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WO2025238187A1 - Immunoconjugués ciblant l1-cam - Google Patents

Immunoconjugués ciblant l1-cam

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Publication number
WO2025238187A1
WO2025238187A1 PCT/EP2025/063464 EP2025063464W WO2025238187A1 WO 2025238187 A1 WO2025238187 A1 WO 2025238187A1 EP 2025063464 W EP2025063464 W EP 2025063464W WO 2025238187 A1 WO2025238187 A1 WO 2025238187A1
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Prior art keywords
sequence
seq
sequence according
antibody
heavy chain
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English (en)
Inventor
Christian GERATHS
Michael HACKEBEIL
Franziska STRÜTT
Daniela Winkler
Dominik BRÜCHER
Christoph Schäfer
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Cis Biopharma Ag
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Cis Biopharma Ag
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Publication of WO2025238187A1 publication Critical patent/WO2025238187A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68037—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a camptothecin [CPT] or derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell

Definitions

  • the present invention relates to immunoconjugates targeting L1-CAM.
  • the immunoconjugates of the present invention and the pharmaceutical compositions described herein are particularly useful in treatment or diagnosis of an L1-CAM (CD171) associated cancer.
  • CAR-modified T cells are no exception to the immunosuppressive tumor microenvironment, where Tregs, tumor associated macrophages and myeloid suppressor cells work in concert to circumvent the anti-tumor properties of CAR modified T-cells.
  • CAR modified T-cells are subject to the same immunosuppressive constraints confronted by classic T-cells, including anergy following engagement of CTLA4 by B7 or PD-1 by PD-L1 (B7-H1) on tumor cells. Clinically effective alternatives to CAR modified T-cell therapy for the treatment of cancer are thus needed.
  • L1-CAM has been shown to be expressed in many human cancers and be often associated with bad prognosis, mostly due to motility and invasion promoting function of L1-CAM (Altevogt et al., International Journal of Cancer, 138, 1565-1576 (2016)). Kiefel and coworkers (Cell Adhesion & Migration 6:4, 374- 384) have described L1-CAM as a major drive for tumour cell invasion and motility.
  • Rached et al have shown that L1-CAM knowck- out radiosensitizes neuroblastoma IMR-32 cells by simultaneously decreasing MycN, but increasing PTEN protein expression (Rached et al., International; Journal of Oncology, 49: 1722-1730, 2016).
  • LI- CAM has also been shown to increase adhesion-mediated proliferation and chemoresistance of retinoblastoma (Jo et al., Oncotarget, 2017, Vol 8, pp 15441-15452).
  • Terraneo et al have shown that LI- CAM confers radioresistance to ovarian cancer and defines a new cancer stem cell population (Terraneo et al., Cancers 2020, 12, 217).
  • Expression profile analysis in multiple human tumors idenfiied L1-CAM as a molecular marker for differential diagnosis and targeted therapy (Huszar et al., Human Pathology (2006), 37, 1000-1008).
  • Therapeutic antibodies targeting L1-CAM have been previously described. Hoefnagel et al (European Journal of Nuclear Medicine, 2001 , 28:359-368) disclose targeting of neuroblastoma with anti L1-CAM antibody mAb chCE7. Therapeutic efficacy in a neuroblastoma xenograft model and imaging of neuroblastoma patients are disclosed. The same antibody chCE7 has been shown to bind to an isoform of L1-CAM present in renal carcinoma cells (Meli et al., International Journal of Cancer, 83, 401-408, 1999). Novak-Hofer et al (J. Nucl.
  • L1-CAM Since L1-CAM is known to be expressed in the peripheral nerve tissue, the side effect of treatment targeting L1-CAM are common. In particular, said side effects include ADCC, CDC and other immune reactions. It is accordingly crucial to provide an antibody (or, if applicable, a fragment thereof) wherein said toxic effects would be diminished. It is particularly desirable in the case of targeted radionuclide therapy to provide an antibody with a low KD, which is related to a low k-off once bound to the tumour, which would allow avoiding its diffusion to the healthy tissue. Document WO 2018/232188 discloses certain anti-L1-CAM antibodies and uses thereof.
  • Amstutz et al. discloses production and characterization of a mouse/human chimeric antibody directed against human neuroblastoma.
  • the antibodies used in the immunoconjugates of the present invention show improved affinity in binding to L1-CAM in comparison to closely related antibodies, in particular to CE7 antibody (Amstutz et al.) that they are derived from.
  • the antibodies used in the immunoconjugates of the present invention are humanised antibodies, and the skilled person would expect the humanization of the antibody to negatively impact the binding affinity of the antibody to its antigen.
  • the present invention is based, at least in part, on an unexpected discovery that antibodies of the present invention, obtained upon humanization of CE7 antibody, exhibit surprisingly improved binding affinity to L1-CAM in comparison to CE7 antibody, as demonstrated, among others, in the SPR study performed by the present inventors.
  • the antibodies used in the immunoconjugates of the present invention show at least comparable (and not worse) affinity in comparison to CE7 antibody that they are derived from through a process of humanization.
  • the present inventors have further demonstrated that the antibodies used in the immunoconjugates of the present invention have surprisingly shown improved stability in comparison to closely related antibodies, in particular to CE7 antibody that they are derived from. Said improved stability is manifested, at least, in a higher aggregation onset temperature as demonstrated in the PANTA studies.
  • the invention will be summarized in the following embodiments.
  • the present invention relates to an immunoconjugate, comprising: a) an antibody that specifically binds to L1-CAM (CD 171), the antibody characterized by: a1 ) a heavy chain comprising a sequence at least 95% identical to a sequence according to a SEQ ID NO.: 2 and a light chain comprising a sequence at least 95% identical to a sequence according to SEQ ID NO.: 6, a2) a heavy chain comprising sequence at least 95% identical to a sequence according to a SEQ ID NO.: 4 and a light chain comprising a sequence at least 95% identical to a sequence according to SEQ ID NO.: 6, a3) a heavy chain comprising a sequence at least 95% identical to a sequence according to a SEQ ID NO.: 10 and a light chain comprising a sequence at least 95% identical to a sequence according to SEQ ID NO.: 8, a4) a heavy chain comprising sequence at least 95% identical to a sequence according to a SEQ ID NO.
  • Li is — (C1-10 alkylene)-CO-, preferably -(CH2)6-CO-, attached through its left empty valence to Zi and through its right empty valence to Lp;
  • Lp is -Gly-Gly-Phe-Gly-, wherein the C-terminus of the Lp is connected to the cytotoxic compound and wherein the N-terminus is connected to Li; c2) a moiety according to formula: -Z 2 -LPEG-LP-LX- wherein wherein the bottom empty valence is connected to LPEG,
  • LPEG is -(CH 2 CH 2 0)n-(Ci-io alkylene)-CO-, wherein n is an integer from 2 to 6, preferably -(CH 2 CH 2 O)4-(CH 2 CH 2 ) 2 -CO-, attached through its left empty valence to Z 2 and through its right empty valence to Lp,
  • Lp is -Gly-Gly-Phe-Gly-, wherein the C-terminus of the Lp is connected to Lx and wherein the N-terminus is connected to LPEG;
  • Lx is -NHCH 2 OCH 2 CO-, wherein the right empty valence of Lx is connected to the cytotoxic compound and wherein the left empty valence of Lx is connected to Lp. wherein the top empty valences are attached to an LN3 moiety of a carrier, wherein the carrier is selected from: d1) a moiety according to formula:
  • the present invention likewise relates, in a further embodiment, to an immunoconjugate comprising: a) an antibody that specifically binds to L1-CAM (CD171), the antibody characterized by: a1 ) a heavy chain comprising a sequence at least 95% identical to a sequence according to a SEQ ID NO.: 2 and a light chain comprising a sequence at least 95% identical to a sequence according to SEQ ID NO.: 6; or a2) a heavy chain with sequence at least 95% identical to a sequence according to a SEQ ID NO.: 4 and a light chain with a sequence at least 95% identical to a sequence according to SEQ ID NO.: 6.
  • cytotoxic compound wherein the cytotoxic compound is Exatecan
  • linker wherein the linker is in a form of a polymeric carrier, which comprises at least two types of repeating unit, wherein at least one type of the repeating unit is covalently attached to an active agent, for example a cytotoxic agent.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the immunoconjugate of the present invention and a pharmaceutically acceptable carrier.
  • the present invention relates to the immunoconjugate of the present invention for use as a medicament.
  • the present invention relates to the immunoconjugate of the present invention for use for use in the treatment of an L1-CAM (CD171) associated cancer.
  • the present invention relates to use of the immunoconjugate of the present invention in the manufacture of a medicament for treatment of an L1-CAM (CD171) associated cancer.
  • the present invention relates to a method of treatment of an L1-CAM (CD171) associated cancer, the method comprising administering the immunoconjugate of the present invention to an individual in need thereof. It is to be understood that a therapeutically effective amount is to be administered.
  • the present invention relates to the immunoconjugate of the present invention for use in diagnosis.
  • the present invention relates to the immunoconjugate of the present invention for use for use in the diagnosis of an L1-CAM (CD171) associated cancer.
  • Figure 1 presents purification profiles of the rAbs (huCE7 Variant 7). Non-reduced PAGE with Coomassie blue staining. MW. Molecular weight marker. IN. Input. FT. Flow through. E. Eluted fractions.
  • Figure 2 presents Final sample QC of (huCE7 Variant 7). Coomassie blue staining. A & B. Reduced and Non-Reduced PAGE analysis. MW. Molecular weight marker. 2 pg loaded per lane; C. SEC-HPLC profile; D. Absorbance profile. E. Agarose gel electrophoresis. Final buffer: PBS, pH 7.5, Purity: 98.38% (measured by SEC-HPLC).
  • Figure 3 shows Target protein purification profile huCE7 Varant 7AG (Batch to Batch Variance): Nonreduced PAGE analysis with Coomassie blue staining. MW. Molecular weight marker. IN. Input. FT. Flow through. W. Washes. E. Eluted fractions.
  • Figure 5 presents A SECprofil of huCE7 Variant 7AG Batch 1 , B SEC profile of huCE7 Variant 7AG Batch 2, C Analysis of SEC peaks (SEC assay conditions: Blank control: dilution (5x) of the sample with DD water, Column: G3000SWXL, TOSOH, 7.8x300mm; HPLC: Waters 2695, Elution gradient: mobile phase isocratic elution, Flow rate: 0.8mL/min; Temperature: 25°C, Injection volume: 30 pL, Detection wavelength: 280nm, Collect date time: 25min, Equilibration with mobile phase 100% for 5 min, Run Samples: Injection of one blank control and subsequently injection of samples, Data Analysis).
  • Blank control dilution (5x) of the sample with DD water, Column: G3000SWXL, TOSOH, 7.8x300mm
  • HPLC Waters 2695
  • Elution gradient mobile phase isocratic elution
  • Flow rate
  • Figure 6 shows Purification profiles of the rAbs (huCE7 LV32). Non-reduced PAGE with Coomassie blue staining.tJWN. Molecular weight marker. IN. Input. FT. Flow through. E. Eluted fractions.
  • Figure 7 presents Final sample QC of (huCE7 LV32). Coomassie blue staining.
  • a & B Reduced and Non-Reduced PAGE analysis. MW. Molecular weight marker. 2 pg loaded per lane;
  • C SEC- HPLC profile;
  • D Absorbance profile.
  • E Agarose gel electrophoresis. Final buffer: PBS, pH 7.5, Purity: 98.69% (measured by SEC-HPLC).
  • Figure 8 presents Purification profiles of the rAbs (huCE7 LV40). Non-reduced PAGE with Coomassie blue staining. MW. Molecular weight marker. IN. Input. FT. Flow through. E. Eluted fractions.
  • Figure 9 presents Final sample QC of (huCE7 LV40). Coomassie blue staining.
  • a & B Reduced and Non-Reduced PAGE analysis. MW. Molecular weight marker. 2 pg loaded per lane;
  • C SEC- HPLC profile;
  • D Absorbance profile.
  • E Agarose gel electrophoresis. Final buffer: PBS, pH 7.5, Purity: 98.38% (measured by SEC-HPLC).
  • Figure 10 presents Purification profiles of the rAbs (huCE7 LV40 & LV42). Non-reduced PAGE with Coomassie blue staining. MW. Molecular weight marker. IN. Input. FT. Flowthrough. E. Eluted fractions.
  • Figure 11 presents the final QC gel (for the purification shown in Figure 10). Coomassie blue staining. Reduced and Non-reduced PAGE analysis. MW. Molecular weight marker. 2 pg loaded per lane.
  • Figure 12 shows Figure 10: A SECprofil of huCE7 LV40, B SEC profile of huCE7 LV42, C Analysis of SEC peaks.
  • SEC assay conditions Blank control: dilution (5x) of the sample with double distilled water Column: G3000SWXL, TOSOH, 7.8x300mm; HPLC: Waters 2695, Elution gradient: mobile phase isocratic elution, Flow rate: 0.8mL/min; Temperature: 25°C, Injection volume: 30 pL, Detection wavelength: 280nm, Collect date time: 25min, Equilibration with mobile phase 100% for 5 min, Run Samples: Injection of one blank control and subsequently injection of samples, Data Analysis.
  • Figure 13 presents A LC-MS spectra, total ion count (TIC) of huCE7 LV32 before and after attachment of NH 2 -(PEG)2-N[PEG3-N 3 ]2 by microbial transglutaminase (mTG) reaction according to protocols presented in example 3 (reaction control, without purification).
  • B Detailed mass spectra of compounds (antibodies LC, HC; mTG & linker educt).
  • Figure 14 presents Characterization of huCE7 LV32-linker & linker drug conjugate by HIC/ Overlay of hydrophobic interaction chromatography (HIC) of huCE7 LV32 modified with 4 azide groups by attached linker (see Example 3 step 1) and linker Drug (DAR 4) (see Example 3 step 2).
  • Drug Deruxtecan (DxD). Shift in HIC indicated change in mAbs hydrophobicity due to linker drug attachment.
  • Figure 15 presents Characterization of huCE7 LV32 linker drug conjugate by LC-MS.
  • DAR antibody ration
  • Figure 16 presents the final sample QC of huCE7 variant 7 after purification. Coomassie blue staining with 2 g protein per lane: A. Reducing B. non-reducing SDS-PAGE C. SEC-HPLC Results: Purity by SEC-HPLC was calculated to be 99.01 % monomer content.
  • Figure 17 presents the final sample QC of the ADC huCE7 V7 7D8 after purification by ultracentrifugation and buffer exchange.
  • A LC-MS Deconvoluted mass spectrum HC
  • B LC-MS Deconvoluted mass spectrum LC
  • C SEC-HPLC determination of purity and aggregates.
  • DAR of huCE7 V7 7D8 was calculated to be 8.0, SEC purity 97.84 % with 2.16 % aggregates.
  • the present invention relates to an immunoconjugate of the present invention.
  • the immunoconjugate of the present invention comprises: a) an antibody that specifically binds to L1-CAM (CD 171); b) a cytotoxic compound; and c) a linker.
  • Said immunoconjugate targets L1-CAM (CD171).
  • said immunoconjugate specifically binds to L1 -CAM (CD171 ).
  • binding of the immunoconjugate to L1-CAM is mediated through an antibody comprised in the immunoconjugate.
  • the immunoconjugate of the present invention comprises a) an antibody that specifically binds to L1-CAM (CD171).
  • L1-CAM refers to a transmembrane protein member of the L1 protein family encoded by the L1-CAM gene, as described in UniProt database under the reference P32004.
  • the L1-CAM protein is a neuronal cell adhesion molecule with a strong implication in cell migration, adhesion, neurite outgrowth, myelination and neuronal differentiation.
  • the L1-CAM protein has also been found to play a role in treatment-resistant cancers.
  • the term “L1-CAM” may be used interchangeably with the term “CD171”, both of which are known to the skilled person.
  • an antibody (or an antigen binding fragment thereof) that specifically binds to L1-CAM may also be referred to as an antibody (or an antigen binding fragment thereof, respectively) that binds to an epitope within L1-CAM, preferably specifically binds to an epitope within L1-CAM.
  • An "antibody that binds to an epitope" within a defined region of a protein is an antibody that requires the presence of one or more of the amino acids within that region for binding to the protein.
  • the antibodies comprised in immunoconjugates according to the present invention preferably bind to Ig6-like domain of L1-CAM and prevent the binding of L1-CAM to integrins. Accordingly, said antibodies preferably bind to the same epitope as chCE7 antibody, referred to herein.
  • specific binding to L1-CAM refers to the situation, wherein, the antibody is capable of binding L1-CAM with sufficient affinity that said antibody is useful as a diagnostic and/or therapeutic agent in targeting L1 -CAM.
  • the extent of binding of such antibody to an unrelated, non-L1 -CAM protein is less than about 10% of the binding of the antibody to L1 -CAM, as measured e.g., in an immunoassay (for example a radioimmunoassay) or in an SPR assay (surfaceplasmon resonance). More preferably, the extent of binding of such antibody to an unrelated, non-L1- CAM protein is less than about 5% of the binding of the antibody to L1-CAM, as measured e.g. in an immunoassay (for example a radioimmunoassay) or in an SPR assay (surface-plasmon resonance, e.g. performed used Biacore).
  • an immunoassay for example a radioimmunoassay
  • SPR assay surface-plasmon resonance
  • binding to defines a binding (interaction) of at least two “antigen-interaction-sites” with each other.
  • antiigen-interaction-site defines, in accordance with the present invention, a motif of a polypeptide, i.e. , a part of the antibody of the present invention, which shows the capacity of specific interaction with a specific antigen or a specific group of antigens of L1-CAM.
  • Cross-reactivity of antibodies under investigation may be tested, for example, by assessing binding of said panel of antibodies under conventional conditions (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1988) and Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1999)) to the (poly)peptide of interest as well as to a number of more or less (structurally and/or functionally) closely related (poly)peptides. Only those constructs (i.e.
  • L1-CAM antibodies, but also antigen-binding fragments thereof, antibody constructs and the like
  • bind to the certain structure of L1-CAM as defined herein e.g., a specific epitope or (poly) peptide/protein of L1-CAM as defined herein but do not or do not essentially bind to any of the other epitope or (poly) peptides of the same L1-CAM, are considered specific for the epitope or (poly) peptide/protein of interest and selected for further studies in accordance with the method provided herein.
  • These methods may comprise, inter alia, binding studies, blocking and competition studies with structurally and/or functionally closely related molecules.
  • binding studies also comprise FACS analysis, surface plasmon resonance (SPR, e.g.
  • an antibody is defined in the following.
  • antibody is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), fully-human antibodies and antibody fragments so long as they exhibit the desired antigen-binding activity.
  • monoclonal antibodies polyclonal antibodies
  • multispecific antibodies e.g., bispecific antibodies
  • fully-human antibodies antibody fragments so long as they exhibit the desired antigen-binding activity.
  • any form of the antibody as apparent to the skilled person such as a form selected from monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and fully-human antibodies
  • an antibody may also refer to an antigen-binding fragment thereof.
  • an "antigen-binding fragment" of an antibody refers to a molecule other than an intact antibody that comprises a portion of an intact antibody and that binds the antigen to which the intact antibody binds.
  • antibody fragments include but are not limited to Fv, Fab, Fab', Fab' -SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.
  • the antibody is not antigen-binding fragment of an antibody.
  • the antibody is a monoclonal antibody, a chimeric antibody, a recombinant antibody, a single chain antibody, a humanized antibody, a bispecific antibody, or a multi-specific antibody.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method described by Kohler, Nature 256 (1975), 495.
  • recombinant antibody includes all antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal (e.g. a mouse) that is transgenic for human immunoglobulin genes, antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial human antibody library, or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Accordingly, the term antibody also relates to recombinant human antibodies, heterologous antibodies and heterohybrid antibodies. Such recombinant human antibodies have variable and constant regions (if present) derived from human germline immunoglobulin sequences.
  • Such antibodies can, however, be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
  • a “heterologous antibody” is defined in relation to the transgenic non-human organism producing such an antibody. This term refers to an antibody having an amino acid sequence or an encoding nucleic acid sequence corresponding to that found in an organism not consisting of the transgenic non-human animal, and generally from a species other than that of the transgenic non-human animal.
  • heterohybrid antibody refers to an antibody having light and heavy chains of different organismal origins.
  • an antibody having a human heavy chain associated with a murine light chain is a heterohybrid antibody.
  • heterohybrid antibodies include chimeric and humanized antibodies.
  • humanized antibodies also relate to humanized antibodies.
  • "Humanized" forms of non-human (e.g. murine or rabbit) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • CDR complementary determining region
  • humanized antibody may comprise residues, which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • the term “antibody” relates to full immunoglobulin molecules as well as to parts of such immunoglobulin molecules (i.e., “antigen-binding fragment thereof’). Furthermore, the term relates, as discussed above, to modified and/or altered antibody molecules. The term also relates to recombinantly or synthetically generated/synthesized antibodies. The term preferably relates to intact antibodies. However, the term may also relate to antibody fragments thereof, like, separated light and heavy chains, Fab, Fv, Fab’, Fab’-SH, F(ab’)2. The term antibody also comprises but is not limited to fully-human antibodies, chimeric antibodies, humanized antibodies, CDR-grafted antibodies and antibody constructs, like single chain Fvs (scFv) or antibody-fusion proteins.
  • scFv single chain Fvs
  • a single chain antibody i.e., “single-chain Fv” or “scFv” antibody fragments have, in the context of the invention, the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain.
  • the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.
  • a “Fab fragment” as used herein is comprised of one light chain and the CH1 and variable regions of one heavy chain.
  • the heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
  • An "Fc" region contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.
  • a "Fab 1 fragment” contains one light chain and a portion of one heavy chain that contains the VH domain and the C H1 domain and also the region between the CH1 and C H2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form a F(ab')2 molecule.
  • a “F(ab')2 fragment” contains two light chains and two heavy chains containing a portion of the constant region between the CH1 and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains.
  • a F (ab')2 fragment thus is composed of two Fab' fragments that are held together by a disulfide bond between the two heavy chains.
  • the "Fv region” comprises the variable regions from both the heavy and light chains, but lacks the constant regions.
  • a bispecific antibody as referred to herein, is an antibody that can simultaneously bind to two different types of antigen, or to two different epitopes of the same antigen. Upon development, bispecific antibodies can be manufactured in several structural formats, which are known to the skilled person. Within the invention, at least one of antigens relates to L1-CAM, as defined herein.
  • a multi-specific antibody as referred to herein, is an antibody that can simultaneously bind to more than two different types of antigen, or to more than two different epitopes of the same antigen.
  • Antibodies, antibody constructs, antibody fragments, antibody derivatives (all being Ig-derived) to be employed in accordance with the invention or their corresponding immunoglobulin chain(s) can be further modified using conventional techniques known in the art, for example, by using amino acid deletion(s), insertion(s), substitution(s), addition(s), and/or recombination(s) and/or any other modification(s) known in the art either alone or in combination. Methods for introducing such modifications in the DNA sequence underlying the amino acid sequence of an immunoglobulin chain are well known to the person skilled in the art; see, e.g., Sambrook (1989), loc. cit.
  • the term “Ig-derived domain” particularly relates to (poly) peptide constructs comprising at least one CDR.
  • Fragments or derivatives of the recited Ig-derived domains define (poly) peptides which are parts of the above antibody molecules and/or which are modified by chemical/biochemical or molecular biological methods.
  • Corresponding methods are known in the art and described inter alia in laboratory manuals (see Sambrook et al., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press, 2nd edition (1989) and 3rd edition (2001); Gerhardt et al., Methods for General and Molecular Bacteriology ASM Press (1994); Lefkovits, Immunology Methods Manual: The Comprehensive Sourcebook of Techniques; Academic Press (1997); Golemis, Protein- Protein Interactions: A Molecular Cloning Manual Cold Spring Harbor Laboratory Press (2002)).
  • the antibody as referred to herein is a monoclonal antibody.
  • the antibody as referred to herein may be an lgG1 , lgG2a or lgG2b, lgG3, lgG4, IgM, lgA1 , lgA2, IgAsec, IgD, IgE.
  • isotype refers to the antibody class (e.g., IgM or lgG1) that is encoded by heavy chain constant region genes.
  • class of an antibody refers to the type of constant domain or constant region possessed by its heavy chain.
  • IgA immunoglobulin A
  • IgD immunoglobulin D
  • IgE immunoglobulin G
  • IgM immunoglobulin M
  • subclasses e.g., lgG1 , lgG2, lgG3, lgG4, lgA1, and lgA2.
  • the heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, 5, £, y, and p, respectively.
  • the monoclonal antibody as described herein is an lgG1 antibody.
  • the antibodies of the present invention can be full length or can include only an antigenbinding fragment such as the antibody constant and/or variable domain of lgG1, lgG2, lgG3, lgG4, IgM, lgA1 , lgA2, IgAsec, IgD or IgE or could consist of a Fab fragment, a F(ab') 2 fragment and a Fv fragment.
  • an antigenbinding fragment such as the antibody constant and/or variable domain of lgG1, lgG2, lgG3, lgG4, IgM, lgA1 , lgA2, IgAsec, IgD or IgE or could consist of a Fab fragment, a F(ab') 2 fragment and a Fv fragment.
  • the antibody comprised a variable heavy chain region, as described hereinbelow, and/or a variable light chain region, as described hereinbelow.
  • the antibody that binds L1-CAM in a) is characterized by: a1) a heavy chain comprising a sequence at least 95% identical to a sequence according to a SEQ ID NO.: 2 and a light chain comprising a sequence at least 95% identical to a sequence according to SEQ ID NO.: 6, a2) a heavy chain comprising sequence at least 95% identical to a sequence according to a SEQ ID NO.: 4 and a light chain comprising a sequence at least 95% identical to a sequence according to SEQ ID NO.: 6, a3) a heavy chain comprising a sequence at least 95% identical to a sequence according to a SEQ ID NO.: 10 and a light chain comprising a sequence at least 95% identical to a sequence according to SEQ ID NO.: 8, a4) a heavy chain comprising sequence at least 95% identical to a sequence according to a SEQ ID NO.
  • the immunoconjugate of the present invention the antibody that binds L1-CAM in a) is as described in any one of points a1), a2), a3), a4), a5), a6), a7), a8), a9), or a10), as provided hereinabove or hereinbelow.
  • Particularly preferred are antibody that binds L1-CAM as described in point a1) or in point a2).
  • the antibody that binds L1-CAM in a) is as described in point a1).
  • the antibody is characterized by a1) a heavy chain comprising a sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a sequence according to a SEQ ID NO.: 2, preferably a heavy chain with sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a SEQ ID NO.: 1 , and a light chain comprising a sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, again more
  • the antibody is characterized by a2) a heavy chain comprising sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a sequence according to a SEQ ID NO.: 4, preferably a heavy chain with sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a SEQ ID N0.:3, and a light chain comprising a sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to
  • the antibody is characterized by a3) a heavy chain comprising a sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a sequence according to a SEQ ID NO.: 10, preferably a heavy chain with sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a SEQ ID NO.: 9, and a light chain comprising a sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to
  • the antibody is characterized by a4) a heavy chain comprising sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a sequence according to a SEQ ID NO.: 12, preferably a heavy chain with sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a SEQ ID NO.: 11 and a light chain comprising a sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a
  • the antibody is characterized by a5) a heavy chain comprising a sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a sequence according to a SEQ ID NO.: 14, preferably a heavy chain with sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a SEQ I D NO.: 13, and a light chain comprising a sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical
  • the antibody is characterized by a6) a heavy chain comprising sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a sequence according to a SEQ ID NO.: 16, preferably a heavy chain with sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a SEQ ID NO.: 15 and a light chain comprising a sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a
  • the antibody is characterized by a7) a heavy chain comprising a sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a sequence according to a SEQ ID NO.: 18, preferably a heavy chain with sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a SEQ I D NO.: 17, and a light chain comprising a sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical
  • the antibody is characterized by a8) a heavy chain comprising sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a sequence according to a SEQ ID NO.: 20, preferably a heavy chain with sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a SEQ ID NO.: 19 and a light chain comprising a sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a
  • the antibody is characterized by a9) a heavy chain comprising a sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a sequence according to a SEQ ID NO.: 22, preferably a heavy chain with sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a SEQ I D NO.: 21 , and a light chain comprising a sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, again more
  • the antibody is characterized by a10) a heavy chain comprising sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a sequence according to a SEQ ID NO.: 24, preferably a heavy chain with sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a SEQ ID NO.: 23 and a light chain comprising a sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a
  • the antibody is characterized by a11) a heavy chain comprising a sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a sequence according to a SEQ ID NO.: 26, preferably a heavy chain with sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a SEQ I D NO.: 25, and a light chain comprising a sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, again more
  • the antibody is characterized by a12) a heavy chain comprising sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a sequence according to a SEQ ID NO.: 28, preferably a heavy chain with sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a SEQ ID NO.: 27 and a light chain comprising a sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to
  • the antibody is characterized by a13) a heavy chain comprising a sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a sequence according to a SEQ ID NO.: 30, preferably a heavy chain with sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a SEQ I D NO.: 29, and a light chain comprising a sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, again more
  • the antibody is characterized by a14) a heavy chain comprising sequence at least 95% identical to a sequence according to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a SEQ ID NO.: 32, preferably a heavy chain with sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a SEQ ID NO.: 31 and a light chain comprising a sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to
  • the antibody is characterized by a15) a heavy chain comprising a sequence at least 95% identical to a sequence according to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a SEQ ID NO.: 34, preferably a heavy chain with sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a SEQ I D NO.: 33, and a light chain comprising a sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, again
  • the antibody is characterized by a16) a heavy chain comprising sequence at least 95% identical to a sequence according to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a SEQ ID NO.: 36, preferably a heavy chain with sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a SEQ ID NO.: 35 and a light chain comprising a sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to
  • the antibody is characterized by a17) a heavy chain comprising a sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a sequence according to a SEQ ID NO.: 38, preferably a heavy chain with sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a SEQ I D NO.: 37, and a light chain comprising a sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, again
  • the antibody is characterized by a18) a heavy chain comprising sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a sequence according to a SEQ ID NO.: 40, preferably a heavy chain with sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a SEQ ID NO.: 39 and a light chain comprising a sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to
  • the antibody is characterized by a19) a heavy chain comprising a sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a sequence according to a SEQ ID NO.: 42, preferably a heavy chain with sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a SEQ I D NO.: 41 , and a light chain comprising a sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5%
  • the antibody is characterized by a20) a heavy chain comprising sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a sequence according to a SEQ ID NO.: 44, preferably a heavy chain with sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a SEQ ID NO.: 43 and a light chain comprising a sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to
  • the antibody is characterized by a21) a heavy chain comprising a sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a sequence according to a SEQ ID NO.: 46, preferably a heavy chain with sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a SEQ I D NO.: 45, and a light chain comprising a sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, again
  • the antibody is characterized by a22) a heavy chain comprising sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a sequence according to a SEQ ID NO.: 48, preferably a heavy chain with sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a SEQ ID NO.: 47 and a light chain comprising a sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to
  • the antibody is characterized by a23) a heavy chain comprising a sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a sequence according to a SEQ ID NO.: 50, preferably a heavy chain with sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a SEQ I D NO.: 49, and a light chain comprising a sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, again
  • the antibody is characterized by a24) a heavy chain comprising sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a sequence according to a SEQ ID NO.: 52, preferably a heavy chain with sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a SEQ ID NO.: 51 and a light chain comprising a sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to
  • the antibody is characterized by a25) a heavy chain comprising a sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a sequence according to a SEQ ID NO.: 54, preferably a heavy chain with sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a SEQ I D NO.: 53, and a light chain comprising a sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, again
  • the antibody is characterized by a26) a heavy chain comprising sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a sequence according to a SEQ ID NO.: 56, preferably a heavy chain with sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a SEQ ID NO.: 55 and a light chain comprising a sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to
  • the antibody is characterized by a27) a heavy chain comprising a sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a sequence according to a SEQ ID NO.: 58, preferably a heavy chain with sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a SEQ I D NO.: 57, and a light chain comprising a sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.
  • the antibody is characterized by a28) a heavy chain comprising sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a sequence according to a SEQ ID NO.: 60, preferably a heavy chain with sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical to a SEQ ID NO.: 59 and a light chain comprising a sequence at least 95% identical to, preferably at least 96% identical to, more preferably at least 97% identical to, even more preferably at least 98% identical to, still more preferably at least 99% identical to, again more preferably at least 99.5% identical to, most preferably identical
  • constructs for expression of heavy chain and light chain of antibodies include additional signal sequences, namely MKHLWFFLLLVAAPRWVLS (SEQ ID NO.: 75) for the heavy chain and MVLQTQVFISLLLWISGAYG (SEQ ID NO.: 76) for the light chain. It is to be understood that these sequences may be absent in the final antibody.
  • a heavy chain amino acid sequence according to SEQ ID NO.: 1 when compared to an amino acid sequence according to SEQ ID NO.: 2, includes an additional N-terminal amino acid sequence according to SEQ ID NO.: 75.
  • a light chain amino acid sequence according to SEQ ID NO.: 6 when compared to an amino acid sequence according to SEQ ID NO.: 5, includes an additional N-terminal amino acid sequence according to SEQ ID NO.: 76.
  • the antibody of the present invention is a humanised antibody.
  • Humanization approaches are well known in the art and in particular described for antibody molecules, e.g. Ig-derived molecules.
  • the term “humanized” refers to humanized forms of non-human (e.g., murine) antibodies or fragments thereof (such as Fv, Fab, Fab’, F(ab’), scFvs, or other antigen-binding partial sequences of antibodies) which contain some portion of the sequence derived from non-human antibody.
  • Humanized antibodies include human immunoglobulins in which residues from a complementary determining region (CDR) of the human immunoglobulin are replaced by residues from a CDR of a non- human species such as mouse, rat or rabbit having the desired binding specificity, affinity and capacity.
  • CDR complementary determining region
  • the humanized antibody will comprise substantially all of at least one, and generally two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR (framework) regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin; see, inter alia, Jones et al., Nature 321 (1986), 522-525, Presta, Curr. Op. Struct. Biol. 2 (1992), 593-596.
  • Fc immunoglobulin constant region
  • a humanized antibody has one or more amino acids introduced into it from a source which is non-human still retain the original binding activity of the antibody.
  • Methods for humanization of antibodies/antibody molecules are further detailed in Jones et al., Nature 321 (1986), 522-525; Reichmann et al., Nature 332 (1988), 323-327; and Verhoeyen et al., Science 239 (1988), 1534-1536.
  • Specific examples of humanized antibodies, e.g. antibodies directed against EpCAM are known in the art, see e.g. (LoBuglio, Proceedings of the American Society of Clinical Oncology Abstract (1997), 1562 and Khor, Proceedings of the American Society of Clinical Oncology Abstract (1997), 847).
  • the antibody of the present invention comprises a variable heavy chain region comprising CDR-H1 according to SEQ ID NO.: 61 (GYFMH), CDR-H2 according to SEQ ID NO.: 62 (EINPSNARTNYNERFQG), and CDR-H3 according to SEQ ID NO.: 63 (DYYGTSYNFDY), and a variable light chain region comprising CDR-L1 according to SEQ ID NO.: 64 (KANEDINARLA), CDR-L2 according to SEQ ID NO.: 65 (GATNLVT), and CDR-L3 according to SEQ ID NO.: 66 (QQYYSTPFT).
  • framework regions also referred to as FR regions are the part of the variable domain of the antibody which is not CDR. Accordingly, in each variable domain sequence of the antibody four framework regions are present, which are separated from each other by hypervariable regions - CDRs.
  • the framework regions typically may make up to 85% of the sequence of the variable domain and act as a scaffold for exposing CDRs so that they can interact with the antigen. As known to the skilled person, variations in the framework region may also affect the binding affinity of the antibody to the antigen specific for CDRs.
  • antibody molecules (or antigen-binding fragments thereof, as applicable) are provided, which are humanized and can successfully be employed in pharmaceutical compositions.
  • Percent (%) amino acid sequence identity with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • the heavy chain of the antibody does not bear a mutation leading do deglycosylation of the antibody.
  • the antibody of the present invention is characterized by a dissociation constant KD to L1-CAM (CD171) not exceeding 10 11 M, more preferably not exceeding 10 12 M. It is to be understood that said KD is preferably measured in a Biacore-based assay.
  • Amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody.
  • Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigenbinding.
  • antibody variants having one or more amino acid substitutions are provided.
  • Sites of interest for substitutional mutagenesis include the CDRs and FRs.
  • Conservative substitutions are shown in Table D1 under the heading of "preferred substitutions.” More substantial changes are provided in Table D1 under the heading of "exemplary substitutions,” and as further described below in reference to amino acid side chain classes.
  • Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.
  • Amino acids may be grouped according to common side-chain properties:
  • Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
  • substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g. a humanized or human antibody).
  • a parent antibody e.g. a humanized or human antibody
  • the resulting variant(s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and/or will have substantially retained certain biological properties of the parent antibody.
  • An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more CDR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g. binding affinity).
  • Alterations may be made in CDRs, e.g., to improve antibody affinity. Such alterations may be made in CDR "hotspots," i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and/or SDRs (a-CDRs), with the resulting variant VH or VL being tested for binding affinity.
  • Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al.
  • affinity maturation diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis).
  • a secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity.
  • Another method to introduce diversity involves CDR-directed approaches, in which several CDR residues (e.g., 4-6 residues at a time) are randomized.
  • CDR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling.
  • CDR H3 and CDR-L3 in particular are often targeted.
  • substitutions, insertions, or deletions may occur within one or more CDRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen.
  • conservative alterations e.g., conservative substitutions as provided herein
  • Such alterations may be outside of CDR "hotspots" or SDRs.
  • each CDR either is unaltered, or contains no more than one, two or three amino acid substitutions.
  • a useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells (1989) Science, 244: 1081-1085.
  • a residue or group of target residues e.g., charged residues such as Arg, Asp, His, Lys, and Glu
  • a neutral or negatively charged amino acid e.g., alanine or polyalanine
  • Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions.
  • a crystal structure of an antigen-antibody complex is used to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution.
  • Variants may be screened to determine whether they contain the desired properties.
  • Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.
  • terminal insertions include an antibody with an N-terminal methionyl residue.
  • Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g. for ADEPT) or a polypeptide which increases the serum half-life of the antibody.
  • an antibody provided herein is altered to increase or decrease the extent to which the antibody is glycosylated.
  • Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.
  • the carbohydrate attached thereto may be altered.
  • Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32 (1997).
  • the oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GIcNAc), galactose, and sialic acid, as well as a fucose attached to a GIcNAc in the "stem" of the biantennary oligosaccharide structure.
  • modifications of the oligosaccharide in an antibody of the invention may be made in order to create antibody variants with certain improved properties.
  • antibody variants having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region.
  • the amount of fucose in such antibody may be from 1 % to 80%, from 1 % to 65%, from 5% to 65% or from 20% to 40%.
  • the amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e. g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example.
  • Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fe region residues); however, Asn297 may also be located about ⁇ 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd).
  • Examples of publications related to "defucosylated” or “fucose deficient” antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621 ; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; W02005/053742; W02002/031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng.
  • Examples of cell lines capable of producing defucosylated antibodies include Led 3 OHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US 2003/0157108 Al, Presta, L; and WO 2004/056312 Al, Adams et al., especially at Example 11), and knockout cell lines, such as alpha-1 , 6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Bioteeh. Bioeng. 87: 614 (2004); Kanda, Y. et al., Bioteehnol. Bioeng., 94(4):680-688 (2006); and W02003/085 I07).
  • Antibodies variants are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GIcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet et al.); US Patent No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.). Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided.
  • Such antibody variants may have improved CDC function.
  • Such antibody variants are described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).
  • one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant.
  • the Fc region variant may comprise a human Fc region sequence (e.g., a human lgG1 , lgG2, lgG3 or lgG4 Fc region) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions.
  • the invention contemplates an antibody variant that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half-life of the antibody in vivo is important yet certain effector functions (such as complement and antibody-dependent cellular cytotoxicity) are unnecessary or deleterious.
  • In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC (complement-dependent cytotoxicity) and/or ADCC activities.
  • Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcyR binding (hence likely lacking ADCC activity), but retains FcRn binding ability.
  • NK cells express Fc(RI II only, whereas monocytes express Fc(RI, Fc(RII and Fc(RIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).
  • Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 (see, e.g. Hellstrom, I. et al. Proc. Nat’l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc.
  • the heavy chain comprises at least one point mutation in Fc part that influences antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), serum half-life and/or glycosylation status of the antibody.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • serum half-life and/or glycosylation status of the antibody it is to be understood to the skilled person that only antigen-binding fragments of said antibody that comprise the heavy chain, or the fragment thereof, may be encompassed herein.
  • the at least one point mutation as referred to herein is preferably selected from L234A, L234F, L235A, L235E, L235Q, G236A, M252Y, S254T, T256E, S267E, H268F, N297A, N297Q, K322A, K322Q, S324T, P331 S, and I332E. More preferably, the at least one point mutation is selected from L234A, L235A, P331 S and N297A. It is to be understood herein that the at least one point mutation may refer to more than one mutation.
  • the heavy chain comprises L234A, L235A, P331 S and N297A point mutations.
  • amino acid positions as recited herein refer to the residue numbering as in I gG1 isotype. Should any other antibody class or isotype be used, the skilled person will be in position to translate the amino acid positions to any other antibody class or isotype known in the art.
  • the heavy chain comprises L234A, L235A, and P331 S point mutations.
  • Such a set of mutations without any mutation to N297, maintains natural glycosylation pattern and is beneficial for the stability of the antibody.
  • the heavy chain comprises N297Q mutation. This mutation enables attachment of 4 cytotoxic molecules through a linker that is conjugated by using transglutaminase attachment strategy.
  • the heavy chain comprises L234A, L235A, P331 S and N297Q point mutations.
  • the skilled person may also envisage including at least one mutation that increases antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • One possible combination is at least one mutation selected from S267E/H268F/S324T/G236A/I332E (EFTAE modification - SEQ ID NO.: 79), preferably the mutations S267E/H268F/S324T/G236A/I332E (EFTAE modification).
  • the skilled person may further envisage including at least one mutation that counters the negative effect of N297A mutation (which allows for the production of deglycosylated antibodies).
  • One possible combination is at least one mutation selected from F241 K, L309D, T307R and T307P, preferably the mutations F241 K, L309D, T307R and T307P.
  • the at least one mutation selected from F241 K, L309D, T307R and T307P, preferably the mutations F241 K, L309D, T307R and T307P may lead to increased thermal stability of the antibody. Without being bound by the theory, said mutation(s) increase the stability of the Fc part of the heavy chain of the antibody.
  • Encompassed by the present invention are further the antibodies as described herein, further comprising in its heavy chain one or more following mutations: G31 D, G31 E, G31 K, G31 P, G31 R, H35K, T28D, T28E, T28K, T28P, T28R, T30D, T30E, T30K, T30P, T30R, W33R, Y27D, Y27E, and Y27K, preferably comprising a mutation selected from G31 D, G31 E, G31 K, G31 P, G31 R, H35K, T28D, T28E, T28K, T28P, T28R, T30D, T30E, T30K, T30P, T30R, W33R, Y27D, Y27E, and Y27K.
  • the antibodies as described herein further comprising in its heavy chain one or more following mutations: T30P, T28K and T30E.
  • at least one mutation selected from G31 D, G31 E, G31 K, G31 P, G31 R, H35K, T28D, T28E, T28K, T28P, T28R, T30D, T30E, T30K, T30P, T30R, W33R, Y27D, Y27E, and Y27K preferably selected from T30P, T28K and T30E stabilize the flanking region of CDR1 and/or may lead to improved affinity of the antibody or its fragment to L1-CAM.
  • the antibodies as described herein further comprising in its heavy chain one or more following mutations: A40R, V68A, and L115T.
  • these mutations correspond to incorporation of the residues originally present in the murine antibody, which are close to the aggregation-prone regions.
  • the at least one mutation selected from A40R, V68A, and L115T may lead to increased thermal stability of the antibody.
  • said mutation(s) increase the stability of the Fc part of the heavy chain of the antibody.
  • the antibody or an antigen binding thereof of the present invention, provided herein may be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available.
  • the moieties suitable for derivatization of the antibody include but are not limited to water soluble polymers.
  • Nonlimiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1 , 3-dioxolane, poly-1 , 3, 6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.
  • PEG polyethylene glycol
  • copolymers of ethylene glycol/propylene glycol carboxymethylcellulose
  • dextran polyvinyl alcohol
  • Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water.
  • the polymer may be of any molecular weight, and may be branched or unbranched.
  • the number of polymers attached to the antibody may vary, and if more than one polymer are attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.
  • water-soluble polymers such as polymers that include acrylic backbone, are described hereinbelow.
  • the antibody or an antigen binding thereof of the present invention may also be further modified to contain additional moieties, thus yielding immunoconjugates comprising the antibody as described herein. Said immunoconjugates are further described herein.
  • Antibodies may be produced using recombinant methods and compositions, e.g., as described in U.S. Patent No. 4,816,567.
  • isolated nucleic acid encoding an antibody described herein is provided.
  • Such nucleic acid may encode an amino acid sequence comprising the VL and/or an amino acid sequence comprising the VH of the antibody (e.g., the light and/or heavy chains of the antibody).
  • isolated nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment.
  • An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extra chromosomally or at a chromosomal location that is different from its natural chromosomal location.
  • isolated nucleic acid encoding an anti-L1-CAM antibody refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.
  • one or more vectors comprising such nucleic acid are provided.
  • the present invention relates to a polynucleotide encoding at least one variable heavy chain sequence and/or at least one variable light chain sequence as described herein. It is to be understood that, unless indicated to the contrary, the terms polynucleotide and (isolated) nucleic acids may be used interchangeably.
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced.
  • Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors”.
  • the present invention further relates to a vector comprising the polynucleotide encoding at least one variable heavy chain sequence and/or at least one variable light chain sequence as described herein.
  • a host cell comprising such nucleic acid is provided.
  • host cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
  • a host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH of the antibody.
  • the host cell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., YO, NSO, Sp20).
  • a method of making an antibody of the present invention comprises culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).
  • nucleic acid encoding an antibody is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell.
  • nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).
  • Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein.
  • antibodies may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed.
  • U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. See also Charlton, Methods in Molecular Biology, Vai. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, describing expression of antibody fragments in E. coli.
  • the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized,” resulting in the production of an antibody with a partially or fully human glycosylation pattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).
  • Suitable host cells for the expression of glycosylated antibody are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera fiugiperda cells.
  • Plant cell cultures can also be utilized as hosts. See, e.g., US Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIESTM technology for producing antibodies in transgenic plants).
  • Vertebrate cells may also be used as hosts.
  • mammalian cell lines that are adapted to grow in suspension may be useful.
  • Other examples of useful mammalian host cell lines are macaque kidney CVI line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham et al., J. Gen Viral. 36:59 (1977)); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described, e.g., in Mather, Biol. Reprod.
  • CV I macaque kidney cells
  • VERO-76 African green macaque kidney cells
  • HELA human cervical carcinoma cells
  • canine kidney cells MDCK; buffalo rat liver cells (BRL 3A); human lung cells (WI38); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., in Mather et al., Annals N. Y Aead. Sei. 383:44-68 (1982); MRC 5 cells; and FS4 cells.
  • Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR CHO cells (Urlaub et al., Proc. Natl. Acad.
  • the antibody may in one embodiment include a further domain or a further amino acid sequence.
  • the antibody may include a localization sequence.
  • suitable include aldehyde tag and sortase recognition motif.
  • the aldehyde tag is an artificial peptide tag recognized by the formylglycine-generating enzyme (FGE).
  • FGE formylglycine-generating enzyme
  • a suitable example of an aldehyde tag is a tag according to sequence LCTPSR (SEQ ID NO.: 67), wherein upon FGE acting on said sequence, the cysteine residue is converted to formylglycine.
  • the sortase recognition motif is according to sequence LPXTG (SEQ ID NO.: 68), wherein X can be any natural amino acid residue.
  • the sortase enzyme for example Staphylococcus aureus sortase, is a transpeptidase that attaches surface proteins to the cell wall; it cleaves between the Gly and Thr of the LPXTG motif and catalyses the formation of an amide bond between the carboxyl-group of threonine and the amino-group of the cell-wall peptidoglycan.
  • sortase recognition motif allows for attachment of further peptidic moieties.
  • the antibody may in one embodiment include further a domain or an amino acid sequence used for cotargeting to tumor microenvironment (e.g., targeting Fibroblast activation protein-a (FAP)); to overcome blood brain barrier (BBB) (e.g., targeting transferrin receptors); or to overcome endothelial cells (EC) barrier, (e.g., caveolae targeting of aminopeptidase P2 (APP2).
  • BBB blood brain barrier
  • EC endothelial cells
  • Said additional targeting domain may thereby be directly included into the sequence of the antibody or the antigen-binding fragment to the C or N-terminus using a spacer peptide (fusion construct) or later attached e.g., by a site-specific functionalization.
  • the antibody may also optionally include one or more non-canonical amino acids to be used for coupling of said antibody with another chemical entity.
  • Said amino acids may include residues that would be reactive in addition reactions known to the skilled person as click chemistry. Suitable examples of such residues include residues comprising azide moiety, or cyclooctyne moiety or a moiety being capable to perform an inverse-demand Diels-Alder cycloaddition reaction e.g., a trans-cyclooctene / tetrazine reaction pair.
  • the invention is not meant to be limited to any of these examples, and other such residues known to the skilled person may also be used.
  • the methods of producing antibodies (or fragments thereof, as applicable) comprising non-canonical amino acid residue(s) using recombinant methods are known to the skilled person.
  • the immunoconjugate of the present invention comprises b) a cytotoxic compound.
  • Said cytotoxic compound may be a cytotoxic agent or a prodrug thereof.
  • Said cytotoxic compound/agent or prodrug may also be referred to as drug or an active agent.
  • the immunoconjugate of the present invention may also be referred to as an antibody-drug conjugate.
  • Antibody-drug conjugates are targeted chemotherapeutic molecules which combine properties of both antibodies and cytotoxic drugs by targeting potent cytotoxic drugs to antigen expressing tumor cells (Teicher, B.A. (2009) Current Cancer Drug Targets 9:982-1004), thereby enhancing the therapeutic index by maximizing efficacy and minimizing off-target toxicity (Carter, and Senter P.D. (2008) The Cancer Jour. 14(3): 154-169; Chari, R.V. (2008) Ace. Chem. Res. 41 :98-107.
  • the ADC compounds of the invention include those with anticancer activity.
  • the ADC compounds include an antibody conjugated, i.e. covalently attached, to the drug moiety.
  • the antibody is covalently attached to the drug moiety through a linker. It is preferred that the antibody is covalently attached to the drug moiety through a linker, as described herein.
  • the antibody-drug conjugates (ADC) of the invention selectively deliver an effective dose of a drug to tumor tissue whereby greater selectivity, i.e. a lower efficacious dose, may be achieved while increasing the therapeutic index ("therapeutic window").
  • Drug moieties may impart their cytotoxic and cytostatic effects by mechanisms including but not limited to tubulin binding, DNA binding or intercalation, and inhibition of RNA polymerase, protein synthesis, and/or topoisomerase.
  • Exemplary drug moieties include, but are not limited to, a maytansinoid, calicheamicin, pyrrolobenzodiazepine (PBD), nemorubicin and its derivatives, PNU-159682, anthracycline, duocarmycin, vinca alkaloid, taxane, trichothecene, CC1065, camptothecin, Deruxtecan, Exatecan, elinafide, and stereoisomers, isosteres, analogs, and derivatives thereof that have cytotoxic activity.
  • PBD pyrrolobenzodiazepine
  • nemorubicin and its derivatives PNU-159682
  • anthracycline duocarmycin
  • vinca alkaloid taxane
  • taxane
  • the drug is a topoisomerase inhibitor, i.e. an inhibitor of topoisomerase I and/or II, for example a derivative of Camptothecin, preferably selected from Exatecan, Deruxtecan, topotecan, irinotecan, SN38, and belotecan.
  • a topoisomerase inhibitor i.e. an inhibitor of topoisomerase I and/or II, for example a derivative of Camptothecin, preferably selected from Exatecan, Deruxtecan, topotecan, irinotecan, SN38, and belotecan.
  • the active agent i.e., the drug moiety, or the cytotoxic compound
  • the active agent is selected from maytansinoid, calicheamicin, pyrrolobenzodiazepine (PBD), nemorubicin and its derivatives, PNU-159682, anthracycline, duocarmycin, vinca alkaloid, taxane, trichothecene, CC1065, camptothecin, elinafide, Exatecan, Deruxtecan, topotecan, irinotecan, SN38, and belotecan. More preferably, the drug is selected from Exatecan, Deruxtecan and SN38.
  • the immunoconjugate of the present invention comprises b) a cytotoxic compound, preferably selected from Exatecan, Deruxtecan and SN-38, more preferably selected from Exacetan and Deruxcetan.
  • the immunoconjugate of the present invention comprises Exatecan.
  • the compound Exatecan is to be understood as the compound according to formula: or a pharmaceutically acceptable salt thereof.
  • Exatecan can be attached to the antibody in any way conceivable to the skilled person. Particularly preferred is attachment through amino group or hydroxy group. Even more preferred is attachment through amino group. Particularly preferred attachment of Exatecan is through the linker of formula
  • Li is— (CH2)6-CO- 3 attached through its left empty valence to Zi and through its right empty valence to Lp;
  • Lp is -Gly-Gly-Phe-Gly- (SEQ ID NO.: 77), wherein the C-terminus of the Lp is connected to the cytotoxic compound (i.e., to amino group of Exatecan, through an amide bond) and wherein the N-terminus is connected to Li.
  • Such particularly preferred attachment corresponds to the attachment of the following compound: upon reaction with -SH group of cysteine of the antibody, with the maleimide moiety. Accordingly, in one embodiment, the immunoconjugate of the present invention is obtainable in the reaction of said compound with -SH group of cysteine of the antibody.
  • the linker is of formula
  • Li is— (CH2)5-CO-, attached through its left empty valence to Zi and through its right empty valence to Lp;
  • Lp is -Gly-Gly-Phe-Gly- (SEQ ID NO.: 77), wherein the C-terminus of the Lp is connected to the cytotoxic compound (i.e., to amino group of Exatecan, through an amide bond) and wherein the N-terminus is connected to Li.
  • Li is — (C1-10 alkylene)-CO-, preferably -(CH2)6-CO-, attached through its left empty valence to Z1 and through its right empty valence to Lp;
  • Lp is -Gly-Gly-Phe-Gly- (SEQ ID NO.: 77), wherein the C-terminus of the Lp is connected to Lx and wherein the N-terminus is connected to Li;
  • Lx is -NHCH2OCH2CO-, wherein the right empty valence of Lx is connected to the cytotoxic compound (i.e., to amino group of Exatecan, through an amide bond)and wherein the left empty valence of Lx is connected to Lp.
  • a linker is according to formula
  • Li is — (C1-10 alkylene)-CO-, preferably -(CH2)5-CO-, attached through its left empty valence to Z1 and through its right empty valence to Lp;
  • Lp is -Gly-Gly-Phe-Gly- (SEQ ID NO.: 77), wherein the C-terminus of the Lp is connected to Lx and wherein the N-terminus is connected to Li; and Lx is -NHCH2OCH2CO-, wherein the right empty valence of Lx is connected to the cytotoxic compound (i.e., to amino group of Exatecan, through an amide bond) and wherein the left empty valence of Lx is connected to Lp.
  • cytotoxic compound i.e., to amino group of Exatecan, through an amide bond
  • the immunoconjugate of the present invention comprises Deruxtecan.
  • Deruxtecan is well known to the skilled person.
  • an immunoconjugate that comprises Deruxtecan is obtainable upon reacting with the antibody or its fragment the compound of formula:
  • the compound of formula as outlined directly above may also be referred to as Deruxtecan.
  • Deruxtecan comprises Exatecan
  • the immunoconjugate comprising Deruxtecan is an immunoconjugate comprising Exatecan.
  • the immunoconjugate of the present invention comprises SN-38.
  • SN-38 is a compound according to formula: or a pharmaceutically acceptable salt thereof. It is to be understood that SN38 can be attached to the antibody in any way conceivable to the skilled person. Particularly preferred is attachment through aliphatic hydroxy group.
  • the cytotoxic compound is selected from Fasudil, Sirolimus, Imatinib, Gefitinib, Erlotinib, Soragenib, Sunitinib, Dasatinib, Lapatinib, Nilotinib, Temsirolimus, Everolimus, Pazopanib, Ruxolitinib, Vandetanib, Vemurafenib, Crizotinib, Icotinib, Axitinib, Tofacitinib, Bosutinib, Cabozantinib, Ponatinib, Regorafenib, Afatinib, Dabrafenib, Trametinib, Ibrutinib, Nintedanib, Idelasilib, Ceritinib, Apatinib, Ripasudil, Alectinib, Cobimetinib, Lenvatinib, Palbociclib, Radotinib, Osi
  • cytotoxic agent refers to a substance thatinhibits or prevents a cellular function and/or causes cell death or destruction.
  • Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At 211 , 1 131 1 125 , Y 90 , Tb 161 ,Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins
  • radioisotope/radionuclide in the scope of this invention are preferably used synonymically and preferably represent an atom that has excess nuclear energy, making it unstable. This excess energy can be used in one of three ways: emitted from the nucleus as gamma radiation; transferred to one of its electrons to release it as a conversion electron; or used to create and emit a new particle (alpha particle or beta particle) from the nucleus.
  • Radioisotope/radionuclide is herein preferably defined as an isotope which has a half-life of less than 10 19 years.
  • the present invention relates to an immunoconjugate, wherein said immunoconjugate comprises a radioisotope as an active agent.
  • said immunoconjugate comprises a radioisotope as an active agent.
  • Such conjugate may also be referred to as antibody-radionuclide conjugate.
  • ARC antibody radionuclide conjugate
  • drug molecule or active molecule represents a radionuclide/radioisotope either covalently bound to the antibody-polymer-conjugate e.g. in case of radioactive iodine or by a metal chelator complex e.g. with radioactive lutetium, actinium or terbium.
  • the so formed ARC is capable to deliver a high amount of radiation to the tumor tissue thereby killing the tumor cells due to the damaging of DNA, essential enzymes etc.
  • ARC also encompasses antibody radionuclide conjugates not comprising polymer carrier, but instead comprising PEG or peptide linkers.
  • PEG polymer carrier
  • peptide linker is (Ac)-Lys-Ala-Tyr-Ala-Lys(Azide)-NH2 [SEQ ID NO.: 78] useful for subsequent functionalization with DBCO-DOTA.
  • certain antibody-radionuclide conjugate may also be used for diagnostic purposes.
  • Certain radionuclides as disclosed herein can be monitored, for example terbium 161 due to its y-emission can be visualized with gamma camera and hence used for detection of cancer tissue or cell-type, as targeted to by the antibody.
  • Terbium-149 which can be used for targeted alpha therapy, has visibility in PET scans and thus can be monitored.
  • fluorine-18, scandium-43, scandium-44, copper-61 , copper-64, gallium-68, zirconium-89, indium-11 1 , iodine-123, terbium-152, terbium-155 are particularly useful in diagnostic application as described herein and may be referred to as radionuclides useful in diagnosis.
  • the radionuclides useful in diagnosis can be monitored by using a suitable method, for example Scintigraphy, Single Photon Emission Computed Tomography (SPE-CT); or Positron emission tomography Computed Tomography (PET-CT).
  • an immunoconjugate wherein the active agent comprises a radionuclide useful for therapeutic application for example selected from copper-67, strontium-89, yttrium-90, iodine-131, samarium-153, lutetium-177, radium-223 and actinium 225, (these radionuclides may be referred to as radionuclides useful in therapy) will have substantially the same biodistribution as the immunoconjugate wherein the active agent is a radionuclide useful for diagnostic application. Therefore, the immunoconjugates of the present invention can preferably be used for monitoring of biodistribution of therapeutic immunoconjugates during therapy.
  • an immunoconjugate comprising an active agent being a radionuclide useful in therapy can be supplemented for this purpose preferably with less than 10 weight% of an immunoconjugate wherein the active agent comprises a radionuclide useful in diagnosis, as defined herein.
  • the immunoconjugate of the present invention for use in the combined therapeutic and diagnostic application may comprise two radionuclides, one radionuclide useful in therapy and one radionuclide useful in diagnosis, for example attached to a single polymeric carrier, to different repeating units thereof.
  • Preferable are combinations wherein a radionuclide useful in therapy and a radionuclide useful in diagnosis are isotopes of the same element.
  • preferred combinations include scandium-43 and scandium-47, copper-61 and copper-67, copper-64 and copper-67, iodine-123 and iodine-131 , terbium-152 and terbium-161 , and terbi um-155 and terbi um- 161 .
  • Further preferred combinations include isotopes of two different elements, for example indium-111 and lutetium-177, and indium-111 and terbium-161.
  • linkers are not to be particularly limited and any linker conceivable to the skilled person as usable in the immunoconjugate, or antibody-drug conjugate, can be used within the scope of the present invention.
  • Exatecan can be attached as shown in the following schemes, wherein R represents, in general, the antibody or the antigen-binding fragment thereof, including the rest of the linkage between said antibody or said fragment thereof and the drug moiety (D):
  • the linker takes the form of a polymeric linker.
  • said polymeric linker includes at least two types of repeating unit, preferably more than two types of a repeating unit.
  • at least one type of the repeating unit is covalently attached to an active agent, for example a cytotoxic agent.
  • at least one type of the repeating unit is free of said active agent, thus accordingly, the polymeric carrier may the active agent incorporated into some of its repeating units. Further accordingly, multiple molecules of the active agent may thus so be attached to a single attachment point on the antibody or an antigen-binding fragment thereof, by using a polymeric carrier.
  • any polymeric carrier attachable to an antibody or to a fragment thereof may be used in the immunoconjugates of the present invention.
  • Examples of polymeric carriers particularly suitable for use in the immunoconjugates of the present invention are described in the patent applications PCT/EP2020/080545 and PCT/EP2019/061769, each of which is herewith incorporated by reference in its entirety.
  • One exemplary polymeric carrier will be defined in the following.
  • the polymeric carrier comprises a repeating unit of a formula (R1 ) wherein R is -H, -CH 3 , -CH2-CH3 or -(CH 2 )2-CH 3 ;
  • X is -NH(CH 2 )4- -NH(CH 2 ) 3 -, -O-C6H4-CH2-, -O-CH2-, - O-CH(CH 3 )-, -S-CH2- or -NH-C6H4-CH2-;
  • L is a spacer, and P comprises an active agent.
  • L may be is a linker/spacer that is non-cleavable under physiological conditions.
  • L may be a linker/spacer that is cleavable under physiological conditions.
  • linkers include cathepsin B-sensitive dipeptide or tripeptide linkers, pH-sensitive hydrazones or cis-aconityl-based linkers, or pyrophosphate diester linkers for lysosomal trafficking and cleavage.
  • L may also be -CO-valine-citrulline-PABC, wherein PABC represents p-aniline-beta-carbamate, valine-lysine, valine-alanine, valine-arginine, or glutamate- valine-citrulline.
  • A is -0-, Z is preferably H.
  • the copolymer comprising the repeating unit of formula (R1) further comprises a repeating unit of formula (R2): wherein R is -H, -CH 3 , -CH2-CH3 or -(CH 2 )2-CH 3 ;
  • X is -NH(CH 2 )4-, -NH(CH 2 ) 3 -, -O-C6H4-CH2-, -O-CH2-, - O-CH(CH 3 )-, -S-CH2- or -NH-C6H4-CH2- ;
  • A is -0- or -NH-, and/or a repeating unit of formula (R3):
  • A is -0-, Z is preferably H.
  • Z can be H and/or Y can be H.
  • Z and/or Y can comprise a second payload molecule.
  • the payload molecule is as defined herein.
  • the polymeric carrier as described herein comprises a further repeating unit obtainable by polymerization of N,N-dimethyl-acrylamide, N-isobutyl-acrylamide, N-tert. butyl-acrylamide, N-hydroxyethyl-acrylamide, N-(2-hydroxypropyl)-acrylamide, N-(3-hydroxypropyl)-acrylamide, N-(3- hydroxypropyl)-methacrylamide, N-(2-hydroxypropyl)-methacrylamide, N-(3-aminopropyl)-acrylamide hydrochloride, or N-(3-aminopropyl)-methacrylamide hydrochloride, or a repeating unit obtained through polymerization of methacrylic acid, 2-hydroxyethyl-acrylate, 2-hydroxypropyl-acrylate, 3-hydroxypropyl- acrylate, 2- hydroxy-1 -methylethyl-acrylate, 2-aminoethyl acrylate hydrochloride, 3-hydroxypropyl-
  • the average number of repeating units according to formula (R1) per molecule of copolymer is 2 to 12, preferably 2 to 8, more preferably 2 to 6.
  • the average number of repeating units according to formulae (R1 ), (R2) or (R3) per molecule of copolymer is 10 to 50, preferably 10 to 40, more preferably 10 to 30.
  • the average number of repeating units according to formulae (R1), (R2) or (R3) per molecule of copolymer is 4 to 20, preferably 4 to 15, more preferably 4 to 10.
  • R is -H, -CH 3 , -CH2-CH3 or -(CH 2 )2-CH 3 ;
  • A is -0-, Z is preferably H.
  • Z can be H and/or Y can be H.
  • Z and/or Y can comprise a second payload molecule.
  • the payload molecule is as defined herein.
  • polymeric carriers to be used in the immunoconjugates of the present invention include the carriers derived from the compounds selected from: wherein each X is independently -NH(CH2)4-, or -NH-CeH4-CH2-;n is 40-120; m is 4-16; o is 0-16; f is -H or -I (it is to be understood that I includes both cold isotopes of iodine as well as hot isotopes of iodine), k and q are designated as an active payload or could be H.
  • the end group, e may be H or may be a modified end group, as discussed and exemplified in the following.
  • the end group e may be introduced via a thiol - reactive group.
  • Suitable reagents include but not limited to:
  • the payload molecules k and q may be introduced via an amino-reactive group.
  • Suitable reagents that could be used for introducing said payload molecules are selected from: p-SCN-Bn-CHX-A”-DTPA: [(R)-2-Amino-3-(4-isothiocyanatophenyl)propyl]-trans-(S,S)-cyclohexane-1 ,2- diamine-pentaacetic acid; i-SCN-Bn-DTPA: S-2-(4-lsothiocyanatobenzyl)-diethylenetriamine pentaacetic acid;
  • DOTA-GA ide 2,2',2”-(10-(2, 6-dioxotetrahydro-2H-pyran-3-yl)-1, 4,7,10-tetraazacyclododecane- 1 ,4,7-triyl)triacetic acid;
  • i-SCN-Bn-DOTA S-2-(4-lsothiocyanatobenzyl)-1 ,4,7,10-tetraazacyclododecane tetraacetic acid;
  • NHS DOTA 1 ,4,7, 10-Tetraazacyclododecane-1 ,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester;
  • i-SCN-Bn-NOTA 2-S-(4-lsothiocyanatobenzyl)- 1 ,4,7-triazacyclononane-1 ,4, 7-triacetic acid;
  • NOTA-NHS ester 2,2'-(7-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7-triazonane-1,4-diyl)diacetic acid;
  • NODA-GA-NHS ester 2,2'-(7-(1-carboxy-4-((2,5-dioxopyrrolidin-1-yl)oxy)-4-oxobutyl)-1 ,4,7-triazonane- 1 ,4-diyl)diacetic acid;
  • Paclitaxcel NHS (1 S,2R)-1 -benzamido-3-(((2aR,4S,4aS,6R,9S, 11 S, 12S, 12aR, 12bS)-6, 12b-diacetoxy- 12-(benzoyloxy)-4, 11 -dihydroxy-4a, 8, 13, 13-tetramethyl-5-oxo-2a,3,4,4a, 5,6,9,10,11 ,12,12a, 12b- dodecahydro-1 H-7, 11 -methanocyclodeca[3,4]benzo[1 ,2-b]oxet-9-yl)oxy)-3-oxo-1 -phenylpropan-2-yl (2,5-dioxopyrrolidin-1-yl) succinate;
  • MMAE-NHS 4-(2-(2-((((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)amino)-3-methylbutanamido)-5- ureidopentanamido)benzyl (1-((1-((1-(2-(3-((1 -hydroxy-1 -phenylpropan-2-yl)amino)-1 -methoxy-2-methyl- 3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan- 2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate;
  • MMAF-NHS N-[6-[(2,5-Dioxo-1-pyrrolidinyl)oxy]-6-oxohexyl]-N-methyl-L-valyl-L-valyl-(3R,4S,5S)-3- methoxy-5-methyl-4-(methylamino)heptanoyl-(aR,pR,2S)-p-methoxy-a-methyl-2-pyrrolidinepropanoyl-L- phenylalanine;
  • DM1-SMCC Maytansinoid-NHS-derivative: N2'-deacetyl-N2'-[3-[[1 -[[4-[[(2, 5-dioxo- 1 - pyrrolidinyl)oxy]carbonyl]cyclohexyl]methyl]-2,5-dioxo-3-pyrrolidinyl]thio]-1-oxopropyl]- Maytansine;
  • SC-VC-PAB-DM1 (CAS: 2259318-47-1);
  • Doxorubicin-SMCC 7,8,9, 10-tetrahydro-6,8,11 -trihydroxy-8-(2-hydroxyacetyl)-1 -methoxy-10-[[2,3,6- trideoxy-3-[[[4-[(2,5-dihydro-2,5-dioxo-1 H-pyrrol-1 -yl)methyl]cyclohexyl]carbonyl]amino]-a-L-lyxo- hexopyranosyl]oxy]-, (8S,1 OS)- 5,12-Naphthacenedione;
  • the linker comprises d ) a moiety according to formula:
  • the linkage to the antibody through Zi may also be referred to as thiol- maleimide linkage, which is formed in the following chemical reaction: wherein Cys is a cysteine residue within the antibody, presenting an -SH moiety, as shown in the scheme.
  • thiol- maleimide linkage is formed in the following chemical reaction: wherein Cys is a cysteine residue within the antibody, presenting an -SH moiety, as shown in the scheme.
  • Li is — (C1-10 alkylene)-CO-.
  • Li is — (C4-8 alkylene)-CO-. More preferably, Li is — (C5-7 alkylene)-CO-. Even more preferably Li is — (Ce alkylene)-CO-. Still more preferably, Li is— (CH2)6- CO-.
  • Li attached through its left empty valence to Z1 and through its right empty valence to Lp.
  • Lp is -Gly-Gly-Phe-Gly-, wherein the C-terminus of the Lp is connected to the cytotoxic compound and wherein the N-terminus of the Lp is connected to Li.
  • the Lp as recited herein or in other occurrences of the present disclosure, is the most preferred peptide portion of the linker. However, as apparent to the skilled person, Lp can also be another peptidic moiety, such as -Val-Citruline-.
  • the linker comprises c2) a moiety according to formula (II):
  • Z2 is connected to LPEG, wherein the top empty valences of Z2 are attached to an LN3 moiety of a carrier.
  • Said carrier will be defined in the following.
  • LPEG is -(CH2CH20)n-(Ci-io alkylene)-CO-, wherein n is an integer from 2 to 6. Accordingly, n can be 2, 3, 4, 5, or 6. Preferably, n is an integer from 3 to 5. More preferably, n is 4.
  • C1-10 alkylene is C1-5 alkylene, more preferably, C1-10 alkylene is C1-3 alkylene, even more preferably, C1-10 alkylene is -CH2CH2-.
  • LPEG is -(CH2CH2O)4-(CH2CH2)-CO-. It is to be understood that, preferably, LPEG is attached through its left empty valence to Z2 and through its right empty valence to Lp.
  • Lp is -Gly-Gly-Phe-Gly-. It is to be understood that the C-terminus of Lp is connected to -Lx and that the N-terminus of Lp is connected to LPEG.
  • Lx is -NHCH2OCH2CO-.
  • the right empty valence of Lx as shown herein, is connected to the cytotoxic compound and the left empty valence of Lx is connected to Lp.
  • the top empty valences of Z2 are attached to an LN3 moiety of a carrier, as discussed herein.
  • the carrier as referred to herein, can be d1) a moiety according to formula (III)
  • n is an integer from 2 to 4. Accordingly, m can be 2, 3 or 4. Preferably m is 2.
  • each o is independently an integer from 2 to 6. Accordingly, each o can be 2, 3, 4, 5 or 6. Preferably, each o is 3.
  • LN3 is a moiety according to formula: wherein the bottom empty valences are attached to Z2.
  • LN3 moiety and Z2 moiety are formed in the course of a 3+2 cycloaddition, which is also referred to as click-chemistry, of a compound comprising an azide moiety (-N3) and a compound comprising a DBCO moiety:
  • the carrier as referred to herein, may be d2) a moiety according to formula (IV):
  • p is an integer from 2 to 10. Accordingly, p can be 2, 3, 4, 5, 6, 7, 8, 9 or 10. Preferably, p is an integer from 4 to 6. More preferably, p is 5.
  • L c is a polymeric moiety comprising a repeating unit according to formula (Va): and a repeating unit according to formula (Vb): wherein LN3 is a moiety according to formula: wherein the bottom empty valences are attached to Z2.
  • L c includes on average 3 to 5 repeating units of formula Vb. Further preferably, the ratio of repeating units according to formula (Vb) to repeating units according to formula (Va) is from 0.06 to 0.14.
  • attachment of the cytotoxic drug may also occur according to the reaction of trans-cyclooctene with tetrazine, according to the following reaction scheme:
  • the present invention further relates to, in one specific embodiment, an immunoconjugate comprising: a) an antibody that specifically binds to L1-CAM (CD 171); b) a cytotoxic compound, wherein the cytotoxic compound is Exatecan; and c) a linker, wherein the linker is in a form of a polymeric carrier, which comprises at least two types of repeating unit, wherein at least one type of the repeating unit is covalently attached to an active agent, for example a cytotoxic agent.
  • an immunoconjugate comprising: a) an antibody that specifically binds to L1-CAM (CD 171); b) a cytotoxic compound, wherein the cytotoxic compound is Exatecan; and c) a linker, wherein the linker is in a form of a polymeric carrier, which comprises at least two types of repeating unit, wherein at least one type of the repeating unit is covalently attached to an active agent, for example a cytotoxic agent.
  • a) is as described herein.
  • a) is as follows: a1 ) a heavy chain comprising a sequence at least 95% identical to a sequence according to a SEQ ID NO.: 2 and a light chain comprising a sequence at least 95% identical to a sequence according to SEQ ID NO.: 6; or a2) a heavy chain with sequence at least 95% identical to a sequence according to a SEQ ID NO.: 4 and a light chain with a sequence at least 95% identical to a sequence according to SEQ ID NO.: 6.
  • the antibody in a) may be characterized by a1 ) a heavy chain with sequence at least 95% identical to a sequence according to a SEQ
  • the antibody in a) is characterized by a1 ) a heavy chain with sequence at least 98% identical to a sequence according to a SEQ ID NO.: 2 and a light chain with a sequence at least 98% identical to a sequence according to SEQ ID NO.: 6, preferably a heavy chain with sequence identical to a sequence according to a SEQ ID NO.: 2 and a light chain with a sequence identical to a sequence according to SEQ ID NO.: 6.
  • a construct for expression of heavy chain may include an additional signal sequence according to SEQ ID NO.: 75
  • a construct for expression of the light chain may include an additional signal sequence according to SEQ ID NO.: 76.
  • the antibody in a) is characterized by a1 ) a heavy chain with sequence at least 98% identical to a sequence according to a SEQ ID NO.: 1 and a light chain with a sequence at least 98% identical to a sequence according to SEQ ID NO.: 5.
  • the antibody in a) is characterized by a1) a heavy chain with sequence identical to a sequence according to a SEQ ID NO.: 1 and a light chain with a sequence identical to a sequence according to SEQ ID NO.: 5.
  • the antibody in a) may be characterized by a2) a heavy chain with sequence at least 95% identical to a sequence according to a SEQ ID NO.: 4 and a light chain with a sequence at least 95% identical to a sequence according to SEQ ID NO.: 6.
  • the antibody in a) may be characterized by a2) a heavy chain with sequence at least 98% identical to a sequence according to a SEQ ID NO.: 4 and a light chain with a sequence at least 98% identical to a sequence according to SEQ ID NO.: 6, preferably a heavy chain with sequence identical to a sequence according to a SEQ ID NO.: 4 and a light chain with a sequence identical to a sequence according to SEQ ID NO.: 6.
  • a construct for expression of heavy chain may include an additional signal sequence according to SEQ ID NO.: 75
  • a construct for expression of the light chain may include an additional signal sequence according to SEQ ID NO.: 76.
  • the antibody in a) is characterized by a2) a heavy chain with sequence at least 98% identical to a sequence according to a SEQ ID NO.: 3 and a light chain with a sequence at least 98% identical to a sequence according to SEQ ID NO.: 5.
  • the antibody in a) is characterized by a2) a heavy chain with sequence identical to a sequence according to a SEQ ID NO.: 3 and a light chain with a sequence identical to a sequence according to SEQ ID NO.:
  • the polymeric carrier comprises a repeating unit of a formula (R1 ) wherein R is -H, -CH 3 , -CH2-CH3 or -(CH 2 )2-CH 3 ;
  • X is -NH(CH 2 )4- -NH(CH 2 ) 3 -, -O-C6H4-CH2-, -O-CH2-, - 0-CH(CH 3 )-, -S-CH2- or -NH-C6H4-CH2-;
  • L is a /spacer
  • P comprises
  • the present invention is further not limited in any way with respect to the cytotoxic compound-antibody ratio (also referred to as drug-antibody ratio, i.e., DAR), and any obtainable ratio can be used in the immunoconjugate of the present invention.
  • the drug/antibody ratio may be between 1 and 8, preferably between 3 and 5, such as 3.0, 3.1 , 3.2, .3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5.0.
  • said ratio is between 3.5 and 4.5.
  • Particularly preferred is ratio of about 4.0, such as 4.0.
  • the drug/antibody ratio is between 5 and 7, such as 5.0, 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0.
  • Particularly preferred ratio is about 6.0, such as 6.0.
  • the drug antibody ratio can also be between 7 and 8, such as 7.0, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0.
  • Particularly preferred ratio is about 8.0, such as 8.0.
  • immunoconjugates of the present inventions can be obtained according to methods and techniques well known to the skilled person.
  • the immunoconjugates of the present inventions are obtainable according to the methods as described in, or analogous to the methods described in, the Examples section.
  • compositions of an immunoconjugate of the present invention as described herein are prepared by mixing such immunoconjugate having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions.
  • Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m- cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arg
  • sHASEGP soluble neutral-active hyaluronidase glycoproteins
  • rHuPH20 HYLENEX®, Baxter International, Inc.
  • Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in US Patent Publication Nos. 2005/0260186 and 2006/0104968.
  • a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.
  • pharmaceutical formulation or “pharmaceutical composition” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
  • a “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject.
  • a pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
  • Exemplary lyophilized immunoconjugate formulations are described in US Patent No. 6,267,958.
  • Aqueous immunoconjugate formulations include those described in US Patent No. 6,171,586 and W02006/044908, the latter formulations including a histidine-acetate buffer.
  • the formulation herein may also contain more than one active ingredient as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
  • Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody or immunoconjugate, which matrices are in the form of shaped articles, e.g. films, or microcapsules.
  • the formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.
  • the present invention relates to the immunoconjugate of the present invention or pharmaceutical composition of the present invention for use as a medicament.
  • the present invention relates to the immunoconjugate of the present invention or pharmaceutical composition of the present invention for use in therapy. It is to be understood thatthe immunoconjugate or the pharmaceutical compositions of the present invention can be used in the treatment of a disease or a disorder.
  • treatment refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • immunoconjugates of the invention are used to delay development of a disease or to slow the progression of a disease.
  • An immunoconjugate of the invention can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional, intrauterine or intravesical administration.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g. by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.
  • Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.
  • Immunoconjugates of the invention would be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.
  • the immunoconjugate need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of immunoconjugate present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate.
  • an immunoconjugate of the invention when used alone or in combination with one or more other additional therapeutic agents, will depend on the type of disease to be treated, the type of immunoconjugate, the severity and course of the disease, whether the immunoconjugate is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the immunoconjugate, and the discretion of the attending physician.
  • the immunoconjugate is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 pg/kg to 15 mg/kg (e.g.
  • 0.1 mg/kg-10 mg/kg of antibody or immunoconjugate can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion.
  • One typical daily dosage might range from about 1 pg/kg to 100 mg/kg or more, depending on the factors mentioned above.
  • the treatment would generally be sustained until a desired suppression of disease symptoms occurs.
  • One exemplary dosage of the immunoconjugate would be in the range from about 0.05 mg/kg to about 10 mg/kg.
  • one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administered to the patient.
  • Such doses may be administered intermittently, e.g. every week or every three weeks (e.g. such that the patient receives from about two to about twenty, or e.g. about six doses of the antibody).
  • An initial higher loading dose, followed by one or more lower doses may be administered.
  • other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
  • the present invention relates to the immunoconjugate of the present invention or the pharmaceutical composition of the present invention for use in the treatment of an L1-CAM (CD171) associated cancer.
  • the L1-CAM (CD171) associated cancer is selected from leukemia, Ewing's sarcoma, neuroblastoma, osteosarcoma, glioblastoma multiforme, ovarian cancer, endometrial cancer, uterine cancer, triple negative breast cancer, quadruple-negative breast cancer, melanoma, clear cell renal cell cancer, pheochromacytoma and paraganglioma, mesothelioma, small cell lung cancer (SCLC), non-small cell lung cancer, NSCLC, pancreatic ductal cancer, colon cancer, pancreatic cancer, hepatocellular carcinoma, gastric cancer, cholangiocarcinoma, carcinoid, neuroendocrine tumors, gastrointestinal stromal tumor (GIST), pheochromocytoma, glioma, pancreatic neuroectodermal cancer, pancreatic adenocarcinoma, colorectal cancer, renal cell carcinoma,
  • Immunoconjugates of the invention can be used either alone or in combination with other agents in a therapy.
  • an immunoconjugate of the invention may be co-administered with at least one additional therapeutic agent.
  • Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case, administration of the immunoconjugate of the invention can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent and/or adjuvant.
  • Immunoconjugates of the invention can also be used in combination with radiation therapy.
  • said immunoconjugate is to be administered to a subject with an additional therapeutic agent, selected from alkylating agents, platinum agents, taxanes, vinca agents, anti-estrogen drugs, aromatase inhibitors, ovarian suppression agents, VEGF/VEGFR inhibitors, EGF/EGFR inhibitors, PARP inhibitors, cytostatic alkaloids, cytotoxic antibiotics, antimetabolites, endocrine/hormonal agents, immune checkpoint inhibitors and bisphosphonate therapy agent.
  • an additional therapeutic agent selected from alkylating agents, platinum agents, taxanes, vinca agents, anti-estrogen drugs, aromatase inhibitors, ovarian suppression agents, VEGF/VEGFR inhibitors, EGF/EGFR inhibitors, PARP inhibitors, cytostatic alkaloids, cytotoxic antibiotics, antimetabolites, endocrine/hormonal agents, immune checkpoint inhibitors and bisphosphonate therapy agent.
  • said immunoconjugate of the invention is to be administered to a subject with an additional therapeutic agent, wherein said additional therapeutic agent may be an agent sensitizing the cells to radiotherapy, for example selected from protein kinase inhibitor and DNA intercalating agent.
  • protein kinase inhibitor is selected from Alisertib, MK1775, MK2206, Saracatinib, Temsirolimus, Crizotinib, Ceritinib, Alectinib, Brigatinib, Bosutinib, Dasatinib, Imatinib, Nilotinib, Ponatinib, Vemurafenib, Dabrafenib, Ibrutinib, Ibrutinib, Palbociclib, Sorafenib, Ribociclib, Crizotinib, Cabozantinib, Gefitinib, Erlotinib, Lapatinib, Vandetanib, Afatinib
  • An agent sensitizing the cells to radiotherapy may also be AZD7648:
  • the present invention further relates to the immunoconjugate of the present invention or the pharmaceutical composition of the present invention for use in the manufacture of a medicament for treating an L1-CAM associated cancer.
  • the present invention further relates to the method of treatment of an L1-CAM associated cancer, the method comprising administering to an individual in need thereof of the immunoconjugate of the present invention or the pharmaceutical composition of the present invention. It is to be understood that the immunoconjugate of the invention or the pharmaceutical composition of the invention, are to be administered in a therapeutically effective amount.
  • mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non human primates such as macaques), rabbits, and rodents (e.g., mice and rats).
  • domesticated animals e.g., cows, sheep, cats, dogs, and horses
  • primates e.g., humans and non human primates such as macaques
  • rabbits e.g., mice and rats
  • rodents e.g., mice and rats.
  • the individual or subject is a human.
  • an "effective amount" of an agent refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
  • the present invention further relates to the immunoconjugate of the present invention for use in diagnosis.
  • the immunoconjugates of the present invention are particularly useful in diagnosis of L1-CAM (CD171) associated cancer, as described herein.
  • the L1-CAM (CD171) associated cancer is selected from leukemia, Ewing's sarcoma, neuroblastoma, osteosarcoma, glioblastoma multiforme, ovarian cancer, endometrial cancer, uterine cancer, triple negative breast cancer, quadruple-negative breast cancer, melanoma, clear cell renal cell cancer, pheochromacytoma and paraganglioma, mesothelioma, small cell lung cancer (SCLC), non-small cell lung cancer, NSCLC, pancreatic ductal cancer, colon cancer, pancreatic cancer, hepatocellular carcinoma, gastric cancer, cholangiocarcinoma, carcinoid, neuroendocrine tumors, gastrointestinal stromal tumor (GIST),
  • the immunoconjugates as described herein may be used in diagnostic methods, in particular in-vitro diagnostic methods, wherein said antibody or said fragment is used for detection of L1-CAM positive cells in tissue, wherein said antibody or said fragment thereof is then detected by a secondary antibody, preferably a monoclonal antibody.
  • immunoconjugates useful in diagnosis comprise an active agent, wherein said active agent is a moiety that enables detection and localization of said immunoconjugate.
  • said active agent while being not particularly limited, as long as it is suitable for a diagnostic purpose, is preferably selected from radionuclide, MRI active compound, ultrasound contrast agent, fluorophore (preferably fluorophore in the far red/near-IR spectral region), marker for PET and SPECT, and Gd-based and Fe particle-based MRI contrast agent.
  • the suitable imaging methods allowing the diagnostic applications are known to the skilled person.
  • said active agent is a radionuclide.
  • said radionuclide is a radionuclide useful in diagnosis, preferably selected from fluorine-18, scandium-43, scandium-44, copper-61 , copper-64, gallium-68, zirconium-89, indium-111 , iodine-123, terbium-152, and terbium-155.
  • radionuclide conjugate of the present invention wherein the radionuclide is a radionuclide useful in diagnosis, preferably selected from fluorine-18, scandium-43, scandium-44, copper-61 , copper-64, gallium-68, zirconium-89, indium-1 11, iodine-123, terbium-152, and terbium-155, as described hereinabove.
  • a radionuclide useful in diagnosis preferably selected from fluorine-18, scandium-43, scandium-44, copper-61 , copper-64, gallium-68, zirconium-89, indium-1 11, iodine-123, terbium-152, and terbium-155, as described hereinabove.
  • DOTA (1 ,4,7, 10-tetraazacyclodecane-1 ,4,7,10-tetraacetic acid
  • EDTA Ethylenediaminetetraacetic acid.
  • PBS Phosphate buffer saline
  • TCEP Tris(2-carboxyethyl)phosphin -hydrochloride
  • huCE7 V7 (heavy chain characterized by sequence of SEQ ID NO.: 3, light chain characterized by sequence of SEQ ID NO.: 5)
  • huCE7 V7AG (heavy chain characterized by sequence of SEQ ID NO.: 1 , light chain characterized by sequence of SEQ ID NO.: 5)
  • huCE7 LV32 (heavy chain characterized by sequence of SEQ ID NO.: 21, light chain characterized by sequence of SEQ ID NO.: 7)
  • huCE7 LV40 (heavy chain characterized by sequence of SEQ ID NO.: 25, light chain characterized by sequence of SEQ ID NO.: 7)
  • Example 1 mAb production (taken from Proteogenix protocol)
  • HC and LC variable regions Humanized variants antibodies resulted from the in-silico humanization step
  • full-length human lgG1 Kappa antibodies harboring mutations L234A, L235A and P331 S (as in case for huCE7 variant 7) were designed.
  • the cDNA coding for the variable regions of the HC and LC were chemically synthesized with optimization for expression in CHO cells and subcloned in ProteoGenix’s proprietary mammalian cells expression vectors containing backbones for human I g G 1 heavy chain constant region and human kappa light chain constant region. Sequences coding for signal peptides were added in 57Nter position. The sequences are illustrated below (exemplary for huCE7 variant 7).
  • IEX CaptoQ-Cytiva
  • huCE7-variant 7 (5 mg/ml) in PBS pH 7.4, TCEP 10 mM (CAS 51805-45-9, supplier Carl Roth, Germany) and EDTA 100 mM (CAS 6381 -92-6, supplier Carl Roth, Germany) were prepared.
  • 1 ml of antibody stock (5 mg, 0.033 pMol, 1 eq) was subsequently mixed with 40 pl TCEP stock solution (12 eq) and 10 pl EDTA stock solution (30 eq.) and incubated for 2h at 37°C under stirring (300 rpm, thermomixer, Eppendorf, Germany).
  • the reduced mAb was afterwards buffer exchanged (3 times) with 100 mM potassium phosphate +1 mM EDTA using ultra-filtration (30 MWCO cut-off). Final protein concentration of reduced mAb was characterize by UV-Vis at 280 nm.
  • Antibody conjugate huCE7 7AG-(MC-GFGG- Exatecan)s was characterized by SEC and LC-MS for purity and DAR.
  • the person skilled in art may also use this protocol for the attachment of MC-GGFG-Deruxtecan (Dxd) (CAS 1599440-13-7), MC-GGFG-SN38 or similar maleimide based or alkyne-based linker-drugs simply by exchanging the linker-drug compound. Procedure can also be used as a model for all disclosed mAb variants.
  • reaction 1 mL/5 mg of antibody was mixed with 342.5 pL (80 eq) linker and 250 pL mTG (2.5 eq per antibody HC) and incubated 19 h at 37°C under shaking (300 rpm, thermomixer, Eppendorf, Germany). After completing the reaction time 10 pL sample was taken and diluted it with 30 pL 0.1 M PBS (pH 7.4) and 1 pL DTT (CAS 3483-12-3, supplier Car Roth, Germany) to determine competition of conjugation reaction by LC-MS. Afterwards the mAb conjugate was purified by a protein A column (supplier, Marvelgent Bioscience) using manufactures protocol. Final protein concentration of mAb-linker conjugate was characterized by UV-Vis at 280 nm.
  • Protocols are presented in example 6.
  • Model carrier synthesis was performed in dependence on protocols & information disclosed in the patent application PCT/EP2019/061769, published as WO 2019/215207 (example 33-35), which is incorporated herein by reference in its entirety, but using a MAL-GGFG-Exatecan instead of the presented NHS-DOTA for functionalization of carrier’s side chain and replacing the mTG tag by additional attachment of a DBCO- NHS to carrier’s head group.
  • step 2 the azide functionalized mAb was modified with the carrier by click reaction using the following procedure:
  • Protocols are presented in example 6.
  • the conjugate was purified with by 6-times buffer exchange with PBS (pH 7.4) using an ultra-filtration (100 MWCO cut-off). Final protein concentration of mAb was characterized by UV- Vis at 280 nm.
  • Antibody conjugate huCE7 7AG-[NH-(CH2CH2O)4-TCO-Tetrazine-Carrier]2 was characterized by GPC and LC-MS for purity and DAR.
  • the so synthesized antibody-polymer conjugate (APC) was in the 3 rd step be functionalized with a DBCO- Linker-active agent e.g. DBCO- (CH2CH2O)4-GGFG-Dxd (as presented and using similar protocols as in example 2) by a click reaction with azide groups in carrier sides chains.
  • Such antibody-polymer conjugate is further useful for a pre-targeting approach were the L1CAM specific antibody-polymer conjugate is given to a cancer patient and binds L1 CAM positive tumor cells.
  • DBCO-(CH2CH2O)4-GGFG-Dxd (stable in plasma) or a similar DBCO modified active agent is given systemically and active agent is immobilized by bio-orthogonal click reaction with the APC on the tumor cells.
  • Protocols are presented in example 6.
  • huCE7 variantX (1 mg/ml in PBS pH 7.5) was mixed with 1 .4 Units of N-Glycosidase (1 Unit/pL, Supplier, Sigma-Aldrich, REF: 11365177001) in a 0.5 ml reaction tube and shook gently for 24 h at 37,5°C. After 24 h the de-glycosylation status was checked by LC-MS analysis using 2 pl of reaction solutions as well as the native antibody as a control. De-glycolyzed mAb was subsequently buffer exchanged to Trizma (50 mM pH 7.4) using ultrafiltration with 50 MWCO cut-off. Final protein concentration of de-glycolyzed mAb was characterized by UV-Vis at 280 nm.
  • a DAR 4 chelator modified huCE7 antibody conjugate was prepared by similar protocols as presented in example 2 with the exception that DBCO-DOTA chelator was used in the click reaction of step 2.
  • the so prepared huCE7 7AG-[NH-(CH2CH2O)2CH2CH2-N-((CH2CH2O)3CH 2 CH2-N3-DBCO-DOTA)2]2 was subsequently radiolabeled with 177Lu to be used as radioligand for targeted radio therapy and/or diagnostic.
  • the radionuclide 177Lu was used one to three days post-specified calibration date.
  • Protocols are presented in example 6.
  • reaction solution 10 pL reaction solution was diluted with 40 pL PBS (pH 7.5) and 1 pL 1 M DTT in a HPLC vial with insert and incubated 0 min at 37°C in a thermal shaker. 2-5 pL of this solution was injected into the system.
  • BMPS can also be used instead of SMCC (CAS Number: 55750-62-4).
  • Dissolve SMCC (CAS: 64987-85-5) in dry DMSO (16 mg, 50 mM).
  • Step 2 Reduction of the disulfide bridges in the antibody
  • Step 3 Conjugation: Antibody + polymer-maleimide
  • the copolymer HS-DMA53AK4 (5.0 mg, 0.79 pmol) was dissolved in DMF (200 pL).
  • Exatecan-2- (aminomethoxy)acetamide-Gly-Phe-Gly-Gly-PEGI-NHS ester (supplier BroadPharm, USA) (5.9 mg, 5.53 pmol, 7 eq) and triethylamine (CAS 121-44-8, supplier Merck, Germany) (5.4 pL, 39.5 pmol, 50 eq) were added and the solution was shaken at 25 °C and 500 rpm overnight (thermomixer, Eppendorf, Germany). After dilution with water (300 pL), the reaction solution was dialysed against water and subsequently lyophilized. The product was obtained as a white solid which was analysed by GPC and LC-MS.
  • the number of drugs conjugated to the carrier can be varied by modifying the reaction stoichiometry.
  • HS-DMA53(AK-Peg1-GGFG-Exatecan-2-(aminomethoxy)acetamide)4 (3.0 mg, 0.28 pmol) was dissolved in water (150 pL) and layered with argon.
  • the DBCO-polymer can then be coupled to an azide modified mAb as described in example 4.
  • All carrier constructs could also be used for PK-modification of an ADC e.g. due to influence of carrier on size, surface charge, hydrophobic balance and hydrodynamic volume.
  • Step 1 Reduction of the disulfide bridges in the antibody huCE7-variant 7 is reduced as described in Example 2.
  • the reduced antibody is combined with a solution of in-situ prepared Azido-PEG3-Maleimide (CAS 1858264-36-4, supplier BroadPharm, USA) in dry DMSO in a 15:1 linker: antibody ratio.
  • the mixture is incubated for 2 h at 25 °C and 500 rpm (thermomixer, Eppendorf, Germany).
  • the conjugate is purified by 5-times buffer exchange with PBS (pH 7.4) using an ultra-filtration (30 MWCO cut-off).
  • Final protein concentration of ADC is characterized by UV-VIS at 280 nm.
  • Azido-PEGs-Maleimide or Azido-PEGi2-Maleimide may be used for bulky carrier constructs.
  • the degree of functionalization may be varied by reducing the amount of linker added.
  • Step 3 Click reaction with DBCO-polymer
  • a stock solution of any DBCO-polymer in DMF is prepared. This solution is added to a solution of azide- modified mAb from step 2 and incubated at 37°C under shaking (300 rpm, thermomixer, Eppendorf, Germany) over night. Subsequently, the conjugate is purified by 6-times buffer exchange with PBS (pH
  • huCE7 V7-[Q295-NH-PEG4-azide]2 was subsequently incubated with a 10 times molar excess of a click reactive DBCO-(PEG2-vc-PAB-MMAE)2 (Supplier, SyntaBio, USA) in PBS pH 7.5 supplemented with 10% (m/v) dimethylacetamide for 16h at room temperature.
  • the ADC was purified by Sephadex G25 column into PBS pH 7.5 followed by activated carbon depletion of residual toxin linker.
  • the resulting ADC (huCE7-V7AG-(MMAE2)2) was concentrated to approximately 3 mg/mL before a final 0.2 pm filtration.
  • the monomer constituted 96.8% of the sample, as determined by SEC.
  • Drug to antibody ration determined by LC-MS was 3.9.
  • Example 17 Tumor cell killing properties of ADCs based on huCE7 V7 and huCE7 V7 AG
  • Step one verification of cell binding & identification of suitable models
  • H-524 showing (High), H-2171 (Medium), H-69 (Low) and H-211 (no) L1 CAM expression were chosen for additional semi-quantitative characterization of L1 CAM protein/cell using the following short protocol:
  • Test principle The assay uses beads labelled with actual fluorochromes to generate a standard curve to quantify the antibodies binding to the cell surface. The assumption is that the antigen, primary antibody (huCE7 V7) and secondary antibody bind with a 1 : 1 : 1 ratio when saturated. The secondary antibody has a fluorochorome/IgG ratio of ⁇ 3.4.
  • the bound antibody (B) is calculated according to the standard curve, the free antibody (F) is calculated by subtracting (B) from total antibody.
  • tumor cells were seeded per well (96 well palate) and the huCE7 V7 antibody was applied in a 12-point dilution curve -2-fold diluted [0.730000 pg/mL, 0.365000 pg/mL, 0.182500 pg/mL, 0.091250 pg/mL, 0.045625 pg/mL, 0.022813 pg/mL, 0.011406 pg/mL, 0.005703 pg/mL, 0.002852 pg/mL, 0.001426 pg/mL, 0.000713 pg/mL, 0.000356]; 100 pL/well and incubated at 4°C for 1 hour.
  • the wells were centrifuged and the supernatant discarded before the secondary antibody (anti human Fc-FITC labelled) was applied. After a short incubation period the cells and the in parallel prepared fluorochrome bead standard curve were analyzed by FACS. As negative control cells incubated with secondary antibody only were used.
  • Cell killing assay was performed with 5 human SCLC cells lines H-2227, H-524, H-2171 , H-69 and H-21 1 having different L1 CAM receptor expression rates (from high to low/ near negative target expression). For this, cells were resuspended in RPMI 1640 medium with 10% FBS and seeded into 96-well clear bottom black plates in a volume of 50 pL 1 day prior to the experiment and culture at 37°C and 5 % CO2.
  • the cells were cultured at 37°C and 5 % CO2 until day 6 and then tested for cell viability using CellTiter-Glo® Luminescent Cell Viability Assay (Promega) according to manufacturer protocols.
  • mice Bodyweight, behavior and tumor size were monitored on a daily basis. Mice were euthanized once tumor reach the threshold of 2500 mm 3 for ethical reasons or if the bodyweight decreased > 20 %. Collection of plasma samples was performed at 24h pre-dose and 5 Days post first administration (4 mice per arm). Samples were later used to determine the influence of treatment on liver function (Liver panel: ALT, AST, ALP, T-Bil-D, TP, ALB, y-GT). Results of the study are presented in Figure 16 and Table 7.
  • Blood samples were taken from 4 mice of each group 1 day pre-treatment and 5 days after the first injection of the drug and frozen. Samples were subsequently analyzed for standard liver function and health parameters.
  • the tumor had a fast growth kinetic and the first 3 mice had to be euthanized on day 25 whereas the last mice were euthanized on day 32 due to reaching the tumor volume threshold.
  • the growth was significantly inhibited (39.52 % on day 21 compared to PBS) in the Cisplatin (SoC) group and a positive effect on survival was seen.
  • the first mice had to be euthanized on day 28 and the last mice on day 39 whereas one mouse was found dead on day 39.
  • the treatment groups with huCE7 V7 7D8 the tumor growth inhibition was significantly stronger than with the SoC.
  • Minimal effective does was 1 mg/kg with 85.21 % tumor growth inhibition on day 21 whereas 3 mg/kg and 10 mg/kg reached 90.33 % and 98.88% on day 21 respectively. This positive trend continued after the last dose was given. Median overall survival was calculated to be 28 days (PBS), and 35 days for (SoC Cisplatin 2mg/kg). A significant improvement compared to SoC was seen in the huCE7 V7 7D8 groups 56 days (1 mg/kg), 65 days (3mg/kg) and 74 days for the 10 mg/kg group. Study was ongoing since one mouse in the 10 mg/kg was still below the threshold of 2500 mm 3 . For the overall survival in the huCE7 V7 7D8 treatment a dosedependent behavior could be detected.
  • Variant 7 LC full length (SEQ ID NO.: 5)
  • Variant 7 LC (SEQ ID NO.: 6)
  • Variant LV29 HC AG (SEQ ID NO.: 10)
  • Variant LV30 HC AG full length (SEQ ID NO.: 13)
  • Variant LV40 HC AG full length (SEQ ID NO.: 25)
  • Variant LV33 HC AG (CE7-1-2-VHB N297A (TH30P; WH33F; GH55A)) (SEQ ID NO.: 30) also referred to as CE7-1 -2-VHB-TH30P-WH33F-GH55A (L234A L235A + P331 S+N297A)
  • Variant LV33 HC full length (CE7-1-2-VHB N297A (TH30P; WH33F; GH55A)) (SEQ ID NO.: 31) also referred to as CE7-1 -2-VHB-TH30P-WH33F-GH55A (L234A L235A + P331 S+N297A)
  • LV33 HC CE7-1-2-VHB N297A (TH30P; WH33F; GH55A)
  • SEQ ID NO.: 32 also referred to as CE7-1 -2-VHB-TH30P-WH33F-GH55A (L234A L235A + P331 S)
  • Variant LV34HC AG full length also referred to as CE7-1 -2-VHB-WH33F-AH40R-GH55A (L234A L235A + P331 S+N297A)
  • Variant LV34HC AG also referred to as CE7-1 -2-VHB-WH33F-AH40R-GH55A (L234A L235A + P331 S+N297A)
  • Variant LV34HC also referred to as CE7-1 -2-VHB-WH33F-AH40R-GH55A (L234A L235A + P331 S)
  • Variant LV35 HO AG full length (SEQ ID NO: 37) also referred to as CE7-1 -2-VHB-WH33F-GH55A-VH68A (L234A L235A + P331 S+N297A)
  • DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Variant LV35 HC AG also referred to as CE7-1 -2-VHB-WH33F-GH55A-VH68A (L234A L235A + P331 S+N297A)
  • Variant LV35 HC full length also referred to as CE7-1 -2-VHB-WH33F-GH55A-VH68A (L234A L235A + P331 S)
  • Variant LV35 HC also referred to as CE7-1 -2-VHB-WH33F-GH55A-VH68A (L234A L235A + P331 S)
  • Variant LV36 HC AG also referred to as CE7-1 -2-VHB-WH33F-GH55A-LH1 15T (L234A L235A + P331 S+N297A)
  • Variant LV36 HC full length (SEQ ID NO.: 43) also referred to as CE7-1 -2-VHB-WH33F-GH55A-LH1 15T (L234A L235A + P331 S)
  • DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Variant LV36 HC also referred to as CE7-1 -2-VHB-WH33F-GH55A-LH1 15T (L234A L235A + P331 S)
  • Variant LV37 HC AG full length also referred to as CE7-1 -2-VHB-WH33F-GH55A-FH241 K (L234A L235A + P331 S+N297A)
  • Variant LV37 HC AG also referred to as CE7-1 -2-VHB-WH33F-GH55A-FH241 K (L234A L235A + P331 S+N297A)
  • Variant LV37 HC also referred to as CE7-1 -2-VHB-WH33F-GH55A-FH241 K (L234A L235A + P331 S)
  • Variant LV 38 HC AG full length also referred to as CE7-1 -2-VHB-WH33F-GH55A-LH309D (L234A L235A + P331 S+N297A)
  • DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Variant LV 38 HC AG also referred to as CE7-1 -2-VHB-WH33F-GH55A-LH309D (L234A L235A + P331 S+N297A)
  • Variant LV 38 HC full length also referred to as CE7-1 -2-VHB-WH33F-GH55A-LH309D (L234A L235A + P331 S)
  • Variant LV 38 HC also referred to as CE7-1 -2-VHB-WH33F-GH55A-LH309D (L234A L235A + P331 S)
  • Variant LV39 HC AG also referred to as CE7-1 -2-VHB-WH33F-GH55A-TH307R (L234A L235A + P331 S+N297A)
  • Variant LV39 HC full-length also referred to as CE7-1 -2-VHB-WH33F-GH55A-TH307R (L234A L235A + P331 S)
  • DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Variant LV39 HC also referred to as CE7-1 -2-VHB-WH33F-GH55A-TH307R (L234A L235A + P331 S)
  • Variant LV42 HC AG full length (SEQ ID NO.: 57)

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  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Cell Biology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne des immunoconjugués ciblant L1-CAM. Les immunoconjugués de la présente invention et les compositions pharmaceutiques décrites ici sont particulièrement utiles dans le traitement ou le diagnostic d'un cancer associé à L1-CAM (CD171).
PCT/EP2025/063464 2024-05-15 2025-05-15 Immunoconjugués ciblant l1-cam Pending WO2025238187A1 (fr)

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EP24176106 2024-05-15

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