Clackson et at., Nature 352, 624-628 (1991) and Marks et at., 3. Mol. Biol. 222, 5811991) 597־).Monoclonal antibodies may be obtained from any suitable source. Thus, for example, monoclonal antibodies may be obtained from hybridomas prepared from murine splenic B cells obtained from mice immunized with an antigen of interest, for instance in form of cells expressing the antigen on the surface, or a nucleic acid encoding an antigen of interest Monoclonal antibodies may also be obtained from hybridomas derived from antibody- expressing cells of immunized humans or non-human mammals such as rats, dogs, primates, etc.In one embodiment, the antibody of the invention is a human antibody. Human monoclonal antibodies directed against HER2 may be generated using transgenic or transchromosoma! mice carrying parts of the human immune system rather than the mouse system. Such transgenic and transchromosomic mice include mice referred to herein as HuMAb mice and KM mice, respectively, and are collectively referred to herein as "transgenic mice".The HuMAb mouse contains a human immunoglobulin gene miniloci that encodes unrearranged human heavy (p and y) and k light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous p and k chain loci (Lonberg, N. et al., Nature 368. 856-859 (1994)). Accordingly, the mice exhibit reduced expression of mouse TgM or k and in response to immunization, the introduced human heavy and light chain transgenes, undergo class switching and somatic mutation to generate high affinity human IgG,K monoclonal antibodies (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N. Handbook of Experimental Pharmacology 113, 49-101 (1994), Lonberg, N. and Huszar, D.f Intern. Rev. Immunol. Vol. 13 65-93 (1995) and Harding, F. and Lonberg, N. Ann. N.Y. Acad. Sci 764 536-546 (1995)). The preparation of HuMAb mice is described in detail in Taylor, L. et aL, Nucleic Adds Research 20, 6287-6295 (1992), Chen, 3. et al., International Immunology 5, 647-656 (1993), Tuaillon et al., J. Immunol. 152, 2912-2920 (1994),Taylor, L. et at., International Immunology 6, 579-591 (1994), Fishwild, D. et at., Nature Biotechnology 14, 845-851 (1996). See also US 5,545,806, US 5,569,825, US 5,625,126,US 5,633,425, US 5,789,650, US 5,877,397, US 5,661,016, US 5,814,318, US 5,874,299, PCT/EP2011/058779 WO 2011/147986 US 5,770,429, US 5,545,807, WO 98/24884, WO 94/25585, WO 93/1227, WO 92/22645, WO 92/03918 and WO 01/09187.The HC07, HC012, HC017 and HC020 mice have a 3KD disruption in their endogenous light chain (kappa) genes (as described in Chen et al, EMBO 3. 12, 821-8(1993)), a CMD disruption in their endogenous heavy chain genes (as described in Example of WO 01/14424), and a KC05 human kappa light chain transgene (as described in Rshwild et al., Nature Biotechnology 14, 845-851 (1996)). Additionally, the Hc07 mice have a HC07 human heavy chain transgene (as described in US 5,770,429), the HC012 mice have a HC012 human heavy chain transgene (as described in Example 2 of WO 01/14424), the HC017 mice have a HC017 human heavy chain transgene (as described in Example 2 of WO 01/09187) and the HC020 mice have a HC020 human heavy chain transgene. The resulting mice express human immunoglobulin heavy and kappa light chain transgenes in a background homozygous for disruption of the endogenous mouse heavy and kappa light chain iod.In the KM mouse strain, the endogenous mouse kappa light chain gene has been homozygously disrupted as described in Chen et al., EMBO 3. 12, 811-820 (1993) and the endogenous mouse heavy chain gene has been homozygously disrupted as described in Example 1 of WO 01/09187. This mouse strain carries a human kappa light chain transgene, KC05, as described in Fishwild et al., Nature Biotechnology 14, 845-851 (1996). This mouse strain also carries a human heavy chain transchromosome composed of chromosome 14 fragment hCF (SC20) as described in WO 02/43478. HCo!2־Balb/C mice can be generated by crossing HC012 to KC05[J/K](Balb) as described in WO/2009/097006.Spienocytes from these transgenic mice may be used to generate hybridomas that secrete human monoclonal antibodies according to well known techniques.Further, human antibodies of the present invention or antibodies of the present invention from other species may be identified through display-type technologies, including, without limitation, phage display, retroviral display, ribosomai display, and other techniques, using techniques well known in the art and the resulting molecules may be subjected to additional maturation, such as affinity maturation, as such techniques are well known in the art (see for instance Hoogenboom et al., 3. Mol. Biol. 227, 381 (1991) (phage display), Vaughan et al., Nature Biotech 14, 309 (1996) (phage display), Hanes and Plucthau, PNAS USA 94, 4937-4942 (1997) (ribosomai display), Parmley and Smith, Gene 73, 305-318 (1988) (phage display), Scott TIBS 17, 241-245 (1992), Cwirla et al., PNAS USA 87, 6378-6382 (1990), Russel etal., Nucl. Acids Research 21, 1081-1085 (1993), Hogenboom et al., Immunol. Reviews 130, 43-68 (1992), Chiswell and McCafferty TIBTECH PCT/EP2011/058779 WO 2011/147986 , 80-84 (1992), and US 5,733,743). If display technologies are utilized to produce antibodies that are not human, such antibodies may be humanized.
Antibodies of cross-block group 1In one aspect of the antibody of the invention, the antibody binds the same epitope on HER2 as one or more of the novel human antibodies of cross-block group 1 described herein.In one embodiment, the antibody cross-blocks the binding to soluble HER2 of trastuzumab, when determined as described in Example 14.
In one embodiment, the antibody binds the same epitope as a reference antibody comprising a VH region comprising the sequence of SEQ ID NO:l and a VL region comprising the sequence of SEQ ID NO:5 (169).In one embodiment, the antibody binds the same epitope as a reference antibody comprising a VH region comprising the sequence of SEQ ID NO:8 and a VL region comprising the sequence of SEQ ID NO: 12 (050).In one embodiment, the antibody binds the same epitope as a reference antibody comprising a VH region comprising the sequence of SEQ ID NO: 15 and a VL region comprising the sequence of SEQ ID NO:84 (084).In one embodiment, the antibody binds to the same epitope as a reference antibody comprising VH and VL regions selected from the group consisting of:a) a VH region comprising the sequence of SEQ ID NO: 77 and a VL region comprising the sequence of SEQ ID NO:78 (049);b) a VH region comprising the sequence of SEQ ID NO:79 and a VL region comprising the sequence of SEQ ID NO:8Q (051);c) a VH region comprising the sequence of SEQ ID NO:81 and a VL region comprising the sequence of SEQ ID NO:82 (055);d) a VH region comprising the sequence of SEQ ID NO:83 and a VL region comprising the sequence of SEQ ID NO:84 (123);e) a VH region comprising the sequence of SEQ ID NO:85 and a VL region comprising the sequence of SEQ ID NO;86 (161); andf) a VH region comprising the sequence of SEQ ID NO:87 and a VL region comprising the sequence of SEQ ID NO:88 (124).
PCT/EP2011/058779 WO 2011/147986 In another additional or alternative aspect of the antibody of the invention, the antibody binds to HER2 and comprises a VH CDR3, VH region and/or VL region sequence similar or identical to a sequence of the novel antibodies described herein.
In one embodiment, the antibody comprises a VH CDR3 region having a sequence selected from the group consisting ofSEQ ID NO:ll (050, 049, 051,055), optionally wherein the VH region is derived from the IgHV3 ־ 21 ־ l germline sequence;SEQ ID No: 130, such as the sequence of SEQ ID NO: 18 (084), optionally wherein the VH region is derived from the IgHVl-69-04 germline sequence;SEQ ID NO:133 (169, 123, 161, 124),such as the sequence of SEQ ID NO: (169),optionally wherein the VH region is derived from the IgHVl-18-1 germline sequence; orIn one embodiment, the antibody comprises a VH CDR3 region of one of antibodies 123, 161, or 124, as shown in Figure 1, optionally׳ wherein the VH region is derived from an IgHVl-18-1 germline.
In one embodiment, the antibody comprises a VH region selected from the group consisting ofa) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:9, 1and 11, such as the CDR1, CDR2 and CDR3 sequences of SEQ ID NOS: 9, 10 and (050); optionally where the VH region is derived from an IgHV3-23-1 germline;b) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:128, 129 and 130, such the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:16, 17 and 18, respectively (084), optionally where the VH region is derived from an IgHVl-69- germline; andc) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs:131, 132, and 133, such as the CRD1, CDR2, and CDR3 sequences of SEQ ID NOs: 2, and 4 (169), respectively, optionally where the VH region is derived from an IgHVl- 181־ germline.
In one embodiment, the antibody comprises a VH region selected from the preceding embodiments (a) or (b) and a VL region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 13, XAS (wherein X is A or V), and SEQ ID No: 155, respectively, such as a CDR1 sequence selected from SEQ ID Nos: 13 or 20, a CDR2 which is AAS or VAS, and a PCT/EP2011/058779 WO 2011/147986 CDR3 sequence selected from SEQ ID NOs:14 and 21 (050, 084); respectively, optionally where the VL region is derived from an IgKVl-12-01 germline.In one embodiment, the antibody comprises a VH region which is the preceding embodiment (c) and a VL region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:6, DXS (wherein X=A or T), and SEQ ID NO:156 (169), respectively, optionally wherein the VL region is derived from IgKV3-11-01.
In one embodiment, the antibody comprises a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:2, 3 and 4, respectively; and a VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:6, DAS, and SEQ ID NO:7, respectively (169).In one embodiment, the antibody comprises a VH region comprising the CDR1, CDRand CDR3 sequences of SEQ ID NOs:9, 10 and 11, respectively; and a VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:13, AAS, and SEQ ID NO: 14, respectively (050).In one embodiment, the antibody comprises a VH region comprising the CDR1, CDRand CDR3 sequences of SEQ ID NOs:16, 17 and 18, respectively; and a VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID N0s:20, VAS, and SEQ ID NO:21, respectively (084).
In separate embodiments, the antibody comprises:a) a VH region comprising the sequence of SEQ ID NO:l and, preferably, a VL region comprising the sequence of SEQ ID NQ:5 (169);b) a VH region comprising the sequence of SEQ ID NO:8 and, preferably, a VL region comprising the sequence of SEQ ID NO:12 (050);c) a VH region comprising the sequence of SEQ ID NO: 15 and, preferably, a VL region comprising the sequence of SEQ ID NO: 19 (084);d) a VH region comprising the sequence of SEQ ID NQ:77 and, preferably, a VL region comprising the sequence of SEQ ID NO:78 (049);e) a VH region comprising the sequence of SEQ ID NO:79 and, preferably, a VL region comprising the sequence of SEQ ID NO:80 (051);f) a VH region comprising the sequence of SEQ ID NO:81 and, preferably, a VL region comprising the sequence of SEQ ID NO:82 (055);g) a VH region comprising the sequence of SEQ ID NO:83 and, preferably, a VL region comprising the sequence of SEQ ID NO:84 (123); PCT/EP2011/058779 WO 2011/147986 h) a VH region comprising the sequence of SEQ ID NO:85 and, preferably, a VL region comprising the sequence of SEQ ID NO:86 (161);i) a VH region comprising the sequence of SEQ ID NO:87 and, preferably, a VL region comprising the sequence of SEQ ID NO:88 (124); and/orj) a variant of any of said antibodies, wherein said variant preferably has at most 1, or 3 amino-acid modifications, more preferably amino-acid substitutions, such as conservative amino acid substitutions and substitutions where the new amino add is one at the same position in an aligned sequence in Figures 1 or 2, particularly at positions indicated by "X" in the corresponding consensus sequence.
Antibodies of cross-block group 2In one aspect of the antibody of the invention, the antibody binds the same epitope on HER2 as one or more of the novel human antibodies of cross-block group 2 described herein.In one embodiment, the antibody cross-blocks the binding to soluble HER2 of pertuzumab, when determined as described in Example 14.
In one embodiment, the antibody binds the same epitope as a reference antibody comprising a VH region comprising the sequence of SEQ ID NO:22 and a VL region comprising the sequence of SEQ ID NO:26 (025).In one embodiment, the antibody binds the same epitope as a reference antibody comprising a VH region comprising the sequence of SEQ ID NO:29 and a VL region comprising the sequence of SEQ ID NO:32 (091).In one embodiment, the antibody binds the same epitope as a reference antibody comprising a VH region comprising the sequence of SEQ ID NO:35 and a VL region comprising the sequence of SEQ ID NO:39 (129).In one embodiment, the antibody binds to the same epitope as a reference antibody comprising VH and VL regions selected from the group consisting of:a) a VH region comprising the sequence of SEQ ID NO:89 and a VL region comprising the sequence of SEQ ID NO:90 (001);b) a VH region comprising the sequence of SEQ ID NO:91 and a VL region comprising the sequence of SEQ ID NO:92 (143);c) a VH region comprising the sequence of SEQ ID NO:93 and a VL region comprising the sequence of SEQ ID NO:94 (019);d) a VH region comprising the sequence of SEQ ID NO:95 and a VL region comprising the sequence of SEQ ID NO:96 (021); PCT/EP2011/058779 WO 2011/147986 e) a VH region comprising the sequence of SEQ ID NO:97 and a VL region comprising the sequence of SEQ ID NO:98 (027);f) a VH region comprising the sequence of SEQ ID NO:99 and a VL region comprising the sequence of SEQ ID NO:100 (032)g) a VH region comprising the sequence of SEQ ID NO: 101 and a VL region comprising the sequence of SEQ ID NO:102 (035);h) a VH region comprising the sequence of SEQ ID NO: 103 and a VL region comprising the sequence of SEQ ID NO: 104 (036);i) a VH region comprising the sequence of SEQ ID NO: 105 and a VL region comprising the sequence of SEQ ID NO: 106 (054); andj) a VH region comprising the sequence of SEQ ID NO: 107 and a VL region comprising the sequence of SEQ ID NO: 108 (094).
In another additional or alternative aspect of the antibody of the invention, the antibody binds to HER2 and comprises a VH CDR3, VH region and/or VL region sequence similar or identical to a sequence of the novel antibodies described herein.
In one embodiment, the antibody comprises a VH CDR3 region having a sequence selected from the group consisting ofSEQ ID NO: 136, such as the sequence of SEQ ID NO:25 (025), optionally wherein the VH region is derived from the IgHV4-34-l germline sequence;SEQ ID NO: 139, such as the sequence of SEQ ID NO:31 (091), optionally wherein the VH region is derived from the IgHV4-34-01 germline sequence; andSEQ ID NO: 142, such as the sequence of SEQ ID NO:38 (129), optionally wherein the VH region is derived from the IgHV3-30־Dl germline sequence.In one embodiment, the antibody comprises a VH CDR3 region of one of antibodies 001, 143, 019, 021, 027, 032, 035, 036, 054 or 094 as shown in Figure 1, optionally wherein the VH region is derived from an IgHV4-34-l germline.
In one embodiment, the antibody comprises a VH region selected from the group consisting ofa) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:134, 135 and 136, such as the CDR1, CDR2 and CDR3 sequences of SEQ ID NOS: 23, and 25 (025); optionally where the VH region is derived from an IgHV4-34־l germiine; 21 PCTOP2011/058779 WO 2011/147986 b) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:137f 138 and 139, such the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:30, 163, and 31, respectively (091), optionally where the VH region is derived from an IgHV4-34-01 germline; andc) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs:140, 141 and 142, such as the CRD1, CDR2, and CDR3 sequences of SEQ ID NOs: 36, and 38 (129), respectively, optionally where the VH region is derived from an IgHV3-30-01 germline.
In one embodiment, the antibody comprises a VH region selected from the preceding embodiment (a) and a VL region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 157, AAS, and SEQ ID No: 164, respectively, such as the CDR1, CDR2, and CDRsequences of SEQ ID Nos:27, AAS, and SEQ ID NO:28 (025); respectively, optionally where the VL region is derived from an IgKVlD-1601־ germline.In one embodiment, the antibody comprises a VH region selected from the preceding embodiment (b) and a VL region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:33, AX!X2 (wherein X! is A or T, preferably A; and X2 is S or F, preferably S), and SEQ ID No: 158, respectively, such as the CDR1, CDR2 and CDR3 sequences of SEQ ID Nos:33, AAS, and SEQ ID NO:34 (091); respectively, optionally where the VL region is derived from an IgKVlD-1601־ germline.In one embodiment, the antibody comprises a VH region which is the preceding embodiment (c) and a VL region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:40, DAS and SEQ ID NO :41 (129), respectively, optionally wherein the VL region is derived from IgKV3״ll"01.
In one embodiment, the antibody comprises a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:23, 24 and 25, respectively; and a VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 27, AAS, and SEQ ID NO:28, respectively (025).In one embodiment, the antibody comprises a VH region comprising the CDR1, CDRand CDR3 sequences of SEQ ID NOs:30, 163 and 31, respectively; and a VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:33, AAS, and SEQ ID NO:34, respectively (091).In one embodiment, the antibody comprises a VH region comprising the CDR1, CDRand CDR3 sequences of SEQ ID NOs:36, 37 and 38, respectively; and a VL region PCT/EP2011/058779 WO 2011/147986 comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:40, DAS, and SEQ ID NO:41, respectively (129).
In separate embodiments, the antibody comprises:a) a VH region comprising the sequence of SEQ ID NO:22 and, preferably, a VL region comprising the sequence of SEQ ID NO:26 (025);b) a VH region comprising the sequence of SEQ ID NO:29 and, preferably, a VL region comprising the sequence of SEQ ID NO:32 (091);c) a VH region comprising the sequence of SEQ ID NO: 35 and, preferably, a VL region comprising the sequence of SEQ ID NO:39 (129);d) a VH region comprising the sequence of SEQ ID NG:89 and, preferably, a VL region comprising the sequence of SEQ ID NO:90 (001);e) a VH region comprising the sequence of SEQ ID NO:91 and, preferably, a VL region comprising the sequence of SEQ ID NO:92 (143);f) a VH region comprising the sequence of SEQ ID NO:93 and, preferably, a VL region comprising the sequence of SEQ ID NO:94 (019);g) a VH region comprising the sequence of SEQ ID NO:95 and, preferably, a VL region comprising the sequence of SEQ ID NO:96 (021);h) a VH region comprising the sequence of SEQ ID NO:97 and, preferably, a VL region comprising the sequence of SEQ ID NO:98 (027);i) a VH region comprising the sequence of SEQ ID NO:99 and, preferably, a VL region comprising the sequence of SEQ ID NO: 100 (032);j) a VH region comprising the sequence of SEQ ID NO: 101 and, preferably, a VL region comprising the sequence of SEQ ID NO: 102 (035);k) a VH region comprising the sequence of SEQ ID NO: 103 and, preferably, a VL region comprising Hie sequence of SEQ ID NO:104 (036);l) a VH region comprising the sequence of SEQ ID NO: 105 and, preferably, a VL region comprising the sequence of SEQ ID NO: 106 (054);m) a VH region comprising the sequence of SEQ ID NO: 106 and, preferably, a VL region comprising the sequence of SEQ ID NO: 108 (094); and/orn) a variant of any of said antibodies, wherein said variant preferably has at most 1, or 3 amino-acid modifications, more preferably amino-acid substitutions, such as conservative amino acid substitutions and substitutions where the new amino add is one at the same position in an aligned sequence in Figures 1 or 2, particularly at positions indicated by "X״ in the corresponding consensus sequence.
PCT/EP2011/058779 WO 2011/147986 Antibodies of cross-block group 3In one aspect of the antibody of the invention, the antibody binds the same epitope on HER2 as one or more of the novel human antibodies of cross-block group 3 described herein.In one embodiment, the antibody cross-blocks the binding to soluble HER2 of Fand/or C5, when determined as described in Example 14.
In one embodiment, the antibody binds the same epitope as a reference antibody comprising a VH region comprising the sequence of SEQ ID NO:46 and a VL region comprising the sequence of SEQ ID NO:49 (127).In one embodiment, the antibody binds the same epitope as a reference antibody comprising a VH region comprising the sequence of SEQ ID NO:49 and a VL region comprising the sequence of SEQ ID NO:53 (159).In one embodiment, the antibody binds the same epitope as a reference antibody comprising a VH region comprising the sequence of SEQ ID NO:56 and a VL region comprising the sequence of SEQ ID NO:60 (098).In one embodiment, the antibody binds the same epitope as a reference antibody comprising a VH region comprising the sequence of SEQ ID NO:63 and a VL region comprising the sequence of SEQ ID NO:67 (153).In one embodiment, the antibody binds the same epitope as a reference antibody comprising a VH region comprising the sequence of SEQ ID NO:70 and a VL region comprising the sequence of SEQ ID NO;74 (132).In one embodiment, the antibody binds to the same epitope as a reference antibody comprising VH and VL regions selected from the group consisting of:k) a VH region comprising the sequence of SEQ ID NO:109 and a VL region comprising the sequence of SEQ ID NO:110 (105);l) a VH region comprising the sequence of SEQ ID NO: 111 and a VL region comprising the sequence of SEQ ID NO: 112 (100);m) a VH region comprising the sequence of SEQ ID NO: 113 and a VL region comprising the sequence of SEQ ID NO:114 (125);n) a VH region comprising the sequence of SEQ ID NOrllS and a VL region comprising the sequence of SEQ ID NO:116 (162);0) a VH region comprising the sequence of SEQ ID N0.117 and a VL region comprising the sequence of SEQ ID NO: 118 (033);p) a VH region comprising the sequence of SEQ ID NO: 119 and a VL region comprising the sequence of SEQ ID NO: 120 (160) PCT/EP2011/058779 WO 2011/147986 q) a VH region comprising the sequence of SEQ ID NO: 121 and a VL region comprising the sequence of SEQ ID NO: 122 (166);r) a VH region comprising the sequence of SEQ ID NO: 123 and a VL region comprising the sequence of SEQ ID NO: 124 (152); ands) a VH region comprising the sequence of SEQ ID NO: 125 and a VL region comprising the sequence of SEQ ID NO: 126 (167).
In another additional or alternative aspect of the antibody of the invention, the antibody binds to HER2 and comprises a VH CDR3, VH region and/or VL region sequence similar or identical to a sequence of the novel antibodies described herein.
In one embodiment, the antibody comprises a VH CDR3 region having a sequence selected from the group consisting ofSEQ ID NO: 148, such as the sequence of SEQ ID NO:48 (127), optionally wherein the VH region is derived from the IgHV5-51-01 germline sequence;SEQ ID NO:52 (t 59), optionally wherein the VH region is derived from the IgHVS- 51-01 germline sequence;SEQ ID NO; 145, such as the sequence of SEQ ID NO:59 (098), optionally wherein the VH region is derived from the IgHV3-23-01 germline sequence;SEQ ID NO; 154, such as the sequence of SEQ ID NO:66 (t 53), optionally wherein the VH region is derived from the IgHV3-30-G301־ germline sequence; andSEQ ID NO: 151, such as the sequence of SEQ ID NO:73 (132), optionally wherein the VH region is derived from the IgHVl-18-01 germitne sequence.In one embodiment, the antibody comprises a VH CDR3 region of one of antibodies 105, 100, 125 or 162 as shown in Figure 1, optionally wherein the VH region is derived from an IgHV3-23־l germline.In one embodiment, the antibody comprises a VH CDR3 region of one of antibodies 033, 160, 166, 152 or 167 as shown in Figure 1, optionally wherein the VH region is derived from an IgHV3-30-301־ germline.
In one embodiment, the antibody comprises a VH region selected from the group consisting ofa) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO$:146, 147 and 148, such as the CDR1, CDR2 and CDR3 sequences of SEQ ID NOS: 43, and 45 (127); optionally where the VH region is derived from an IgHV5-51-germ line; PCT/EP2011/058779 WO 2011/147986 b) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 149, and 52, such as the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:50, 51 and 52, respectively (159), optionally where the VH region is derived from an IgHV5-51- germline;c) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs :143, 144 and 145, such as the CRD1, CDR2, and CDR3 sequences of SEQ ID NOs: 57, and 59 (09 8), respectively, optionally where the VH region is derived from an IgHV323-01־ germline;d) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 152, 153 and 154, such as the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:64, and 66, respectively (153), optionally where the VH region is derived from an IgHV301 ־ 03 ־ 30 ־ germline; ande) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs:71, 150 and 151, such as the CRD1, CDR2, and CDR3 sequences of SEQ ID NOs: 71, and 73 (132), respectively, optionally where the VH region Is derived from an IgHVl-18-01 germline.
In one embodiment, the antibody comprises a VH region selected from the preceding embodiment (a) and a VL region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:47, AAS and SEQ ID NO:48, respectively (127); respectively, optionally where the VL region is derived from an IgKVlD-8-01 germline.In one embodiment, the antibody comprises a VH region selected from the preceding embodiment (b) and a VL region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:54, AAS, and SEQ ID No;55 (159); respectively, optionally where the VL region is derived from an IgKVlD-16-01 germline.In one embodiment, the antibody comprises a VH region which is the preceding embodiment (c) and a VL region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 159, AAS and SEQ ID NO: 160, respectively, such as the VL CDR1, CDR2 and CDRsequences of SEQ ID NOS: 61, AAS and SEQ ID NO:62 (098), optionally wherein the VL region is derived from IgKVlD~1601־.In one embodiment, the antibody comprises a VH region which is the preceding embodiment (d) and a VL region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 161, XAS (wherein X=D or A, preferably D), and SEQ ID NO: 162 (153), respectively, such as the VL CDR sequences of SEQ ID NO:68, DAS, and 69, optionally wherein the VL region is derived from IgKVlD-16-01.
PCT/EP2G11/058779 WO 2011/147980 In one embodiment, the antibody comprises a VH region which is the preceding embodiment (e) and a VL region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:75, DAS and SEQ ID NO:76 (1 32), respectively, optionally wherein the VL region is derived from IgKV3־ll01־.
In one embodiment, the antibody comprises a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:43, 44 and 45, respectively; and a VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:47, AAS, and SEQ ID NO:48, respectively (127).In one embodiment, the antibody comprises a VH region comprising the CDR1, CDRand CDR3 sequences of SEQ ID NOs;5Q, 51 and 52, respectively; and a VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:54, AAS, and SEQ ID NO:55, respectively (159).In one embodiment, the antibody comprises a VH region comprising the CDR1, CDRand CDR3 sequences of SEQ ID NOs:57, 58 and 59, respectively; and a VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:6Q, AAS, and SEQ ID NO:61, respectively (098).In one embodiment, the antibody comprises a VH region comprising the CDR1, CDRand CDR3 sequences of SEQ ID NOs:64, 65 and 66, respectively; and a VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:68, DAS, and SEQ ID NO:69, respectively (153).In one embodiment, the antibody comprises a VH region comprising the CDR1, CDRand CDR3 sequences of SEQ ID NOs:71, 72 and 73, respectively; and a VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:75, DAS, and SEQ ID NO:76, respectively (132).
In separate embodiments, the antibody comprises:a) a VH region comprising the sequence of SEQ ID NO:46 and, preferably, a VL region comprising the sequence of SEQ ID NO:49 (127);b) a VH region comprising the sequence of SEQ ID NO:49 and, preferably, a VL region comprising the sequence of SEQ ID NO: 53 (159);c) a VH region comprising the sequence of SEQ ID NO:56 and, preferably, a VL region comprising the sequence of SEQ ID NO:60 (098);d) a VH region comprising the sequence of SEQ ID NO:63 an, preferably, a VL region comprising the sequence of SEQ ID NO:67 (153); PCT/EP2011/058779 WO 2011/147986 e) a VH region comprising the sequence of SEQ ID NO:70 and, preferably, a VL region comprising the sequence of SEQ ID NO:74 (132);f) a VH region comprising the sequence of SEQ ID NO: 109 and, preferably, a VL region comprising the sequence of SEQ ID NO: 110 (105);g) a VH region comprising the sequence of SEQ ID NO:111 and, preferably, a VL region comprising the sequence of SEQ ID NO: 112 (100);h) a VH region comprising the sequence of SEQ ID NO: 113 and, preferably, a VL region comprising the sequence of SEQ ID NO: 114 (125);i) a VH region comprising the sequence of SEQ ID NO: 115 and, preferably, a VL region comprising the sequence of SEQ ID NO: 116 (162);j) a VH region comprising the sequence of SEQ ID NO: 117 and, preferably, a VL region comprising the sequence of SEQ ID NO: 118 (033);k) a VH region comprising the sequence of SEQ ID NO: 119 and, preferably, a VL region comprising the sequence of SEQ ID NO: 120 (160)l) a VH region comprising the sequence of SEQ ID NO: 121 and, preferably, a VL region comprising the sequence of SEQ ID NO:122 (166);m) a VH region comprising the sequence of SEQ ID NO: 123 and, preferably, a VL region comprising the sequence of SEQ ID NO: 124 (152);o) a VH region comprising the sequence of SEQ ID NO: 125 and, preferably, a VL region comprising the sequence of SEQ ID NO: 126 (167); and/orp) a variant of any of said antibodies, wherein said variant preferably has at most 1, or 3 amino-acid modifications, more preferably amino-acid substitutions, such as conservative amino acid substitutions and substitutions where the new amino acid is one at the same position in an aligned sequence in Figures 1 or 2, particularly at positions indicated by "X״ in the corresponding consensus sequence.
Bispecific antibodiesIn one embodiment, the antibody is a bispecific antibody, comprising (i) a first antibody comprising antigen-binding region of an antibody as defined herein, e.g. an antibody of cross-block 1, 2 or 3, or the VH and VL region comprising the sequences of (005), and (ii) a second antibody comprising an antigen-binding region of an antibody which binds to CD3.In one embodiment, the antibody is a bispecific antibody, comprising (i) a first antibody comprising an antigen-binding region of an antibody as defined herein or the VH and VL region comprising the sequences of (005), and (ii) a second antibody comprising antigen-binding region of an antibody as defined herein or the VH and VL region comprising FCT/EP2011/OS8779 WO 2011/147986 sequences of (005), wherein the first antigen-binding region binds to a different epitope than the second antigen-binding region.In one embodiment the first antibody comprises a VH region comprising a CDRsequence of an antibody of cross-block 1, 2 or 3 as defined herein, such as SEQ ID NO: 4, or 66 (169, 025 or 153), ora CDR3 sequence of SEQ ID NO:168 (005).In one embodiment the first antibody comprises a VH region comprising CDR1, CDRand CDR3 sequences of an antibody of cross-block 1, 2 or 3 as defined herein, such as CDR1, CDR2, and CDR3 sequences SEQ ID NOs: 2, 3 and 4 (169), or CDR1, CDR2 and CDR3 sequences of SEQ ID NOs; 23, 24 and 25 (025), or CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 64, 65 and 66 (153), or CDR1, CDR2 CDR3 sequence of SEQ ID NOs: 166, 167 and 168 (005).In a further or alternative embodiment the first antibody comprises a VH region comprising a CDR3 sequence of an antibody of cross-block 1, 2 or 3 as defined herein, such as CDR3 sequence an antibody of cross-block 1 of SEQ ID NO: 11 (050), or SEQ ID NO: (084); or a CDR3 sequence of an antibody of cross-block 2 of SEQ ID NO: 31 (091), or SEQ ID NO: 38 (129), or a CDR3 sequence of an antibody of cross-block 3 of SEQ ID NO: (127), or SEQ ID NO:52 (159), or SEQ ID NO:59 (098), or SEQ ID NO:73 (1 32).In one embodiment the first antibody comprises a VH region comprising CDR1, CDRand CDR3 sequences of an antibody of cross-block 1, 2 or 3 as defined herein, such as CDR1, CDR2, and CDR3 sequences SEQ ID NOs: 2, 3 and 4 (169), or CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:23, 24 and 25 (025), or CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 64, 65 and 66 (1 53), or CDR1, CDR2 CDR3 sequence of SEQ ID NOs: 166, 167 and 168 (005).In one embodiment the first antibody comprises a VH region comprising CDR1, CDRand CDR3 sequences of an antibody of cross-block 1, 2 or 3 as defined herein a VL region comprising CDR1, CDR2 and CDR3 sequences of an antibody of cross-block 1, 2 or 3 as defined herein.In a further or alternative embodiment the first antibody comprises a VH region comprising CDR1, CDR2 and CDR3 sequences of an antibody of cross-block 1, 2 or 3 as defined herein, such as CDR1, CDR2, and CDR3 sequences of an antibody of cross-block of SEQ ID NOs: 9, 10 and 11 (050), or SEQ ID NOs: 16, 17 and 18 (084); or CDR1, CDR2, and CDR3 sequences of an antibody of cross-block 2 of SEQ ID NOs: 30, 163 and 31 (091), or SEQ ID NOs: 36, 37 and 38 (129), or CDR1, CDR2, and CDR3 sequences of an antibody of cross-block 3 SEQ ID NOs: 43, 44 and 45 (127), or SEQ ID NOs:50, 51 and 52 (159), or SEQ ID NOs:57, 58 and 59 (098), or SEQ ID NOs:71, 72 and 73 (1 32).
PCT/EP2011/058779 WO 2011/147986 In one embodiment the first antibody׳ comprises a VH region and a VL region selected from the group consisting of:a) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 2, 3 and 4; and a VL region comprising the CDR1, CDR2 and CDRsequences of SEQ ID: 6, GAS and SEQ ID NO:7, respectively (169);b) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 23, 24 and 25; and a VL region comprising the CDR1, CDR2 and CDRsequences of SEQ ID NO: 27, AAS and SEQ ID NO; 28, respectively (025);c) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 64, 65 and 66; and a VL region comprising the CDR1, CDR2 and CDRsequences of SEQ ID NO: 68, DAS and SEQ ID NO:69 (153); andd) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 166, 167 and 168; and a VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 169, GAS and SEQ ID NO:170 (005).In a further or alternative embodiment the first antibody comprises a VH region and a VL region selected from the group consisting of:a) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NQs:9, 127 and 11, such as the CDR1, CDR2 and CDR3 sequences of SEQ ID NOS: 9, 10 and 11 (050); optionally where the VH region is derived from an IgHV3 ־ 23 ־ l germline;b) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 128, 129 and 130, such the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 16, 17 and 18, respectively (084), optionally where the VH region is derived from an IgHVl-69-04 germline; andc) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 137, 138 and 139, such the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:30, 163, and 31, respectively (091), optionally where the VH region is derived from an IgHV434-01״ germline; andd) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 140, 141 and 142, such as the CRD1, CDR2, and CDR3 sequences of SEQ ID NOs: 36, 37 and 38 (129), respectively, optionally where the VH region is derived from an IgHV3-30-01 germline.e) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 146, 147 and 148, such as the CDR1, CDR2 and CDR3 sequences of SEQ ID NOS: 43, 44 and 45 (127); optionally where the VH region is derived from an IgHV5-51-01 germiine; PCT/EP2011/058779 WO 2011/147986 f) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 149, 51 and 52, such as the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:5Q, 51 and 52, respectively (159),optionally where the VH region is derived from an IgHV5-5101־ germ line;g) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs:143, 144 and 145, such as the CRD1, CDR2, and CDR3 sequences of SEQ ID NOs: 57, 58 and 59 (098), respectively, optionally where the VH region is derived from an IgHV3-2301־ germline;h) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs:71, 150 and 151, such as the CRD1, CDR2, and CDR3 sequences of SEQ ID NOs: 71, 72 and 73 (132), respectively, optionally where the VH region is derived from an IgHVl-18-01 germline.
In one embodiment the second antibody is one of the previous embodiment described for the first antibody, but wherein the second antibody binds to a different epitope than the first antibody.In one embodiment the second antibody is a CD3 antibody. In one embodiment a CD3 antibody may be an antibody comprising a VH region comprising the sequence of SEQ ID NO: 171 (YTH12.5)and VL region comprising the sequence of SEQ ID NO: 1 (YTH12.5).Another example of a CD3 antibody is an antibody comprising a VH region comprising the sequence of SEQ ID NO: 173 (huCLB־T3/4) and VL region comprising the sequence of SEQ ID NO:174 (huCLB־T3/4).In one embodiment, the antibody is a bispecific antibody, comprising (i) a first antibody having an Fc region and VH and VL sequences, which antibody comprises the VH and VL region sequences of (005), (025), (153) or (189), and which antibody comprises an IgGl wildtype Fc region, wherein the CH3 region contains lie at position 350, a Thr at position 370, and a Leu at position 405 and (ii) a second antibody having an Fc region and VH and VL sequences, which antibody comprising the VH and VL region sequences of (005), (025), (153) or (169), and which antibody comprises a IgGl wildtype Fc region, wherein the CH3 region contains an Arg at position 409. Specific embodiments are disclosed in examples.In one embodiment, the antibody is a bispecific antibody, comprising (i) a first antibody having an Fc region and VH and VL sequences, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 164, and the VL region comprises the amino acid sequence of SEQ ID NO: 165 (005), optionally wherein the first antibody comprises an IgGl,K Fc region, wherein the CH3 region contains lie at position 350, a Thr at position 370, PCT/EP2011/058779 WO 2011/147986 and a Leu at position 405; and (1i) a second antibody having an Fc region and VH and VL sequences ,wherein the VH region comprises the amino add sequence of SEQ ID HO: 1 and the VL region comprises the amino acid sequence of SEQ ID NO:5 (!69), optionally wherein the second antibody comprises an IgGl,K Fc region having an Arg at position 409.In one embodiment, the antibody is a bispecific antibody, comprising (i) a first antibody having an Fc region and VH and VL sequences, wherein the VH region comprises the amino acid sequence of SEQ ID NO:22, and the VL region comprises the amino add sequence of SEQ ID NO:26 (025), optionally wherein the first antibody comprises an IgGl,K Fc region, wherein the CH3 region contains lie at position 350, a Thr at position 370, and a Leu at position 405; and (ii) a second antibody having an Fc region and VH and VL sequences ,wherein the VH region comprises the amino acid sequence of SEQ ID NO: 1and die VL region comprises the amino add sequence of SEQ ID NO:165 (005), optionally wherein the second antibody comprises an IgGl,K Fc region having an Arg at position 409.In one embodiment, the antibody is a bispecific antibody, comprising (i) a first antibody having an Fc region and VH and VL sequences, wherein the VH region comprises the amino acid sequence of SEQ ID NO:22, and the VL region comprises the amino add sequence of SEQ ID NO:26 (025), optionally wherein the first antibody comprises an IgGl,K Fc region, wherein the CH3 region contains lie at position 350, a Thr at position 370, and a Leu at position 405; and (ii) a second antibody having an Fc region and VH and VL sequences ,wherein the VH region comprises the amino acid sequence of SEQ ID NO:63 and the VL region comprises the amino add sequence of SEQ ID NO:37 (153), optionally wherein the second antibody comprises an IgGl,K Fc region having an Arg at position 409.In one embodiment, the antibody is a bispecific antibody, comprising (i) a first antibody having an Fc region and VH and VL sequences, wherein the VH region comprises the amino add sequence of SEQ ID NO:22, and the VL region comprises the amino acid sequence of SEQ ID NO:26 (025), optionally wherein the first antibody comprises an IgGl,K: Fc region, wherein the CH3 region contains lie at position 350, a Thr at position 370, and a Leu at position 405; and (ii) a second antibody having an Fc region and VH and VL sequences ,wherein the VH region comprises the amino add sequence of SEQ ID NO:l and the VL region comprises the amino add sequence of SEQ ID NO:5 (169), optionally wherein the second antibody comprises an IgGl,K Fc region having an Arg at position 409.In one embodiment, the antibody is a bispecific antibody, comprising (!) a first antibody having an Fc region and VH and VL sequences, wherein the VH region comprises the amino acid sequence of SEQ ID NQ:63, and the VL region comprises the amino acid sequence of SEQ ID NO:67 (153), optionally wherein the first antibody comprises an IgGl,K Fc region, wherein the CH3 region contains lie at position 350, a Thr at position 370, and a PCT/EP2011/058779 WO 2011/147986 Leu at position 405; and (ii) a second antibody having an Fc region and VH and VL sequences ,wherein the VH region comprises the amino acid sequence of SEQ ID NO: 1and the VL region comprises the amino acid sequence of SEQ ID NO: 165 (005),optionally wherein the second antibody comprises an IgGl,K Fc region having an Arg at position 409.In one embodiment, the antibody is a bispecific antibody, comprising (i) a first antibody having an Fc region and VH and VL sequences, wherein the VH region comprises the amino acid sequence of SEQ ID NO:63, and the VL region comprises the amino acid sequence of SEQ ID NO:67 (153},optionally wherein the first antibody comprises an IgGl,K Fc region, wherein the CH3 region contains lie at position 350, a Thr at position 370, and a Leu at position 405; and (ii) a second antibody having an Fc region and VH and VL sequences ,wherein the VH region comprises the amino acid sequence of SEQ ID NO: 1 and the VL region comprises the amino add sequence of SEQ ID NO:5 (169),optionalJy wherein the second antibody comprises an IgGl,K Fc region having an Arg at position 409.In one embodiment, the antibody is a bispecific antibody, comprising (i) a first antibody having an Fc region and VH and VL sequences, wherein the VH region comprises the amino acid sequence of SEQ ID NO:63, and the VL region comprises the amino acid sequence of SEQ ID NO:67 (153),optionally wherein the first antibody comprises an IgGl,K Fc region having Arg at position 409, or Gin at position 297, or Arg at position 409 and Gin at position 297; and (ii) a second antibody having an Fc region and VH and VL sequences ,wherein the VH region comprises the amino acid sequence of SEQ ID NO: 171 and the VL region comprises the amino add sequence of SEQ ID NO: 172 (YTH12.5), optionally wherein the second antibody comprises an IgGl,K Fc region having an Gin at position 297, or Leu at position 405, or Gin at position 297 and Leu at position 405.In one embodiment, the antibody is a bispecific antibody, comprising (i) a first antibody having an Fc region and VH and VL sequences, wherein the VH region comprises the amino acid sequence of SEQ ID NO:l, and the VL region comprises the amino acid sequence of SEQ IDNO: 5 (1 69),optionally wherein the first antibody comprises an IgGl,K Fc region having Arg at position 409; and (ii) a second antibody having an Fc region and VH and VL sequences ,wherein the VH region comprises the amino acid sequence of SEQ ID NO:171 and the VL region comprises the amino acid sequence of SEQ ID NO:1(YTH12.5),, optionally wherein the second antibody comprises an IgGl,K Fc region having an Gin at position 297, or Leu at position 405, or Gin at position 297 and Leu at position 405.In one embodiment, the antibody is a bispecific antibody, comprising (i) a first antibody having an Fc region and VH and VL sequences, wherein the VH region comprises the amino acid sequence of SEQ ID NO:63, and the VL region comprises the amino acid PCT/EP2011/058779 WO 2011/147986 sequence of SEQ ID NG:67 (153), optionally wherein the first antibody comprises an IgGl,x Fc region having Arg at position 409, or Gin at position 297, or Arg at position 409 and Gin at position 297; and (ii) a second antibody having an Fc region and VH and VL sequences ,wherein the VH region comprises the amino acid sequence of SEQ ID NO: 173 and the VL region comprises the amino add sequence of SEQ ID NO: 174 (huCLB-T3/4), optionally wherein the second antibody comprises an IgGl,K Fc region having an Gin at position 297, or Leu at position 405, or Gin at position 297 and Leu at position 405,In one embodiment, the antibody is a bispecific antibody, comprising (i) a first antibody having an Fc region and VH and VL sequences, wherein the VH region comprises the amino acid sequence of SEQ ID NO:l, and the VL region comprises the amino acid sequence of SEQ IP NO:5 (169), optionally wherein the first antibody comprises an IgGl,K Fc region having Arg at position 409; and (ii) a second antibody having an Fc region and VH and VL sequences ,wherein the VH region comprises the amino acid sequence of SEQ ID NO:173 and the VL region comprises the amino acid sequence of SEQ ID NO: 174 (huCLB- T3/4), optionally wherein the second antibody comprises an IgGl,K Fc region having an Gin at position 297, or Leu at position 405, or Gin at position 297 and Leu at position 405,A CD3 antibody is an antibody with a VH region comprising the sequence of SEQ ID NO: 171 (VH YTH12.5) and VL region comprising the sequence of SEQ ID NO:172 (VL YTH12.5). Another example is a CD3 antibody with a VH region comprising the sequence of SEQ ID NO: 173 (VH huCLB-T3/4) and VL region comprising the sequence of SEQ ID NO: 174 (VL huCLB-T3/4).In one embodiment a bispecific antibody of the present invention may be selected from the group consisting of: IgGl ־ 005 ־ ITL x IgGl-169-K409R, IgGl ־ 025 ־ ITL x IgGl-005- K409R, IgGl-025-ITL x IgGl-153-K409R, IgGl-025-ITL x IgGl-169-K409R, IgGl-153-ITL x IgGl0־O5־K4O9R; and IgGl ־ 153 ־ ITL x IgGl ־ 169 ־ K409R, wherein rgGl-005-UL means 005 IgGl,K having He at position 350, Thr at position 370, and Leu at position 405, IgGl-005- K409R means 005IgGl,K having an Arg at position 409, IgGl-025-ITL means 025IgGl,x having lie at position 350, Thr at position 370, and Leu at position 405, IgGl-153־ITL means 153IgGl,K having contains lie at position 350, Thr at position 370, and Leu at position 405, IgGl-153-K409R means 153IgGl,x having an Arg at position 409, IgGl-169- K409R means 169IgGl,K having an Arg at position 409, and wherein the bold numbers refer to antibodies described herein with the VH and VL regions comprising the sequences described in table 1 and for 005in example 21; i.e. SEQ ID NOs;164 and 165.In one embodiment the bispecific antibody may be selected from the group consisting of: IgGl-HER2-153-K409R x IgGl-YTH12.5־F405L, IgGl־HER2-153־K409R x IgGl-YTH12.5־N297Q־F405L, IgGl-HER2-153-K409R x IgGl-hu-CLB־T3/4-F405L, IgGl- PCT/EP2011/058779 WO 2011/147986 HER2-153-K409R x IgGl-hU'-CLB-T3/4־N297Q-F405L, IgGl־HER2 ־ 153 ־ N297Q-K409R x IgGl-YTH12.5-F405L, IgGl־HER2-153-N297Q-K409R x IgGl־YTH12.5-N297Q-F405L, IgGl- HER2-153-N297Q-K409R x IgGl-hu־CLB־T3/4-F405L, IgGl- HER2-153-N297Q-K409R x IgGl-hu־CLB־T3/4-N297Q-F405L, IgGl־HER2-169־K409R x IgGl-hu־CLB״T3/4-F405L, IgGl-HER2169־-K409R x IgGl-hu־CLB-T3/4־N297Q-F405L, IgGl־HER2 ־ 169 ־ K409R x IgGl- YTH12.5-F405L and IgGl־HER2-169־K409R x IgGl-YTH12.5־N297Q-F405L.
Functional properties of Group 1. 2 and 3 antibodies and bispecific antibodies In another aspect of the antibody of the invention, the antibody binds to the same HERepitope as one or more of the novel Group 1, 2 or 3 antibodies described herein, preferably when determined as described in Example 14; and is further characterized by one or more properties determined as described in Examples 12, 13, 15, 16, 17, 18 and 19.
In one embodiment, the HER2 antibody has a lower EC50 value (half maximal effective concentration) than trastuzumab in binding to A431 cells, preferably an EC50 value lower than 0.80 pg/ml, 0.50 pg/ml, or 0.30 ng/ml, when determined as described in Example 12, and preferably binds the same epitope as at least one reference antibody comprising the VH and VL regions selected from the group consisting ofa) a VH region comprising the sequence of SEQ ID NO:l and a VL region comprising the sequence of SEQ ID NO:5 (169);b) a VH region comprising the sequence of SEQ ID NO: 15 and a VL region comprising the sequence of SEQ ID NO: 19 (084);c) a VH region comprising the sequence of SEQ ID NO:22 and a VL region comprising the sequence of SEQ ID NO:26 (025);d) a VH region comprising the sequence of SEQ ID NO: 29 and a VL region comprising the sequence of SEQ ID NO:32 (091);e) a VH region comprising the sequence of SEQ ID NO:46 and a VL region comprising the sequence of SEQ ID NO:49 (127);f) a VH region comprising the sequence of SEQ ID NO:49 and a VL region comprising the sequence of SEQ ID NO:53 (159);g) a VH region comprising the sequence of SEQ ID NO: 56 and a VL region comprising the sequence of SEQ ID NO:60 (098);h) a VH region comprising the sequence of SEQ ID NO:63 and a VL region comprising the sequence of SEQ ID NO:67 (153); andi) a VH region comprising the sequence of SEQ ID NO:70 and a VL region comprising the sequence of SEQ ID NO:74 (132).
PCT/EP2011/058779 WO 2011/147986 In an additional or alternative embodiment, the anti-HER2 antibody specifically binds HER2- positive Rhesus epithelial cells, when determined as described in Example 13, and preferably binds the same epitope as at least one reference antibody comprising the VH and VL regions selected from the group consisting of the VH and VI regions of any of antibodies 169, 050, 084, 025, 091, 129, 127, 159, 098, 153 and 132.
In an additional or alternative embodiment, the anti־HER2 antibody efficiently induces ADCC (antibody-dependent cell-mediated cytotoxicity), preferably achieving a specific 51Cr-reiease of at least 30%, more preferably at least 40%, when determined as described in Example 15, and preferably binds the same epitope as at least one reference antibody comprising the VH and VL regions selected from the group consisting of:a) a VH region comprising the sequence of SEQ ID NO:l and a VL region comprising the sequence of SEQ ID NO:5 (169);b) a VH region comprising the sequence of SEQ ID NO:8 and a VL region comprising the sequence of SEQ ID NO: 12 (050);c) a VH region comprising the sequence of SEQ ID NO: 15 and a VL region comprising the sequence of SEQ ID NO: 19 (084);d) a VH region comprising the sequence of SEQ ID NO: 22 and a VL region comprising the sequence of SEQ ID NO:26 (025);e) a VH region comprising the sequence of SEQ ID NO:29 and a VL region comprising the sequence of SEQ ID NO:32 (091);f) a VH region comprising the sequence of SEQ ID NO: 35 and a VL region comprising the sequence of SEQ ID NO:39 (129); andg) a VH region comprising the sequence of SEQ ID NO:63 an, preferably, a VL region comprising the sequence of SEQ ID NO:67 (153).
In an additional or alternative embodiment, the HER2 antibody specifically binds HER2- expressing AU565 cells but promotes ligand-independent proliferation of the cells less than any of F5 and Cl when determined as described in Example 16, and preferably binds the same epitope as at least one reference antibody comprising the VH and VL regions selected from the group consisting ofa) a VH region comprising the sequence of SEQ ID NO:l and a VL region comprising the sequence of SEQ ID NO: 5 (169);b) a VH region comprising the sequence of SEQ ID NO:8 and a VL region comprising the sequence of SEQ ID NO: 12 (050); PCT/EP2011/058779 WO 2011/147986 c) a VH region comprising the sequence of SEQ ID NO: 15 and a VL region comprising the sequence of SEQ ID NO:19 (084);d) a VH region comprising the sequence of SEQ ID NO:22 and a VL region comprising the sequence of SEQ ID NO:26 (025);e) a VH region comprising the sequence of SEQ ID NO:29 and a VL region comprising the sequence of SEQ ID NO:32 (091);f) a VH region comprising the sequence of SEQ ID NO:35 and a VL region comprising the sequence of SEQ ID NO:39 (129);g) a VH region comprising the sequence of SEQ ID NO:46 and a VL region comprising the sequence of SEQ ID NO:49 (127);h) a VH region comprising the sequence of SEQ ID NO:49 and a VL region comprising the sequence of SEQ ID NO:53 (159);i) a VH region comprising the sequence of SEQ ID NO:56 and a VL region comprising the sequence of SEQ ID NO:60 (098);j) a VH region comprising the sequence of SEQ ID NO: 63 and a VL region comprising the sequence of SEQ ID NO:67 (153); andk) a VH region comprising the sequence of SEQ ID NO:70 and a VL region comprising the sequence of SEQ ID NO:74 (132).
In an additional or alternative embodiment, the HER2 antibody specifically binds HER2- expressing AU565 cells and inhibits ligand-independent proliferation of the cells, preferably inhibiting proliferation by at least 20%, more preferably at least 25%, when determined as described in Example 16, and preferably binds the same epitope as at least one reference antibody comprising the VH and VL regions selected from the group consisting of:a) a VH region comprising the sequence of SEQ ID NO:l and 8 VL region comprising the sequence of SEQ ID NO:5 (169); andb) a VH region comprising the sequence of SEQ ID NO:8 and a VL region comprising the sequence of SEQ ID NG:12 (050).
In an additional or alternative embodiment, the HER2 antibody specifically binds HER2- expressing AU565 ceils but has no significant effect on, or does not promote, ligand-induced proliferation of the cells, preferably inhibiting proliferation by no more than 25%, more preferably by no more than 15%, when determined as described in Example 17, and binds the same epitope as at least one reference antibody comprising the VH and VL regions selected from the group consisting of: PCT/EP2011/058779 WO 2011/147986 a) a VH region comprising the sequence of SEQ ID NO:l and a VL region comprising the sequence of SEQ ID NO:5 (169);b) a VH region comprising the sequence of SEQ ID NO;8 and a VL region comprising the sequence of SEQ ID NO:12 (050);c) a VH region comprising the sequence of SEQ ID NO: 15 and a VL region comprising the sequence of SEQ ID NO:19 (084); andd) a VH region comprising the sequence of SEQ ID NO: 56 and a VL region comprising the sequence of SEQ ID NO:60 (098).
In an additional or alternative embodiment, the HER2 antibody specifically binds HER2- expressing MCF-7 cells and inhibits ligand-induced proliferation, e.g. it may completely inhibit the ligand-induced effect or inhibit the total proliferation by 50%, e.g. 60% or 70% or 80%, of the cells when determined as described in Example 17, and binds the same epitope as at least one reference antibody comprising the VH and VL regions selected from the group consisting of:a) a VH region comprising the sequence of SEQ ID NO: 22 and a VL region comprising the sequence of SEQ ID NO:26 (025);b) a VH region comprising the sequence of SEQ ID NO:29 and a VL region comprising the sequence of SEQ ID NO:32 (091);c) a VH region comprising the sequence of SEQ ID NO:35 and a VL region comprising the sequence of SEQ ID NO:39 (129); andd) a VH region comprising the sequence of SEQ ID NO:63 an, preferably, a VL region comprising the sequence of SEQ ID NO: 67 (153).
In an additional or alternative embodiment, the antibody, when conjugated directly or indirectly to a therapeutic moiety such as a truncated form of the pseudomonas-exotoxin A, is more effective than trastuzumab in killing AU565 cells, A431 cells, or both AU565 and A431 ceils, when determined as described in Example 18.In one embodiment, the conjugated antibody has an ECso value of less than 70 ng/ml, less than 50 ng/ml, or less than 30 ng/ml in killing AU565 cells and/or A431 cells, when determined as described in Example 18, and binds the same epitope as at least one reference antibody comprising the VH and VL regions of an antibody selected from the group consisting of 169, 091, 050, 084, 098, 05, 153, 129, 132, 127 and 159; preferably selected from antibodies 153, 129, 098, 091 and 025.In one embodiment, the conjugated antibody has or results in a higher percentage of killed AU565 cells than trastuzumab and pertuzumab when determined as described in PCT/EP2011/058779 WO 2011/147986 Example 18, preferably killing at least 49%, more preferably at least 60% of the AU5cells, and binds the same epitope as at least one reference antibody comprising the VH and VL regions of an antibody selected from the group consisting of 169, 091, 050, 084, 098, 025, 153, 129, 132, 127 and 159; preferably selected from antibodies 153, 132, 127, 129, 159 and 025.In a preferred embodiment, the conjugated antibody binds to the same epitope as a reference antibody comprising a VH region comprising the sequence of SEQ ID NO:49 and a VL region comprising the sequence of SEQ ID NO: 53 (159).In one embodiment, the conjugated antibody has a higher percentage of killed AU431 cells than trastuzumab and pertuzumab when determined as described in Example 18, preferably killing at least 50%, more preferably at least 70%, and binds the same epitope as at least one reference antibody comprising the VH and VL regions of an antibody selected from the group consisting of 025, 084, 091, 098, 129 and 153; preferably selected from antibodies 025, 091, 098, 129 and 153.In a preferred embodiment, the conjugated antibody binds to the same epitope as a reference antibody comprising a VH region comprising the sequence of SEQ ID NO: 56 and a VL region comprising the sequence of SEQ ID NO:60 (098).
Tn an additional or alternative embodiment, the antibody is internalized by tumor cells expressing HER2, such as AU565 cells, to a higher degree than trastuzumab and pertuzumab, preferably more than twice or three times the amount of internalized trastuzumab, preferably when determined according to Example 18, and binds to the same epitope as an antibody comprising VH and VL regions selected from the group consisting of:a) a VH region comprising the sequence of SEQ ID NO:46 and a VL region comprising the sequence of SEQ ID NO:49 (127);b) a VH region comprising the sequence of SEQ ID NO:49 and a VL region comprising the sequence of SEQ ID NO:53 (159);c) a VH region comprising the sequence of SEQ ID NO:56 and a VL region comprising the sequence of SEQ ID NO:60 (098);d) a VH region comprising the sequence of SEQ ID NO: 63 and a VL region comprising the sequence of SEQ ID NO;67 (153); ande) a VH region comprising the sequence of SEQ ID NO:70 and a VL region comprising the sequence of SEQ ID NO:74 (1 32).Preferably, the antibody binds to the same epitope as an antibody comprising VH and VL regions selected from PCT/EP2011/058779 6 WO 2011/147986 a) a VH region comprising the sequence of SEQ ID NO:46 and a VL region comprising the sequence of SEQ ID NO:49 (127) andb) a VH region comprising the sequence of SEQ ID NO: 56 and a VL region comprising the sequence of SEQ ID NO:60 (098).
In a further embodiment, the antibody binds to Domain II or IV of HER2, preferably wherein the antibody does not significantly promote proliferation of HER2 expressing cells, and is more efficiently internalized, or is internalized to a higher degree, than trastuzumab or pertuzumab into HER2-expressing tumor cells, preferably when determined as described in the Examples, e.g. examples 16 and 19, respectively.
In a further embodiment the antibody enhanced HER2 downmodulation more than trastuzumab, e.g. the antibody enhanced HER2 downmodulation by more 30%, such as more than 40% or more than 50% when determined as described in example 22, preferably wherein the antibody binds to the same epitope as an antibody of cross-block group 3 of the present invention, e.g. an antibody binding to the same epitope as an antibody comprising VH and VL regions selected from the group consisting of:a) a VH region comprising the sequence of SEQ ID NO:56 and a VL region comprising the sequence of SEQ ID NQ:60 (098);b) a VH region comprising the sequence of SEQ ID NO:63 and a VL region comprising the sequence of SEQ ID NO:67 (153).
In another or alternative embodiment the antibody decreased tumour growth and improved survival in vivo more than trastuzumab, when determined as described in example 29, preferably wherein the antibody binds to the same epitope as an antibody of cross-block or cross-block 2 of the present invention, e.g. an antibody binding to the same epitope as an antibody comprising VH and VL regions selected from the group consisting of:a) a VH region comprising the sequence of SEQ ID NO:l and a VL region comprising the sequence of SEQ ID NO: 5 (169);b) a VH region comprising the sequence of SEQ ID NO: 15 and a VL region comprising the sequence of SEQ ID NO: 19 (084); andc) a VH region comprising the sequence of SEQ ID NO: 29 and a VL region comprising the sequence of SEQ ID NO:32 (091).
In another or alternative embodiment the antibody decreased tumour growth and improved survival in vivo more than trastuzumab, when determined as described in example 30, PCT/EP2011/058779 WO 2011/147986 preferably wherein the antibody binds to the same epitope as an antibody of cross-block or cross-block 3 of the present invention, e.g. an antibody binding to the same epitope as an antibody comprising VH and VL regions selected from the group consisting of:a) a VH region comprising the sequence of SEQ ID NO:22 and a VL region comprising the sequence of SEQ ID NO:26 (025);b) a VH region comprising the sequence of SEQ ID NO:29 and a VL region comprising the sequence of SEQ ID NO:32 (091);c) a VH region comprising the sequence of SEQ ID NO:35 and a VL region comprising the sequence of SEQ ID NO:39 (129); andd) a VH region comprising the sequence of SEQ ID NO:63 and a VL region comprising the sequence of SEQ ID NO:67 (153).More particularly, wherein the antibody binds to the same epitope as an antibody comprising VH and VL regions selected from the group consisting of:a) a VH region comprising the sequence of SEQ ID NO: 22 and a VL region comprising the sequence of SEQ ID NO:26 (025); andb) a VH region comprising the sequence of SEQ ED NO:29 and a VL region comprising the sequence of SEQ ID NO:32 (091).
In one embodiment the antibody is a bispecific antibody.In a further embodiment the antibody is a bispecific antibody which enhanced HERdownmodulation, in particular more than their monospecific counterparts, e.g. the antibody enhanced HER2 downmodulation by more 20%, such as more than 30% or more than 40% when determined as described in example 22, preferably wherein the antibody binds to the same epitopes as bispecific antibody selected from the group consisting of IgGl-005-UL x IgGl-169-K409R, IgGl-025-ITL x IgGl-005-K409R, IgGl-025־ITL x IgGl-153־K409R׳ IgGl-025־ITL x IgGl ־ 169 ־ K409R, IgGl-153־ITL x IgGl-0Q5-K409R; and IgGl-153-ITL x IgGl-169-K409R.
In an additional or alternative embodiment, the bispecific antibody specifically binds HER2- expressing AU565 cells and inhibits ligand-induced proliferation of the cells when determined as described in Example 24, and binds the same epitopes as at least one bispecific antibody selected from the group consisting of: IgGl005־-ITL x IgGl-169-K409R, IgGl-025־ITL x IgGl-005-K4Q9R, IgGl-025-ITL x IgGl-153-K409R, lgGl-025-ITL x IgGl- 169-K409R, IgGl153־-ITL x IgGl-0Q5-K409R; and IgGl-153-ITL x lgGl-169-K409R. In particular the bispecific antibody inhibits proliferation of the AU565 cells more than their PCT/EP2011/058779 WO 2011/147986 monospecific counterparts and is selected from the group consisting of IgGl-005-rTL x IgGl־T69־K409R and IgGl-025־ITL x IgGl-005-K409R.
In an additional or alternative embodiment the bispecific antibody is a HER2 x CD3 bispecific antibody induce T ceil mediated cytotoxicity of AU565 as described in example 25, and binds the same epitopes as at least one of the bispecific antibodies seletected from the group consisting of: Duo huCLB~Q/153-Q, Duo huCLB-Q/B12-Q, Duo YTH12.5/153-Q and Duo YTH12.5/B12-Q (Duo indicating bispecific antibody).
Antibody formatsThe present invention provides HER2 antibodies which efficiently bind to and internalize into HER2־expressing tumor cells, typically without significantly promoting ligand-independent proliferation of the cells. Depending on the desired functional properties for a particular use, particular antibodies can be selected from the set of antibodies provided in the present invention and/or their format can be adapted to change these properties, as described below.The antibody of the invention can be of any isotype. The choice of isotype typically will be guided by the desired effector functions, such as ADCC induction. Exemplary isotypes are IgGl, IgG2, IgG3, and IgG4. Either of the human light chain constant regions, kappa or lambda, may be used. If desired, the class of a HER2 antibody of the present invention may be switched by known methods. For example, an antibody of the present invention that was originally IgM may be class switched to an IgG antibody of the present invention. Further, class switching techniques may be used to convert one IgG subclass to another, for instance from IgGl to IgG2. Thus, the effector function of the antibodies of the present invention may be changed by isotype switching to, e.g., an IgGl, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody for various therapeutic uses. In one embodiment an antibody of the present invention is an IgGl antibody, for instance an IgGl,K.In a further embodiment, the antibody of the invention is glyco-engineered to reduce fucose and thus enhance ADCC, e.g. by addition of compounds to the culture media during antibody production as described in US2009317869 or as described in van Berkel et a(. (2010) Biotechnol. Bioeng. 105:350 or by using FUT8 knockout cells, e.g. as described in Yamane-Ohnuki et al (2004) Biotechnol. Bioeng 87:614. ADCC may alternatively be optimized using the method described by Umafia ef al. (1999) Nature Biotech 17:176.In a further embodiment, the antibody of the invention has been engineered to enhance complement activation, e.g. as described in Natsume et al. (2009) Cancer Sci. 100:2411.
PCT/EP2011/058779 WO 2011/147986 In one embodiment, the antibody of the invention is a full-length antibody, preferably an IgGl antibody, in particular an IgGl,K antibody. In another embodiment, the antibody of the invention is an antibody fragment or a single-chain antibody.Antibody fragments may e.g. be obtained by fragmentation using conventional techniques, and the fragments screened for utility in the same manner as described herein for whole antibodies. For example, F(ab')2 fragments may be generated by treating an antibody with pepsin. The resulting F(ab')a fragment may be treated to reduce disulfide bridges with a reducing agent, such as dithiothreitol, to produce Fab' fragments. Fab fragments may be obtained by treating an antibody with papain. A F(ab')2 fragment may also be produced by binding Fab' fragments via a thioether bond or a disulfide bond. Antibody fragments may also be generated by expression of nucleic adds encoding such fragments in recombinant cells (see for instance Evans ef a/., J. Immunol. Meth. 184. 123-38 (1995)). For example, a chimeric gene encoding a portion of an F(ab')2 fragment could include DNA sequences encoding the CH1 domain and hinge region of the H chain, followed by a translational stop codon to yield such a truncated antibody fragment molecule.As explained above, in one embodiment, the HER2 antibody of the invention is a bivalent antibody, i.e. an antibody capable of binding two antigens or epitopes on the same antigen.In another embodiment, the HER2 antibody of the invention is a monovalent antibody.In one embodiment, the antibody of the invention is a Fab fragment or a one-armed antibody, such as described in US20080063641 (Genentech) or other monovalent antibody, e.g. such as described in W02007048037 (Amgen).In a preferred embodiment, a monovalent antibody has a structure as described in W02007059782 (Genmab) (incorporated herein by reference) having a deletion of the hinge region. Accordingly, in one embodiment, the antibody is a monovalent antibody, wherein said HER2 antibody is constructed by a method comprising:i) providing a nucleic acid construct encoding the light chain of said monovalent antibody, said construct comprising a nucleotide sequence encoding the VL region of a selected antigen specific HER2 antibody and a nucleotide sequence encoding the constant CL region of an Ig, wherein said nucleotide sequence encoding the VL region of a selected antigen specific antibody and said nucleotide sequence encoding the CL region of an Ig are operably finked together, and wherein, in case of an IgGl subtype, the nucleotide sequence encoding the CL region has been modified such that the CL region does not contain any amino adds capable of forming disulfide bonds or covalent bonds with other peptides PCT/EP2011/058779 WO 2011/147986 comprising an identical amino acid sequence of the CL region in the presence of polyclonal human IgG or when administered to an animal or human being;ii) providing a nucleic add construct encoding the heavy chain of said monovalent antibody, said construct comprising a nucleotide sequence encoding the VH region of a selected antigen specific antibody and a nucleotide sequence encoding a constant CH region of a human Ig, wherein the nucleotide sequence encoding the CH region has been modified such that the region corresponding to the hinge region and, as required by the Ig subtype, other regions of the CH region, such as the CH3 region, does not comprise any amino acid residues which participate in the formation of disulphide bonds or covalent or stable non-covalent inter-heavy chain bonds with other peptides comprising an identical amino add sequence of the CH region of the human Ig in the presence of polyclonal human IgG or when administered to an animal human being, wherein said nucleotide sequence encoding the VH region of a selected antigen specific antibody and said nucleotide sequence encoding the CH region of said Ig are operably linked together;iii) providing a cel! expression system for producing said monovalent antibody;iv) producing said monovalent antibody by co-expressing the nucleic acid constructs of (i) and (ii) in cells of the cell expression system of (iii).Similarly, in one embodiment, the HER2 antibody is a monovalent antibody, which comprises(i) a variable region of an antibody of the invention as described herein or an antigen binding part of the said region, and(ii) a CH region of an immunoglobulin or a fragment thereof comprising the CHand Ch3 regions, wherein the CH region or fragment thereof has been modified such that the region corresponding to the hinge region and, if the immunoglobulin is not an IgG4 subtype, other regions of the CH region, such as the CH3 region, do not comprise any amino acid residues, which are capable of forming disulfide bonds with an identical CH region or other covalent or stable non-covalent inter-heavy chain bonds with an identical CH region in the presence of polyclonal human IgG.In a further embodiment hereof, the heavy chain of the monovalent HER2 antibody has been modified such that the entire hinge has been deleted.In another further embodiment, the immunoglobulin referred to in step ii) above is of the IgG4 subtype.In another further embodiment, said monovalent antibody is of the IgG4 subtype, but the Ch3 region has been modified so that one or more of the following amino acid substitutions have been made; PCT/EP2011/058779 WO 20H/147986 Numbering of CH3 mutations KABAT* EU index G4* Mutations E378 E357 E357A or E357T or E357V or E357IS387 S364 S364R or S364KT389 T366 T366A or T366R or T366K or T366NL391 L368 L368A or L368V or L368E or L368G or L368S or L368TD427 D399 D399A or D399T or D399SF405A or F405L or F405T or F405D or F405R or F405QF436 F405 or F405K or F405YY438 Y407 Y407A or Y407E or Y407Q or Y407K or Y407FF436 and Y438 F405 and Y407 (F405T and Y407E) or (F405D and Y407E)(D399S and Y407Q) or (D399S and Y407K) or (D399SD427 and Y438 D399 and Y407 and Y407E)*KABAT indicates amino acid numbering according to Kabat (Kabat et al., Sequences of Proteins of immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991). EU index indicates amino acid numbering according to EUindex as outlined in Kabat et al., (supra).In another further embodiment/ the sequence of said monovalent antibody has been modified so that it does not comprise any acceptor sites for N-linked giycosylation.
HER2 antibodies of the invention also include single chain antibodies. Single chain antibodies are peptides in which the heavy and light chain Fv regions are connected. In one embodiment/ the present invention provides a single-chain Fv (scFv) wherein the heavy and light chains in the Fv of a HER2 antibody of the present invention are joined with a flexible peptide linker (typically of about 10, 12, 15 or more amino acid residues) in a single peptide chain. Methods of producing such antibodies are described in for instance US 4,946,778, Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994), Bird et a!.r Science 242. 423426־ (1988), Huston et at., PNAS USA 5879-5883 (1988) and McCafferty et atNature 348. 552-554 (1990). The single chain antibody may be monovalent, if only a single VH and VL are used, bivalent, if two VH and VL are used, or polyvalent, if more than two VH and VL are used.In one embodiment, the HER2 antibody of the invention is an effector-function- deficient antibody. In one embodiment, the effector-functian-deficient HER2 antibody is a human stabilized IgG4 antibody, which has been modified to prevent Fab-arm exchange (van der Neut Kolfschoten et al. (2007) Science 317(5844):15547־). Examples of suitable45 PCT/EP2011/058779 WO 2011/147986 human stabilized IgG4 antibodies are antibodies, wherein arginine at position 409 in a heavy chain constant region of human IgG4, which is indicated in the EU index as in Kabat et ah, is substituted with lysine, threonine, methionine, or leucine, preferably lysine (described in W02006033386 (Kirin)) and/or wherein the hinge region has been modified to comprise a Cys-Pro-Pro-Cys sequence.In one embodiment, the stabilized IgG4 HER2 antibody is an IgG4 antibody comprising a heavy chain and a tight chain, wherein said heavy chain comprises a human IgG4 constant region having a residue selected from the group consisting of: Lys, Ala, Thr, Met and Leu at the position corresponding to 409 and/or a residue selected from the group consisting of: Ala, Val, Gly, He and Leu at the position corresponding to 405, and wherein said antibody optionally comprises one or more further substitutions, deletions and/or insertions, but does not comprise a Cys-Pro-Pro-Cys sequence in the hinge region. Preferably, said antibody comprises a Lys or Aia residue at the position corresponding to 409 or the CH3 region of the antibody has been replaced by the CH3 region of human IgGl, of human IgG2 or of human IgG3. See also WO2008145142 (Genmab).In an even further embodiment, the stabilized IgG4 HER2 antibody is an IgGantibody comprising a heavy chain and a light chain, wherein said heavy chain comprises a human IgG4 constant region having a residue selected from the group consisting of: Lys, Ala, Thr, Met and Leu at the position corresponding to 409 and/or a residue selected from the group consisting of: Ala, Val, Gly, He and Leu at the position corresponding to 405, and wherein said antibody optionally comprises one or more further substitutions, deletions and/or insertions and wherein said antibody comprises a Cys-Pro-Pro-Cys sequence in the hinge region. Preferably, said antibody comprises a Lys or Ala residue at the position corresponding to 409 or the CH3 region of the antibody has been replaced by the CHregion of human IgGl, of human IgG2 or of human IgG3.In a further embodiment, the effector-function־deficient HER2 antibody is an antibody of a non-IgG4 type, e.g. IgGl, IgG2 or IgG3 which has been mutated such that the ability to mediate effector functions, such as ADCC, has been reduced or even eliminated. Such mutations have e.g. been described in Dall'Acqua WP e! a!., J Immunol. 122(2): 1129-1138 (2006) and Hezareh M, J Virol. ;25(24): 121612001) 12168־).
ConjugatesIn a further embodiment, the present invention provides a HER2 antibody or an HER2 bispecific antibody linked or conjugated to a therapeutic moiety, such as a cytotoxin, a chemotherapeutic drug, a cytokine, an immunosuppressant, or a radioisotope. Such PCT/EP2011/058779 WO 2011/147986 conjugates are referred to herein as "immunocanjugates". rmmunoconjugates which include one or more cytatoxins are referred to as "immunotoxins".A cytotoxin or cytotoxic agent includes any agent that is detrimental to (e.g.f kills) cells. Suitable therapeutic agents for forming immunoconjugates of the present invention include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, maytansine or an analog or derivative thereof, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin,; calicheamicin or analogs or derivatives thereof; antimetabolites (such as methotrexate, 6־mercaptopurine, 6-thioguanine, cytarabine, fludarabin, 5־f!uorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, cladribine), alkylating agents (such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin and other platinum derivatives, such as carboplatin; as well as duocarmycin A, duocarmycin SA, CC-10(a.k.a. rachelmycin), or analogs or derivatives of CC-1065), antibiotics (such as dactinomycin (formerly actinomycin), bleomycin, daunorubicin (formerly daunomycin), doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC)), anti-mitotic agents (e.g., tubulin-inhibitors) such as monomethyl auristatin E, monomethyl auristatin F, or other analogs or derivatives of dolastatin 10; diphtheria toxin and related molecules (such as diphtheria A chain and active fragments thereof and hybrid molecules); ricin toxin (such as ricin A or a deglycosylated ricin A chain toxin), cholera toxin, a Shiga-like toxin (SLT-I, SLT-II, SLT-IIV), LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman-Btrk protease inhibitor, Pseudomonas exotoxin, alorin, saporin, modeccin, gelanin, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, and enomycin toxins. Other suitable conjugated molecules include antimicrobial/lytic peptides such as CLIP, Magainin 2, mellitin, Cecropin, and P18; ribonuclease (RNase), DNase I, Staphylococcal enterotaxin-A, pokeweed antiviral protein, diphtherin toxin, and Pseudomonas endotoxin. See, for example, Pastan et a/., Cell 47. 641 (1986) and Goldenberg, Calif. A Cancer Journal for Clinicians 441994) 43 ׳). Therapeutic agents that may be administered in combination with a HER2 antibody of the present invention as described elsewhere herein, such as, e.g., anti-cancer cytokines or chemokines, are also candidates for therapeutic moieties useful for conjugation to an antibody of the present invention.
PCT/EP2011/058779 WO 2011/147986 In one embodiment, a HER2 antibody of the invention comprises a conjugated nucleic acid or nucleic acid-associated molecule. In one such embodiment, the conjugated nucleic add is a cytotoxic ribonuclease, an antisense nucleic acid, an inhibitory RNA molecule (e.g., a si RNA molecule) or an immunostimulatory nucleic acid (e.g.r an immunostimulatory CpG motif-containing DNA molecule). In another embodiment, a HERantibody of the invention is conjugated to an aptamer or a ribozyme.In one embodiment, HER2 antibodies comprising one or more radiolabeled amino acids are provided. A radiolabeled HER2 antibody may be used for both diagnostic and therapeutic purposes (conjugation to radiolabeled molecules is another possible feature). Non-limiting examples of labels for polypeptides include 3H, 14C, 15N, 35S, 90Y, 99Tc, and 1251, 1311, and 186Re.In one embodiment, the antibody is conjugated to a radioisotope or to a radioisotope-containing chelate. For example, the antibody can be conjugated to a chelator linker, e.g. DOTA, DTPA or tiuxetan, which allows for the antibody to be complexed with a radioisotope. The antibody may also or alternatively comprise or be conjugated to one or more radiolabeled amino acids or other radiolabeled molecule. A radiolabeled CD74 Ab may be used for both diagnostic and therapeutic purposes. Non-limiting examples of radioisotopes include 3H, 14C, 15N, 35S, 90Y, ״Tc, i2sI, 1uln, 131I, 186Re, 213Bs, 225Ac and 227Th.HER2 antibodies may also be chemically modified by covalent conjugation to a polymer to for instance increase their circulating half-life. Exemplary polymers, and methods to attach them to peptides, are illustrated in for instance US 4,766,106, US 4,179,337, US 4,495,285 and US 4,609,546. Additional polymers include polyoxyethylated polyols and polyethylene glycol (PEG) (e.g., a PEG with a molecular weight of between about 1,000 and about 40,000, such as between about 2,000 and about 20,000).Any method known in the art for conjugating the HER2 antibody to the conjugated molecule(s), such as those described above, may be employed, including the methods described by Hunter ef a/., Nature 144. 945 (1962), David ef a/., Biochemistry 12, 10(1974), Pain et a(., 3. Immunol. Meth. 40, 219 (1981) and Nygren, J. Histochem. and Cytochem. 20, 407 (1982). Such antibodies may be produced by chemically conjugating the other moiety to the N־terminal side or C-terminai side of the HER2 antibody or fragment thereof (e.g., a HER2 antibody H or L chain) (see, e.g., Antibody Engineering Handbook, edited by Osamu Kanemitsu, published by Chijin Shokan (1994)). Such conjugated antibody derivatives may also be generated by conjugation at internal residues or sugars, where appropriate.The agents may be coupled either directly or indirectly to a HER2 antibody of the present invention. One example of indirect coupling of a second agent is coupling via a PCT/EP2011/058779 WO 2011/147986 spacer moiety to cysteine or lysine residues in the antibody. In one embodiment, a HERantibody is conjugated to a prodrug molecule that can be activated in vivo to a therapeutic drug via a spacer or linker. After administration, the spacers or linkers are cleaved by tumor-ceil associated enzymes or other tumor-specific conditions, by which the active drug is formed. Examples of such prodrug techologies and linkers are described in W002083180, W02004043493, W020Q7018431, WO2007089149, and W02009017394 by Syntarga BV, et ai. Suitable antibody-prod rug technology and duocarmycin analogs can also be found in U.S. Patent No. 6,989,452 (Medarex).In one embodiment, the HER2 antibody of the present invention is attached to a chelator linker, e.g. tiuxetan, which allows for the antibody to be conjugated to a radioisotope.
Bisoecific antibodiesIn a further aspect, the invention relates to a bispecific molecule comprising a first antigen binding site from a HER2 antibody of the invention as described herein above and a second antigen binding site with a different binding specificity, such as a binding specificity for a human effector cell, a human Fc receptor, a T cell receptor, a B cell receptor or a binding specificity for a non-overlapping epitope of HER2, i.e. a bispecific antibody wherein the first and second antigen binding sites do not cross-block each other for binding to HER2, e.g, when tested as described in Example 14.Exemplary bispecific antibody molecules of the invention comprise (i) two antibodies, one with a specificity to HER2 and another to a second target that are conjugated together, (ii) a single antibody that has one chain or arm specific to HER2 and a second chain or arm specific to a second molecule, (iii) a single chain antibody that has specificity to HER2 and a second molecule, e.g., via two scFvs linked in tandem by an extra peptide linker; (iv) a dual-variable-domain antibody (DVD-Ig), where each light chain and heavy chain contains two variable domains in tandem through a short peptide linkage (Wu et ai, Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Igw) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg (2010)); (v) a chemically-linked bispecific (Fab2(׳ fragment; (vi) a Tandab, which is a fusion of two single chain diabodies resulting in a tetravalent bispecific antibody that has two binding sites for each of the target antigens; (vii) a flexibody, which is a combination of scFvs with a diabody resulting in a multivalent molecule; (viii) a so called "dock and lock" molecule, based on the "dimerization and docking domain״ in Protein Kinase A, which, when applied to Fabs, can yield a trivalent bispecific binding protein consisting of two identical Fab fragments linked to a different Fab fragment; (ix) a so-called Scorpion molecule, comprising, e.g., two scFvs fused to both termini of a human Fc-region; and (x) a diabody. In one embodiment, the bispecific PCT/EP2011/058779 WO 2011/147986 antibody of the present invention is a diabody, a cross-body, or a bispecific obtained via a controlled Fab arm exchange as those described in the present invention.Examples of platforms useful for preparing bispecific antibodies include but are not limited to BiTE (Micromet), DART (MacroGenics), Fcab and Mab2 (F-star) , Fc-engineered IgGl (Xencor) or DuoBody (based on Fab arm exchange, Genmab, this application).Examples of different classes of bispecific antibodies include but are not limited to• asymmetric IgG-like molecules, wherein the one side of the molecule contains the Fab region or part of the Fab region of at least one antibody, and the other side of the molecule contains the Fab region or parts of the Fab region of at least one other antibody; in this class, asymmetry in the Fc region could also be present, and be used for specific linkage of the two parts of the molecule;• symmetric IgG-like molecules, wherein the two sides of the molecule each contain the Fab region or part of the Fab region of at least two different antibodies;• IgG fusion molecules, wherein full length IgG antibodies are fused to extra Fab regions or parts of Fab regions;• Fc fusion molecules, wherein single chain Fv molecules or stabilized diabodies are fused to Fey regions or parts thereof,;• Fab fusion molecules, wherein different Fab-fragments are fused together;• ScFv-and diabody-based molecules wherein different single chain Fv molecules or different diabodies are fused to eachother or to another protein or carrier molecule.Examples of asymmetric IgG-like molecules include but are not limited to the Triomab/Quadroma (Trion Pharma/Fresenius Biotech), the Knobs-into-Holes (Genentech), CrossMAbs (Roche) and the electrostatically-matched (Amgen), the LUZ-Y (Genentech), the Strand Exchange Engineered Domain body (EMD Serono), the Biclonic (Merus) and the DuoBody (Genmab A/S).Example of symmetric IgG-like molecules include but are not limited to Dual Targeting (DT)-Ig (GSK/Domantis), Two-in-one Antibody (Genentech), Cross-linked Mabs (Karmanos Cancer Center), mAb2 (F-Star) and CovX-body (CovX/Pfizer).Examples of IgG fusion molecules include but are not limited to Dual Variable Domain (DVD)-Ig (Abbott), IgG-like Bispecific (ImCIone/Eli Lilly), Ts2Ab (Medlmmune/AZ) and BsAb (Zymogenetics), HERCULES (Biogen Idee) and TvAb (Roche).Examples of Fc fusion molecules include but are not limited to ScFv/Fc Fusions (Academic Institution), SCORPION (Emergent BioSolutions/Trubion, Zymogenetics/BMS), Dual Affinity Retargeting Technology (Fc־DART) (MacroGenics) and Dual(ScFv)2-Fab (National Research Center for Antibody Medicine - China).
PCT/EP2011/058779 WO 2011/147986 Examples of class V bispecific antibodies include but are not limited to F(ab)(Medarex/AMGEN), Dual-Action or Bis-Fab (Genentech), Dock-and-Lock (DNL) (ImmunoMedics), Bivalent Bispecific (Biotecnol) and Fab-Fv (UCB-Celltech).Examples of ScFv-and diabody-based molecules include but are not limited to Bispecific T Cell Engager (BiTE) (Micromet9, Tandem Diabody (Tandab) (Affimed), Dual Affinity Retargeting Technology (DART) (MacroGenics), Single-chain Diabody (Academic), TCR-like Antibodies (AIT, ReceptorLogics), Human Serum Albumin ScFv Fusion (Merrimack) and COMBODY (Epigen Biotech).In one embodiment, the second molecule is a cancer antigen/tumor-associated antigen such as carcinoembryonic antigen (CEA), prostate specific antigen (PSA), RAGE (renal antigen), a-fetoprotein, CAMEL (CTL-recognized antigen on melanoma), CT antigens (such as MAGE-B5, -B6, -C2, -C3, and D; Mage-12; CT10; NY-ESO-1, SSX-2, GAGE, BAGE, MAGE, and SAGE), mucin antigens (e.g., MUC1, mucin-CA125, etc.), ganglioside antigens, tyrosinase, gp75, c־Met, C-myc, Marti, MelanA, MUM-1, MUM-2, MUM-3, HLA-B7, Ep-CAM or a cancer-associated integrin, such as a5p3 integrin. In another embodiment, the second molecule is a T cell and/or NK cell antigen, such as CD3 or CD16. In another embodiment, the second molecule is an angiogenic factor or other cancer-associated growth factor, such as a vascular endothelial growth factor, a fibroblast growth factor, epidermal growth factor, angiogenin or a receptor of any of these, particularly receptors associated with cancer progression (for instance another one of the HER receptors; HER1, HER3, or HER4). In one embodiment, the second antigen-binding site binds a different, preferably non-blocking, site on HER2 than the one bound by the antibody of the invention. For example, the second molecule may be derived from, or cross-block HER2-binding of, trastuzumab, pertuzumab, F5, or Cl.Methods of preparing bispecific antibodies include those described in WO 2008119353 (Genmab) and reported van der Neut-Kolfschoten et al. (Science. 2007 Sep 14;317(5844):1554-7) and it may for example be performed as described in example 20 of the present invention.
Nucleic add sequences, vectors and host cellsIn a further aspect, the invention relates to nucleic acid sequences, such as DNA sequences, encoding heavy and light chains of an antibody of the invention.In one embodiment, the nucleic acid sequence encodes an amino acid sequence selected from the group consisting of: SEQ ID NO: 1, 5, 8, 12, 15, 19, 22, 26, 29, 32, 35, 39, 42, 46, 49, 53, 56, 6Q88 ,87 ,86 ,85 ,84 ,83 ,82 ,81 ,80 ,79 ,78 ,77 ,74 ,70 ,67 ,63 ״, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, PCT/EP2011/058779 WO 2011/147986 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125 and 126.In another particular embodiment, the nucleic acid sequence encodes a VH amino add sequence selected from the group consisting of: SEQ ID NO: 1, 8, 15, 22, 29, 35, 42, 49, 56, 63, 70, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,111, 113, 115, 117, 119, 121, 123, and 125.In another particular embodiment, the nucleic acid sequence encodes a VL amino acid sequence selected from the group consisting of: SEQ ID NO: 5, 12, 19, 26, 32, 39, 46, 53, 60, 67, 74, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110,112, 114, 116, 118, 120, 122, 124, and 126.In an even further aspect, the invention refates to an expression vector, or a set of expression vectors, encoding an antibody of the invention. The heavy and light chain of the antibody may be encoded by the same vector or by different vector.Such expression vectors may be used for recombinant production of antibodies of the invention.In one embodiment, the expression vector of the invention comprises a nucleotide sequence encoding one or more of the amino acid sequences selected from the group consisting of: SEQ ID NO: 1, 5, 8, 12, 15, 19, 22, 26, 29, 32, 35, 39, 42, 46, 49, 53, 56, 60,, 63, 67, 70, 74, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125 and 126.In another particular embodiment, the expression vector of the invention comprises a nucleotide sequence encoding one or more of the VH amino acid sequences selected from the group consisting of: SEQ ID NO: 1, 8, 15, 22, 29, 35, 42, 49, 56, 63, 70, 77, 79, 81,83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, and 125.In another particular embodiment, the expression vector of the invention comprises a nucleotide sequence encoding one or more of the VL amino acid sequences selected from the group consisting of: SEQ ID NO: 5, 12, 19, 26, 32, 39, 46, 53, 60, 67, 74, 78, 80, 82,84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, and 126.In a further embodiment, the expression vector further comprises a nucleotide sequence encoding the constant region of a light chain, a heavy chain or both light and heavy chains of an antibody, e.g. a human antibody.An expression vector in the context of the present invention may be any suitable vector, including chromosomal, non-chromosomal, and synthetic nucleic acid vectors (a PCT/EP2011/058779 WO 2011/147986 nucleic acid sequence comprising a suitable set of expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, a HER2 antibody-encoding nucleic acid is comprised in a naked DNA or RNA vector, including, for example, a linear expression element (as described in for instance Sykes and Johnston, Nat Biotech 17, 355-59 (1997)), a compacted nucteic acid vector (as described in for instance US 6,077, 835 and/or WO 00/70087), a plasmid vector such as pBR322, pUC 19/18, or pUC 118/119, a "midge" minimally-sized nucleic acid vector (as described in for instance Schakowski et al., Mo! Ther 3, 793-800 (2001)), or as a precipitated nucleic acid vector construct, such as a CaP04- precipitated construct (as described in for instance WO 00/46147, Benvenisty and Reshef, PNAS USA 83, 9551-55 (1986), Wigler et al., Cel! 14, 725 (1978), and Coraro and Pearson, Somatic Cell Genetics 7, 603 (1981)). Such nucleic acid vectors and the usage thereof are well known in the art (see for instance US 5,589,466 and US 5,973,972).Exemplary expression vectors for the antibodies of the invention are also described in Examples 2 and 3.In one embodiment, the vector is suitable for expression of the HER2 antibody in a bacterial cell. Examples of such vectors include expression vectors such as BlueScript (Stratagene), pIN vectors (Van Heeke & Schuster, 1 Biol Chem 264, 5503-5509 (1989), pET vectors (Novagen, Madison WI) and the like).An expression vector may also or alternatively be a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system may be employed. Suitable vectors include, for example, vectors comprising constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH (reviewed in: F. Ausubel et ai.r ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience New York (1987), and Grant et al., Methods in Enzymol 153. 516-544 (1987)).An expression vector may also or alternatively be a vector suitable for expression in mammalian cells, e.g. a vector comprising glutamine synthetase as a selectable marker, such as the vectors described in Bebbington (1992) Biotechnology (NY) 10:169175־.A nucleic acid and/or vector may also comprises a nucleic acid sequence encoding a secretion/localization sequence, which can target a polypeptide, such as a nascent polypeptide chain, to the periplasmic space or into cell culture media. Such sequences are known in the art, and include secretion leader or signal peptides.In an expression vector of the invention, HER2 antibody-encoding nucleic acids may comprise or be associated with any suitable promoter, enhancer, and other expression- facilitating elements. Examples of such elements include strong expression promoters (e. g., PCT/EP2011/058779 WO 2011/147986 human CMV IE promoter/enhancer as well as RSV, SV40, SL3-3, MMTV, and HIV LTR promoters), effective poly (A) termination sequences, an origin of replication for plasmid product in E. coJi, an antibiotic resistance gene as selectable marker, and/or a convenient cloning site (e.g.f a polylinker). Nucleic adds may also comprise an inducible promoter as opposed to a constitutive promoter such as CMV IE.In one embodiment, the HER2 antibody-encoding expression vector may be positioned in and/or delivered to the host cell or host animal via a viral vector.In an even further aspect, the invention relates to a recombinant eukaryotic or prokaryotic host ceil, such as a transfectoma, which produces an antibody of the invention as defined herein. Examples of host cells include yeast, bacterial, and mammalian ceils, such as CHO or HEK ceils. For example, in one embodiment, the present invention provides a cell comprising a nucleic acid stably integrated into the cellular genome that comprises a sequence coding for expression of a HER2 antibody of the present invention. In another embodiment, the present invention provides a cell comprising a non-integrated nucleic acid, such as a plasmid, cosmid, phagemid, or linear expression element, which comprises a sequence coding for expression of a HER2 antibody of the invention.In a further aspect, the invention relates to a hybridoma which produces an antibody of the invention as defined herein. In an even further aspect, the invention relates to a transgenic non-human animal or piant comprising nucleic acids encoding a human heavy chain and a human light chain, wherein the animal or piant produces an antibody of the invention of the invention.In a further aspect, the invention relates to a method for producing a HER2 antibody of the invention, said method comprising the steps ofa) culturing a hybridoma or a host cell of the invention as described herein above, andb) purifying the antibody of the invention from the culture media.
CompositionsIn a further main aspect, the invention relates to a pharmaceutical composition comprising:- a HER2 antibody as defined herein, and- a pharmaceutically-acceptabie carrier.The pharmaceutical composition of the present invention may contain one antibody of the present invention or a combination of different antibodies of the present invention.The pharmaceutical compositions may be formulated in accordance with conventional techniques such as those disclosed in Remington: The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 1995. A pharmaceutical PCT/EP2011/058779 WO 2011/147986 composition of the present invention may e.g. include diluents, fillers, salts, buffers, detergents (e, g., a nonionic detergent, such as Tween-20 or Tween-80), stabilizers (e. g., sugars or protein-free amino acids), preservatives, tissue fixatives, solubilizers, and/or other materials suitable for inclusion in a pharmaceutical composition.Pharmaceutically acceptable carriers include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity agents, antioxidants and absorption delaying agents, and the like that are physiologically compatible with a compound of the present invention. Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the present invention include water, saline, phosphate buffered saline, ethanol, dextrose, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, carboxymethyl cellulose colloidal solutions, tragacanth gum and injectable organic esters, such as ethyl oleate, and/or various buffers. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. Proper fluidity may 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.Pharmaceutical compositions of the present invention may also comprise pharmaceutically acceptable antioxidants for instance (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium roetabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, lecithin, propyl galiate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.Pharmaceutical compositions of the present invention may also comprise isotonicity agents, such as sugars, pofyalcohols, such as mannitol, sorbitol, glycerol or sodium chloride in the compositions.The pharmaceutical compositions of the present invention may also contain one or more adjuvants appropriate for the chosen route of administration such as preservatives, wetting agents, emulsifying agents, dispersing agents, preservatives or buffers, which may enhance the shelf life or effectiveness of the pharmaceutical composition. The compounds of the present invention may be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Such carriers may include gelatin, glyceryl monostearate, glyceryl distearate, biodegradable, biocompatrble polymers such as PCT/EP2011/058779 WO 2011/147986 ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and poiylactic acid alone or with a wax, or other materials well known in the art, Methods for the preparation of such formulations are generally known to those skilled in the art.Sterile injectable solutions may be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients e.g. as enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients e.g. from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, examples of methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.The actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the pa bent. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions 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 pharmaceutical composition may be administered by any suitable route and mode. In one embodiment, a pharmaceutical composition of the present invention is administered parenterally. "Administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and include epidermal, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbitai, intracardiac, intradermal, intraperitoneaf, intratendinous, transtracheal, subcutaneous, subcuticular, intraartccular, subcapsular, subarachnoid, intraspinal, intracranial, intrathoracic, epidural and intrasternal injection and infusion.In one embodiment that pharmaceutical composition is administered by intravenous or subcutaneous injection or infusion.
Uses PCT/EP20X1/058779 WO 2011/147986 In a further main aspect, the invention relates to a HER2 antibody׳ of the invention for use as a medicament.The HER2 antibodies of the invention may be used for a number of purposes. In particular, the antibodies of the invention may be used for the treatment of various forms of cancer, including metastatic cancer and refractory cancer.In one embodiment, the HER2 antibodies of the invention are used for the treatment of breast cancer, including primary, metastatic, and refractory breast cancer.In one embodiment, the HER2 antibodies of the invention are used for die treatment of a form of cancer selected from the group consisting of prostate cancer, non-small cell lung cancer, bladder cancer, ovarian cancer, gastric cancer, colorectal cancer, esophageal cancer, squamous cell carcinoma of the head & neck, cervical cancer, pancreatic cancer, testis cancer, malignant melanoma and a soft-tissue cancer (e.g. synovial sarcoma).Similarly, the invention relates to a method for killing a tumor cell expressing HER2, comprising administration, to an individual in need thereof, of an effective amount of an antibody of the invention, such as an antibody drug-conjugate (ADC).In one embodiment, said tumor cel! is involved in a form of cancer selected from the group consisting of; breast cancer, prostate cancer, non-small cell lung cancer, bladder cancer, ovarian cancer, gastric cancer, colorectal cancer, esophageal cancer and squamous cell carcinoma of the head & neck, cervical cancer, pancreatic cancer, testis cancer, malignant melanoma, and a soft-tissue cancer (e.g., synovial sarcoma).In one embodiment, the tumor cell is one that co-expresses HER2 and at least one other member of the EGFR family, preferably EGFR, HER3, or both of EGFR and HER3, and is a tumor cell involved in breast cancer, colorectal cancer, endometrial/cervical cancer, lung cancer, malignant melanoma, ovarian cancer, pancreatic cancer, prostate cancer, testis cancer, a soft-tissue tumor (e.g., synovial sarcoma), or bladder cancer.In one aspect, the invention relates to a method for treating cancer in a subject, comprising selecting a subject suffering from a cancer comprising tumor cells co-expressing HER2 and EGFR and/or HER3, and administering to the subject an antibody of the invention, optionally in the form of an antibody conjugated to a cytotoxic agent or drug. In one embodiment, the subject suffers from a cancer selected from the group consisting of breast cancer, colorectal cancer, endometrial/cervical cancer, lung cancer, malignant melanoma, ovarian cancer, pancreatic cancer, prostate cancer, testis cancer, a soft-tissue tumor (e.g., synovial sarcoma), or bladder cancer.Also, the invention relates to the use of a monoclonal antibody that binds to human HER2 for the preparation of a medicament for the treatment of cancer, such as one of the specific cancer indications mentioned above.
PCT/EP2011/058779 WO 2011/147980 The invention further relates to a monoclonal antibody for use in the treatment of cancer, such as one of the cancer indications mentioned above.In a further embodiment of the methods of treatment of the present invention, the efficacy of the treatment is being monitored during the therapy, e.g. at predefined points in time, by determining tumor burden or HER2 expression levels on the relevant tumor cells.Dosage regimens in the above methods of treatment and uses are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. Parenteral compositions may be formulated in dosage unit form for ease of administration and uniformity of dosage.The efficient dosages and the dosage regimens for the HER2 antibodies depend on the disease or condition to be treated and may be determined by the persons skilled in the art. An exemplary, non-limiting range for a therapeutically effective amount of a compound of the present invention is about 0.1-100 mg/kg, such as about 0.1-50 mg/kg, for example about 0.1-20 mg/kg, such as about 0.1-10 mg/kg, for instance about 0.5, about such as 0.3, about l, about 3, about 5, or about 8 mg/kg.A physician or veterinarian having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the HER2 antibody employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable daily dose of a composition of the present invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Administration may e.g. be parenteral, such as intravenous, intramuscular or subcutaneous. In one embodiment, the HER2 antibodies may be administered by infusion in a weekly dosage of from 10 to 500 mg/m2, such as of from 200 to 400 mg/m2. Such administration may be repeated, e.g,, 1 to 8 times, such as 3 to 5 times. The administration may be performed by continuous infusion over a period of from 2 to 24 hours, such as of from 2 to 12 hours. In one embodiment, the HER2 antibodies may be administered by slow continuous infusion over a long period, such as more than 24 hours, in order to reduce toxic side effects.In one embodiment the HER2 antibodies may be administered in a weekly dosage of from 250 mg to 2000 mg, such as for example 300 mg, 500 mg, 700 mg, 1000 mg, 15mg or 2000 mg, for up to 8 times, such as from 4 to 6 times when given once a week. Such regimen may be repeated one or more times as necessary, for example, after 6 months or PCT/EP2011/058779 WO 2011/147986 12 months. The dosage may be determined or adjusted by measuring the amount of compound of the present invention in the blood upon administration by for instance taking out a biological sampie and using anti-idiotypic antibodies which target the antigen binding region of the HER2 antibodies of the present invention.The efficient dosages and the dosage regimens for the bispecific antibodies depend on the disease or condition to be treated and may be determined by the persons skilled in the art. An exemplary, non-limiting range for a therapeutically effective amount of a bispecific antibody of the present invention is about 0.1-100 mg/kg, such as about 0.1-mg/kg, for example about 0.1-20 mg/kg, such as about 0.1-10 mg/kg, for instance about 0.5, about such as 0.3, about 1, about 3, about 5, or about 8 mg/kg.In one embodiment, the HER2 antibodies may be administered by maintenance therapy, such as, e.g.f once a week for a period of 6 months or more.A HER2 antibody may also be administered prophyiacticaily in order to reduce the risk of developing cancer, delay the onset of the occurrence of an event in cancer progression, and/or reduce the risk of recurrence when a cancer is in remission.
HER2 antibodies may also be administered in combination therapy, i.e., combined with other therapeutic agents relevant for the disease or condition to be treated. Accordingly, in one embodiment, the antibody-containing medicament is for combination with one or more further therapeutic agent, such as a cytotoxic, chemotherapeutic or anti-angiogenic agent.Such combined administration may be simultaneous, separate or sequential. For simultaneous administration the agents may be administered as one composition or as separate compositions, as appropriate. The present invention thus also provides methods for treating a disorder involving ceils expressing HER2 as described above, which methods comprise administration of a HER2 antibody of the present invention combined with one or more additional therapeutic agents as described below.In one embodiment, the present invention provides a method for treating a disorder involving cells expressing HER2 In a subject, which method comprises administration of a therapeutically effective amount of a HER2 antibody of the present invention, and optionally at least one additional therapeutic agent, or an antibody binding to a different epitope than said HER2 antibody, to a subject in need thereof.In one embodiment, the present invention provides a method for treating or preventing cancer, which method comprises administration of a therapeutically effective amount of a HER2 antibody of the present invention and at least one additional therapeutic agent to a subject in need thereof.
PCT/EP2G11/058779 WO 2011/147986 1ח one embodiment, such an additional therapeutic agent may be selected from an antimetabolite, such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine or cladribine.In another embodiment, such an additional therapeutic agent may be selected from an alkylating agent, such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin and other platinum derivatives, such as carbopiatin.In another embodiment, such an additional therapeutic agent may be selected from an anti-mitotic agent, such as taxanes, for instance docetaxel, and paclitaxel, and vinca alkaloids, for instance vindesine, vincristine, vinblastine, and vinorelbine.In another embodiment, such an additional therapeutic agent may be selected from a topoisomerase inhibitor, such as topotecan or irinotecan, or a cytostatic drug, such as etoposide and teniposide.In another embodiment, such an additional therapeutic agent may be selected from a growth factor inhibitor, such as an inhibitor of ErbBl (EGFR) (such as an EGFR antibody, e.g. zalutumumab, cetuximab, panitumumab or nimotuzumab or other EGFR inhibitors, such as gefitinib or erlotinib), another inhibitor of ErbB2 (HER2/neu) (such as a HERantibody, e.g. trastuzumab, trastuzumab-DMl or pertuzumab) or an inhibitor of both EGFR and HER2, such as lapatinib).In another embodiment, such an additional therapeutic agent may be selected from a tyrosine kinase inhibitor, such as imatinib (Glivec, Gleevec STI571) or lapatinib, PTK787/ZK222584.In another embodiment, the present invention provides a method for treating a disorder involving cells expressing HER2 in a subject, which method comprises administration of a therapeutically effective amount of an HER2 antibody of the present invention and at least one inhibitor of angiogenesis, neovascularization, and/or other vascularization to a subject in need thereofExamples of such angiogenesis inhibitors are urokinase inhibitors, matrix metalloprotease inhibitors (such as marimastat, neovastat, BAY 129566־, AG 3340, BMS-275291 and similar agents), inhibitors of endothelial cell migration and proliferation (such as TNP-470, squalamine, 2-methoxyestradiol, combretastatins, endostatin, angiostatin, penicillamine, SCH66336 (Schering-Plough Corp, Madison, N3), R1157(Janssen Pharmaceutica, Inc, Titusville, NJ) and similar agents), antagonists of angiogenic growth factors (such as such as ZD6474, SU6668, antibodies against angiogenic agents PCT/EP2O11/058779 WO 2011/147986 and/or their receptors (such as VEGF (e.g. bevadzumab), bFGF, and angiopoietin-1), thalidomide, thalidomide analogs (such as CC-5013), Sugen 5416, SU5402, antiangiogenic ribozyme (such as angiozyme), interferon a (such as interferon a2a), suramin and similar agents), VEGF-R kinase inhibitors and other anti-angiogenic tyrosine kinase inhibitors (such as SU011248), inhibitors of endothelial-specific integrin/survival signaling (such as vitaxin and similar agents), copper antagonists/chelators (such as tetrathiomolybdate, captopril and similar agents), carboxyamido-triazole (CAI), ABT-627, CM101, interleu kin-12 (IL-12), IM862, PNU145156E as well as nucleotide molecules inhibiting angiogenesis (such as antisense-VEGF-cDNA, cDNA coding for angiostatin, cDNA coding for p53 and cDNA coding for deficient VEGF receptor-2).Other examples of such inhibitors of angiogenesis, neovascularization, and/or other vascularization are anti-angiogenic heparin derivatives (e.g., heperinase III), temozolomide, NK4, macrophage migration inhibitory factor, cyclooxygenase-2 inhibitors, inhibitors of hypoxia-inducible factor 1, anti-angiogenic soy isoflavones, oitipraz, fumagillin and analogs thereof, somatostatin analogues, pentosan polysulfate, tecogalan sodium, dalteparin, tumstatin, thrombospondin, NM-3, combrestatin, canstatin, avastatin, antibodies against other targets, such as anti-alpha-v/beta3־ integrin and anti-kininostatin antibodies.In one embodiment, a therapeutic agent for use in combination with a HERantibody for treating the disorders as described above may be an anti-cancer immunogen, such as a cancer antigen/tumor-associated antigen (e.g., epithelial cell adhesion molecule (EpCAM/TACSTDl), mucin 1 (MUC1), carcinoembryonic antigen (CEA), tumor-associated glycoprotein 72 (TAG-72), gplOO, Melan-A, MART-1, KDR, RCAS1, MDA7, cancer-associated viral vaccines (e.g., human papillomavirus vaccines) or tumor-derived heat shock proteins,In one embodiment, a therapeutic agent for use in combination with a HERantibody for treating the disorders as described above may be an anti-cancer cytokine, chemokine, or combination thereof. Examples of suitable cytokines and growth factors include IFNy, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNa (e.g., INF02b), IFNp, GM-CSF, CD40L, F(t3 ligand, stem cell factor, ancestim, and TNFa. Suitable chemokines may include Giu-Leu-Arg (ELR)- negative chemokines such as IP-10, MCP-3, MIG, and SDF-la from the human CXC and C-C chemokine families. Suitable cytokines include cytokine derivatives, cytokine variants, cytokine fragments, and cytokine fusion proteins.In one embodiment, a therapeutic agent for use in combination with a HERantibody for treating the disorders as described above may be a cell cycle control/apoptosis regulator (or "regulating agent"). A ceil cycle control/apoptosis regulator may include molecules that target and modulate ceil cycle control/apoptosis regulators such as (i) PCT/EP2011/058779 WO 2011/147986 cdc-25 (such as NSC 663284), (ii) cyciin-dependent kinases that overstimulate the cel! cycle (such as flavopiridol (L868275, HMR1275), 7-hydroxystaurosparine (UCN-01, KW-2401), and roscovitine (R-roscovitine, CYC202)), and (ill) telomerase modulators (such as BIBR1532, SOT-095, GRN163 and compositions described in for instance US 6,440,735 and US 6,713,055). Non-limiting examples of molecules that interfere with apoptotic pathways include TNF-related apoptosis-inducing ligand (TRAIL)/apoptosis-2 ligand (Apo-2L), antibodies that activate TRAIL receptors, IFNs* and anti-sense Bcl-2.In one embodiment, a therapeutic agent for use in combination with a HERantibody for treating the disorders as described above may be a hormonal regulating agent, such as agents useful for anti-androgen and anti-estrogen therapy. Examples of such hormonal regulating agents are tamoxifen, idoxifene, fulvestrant, draloxifene, toremifene, raloxifene, diethylstilbestrol, ethinyl estradiol/estinyl, an antiandrogene (such as ffutaminde/eulexin), a progestin (such as such as hydroxyprogesterone caproate, medroxy- progesterone/provera, megestrol acepate/megace), an adrenocorticosteroid (such as hydrocortisone, prednisone), luteinizing hormone-releasing hormone (and analogs thereof and other LHRH agonists such as buserelin and goserelin), an aromatase inhibitor (such as anastrazole/arimidex, aminoglutethimide/cytraden, exemestane) or a hormone inhibitor (such as octreotide/sandostatin).In one embodiment, a therapeutic agent for use in combination with a HERantibody for treating tt»e disorders as described above may be an anti-anergic agent, such ascompounds are molecules that block the activity of CTLA-4, e.g. ipilimumab.In one embodiment, a therapeutic agent for use in combination with a HERantibody for treating the disorders as described above may be an anti-cancer nucleic acid or an anti-cancer inhibitory RNA molecule.Examples of other anti-cancer agents, which may be relevant as therapeutic agents for use in combination with a HER2 antibody for treating the disorders as described above are differentiation inducing agents, retinoic acid analogues (such as all trans retinoic acid, 13־cis retinoic acid and similar agents), vitamin D analogues (such as seocalcitol and similar agents), inhibitors of ErbB3, ErbB4, IGF-IR, insulin receptor, PDGFRa, PDGFRbeta, Flk2,F!t4, FGFR1, FGFR2, FGFR3, FGFR4, TRKA, TRKC, RON (such as an ant»-RON antibody), Sea, Tie, Tie2, Eph, Ret, Ros, Aik, LTK, PTK7 and similar agents.Examples of other anti-cancer agents, which may be relevant as therapeutic agents for use in combination with a HER2 antibody for treating the disorders as described above are estramustine and epirubicin.Examples of other anti-cancer agents, which may be relevant as therapeutic agents for use in combination with a HER2 antibody for treating the disorders as described above PCT/EP2011/058779 WO 2011/147986 are a HSP90 inhibitor like 17-allyl amino geid-anamycin, antibodies directed against a tumor antigen such as PSA, CA125, KSA, integrins, e.g. integrin pi, or inhibitors of VCAM.Examples of other anti-cancer agents, which may be relevant as therapeutic agents for use in combination with a HER2 antibody for treating the disorders as described above are calcineurin-inhibitors (such as valspodar, PSC 833 and other MDR-1 or p~glycoprotein inhibitors), TOR-inhibitors (such as siroiimus, everolimus and rapamcyin). and inhibitors of "lymphocyte homing״ mechanisms (such as FTY72Q), and agents with effects on cell signaling such as adhesion molecule Inhibitors (for instance anti-LFA),In one embodiment, the HER2 antibody of the invention is for use in combination with one or more other therapeutic antibodies, such as ofatumumab, zanolimumab, daratumumab, ranibizumab, nimotuzumab, panitumumab, hu806, daciizumab (Zenapax), basiliximab (Simuiect), infliximab (Remicade), adalimumab (Humira), natalizumab (Tysabri), omaiizumab (Xolair), efalizumab (Raptiva) and/or rituximab.In another embodiment, two or more different antibodies of the invention as described herein are used in combination for the treatment of disease. Particularly interesting combinations include two or more non-blocking antibodies. Such combination therapy may lead to binding of an increased number of antibody molecules per cell, which may give increase efficacy, e.g. via activation of complement-mediated lysis.In addition to the above, other embodiments of combination therapies of the invention include the following:For the treatment of breast cancer, a HER.2 antibody or a therapeutic conjugate thereof, in combination with methotrexate, paclitaxel, doxorubicin, carboplatin, cyclophosphamide, daunorubicin, epirubicin, 5-fluorouracil, gemcitabine, ixabepilone, mutamycin, mitoxantrone, vinorelbine, docetaxel, thiotepa, vincristine, capecitabine, an E6FR antibody (e.g. zalutumumab, cetuximab, panitumumab or nimotuzumab) or other EGFR inhibitor (such as gefitinib or erlotinib), another HER2 antibody or -conjugate (such as, e.g., trastuzumab, trastuzumab־DMl or pertuzumab), an inhibitor of both EGFR and HER2 (such as lapatinib), and/or in combination with a HER3 inhibitor.For the treatment of non-small-cell lung cancer, a HER2 antibody in combination with EGFR inhibitors, such as an EGFR antibody, e.g. zalutumumab, cetuximab, panitumumab or nimotuzumab or other EGFR inhibitors (such as gefitinib or erlotinib), or in combination with an another HER2 agent (such as a HER2 antibody, e.g. trastuzumab, trastuzumab״DMl or pertuzumab) or in combination with an inhibitor of both EGFR and HER2, such as lapatinib, or in combination with a HER3 Inhibitor.For the treatment of colorectal cancer a HER2 antibody in combination with one or more compounds selected from: gemcitabine, bevacizumab, FOLFOX, FOLFIRI, XELOX, IFL, PCT/EP2011/058779 WO 2011/147986 oxaliplatin, irinotecan, 5-FU/LV, Capecitabine, UFT, EGFR targeting agents, such as cetuximab, panitumumab, zalutumumab; VEGF inhibitors, or tyrosine kinase inhibitors such as sunitinib.For the treatment of prostate cancer a HER2 antibody in combination with one or more compounds selected from: hormonai/antihormonal therapies; such as antiandrogens, Luteinizing hormone releasing hormone (LHRH) agonists, and chemotherapeutics such as taxanes, mitoxantrone, estramustine, 5FU, vinblastine, and ixabepilone.
Radiotherapy - suraervIn one embodiment, the present invention provides a method for treating a disorder involving ceils expressing HER2 in a subject, which method comprises administration of a therapeutically effective amount of a HER2 antibody, such as a HER2 antibody of the present invention, and radiotherapy to a subject in need thereof.In one embodiment, the present invention provides a method for treating or preventing cancer, which method comprises administration of a therapeutically effective amount of a HER2 antibody, such as a HER2 antibody of the present invention, and radiotherapy to a subject in need thereof.In one embodiment, the present invention provides the use of a HER2 antibody, such as a HER2 antibody of the present invention, for the preparation of a pharmaceutical composition for treating cancer to be administered in combination with radiotherapy.Radiotherapy may comprise radiation or associated administration of radiopharmaceuticals to a patient is provided. The source of radiation may be either external or internal to the patient being treated (radiation treatment may, for example, be in the form of external beam radiation therapy (EBRT) or brachytherapy (BT)). Radioactive elements that may be used in practicing such methods include, e.g., radium, cesium-137, iridium-192, americium-241, gold-198, cobalt-57, copper-67, technetium-99, iodide-123, iodide-131, and indium-111.In a further embodiment, the present invention provides a method for treating or preventing cancer, which method comprises administration to a subject in need thereof of a therapeutically effective amount of a HER2 antibody, such as a HER2 antibody of the present invention, in combination with surgery.
Diagnostic usesThe HER2 antibodies of the invention may also be used for diagnostic purposes. Thus, in a further aspect, the invention relates to a diagnostic composition comprising a HER2 antibody as defined herein.
PCT/EP2011/058779 WO 2011/147986 In one embodiment, the HER2 antibodies of the present invention may be used in vivo or in vitro for diagnosing diseases wherein activated ceils expressing HER2 play an active role in the pathogenesis, by detecting levels of HER2, or levels of cells which contain HER2 on their membrane surface. This may be achieved, for example, by contacting a sample to be tested, optionally along with a control sample, with the HER2 antibody under conditions that allow for formation of a complex between the antibody and HER2.Thus, in a further aspect, the invention relates to a method for detecting the presence of HER2 antigen, or a cell expressing HER2, in a sample comprising:- contacting the sample with a HER2 antibody of the invention under conditions that allow for formation of a complex between the antibody and HER2; and־ analyzing whether a complex has been formed.In one embodiment, the method is performed in vitro.More specifically, the present invention provides methods for the identification of, and diagnosis of invasive cells and tissues, and other cells targeted by HER2 antibodies of the present invention, and for the monitoring of the progress of therapeutic treatments, status after treatment, risk of developing cancer, cancer progression, and the like.Suitable labels for the HER2 antibody and/or secondary antibodies used in such techniques are well-known in the art.In a further aspect, the invention relates to a kit for detecting the presence of HERantigen, or a cell expressing HER2, in a sample comprising- a HER2 antibody of the invention or a bispecific molecule of the invention; and- instructions for use of the kit.In one embodiment, the present invention provides a kit for diagnosis of cancer comprising a container comprising a HER2 antibody, and one or more reagents for detecting binding of the HER2 antibody to HER2, Reagents may include, for example, fluorescent tags, enzymatic tags, or other detectable tags. The reagents may also include secondary or tertiary antibodies or reagents for enzymatic reactions, wherein the enzymatic reactions produce a product that may be visualized.
Anti-idiotypic antibodiesIn a further aspect, the invention relates to an anti-idiotypic antibody which binds to a HER2 antibody of the invention as described herein.An anti-idiotypic (Id) antibody is an antibody which recognizes unique determinants generally associated with the antigen-binding site of an antibody. An Id antibody may be prepared by immunizing an animal of the same species and genetic type as the source of a HER2 mAb with the mAb to which an anti-id is being prepared. The immunized animal PCT/EP2011/058779 WO 2011/147986 typically cap recognize and respond to the idiotypic determinants of the immunizing antibody by producing an antibody to these idiotypic determinants (the anti-id antibody).An anti-id antibody may also be used as an "immunogen" to induce an immune response in yet another animal, producing a so-called anti־anti-Id antibody. An anti-anti-Id may be epitopicaliy identical to the original mAb, which induced the anti-id. Thus, by using antibodies to the idiotypic determinants of a mAb, it is possible to identify other clones expressing antibodies of identical specificity.The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention.
The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention, EXAMPLES Example 1 ־ Expression constructs lor HER2 and HER2 variants Fully codon-optimized constructs for expression of full length HER2 (1255 aa, Swissprot P04626), the extracellular domain (ECD) of HER2 (Her2-ECDHis, aa 1653־ with a C־terminal His6 tag), the naturally occurring HER2 splice variant (Her2״delexl6, resulting from exon deletion and iacking aa 633648־) and a truncated form of the HER2 receptor (Her2-stumpy, aa 6481256־), were generated. The construct contained suitable restriction sites for cloning and an optimal Kozak sequence (Kozak, M., Gene 1999;234(2); 187208־.). The constructs were cloned in the mammalian expression vector pEE13.4 (Lonza Biologies; Bebbington, C.R., et al., Biotechnology (N Y) 1992;10(2): 16975־) and fully sequenced to confirm the correctness of the construct.
Example 2 ־ Expression constructs for Pertuzumab, C1 and F5 Fully codon-optimized constructs for expression of the heavy chain (HC) and the light chain (LC) of the IgGl antibodies pertuzumab, Cl and F5 in HEK cells, were generated. The variable regions encoded by these constructs are identical to those described in U.S. Patent No. 6,949,245 for pertuzumab heavy chain and light chain and U.S. Patent No. 7,244,8for Cl and F5 heavy and light chain. For Cl and F5, the mammalian expression vectors p33Glf and p33K or p33L (pcDNA3.3 (Invitrogen)) containing the fully codon optimized constant region for the human IgGl heavy chain (allotype f), the human kappa light chain or the human lambda light chain, respectively, were used. For pertuzumab, the mammalian expression vectors pGlf (pEE12.4 (Lonza Biologies) and pKappa (pEE6.4 (Lonza Biologies), containing the fully codon-optimized constant region for the human IgGl heavy chain(allotype f) and the human kappa light chain, respectively, were used.66 PCT/EP2011/058779 WO 2011/147986 Trastuzumab (Herceptin®) can be produced in the same manner, using the heavy and light chain sequences described in, e.g., U.S. Patent No. 7,632,924.
Example 3 - Transient expression in HEK-293 or CHO cells Freestyle™ 293-F (a HEK-293 subclone adapted to suspension growth and chemically defined Freestyle medium, (HEK-293F)) cells were obtained from Invitrogen and transfected with the appropriate plasmid DNA, using 293fecttn (Invitrogen) according to the manufacturer's instructions. In the case of antibody expression, the appropriate heavy chain and light chain expression vectors were co-expressed.pEE13.4Her2, pEE13.4Her2״delexl6 and pEE13.4Her2-stumpy were transiently transfected in the Freestyle™ CHO-S (Invitrogen) cel! line using Freestyle MAX transfection reagent (Invitrogen). Expression of HER2 and Her2־delexl6 was tested by means of FACS analysis as described below.
Example 4 - Stable polyclonal pool expression in NSO pEE13.4Her2, pEE13.4Her2־delexl6 and pEE13.4Her2-stumpy were stably transfected in NSO cells by nucleofection (Amaxa).A pool of stably transfected cells was established after selection on glutamine dependent growth, based on the integrated glutamine synthetase selection marker (Barnes, L.M., et aL, Cytotechnology 2000;32(2):109123־).
Example 5 - Purification of Hls-tagged HER2 Her2ECDHis was expressed in HEK-293F cells. The His-tag in Her2ECDHis enabled purification with immobilized metal affinity chromatography, since the His-tagged protein binds strongly to the resin beads, while other proteins present in the culture supernatant do not bind strongly.In this process, a chelator fixed onto the chromatographic resin was charged with Co2+ cations. Her2ECDHis containing supernatant was incubated with the resin in batch mode (i.e. solution). After incubation, the beads were retrieved from the supernatant and packed into a column. The column was washed in order to remove weakly bound proteins. The strongly bound Her2ECDHis proteins were then eluted with a buffer containing imidazole, which competes with the binding of His to Co2 The eluent was removed from the protein by buffer exchange on a desalting column.
Example 6 - Immunization procedure of transgenic mice Antibodies 001, 019, 021, 025, 027, 032, 033, 035, 036, 049, 050, 051, 054, 055, 084,091, 094, 098, 100, 105, 123 and 124 were derived from the following immunization: three PCT/EP2011/058779 WO 2011/147986 female HC012 mice, one male and two female HC012־Balb/C mice, one male HC017 mouse and one male HC020 mouse (Medarex, San lose, CA, USA) were Immunized alternating with 5x10s NSO cells transiently transfected with Her2ECD intraperitoneal (IP) and 20 jjg Her2ECDHis protein coupled to the hapten Keyhole Limpet Hemocyanin (KLH) subcutaneous (SC) at the tail base, with an interval of fourteen days, A maximum of eight immunizations was performed per mouse (four IP and four SC immunizations). The first immunization with cells was done in complete Freunds' adjuvant (CFA; Difco Laboratories, Detroit, MI, USA). For all other immunizations, cells were injected IP in PBS and KLH coupled Her2ECD was injected SC using incomplete Freunds' adjuvant (IFA; Difco Laboratories, Detroit, MI, USA).Antibodies 125, 127, 129, 132, 152, 153 and 159 were derived from the following immunization: one male and two female HC012-Balb/C mice, one female HC020 mouse, and one female HC012 mouse (Medarex) were immunized alternating with 5xlQ6 NSO cells transiently transfected with Her2delexl6 IP and 20 pg Her2ECDHis protein coupled to the hapten Keyhole Limpet Hemocyanin (KLH) SC at the tail base, with an Interval of fourteen days. A maximum of eight immunizations was performed per mouse (four IP and four SC immunizations). The first immunization with cells was done in complete Freunds' adjuvant (CFA; Difco Laboratories, Detroit, MI, USA). For all other immunizations, cells were injected IP in PBS and KLH coupled Her2ECD was injected SC using Incomplete Freunds' adjuvant (IFA; Difco Laboratories, Detroit, MI, USA).Antibody 143, 160, 161, 162, 166 and 169 were derived from the following immunization: one female and one male Hcol2 mouse, one female Hcol2־Balb/C mouse, one male HC017 mouse and one male HC020 mouse (Medarex) were immunized alternating with 20 pg Her2ECDHis protein coupled to the hapten Keyhole Limpet Hemocyanin (KLH), alternating IP and SC at the tail base with an interval of fourteen days. A maximum of eight immunizations was performed per mouse (four IP and four SC immunizations). The first immunization was done IP in complete Freunds' adjuvant (CFA; Difco Laboratories, Detroit, MI, USA). The other immunizations were injected using incomplete Freunds' adjuvant (IFA; Difco Laboratories, Detroit, MI, USA).Mice with at least two sequential titers against TC1014-Her2, TC1Q14-Her2delexlor TC1014־Her2stumpy in the antigen specific FMAT screening assay (as described in example 7), were considered positive and fused.
Example 7 ־ Homogeneous antigen specific screening assay The presence of HER2 antibodies in sera of immunized mice or HuMab (human monoclonal antibody) hybridoma or transfectoma culture supernatant was determined by homogeneous antigen specific screening assays (four quadrant) using Fluorometric Micro volume Assay PCT/EP2011/058779 WO 2011/147986 Technology (FMAT; Applied Biosystems, Foster City, CA, USA). For this, a combination of cell based assays was used. Binding to TC1014-Her2 (HEK-293F cells transiently expressing the HER2 receptor; produced as described above), TC1014-Her2delex 16 (HEK-293F cells transiently expressing the extracellular domain of Her2-delex (a 16 amino acid deletion mutant of the HER2 receptor; produced as described above) and TC1014׳-Her2stumpy (HEK-293F cells transiently expressing the extracellular stumpy domain of the HERreceptor; produced as described above) as well as HEK293 wild type cells (negative control cells which do not express HER2) was determined. Samples were added to the cells to allow binding to HER2. Subsequently, binding of HuMab was detected using a fluorescent conjugate (Goat anti-Human IgG-Cy5; Jackson ImmunoResearch). TH1014-Pertuzumab (produced in HEK-293F cells) was used as a positive control and HuMab-mouse pooled serum and HuMab-KLH were used as negative controls. The samples were scanned using an Applied Biosystems 8200 Cellular Detection System (8200 CDS) and 'counts x fluorescence׳ was used as read-out. Samples were stated positive when counts were higher than 50 and counts x fluorescence were at least three times higher than the negative control.
Example 8 - HuMab hybrldoma generation HuMab mice with sufficient antigen-specific titer development (defined as above) were sacrificed and the spleen and lymph nodes flanking the abdominal aorta and vena cava were collected. Fusion of splenocytes and lymph node cells to a mouse myeloma cell line was done by electrofusion using a CEEF 50 Electrofusion System (Cyto Pulse Sciences, Glen Burnie, MD, USA), essentially according to the manufacturer's instructions. Next, the primary wells were sub cloned using the ClonePix system (Genetix, Hampshire, UK). To this end specific primary well hybridoma's were seeded in semisolid medium made from 40% CloneMedia (Genetix, Hampshire, UK) and 60% HyQ 2x complete media (Hyclone, Waltham, USA). The sub clones were retested in the antigen-specific binding assay as described in Example 7 and IgG levels were measured using an Octet (Fortebio, Menlo Park, USA) in order to select the most specific and best producing done per primary well for further expansion. Further expansion and culturing of the resulting HuMab hybridomas were done based upon standard protocols (e.g. as described in Coligan J.E., Bierer, B.E., Margulies, D.H., Shevach, E.M. and Strober, W., eds. Current Protocols in Immunology, John Wiley & Sons, Inc., 2006). Clones derived by this process were designated PC1014.
Example 9 י Mass Spectrometry of purified antibodies Small aliquots of 0.8 ml antibody containing supernatant from 6-well or Hyperflask stage were purified using PhyTip columns containing Protein G resin (PhyNexus Inc., San Jose, PCT/EP2011/058779 WO 2011/147986 USA) סח a Sciclone ALH 3000 workstation (Caliper Lifeseiences, Hopkinton, USA). The PhyTip columns were used according to manufacturer's instructions, although buffers were replaced by: Binding Buffer PBS (B.Braun, Medical B.V., Oss, Netherlands) and Elution Buffer 0.1M Glycine-HCl pH 2.7 (Fluka Riedel-de Haen, Buchs, Germany). After purification, samples were neutralized with 2M Tris-HCi, pH 9.0 (Sigma-Aldrich, Zwijndrecht, Netherlands). Alternatively, in some cases larger volumes of culture supernatant were purified using MabSelect SuRe.After purification, the samples were placed in a 384־well plate (Waters, 100 pi square well plate, part# 186002631). Samples were deglycosyiated overnight at 37°C with N-glycosidase F (Roche cat no 11365177001. DTT (15 mg/mL) was added (1 pL/well) and incubated for 1 h at 37°C. Samples (5 or 6 pL) were desalted on an Acquity UPLC™(Waters, Milford, USA) with a BEH300 08, 1.7pm, 2.lx 50 mm column at 60 °C. MQ water and LC־MS grade acetonitrile (Biosolve, cat no 01204101, Valkenswaard, The Netherlands) with both 0.1% formic acid (Fluka, cat no 56302, Buchs, Germany), were used as Eluens A and B, respectively. Time-of-flight electrospray ionization mass spectra were recorded on- line on a micrOTOF™ mass spectrometer (Broker, Bremen, Germany) operating in the positive ion mode. Prior to analysis, a 9003000־ m/z scale was calibrated with ES tuning mix (Agilent Technologies, Santa Clara, USA). Mass spectra were deconvolved with DataAnalysIs™ software v. 3.4 (Broker) using the Maximal Entropy algorithm searching for molecular weights between 5 and 80 kDa.After deconvolution, the resulting heavy and light chain masses for all samples were compared in order to find duplicate antibodies. This was sometimes due to the presence of an extra light chain, but in the comparison of the heavy chains, the possible presence of C- terminal lysine variants was also taken into account. This resulted in a list of unique antibodies, i.e., a unique combination of specific heavy and light chains. In case duplicate antibodies were found, one unique antibody was selected based on results from other tests.
Example 10 ־ Sequence analysis of the HER2 antibody variable domains and cionfng in expression vectors Total RNA of the HER2 HuMabs was prepared from 5xl06 hybridoma cells and 5'־RACE־ Complementary DNA (cDNA) was prepared from 100 ng total RNA, using the SMART RACE cDNA Amplification kit (Clontech), according to the manufacturer's instructions. VH and VL coding regions were amplified by PCR and cloned directly, in frame, in the pGlf and pKappa expression vectors, by ligation independent cloning (Aslanidis, C. and PJ. de long, Nucleic Acids Res 1990:18(20): 606974־). Clones derived by this process were designated TH1014. For each antibody, 16 VL clones and 8 VH clones were sequenced. Clones which predicted 70 PCT/EP2011/058779 WO 2011/147986 heavy and light chain mass in agreement with the mass of the hybridoma derived material of the same antibody (as determined by mass spectrometry) were selected for further study and expression.The resulting sequences are shown in Figures 1 and 2 and in the Sequence Listing. Selected sequences are also described in more detail below. CDR sequences were defined according to IMGT (Lefranc MP. et a/., Nucleic Acids Research, 27, 2091999 ,212־ and Brochet X. Nuci. Acids Res. 36, W503-508 (2008)). Table 1, Table 2 and Table 3 give an overview of antibody sequence information or germline sequences, and Table 4 shows consensus sequences.
Table 1: Heavy chain variable region (VH), light chain variable region (VL) and CDR sequences of HuMabs 169, 050, 084, 025, 091, 129, 127, 159, 098, 153, and 132.
SEQ ID N0:1 VH 169 QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGISWVRQAPGQGLEWMGWLSAYSGNTIYAQKLQGRVTMTTDTSTTTAYMELRSLRSDDTAVYYCARDRIWRPDYFDYWGQGTLVTVSSSEQ ID No: 2 VH 169, CDR1 GYTFTNYGSEQ ID No:3 VH 169, CDR2 LSAYSGNTSEQ ID No;4 VH 169, CDR3 ARDRIVVRPDYFDYSEQ ID No :5 VL 169 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPRTFGQGTKVEIKSEQ ID No:6 VL 169, CDR1 QSVSSYVL 169, CDR2 DASSEQ ID No:7 VL 169, CDR3 QQRSNWPRTSEQ ID No:8 VH 050 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKG LE WVS AISGRGGTTYY ADSVKG RFTISRDNSKNTLYLQMSSLRAEDTAVYYCAKARANWDYFDYWGOGTLVTVSSSEQ ID No:9 VH 050, CDR1 GFTFSSYASEQ ID No: 10 VH 050, CDR2 ISGRGGTTSEQ ID No: 11 VH 050, CDR3 AKARANWDYFDYSEQ ID No: 12 VL 050 DIQMTQSPSSVSASVGD RVTITCRASQGISS W LAWYQHKPGKAPKLLIYAASILQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPITFGQGTRLEIKSEQ ID No: 13 VL 050, CDR1 QGISSWVL 050, CDR2 AASSEQ ID No: 14 VL 050, CDR3 QQANSFPIT PCT/EP2011/058779 WO 2011/147986 SEQ ID No: 15 VH 084 QVQLVQSGAEVKKPGSSVKVSCKASGGTFRTYAINWVRQAPGQGLEWMGRINTVLGIVNHAQKFQGRVTITADKSTNTAYMELNSLRSEDTAVYYCAREKGVDYYYGIEVWGQGTTVTVSSSEQ ID No: 16 VH 084, CDR1 GGTFRTYASEQ ID No: 17 VH 084, CDR2 INTVLGIVSEQ ID No: 18 VH 084, CDR3 AREKGVDYYYGIEVSEQ ID No: 19 VL 084 DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQHKPGKAPKLLIYVASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPLTFGGGTKVEIKSEQ ID No:20 VL 084, CDR1 QGISSWVL 084, CDR2 VASSEQ ID No: 21 VL 084, CDR3 QQANSFPLTSEQ ID No:22 VH 025 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKGLEWIGEIHHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGYYDSGVYYFDYWAQGTLVTVSSSEQ ID No:23 VH 025, CDR1 GGSFSDYYSEQ ID No: 24 VH 025, CDR2 IHHSGSTSEQ ID No:25 VH 025, CDR3 ARGYYDSGVYYFDYSEQ ID No :26 VL 025DIQMTQSPSSLSASVGDRVTITCRASQGISRWLAWYQQKPEKAPKSUYAASSLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPITFGQGTRLEIKSEQ ID No:27 VL 025, CDR1 QGISRWVL 025, CDR2 AASSEQ ID No :28 VL 025, CDR3 QQYNSYPITSEQ ID No:29 VH 091 QVQLQQWGAGLLKPSETLSLTCAVSGGSFSGYYWTWIRQPPGKGLEWIGEIYHSGDTNYNPSLKSRVTISVDTSKNQFSLKLYSVTAADTAVYYCARLYFGSGIYYLDYWGQGTLVTVSSSEQ ID No:30 VH 091, CDR1 GGSFSGYYSEQ ID No: 163 VH 091, CDR2 IYHSGDTSEQ ID No: 31 VH 091, CDR3 ARLYFGSGIYYLDYSEQ ID No: 32 VL 091 DIQHTQSPSSLSASVGDRVTITCRASQGISSWLVWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSFPPTFGQGTKVEIKSEQ ID No: 33 VL 091, CDR1 QGISSWVL 091, CDR2 AASSEQ ID No:34 VL 091, CDR3 QQYNSFPPTSEQ ID No:35 VH 129 QVQLVESGGGWQPGRSLRLSCAASGFTFSTFAIHWV RQ APGKG LE WV AVISYDGG H KFYADS V KG RFTIS R DNSKNTLYLQMNSLRAEDTAMYYCARGLGVWGAFD YWGQGTLVTVSSSEQ ID No:36 VH 129, CDR1 GFTFSTFA 72 PCT/EP2011/058779 WO 2011/147986 SEQ ID No:37 VH 129, CDR2 ISYDGGHKSEQ ID No: 38 VH 129, CDR3 ARGLGVWGAFDYSEQ ID No: 39 VL 129 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTTSSLEPEDFAVYYCQQRSNWWTFGQGTKVE1KSEQ ID No;40 VL 129, CDR1 QSVSSYVL 129, CDR2 DASSEQ ID No :41 VL 129, CDR3 QQRSNWWTSEQ ID No:42 VH 127 EVQLVQSGAEVKKPGESLTISCKGSGYSFSIYWIGWVRQMPGKGLEWMGIIFPGDSDIRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARQPGDWSPRHWYFDLWGRGTLVTVSSSEQ ID No:43 VH 127, CDR1 GYSFSIYWSEQ ID No ;44 VH 127, CDR2 IFPGDSDISEQ ID No:45 VH 127, CDR3 ARQPGDWSPRHWYFDLSEQ ID No:46 VL 127 VIWMTQSPSLLSASTGDRVTISCR M SQGIS SYLAWY QQK PGKAPELLIYAASTLQSGV PSRFSGSGSGTDFTL ’ TISYLQSEDFATYYCQQYYSFPLTFGGGTKVEIKSEQ ID No:47 VL 127, CDR1 QGISSYVL 127, CDR2 AASSEQ ID No :48 VL 127, CDR3 QQYYSFPLTSEQ ID No:49 VH 159 EVQLVQSGAEVKKPGESLKISCKGSGYNFTSYWIGWVRQHPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARWGTYYDILTGYFNWFDPWGQGTLVTVSSSEQ ID No: 50 VH 159, CDR1 GYNFTSYWSEQ ID No: 51 VH 159, CDR2 IYPGDSDTSEQ ID No: 52 VH 159, CDR3 ARWGTYYDILTGYFNSEQ ID No: 53 VL 159 DIQMTQSPSSLSASVGDRVTTTCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPWTFGQGTKVEIKSEQ ID No: 54 VL 159, CDR1 QGISSWVL 159, CDR2 AASSEQ ID No: 55 VL 159, CDR3 QQYYIYPWTSEQ ID No:56 VH 098evqllesggglvqpggsLrlscaasgftfsnygmswVRQAPGKGLEWVSAISGSAYSTYYADSVKGRFTISRDNSKNTLWLQMNSLRAEDTAVYYCAKAHYHGSGSYYTLFDYWGQGTLVTVSSSEQ ID No:57 VH 098, CDR1 GFTFSNYGSEQ ID No :58 VH 098, CDR2 ISGSAYSTSEQ ID No :59 VH 098, CDR3 AKAHYHGSGSYYTLFDY PCT/EP2011/058779 WO 2011/147986 SEQ ID No :60 VL 098 DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIKSEQ ID No:61 VL 098, CDR1 QGISSWVL 098, CDR2 AASSEQ ID No :62 VL 098, CDR3 QQYNSYPYTSEQ ID No:63 VH 153 QVQLVESGGGVVQPGRSLRLSCAASGFTFSDYVIHWVRQAPGKGLEWVTVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLSAEDTAMYYCARGGITGTTGVFDYWGQGTLVTVSSSEQ ID No:64 VH 153, CDR1 GFTFSDYVSEQ ID No: 65 VH 153, CDR2 ISYDGSNKSEQ ID No: 66 VH 153, CDR3 ARGGITGTTGVFDYSEQ ID No :67 VL 153 DIQMTQSPSSISASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYDASSLQSGVPSRFSGSGYGTDFSLTISSLQPEDFAIYYCQQYKSYPITFGOGTRLEIKSEQ ID No :68 VL 153, CDR1 QGISSWVL 153, CDR2 DASSEQ ID No: 69 VL 153, CDR3 QQYKSYPITSEQ ID No: 70 VH 132 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISW VRQAPGQGLEWMGWISAYNGNSNYVQKFQGRVTM TTDTTTSTAYM ELRSLTSDDTAVYYCAREYSYDSGTY FYYGM D VWGQGTTVTVSSSEQ ID No :71 VH 132, CDR1 GYTFTSYGSEQ TD No:72 VH 132, CDR2 ISAYNGNSSEQ ID No:73 VH 132, CDR3 AREYSYDSGTYFYYGM DVSEQ ID No: 74 VL 132 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQ QKPGQAPRLLEYDASN RAT GIPARFSGSGSGTD FTLTI SSLEPEDFAVYYCQQRSNWPMYTFGQGTKLEIKSEQ ID No:75 VL 132, CDR1 QSVSSYVL 132, CDR2 DASSEQ ID No:76 VL 132, CDR3 QQRSNWPMYT Table 2: Mouse origin and heavy and light chain sequence homologies of selected HuMabs.
HuMab: Mouse: Strain: Germline VH: Germline VL: 169 361494 HC020 IgHVl-18-01 IgKV3-ll~01 050 350633 HC012 IgHV3-23-01 IgKVl-12-01 084 350615 HC012—BalbC IgHVl-69-04 IgKVl-12-Ol 025 350631 HC012 IgHV4-34-01 IgKVID—16-01 091 350630 HC012 IgSV4-34-01 IgKVlD-16-01 PCT/EP2011/058779 WO 2011/147986 129 359783 HCo!2~BalbC IgHV3-30-3-01 IgKV3-ll-01 127 359783 HC012~BalbC IgHV5-51~01 IgKVlD-8-01 159 363503 HC012 IgHV5-51-01 IgKVlD-16-01 098 350659 HCol? IgHV3-23~01 IgKVlD-16-01 153 359785 HC012~BalbC IgHV3-30-3-01 IgKVlD-16-01 132 361487 HC020 IgHVl-18-01 IgKV3-ll-01 Table 3: Heavy chain variable region (VH), light chain variable region (VI) sequences of HuMabs 049, 051, 055, 123, 161, 124, 001, 143, 019, 021, 027, 032, 035, 036, 054, 094. The respective CDRs correspond to those underlined in Figures 1 and 2, for VH and VL sequences, respectively.
SEQ IO No: 77 VH 049 EVQLLESGGDLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGRGGTTYYADSVKGRFnSRDNSKSTLCLQMNSLRAEDTAVYYCAKARANWDYFDYWGQGTLVTVSSSEQ ID No: 78 VL 049 DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQHKP6KAPKLLIYAASILQSGVPSRFSGSGSGTDFTLTISSLRPEDFATYYCQQANSFPITFGQGTRLEIKSEQ ID No: 79 VH 051 EVQLLESGGDLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGRGGTTYYADSVKGRFTISRDNSKSTLCLQMNSLRAEDTAVYYCAKARANWDYFDYWGQGTLVTVSSSEQ ID No: 80 VL 051 DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQHKPGKAPKLLIYAASILQSGVPSRFSGSGSGTDFTLTISSLRPEDFATYYCQQANSFPITFGQGTRLEIKSEQ ID No: 81 VH 055 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSAISGRGGTTYYADSVKGRFTISRDNSKSTLCLQMNSLRAEDTAVYYCAKARANWDYFDYWGQGTLVTVSSSEQ ID No: 82 VL 055 DIQMT QSPSS VS AS VG DRVTITCR ASQGISS WLA W Y QH KPG K APKLLIYAASILQSGVPSRFSGSGSGTDFTLTISSLRPEDFATYY CQQANSFPITFGQGTRLEIKSEQ ID No: 83 VH 123 QVQLVQSGAEVKKPGASVKVSCKAAGYTFTNYGISWVRQAPGQALEWMGWITTYSSNTIYAQKLQGRVTMTTDTSTSTAYHELRSLRSDDTAVYYCARDRVWRPDYFDYWGQGTLVTVSSSEQ ID No: 84 VL 123 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDTSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSHWPRTFGQGTKVEIKSEQ ID No: 85 VH 161 QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGISWVRQAPG QG LE WMG WLSAY SG NTIYAQKLQGRVTMTTDTSTTT AYM ELR SLRSDDTAVYYCARDRIVVRPDYFDYWGQGTLVTVSSSEQ ״ID No: 86VL 161 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAP RLLIYDASNRATGIPARFSG5GSGTDFTLTISSLEPEDFAVYYCQ QRS N W PRTFGQGTKVEIKSEQ ID No: 87 VH 124 QVQLVQSGAEVKKPGASVKVSCKAAGYTFTNYGISWVRQAPG QG LEWHGWIITYNGNTIYAQRFQDRVTMTTDTSTSTAYM ELRS LRSD DTAVYYCARD RIIVRPDY FDY WGQGTLVTVSSSEQ ID No: 88 VL 124 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPRTFGQGTKVEIK75 PCT/EP2011/058779 WO 2011/547986 SEQ ID No: 89 VH 001 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWNWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVnSVDTSKNQFSLKLSSVTAADTAVYYCARGNYGSGYYYFDLWGRGTQVTVSSSEQ ID No: 90 VL 001 DIQNTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIFAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCGQYISFPITFGQGTRLEIKSEQ ID No: 91 VH 143 QVQLQQWGAGLLKPSETISLTCAVYGGSFSGYYWNWIRQPPGKGLEWIGEIHHSGSANYNPSLMSRVTISVDTSKNQFSLQLSSVTAADTAVYYCARGYYGSGYYYFDYWGQGTLVTVSSSEQ ID No: 92 VL 143 DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSUYAASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFA1YYCQOYNSYPUFGQGTRLEIKSEQ ID No: 93 VH 019 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWrRQPPGKGLEWIGEIHHVGSTNYNPSLKSRVTISVDTSKSQFSLKLSSVTAADTAVYYCARGYYDSGVYYFDYWAOGTLVTVSSSEQ ID No: 94 VL 019 DIQMTQSPSSLSASVGDRVTrrCRASQGISRWLAWYQQKPEKAPKSLIYAASSLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPITFGOGTRLEIKSEQ ID No: 95 VH 021 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKGLEWIGEIHHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGYYASGVYYFDYWGQGTLVTVSSSEQ ID No: 96 VL 021 DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEK APKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQYNSYPITFGQGTRLEIKSEQ ID No: 97 VH 027 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYFWNWIRQPPG KGLE WIGEIH H SG STNYN PS LKSRVTIS VDTSKNQFS LN LSSVT AADTAVYYCARGLIGSGYYYFDYWDQGTLVTVSSSEQ ID No: 98 VL 027 DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPITFGOGTRLEIKSEQ ID No: 99 VH 032 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGDTNYNPSLTSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLFYGSGIYYFDYWGQGTLVTVSSSEQ ID No: 100 VL 032 DIQMTQSFSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYATFRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSFPPTFGQGTKVEIKSEQ ID No: 101 VH 035 QVQLQQWGAGLLKPSETLSLTCAIYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGDTNYNPSLTSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLFYGSGIYYFDYWGQGTLVTVSSSEQ ID No: 102 VL 035 DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYATFRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSFPPTFGQGTKVEIKSEQ ID No: 103 VH 036 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLYYGSGTYYFDYWGQGTLVTVSSSEQ ID No: 104 VL 036 DIQMTQSPSSLSASVGDRVTITCRASQGISSWLTWYQQKPEKAPKSLIYAASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSFPPTFGQGTKVEIKSEQ ID No: 105 VH 054 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIHHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLWYGSGSYYFDYWGQGTLVTVSSSEQ ID No: 106 VL 054 DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSFPPTFGGGTKVEIK76 PCT/EP201 i/058779 WO 2011/147986 SEQ ID No: 107 VH 094 QVQLQQWGAGLLKPSETLSLTCAVSGGSFSGYYWTWIRQPPGKGLEWIGEIYHSGDTNYNPSLKSRVTfSVDTSKNQFSLKLYSVTAADTAVYYCARLYFGSGIYYLDYWGQGTLVTVSSSEQ ID No: 108 VL 094 DIQHTQSPSSLSASVGDRVTITCRASQGISSWLVWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSFPPTFGQGTKVEIKSEQ ID No: 109 VH 105 EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWVSAISGSAYSTYYADSVKGRFTISRDNSKNTLWLQMNSLRAEDTAVYYCAKAHYHGSGSYYTLFDYWGQGTLVTVSSSEQ ID No: 110 VL 105 DIQMTQSPSSLSASVGDRVTTTCRASQGISSWLAWYQQKPEK APKSLIYAASSLQSGVPSRFSGSG SGTD FTLTISSLQPEDFATYY CQGYNSYPYTFGGGTKLEIKSEQ ID No: 111 VH 100 EVQLLESGGGLVQPGGS LRLSCAASGFTFN NYG M N WVRQ APG KGLEWVSAISGTGYSTYYADSVKGRFTISRDNSKNTLYLQMNS LRAEDTAVYYCAKAHYFGSGSYYTLFDYWGOGTLVTVSSSEQ ID No: 112 VL 100 DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEK APKSLIYAASSLQSGVPSRFSGSGSGTD FTLTISSLQPEDFATYY CQOYNSYPYTFGOGTKLEIKSEQ ID No: 113 VH 125 EVQLLESGGGLVQPGGSLRLSCAASGFTFTDYAMNWVRQAPGKGLEWVSTISGSGYATYYADSVKGRFTISRDNSKTTLYLQMNSLRAEDTAVYYCAKGHTLGSGSYYTLFDYWGOGTLVTVSSSEQ ID No: 114 VL 125 DIQMTQSPSSLSASVGDRVTTTCRASQGINSWLAWYQQKPEK APKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQYNS YPYTFGGGTK LEI KSEQ ID No: 115 VH 162 EVQLWESGGGSVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLEWVSGISGSGYSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGYYHGSGSYYTSFDYWGQGTLVTVSSSEQ ID No: 116 VL 162 DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGGGTKVEIKSEQ ID No: 117 VH 033 QVQLVESGGGVVQTGRSLRLSCAASGFTFSSHAMHWVRQAPGKGLEWVAAISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGDYISSSGVFDYWGQGTLVTVSSSEQ ID No: 118 VL 033 DIQMTQS PSSLSASVGD RVTITCRASQGISSW LAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPITFGQGTRLEIKSEQ ID No; 119 VH 160 QVQLVESGGGWQPGRSLRLSCAASGFTFSSHAMHWVRQAPGKGLEWVAAISYDGSNKYYADSVKGRFTISRDNSKNTMYLQMNSLRAEDTAMCYCARGSITGSTGVFDYWGQGTLVTVSSSEQ ID No: 120 VL 160 DIQMTQSPSSLSASVGDRVTITCRASQDISSWLAWYQQKPEK APKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYY COO YNSYPITFGQGTRLEI KSEQ ID No: 121 VH 166 QVQLVESGGGWQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISYDGSNEYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSIIGSTGVFDYWGOGTLVTVSSSEQ ID No: 122 VL 166 DIQMTQSPSSLSASVGDRVTrrCRASQGISNWLAWYQQKPEKAPKSLIYDASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPITFGQGTRLEIKSEQ ID No: 123 VH 152 QVQWESGGGWQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISYDGSYKYYADSVKGRFTISRDNSKNTLYLQMIMSLRAEDTAVYYCARGSITGSTGVFDYWGOGTLVTVSSSEQ ID No: 124 VL 152 DIQMTQSPSSLSASVGDRVTITCRASQGINSWLAWYQQKPEKAPKSLIYDASSLQSGVPSRFSGSGSGTDFTLTISSLQPENFATYYCQQYNSYPITFGQGTRLEIK77 PCT/EP2011/058779 WO 2011/147986 SEQ ID No: 125 VH 167 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAIHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMN5lraedtavyycargsitgstgvfdywgqgtlvtvssSEQ ID No: 126 VI167DIQMTQSPSSLSASVGDRSmTCRASQGISNWLAWYQQKPEkAPKSLIYDASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCOOYNSYPITFGOGTRLEIK Table 4: Consensus CDRs based on sequence alignments shown in Figures 1 and 2.
SEQ ID No: 9050-049-051-055 IgHV3-23־l VHCDR1GFTFSSYA SEQ ID No: 127־ 050-049-051055 IgHV3-23-l VHCDR2ISGX1GGX2T Wherein X1=R or S, andX2-T or S־ preferably,wherein X1־=R and X2-TSEQ ID No: 11050-049-051-055 IgHV3 ־ 23 ־ l VHCDR3AKARANWDYFDY SEQ ID No: 128084IgHVl-69-04 VHCDR1GGTFX1X2YA Wherein X1=R or S, andX2=T or S; preferably,wherein X1=R and X2=TSEQ ID No: 129084IgHVl-6904״ VHCD R2IX2X3X3LGIX4 Wherein XI-N or I, X2=T orP, X3=V or I, and X4=V or A,preferably, wherein X1=N,X2־־T, X3=V, and X4=VSEQ ID No: 130084IgHVl-69-04 VHCDR3AREKGVDYYYGX1X2Wherein XI=1 or M, X2=E orD; preferably, wherein XI=1,X2=ESEQ ID No: 131־ 169-123-161124 IgHVl-18-1 VHCDR1GYTFTXYG Wherein X=N or S, preferablyN SEQ TD No: 132- 161 ־ 123 ־ 169124 IgHVl-18-1 VHCD R2IX1X2YX3GNT Wherein XI=S, Tr or I; X2=Aor T; X3=S or N; preferably,wherein X1=S, X2=A, andX3=S PCT/EF2011/058779 WO 2011/147986 SEQ ID No: 133169-123-161-124 IgHVl ־ 18 ־ l VHCDR3ARDRX1X2VRPDYFDYWherein XI=1 or V, X2=V orI; preferably, wherein X1=Iand X2=VSEQ ID No: 134־ 025-001-143027 ־ 019-021 IgHV4-34-01 VHCDR1GGSFSX1YX2 Wherein X1=D or G andX2=Y or F; preferably,wherein X1=D and X2=YSEQ ID No: 135025-001-143-019-021-027 IgHV4-34-01 VHCDR2IX1HX2GSX3 Wherein X1=H or N, X2=S orV, and X3=T or A; preferably,wherein X1=H, X2=S, andX3=TSEQ ID No: 136- 143 ־ 025-001019-021-027 IgHV401~34־ VHCDR3ARGX1X2X3SGX4YYFDX5Wherein XI =Y, N or L; X2=Yor I, X3=D, G or A; X4=V0rY; and X5=Y or L; preferably,wherein X1=Y, X2=Y, X3=D,X4־V, and X5=YSEQ ID No: 137- 035 ־ 032 ־ 091036-054-094 IgHV4 ־ 34 ־ Ql VHCDR1GGSFSX1YY Wherein XI=G or D,preferably G SEQ ID No: 138091-032-035-036-054-094 IgHV4-34-01 VHCDR2IX1HSGX2T Wherein X1=Y, N or H■ andX2=D or S; preferably,wherein X1=Y and X2=DSEQ ID No: 139091-032-035-036-054-094 IgHV4-34-01 VHCDR3ARLX1X2GSGX3YYX4DYWherein X1=Y, F or W; X2=־For Y; X3=I, T or S; and X4=־Lor F; preferably, whereinXl-Y, X2=F, X3=I, and X4־LSEQ ID No: 140129IgHV330-01־ VHCDR1GFTFSX1X2A Wherein X1=T or F, X2=F orY; preferably, wherein XI=Tand X2=FSEQ ID No: 141129IgHV3-3001־ VHCDR2ISYDGX1X2K Wherein XI=G or S, X2=H orN; preferably, wherein X1=Gand X2=HSEQ ID No :142129IgHV33־Q01־ VHCDR3ARGLGVWGX1FDYWherein XI =A or Y,preferably A PCT/EP2011/058779 WO 2011/147986 SEQ ID No: 143098-105-100-125-162 IgHV3-2301־ VHCDR1GFTFX1X2YX3 Wherein X1־=S, N orT; X2=N,D or S; and X3=G or A;preferably, wherein X1=S,X2=N and X3=GSEQ ID No: 144־ 100 ־ 105 ־ 098162 ־ 125 IgHV301 ־ 23 ־ VHCDR2ISGX1X2X3X4T Wherein X1=־S or T, X2=A orG, X3=Y or G, X4=S or A;preferably, wherein X1=S,X2=A, X3=Y, X4-SSEQ ID No: 145- 100 ־ 098-105125-162 IgHV301 ־ 23 ־ VHCDR3AKX1X2X3X4GSGSYYTX5FDYWherein Xl-A or G; X2=H orY; X3־־Y orT; X4=H, F or L;X5=L or S; preferably,wherein X1=A; X2־=H; X3=Y;X4=H;X5-LSEQ ID No: 146127IgHV5-5101־ VHCDR1GYSFX1X2YW Wherein XI=S or T, X2=I orS; preferably, wherein X1=S,X2=ISEQ ID No: 147127IgHV501 ־ 51 ־ VHCDR2IX1PGDSDX2 Wherein X1=־F or Y, X2=I orT; preferably, wherein XI=F,X2=ISEQ ID No: 148127IgHV551-01־ VHCDR3ARQPGDWSPRHWYFDLSEQ ID No :149159IgHV5-5101־ VHCDR1GYXFTSYW Wherein X=N or S, preferablyNSEQ ID No: 51159IgHV5-5101־ VHCDR2IYPGDSDT SEQ ID No:52159IgHV501 ־ 51 ־ VHCDR3ARWGTYYDILTGYFNSEQ ID No:71132lgHVl-1801־ VHCDR1GYTFTSYG SEQ ID No: 150132IgHVl-1801־ VHCDR2ISAYNGNX Wherein X=S or T, preferablySSEQ ID No: 151132IgHVl-18-01 VHCDR3AREYSYDSGTYFYYGMDVSEQ ID No:152- 160 ־ 153-033167 ־ 152 ־ 166 IgHV3-30-03-01VHCDR1GFTFSX1X2X3 Wherein XI-D or S, X2=Y orH, X3=V or A; preferably,wherein XI = D, X2=Y, X3=V 80 PCT/EP2011/058779 WO 2011/147986 SEQ ID No: 153153-033-160-166-152-167 IgHV3-30-03-01VHCDR2ISYDGSX1X2 Wherein X1=N or Y, X2=K orE, preferably wherein XI-Nand X2=KSEQ ID No :154153-033-160-166-152-167 IgHV3-30-־ 03VHCDR3AR.GX1X2X3X4X5X6GX7FDYWherein X1=G, D or S; X2=Ior Y; X3=T or I; X4=G or S;X5=T or S; X6=T or S; X7־־Yor V; preferably, whereinX1=G; X2=I; X3=T; X4=G;X5=T; X6=T; and X7=VSEQ ID No:13050-084-049-051-055 IgKVl-12-01 VLCDR1QGISSW - 049 ־ 050-084051-055IgKVl-12-01 VLCDR2XAS Wherein X=A or V SEQ ID No: 155050-084-049-051-055 IgKVl-12-01 VLCDR3QQANSFPXT Wherein X=I or L SEQ ID No:6169-124-161-123 IgKV3-ll-01 VLCDR1QSVSSY 169-124-161-123IgKV3-il-01 VLCDR2DXS Wherein X=A orT, preferablyASEQ ID No: 156169-124-161-123 IgKV3-1161־VLCDR3QQRSXWPRT Wherein X=N or H, preferablyN SEQ ID No: 157025-001-019-143-021-027 IgKVlD-16-VLCDR1QGISXW Wherein X=R or S, preferablyR - 019 ־ 025-001143-021-027IgKVlD-16-VICDR2AAS SEQ ID No: 164025-001-019-143-021-027 IgKVlD-16-VLCDR3QQYNSXPIT Wherein X=Y or F, preferablyY SEQ ID No:33־ 091-032-035094 ־ 054 ־ 036 IgKVlD-16-VLCDR1QGISSW PCT/EP2011/058779 WO 2011/147986 - 035 ־ 091-032036-054-094IgKVlD-16-VLCDR2AX1X2 Wherein X1=A or T, andX2=S or F; preferably,wherein X1־A and X2־=SSEQID No: 158091-032-035-036-054-094 IgKVlD-16-VLCDR3QQYNSFPPT SEQ ID No: 159098-100-105-162 ־ 125 IgKVlD-16-VLCDR1QGIXSW Wherein X=S or N, preferablyS - 105 ־ 098-100125-162IgKVlD-16-VLCDR2AAS SEQ ID No :160098-100-105-125-162 IgKVlD-16-VLCDR3QQYNSYPXT Wherein X=Y or L, preferablyY SEQ ID No: 161- 166 ־ 153-152167-160-033 IgKVlD-16-VLCDR1QGIX1X2W Wherein X1=S or N; X2=S orN; preferably, whereinX1=X2=S- 153-152-166033 ־ 167-160IgKVlD-16-VLCDR2XAS Wherein X=D or A, preferablyDSEQ ID No: 162153-152-166-167-160-033 IgKVlD-16-VLCDR3QQYXSYPIT Wherein X=K or N, preferablyK Example 11 - Purification of antibodies Culture supernatant was filtered over 0.2 pm dead-end filters, loaded on 5 ml MabSelect SuRe columns (GE Health Care) and eluted with 0.1 M sodium cltrate-NaOH, pH 3. The eluate was immediately neutralized with 2M Tris-HCI, pH 9 and dialyzed overnight to 12.mM NaH2P04, 140 mM NaCI, pH 7.4 (B.Braun). Alternatively, subsequent to purification, the eluate was loaded on a HiPrep Desalting column and the antibody was exchanged into 12.6 mM IMaH2P04, 140 mM NaCI, pH 7.4 (B.Braun) buffer. After dialysis or exchange of buffer, samples were sterile filtered over 0.2 pm dead-end filters. Purity was determined by SDS-PAGE and concentration was measured by nephelometry and absorbance at 280 nm. Purified antibodies were stored at 4°C. Mass spectrometry was performed to identify the molecular mass of the antibody heavy and light chains expressed by the hybridomas as described in Example 9. 82 PCT/EP2011/058779 WO 2011/147986 Example 12 - Binding of HER2 clones to tumor cells expressing membrane-bound HER2 measured by means of FACS analysis The binding of HER2 antibodies to AU565 cells (purchased at ATCC, CRL-2351) and A4cells (purchased at ATCC, CRL-1555), was tested using flow cytometry (FACS Canto II, BD Biosciences). Qifi analysis (Dako, Glostmp, Denmark) revealed that AU565 celts expressed on average 1,000,000 copies of HER2 protein per cell, whereas A431 cells expressed on average 15,000 copies per cell. Binding of HER2 antibodies was detected using a Phycoerythrin (PE)-conjugated goat-anti-human IgG antibody (Jackson). Trastuzumab (clinical-grade Herceptin0) was used as positive control antibody, and an isotype control antibody was used as negative control antibody. ECso values were determined by means of non-linear regression (sigmoidal dose-response with variable slope) using Graph Pad Prism V4.03 software (GraphPad Software, San Diego, CA, USA),As shown in Figure 3, all tested HER2 antibodies bound to HER2 expressed on both AU565 and A431 cells in a dose-dependent manner. The EC50 values for binding varied between 0-336-2.290 pg/mL for AU565 cells and 0.068-1.135 pg/mL for A431 cells. Especially on A431 cells, large differences in EC5o values were observed between the tested antibodies. However, antibody 098 had the best (i.e., lowest) ECso value on both types of cells. Also some differences in maximum binding levels were observed between different antibodies, on both AU565 and A431 cells. Of the tested antibodies, antibody 098 also had the highest maximum binding level on AU565 cells, whereas antibody 025 had the highest maximum binding level on A431 cells.
Example 13 ־ Binding of HER2 antibodies to membrane-bound HER2 expressed on Rhesus epithelial cells measured by means of FACS analysis To determine cross-reactivity with Rhesus HER2, the binding of HER2 antibodies to HER2- positive Rhesus epithelial cells (4M8r-5 purchased at ATCC) was tested using flow cytometry (FACS Canto II, BD Biosciences). A Phycoerythrin-conjugated goat-anti-human IgG antibody (Jackson) was used as a secondary conjugate. An isotype control antibody was used as negative control antibody.As shown in Figure 4, all tested HER2 antibodies were cross-reactive with Rhesus monkey HER2. At both tested concentrations (1 pg/mL and 10 pg/mL), the HER2 antibodies were able to bind specifically to Rhesus monkey HER2. Antibody 127 demonstrated poor binding at 1 pg/mL concentration, but showed good binding at 10 pg/mL concentration. Antibody 098 had the highest binding level at both antibody concentrations. No binding was observed with the isotype control antibody.
PCT/EP2011/058779 WO 2011/147986 Example 1 4 - Competition of HER2 antibodies for binding to soluble Her2ECDHis measured in sandwich-ELI SA The optimal coating concentrations of the tested HER2 antibodies and optimal Her2ECDHis concentration were determined in the following manner: ELISA wells were coated overnight at 4°C with HER2 HuMabs serially diluted in PBS (0.125-8 pg/mL in 2-fold dilutions). Next, the ELISA wells were washed with PBST (PBS supplemented with 0.05% Tween-20 [Sigma- Aldrich, Zwijndrecht, The Netherlands]) and blocked for one hour at room temperature (RT) with PBSTC (PBST supplemented 2% [v/v] chicken serum [Gibco, Paisley, Scotfand]). The ELISA wells were then washed with PBST and incubated for one hour at RT with Her2ECDHis serially diluted in PBSTC (0.25-2 pg/mL in 2-fold dilutions). Unbound Her2ECDHis was washed away with PBST, and bound Her2ECDHis was incubated for one hour at RT with 0.25 pg/mL biotinylated rabbit-anti-6xhis-biot (Abeam, Cambridge, UK), The plate was thereafter washed with PBST and incubated for one hour with 0.1 pg/mL Streptavidin-poly- HRP (Sanquin, Amsterdam, The Netherlands) diluted in PBST. After washing, the reaction was visualized through a 15 minutes incubation with 2,2'-azino-bis (3-ethylbenzothiazoiine- 6-sulfonic add (ABTS: one ABTS tablet diluted in 50 mL ABTS buffer (Roche Diagnostics, Almere, The Netherlands)) at RT protected from light. The colorization was stopped by adding an equal volume of oxalic add (Sigma-Aldrich, Zwijndrecht, The Netherlands). Fluorescence at 405 nm was measured on a microtiter plate reader (Biotek Instruments, Winooski, USA). The antibody concentrations that resulted in sub-optimal binding of each antibody were determined and used for the following cross-block experiments.Each HER2 antibody was coated to the ELISA wells at the sub-optimal dose that was determined as described above. After blocking of the ELISA wells, the wells were incubated with the predetermined concentration of 1 pg/mL biotinylated Her2ECDHis in the presence or absence of an excess of a second (competitor) HER2 antibody. The ELISA was then performed as described above. Residual binding of Her2ECDHis to the coated antibody was expressed as a percentage relative to the binding observed in the absence of competitor antibody. Percentage competition was then determined as 100 minus the percentage of inhibition. 75% competition was considered as complete cross-block, whereas 25-74% competition was considered as partial cross-block, and 024%־ competition was considered non-blocking.As shown in Table 5, all HER2 antibodies were found to be able to block binding to Her2ECDHis, at least partially, for themselves. After dividing the antibodies into 3 major cross-block groups, all antibodies were tested for competition with at feast one representative antibody from each group.
PCT/EP2011/058779 WO 2011/147986 The first group comprised trastuzumab and antibodies 169, 050 and 084, which blocked each other for binding to Her2ECDHis, but did not cross-block antibodies from other groups.The second group comprised pertuzumab and antibodies 025, 091 and 129, which blocked each other for binding to Her2ECDHis, except for antibodies 129 and 091 which both cross-blocked pertuzumab and 025, but not each other. None of the antibodies of group 2 blocked antibodies from other groups.A third group comprised antibodies Cl, F5, 127, 098, 132, 153 and 159, which did not cross-block any antibody from the other groups. Within this group 3, some variation was observed. Antibody 127 was the only antibody that was able to cross-block all other antibodies in this group for binding to Her2ECDHis; antibody 159 cross-blocked all other antibodies within this group, except 132; done 098 cross-blocked all antibodies of group 3, except 132 and 153; antibody 153 cross-blocked 127, 132 and 159 for binding to Her2ECDHis, but not 098, Cl or F5; clone 132 cross-blocked 127, 132 and 153. When added as competitor antibodies, F5 and Cl only demonstrated cross-blocking of each other. However, the reverse reaction aiso revealed competition with antibodies 127, 098 and 159, but not 153 and 132. Possibly, these differences may have resulted from lower affinities of antibodies Cl and F5 for Her2ECDHis.Values higher than 100% can be explained by avidity effects and the formation of antibody-Her2ECDHis complexes containing two non-competing antibodies.
Table 5: Competition and cross-blocking of HER2 antibodies for binding to Her2ECDHis PCT/EP2011/058779WO 2011/147986 TH1014-098 107 102 100 103 104 108 104 107 125 96n uung 110 142 TH1014-153 134 111 103 107 121 97 102 106 257 96 iiBgff 115ו!ו TH1014-132 353 ND ND ND 288 ND ND ND 422 379iH!131 309!||1Cross-blockgroup1 1 1 2 2 2 2b 3a 3a 3a 3a 33 3b 3b k) - 24% competition! Depicted values are mean percentages of binding relative to the binding observed in the absence of competitor antibody, of two independent experiments. Competition experiments with HEK produced TH1Q14-C1 and TH1014-F5 were performed once. Trastuzumab (clinical grade Herceptin®) and HEK-produced pertuzumab (TH1014-pert) were also tested.
Example 15 • Antibody-dependent cell-mediated cytotoxicity (ADCC) SK-BR-3 cells (purchased at ATCC, HTB-30) were harvested (5xl06 cells), washed (twice in PBS, 1500 rpm, 5 min) and collected in 1 mL RPMI 1640 medium supplemented with 10% cosmic calf serum (CCS) (HyClone, Logan, UT, USA), to which 200 pCi slCr (Chromium-51; Amersham Biosciences Europe GmbH, Roosendaal, The Netherlands) was added. The mixture was incubated in a shaking water bath for 1.5 hours at 37°C. After washing of the cells (twice in PBS, 1500 rpm, 5 min), the cells were resuspended in RPMI 1640 medium supplemented with 10% CCS, counted by trypan blue exclusion and diluted to a concentration of 1x105 ceils/mL.Meanwhile, peripheral blood mononuclear cells (PBMCs) were isolated from fresh buffy coats (Sanquin, Amsterdam, The Netherlands) using standard Ficoll density centrifugation according to the manufacturer's instructions (lymphocyte separation medium; Lonza, Verviers, France). After resuspension of cells in RPMI 1640 medium supplemented with 10% CCS, cells were counted by trypan blue exclusion and concentrated to lxlQceiis/mLTrastuzumab was produced in CHO cells resulting in an (increased) non-core fucosylation grade of 12.4%, whereas the other HER2 antibodies were produced in HEK cells, resulting on average in 4% non-core fucosylation.For the ADCC experiment, 50 pL 51Cr-labeled SK-BR-3 cells (5.000 cells) were pre- incubated with 15 pg/mL HER2 antibody (IgGl,x) in a total volume of 100 pL RPMI medium supplemented with 10% CCS in a 96-well microtiter plate. After 15 min at RT, 50 pL PBMCs (500,000 cells) were added, resulting in an effector to target ratio of 100:1. The maximum86 PCT/EP2011/058779 WO 2011/147986 amount of cell lysis was determined by incubating 50 pLslCr-labeled SK-BR-3 cells (50ceiis) with 100 pL 5% Triton-XIOO. The amount of spontaneous lysis was determined by incubating 5000 slCr־labeied SK-BR-3 cells in 150 pL medium, without any antibody or effector cells. The level of antibody-independent cell lysis was determined by incubating 5000 SK-BR-3 cells with 500,000 PBMCs without antibody. Subsequently, the cells were incubated 4 hr at 37°C, 5% C02- To determine the amount of cell lysis, the cells were centrifuged (1200 rpm, 3 min) and 75 pL of supernatant was transferred to micronic tubes, after which the released 51Cr was counted using a gamma counter. The measured counts per minute (cpm) were used to calculate the percentage of antibody-mediated lysis as follows:(cpm sample - cpm Ab-independent fysis)/(cpm max. lysis • cpm spontaneous lysis)x 100% As shown in Figure 5, HER2 antibodies from cross-block groups 1 and 2 induced efficient lysis of SK-BR-3 cells through ADCC. From group 3, antibody 153 was the only antibody that induced efficient ADCC, antibody 132 induced about 10% ADCC, and clones 098, 1and 127 did not induce ADCC.
Example 16 - Inhibition of ligand-independent proliferation of AU565 cells HER2 antibodies were tested for their ability to inhibit proliferation of AU565 cells in vitro. Due to the high HER2 expression levels on AU565 cells (~ 1,000,000 copies per cell as described in Example 12), HER2 is constitutive!y active in these cells and thus not dependent on ligand-induced heterodimerization.In a 96-well tissue culture plate (Greiner bio-one, Frickenhausen, Germany), 90AU565 cells were seeded per well in the presence of 10 pg/mL HER2 antibody in serum-free cell culture medium. As a control, cells were seeded in serum-free medium without antibody. After 3 days, the amount of viable cells was quantified with Alamarblue (BioSource International, San Francisco, US) according to the manufacturer's instructions. Fluorescence was monitored using the EnVision 2101 Muitilabel reader (PerkinElmer, Turku, Finland) with standard Alamarblue settings. The Alamarblue signal of antibody-treated cells was plotted as a percentage relative to untreated cells. Dunnett's test was applied for statistical analysis.The results are shown in Figure 6, depicting the percentage proliferation of AU5cells after HER2 antibody treatment compared to untreated cells, which was set to 100%.Of the tested Group 1 antibodies, trastuzumab, 050 and 169 demonstrated significant inhibition of AU565 cell proliferation (P<0.05), whereas 084 had no effect. None of the tested antibodies from group 2 (Pertuzumab, 025, 092 and 129) was able to inhibit AU565 PCT/EP2011/058779 WO 2011/147986 cell proliferation. The tested antibodies from group 3 (098 and 153) did not inhibit AU5proliferation. In contrast, both antibodies induced enhanced proliferation of AU565 cells compared to untreated cells (098 more than 153). Enhancing proliferation can be an advantage in some therapeutic applications of ADC-conjugates, e.g,, where the cytotoxic action of the drug relies on, or is enhanced by, cell proliferation. For trastuzumab and pertuzumab, this was in accordance with the results described by !untilia et at. (Cancer Cell 2009; 15(5): 353-355).
Example 17 - Inhibition of ligand-induced proliferation of MCF-7 cells Since HER2 is an orphan receptor, its signaling is mainly dependent on activation of other ErbB-family members such as EGFR and Her3. Upon ligand binding, these two receptors can bind to and activate the HER2 receptor, resulting in e.g. proliferation. Various publications describe that pertuzumab efficiently inhibits Heregulin-pi-induced proliferation (Franklin MC. Cancer Cell 2004 / Landgraf R. BCR 2007). For trastuzumab, it has been described that it has little effect on Heregulin-pi-induced HER2/HER3 heterodimerization and proliferation (Larsen SS.׳ et at., Breast Cancer Res Treat 2000;58:41-56; Agus DB., ef a/., Cancer Cell 2002;2:127-137; Wehrman et at. (2006), supra).To investigate the ability of the present human HER2 antibodies to interfere with Heregulin-pi-induced HER2/HER3 heterodimers, a Heregulin-pl-induced proliferation assay was performed. Therefore, MCF7 cells (purchased at ATCC, HTB-22) expressing 20.000׳״ HER2 molecules per cell, were seeded in a 96-weils tissue culture plate (Greiner bio-one) (2.500 ceils/well) in complete cell culture medium. After 4 hours, the cell culture medium was replaced with starvation medium containing 1% Cosmic Calf Serum (CCS) and gg/mL HER2 antibody. Next, Heregulin-pl (PeproTech, Princeton Business Park, US) diluted in 1% CCS containing starvation medium was added to the wells to a final concentration of 1.5 ng/ml. After 4 days incubation, the amount of viable cells was quantified with Alamarblue (BioSource International) according to the manufacturer's instructions. Fluorescence was monitored using the EnVision 2101 Multilabel reader (PerkinElmer) with standard Alamarblue settings. The Alamarblue signal of HER2 antibody-treated ligand- induced cells was plotted as a percentage signal compared to ligand-induced cells incubated without HER2 antibody. Dunnett's test was applied for statistical analysis.Figure 7 shows the percentage of viable MCF7 cells stimulated with Heregulin-pi and treated with the indicated HER2 antibody, relative to the viable cells after stimulation with Hereguiin~p1 in the absence of HER2 antibody, which was set to 100%. MCF-7 proliferation in absence of both Heregulin-pl and antibody was also depicted (none). Antibodies 025, 091, 129, 153 and pertuzumab (TH1014־pert) demonstrated significant inhibition of 88 PCT/EP2011/058779 WO 2011/147986 Hereguiirv-pi-induced MCF-7 proliferation (P<0.05). Also trastuzumab showed some inhibition of Heregulin-pl-mduced proliferation of MCF-7 cells, although not as efficient as the other tested HER2 antibodies. It has been reported that domain IV of HER2 is involved in the stabilization of EGFR/HER2 heterodimers, but without details on its contribution to HER2/HER3 heterodimers (Wehrman ef a/., supra). Antibodies 050, 084, 169 and 098 had no statistically significant effect on Heregulin-pl-induced proliferation of MCF-7 cells. Without being limited to theory, this suggests that these antibodies do not inhibit ligand- induced HER2/HER3 heterodimerization.
Example 18 - Anti-kappa-ETA' assay To investigate the suitability of HER2 antibodies for an antibody-drug conjugate approach, a generic in vitro cell-based killing assay using kappa-directed pseudomonas-exotoxin A (anti- kappa-ETA') was developed. The assay makes use of a high affinity anti-kappa domain antibody conjugated to a truncated form of the pseudomonas-exotoxin A. Upon internalization, the anti-kappa-ETA' domain antibody undergoes proteolysis and disulfide- bond reduction, separating the catalytic from the binding domain. The catalytic domain is transported from the Golgi to the endoplasmic reticulum via the KDEL retention motif, and subsequently translocated to the cytosol where it inhibits protein synthesis and induces apoptosis (ref. Kreitman Rl. BioDrugs 2009;23(1):1-13). In this assay, to identify HERantibodies that enable internalization and killing through the toxin, HER2 antibodies are preconjugated with the anti-kappa-ETA' before incubation with HER2-positive cells.First, the optimal concentration of anti-kappa-ETA׳ was determined for each ceil line, i.e. the maximally tolerated dose that does not lead to induction of non-specific cell death. AU565 cells (7500 cells/well) and A431 ceils (2500 cells/well) were seeded in normal cell culture medium in 96-wells tissue culture plate (Greiner bio-one) and allowed to adhere for at least 4 hours. Next, cells were incubated with 100, 10, 1, 0.1, 0.01, 0.001 and 0 pg/mL anti-kappa-ETA׳ dilutions in normal ceil culture medium. After 3 days, the amount of viable cells was quantified with Alamarblue (BioSource International, San Francisco, US) according to the manufacturer's instruction. Fluorescence was monitored using the Envision 21Multilabel reader (PerkinElmer, Turku, Finland) with standard Alamarblue settings. The highest concentration anti-kappa-ETA' that did not kilt the cells by itself was used for following experiments (0.5 p9/mL for AU565 and 1 pg/mL for A431).Next, antibody-mediated internalization and killing by the toxin was tested for different HER2 antibodies. Cells were seeded as described above. Dilution-series of HERantibodies were pre-incubated for 30 minutes with the predetermined concentration anti- kappa-ETA׳ before adding them to the cells. After 3 days of incubation, the amount of viable PCT/EP2011/058779 WO 2011/147986 cells was quantified as described above. The Alamarblue signal of cells treated with anti- kappa-ETA' conjugated antibodies was plotted compared to cells treated with antibody alone. 23.4 pg/mL Staurosporin was used as positive control for ceil killing. An isotype control antibody was used as negative control.As shown in Figure 8A,B and Table 6, all anti - ka ppa - ETA'- conj ug ated HERantibodies were able to kill AU565 cells in a dose-dependent manner. All tested anti-kappa- ETA'-conjugated HER2 antibodies demonstrated better killing of AU565 cells compared to both anti-kappa-ETA'-conjugated trastuzumab and anti-kappa-ETA'-conjugated pertuzumab (TH1014-pert). Moreover, the percentage of killed AU565 cells was higher for anti-kappa- ETA'-conjugated HER2 antibodies (70.3 - 49,9 %), compared to anti-kappa-ETA'- conjugated trastuzumab (31.9 %) and anti-kappa-ETA'-conjugated pertuzumab (47.51 %), and the ECSo values were increased. EC50 values for anti-kappa-ETA'-conjugated HERantibodies ranged between 12.12 ng/mLand 46.49 ng/ml) compared to 78.49 ng/mLfor anti-kappa-ETA'-conjugated trastuzumab and 117.8 ng/ml for anti-kappa-ETA'-conjugated pertuzumab. Antibody 159 had the highest percentage of cell-kill, and 098 the lowest EC50.
Table 6: Data shown are EC50 values and maximal percentage cell kill of AU565 cells treated with anti-kappa-ETA'-conjugated HER2 antibodies, measured in one representative experiment. Cell-kill induced by Staurosporin was set as 100% and MFI of untreated cells was set as 0%. Ndet = not detected. antibody % ceils Killed fcCsu ng/mLPC1014-159 70.3 34.93PC1G14-127 69.0 34.46PC1014-132 61,6 39.35PC1014-129 60.8 30.85PC1014-153 60.3 32.26PC1014025־ 60.0 16.71PC 1014098־ 58.7 12.12PC1014084־ 58.1 26.97PC1014-050 52.4 12.71PC1014-Q91 50.6 46.49PC1014169־ 49.9 35.62TH1014־pert 47.5 117.8trastuzumab 31.9 78.49isotype control Ndet Ndet As shown in Figure 8C,D and Table 7, antibodies 025, 091, 098, 129 and 153 were able to induce effective killing of A431 cells (>75%). The highest percentage of cell-kill, and lowest ECso was shown by antibody 098. When conjugated to anti-kappa-ETA׳, trastuzumab and 90 PCT/EP2011/058779 WO 2011/147986 isotype control antibody did not induce killing of A431 cells. Antibodies 169, 084 and pertuzumab induced percentages of cell kill of no more than about 50%. No cell kill was observed with non-conjugated HER2 antibodies.
Table 7: Data shown are EC50 values and maximal percentage ceil kill of A431 cells treated with anti-kappa-ETA'-conjugated HER2 antibodies, measured in one representative experiment. Ceil kill induced by Staurosparin was set as 100% and MFI of untreated cells was set as 0%. "NDet" means not detected. % cells killed tc5u ng/mLPC 1014025־ 86.7 9.77 ״PC1014084־ 50.5 NDPC1014-091 83.3 9.86 ״PC1014098־ 87.2 1.65PC1014-129 75.9 10.60 ״PC1014153־ 82.4 10.11 ״PC1014-169 34.0 NOTH1014־ pert 37.0 61.58trastuzumab Ndet Ndetisotype control NDet NDet Example 19 - Internalization of HER2 antibodies measured with an FMAT-based fab- CypHerSE assay To investigate whether the enhanced killing of AU565 cells observed in the kappa-toxin-ETA' assay described in the previous Example correlated with enhanced internalization of HERantibodies, a fab-CypHer5E־based internalization assay was performed. CypHerSE is a pH sensitive dye which is non-fluorescent at basic pH (extracellular: culture medium) and fluorescent at acidic pH (intracellular: lysosomes), with an acid dissociation constant (pKa) of 7.3.AU565 cells were seeded in 384-well tissue culture plates (Greiner bio-one), at a density of 3000 cells/well in normal cell culture medium supplemented with 240 ng/mL fab- CypHerSE (conjugation of Goat-fab-anti-Human IgG [Jackson] with CypHerSE [GE Healthcare, Eindhoven, The Netherlands] was made according to manufacturer's instructions). Next, HER2 antibodies were serially diluted in normal cell culture medium, added to the cells and left at room temperature for 9 hours. Mean fluorescent intensities (MFI) of intracellular CypHer5E were measured using the 8200 FMAT (Applied Biosystems, Nieuwerkerk A/D Dssel, The Netherlands) and 'counts x fluorescence׳ was used as read-out. An isotype control antibody was used as negative control antibody. ECS0 values and maximal PCT/EP2011/058779 WO 2011/147986 MFI were determined by means of non-linear regression (sigmoidal dose-response with variable slope) using GraphPad Prism V4.03 software (GraphPad Software, San Diego, CA, USA).The results are shown in Table 8, depicting the EC50 and maximal MFI values for all tested HER2 antibodies in the CypHer5E internalization assay with AU565 cells. The maximal MFI values indicate how many HER2 receptors are internalized upon antibody binding. All HER2 antibodies showed higher maximal MFI values (137,904 - 38,801) compared to trastuzumab (35,000) and pertuzumab (TH1014־pert) (32,366), indicating that the tested HER2 antibodies induced enhanced receptor internalization. Notably, antibodies that did not compete with trastuzumab or TH1014־pert induced more receptor internalization compared to antibodies that did compete with trastuzumab and TH1014-pert, with the highest MFI achieved by antibodies 098 and 127. Without being limited to theory, this might be inherent to an inability to inhibit HER2 heterodimerization.
Table 8: Cypher-5-based internalization assay of HER2 antibodies. Data shown are MFI and ECso values of one representative experiment of two experiments with AUS65 cells treated with fab-CypHer5E~labeled HER2 antibodies. Some ECSo values could not be calculated (ND). mAbs that compete with Herceptin mAbs that compete with TH1014-pert mAbs that compete with TH1014-F5 Non-comoetina mAbs m₪₪₪M ■■■■■Antibody ECso ng/ mL Maximal MFIPC1014-025 30.05 63428PC1014-091 32.99 50711PC1014-129 7.15 60302TH1014-pert 530 32366PC1Q14-169 ND 38801PC1014-084 30.51 71059trastuzumab 21.70 35000PC1014-098 13.77 134575PC1014-127 9.68 ״ 137904PC1014-159 ND 92427TH1014-F5 22.65 113116PC1014-132 11.42 112270PC1014-153 14.91 ״ 87531 PCT/EP2011/058779WO 2011/147986 Example 20: Generation of bispecific antibodies by 2-MEA-induced Fab-artn exchange An in vitro method for producing bispecific antibodies is described in WO 20081193(Genmab) and reported van der Neut-Kolfschoten et al. (Science. 2007 Sep 14*,317(5844): 1554-7). Herein, a bispecific antibody is formed by "Fab-arm״ or "half- molecule" exchange (swapping of a heavy chain and attached light chain) between two monospecific IgG4- or IgG4־iike antibodies upon incubation under mildly reducing conditions. This Fab-arm exchange reaction is the result of a disulfide-bond isomerization reaction wherein the inter heavy-chain disulfide bonds in the hinge regions of monospecific antibodies are reduced and the resulting free cysteines form a new inter heavy-chain disulfide bond with cysteine residues of another antibody molecule with a different specificity. The resulting product is a bispecific antibody having two Fab arms with different sequences.In a novel invention the knowledge of this natural IgG4 Fab-arm exchange is adapted to generate a method to produce stable IgGl-based bispecific antibodies. The bispecific antibody product generated by this method described below will no longer participate in IgG4 Fab-arm exchange. The basis for tbis method is the use of complimentary CHdomains, which promote the formation of heterodimers under specific assay conditions. To enable the production of bispecific antibodies by this method, IgGl molecules carrying certain mutations in the CH3 domain were generated: in one of the parental IgGl antibody T350I, K370T and F405L mutations in the other parental IgGl antibody the K4Q9R mutation.To generate bispecific antibodies, these two parental antibodies, each antibody at a final concentration of 0.5 mg/mL (equimolar concentration), were incubated with 25 mM 2- mercaptoethylamine-HCI (2-MEA) in a total volume of 100 pL TE at 37°C for 90 min. The reduction reaction is stopped when the reducing agent 2-MEA is removed by using spin columns (Microcon centrifugal filters, 30k, Milfipore) according to the manufacturer's protocol.
Example 21 - HER2 x HER2 bispecific antibodies tested in an in vitro kappa- directed ETA’ killing assay Hie example shows that HER2 x HER2 bispecific antibodies can deliver a cytotoxic agent into tumor cells after internalization in a generic in vitro cell-based killing assay using kappa-directed pseudomonas-exotoxin A (anti-kappa-ETA׳). This assay makes use of a high affinity anti-kappa domain antibody conjugated to a truncated form of the pseudomonas- exotoxin A. Similar fusion proteins of antibody binding proteins (IgG-binding motif from PCT/EP2011/058779 WO 2011/147986 Streptococcal protein A or protein G) and diphtheria toxin or Pseudomonas exotoxin A have previously been (Mazor Y. efe ah, J. Immunol. Methods 2007; 321:4159־); Kuo SR. et a!., 2009 Bioconjugate Chem. 2009; 20:1975-1982). These molecules in contrast to anti-kappa- ETA' bound the Fc part of complete antibodies. Upon internalization and endocytic sorting the antl-kappa-ETA' domain antibody undergoes proteolysis and disulfide-bond reduction, separating the catalytic from the binding domain. The catalytic domain is then transported from the Golgi to the endoplasmic reticulum via a KDEL retention motif, and subsequently translocated to the cytosol where it inhibits protein synthesis and induces apoptosis (Kreitman RJ. et. al., BioDrugs 2009; 23:1-13).The anti־HER2 antibodies used in this example are 025,153 and 169. In addition a fully human monoclonal IgGl,1c antibody 005 of the following sequence was used: 005:SEQ IDNO :164VH 005 EVQLVQSGAEVKKPGESLKISCKASGYSFHFYWIGWVRQMPGKGLEWMGSIYPGDSDTRYRPSFQGQVTISADKSISTAYLQWTSLKASDTAIYYCARORGDYYYFYGMDVWGOGTTVTVSSSEQ IDNO: 165VL 005 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQVPRLLIYGASSRATGIPDRFSGSGSGTDFTLTTSRLEPEDFAVYYCQQYGSSLTFGGGTKVEIKSEQ IDNO :166VHCDR1GYSFHFYWSEQ ID NO:167VHCDR2YPGDSDTSEQ IDNO: 168VHCDR3ARQRGDYYYFYGMDVSEQ IDNO: 169VLCDR1QSVSSSYVLCDR2GASSEQ IDNO :170VLCDR3QQYGSSLT The following antibodies were used as starting materials: IgGl-005-m = 005 IgGl,K having He at position 350, Thr at position 370, and Leu at position 405IgGl־Q05־K409R = 005 IgGl,K having an Arg at position 409IgGl-025-ITL = 025 IgGl,K having lie at position 350, Thr at position 370, and Leu at position 405IgGl-153-lTL =153 IgGl,K having contains lie at position 350, Thr at position 370, and Leu at position 405IgGl-153-K409R =153 IgGl,K having an Arg at position 40994 PCT/EP2011/058779 WO 2011/147986 rgGl-169־K409R =169 IgGi,K having an Arg at position 409 The following b!specific antibodies were generated in analogy with the below procedure: IgGl ׳ 005 ־ rTL x IgGl ־ 169 ־ K409R IgGl ־ 025 ־ rTL x IgGl ־ 005 ־ K409R IgGl-025־ITL x IgGl-153-K409R IgGl-025-ITL x IgGl-169-K409R IgGl-153-ITL x IgGl-005-K409R IgGl ־ 153 ־ ITL x IgGl ־ 169 ־ K409R The bispecific antibodies were produced according to the procedure described in example 20 . The antibody mixtures, containing each antibody at a final concentration of 0.5 mg/mL, was incubated with 25 mM 2-mercaptoethylamine HCI (2-MEA) in a total volume of 100 jiLTE at 37°C for 90 min. To stop the reduction reaction, the reducing agent 2-MEA was removed by desalting the samples using spin columns (Microcon centrifugal filters, 30k, Millipore) according to the manufacturer's recommendations.The HER2 x HER2 bispecific antibodies were pre-incubated with the anti-kappa-ETA' before incubation with A431 cells. A431 cells express ~ 15,000 HER2 antibodies per cell (determined via Qift analysis) and are not sensitive to treatment with 'naked' HER2- antibodies.First, the optimal concentration of anti־kappa־ETA׳ was determined for each cell line, i.e. the maximally tolerated dose that does not lead to induction of non-specific cell death. A431 cells (2500 cells/well) were seeded in normal cell culture medium in a 96-wells tissue culture plate (Greiner bio-one) and allowed to adhere for at least 4 hours. These cells were incubated with an anti-kappa-ETA' dilution series, 100, 10, 1, 0.1, 0.01, 0.001 and 0 pg/mL In normal cell culture medium. After 3 days, the amount of viable cells was quantified with Alamarblue (BioSource International, San Francisco, US) according to the manufacturer's instruction. Fluorescence was monitored using the Envision 2101 Multilabel reader (PerkinElmer, Turku, Finland) with standard Alamarblue settings. The highest concentration anti-kappa-ETA' that did not kill the cells by itself (1 pg/mL for A431 cells) was used for following experiments.Next, the effect of HER2 x HER2 bispecific antibodies and HER2 monospecific antibodies pre-incubated with anti-kappa-ETA׳ was tested for their ability to induce cell kill. A431 cells were seeded as described above. A dilution series of the HER2 specific antibodies PCT/EP20U/0S8779 WO 2011/147986 (monospecific and bispecific antibodies) was made and pre-incubated for 30 min with the predetermined concentration of anti-kappa-ETA׳ before adding them to the ceils. After days incubation at 37°C, the amount of viable cells was quantified as described above. The Alamarblue signal of ceils treated with anti-kappa-ETA׳ pre-incubated with the antibodies was plotted compared to cells treated without antibody treatment. EC50 values and maxima! cell death were calculated using GraphPad Prism 5 software. Staurosporin (23.4 pg/mL) was used as positive control for cell killing. An isotype control antibody (IgGl/kappa; IgGl-3G8- Q[TL) was used as negative control.Figure 9 and table 9 shows that ail anti-kappa-ETA' pre-incubated HER2 bispecific antibodies were able to kill A431 cells in a dose-dependent manner. These results demonstrate that most HER2 bispecific antibodies tested were more effective than the monospecific antibody present in the combination in this anti-kappa-ETA׳ assay. In addition, the efficacy of bispecific antibody 005X169, 025X169 and 153X169 showed that the efficacy of a monospecific antibody which lacks activity in this in vitro kappa-directed ETA' killing, HER2 specific antibody (169), can be increased through bispecific combination with another HER2 specific antibody.
Table 9: ECS0 values and maximal percentage cell kill of AU565 cells treated with anti- kappa-ETA'-conjugated HER2 x HER2 bispecific antibodies. "Ndet" means not detected. antiDoay percentage kin fcCSu[ng/rriLjHerceptin 2.79 IMdetIgGl-005-ITL 79.34 2.57IgGl ־ 005 ־ K409R 79,83 2.87IgGl-025-ITL 69.81 3.76IgGl-153־ITl 70.66 12.45IgGl ־ 153 ־ K4Q9R 72.84 15.47IgGl ־ 169 ־ K409R 16.45 3.45IgGl-005-ITL x IgGl ־ 169 ־ K409R 59.94 4.28IgGl-025-ITL x IqGl-005-K409R 63,45 4.27IgGl-025-UL x IqGl-153-K409R 80.82 7.66IgGl-025-ITL x IgGl ־ 169 ־ K4Q9R 45.88 7.97IgGl-153-ITl x IgGl-005-K409R 80.05 4.51IqGl ־ 153 ־ ITL x IqGl ־ 169 ־ K409R 84.68 29.14 Example 22 - HER2 receptor downmodulation by incubation with bispecific antibodies targeting different HER2 epitopes HER2 x HER2 bispecific antibodies may bind two different epitopes on two spatially different HER2 receptors. This may allow other HER2 x HER2 bispecific antibodies to bind to the remaining epitopes on these receptors. This could result in multivalent receptor cross-96 PCT/EP2011/058779 WO 2011/147986 linking (compared to dimerization induced by monovalent antibodies) and consequently enhance receptor downmodulation. To investigate whether HER2 x HER2 bispecific antibodies induce enhanced downmodulation of HER2, AU565 cells were incubated with antibodies and bispecific antibodies for three days. Total levels of HER2 and levels of antibody bound HER2 were determined.AU565 cells were seeded in a 24-weli tissue culture plate (100.000 cells / well) in normal cell culture medium and cultured for three days at 37°C in the presence of 10 pg/mL HER2 antibody with either the ITL or the K409R mutation or HER2 x HER2 bispecific antibodies. As a control, the combination of two monospecific HER2 antibodies, with unmodified IgGl backbones, was also tested (1:1), at a final concentration of 10 pg/mL. After washing with PBS, cells were lysed by incubating them for 30 min at room temperature with 25 pL Surefire Lysis buffer (Perkin Elmer, Turku, Finland). Total protein levels were quantified using bicinchoninic acid (BCA) protein assay reagent (Pierce) following manufacturer's protocol. HER2 protein levels in the lysates were analyzed using a HER2־specific sandwich ELISA. Rabbit-anti-humaח HER2 intracellular domain antibody (Cell Signaling) was used to capture HER2 and biotinylated goat-anti ־human HER2 polyclonal antibody R&D systems, Minneapolis, USA), followed by streptavidin-poly-HRP, were used to detect bound HER2. The reaction was visualized using 2,2'-azino־bis 3-ethylbenzothiazoline- 6-sulfonic acid (one ABTS tablet diluted in 50 mL ABTS buffer [Roche Diagnostics, Aim ere, The Netherlands]) and stopped with oxalic acid (Sigma-Aldrich, Zwijndrecht, The Netherlands). Fluorescence at 405 nm was measured on a microtiter plate reader (Biotek Instruments, Winooski, USA) and the amount of HER2 was expressed as a percentage relative to untreated cells.The results are shown in Figure 10 and Table 10 which demonstrates that all the tested HER2 x HER2 bispecific antibodies induced >40% HER2 downmodulation. Interestingly, all HER2 x HER2 bispecific antibodies demonstrated increased HERdownmodulation compared to both of their monospecific counterparts.
Table 10: HER2 x HER2 bispecific induced downmodulation of HER2 depicted as percentage HER2 compared to untreated cells PCT/EP2011/058779 WO 2011/147986 antibody % Htki compared to untreated cellsHerceptin 71IgGl-005-ITL 54IgGl-005־K409R 50IgGl-025-ITL 64IgGl-153-ITL 43IqGl-153-K409R 40IgGl ־ 169 ־ K409R 64IgGl005־-ITL x IqGl ־ 169 ־ K409R 29[qGl-025־ETL x IgGl-005-K409R 38IgGl ־ 025 ־ rTL x EqGl-153-K409R 29EgGl-025-ETL x EgGl-169-K409R 34EgGl-153-ETL x EqGl ־ 005 ־ K409R 23IgGl-153~ITL x EqGl-169-K409R 28rgGl-005 + IgGl-169 28IgGl-025 + IgGl-005 28EgGl-025 + IgGl-153 23IgGl-025 ■f IqGl-169 25IgGl-153 + IqGl-005 23IgGl-153 + IqGl-169 23isotype control 108 Example 23 - Colocalization of HER2 x HER2 bispecific antibodies with lysosomal marker LAMP1 analyzed by confocal microscopy The HER2 downmodulation assay as described in Example 22 indicated that HER2 x HER2 bispecific antibodies were able to increase lysosomal degradation of HER2. To confirm these findings, confocal microscopy technology was applied. AU565 cells were grown on glass coverslips (thickness 1.5 micron, Thermo Fisher Scientific, Braunschweig, Germany) in standard tissue culture medium at 37°C for 3 days. Cells were pre-incubated for 1 hour with pg/mL leupeptin (Sigma) to block lysosomal activity after which 10 pg/mL HERmonospecific antibodies or HER2 x HER2 bispecific antibodies were added. Also the combination of two monospecific IgGl antibodies (1:1) was tested at a final concentration of 10 pg/mL. The cells were incubated for an additional 3 or 18 hours at 37״C. Hereafter the ceils were washed with PBS and incubated for 30 min. at room temperature with 4% formaldehyde (Klinipath). Slides were washed with blocking buffer (PBS supplemented with 0.1% saponin [Roche] and 2% BSA [Roche]) and incubated for 20 min with blocking buffer containing 20 mM NH4CI to quench formaldehyde. Slides were washed again with blocking buffer and incubated for 45 min at room temperature with mouse-anti-human CD107a (LAMP1) (BD Pharmingen) to stain/identify lysosomes. Following washing with blocking buffer, the slides were incubated 30 min at room temperature with a cocktail of secondary antibodies; goat-anti-mouse IgG-Cy5 (Jackson) and goat-anti-human IgG-FITC (Jackson). Slides were washed again with blocking buffer and mounted overnight on microscope slides using 20 pL mounting medium (6 gram Glycerol [Sigma] and 2.4 gram Mowiol 488־98 PCT/EP2011/058779 WO 2011/147986 [Omnilabo] was dissolved in 6 mL distilled water to which 12 mL 0.2M Tris [Sigma] pH8.was added followed by incubation for 10 min at 50-60°C. Mounting medium was aliquoted and stored at -20°C.). Slides were imaged with a Leica SPE-II confocal microscope (Leica Microsystems) equipped with a 63x 1.32-0.6 oil immersion objective lens and LAS-AF software. To allow for quantification of overlapping pixel intensities, saturation of pixels should be avoided. Therefore the FITC laser intensity was decreased to 10%, smart gain was set at 830 V and smart offset was set at -9.48 %. By using these settings, the bispecific antibodies were dearly visualized without pixel saturation, but the monospecific antibodies were sometimes difficult to detect To compare lysosomal colocalization between monospecific and bispecific antibodies, these settings were kept the same for all analyzed confocal slides.12-bit grayscale TIFF images were analyzed for colocalisation using MetaMorph® software (version Meta Series 6.1, Molecular Devices Inc, Sunnyvale California, USA). FITC and Cy5 images were imported as stacks and background was subtracted. Identical thresholds settings were used (manually set) for all FITC images and all Cy5 images. Colocalisation was depicted as the pixel intensity of FITC in the region of overlap (ROI), were the ROI is composed of all Cy5 positive regions. To compare different slides stained with several HER2 antibodies, HER2 x HER2 bispecific antibodies or the combination of two different monospecific antibodies the images were normalized using the pixel intensity of Cy5. Goat-anti-mouse IgG-Cy5 was used to stain the lysosomal marker LAM PI (CD107a). The pixel intensity of LAMP1 should not differ between various HER2 antibodies or the HERx HER2 bispecific antibodies tested (one cell had a pixel intensity of Cy5 of roughly 200 . 000 ).
Normalized values for colocalization of FITC and Cy5 =[(TPI-FITC x percentage FITC-CyS colocalization )/100] x [200.000/TPI-Cy5] In this formula, TPI stands for Total Pixel Intensity.
Figure 11 and Table 11 present percentage of viable cells, as measured by the FITC pixel intensity overlapping with CyS for various monospecific HER2 antibodies and HER2 x HERbispecific antibodies. For each antibody or bispecific molecule depicted, three different images were analyzed from one slide containing 3 ,1 ״ or >5 cells. Significant variation was observed between the different images within each slide. However, it was evident that ail HER2 x HER2 bispecific antibodies demonstrate increased colocalisation with the lysosomal marker LAM PI, when compared with their monospecific counterparts. These results indicate PCT/EP2011/058779WO 2011/147986 that once internalized, HER2 x HER.2 bispecific antibodies are efficiently sorted towards lysosomal compartments, making them suitable for a bispecific antibody drug conjugate approach.
Table 11: Mean FITC pixel intensities overlapping with Cy5 depicted as arbitrary unitsantibody FITC pixel intensity in lysosomes [arbitrary units]Herceptin 0.218IqGl-005־m 0.070IqGl-025־ ITL 0.268IgGl-153-FrL 0.102IgGl- 169-K409R 0.220IqGl-005-ITL x IqGl ־ 169 ־ K409R 0.531IqGl-025־ rTL x IqGl-005־K4Q9R 0.347IgGl-025־ ITL x IqGl-153-K409R 0.582IqGl-025-ITL x IqG1-169-K409R 0.439IqGl-153-ITL x IqGl ־ 005 ־ K409R 0.494IgGl-153־ ITL x IqGl-169- K409R 0.604IgGl-025 + IgGl-169 0.576IqGl-153 + IqGl005 ״ 0.636IqGl-153 + IqGl-169 0.626 Example 24 - Inhibition of proliferation of AU565 cells upon incubation with HER2 monospecific or HER2 X HER2 bispecific antibodies The HER2 x HER2 bispecific antibodies were tested for their ability to inhibit proliferation of AU565 cells in vitro. Due to the high HER2 expression levels on AU565 cells (1,000.000׳"■׳ copies per cell as determined with Qifi-kit), HER2 is constitutively active rn these cells and thus not dependent on ligand-induced heterodimerization. In a 96-weils tissue culture plate (Greiner bio-one, Frickenhausen, Germany), 9.000 AU565 cells were seeded per well in the presence of 10 pg/mL HER2 antibody or HER2 x HER2 bispecific antibodies in serum-free cell culture medium. As a control, cells were seeded in serum-free medium without antibody or bispecific antibodies. After three days, the amount of viable cells was quantified with Alamarbiue (BioSource International, San Francisco, US) according to the manufacturer's instructions. Fluorescence was monitored using the EnVision 21Multilabel reader (PerkinElmer, Turku, Finland) with standard Alamarbiue settings. The Alamarbiue signal of antibody-treated cells was plotted as a percentage relative to untreated cells.Figure 12 and table 12 depicts the fluorescent intensity of Alamarbiue of AU565 cells after incubation with HER2 antibodies and HER2XHER2 bispecific antibodies. Herceptin® (trastuzumab) was included as positive control and demonstrated inhibition of proliferation as described by Juntiila TT. et a!., Cancer Cel! 2009; 15: 429-440. AH HER2 x HERbispecific antibodies were able to inhibit proliferation of AU565 cells, Bispecific antibodies: 100 PCT/EP2011/058779 WO 2011/147986 IgGl-005־m x rgGl ־ 169 ־ K409R and IgGl-025־ETL x rgGl-005-K409R were more effective compared to their monospecific antibody counterparts in this assay. bispecific Table 12: Percentage viable AU565 cells after treatment with HER2 x HER2antibodies.antibody percentage viable censHerceptin 62IgGl-005-ITL 91IgGl ־ 005 ־ K409R 96IgGl-025־ITL 79IqGl-153־ITL 98IgGl-153־K4G9R 97lgGl-169־K409R 63IgGl-005-ITL x IgGl-169-K409RIgGl025־-m x IgGl-005-K409R 61IgGl-025-m x IgGl-153-K409R 74IgGl ־ 025 ־ ITL x IqGl-169-K409R 76IgGl-153־ITLx IgGl-005-K409R 71IgGl ־ 153 ־ ITLx IgGl ־ 169 ־ K409R 77isotype control 95 Example 25 ־ Her2 x CD3 bispecific antibodies tested in an in vitro cytotoxicity assay CD3 is a co-receptor in the T cell receptor complex expressed on mature T cells. Combination of a CD3 specific antibody Fab-arm with a tumor antigen specific antibody Fab- arm in a bispecific antibody would result in the specific targeting of T cells to tumor cells, leading to T cell mediated tumor cell lysis. Likewise, CD3 positive T cells could be targeted to other derailed cells in the body, to infected cells or directly to pathogens.HER2 x CD3 bispecific antibodies were generated. Heavy and light chain variable region sequences for the HER2 specific Fab-arm were as indicated for antibody 153 and 1in Example 21. The following heavy and light chain variable region sequences for the CDspecific Fab-arm were used: YTH12.5 (Sequence as described by Routiedge et aL, Eurl Immunol. 1991, 21(11):2717- 25.)SEQIDNO:171 VHYTH12.5EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFPMAWVRQAPGKGLEWVSTISTSGGRTYYRDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKFRQYSGGFDYWGQGTLVTVSSSEQIDNO:172 VLYTH12.5DIQLTQPNSVSTSLGSTVKLSCTLSSGNIENNYVHWYQLYEGRSPTTMIYDDDKRPDGVPDRFSGSIDRSSNSAFLTIHNVAIEDEAIYFCHSYVSSFNVFGGGTKLTVL 101 PCT/EP2011/058779 WO 2011/147986 huCLB־T3/ 4 (Sequence as described by Parren et al., Res Immunol. 1991, 142{9):749-63. Minor amino acid substitutions were introduced to make the sequence resemble the closesthuman germline.)SEQroNO:173 VHhuCLB-T3/4EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYGMFWVRQAPGKGLEWVATISRYSRYIYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRPLYGSSPDYWGQGTLVTVSSSEQIDNO:174 VLhuCLB-T3/4EIVLTQSPATLSLSPGERATLSCSASSSVTYVHWYQQKPGQAPRLLIYDTSKLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCFQGSGYPLTFGSGTKLEMR All antibodies were expressed as IgGl,1c being modified in their Fc regions as follows: IgGl-HER2-153-K409R and IgGl- HER2-153-N297Q-K409R, IgGl-HER2-169-K409R, IgGl- hu-CLB-T3/4-F405L and IgGl-hu-CLB-T3/4-N297Q-F405L, [gGl-YTH12.5-F405L and IgGl- YTH12.5-N297Q-F405L.Bispecific antibodies from these HER2 and CD3 specific antibodies were generated as described in Example 20 and tested in an in vitro cytotoxicity assay using AU565 cells. AU565 cells were cultured to near confluency. Cells were washed twice with PBS, and trypsinized for 5 minutes at 37°C. 12 ml culture medium was added to inactivate trypsin and cells were spun down for 5 min, 800 rpm. Cells were resuspended in 10 mL culture medium and a single cell suspension was made by passing the cells through a cellstrainer. 100 pL of a 5x10s cells/mL suspension was added to each well of a 96-well culture plate, and cells were incubated at least 3 hrs at 37°C, 5% C02 to allow adherence to the plate. Peripheral blood mononuclear cells (PBMC) were isolated from blood from healthy volunteers using Leucosep 30 mL tubes, according to the manufacturer's protocol (Greiner Bio-one). T cells were isolated from PBMC preparations by negative selection using the Untouched Human T-celis Dynabead kit (Dynal). Isolated cells were resuspended in culture medium to a final concentration op 7x10® cells/mL.Culture medium was removed from the adhered AU565 cells, and replaced with pl/well 2x concentrated antibody-dilution and 50 pi/well 7xl06 T cells/mL (ratio effector:target = 7:1). Plates were incubated for 3 days at 37°C, 5% C02- Supernatants were removed and plates were washed twice with PBS. To each well 150 pL culture medium and 15 pL Alamar blue was added. Plates were incubate for 4 hours at 37°C, 5% C02, and absorbance was measured (Envision, Perkin Elmer).Figure 13 and table 13 shows that whereas control antibodies (Her2 monospecific IgGl-Herceptin®, CD3 monospecific IgGl-YTH12.5 and monospecific IgGl-huCLB-T3/4, irrelevant antigen monospecific IgGl-bl2, and CD3 x bl2 bispecific antibodies) did not102 PCT/EP2011/058779WO 2011/147986 induce T cell mediated cytotoxicity, bispecific (Duo) Her2 x CD3 antibodies huCLB/Her2- 153, huCLB/Her2-169, YTH12.5/Her2-153 and YTH12.5/Her2-169 induced dose dependent T cell mediated cytotoxicity of AU565 cells. Bispecific antibodies containing Her2-169 were more potent than those containing Her2153־.Mutants of IgGl-hu־CLB־T3/4, IgGl-YTH12.5 and Her2-153 were made containing a N297Q mutation to remove a glycosylation site; glycosyiation at this site is critical for IgG- Fcgamma receptor interactions (Bolt S et al., Eur J Immunol 1993, 23:403-411). Figure shows that N297Q mutation and therefore absence of Fc glycosylation of Her2 x CDbispecific antibodies YTH12.5/Her2-153 and huCLB/Her2-153 did not impact the potential to induce dose dependent T cell mediated cytotoxicity of AU565 cells.
Table 13: EC50 values of cell kill induced by HER2 x CD3 bispecific antibodies. "Ndet" means not detected.antibody EC5UTng7rhT|Herceptin NdetDuo huCLB-Q/153-Q 10.55Duo huCLB-Q/Bl2־Q NdethuCLB-Q NdetB12-Q NdetDUO YTH12.5-Q/153-Q 10.73Duo YTH12.5-Q/B12-Q NdetrrH12.50״ NdetB12-Q Ndet Example 26 - HER2 downmodulation To investigate if enhanced HER2 internalization induced by Group 3 antibodies 098 and 1also results in enhanced receptor downmodulation, AU565 cells were incubated with HERantibodies for 3 days, and analyzed for presence of HER2. AU565 cells were seeded in a 24- weds tissue culture plate (100.000 cells/well) in normal cell culture medium and cultured for days at 37°C in the presence of 10 pg/mL HER2 antibody. After washing with PBS, cells were lysed by incubating 30 min at room temperature with 25 pL Surefire Lysis buffer (Perkin Elmer, Turku, Finland). Total protein levels were quantified using bicinchoninic acid (BCA) protein assay reagent (Pierce) according to the manufacturer's protocol. HER2 protein levels in the lysates were analyzed using a HER2-specif1c sandwich ELISA. Rabbit-anti- human HER2 intracellular domain antibody (Cell Signaling) was used to capture HER2 and biotinylated goat-antr-human HER2 polyclonal antibody (R&D), followed by streptavidin- poly-HRP, were used to detect bound HER2. The reaction was visualized using 2,2'-azino-bis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS: dilute one ABTS tablet in 50 mL ABTS buffer (Roche Diagnostics, Almere, The Netherlands]) and stopped with oxalic add (Sigma-Aldrich,103 PCT/EP2011/058779 WO 2011/147986 Zwijndrecht, The Netherlands). Fluorescence at 405 nm was measured סח a microtiter plate reader (Biotek Instruments, Winooski, USA) and the amount of HER2 was expressed as a percentage relative to untreated ceils.The results shown in Figure 14 and Table 14 demonstrate that both Group 3 antibodies (0and 153) induced more than 50% HER2 downmodulation. In contrast, antibodies 025, 1and Herceptin barely induced downmodulation (approximately 20% of untreated cells). This was in line with enhanced internalization observed by antibodies 098 and 153.
Table 14: Antibody induced downmodulation of HER2 depicted as percentage HER2compared to untreated cellsantibody % HER2 compared to untreated cellsHerceptin 80IqGl-1014-169 82IqGl-1014-025 85IqGl-1014098 ־ 44IqGl-1014-153 50isotype control 108 Example 27 - Colocalization of HER2 antibodies with lysosomal marker LAMP1 analyzed by confocal microscopy. The HER2 downmodulation assay as described in example 26 and the CypHer-5E based internalization assay as described in example 19 indicated that HER2 antibodies from group were more efficiently internalized and targeted towards lysosomes compared to antibodies from Groups 1 and 2. However, in these experiments the confocal imaging was done with settings that allowed discriminating between monospecific and bispecific antibodies but not between different monospecific antibodies, in fact, with these settings monospecific antibodies could haridy be detected. To be able to compare between the different monospecific antibodies, the confocal slides were measured again with increased gain settings, to enhance fluorescence intensity. Ail other steps of the procedure were the same as described in example 23.The results are depited in Figure 15 and Table 15, and show that the FITC pixel intensity overlapping with Cy5 for various monospecific HER2 antibodies. From each slide three different images were analyzed containing ~ 1, 3 or >5 cells. Significant variation was observed between the different images within each slide. Still, it was evident that antibodies 098 and 153 were more efficiently targeted towards lysosomal compartments, compared to 025, pertuzumab, 169 and Herceptin. This correlated well with the enhanced internalization and receptor degradation induced by these antibodies. 104 PCTOP2011/058779 WO 2011/147986 Table 15: Mean FITC pixel intensities overlapping with Cy5 depicted as arbitrary unitsantibody FITC pixel intensity in lysosomes [arbitrary units]TH1014-098 0.522TH1014-153 0.409TH1014- 025 0.248TH1014-pert 0.214TH1014-169 0.255Herceptin 0.236 Example28 - HER2 extracellular domain shuffle human-to-chicken To further define the HER2 binding regions recognized by antibodies from the four different cross-competition groups, a HER2 extracellular domain shuffle experiment was performed. To this end, a small gene-synthesis library with five constructs was generated, swapping the sequences of domain I, II, III or IV of the extracellular domain of human HER2 to the corresponding sequence of chicken HER2 (Gallus gallus isoform B NCBI: IMP_001038126.1): 1) fully human HER2 (Uniprot P04626) hereafter named hu-HER2, 2) hu-HER2 with chicken domain I (replacing amino acids (aa) 1203־ of the human Her2 with the corresponding chicken Her2 region) hereafter named hu־HER2-ch(I), 3) hu־HER2 with chicken domain II (replacing amino adds (aa) 204330־ of the human Her2 with the corresponding chicken Her2 region) hereafter named hu-HER2־ch(II), 4) hu-HER2 with chicken domain III (replacing aa 331-507 of the human Her2 with the corresponding chicken Her2 region) hereafter named hu-HER2-ch(III) and 5) hu-HER2 with chicken domain IV (replacing aa 508-651 of the human Her2 with the corresponding chicken Her2 region) hereafter named hu-HER2-ch(IV). The human and chicken HER2 orthologs show 67% homology in their extracellular domain with 62% homology in domain I, 72% homology in domain II, 63% homology in domain III and 68% homology in domain IV. The constructs were transiently transfected in the Freestyle™ CHO-S (Invitragen) cell line using Freestyle MAX transfection reagent (Invitrogen) according to the instructions of the manufacturer, and transfected cells were cultured for 20 hours. HER2 antibody binding to the transfected cells was analyzed by means of flow cytometry: The transfected CHO-S cells were harvested, washed with FACS buffer and incubated with 10 pg/mL HER2 antibody (30 minutes on ice). Binding of HERantibodies was detected using a Phycoerythrin (PE)-conjugated goat-anti-human IgG antibody (Jackson). To check if expression between different batches was the same, cells were fixed and permeabilized using Cytofix/Cytoperm solution (BD) according manufacturer's instruction and stained with a rabbit-anti-human intracellular HER2 antibody (DAKO) in combination with a secondary PE-conjugated goat-anti-rabbit antibody (Jackson). An isotype control antibody was used as negative control. Fluorescence was measured on a FACSCanto-II (BD) and binding curves were made by means of non-linear regression105 PCT/EP2011/058779 WO 2011/147986 (sigmoidal dose-response with variable slope) using GraphPad Prism V4.03 software (GraphPad Software, San Diego, CA, USA). Loss of binding was used as read out to identify which HER2 domains were recognized by the different antibodies.Exemplary binding curves for antibody 153 are shown in figure 16. All binding results are shown in Table 16. Group 1 HER2 antibodies 050, 084, 169 and Herceptin showed loss of binding to Hu-HER2-ch(IV), but not to the proteins with one of the remaining domains shuffled, demonstrating that the epitopes of Group 1 mAbs reside in HER2 domain IV. Group 2 antibodies 025, 091, 129 and pertuzumab showed only loss of binding to Hu-HER2- ch(II), indicating that the epitope resides in HER2 domain II. Antibodies 098 and 153 were both defined to Group 3 in the cross-competition assay but showed some variation in the shuffle experiment. Antibody 098 dearly showed loss of binding to Hu-HER2-ch(I) and a minor decrease in binding to Hu-HER2-ch(II), while 153 showed only loss of binding to Hu״ HER2-ch(II). These data suggest that Group 3 mAbs 098 and 153 can also bind, at least partially, to the HER2 domain II, with epitopes that possibly extend into HER2 domain I, as is the case for 098.
Table 16: Summary of HER2 antibody binding to different HER2ECD receptor constructs. FL; hu-HER2, I; hu-HER2-ch(I), II; hu-HER2-ch(II), III; hu-HER2-ch(III), IV; hu-HER2- ch(IV). +++ indicates normal binding, ++ indicates reduced EC50 but the similar maximal binding compared to binding observed to hu-HER2, + indicates reduced EC50 and reduced maximal binding detected compared to binding observed to hu-HER2, ־ indicates no binding.
Example 29 - In vivo efficacy of HER2 HuMabs 091} 084 and 169 in NCI-N87 human gastric carcinoma xenografts in SCI D mice 106 PCT/EP2011/058779 WO 2011/147986 The in vivo effect of HER2-HuMabs 091 (cross-competition-^iroup 2)f 084 and 169 (both cross-competition Group 1) on tumor growth and survivhf^in a NCI-N87 human gastric carcinoma xenograft model in female CB.17 severe combined immunodeficiency (SCID) mice was determined. lOxlO6 NCI-N87 tumor cells in 50% matrigel were injected s.c. in female SCID mice, 10 mice per group. Eight days after tumor inoculation, intravenous treatment with HER2-HuMabs 091, 084, and 169 or control antibody HuMab-HepC was started. In figure 17 (A) this is indicated as day 1, day of treatment initiation. The first dose was at 40 mg/kg, followed by 10 mg/kg von days 4, 8, 11, 15, 18, 22, and 25 after treatment initiation.Tumor volume was determined at least 2 times per week. Volumes (mm3) were calculated from caliper (PLEXX) measurements as (width2 x length)/2.The results are depicted in Figure 17A and 17B which shows that the mice administered with HuMab 084, 169 and 091 demonstrated slower tumor growth (A) and better survival (B) than the mice that received negative control antibody HuMab-HepC. All treatments were well-tolerated.

Claims (44)

1./ 1 CLAIMS 1. A monoclonal antibody which binds human epidermal growth factor receptor 2 (HER2) and which comprises a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:23, 24 and 25, respectively; and a VL region comprising the CDR1, CDR2 and CDRsequences of SEQ ID NOs:27, AAS, and SEQ ID NO:28, respectively ( 025 ).
2. The antibody of claim 1 comprising a VH region comprising the sequence of SEQ ID NO:22 and, preferably, a VL region comprising the sequence of SEQ ID NO:26 ( 025 ).
3. The antibody of any one of the preceding claims, which has an EC50 value for binding to HER2-expressing cells lower than 0.80 µg/ml, preferably lower than 0.50 µg/ml, when determined as described in Example 12.
4. The antibody of any one of the preceding claims, which induces ADCC, preferably achieving a Cr-release of at least 30%, more preferably at least 40%, when determined as described in Example 15.
5. The antibody of any one of the preceding claims, which promotes ligand-independent proliferation of HER2-expressing cells less than F5 when determined as described in Example 16.
6. The antibody of any one of the preceding claims, which inhibits ligand-independent proliferation of HER2-expressing cells when determined as described in Example 16.
7. The antibody of any one of the preceding claims, which does not promote ligand-induced proliferation of HER2-expressing cells when determined as described in Example 17.
8. The antibody of any one of the preceding claims, which inhibits ligand-induced proliferation of HER2-expressing cells when determined as described in Example 17.
9. The antibody of any one of the preceding claims, which, when conjugated directly or indirectly to a therapeutic moiety such as a truncated form of the pseudomonas-exotoxin A, kills at least 49%, preferably at least 60%, of HER2-expressing cells when determined as described in Example 18.
10. The antibody of any one of the preceding claims, wherein the HER2-expressing tumor cell-line expresses less than an average of 30000 HER2 molecules per cell. 267022/ 1
11. The antibody of any one of the preceding claims, wherein a higher amount of the antibody than trastuzumab is internalized by a HER2-expressing tumor cell-line when determined according to Example 18.
12. The antibody of any one of the preceding claims, wherein the antibody is a bispecific antibody, comprising an antigen-binding region of an antibody as defined in any one of the preceding claims, and a second antigen-binding site having a different binding specificity, such as a binding specificity for a human effector cell, a human Fc receptor, a T cell receptor or a non-blocking epitope of HER2.
13. A bispecific antibody comprising (i) a first antibody and a (ii) second antibody, wherein the first antibody is selected from the group consisting of: an antibody according to any one of claims 1-13, and wherein the second antibody binds to a different epitope than the first antibody.
14. A bispecific antibody according to claim 14, wherein the second antibody is a CDantibody.
15. A bispecific antibody according to any one of claims 13-14, wherein the second antibody comprises a VH region comprising CDR1, CDR2 and CDR3 sequences of SEQ ID Nos: 166, 167 and 168, respectively ( 005 ).
16. A bispecific antibody according to claim 15, wherein the second antibody comprises a VH region comprising CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 166, 167 and 168, respectively; and a VL region comprising CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 169, GAS and SEQ ID NO: 170, respectively ( 005 ).
17. A bispecific antibody according to claim 16, wherein the second antibody comprises a VH region comprising the sequence of SEQ ID NO:164, and a VL region comprising the sequence of SEQ ID NO:165 ( 005 ).
18. A bispecific antibody according to any one of claims 14-17, wherein the CD3 antibody comprises a) a VH region comprising the sequence of SEQ ID NO:171 and a VL region comprising the sequence of SEQ ID NO:172 ( YTH12.5 ); or b) a VH region comprising the sequence of SEQ ID NO: 173 and a VL region comprising the sequence of SEQ ID NO: 1( huCLB-T3/4B ).
19. The antibody of any one of the preceding claims, wherein the antibody is a full-length antibody, preferably an IgG1 antibody, in particular an IgG1,κ antibody. 267022/ 1
20. The antibody of any one of the preceding claims, wherein the antibody is conjugated to another moiety, such as a cytotoxic moiety, a radioisotope, a drug, or a cytokine.
21. The antibody of claim 20, wherein the cytotoxic moiety is selected from the group consisting of taxol; cytochalasin B; gramicidin D; ethidium bromide; emetine; mitomycin; etoposide; tenoposide; vincristine; vinblastine; colchicin; doxorubicin; daunorubicin; dihydroxy anthracin dione; a tubulin-inhibitor such as maytansine or an analog or derivative thereof; mitoxantrone; mithramycin; actinomycin D; 1–dehydrotestosterone; a glucocorticoid; procaine; tetracaine; lidocaine; propranolol; puromycin; calicheamicin or an analog or derivative thereof; an antimetabolite; an alkylating agent; an antibiotic; an antimitotic agent such as monomethyl auristatin E or F or an analog or derivative thereof; diphtheria toxin; diphtheria A chain and active fragments thereof and hybrid molecules; ricin toxin such as ricin A or a deglycosylated ricin A chain toxin; cholera toxin; a Shiga-like toxin; LT toxin; C3 toxin; Shiga toxin; pertussis toxin; tetanus toxin; soybean Bowman-Birk protease inhibitor; Pseudomonas exotoxin; alorin; saporin; modeccin; gelanin; abrin A chain; modeccin A chain; alpha-sarcin; Aleurites fordii proteins; dianthin proteins; Phytolacca americana proteins; momordica charantia inhibitor; curcin; crotin; sapaonaria officinalis inhibitor; gelonin; mitogellin; restrictocin; phenomycin; and enomycin toxins; ribonuclease (RNase); DNase I; Staphylococcal enterotoxin A; pokeweed antiviral protein; diphtherin toxin; and Pseudomonas endotoxin.
22. The antibody of claim 21, wherein the antimetabolite is selected from the group consisiting of methotrexate, 6 mercaptopurine, 6 thioguanine, cytarabine, fludarabin, fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, and cladribine.
23. The antibody of claim 21, wherein the alkylating agent is selected from the group consisiting of mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin, carboplatin, duocarmycin A, duocarmycin SA and rachelmycin (CC-1065).
24. The antibody of claim 21, wherein the antibiotic is selected from the group consisiting of dactinomycin, bleomycin, daunorubicin, doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC).
25. The antibody of claim 21, wherein the Shiga-like toxin is selected from the group consisiting of SLT I, SLT II and SLT IIV. 267022/ 1
26. The antibody of claim 21, wherein the Phytolacca americana protein is selected from the group consisiting of such as PAPI, PAPII, and PAP S.
27. The antibody of claim 20, which is conjugated to a cytotoxic moiety selected from the group consisting of maytansine, calicheamicin, duocarmycin, rachelmycin (CC-1065), monomethyl auristatin E, monomethyl auristatin F or an analog, derivative, or prodrug of any thereof.
28. The antibody of claim 20, which is conjugated to a cytokine selected from the group consisting of IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNα, IFNβ, IFNγ, GM-CSF, CD40L, Flt3 ligand, stem cell factor, ancestim, and TNFα.
29. The antibody of claim 20, which is conjugated to a radioisotope, such as an alpha emitter.
30. A nucleotide sequence encoding encoding a VH region and a VL region of an antibody as defined in any one of claims 1-15, said VH and VL regions being defined by SEQ ID NO: and 26, respectively.
31. An expression vector comprising a nucleotide sequence according to claim 30, wherein the vector further encodes an operately-linked constant region of a light chain, constant region of a heavy chain or both light and heavy chains of an antibody.
32. A recombinant eukaryotic or prokaryotic host cell which produces an antibody as defined in any one of claims 1 to 19.
33. A pharmaceutical composition comprising an antibody as defined in any one of claims to 19 and a pharmaceutically acceptable carrier.
34. A pharmaceutical comprising an antibody as defined in any one of claims 20 to 29, and a pharmaceutically acceptable carrier.
35. The antibody of any one of claims 1 to 19 for use as a medicament.
36. The antibody of any one of claims 20 to 29 for use as a medicament.
37. The antibody of any one of claims 1 to 19 for use in the treatment of cancer.
38. The antibody of any one of claims 20 to 29 for use in the treatment of cancer. 267022/ 1
39. The antibody of any one of claims 37 and 38, wherein the cancer is selected from the group consisting of breast cancer, prostate cancer, non-small cell lung cancer, bladder cancer, ovarian cancer, gastric cancer, colorectal cancer, esophageal cancer, squamous cell carcinoma of the head and neck, cervical cancer, pancreatic cancer, testis cancer, malignant melanoma and soft-tissue cancer.
40. The antibody for the use of any one of claims 37 to 38, wherein the antibody is for the treatment of cancer in combination with one or more further therapeutic agent, such as a chemotherapeutic agent.
41. The antibody of any one of the claims 1 to 29 for use in the manufacture of a medicament for treatment of cancer.
42. A method for producing an antibody of any one of claims 1 to 29, said method comprising the steps of a) culturing a host cell of claim 31, and b) purifying the antibody from the culture media.
43. A method for detecting the presence of HER2 in a sample, comprising: - contacting the sample with an antibody of any one of claims 1 to 29 under conditions that allow for formation of a complex between the antibody and HER2; and - analyzing whether a complex has been formed.
44. A kit for detecting the presence of HER2 in a sample comprising - an antibody of any one of claims 1 to 29; and - instructions for use of the kit. For the Applicant , REINHOLD COHN AND PARTNERS By:
IL267022A 2010-05-27 2019-06-02 Monoclonal antibodies aganist her2 IL267022B2 (en)

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Citations (3)

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WO2001009187A2 (en) * 1999-07-29 2001-02-08 Medarex, Inc. Human monoclonal antibodies to her2/neu
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WO2001009187A2 (en) * 1999-07-29 2001-02-08 Medarex, Inc. Human monoclonal antibodies to her2/neu
WO2008150485A2 (en) * 2007-05-29 2008-12-11 Wyeth Erbb2 binding proteins and use thereof
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