EP2408819A2 - Anticorps anti-peptide ny-eso-1 de type récepteur des cellules t de haute affinité, procédés, et leurs utilisations - Google Patents

Anticorps anti-peptide ny-eso-1 de type récepteur des cellules t de haute affinité, procédés, et leurs utilisations

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Publication number
EP2408819A2
EP2408819A2 EP10715349A EP10715349A EP2408819A2 EP 2408819 A2 EP2408819 A2 EP 2408819A2 EP 10715349 A EP10715349 A EP 10715349A EP 10715349 A EP10715349 A EP 10715349A EP 2408819 A2 EP2408819 A2 EP 2408819A2
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EP
European Patent Office
Prior art keywords
antibody
eso
cancer
specific binding
antibodies
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP10715349A
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German (de)
English (en)
Inventor
Christoph Renner
Andreas Wadle
Vincenzo Cerundolo
Guillaume Stewart-Jones
E. Yvonne Jones
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universitaet Zuerich
Ludwig Institute for Cancer Research Ltd
University of Oxford
Ludwig Institute for Cancer Research New York
Original Assignee
Universitaet Zuerich
Ludwig Institute for Cancer Research Ltd
University of Oxford
Ludwig Institute for Cancer Research New York
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Publication date
Application filed by Universitaet Zuerich, Ludwig Institute for Cancer Research Ltd, University of Oxford, Ludwig Institute for Cancer Research New York filed Critical Universitaet Zuerich
Publication of EP2408819A2 publication Critical patent/EP2408819A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2833—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against MHC-molecules, e.g. HLA-molecules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/32—Immunoglobulins specific features characterized by aspects of specificity or valency specific for a neo-epitope on a complex, e.g. antibody-antigen or ligand-receptor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to specific binding members, particularly antibodies and fragments thereof, which bind to NY-ESO-I tumor antigen peptide/MHC molecule complexes, particularly in a manner like T cell receptors and with greater affinity than T cell receptors recognizing NY-ESO-I peptide/HLA complexes.
  • These antibodies, particularly human antibodies are useful in the diagnosis and treatment of cancer, including melanoma, lung, esophageal, liver, gastric, prostate, ovarian, bladder and synovial sarcoma.
  • the antibodies and fragments thereof of the present invention may also be used in therapy in combination with chemotherapeutics, immune modulators, or anti-cancer agents and/or with other antibodies or fragments thereof.
  • NY-ESO-I is a germ cell antigen that is aberrantly expressed by different tumor types
  • NY-ESO-I was discovered based on its capacity to induce an antibody response in vivo in cancer patients. This humoral response is restricted to cancer patients and not seen in healthy individuals (Stockert, E. et al (1998) J. Exp. Med. 187: 1349- 1354).
  • CTAGl The gene coding for NY-ESO-I, known as CTAGl, is a gene with expression limited to germ cells and is not expressed in normal somatic tissue.
  • NY-ESO-I is frequently expressed in cancer and normal testis, and is thus termed a cancer testis (CT) antigen (Scanlan, MJ. et al (2004) Cancer Immunol 4:9).
  • CT cancer testis
  • the NY-ESO-I gene maps to the Xq28 region of the X chromosome and codes for several products (Chen, Y.T. et al (1997) Proc Natl Acad Sci 94: 1914-1918).
  • the main product of NY-ESO-I is a 180 amino acid long 18 KDa protein, with a glycine-rich N-terminal region and an extremely hydrophobic C-terminal region, which is very insoluble and can be confused for a ' transmembrane domain.
  • the -function of NY-ESO-I remains unknown.
  • NY-ESO-I monoclonal antibodies ES121, E978 and B9.8 have been developed and confirm its expression in early spermatagonia with loss of expression upon sperm cell differentiation (Jungbluth, A.A. et al (2001) In J. Cancer 92:856-860; Schulz-Thater, E. et al. (2000) Br J. Cancer 83:204-208; Vaughan, H.A. et al (2004) Clin Cancer Res 10:8396-8404).
  • NY-ESO-I expression in primary or metastatic cancer tissue has been assessed by RT-PCR analysis, immunohistochemistry or Western blot.
  • NY-ESO- 1 is seen in approximately one-third to one-quarter of all melanoma, lung, esophageal, liver, gastric, prostate, ovarian and bladder cancers (Gnjatic, S. et al (2004) Ado Cancer Res 95: 1-30). Eighty percent of synovial sarcomas express NY-ESO-I (Jungbluth, A. et al (2001) In J. Cancer 94:252-256).
  • HLA-DR4 Several epitopes restricted by HLA-DR4 in CD4 T cell responses have been demonstrated, and responses against peptide 157-170 were restricted by HLA-DP4, an allele found in the majority of Caucasians (Jager et al (2000) J Exp Med 191 :625-630; Zang et al (2001) Proc Natl Acd Sci 98:3964-3969).
  • the NY-ESO-I antigen has been evaluated in immunotherapy as a cancer vaccine candidate, including based on full length protein, various peptide fragments, DNA, and recombinant virus-based immunogens.
  • Analysis of CD8 + T cells generated in vaccine trials using NY-ESO-I derived peptides (157-165 and 157-167) showed that the dominant immune response was directed against a cryptic epitope (159-167), which diverted the immune response from tumor recognition.
  • CTLs reactive to the NY-ESO- 1 157 - 165 peptide were capable of lysing NY-ESO-l/HLA-A0201-expressing tumor cells. Therefore, a ligand binding specifically to NY-ESO-I 157 - I65 / HLA complex has been recognized as a potentially useful cancer agent.
  • Phage display technology was utilized to isolate Fab antibodies with T-cell receptor-like specificity, and Fab Abs recognizing the NY-ESO-I i 57 . ! 65 peptide in the HLA-A0201 context were isolated.
  • Nine different antibodies were identified with TCR-like specificity and all bound specifically to NY-ESO- l i 57 -i 65 /HLA-2 complexed by ELISA or FACS analysis on the surface of peptide-pulsed T2 cells.
  • Representative clones included 3M4E5, 2M4B2, 3M4H7, and 2M4D10 (Held G. et al (2004) Eur J Immunol 34:2919-2929).
  • Clone 3M4E5 was initially chosen for further analysis because it displayed a substantial shift in FACS analysis.
  • Fab-3M4E5 binds to the NY-ESO- 1 157 .
  • I65 /HLA-2 complex displayed on mammalian cells after endogenous processing and blocks T-cell reactivity to the epitope NY-ESO-1 157 - I65 displayed by target cells in the HLA-A2 context.
  • Melanoma cell line SK-MEL-37 which expresses both HLA-A2 and NY-ESO-I, is stained by Fab-3M4E5 only weakly (Held, G. et al (2004) Eur J Immunol 34:2919-2929).
  • the invention provides affinity matured high affinity antibodies mimicking a TCR that are useful in targeting specific pMHC complexes for diagnostic and therapeutic purposes.
  • high affinity antibodies targeting NY-ESO-I peptide MHC complexes are provided, wherein said antibodies have a peptideMHC affinity which is greater than that of corresponding TCRs.
  • Fab antibodies and single chain antibodies are particularly provided herein.
  • the antibodies of the present invention have diagnostic and therapeutic use and application in cancers, particularly in cancers wherein NY-ESO-I is involved or otherwise presented during the cancer or metastatic process or is a part of the humoral and/or cellular response to cancer, oncogenesis, or other disease pathogenesis.
  • the antibodies of the invention are applicable in cancers, including melanoma, lung, esophageal, liver, gastric, prostate, ovarian, bladder and synovial sarcoma.
  • the present invention provides T cell receptor-like antibodies directed against NY-ESO- 1 peptide antigen(s) in ' complex with MHC molecule(s) and having affinity for peptideMHC complexes which is greater than the relative affinity of T cell receptor(s).
  • the present invention provides an isolated specific binding member, particularly an antibody or fragment thereof, including an Fab fragment and a single chain or domain antibody, which recognizes an epitope on NY-ESO- 1 antigen in combination with MHC peptide.
  • the present invention provides an antibody or fragment thereof, which recognizes NY-ESO-I peptide 157-165/Class I MHC epitope which is found in tumorigenic, hyperproliferative or abnormal cells and is not detectable in normal somatic cells.
  • the NY-ESO-1 157 - 105 immunodominant peptide corresponds in sequence to SLLMWITQV.
  • the antibody or fragment of the invention is specific for NY-ESO- I peptide MHC molecules and is a T cell receptor like antibody, recognizing peptideMHC molecules with affinity that is greater than that of the T-cell receptor (TCR).
  • the antibody of the invention has a binding affinity for NY-ESO-I peptide MHC molecules, particularly for NY-ESO-1 157 -1 65 /HLA complex, of less than 10 nm.
  • the affinity of antibodies of the present invention for NY-ESO-I i 57 _ 165 /HLA-A * 0201 target is on the order of 2-4nm.
  • the affinity of antibodies of the present invention for NY-ESO-1157.165 /HLA-A * 0201 target exceeds the affinity of TCR by about 1000 fold.
  • the present inventors have discovered novel high affinity T cell receptor-like antibodies, exemplified herein by the antibodies designated Tl, T2 and T3, which specifically and with high affinity recognize NY-ESO-I peptide MHC complexes, particularly NY-ESO-I peptide 157-165/MHC molecules, particularly NY-ESO-1 157 - 165 /HLA-A * 0201.
  • the antibodies exemplified herein include Fab antibodies and single chain antibodies based thereon.
  • the antibodies have the amino acid sequences as set out herein and in Figures 10, 12 and 13.
  • the antibodies of the present invention can specifically categorize the nature of cancer and tumor cells, by binding, staining or otherwise recognizing those cells wherein NY-ESO-I peptide MHC complexes, particularly NY-ESO-I peptide 157-165/MHC molecules, is present or presented for immune system recognition and modulation. Further, the antibodies of the present invention, as exemplified by antibodies Tl, T2 and T3, when converted into scFv fragments and cloned into vectors to generate recombinant single chain TCRs, demonstrate specific recognition and killing of T cells presenting the NY- ESO-1 157 . 165 peptide and not others.
  • the unique specificity and high affinity of the antibodies and fragments of the invention provides diagnostic and therapeutic uses to identify, characterize and target a number of tumor types, for example, melanoma, lung, esophageal, liver, gastric, prostate, ovarian, bladder and synovial sarcoma, without the problems associated with normal tissue uptake.
  • the antibody is one which has the characteristics of the antibody which the inventors have identified and characterized, in particular recognizing NY-ESO-I peptide MHC complexes, particularly NY-ESO-I peptide 157-165/MHC molecules.
  • the antibody is Tl, T2 or T3, or active fragments thereof.
  • the antibody of the present invention comprises the VH and VL amino acid sequences depicted in Figures 10, 12 and 13.
  • the antibody of the invention comprises the CDR sequences depicted in Figure 10, Figure 12 or Figure 13.
  • the antibody is Tl and comprises the variable region sequences set out in Figure 10.
  • the antibody is Tl and comprises the CDR region sequences set out in Figure 10.
  • CDRs complementarity- determining regions
  • specific binding members based on the CDR regions of the heavy or light chain, and preferably both, of the antibodies of the invention, particularly of Tl, T2, and/or T3, will be useful specific binding members for therapy and/or diagnostics.
  • the CDRs of the antibodies are depicted in Figures 10, 12 and 13.
  • Antibody Tl comprises heavy chain CDR sequences GFTFSTY, IVSSGGST and AGELLPYYGMDV and light chain CDR sequences ERDVGGNY, DVI and WSFAGGYYV, as set out in Figure 10.
  • Antibody T2 comprises heavy chain CDR sequences GFTFSTY, ILSSGGET and AGMLLPYYGMDV and light chain CDR sequences ERDVGGNY, DVI and WSFAGGYYV, as set out in Figure 12.
  • Antibody T3 comprises heavy chain CDR sequences GFTFSTY, IASSGGET and AGSLLPYYGMDV and light chain CDR sequences ERDVGGNY, DVI and WSFAGGYYV, as set out in Figure 13.
  • specific binding proteins such as antibodies which are based on the CDRs of the antibody(ies) identified herein will be useful for targeting NY-ESO-I cancers and/or NY-ESO-I /MHC complexes, and/or NY-ESO-I peptides presented by antigen presenting cells.
  • the antibodies of the invention do not bind significantly to normal somatic cells or to cells presenting alternative or distinct antigens, and have peptide MHC affinities greater than TCRs, it is anticipated that there will not be significant uptake in normal tissue and there will be suitable affinity for the complexes, a limitation of other antibodies.
  • the present invention provides an isolated antibody or fragment thereof capable of binding an antigen, wherein said antibody or fragment thereof comprises a polypeptide binding domain comprising an amino acid sequence substantially as set out in Figures 10, 12 and 13.
  • the invention provides an isolated nucleic acid which comprises a sequence encoding a specific binding member as defined above, and methods of preparing specific binding members of the invention which comprise expressing said nucleic acids under conditions to bring about expression of said binding member, and recovering the binding member.
  • a nucleic acid encoding antibody variable region sequence having the amino acid sequences as set out in Figures 10, 12 or 13 is provided or an antibody having CDR domain sequences as set out in Figures 10, 12 or 13 is provided.
  • a nucleic acid of Figure 1 1 is provided.
  • the present invention also relates to a recombinant DNA molecule or cloned gene, or a degenerate variant thereof, which encodes an antibody of the present invention; preferably a nucleic acid molecule, in particular a recombinant DNA molecule or cloned gene, encoding the antibody VH and VL, particularly the CDR region sequences, which has a sequence or is capable of endcoding a sequence shown in FIGURE 10, 12 or 13.
  • the antibodies, fragments thereof and recombinant single chain TCRs according to the invention may be used in a method of treatment or diagnosis of the human or animal body, such as a method of treatment of a tumor in a human patient which comprises administering to said patient an effective amount of the antibodies, fragments thereof and recombinant single chain TCRs of the invention.
  • the present invention also includes polypeptides or antibodies having the activities noted herein, and that display the amino acid sequences set forth and described above and in Figures 10, 12 or 13 hereof, or are antibodies having a heavy chain and a light chain wherein the complementarity determining regions (CDRs) of the heavy and light chain comprise the amino acid sequences depicted in each or any of Figures 10, 12 and 13.
  • CDRs complementarity determining regions
  • the diagnostic utility of the present invention extends to the use of the antibodies of the present invention in assays to characterize tumors or cellular samples or to screen for tumors or cancer, including in vitro and in vivo diagnostic assays.
  • a control quantity of the antibodies, or the like may be prepared and labeled with an enzyme, a specific binding partner and/or a radioactive element, and may then be introduced into a cellular sample. After the labeled material or its binding partner(s) has had an opportunity to react with sites within the sample, the resulting mass may be examined by known techniques, which may vary with the nature of the label attached.
  • Specific binding members of the invention may carry a detectable or functional label.
  • the specific binding members may carry a radioactive label, such as the isotopes 3 H, 14 C, 32 P, 35 S, 36 Cl, 51 Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 121 I, ' 24 I, 125 I, 131 I, 111 In, 117 Lu, 211 At, 198 Au, 57 Cu, 225 Ac, 213 Bi, 99 Tc and 186 Re.
  • radioactive labels known currently available counting procedures may be utilized to identify and quantitate the specific binding members.
  • detection may be accomplished by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques known in the art.
  • radiolabeled specific binding members are useful in in vitro diagnostics techniques and in in vivo radioimaging techniques.
  • radiolabeled specific binding members, particularly antibodies and fragments thereof, particularly radioimmunoconjugates are useful in radioimmunotherapy, particularly as radiolabeled antibodies for cancer therapy.
  • the radiolabeled specific binding members, particularly antibodies and fragments thereof are useful in radioimmuno-guided surgery techniques, wherein they can identify and indicate the presence and/or location of cancer cells, precancerous cells, tumor cells, and hyperproliferative cells, prior to, during or following surgery to remove such cells.
  • Immunoconjugates or antibody fusion proteins of the present invention wherein the specific binding members, particularly antibodies and fragments thereof, of the present invention are conjugated or attached to other molecules or agents further include, but are not limited to binding members conjugated to a chemical ablation agent, toxin, immunomodulator, cytokine, cytotoxic agent, chemotherapeutic agent or drug.
  • the present invention includes an assay system which may be prepared in the form of a test kit for the quantitative analysis of the extent of the presence of, for instance, NY-ESO-I peptide/MHC complexes.
  • the system or test kit may comprise a labeled component prepared by one of the radioactive and/or enzymatic techniques discussed herein, coupling a label to the antibody, and one or more additional immunochemical reagents, at least one of which is a free or immobilized components to be determined or their binding partner(s).
  • the present invention relates to certain therapeutic methods which would be based upon the activity of the binding member, antibody, or active fragments thereof, or upon agents or other drugs determined to possess the same activity.
  • a first therapeutic method is associated with the prevention or treatment of cancer, including but not limited to melanoma, lung, esophageal, liver, gastric, prostate, ovarian, bladder and synovial sarcoma.
  • the binding members and antibodies of the present invention can be prepared in pharmaceutical compositions, including a suitable vehicle, carrier or diluent, for administration in instances wherein therapy is appropriate, such as to treat cancer.
  • Such pharmaceutical compositions may also include methods of modulating the half-life of the binding members, antibodies or fragments by methods known in the art such as pegylation.
  • Such pharmaceutical compositions may further comprise additional antibodies or therapeutic agents.
  • a composition of the present invention may be administered alone or in combination with other treatments, therapeutics or agents, either simultaneously or sequentially dependent upon the condition to be treated.
  • the present invention contemplates and includes compositions comprising the binding member, particularly antibody or fragment thereof, herein described and other agents or therapeutics such as anti-cancer agents or therapeutics, anti-mitotic agents, apoptotic agents or antibodies, or immune modulators. More generally these anti-cancer agents may be tyrosine kinase inhibitors or phosphorylation cascade inhibitors, post-translational modulators, cell growth or division inhibitors (e.g. anti-mitotics), inhibitors or signal transduction inhibitors.
  • compositions may be administered with suitable doses of pain relief drugs such as non-steroidal antiinflammatory drugs (e.g. aspirin, paracetamol, ibuprofen or ketoprofen) or opiates such as morphine, or anti-emetics.
  • pain relief drugs such as non-steroidal antiinflammatory drugs (e.g. aspirin, paracetamol, ibuprofen or ketoprofen) or opiates such as morphine, or anti-emetics.
  • the composition may be administered with immune modulators, such as interleukins, tumor necrosis factor (TNF) or other growth factors, colony stimulating factors, cytokines or hormones such as dexamethasone which stimulate the immune response and reduction or elimination of cancer cells or tumors.
  • TNF tumor necrosis factor
  • cytokines cytokines
  • the composition may also be administered with, or may include combinations along with other anti-N Y-ESO-I antibodies or other anti-tunor antigen antibodies.
  • the present invention also includes antibodies and fragments thereof, which are covalently attached to or otherwise associated with other molecules or agents.
  • molecules or agents include, but are not limited to, molecules (including antibodies or antibody fragments) with distinct recognition characteristics, toxins, ligands, and chemotherapeutic agents.
  • FIGURE 1A-1F Structural comparison of TCR and Fab.
  • C Schematic of CDR loops of 1G4 TCR in complex with HLA-A*0201/NY-ESO-l , 57 -i67 with loops coloured as follows: Va CDRl, green; Va CDR2, red; Va CDR3, blue; V ⁇ CDRl, magenta; V ⁇ CDR2, orange; V ⁇ CDR3, cyan. Water molecules within 4A of both TCR and peptide are indicated as red spheres.
  • FIG. 1 Diagram of CDR loops of 3M4E5 Fab in complex with HLA- A*0201/NYESO-I 157-167 with loops coloured as follows: VH CDRl, green; VH CDR2, red; VH CDR3, blue; VL CDRl, magenta; VL CDR2, orange; VL CDR3, cyan. Water molecules within 4A of both TCR and peptide are indicated as red spheres.
  • E Structure of the 1G4 TCR residues forming the 'roof residues in the cavity that binds the peptide (cyan) MW side chains.
  • FIGURE 2 Simulated annealing omit electron density map showing the peptide MW residues bound by the 3M4F5 Fab in yellow chicken wire. Fab residues are coloured according to the CDR colour as in Fig. ID. Hydrogen bonds between the Fab and the MW residues are shown in black. The map is contoured at 3 ⁇ .
  • FIGURE 3A-3C (A) Superposition, based on the MHC ⁇ l/ ⁇ 2 helices, of the two 3M4E5 and four 3M4F4 Fab complexes found in the crystallographic asymmetric unit, illustrating the variable positioning of the Fabs in the different crystal contexts. Colours are for 3M4E5 chains H/L, green; K/M, red; and for 3M4F4 H/L, blue; G/I, magenta; N/O, cyan and S/T coral (chain labels correspond to those for the deposited PDB files). MHC is coloured grey and the NYESO-1 157 _ 165 peptide in yellow.
  • (B) Superposition, based on the VH domains, of all 6 bound Fab structures (3M4E5 chains H and K, 3M4F4 chains H, I, O, T), illustrating the 'down' conformation of the CDR2 VH loop found in chains H and I of the 3M4F4 Fab, where in all the other Fab structures, the CDR2 adopts the 'up' conformation.
  • (C) Superposition of the VH domains from representative 3M4F4 structures for the two CDR2 VH conformations, chains H (blue) and T (coral), illustrating the 7A main chain change and side chain reorientations.
  • FIGURE 4A-4F Conformational changes on binding for (A) the 1G4 TCR (alpha chain is green and beta chain is red, unliganded structures in light colours) and (B) the 3M4F5 Fab (heavy chain is green and light chain is red, unliganded structures in light colours) where minor conformational adjustments are apparent for the 1G4 TCR compared to almost no significant main chain conformational adjustment of the 3M4E5 Fab CDR loops.
  • the peptide MW residues are illustrated in yellow.
  • FIGURE 5A-5D Structural basis for a second generation library.
  • FIGURE 6 Classification of 3M4E5 mutants by phylogenetic tree analysis. Amino acid sequences of mutated regions were subjected to cluster analysis using CLUSTAL W software for 123 mutant clones. The following conditions were used: matrix, BLOSUM; gap opening penalty, 10.0; gap extension penalty, 0.05. Results are displayed as a phylogenetic tree, which indicates the genetic distance to the parental 3M4E5 sequence. The three types of mutants that were repeatedly identified are indicated in red.
  • FIGURE 7A-7D Binding characteristics of 3M4E5-evolved Fab antibodies T 1-3.
  • IFN- ⁇ response of NY-ESO- l , 57 _ 165 -specific CTL to T2 cells pulsed with SLLMWITQC peptide (10 "6 M) was detected by ELISPOT. All assays were done in triplicate.
  • FIGURE 8 Tetramer staining of transduced T-cells. Binding specificity of Tl(*), wt ( ⁇ ) and control (A) sc-TCRs on transduced CD3+ T-cells were analysed by incubation with indicated amounts of PE conjugated HLA-A*0201/NY-ESO-I i 57 - I6S tetramers using flow cytometry. Assay was done in triplicates and standard deviation is depicted.
  • FIGURE 9 Fab activity as recombinant scTCRs.
  • AU assays were performed at least in triplicate with Tl always achieving a significantly (p ⁇ 0.05) higher cytotoxic activity.
  • FIGURE 10 depicts the amino acid sequence of the heavy and light chain variable region of antibody Tl compared with antibody 3M4E5.
  • the CDR regions are depicted in blue. Amino acid differences are shown in red.
  • FIGURE 11 depicts the nucleic acid sequence encoding the heavy and light chain variable region of antibody Tl compared with antibody 3M4E5. The CDR regions are depicted in blue. Nucleotide differences are shown in red.
  • FIGURE 12 depicts the amino acid sequence of the heavy and light chain variable region of antibody T2 compared with antibody 3M4E5.
  • the CDR regions are depicted in blue. Amino acid differences are shown in red.
  • FIGURE 13 depicts the amino acid sequence of the heavy and light chain variable region of antibody T3 compared with antibody 3M4E5.
  • the CDR regions are depicted in blue. Amino acid differences are shown in red.
  • the term "specific binding member” describes a member of a pair of molecules which have binding specificity for one another.
  • the members of a specific binding pair may be naturally derived or wholly or partially synthetically produced.
  • One member of the pair of molecules has an area on its surface, or a cavity, which specifically binds to and is therefore complementary to a particular spatial and polar organisation of the other member of the pair of molecules.
  • the members of the pair have the property of binding specifically to each other.
  • types of specific binding pairs are antigen-antibody, biotin-avidin, hormone-hormone receptor, receptor-ligand, enzyme-substrate. This application is concerned with antigen-antibody type reactions.
  • antibody describes an immunoglobulin whether natural or partly or wholly synthetically produced.
  • the term also covers any polypeptide or protein having a binding domain which is, or is homologous to, an antibody binding domain.
  • CDR grafted antibodies are also contemplated by this term.
  • An “antibody” is any immunoglobulin, including antibodies and fragments thereof, that binds a specific epitope.
  • the term encompasses polyclonal, monoclonal, and chimeric antibodies, the last mentioned described in further detail in U.S. Patent Nos. 4,816,397 and 4,816,567.
  • antibody(ies) includes a wild type immunoglobulin (Ig) molecule, generally comprising four full length polypeptide chains, two heavy (H) chains and two light (L) chains, or an equivalent Ig homologue thereof (e.g., a camelid nanobody, which comprises only a heavy chain); including full length functional mutants, variants, or derivatives thereof, which retain the essential epitope binding features of an Ig molecule, and including dual specific, bispecific, multispecif ⁇ c, and dual variable domain antibodies; Immunoglobulin molecules can be of any class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), or subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2).
  • Ig immunoglobulin
  • an “antibody fragment” means a molecule comprising at least one polypeptide chain that is not full length, including (i) a Fab fragment, which is a monovalent fragment consisting of the variable light (VL), variable heavy (VH), constant light (CL) and constant heavy 1 (CHl) domains; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a heavy chain portion of an Fab (Fd) fragment, which consists of the VH and CHl domains; (iv) a variable fragment (Fv) fragment, which consists of the VL and VH domains of a single arm of an antibody, (v) a domain antibody (dAb) fragment, which comprises a single variable domain (Ward, E.S.
  • antibody should be construed as covering any specific binding member or substance having a binding domain with the required specificity.
  • this term covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. Cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP-A-0125023 and U.S. Patent Nos. 4,816,397 and 4,816,567.
  • an "antibody combining site” is that structural portion of an antibody molecule comprised of light chain or heavy and light chain variable and hypervariable regions that specifically binds antigen.
  • the phrase "antibody molecule” in its various grammatical forms as used herein contemplates both an intact immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule.
  • Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin molecule that contains the paratope, including those portions known in the art as Fab, Fab', F(ab') 2 and F(v), which portions are preferred for use in the therapeutic methods described herein.
  • Antibodies may also be bispecific, wherein one binding domain of the antibody is a specific binding member of the invention, and the other binding domain has a different specificity, e.g. to recruit an effector function or the like.
  • Bispecific antibodies of the present invention include wherein one binding domain of the antibody is a specific binding member of the present invention, including a fragment thereof, and the other binding domain is a distinct antibody or fragment thereof, including that of a distinct anticancer or anti-tumor specific antibody.
  • the other binding domain may be an antibody that recognizes or targets a particular cell type, as in a neural or glial cell-specific antibody.
  • the one binding domain of the antibody of the invention may be combined with other binding domains or molecules which recognize particular cell receptors and/or modulate cells in a particular fashion, as for instance an immune modulator (e.g., interleukin(s)), a growth modulator or cytokine (e.g. tumor necrosis factor (TNF), and particularly, the TNF bispecific modality demonstrated in U.S. S.N. 60/355,838 filed February 13, 2002 incorporated herein in its entirety) or a toxin (e.g., ricin) or anti-mitotic or apoptotic agent or factor.
  • an immune modulator e.g., interleukin(s)
  • a growth modulator or cytokine e.g. tumor necrosis factor (TNF)
  • TNF tumor necrosis factor
  • the phrase "monoclonal antibody” in its various grammatical forms refers to an antibody having only one species of antibody combining site capable of immunoreacting with a particular antigen.
  • a monoclonal antibody thus typically displays a single binding affinity for any antigen with which it immunoreacts.
  • a monoclonal antibody may also contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a different antigen; e.g., a bispecific (chimeric) monoclonal antibody.
  • an antigen binding domain describes the part of an antibody which comprises the area which specifically binds to and is complementary to part or all of an antigen. Where an antigen is large, an antibody may bind to a particular part of the antigen only, which part is termed an epitope.
  • An antigen binding domain may be provided by one or more antibody variable domains.
  • an antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).
  • Immunoconjugates or antibody fusion proteins of the present invention wherein the antibodies, antibody molecules, or fragments thereof, of use in the present invention are conjugated or attached to other molecules or agents further include, but are not limited to such antibodies, molecules, or fragments conjugated to a chemical ablation agent, toxin, immunomodulator, cytokine, cytotoxic agent, chemotherapeutic agent, antimicrobial agent or peptide, cell wall and/or cell membrane disrupter, or drug.
  • the term "specific” may be used to refer to the situation in which one member of a specific binding pair will not show any significant binding to molecules other than its specific binding partner(s).
  • the term is also applicable where e.g. an antigen binding domain is specific for a particular epitope which is carried by a number of antigens, in which case the specific binding member carrying the antigen binding domain will be able to bind to the various antigens carrying the epitope.
  • the term "consisting essentially of” refers to a product, particularly a peptide sequence, of a defined number of residues which is not covalently attached to a larger product.
  • a product particularly a peptide sequence
  • minor modifications to the N- or C- terminal of the peptide may however be contemplated, such as the chemical modification of the terminal to add a protecting group or the like, e.g. the amidation of the C-terminus.
  • isolated refers to the state in which specific binding members of the invention, or nucleic acid encoding such binding members will be, in accordance with the present invention.
  • Members and nucleic acid will be free or substantially free of material with which they are naturally associated such as other polypeptides or nucleic acids with which they are found in their natural environment, or the environment in which they are prepared (e.g. cell culture) when such preparation is by recombinant DNA technology practised in vitro or in vivo.
  • nucleic acid may be formulated with diluents or adjuvants and still for practical purposes be isolated - for example the members will normally be mixed with gelatin or other carriers if used to coat microtitre plates for use in immunoassays, or will be mixed with pharmaceutically acceptable carriers or diluents when used in diagnosis or therapy.
  • pg means picogram
  • ng means nanogram
  • ug or “ ⁇ g” mean microgram
  • mg means milligram
  • ul or “ ⁇ l” mean microliter
  • ml means milliliter
  • “1" means liter
  • antibody Tl immunoglobulin T2
  • antibody T3 any variants not specifically listed, may be used herein interchangeably, and as used throughout the present application and claims refer to proteinaceous material including single or multiple proteins, and extends to those proteins having the amino acid sequence data described herein and presented in Figures 10, 12 and 13 and the profile of activities set forth herein and in the Claims. Accordingly, proteins displaying substantially equivalent or altered activity are likewise contemplated. These modifications may be deliberate, for example, such as modifications obtained through site-directed mutagenesis, or may be accidental, such as those obtained through mutations in hosts that are producers of the complex or its named subunits.
  • antibody T cell receptor-like antibody
  • NY-ESO-I peptide/MHC antibody antibody Tl
  • antibody T2 antibody T3
  • antibody T3 is intended to include within their scope proteins specifically recited herein as well as all substantially homologous analogs and allelic variations.
  • amino acid residues described herein are preferred to be in the "L" isomeric form.
  • residues in the "D" isomeric form can be substituted for any L-amino acid residue, as long as the desired fuctional property of immunoglobulin-binding is retained by the polypeptide.
  • NH 2 refers to the free amino group present at the amino terminus of a polypeptide.
  • COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide.
  • a "replicon” is any genetic element ⁇ e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo; i.e., capable of replication under its own control.
  • a "vector” is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.
  • DNA molecule refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single stranded form, or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules ⁇ e.g., restriction fragments), viruses, plasmids, and chromosomes.
  • sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA ⁇ i.e., the strand having a sequence homologous to the mRNA).
  • An "origin of replication" refers to those DNA sequences that participate in DNA synthesis.
  • a DNA "coding sequence” is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus.
  • a coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences.
  • a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.
  • Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
  • a "promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
  • the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • a transcription initiation site (conveniently defined by mapping with nuclease Sl), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT” boxes.
  • Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the -10 and -35 consensus sequences.
  • An "expression control sequence” is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence.
  • a coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence.
  • a "signal sequence” can be included before the coding sequence. This sequence encodes a signal peptide, N-terminal to the polypeptide, that communicates to the host cell to direct the polypeptide to the cell surface or secrete the polypeptide into the media, and this signal peptide is clipped off by the host cell before the protein leaves the cell. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes.
  • oligonucleotide as used herein in referring to the probe of the present invention, is defined as a molecule comprised of two or more ribonucleotides, preferably more than three. Its exact size will depend upon many factors which, in turn, depend upon the ultimate function and use of the oligonucleotide.
  • primer refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH.
  • the primer may be either single- stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent.
  • the exact length of the primer will depend upon many factors, including temperature, source of primer and use of the method.
  • the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.
  • the primers herein are selected to be “substantially" complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the strand to hybridize therewith and thereby form the template for the synthesis of the extension product.
  • restriction endonucleases and “restriction enzymes” refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.
  • a cell has been "transformed” by exogenous or heterologous DNA when such DNA has been introduced inside the cell.
  • the transforming DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.
  • the transforming DNA may be maintained on an episomal element such as a plasmid.
  • a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA.
  • a "clone” is a population of cells derived from a single cell or common ancestor by mitosis.
  • a "cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.
  • Two DNA sequences are "substantially homologous" when at least about 75% (preferably at least about 80%, and most preferably at least about 90 or 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Maniatis et al., supra; DNA Cloning, VoIs. I & II, supra; Nucleic Acid Hybridization, supra.
  • DNA sequences encoding specific binding members (antibodies) of the invention which code for e.g. an antibody having the same amino acid sequence as provided in Figure 10, 12 or 13, or comprising the CDR domain region sequences ste out in Figures 10, 12 or 13 but which are degenerate thereto.
  • degenerate to is meant that a different three-letter codon is used to specify a particular amino acid. It is well known in the art that the following codons can be used interchangeably to code for each specific amino acid:
  • Histidine His or H
  • CAC CAC Glutamine
  • Lysine (Lys or K) AAA or AAG
  • Glutamic Acid GAA or GAG
  • Arginine (Arg or R) CGU or CGC or CGA or CGG or AGA or AGG
  • Glycine GGU or GGC or GGA or GGG
  • codons specified above are for RNA sequences.
  • the corresponding codons for DNA have a T substituted for U.
  • Mutations can be made in the sequences encoding the amino acids set out in Figures 10, 12 or 13, or in the sequence of Figure 11, such that a particular codon is changed to a codon which codes for a different amino acid. Such a mutation is generally made by making the fewest nucleotide changes possible.
  • a substitution mutation of this sort can be made to change an amino acid in the resulting protein in a non-conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to another grouping) or in a conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to the same grouping).
  • a conservative change generally leads to less change in the structure and function of the resulting protein.
  • a non-conservative change is more likely to alter the structure, activity or function of the resulting protein.
  • the present invention should be considered to include sequences containing conservative changes which do not significantly alter the activity or binding characteristics of the resulting protein.
  • Aspartic acid Glutamic acid Basic amino acids (positively charged at pH 6.0) Lysine, Arginine, Histidine (at pH 6.0)
  • Another grouping may be those amino acids with phenyl groups:
  • AAnnootthheerr g j grroouup] ing may be according to molecular weight (i.e., size of R groups):
  • Amino acid substitutions may also be introduced to substitute an amino acid with a particularly preferable property.
  • a Cys may be introduced a potential site for disulfide bridges with another Cys.
  • a His may be introduced as a particularly "catalytic" site (i.e., His can act as an acid or base and is the most common amino acid in biochemical catalysis).
  • Pro may be introduced because of its particularly planar structure, which induces ⁇ -turns in the protein's structure.
  • Two amino acid sequences are "substantially homologous" when at least about 70% of the amino acid residues (preferably at least about 80%, and most preferably at least about 90 or 95%) are identical, or represent conservative substitutions.
  • a "heterologous" region of the DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature. Thus, when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism.
  • heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein.
  • a DNA sequence is "operatively linked" to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of that DNA sequence.
  • the term "operatively linked” includes having an appropriate start signal (e.g., ATG) in front of the DNA sequence to be expressed and maintaining the correct reading frame to permit expression of the DNA sequence under the control of the expression control sequence and production of the desired product encoded by the DNA sequence. If a gene that one desires to insert into a recombinant DNA molecule does not contain an appropriate start signal, such a start signal can be inserted in front of the gene.
  • standard hybridization conditions refers to salt and temperature conditions substantially equivalent to 5 x SSC and 65 0 C for both hybridization and wash.
  • standard hybridization conditions are dependent on particular conditions including the concentration of sodium and magnesium in the buffer, nucleotide sequence length and concentration, percent mismatch, percent formamide, and the like.
  • Also important in the determination of “standard hybridization conditions” is whether the two sequences hybridizing are RNA-RNA, DNA-DNA or RNA-DNA.
  • standard hybridization conditions are easily determined by one skilled in the art according to well known formulae, wherein hybridization is typically 10-20 0 C below the predicted or determined T m with washes of higher stringency, if desired.
  • agent means any molecule, including polypeptides, antibodies, polynucleotides, chemical compounds and small molecules.
  • agent includes compounds such as test compounds or drug candidate compounds.
  • the term 'agonist' refers to a ligand that stimulates the receptor the ligand binds to in the broadest sense.
  • the term 'assay' means any process used to measure a specific property of a compound.
  • a 'screening assay' means a process used to characterize or select compounds based upon their activity from a collection of compounds.
  • the term 'preventing' or 'prevention' refers to a reduction in risk of acquiring or developing a disease or disorder (i.e., causing at least one of the clinical symptoms of the disease not to develop) in a subject that may be exposed to a disease-causing agent, or predisposed to the disease in advance of disease onset.
  • prophylactic measures may include the administration of vaccines; the administration of low molecular weight heparin to hospital patients at risk for thrombosis due, for example, to immobilization; and the administration of an anti-malarial agent such as chloroquine, in advance of a visit to a geographical region where malaria is endemic or the risk of contracting malaria is high.
  • 'Therapeutically effective amount means that amount of a drug, compound, antimicrobial, antibody, or pharmaceutical agent that will elicit the biological or medical response of a subject that is being sought by a medical doctor or other clinician.
  • the term "effective amount" is intended to include an effective amount of a compound or agent that will bring about a biologically meaningful decrease in the amount of or extent of infection of gram-positive bacteria, including having a bacteriocidal and/or bacteriostatic effect.
  • terapéuticaally effective amount is used herein to mean an amount sufficient to prevent, and preferably reduce by at least about 30 percent, more preferably by at least 50 percent, most preferably by at least 90 percent, a clinically significant change in the growth or amount of infectious bacteria, or other feature of pathology such as for example, elevated fever or white cell count as may attend its presence and activity.
  • the term 'treating' or 'treatment' of any disease or infection refers, in one embodiment, to ameliorating the disease or infection (i.e., arresting the disease or growth of the infectious agent or bacteria or reducing the manifestation, extent or severity of at least one of the clinical symptoms thereof).
  • 'treating' or 'treatment' refers to ameliorating at least one physical parameter, which may not be discernible by the subject.
  • 'treating' or 'treatment' refers to modulating the disease or infection, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both.
  • 'treating' or 'treatment' relates to slowing the progression of a disease or reducing an infection.
  • phrases “pharmaceutically acceptable” refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
  • pg means picogram
  • ng means nanogram
  • ug means microgram
  • mg means milligram
  • ul means microliter
  • ml means milliliter
  • 1 means liter.
  • the present invention provides novel high affinity antibodies mimicking a TCR that are useful in targeting specific pMHC complexes for diagnostic and therapeutic purposes.
  • high affinity antibodies targeting NY-ESO-I peptide MHC complexes are provided, wherein said antibodies have a peptideMHC affinity which is greater than that of corresponding TCRs.
  • the invention provides an antibody or fragment thereof, which recognizes NY-ESO-I peptide 157-165/MHC complexes with high and specific affinity.
  • the antibodies of the present invention have diagnostic and therapeutic use and application in cancers, particularly in cancers wherein NY-ESO-I is involved or otherwise presented during the cancer or metastatic process or is a part of the humoral and/or cellular response to cancer, oncogenesis, or other disease pathogenesis.
  • the antibodies of the invention are applicable in cancers, including melanoma, lung, esophageal, liver, gastric, prostate, ovarian, bladder and synovial sarcoma.
  • the unique specificity of the antibodies or fragments thereof of the present invention recognizing tumor antigen peptide/MHC complexes, particularly NY-ESO- 1/MHC complexes, with specificity and with greater affinity than TCRs, provides diagnostic and therapeutic in cancer and immune- mediated cancer therapy.
  • the antibodies of the present invention can thus specifically categorize the nature of tumors or tumorigenic cells, by staining or otherwise recognizing those tumors or cells wherein NY- ESO-I or NY-ESO- 1/MHC complexes are present or presented in or on cells.
  • Panels of monoclonal antibodies recognizing NY-ESO-I or NY-ESO-1/MHC complexes can be screened for various properties; i.e., isotype, epitope, affinity, etc.
  • Such antibodies can be readily identified and/or screened in specific binding member activity assays.
  • the CDR regions comprising amino acid sequences substantially as set out as the CDR regions of Figures 10, 12 and 13 will be carried in a structure which allows for binding of the CDR regions to an tumor antigen, particularly to a tumor antigen peptide/MHC complex in a T cell receptor-like fashion.
  • variable region sequences, and/or particularly the CDR sequences, of the invention will be either identical or highly homologous to the specified regions of Figure 10, 12 or 13.
  • highly homologous it is contemplated that only a few substitutions, preferably from 1 to 8, preferably from 1 to 5, preferably from 1 to 4, or from 1 to 3, or 1 or 2 substitutions may be made in the variable region sequence and/or in the CDR sequences.
  • substitutions may be made in the variable region sequence outside of the CDRs so as to retain the CDR sequences.
  • changes in the variable region sequence or alternative non-homologous or veneered variable region sequences may be introduced or utilized, such that the CDR sequences are maintained and the remainder of the variable region sesuence may be substituted.
  • substitutions may be made particularly in the CDRs.
  • CDR sequences for the antibodies of the present invention are set out herein including in Figures 10, 12 and 13.
  • Particularly preferred amino acids for substitution are VL residues 26S, 27R, 32Y, 95G, 96S, 97Y and VH Q33, S35, G50, V52, S57, A59, E99, as set out herein and in the figures.
  • Antibodies of the invention having substitutions as above described and contemplated are selected to maintain the activities, specificity, and affinity commensurate with or greater than the exemplary antibodies, including Tl, T2 and T3 and having the characteristics as set out herein and in the claims.
  • the structure for carrying the CDRs of the invention will generally be of an antibody heavy or light chain sequence or substantial portion thereof in which the CDR regions are located at locations corresponding to the CDR region of naturally occurring VH and VL antibody variable domains encoded by rearranged immunoglobulin genes.
  • the structures and locations of immunoglobulin variable domains may be determined by reference to Kabat, E.A. et al, Sequences of Proteins of Immunological Interest. 4th Edition. US Department of Health and Human Services. 1987, and updates thereof, now available on the Internet (http://immuno.bme.nwu.edu)).
  • variable domains may be derived from any germline or rearranged human variable domain, or may be a synthetic variable domain based on consensus sequences of known human variable domains.
  • the CDR-derived sequences of the invention as defined in the preceding paragraph, may be introduced into a repertoire of variable domains lacking CDR regions, using recombinant DNA technology.
  • Marks et al describe methods of producing repertoires of antibody variable domains in which consensus primers directed at or adjacent to the 5' end of the variable domain area are used in conjunction with consensus primers to the third framework region of human VH genes to provide a repertoire of VH variable domains lacking a CDR/CDRs. Marks et al further describe how this repertoire may be combined with a CDR of a particular antibody.
  • the repertoire may then be displayed in a suitable host system such as the phage display system of WO92/01047 so that suitable specific binding members may be selected.
  • a repertoire may consist of from anything from 10 4 individual members upwards, for example from 10 6 to 10 8 or 10 10 members.
  • a further alternative is to generate novel VH or VL regions carrying the CDR-derived sequences of the invention using random mutagenesis of, for example, the Ab VH or VL genes to generate mutations within the entire variable domain.
  • random mutagenesis of, for example, the Ab VH or VL genes to generate mutations within the entire variable domain.
  • Another method which may be used is to direct mutagenesis to CDR regions of VH or VL genes.
  • Such techniques are disclosed by Barbas et al, (1994, Proc. Natl. Acad. Sci., USA, 91:3809-3813) and Schier et al (1996, J. MoI. Biol. 263:551-567).
  • a substantial portion of an immunoglobulin variable domain will comprise at least the three CDR regions, together with their intervening framework regions. Preferably, the portion will also include at least about 50% of either or both of the first and fourth framework regions, the 50% being the C- terminal 50% of the first framework region and the N-terminal 50% of the fourth framework region. Additional residues at the N-terminal or C-terminal end of the substantial part of the variable domain may be those not normally associated with naturally occurring variable domain regions.
  • construction of specific binding members of the present invention made by recombinant DNA techniques may result in the introduction of N- or C-terminal residues encoded by linkers introduced to facilitate cloning or other manipulation steps.
  • Other manipulation steps include the introduction of linkers to join variable domains of the invention to further protein sequences including immunoglobulin heavy chains, other variable domains (for example in the production of diabodies) or protein labels as provided herein and/or known to those of skill in the art.
  • binding domains based on either of these sequences form further aspects of the invention.
  • binding domains based on the sequence substantially set out in Figure 10, 12 and/or 13 such binding domains may be used as targeting agents for tumor antigens since it is known that immunoglobulin VH domains are capable of binding target antigens in a specific manner.
  • these domains may be used to screen for complementary domains capable of forming a two-domain specific binding member which has in vivo properties as good as or equal to the Ab antibody disclosed herein.
  • Specific binding members of the present invention may further comprise antibody constant regions or parts thereof.
  • specific binding members based on the sequences of Figures 10, 12 and 13 may be attached at their C-terminal end to antibody light chain constant domains including human CK or C ⁇ chains, preferably C ⁇ chains.
  • specific binding members based on the sequences of Figures 10, 12 or 13 may be attached at their C-terminal end to all or part of an immunoglobulin heavy chain derived from any antibody isotype, e.g. IgG, IgA, IgE, IgD and IgM and any of the isotype subclasses, particularly IgGl, IgG2b, and IgG4. IgGl is preferred.
  • the antibodies, or any fragments thereof may be conjugated or recombinantly fused to any cellular toxin, bacterial or other, e.g. pseudomonas exotoxin, ricin, or diphtheria toxin.
  • the part of the toxin used can be the whole toxin, or any particular domain of the toxin.
  • Such antibody-toxin molecules have successfully been used for targeting and therapy of different kinds of cancers, see e.g. Pastan, Biochim Biophys Acta. 1997 Oct 24;1333(2):Cl-6; Kreitman et al., N Engl J Med. 2001 JuI 26;345(4):241-7; Schnell et al., Leukemia. 2000 Jan;14(l): 129-35; Ghetie et al., MoI Biotechnol. 2001 Jul; 18(3):251-68.
  • Bi- and tri-specific multimers can be formed by association of different scFv molecules and have been designed as cross-linking reagents for T-cell recruitment into tumors (immunotherapy), viral retargeting (gene therapy) and as red blood cell agglutination reagents (immunodiagnostics), see e.g. Todorovska et al., J Immunol Methods. 2001 Feb l;248(l-2):47-66; Tomlinson et al., Methods Enzymol. 2000;326:461-79; McCaIl et al., J Immunol. 2001 May 15;166(10):6112-7.
  • Fully human antibodies can be prepared by immunizing transgenic mice carrying large portions of the human immunoglobulin heavy and light chains. These mice, examples of such mice are the XenomouseTM (Abgenix, Inc.) (US Patent Nos. 6,075,181 and 6,150,584), the HuMAb-MouseTM (Medarex, Inc./GenPharm) (US patent 5545806 and 5569825), the TransChromo MouseTM (Kirin) and the KM MouseTM (Medarex/Kirin), are well known within the art. Antibodies can then be prepared by, e.g. standard hybridoma technique or by phage display. These antibodies will then contain only fully human amino acid sequences.
  • Fully human antibodies can also be generated using phage display from human libraries.
  • Phage display may be performed using methods well known to the skilled artisan, and as provided herein as in Hoogenboom et al and Marks et al (Hoogenboom HR and Winter G. (1992) J MoI Biol. 227(2):381-8; Marks JD et al (1991) J MoI Biol. 222(3):581-97; and also U.S. Patents 5885793 and 5969108).
  • Antibodies of the invention may be labelled with a detectable or functional label.
  • Detectable labels include, but are not limited to, radiolabels such as the isotopes 3 H, 14 C, 32 P, 35 S, 36 Cl, 51 Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 121 I, 124 I, 125 I, 131 I, 111 In, 117 Lu, 211 At, 198 Au, 67 Cu, 225 Ac, 213 Bi, 99 Tc and 186 Re, which may be attached to antibodies of the invention using conventional chemistry known in the art of antibody imaging.
  • Labels also include fluorescent labels (for example fluorescein, rhodamine, Texas Red) and labels used conventionally in the art for MRI-CT imaging. They also include enzyme labels such as horseradish peroxidase, ⁇ -glucoronidase, ⁇ -galactosidase, urease. Labels further include chemical moieties such as biotin which may be detected via binding to a specific cognate detectable moiety, e.g. labelled avidin. Functional labels include substances which are designed to be targeted to the site of a tumor to cause destruction of tumor tissue. Such functional labels include cytotoxic drugs such as 5-fluorouracil or ricin and enzymes such as bacterial carboxypeptidase or nitroreductase, which are capable of converting prodrugs into active drugs at the site of a tumor.
  • fluorescent labels for example fluorescein, rhodamine, Texas Red
  • enzyme labels such as horseradish peroxidase, ⁇ -glucoronidase, ⁇ -galact
  • antibodies including fragments thereof, and drugs that modulate the production or activity of the specific binding members, antibodies and/or their subunits may possess certain diagnostic applications and may for example, be utilized for the purpose of detecting and/or measuring conditions such as cancer, precancerous lesions, conditions related to or resulting from hyperproliferative cell growth or the like.
  • the specific binding members, antibodies or their subunits may be used to produce both polyclonal and monoclonal antibodies to themselves in a variety of cellular media, by known techniques such as the hybridoma technique utilizing, for example, fused mouse spleen lymphocytes and myeloma cells.
  • small molecules that mimic or antagonize the activity(ies) of the specific binding members of the invention may be discovered or synthesized, and may be used in diagnostic and/or therapeutic protocols.
  • the radiolabeled specific binding members are useful in in vitro diagnostics techniques and in in vivo radioimaging techniques and in radioimmunotherapy.
  • the specific binding members of the present invention may be conjugated to an imaging agent rather than a radioisotope(s), including but not limited to a magnetic resonance image enhancing agent, wherein for instance an antibody molecule is loaded with a large number of paramagnetic ions through chelating groups.
  • chelating groups include EDTA, porphyrins, polyamines crown ethers and poly ⁇ ximes.
  • radiolabeled specific binding members particularly antibodies and fragments thereof, particularly radioimmunoco ⁇ jugates
  • radioimmunotherapy particularly as radiolabeled antibodies for cancer therapy.
  • the radiolabeled specific binding members, particularly antibodies and fragments thereof are useful in radioimmuno-guided surgery techniques, wherein they can identify and indicate the presence and/or location of cancer cells, precancerous cells, tumor cells, and hyperproliferative cells, prior to, during or following surgery to remove such cells.
  • Immunoconjugates or antibody fusion proteins of the present invention wherein the specific binding members, particularly antibodies and fragments thereof, of the present invention are conjugated or attached to other molecules or agents further include, but are not limited to binding members conjugated to a chemical ablation agent, toxin, immunomodulator, cytokine, cytotoxic agent, chemotherapeutic agent or drug.
  • Radioimmunotherapy has entered the clinic and demonstrated efficacy using various antibody immunoconjugates.
  • 131 I labeled humanized anti-carcinoembryonic antigen (anti-CEA) antibody hMN-14 has been evaluated in colorectal cancer (Behr TM et al (2002) Cancer 94(4Suppl): 1373-81) and the same antibody with 90 Y label has been assessed in medullary thyroid carcinoma (Stein R et al (2002) Cancer 94(1):51-61).
  • Radioimmunotherapy using monoclonal antibodies has also been assessed and reported for non-Hodgkin's lymphoma and pancreatic cancer (Goldenberg DM (2001) Crit Rev Oncol Hematol 39(1-2): 195-201; Gold DV et al (2001) Crit Rev Oncol Hematol 39 (1-2) 147-54). Radioimmunotherapy methods with particular antibodies are also described in U.S. Patent 6,306,393 and 6,331, 175.
  • Radioimmunoguided surgery has also entered the clinic and demonstrated efficacy and usefulness, including using anti-CEA antibodies and antibodies directed against tumor-associated antigens (Kim JC et al (2002) Int J Cancer 97(4):542-7; Schneebaum S et al (2001) World J Surg 25(12): 1495-8; Avital S et al (2000) Cancer 89(8): 1692-8; Mclntosh DG et al (1997) Cancer Biother Radiopharm 12 (4):287-94).
  • animal models of cancer or animal xenograft studies may be utilized by the skilled artisan to further or additionally screen, assess, and/or verify the specific binding members and antibodies or fragments thereof of the present invention, including further assessing NY-ESO- 1 modulation and immunotherapy in vivo.
  • animal models include, but are not limited to models of melanoma, lung, esophageal, liver, gastric, prostate, ovarian, bladder cancer and synovial sarcoma.
  • Antibodies of the present invention may be administered to a patient in need of treatment via any suitable route, including by injection into the bloodstream or CSF, or directly into the site of the tumor.
  • the precise dose will depend upon a number of factors, including whether the antibody is for diagnosis or for treatment, the size and location of the tumor, the precise nature of the antibody (whether whole antibody, fragment, diabody, etc), and the nature of the detectable or functional label attached to the antibody.
  • a radionuclide is used for therapy, a suitable maximum single dose may be about 45 mCi/m 2 , to a maximum of about 250 mCi/m 2 .
  • Preferable dosage is in the range of 15 to 40 mCi, with a further preferred dosage range of 20 to 30 mCi, or 10 to 30 mCi.
  • Such therapy may require bone marrow or stem cell replacement.
  • a typical antibody dose for either tumor imaging or tumor treatment will be in the range of from 0.5 to 40 mg, preferably from 1 to 4 mg of antibody in F(ab')2 form.
  • Naked antibodies are preferably administered in doses of 20 to 1000 mg protein per dose, or 20 to 500 mg protein per dose, or 20 to 100 mg protein per dose. This is a dose for a single treatment of an adult patient, which may be proportionally adjusted for children and infants, and also adjusted for other antibody formats, in proportion for example to molecular weight. Treatments may be repeated at daily, twice-weekly, weekly or monthly intervals, at the discretion of the physician.
  • compositions according to the present invention may comprise, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • a pharmaceutically acceptable excipient such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • the precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. intravenous, or by deposition at a tumor site.
  • compositions for oral administration may be in tablet, capsule, powder or liquid form.
  • a tablet may comprise a solid carrier such as gelatin or an adjuvant.
  • Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
  • the active ingredient may be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection.
  • Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
  • a composition may be administered alone or in combination with other treatments, therapeutics or agents, either simultaneously or sequentially dependent upon the condition to be treated.
  • compositions comprising the binding member, particularly antibody or fragment thereof, herein described and other agents or therapeutics such as anticancer agents or therapeutics, hormones, anti-mitotic agents, anti-apoptotic agents, antibodies, or immune modulators. More generally these anti-cancer agents may be but are not limited to tyrosine kinase inhibitors or phosphorylation cascade inhibitors, post-translational modulators, cell growth or division inhibitors (e.g. anti-mitotics), or signal transduction inhibitors. Other treatments or therapeutics may include the administration of suitable doses of pain relief drugs such as non-steroidal anti-inflammatory drugs (e.g.
  • composition can be administered in combination (either sequentially (i.e. before or after) or simultaneously) with tyrosine kinase inhibitors (including, but not limited to AG1478 and ZD1839, STI571, OSI-774, SU-6668), doxorubicin, temozolomide, cisplatin, carboplatin, nitrosoureas, procarbazine, vincristine, hydroxyurea, 5-fluoruracil, cytosine arabinoside, cyclophosphamide, epipodophyllotoxin, carmustine, lomustine, and/or other chemotherapeutic agents.
  • tyrosine kinase inhibitors including, but not limited to AG1478 and ZD1839, STI571, OSI-774, SU-6668
  • doxorubicin including, but not limited to AG1478 and ZD1839, STI571, OSI-774, SU-6668
  • these agents may be specific anti-cancer agents, or immune cell response modulators or may be more general anticancer and anti-neoplastic agents such as doxorubicin,cisplatin, temozolomide, nitrosoureas, procarbazine, vincristine, hydroxyurea, 5-fluoruracil, cytosine arabinoside, cyclophosphamide, epipodophyllotoxin, carmustine, or lomustine.
  • doxorubicin,cisplatin temozolomide
  • nitrosoureas nitrosoureas
  • procarbazine vincristine
  • hydroxyurea 5-fluoruracil
  • cytosine arabinoside cyclophosphamide
  • epipodophyllotoxin carmustine, or lomustine.
  • compositions may be administered with hormones such as dexamethasone, immune modulators, such as interleukins, tumor necrosis factor (TNF) or other growth factors, colony stimulating factors, or cytokines which stimulate the immune response and reduction or elimination of cancer cells or tumors.
  • hormones such as dexamethasone
  • immune modulators such as interleukins, tumor necrosis factor (TNF) or other growth factors, colony stimulating factors, or cytokines which stimulate the immune response and reduction or elimination of cancer cells or tumors.
  • TNF tumor necrosis factor
  • cytokines which stimulate the immune response and reduction or elimination of cancer cells or tumors.
  • the composition may also be administered with, or may include combinations along with other anti-tumor antigen antibodies.
  • the present invention contemplates and includes therapeutic compositions for the use of the binding member in combination with conventional radiotherapy.
  • a subject therapeutic composition includes, in admixture, a pharmaceutically acceptable excipient (carrier) and one or more of a specific binding member, polypeptide analog thereof or fragment thereof, as described herein as an active ingredient.
  • the composition comprises an antigen capable of modulating the specific binding of the present binding member/antibody with a target cell.
  • compositions which contain polypeptides, analogs or active fragments as active ingredients is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions. However, solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared.
  • the preparation can also be emulsified.
  • the active therapeutic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
  • the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents which enhance the effectiveness of the active ingredient.
  • a polypeptide, analog or active fragment can be formulated into the therapeutic composition as neutralized pharmaceutically acceptable salt forms.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide or antibody molecule) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • the therapeutic antibody- or active fragment-containing compositions are conventionally administered intravenously, as by injection of a unit dose, for example.
  • unit dose when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosage for humans, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle.
  • compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount.
  • quantity to be administered depends on the subject to be treated, capacity of the subject's immune system to utilize the active ingredient, and degree of peptide/MHC or tumor antigen binding capacity desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. Suitable regimes for initial administration and follow on administration are also variable, and may include an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration. Alternatively, "continu intravenous infusion sufficient to maintain appropriate and sufficient concentrations in the blood or at the site of desired therapy are contemplated.
  • compositions for oral administration may be in tablet, capsule, powder or liquid form.
  • a tablet may comprise a solid carrier such as gelatin or an adjuvant.
  • Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
  • the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection.
  • Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
  • the present invention also relates to a variety of diagnostic applications, including methods for detecting the presence of NY-ESO-I /MHC complexes, NY-ESO-I antigen, NY-ESO-I mediated cancer, or canced more generally, by reference to their ability to be recognized by the present specific binding member(s).
  • Peptide complexes can be identified, targeted, labeled, and/or quantitated on dendritic cells or other antigen presenting cells and/or tumor cells.
  • Diagnostic applications of the specific binding members of the present invention include in vitro and in vivo applications well known and standard to the skilled artisan and based on the present description. Diagnostic assays and kits for in vitro assessment and evaluation of tumor and cancer status, may be utilized to diagnose, evaluate and monitor patient samples including those known to have or suspected of having cancer, a precancerous condition, a condition related to hyperproliferative cell growth or from a tumor sample.
  • the assessment and evaluation of cencer, tumor and metastatic disease status is also useful in determining the suitability of a patient for a clinical trial of a drug or for the administration of a particular chemotherapeutic agent or specific binding member, particularly an antibody, of the present invention, including combinations thereof, versus a different agent or binding member.
  • This type of diagnostic monitoring and assessment is already in practice utilizing antibodies against the HER2 protein in breast cancer (Hercep Test, Dako Corporation), where the assay is also used to evaluate patients for antibody therapy using Herceptin.
  • In vivo applications include imaging of tumors or assessing cancer status of individuals, including radioimaging.
  • the antibody used in the diagnostic methods of this invention is human antibody. More preferably, the antibody is a single chain chain antibody or domain antibody.
  • the antibody molecules used herein can be in the form of Fab, Fab', F(ab') 2 or F(v) portions of whole antibody molecules, particularly Fab.
  • antibody(ies) to the NY-ESO-I /MHC molecule complex(es) can be produced and isolated by standard methods including the phage display techniques and mutagenesis and recombinant techniques.
  • kits suitable for use by a medical specialist may be prepared to determine the presence or absence of aberrant expression of including but not limited to amplified and/or an mutation, in suspected target cells.
  • one class of such kits will contain at least the labeled or its binding partner, for instance an antibody specific thereto, and directions, of course, depending upon the method selected, e.g., "competitive,” “sandwich,” “DASP” and the like.
  • the kits may also contain peripheral reagents such as buffers, stabilizers, etc.
  • a test kit may be prepared for the demonstration of the presence of tumor antigen peptide/MHC complexes, comprising:
  • a test kit may be prepared for the demonstration of the presence of cancer, particularly selected from melanoma, lung, esophageal, liver, gastric, prostate, ovarian, bladder cancer, and synovial sarcoma, comprising:
  • an assay system for screening potential drugs effective to modulate the presence or activity of the tumor antigen peptide/MHC complex and/or the activity or binding of the antibody of the present invention may be prepared.
  • the antigen peptide or the binding member or antibody may be introduced into a test system, and the prospective drug may also be introduced into the resulting cell culture, and the culture thereafter examined to observe any changes in the activity of the cells, binding of the antibody, or amount and extent of tumor antigen peptide/MHC complex due either to the addition of the prospective drug alone, or due to the effect of added quantities of the known agent(s).
  • the present invention further provides an isolated nucleic acid encoding a specific binding member of the present invention.
  • Nucleic acid includes DNA and RNA.
  • the present invention provides a nucleic acid which codes for a polypeptide of the invention as defined above, including a polypeptide as set out in Figures 10, 12 or 13 or capable of encoding the CDR regions thereof.
  • the invention provides exemplary nucleic acid of Figure 1 1 which encodes the amino acid of Figure 10.
  • the present invention also provides constructs in the form of plasmids, vectors, transcription or expression cassettes which comprise at least one polynucleotide as above.
  • the present invention also provides a recombinant host cell which comprises one or more constructs as above.
  • a nucleic acid encoding any specific binding member as provided itself forms an aspect of the present invention, as does a method of production of the specific binding member which method comprises expression from encoding nucleic acid therefor. Expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the nucleic acid. Following production by expression a specific binding member may be isolated and/or purified using any suitable technique, then used as appropriate.
  • nucleic acid molecules and vectors according to the present invention may be provided isolated and/or purified, e.g. from their natural environment, in substantially pure or homogeneous form, or, in the case of nucleic acid, free or substantially free of nucleic acid or genes origin other than the sequence encoding a polypeptide with the required function.
  • Nucleic acid according to the present invention may comprise DNA or RNA and may be wholly or partially synthetic.
  • Suitable host cells include bacteria, mammalian cells, yeast and baculovirus systems.
  • Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells and many others.
  • a common, preferred bacterial host is E.coli.
  • the expression of antibodies and antibody fragments in prokaryotic cells such as E.coli is well established in the art.
  • Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
  • Vectors may be plasmids, viral e.g. 'phage, or phagemid, as appropriate.
  • plasmids viral e.g. 'phage, or phagemid, as appropriate.
  • a further aspect of the present invention provides a host cell containing nucleic acid as disclosed herein.
  • a still further aspect provides a method comprising introducing such nucleic acid into a host cell.
  • the introduction may employ any available technique.
  • suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g. vaccinia or, for insect cells, baculovirus.
  • suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage.
  • the introduction may be followed by causing or allowing expression from the nucleic acid, e.g. by culturing host cells under conditions for expression of the gene.
  • the present invention also provides a method which comprises using a construct as stated above in an expression system in order to express a specific binding member or polypeptide as above.
  • DNA sequences may be expressed by operatively linking them to an expression control sequence in an appropriate expression vector and employing that expression vector to transform an appropriate unicellular host.
  • a wide variety of host/expression vector combinations may be employed in expressing the DNA sequences of this invention.
  • Useful expression vectors may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences. Suitable vectors include derivatives of SV40 and known bacterial plasmids, e.g., E.
  • phage DNAs e.g., the numerous derivatives of phage ⁇ , e.g., NM989, and other phage DNA, e.g., M13 and filamentous single stranded phage
  • any of a wide variety of expression control sequences ⁇ sequences that control the expression of a DNA sequence operatively linked to it — may be used in these vectors to express the DNA sequences of this invention.
  • useful expression control sequences include, for example, the early or late promoters of SV40, CMV, vaccinia, polyoma or adenovirus, the lac system, the trp system, the TA C system, the TRC system, the LTR system, the major operator and promoter regions of phage ⁇ , the control regions of fd coat protein, the promoter for 3 -phosphogly cerate kinase or other glycolytic enzymes, the promoters of acid phosphatase (e.g., Pho5), the promoters of the yeast -mating factors, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.
  • a wide variety of unicellular host cells are also useful in expressing the DNA sequences of this invention.
  • These hosts may include well known eukaryotic and prokaryotic hosts, such as strains of E. coli, Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and animal cells, such as CHO, YB/20, NSO, SP2/0, RU, B-W and L-M cells, African Green Monkey kidney cells (e.g., COS 1, COS 7, BSCl, BSC40, and BMTlO), insect cells (e.g., Sf9), and human cells and plant cells in tissue culture.
  • eukaryotic and prokaryotic hosts such as strains of E. coli, Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and animal cells, such as CHO, YB/20, NSO, SP2/0, RU, B-W and L-M cells, African Green Monkey
  • Suitable unicellular hosts will be selected by consideration of, e.g., their compatibility with the chosen vector, their secretion characteristics, their ability to fold proteins correctly, and their fermentation requirements, as well as the toxicity to the host of the product encoded by the DNA sequences to be expressed, and the ease of purification of the expression products. Considering these and other factors a person skilled in the art will be able to construct a variety of vector/expression control sequence/host combinations that will express the DNA sequences of this invention on fermentation or in large scale animal culture.
  • a DNA sequence encoding a specific binding member can be prepared synthetically rather than cloned.
  • the DNA sequence can be designed with the appropriate codons for the specific binding member amino acid sequence. In general, one will select preferred codons for the intended host if the sequence will be used for expression.
  • the complete sequence is assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge, Nature, 292:756 (1981); Nambair et al., Science, 223:1299 (1984); Jay et al., J. Biol. Chem., 259:6311 (1984).
  • DNA sequences allow convenient construction of genes which will express specific binding member analogs or "muteins".
  • DNA encoding muteins can be made by site-directed mutagenesis of native specific binding member genes or cDNAs, and muteins can be made directly using conventional polypeptide synthesis.
  • T-cell interaction with a target cell is a key event in adaptive immune response and primarily driven by T-cell receptor (TCR) recognition of peptide-MHC (pMHC) complexes.
  • TCR avidity for a given pMHC is determined by number of MHC molecules, availability of co-receptors and TCR affinity for MHC or peptide, respectively, with peptide recognition being the most important factor to confer target specificity.
  • SLLMWITQV immunodominant NY-ESO-I I57-I6S peptide analogue
  • T-cell receptors as natural ligands of pMHC complexes can target the pMHC efficiently (2) but generally have low binding affinities preventing their use as therapeutic reagents (3). Therefore, high affinity recombinant TCRs would be ideal candidates for diagnostic and even therapeutic purposes (4). Indeed, affinity maturation strategies have been successfully developed and demonstrated dramatic increases of TCR affinity (5, 6).
  • the 1G4 TCR binds in a classical diagonal orientation with respect to the peptide axis, it notably centres on the peptide's hydrophobic prominently exposed methionine (M) and tryptophan (W) side chains at residues 4 and 5 of the NY-ESO-I i 5 7_i 65 peptide.
  • M methionine
  • W tryptophan
  • This MW motif is enveloped by the TCR's CDR loops and contributes approximately 50% of the buried surface area contacted between TCR and peptide.
  • Both M and W are encoded by single codons each, making the combination of these amino acids rare in the human proteome and an uncommon motif found in peptides presented by MHC molecules.
  • phage display libraries have recently enabled the rapid isolation of human Fab fragments highly specific to pMHC molecules (11).
  • the structure of one Fab fragment (Hyb3) bound to the HLA-A* 1 -MAGE-Al peptide complex has been solved demonstrating considerable deviation form a normal TCR-like binding footprint (12) although, detailed comparison of Hyb3 and HLA-A* 1 -MAGE-Al specific TCR binding modes was precluded because of the lack of a representative TCR-pMHC structure.
  • Hyb3 was generated by random affinity maturation raising the question that the recognition focus on the MHC alpha-1 ( ⁇ l) helix, rather than peptide, might be the result of this process.
  • TCR CDR3 ⁇ residue YlOO and Fab VH CDR3 residue Y103 made hydrogen bonds to the peptide main chain carbonyl oxygen of M4 and their aromatic tyrosine rings interacted closely with the hydrophobic MW motif (FIGURE 2).
  • Fabs and 1G4 TCR shared many contact residues with HLA-A*0201 (R65, K66, A69, Q72, T73, A150, and Q155), including hydrogen bonds to the same atoms (R65 NE, T73 OG, A150 O and Q 155 El).
  • Residues orientated away from the peptide or towards the MHC helices were not included to avoid ineffective changes or unwanted increase of affinity to the MHC helices.
  • the structural data suggested residues 26S, 27R, 32Y, 95G, 96S and 97Y as candidates for variation.
  • Va CDRl Y31 Q155 VaCDR2 Q51 A150, H151, Q155 VaCDR2 S52 Q155 VaCDR2 S53 Q155, E154 VaCDR2 S54 H151.
  • E154 Va CDR3 G97 R65 Va CDR3 G98 R65, K66 Va CDR3 S99 R65 Va CDR3 YlOO K66 V ⁇ CDRl E29 Q72, T73 V ⁇ CDR2 Y47 R65 V ⁇ CDR2 V49 R65, Q72 V ⁇ CDR2 G50 Q72 V ⁇ CDR2 A51 Q72 V ⁇ CDR2 153 K68 V ⁇ CDR2 D55 R65 V ⁇ CDR2 T70 Q72 V ⁇ CDR3 V95 T73 V ⁇ CDR3 N97 A150
  • VH CDR2 S57 T163, Y159
  • the final antibody library contained 10 8 independent clones and after the 3 r round of selection, 480 candidate clones were identified of which 172 revealed, as soluble phage particles, specific binding to the HLA-A*0201/NY- ESO-1 157 . I65 complex. Sequence data of strong binders were grouped by cluster analysis and three types (Fab Tl-3) of repeatedly selected mutants could be identified by phylogenetic tree analysis (FIGURE 6). The most dominant mutations present in all three types of mutants included S26E (LC CDRl) and S96G (LC CDR3), respectively. Fab Tl contained only these amino acid (AA) changes, whereas Fab T2 and T3 had additional heavy chain mutations (TABLE 4). The amino acid sequences of the heavy chain and light chain variable regions of the Tl, T2 and T3 antibodies are shown in Figures 10, 12 and 13 respectively, with the CDR regions depicted in blue.
  • FIGURE 7C using non-saturating amounts of biotinylated 3M4E5 tetramer indicated differences in binding affinities for T1-T3.
  • Fab Tl demonstrated the highest binding affinity by achieving a significantly stronger inhibitory signal when compared to Fab 3M4E5 or Fab T2 and T3, respectively.
  • the same pattern of inhibition was observed in a cellular assay with the strongest T-cell inhibition capacity, for Fab Tl, significantly exceeding the parental Fab 3M4E5 (FIGURE 7D).
  • the Fab By centering on the peptide MW motif, the Fab is positioned similarly to the TCR, and adopts a diagonal binding orientation that generates very similar MHC contact points on the helices.
  • the structures contrast with the HLA-A*01 -MAGE-A 1-Hyb3 complex which binds more to the MHC ⁇ l helix, and samples a smaller portion of the peptide (12).
  • the constraints on MHC class I binding for Fabs derived from phage libraries seem therefore less stringent than for TCRs, although the 3M4E5 and 3M4F4 Fabs adopt similar docking positions as the HLA-A*0201 -NY-ESO-I specific 1G4 TCR.
  • the NY-ESO-I peptide in HLA-A*0201 is a relatively unique epitope, in that it presents the M and W side chains for recognition by incoming receptors (8). These side chains form a much larger available binding surface area than conventional HLA-A*0201 peptides, where often one bulky residue forms a key central motif exploited for functional recognition.
  • the large MW motif therefore, assists in generating peptide-specific interactions, and is an excellent focal feature against which to develop therapeutic Fabs with high specificity and affinity.
  • the footprint of TCR and Fab binding to the MHC backbone and the NY-ESO-I J57-I65 peptide directed the design of the second generation library as described before.
  • Our strategy of keeping key MW motif contact residues conserved whilst randomizing only individual side chains interacting with the peptide but not with the MHC backbone was proven experimentally to be correct.
  • the final affinity matured antibody achieved its 20 fold improved affinity by just two light chain mutations.
  • NY-ESO-I in particular is an attractive target antigen since its expression pattern is restricted to germ cells (lacking MHC class I molecules) and a wide variety of hematological and solid organ tumors (24). However, it is expressed in the nucleus and cytoplasm and, therefore, not accessible for most targeted approaches (25).
  • NY-ESO-I derived peptides such as the NY-ESO-I i 57 . 165 peptide described here are unique cell surface markers when expressed in the appropriate MHC context (14).
  • the rational, peptide focused approach as described resulted in Fab fragments with low nanomolar affinities and will hopefully be a model for the generation of pMHC specific diagnostic and therapeutic reagents.
  • HLA-A*0201 heavy chain with a C-terminal biotinylation tag, B2m, and the respective HLA-A*0201 -restricted peptides were refolded by dilution as previously described (14, 26).
  • HLA-A*0201 heavy chain without the biotinylation tag was used for refolding, and purified as described (27).
  • the 3M4F4-A2-NYESO-1 complex crystallised in 12% PEG 8000 5OmM MES pH 6.9 with crystal dimensions of approximately 50 ⁇ m by 35 ⁇ m by lO ⁇ m.
  • the 3M4E5 Fab crystallised in 14% PEG 8000 5OmM MES pH 6.5 with crystal dimensions of about 50 ⁇ m by 40 ⁇ m by 20 ⁇ m. Crystals were harvested and briefly soaked sequentially in reservoir solutions containing 10% and 20% glycerol, then flash-cooled and maintained at IOOK in a cryostream (Oxford Cry osy stems).
  • Phage display selection For the library construction, an antibody phagemid library was generated by gene-synthesis (GENART, Regensburg, Germany) including random codon mutations (to NNB) at indicated positions and subcloned into the pCES vector. Phage Fab particles were produced and incubated with HLA-A*0201/NY-ESO- I 157 - I65 complex in the presence of Fab 3M4E5 protein (lOO ⁇ g/ml) following the selection procedure as previously described (14). Bound phages were eluted with 100 mM Triethylamin and neutralized with Tris-HCl pH 7.2. Phages were used to infect E.
  • coli periplasmic fraction as described (14) and bound Fab-molecules were detected by murine anti-myc antibody 9E10 (0.3 ⁇ g/ml, Roche, Mannheim, Germany).
  • M13 antibody 0.3 ⁇ g/ml (Amersham Pharmacia Biotech, Sweden) was used for the detection of phage particles.
  • a horseradish peroxidase-conjugated antibody (anti-mouse IgG, 1:2000; Dako, Denmark) was used as secondary reagent. Tetramethylbenzidine was used as substrate (Sigma, Kunststoff, Germany).
  • Kinetic constants were derived using the curve-fitting facility of the BIAevaluation program (Version 3.0, BIAcore) and rate equations derived from the simple 1 : 1 Langmuir binding model (A+B ⁇ AB). Duplicates of each measurement were performed and averaged.
  • PBS-BSA phosphate-buffered saline-bovine serum albumin
  • 3M4E5-b tetramer 3M4E5-b tetramer. 3M4E5-Fab-tetramers were assembled by incubating 60 ⁇ g biotinylated 3M4E5-Fab- monomers with 80 ⁇ g of Streptavidin-conjugated R-PE [Invitrogen, Leiden, Netherland; (45min, 37°C or ON, 4 0 C)] at an optimal stoichometric ratio of 1:4. The suboptimal 3M4E5 tetramer concentration was determined on T2-lb minigene cells or T2 cells pulsed with the respective NY-ESO-I peptide (SLLMWITQV) (pulsing conditions: 2h 37°C, peptide concentration: 10 ⁇ 5 -10 ⁇ 10 M).
  • SLLMWITQV NY-ESO-I peptide
  • T-cell inhibition Assays were performed in a modified ELISPOT assay in triplicates on nitrocellulose-lined 96-well plates (MAHA S45 by Millipore, Bedford MA, USA). Wells were pre-coated overnight with an anti-IFN- ⁇ capture antibody as recommended (Mabtech AB, Nacka, Sweden) and blocked (Ih, 37°C) with RPMI containing 10% human serum.
  • the CD8 + HLA-A2/ NY-ESO-I 157-I65 specific T-cell clone was cultured as previously described (36).
  • Target T2 cells were pulsed with 0.1 ug/ml of NY-ESO-1 , 57 - 165 peptide (Ih, 37°C), stringently washed and incubated (Ih, 37°C) with different concentrations (30, 3, 0.3, 0.003, 0.0003, 0.00003, 0 ⁇ g/ml) of HLA-A0201*/NY-ESO-l 157 -i 65 complex specific or irrelevant Fab antibodies.
  • CD8+T-cells were co-cultured with target cells for 16h at 37°C (E:T of 1 :1, ie. 8000:8000 cells per well). Plates were evaluated using an automated ELISPOT reader (Bioreader 3000, BioSys, Germany).
  • T-cells grafted with the recombinant immunoreceptors were co-cultivated in round bottom 96 well micro-titre plates (1.25-106104 grafted T cells/well) with HLA-A2/NY-ESO-1 157 - I65 positive and negative T2 cells (5000 cells/well). After 48 hours, culture supernatants were analysed for IFN- ⁇ release using a sandwich ELISA [(coat mAb NIB42 (1 ⁇ g/ml) (Pierce, Bonn Germany), detection by biotinylated mAb 4S.B3 (0.5 ⁇ g/ml) (BD Bioscience, Heidelberg, Germany)].
  • reaction was visualised by peroxidase-streptavidin (1: 10,000) and ABTS (both Roche Diagnostics). Specific cytotoxicity of receptor grafted T cells against target cells was analysed using a colorimetric tetrazolium salt based assay indicating cell viability (EZ4U, Biomedica, Austria) as described (18).
  • Greenspan NS (2001) Affinity, complementarity, cooperativity, and specificity in antibody recognition. Curr Top Microbiol Immunol 260:65-85.
  • HLA human leukocyte antigen
  • EL4 mouse lymphoma cell line is transfected with human HLA- A2 and with NY-ESO-I. These lymphoma cells are then capable of expressing the target peptide 157-165 in the context of human HL-A2.
  • BL6 mice are challenged with the EL4-NY-ESO-1/HLA-A2 tumor cells.
  • the 3M4E5 and Tl Fab antibodies are converted into fully human IgGl antibodies and are used in these mice for tumor targeting using 111 Indium as tracer and in radioimmunotherapy using 177 Luthetium for cell killing.
  • T cell receptor-like Fab antibodies specific for melanoma-associated antigen MART-l(26-35) or gp 100(280-288) presented by HLA- A201 were fused to truncated Pseudomonas exotoxin (PE38KDEL) and the immunotoxins used to target and kill HLA-A201 melanoma MART- 1(+) and gpl 00(+) cell lines.

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Abstract

La présente invention concerne des éléments de liaison spécifiques, notamment des anticorps et des fragments de ceux-ci, qui se lient à des complexes peptide d'antigène tumoral NY-ESO-1/molécule du CMH, notamment d'une manière similaire aux récepteurs des cellules T avec une plus grande affinité que les récepteurs des cellules T reconnaissant les complexes peptides NY-ESO-1/HLA. Ces anticorps, en particulier les anticorps humains, sont utiles dans le diagnostic et le traitement du cancer, comprenant un mélanome, un sarcome pulmonaire, oesophagien, hépatique, gastrique, de la prostate, ovarien, de la vessie et synovial. Les anticorps de la présente invention et leurs fragments peuvent également être utilisés en thérapie en combinaison avec des agents chimiothérapeutiques, des modulateurs immunitaires, ou des agents anticancéreux et/ou avec d'autres anticorps ou fragments de ceux-ci. Des exemples des anticorps de ce type sont les nouveaux anticorps T1, T2 et T3 dont les séquences sont proposées dans le présent document.
EP10715349A 2009-03-20 2010-03-19 Anticorps anti-peptide ny-eso-1 de type récepteur des cellules t de haute affinité, procédés, et leurs utilisations Withdrawn EP2408819A2 (fr)

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EA201391159A1 (ru) * 2011-02-11 2014-02-28 Мемориал Слоан-Кеттеринг Кэнсер Сентер Hla-рестриктированные пептидоспецифические антигенсвязывающие белки
AU2014262282B2 (en) * 2011-02-11 2016-10-27 Memorial Sloan-Kettering Cancer Center HLA-restricted, peptide-specific antigen binding proteins
EP2971045B1 (fr) 2013-03-13 2019-06-19 Health Research, Inc. Compositions et procédés pour l'utilisation de récepteurs de lymphocytes t recombinants pour la reconnaissance directe d'un antigène tumoral
AU2016283133A1 (en) * 2015-06-24 2018-01-04 Eureka Therapeutics, Inc. Constructs targeting NY-ESO-1 peptide/MHC complexes and uses thereof
CN106632660B (zh) * 2015-11-04 2021-01-29 广东香雪精准医疗技术有限公司 识别ny-eso-1抗原短肽的tcr
CN107686522A (zh) * 2016-12-28 2018-02-13 天津天锐生物科技有限公司 一种识别hla‑a2/sllmwitqc的单域抗体
MA47265A (fr) 2017-01-13 2019-11-20 Agenus Inc Récepteurs de lymphocytes t qui se lient à ny-eso-1 et méthodes d'utilisation de ces derniers
CN107557338A (zh) * 2017-09-19 2018-01-09 深圳市北科生物科技有限公司 特异性识别ny‑eso‑1的t细胞及其与细胞因子的联合的应用
CN107502596A (zh) * 2017-09-19 2017-12-22 深圳市北科生物科技有限公司 表达ny‑eso‑1特异性tcr的t细胞及其应用
BR112021026376A2 (pt) 2019-06-25 2022-05-10 Gilead Sciences Inc Proteínas de fusão flt3l-fc e métodos de uso
MA56468A (fr) * 2019-07-03 2022-05-11 Regeneron Pharma Protéines de liaison à l'antigène de carcinome 1 à cellules squameuses de l'oesophage anti-new york (ny-eso-1) et leurs procédés d'utilisation
US11692038B2 (en) 2020-02-14 2023-07-04 Gilead Sciences, Inc. Antibodies that bind chemokine (C-C motif) receptor 8 (CCR8)
MX2022010751A (es) 2020-03-30 2022-09-23 Univ Mie Anticuerpo biespecifico.
TW202406932A (zh) 2020-10-22 2024-02-16 美商基利科學股份有限公司 介白素2-Fc融合蛋白及使用方法
TW202313094A (zh) 2021-05-18 2023-04-01 美商基利科學股份有限公司 使用ＦＬＴ３Ｌ—Ｆｃ融合蛋白之方法
US20230242508A1 (en) 2021-12-22 2023-08-03 Gilead Sciences, Inc. Ikaros zinc finger family degraders and uses thereof
US20240124412A1 (en) 2021-12-22 2024-04-18 Gilead Sciences, Inc. Ikaros zinc finger family degraders and uses thereof
TW202340168A (zh) 2022-01-28 2023-10-16 美商基利科學股份有限公司 Ｐａｒｐ７抑制劑
TW202346277A (zh) 2022-03-17 2023-12-01 美商基利科學股份有限公司 Ｉｋａｒｏｓ鋅指家族降解劑及其用途
TW202400138A (zh) 2022-04-21 2024-01-01 美商基利科學股份有限公司 Ｋｒａｓ ｇ１２ｄ調節化合物
US20240116928A1 (en) 2022-07-01 2024-04-11 Gilead Sciences, Inc. Cd73 compounds

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009026547A1 (fr) * 2007-08-22 2009-02-26 Receptor Logi, Ltd. Procédés d'analyse de l'activité de vaccin

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2010106431A2 *

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