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WO2019016784A1 - Anticorps anti-nucléoline - Google Patents

Anticorps anti-nucléoline Download PDF

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Publication number
WO2019016784A1
WO2019016784A1 PCT/IB2018/055471 IB2018055471W WO2019016784A1 WO 2019016784 A1 WO2019016784 A1 WO 2019016784A1 IB 2018055471 W IB2018055471 W IB 2018055471W WO 2019016784 A1 WO2019016784 A1 WO 2019016784A1
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Prior art keywords
antibody
nucleolin
antigen binding
binding fragment
cell
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PCT/IB2018/055471
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English (en)
Inventor
João Nuno SERENO DE ALMEIDA MOREIRA
Sofia Pereira CONSTANTINO ROMANO
Vera Lúcia DANTAS NUNES CALDEIRA DE MOURA
Sérgio Paulo DE MAGALHÃES SIMÕES
João Manuel BRAZ GONÇALVES
Original Assignee
Universidade De Coimbra
Centro De Neurociências E Biologia Celular Da Universidade De Coimbra
Faculdade De Farmácia Da Universidade De Lisboa
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Publication of WO2019016784A1 publication Critical patent/WO2019016784A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]

Definitions

  • Cancer is currently the second leading cause of death worldwide, only slightly surpassed by heart diseases. Surgery, radiotherapy and chemotherapy are the most common prescribed treatment modalities. The majority of cancer subtypes are associated with poor clinical outcomes owing to the ineffectiveness of treatments targeting metastases, the lack of tumor specificity and the development of drug resistance (Torre et al. 2015. A Cancer Journal for Clinicians., 65(2), pp.87-108).
  • Antibody-based therapy is considered a major breakthrough for better therapeutic outcomes in several types of tumors, from hematological malignancies to solid tumors.
  • Antibodies combine the capacity to promote cell death by mechanisms ranging from direct cell killing to immune -mediated cell death, with activity against specific targets, thus potentially decreasing the associated side effects (Scott et al. 2012. Nature reviews. Cancer, 12(4), pp.278- 87).
  • the disclosure relates to antibodies targeting the N-terminal domain of nucleolin, including antibodies comprising part or all of the F3 peptide, and having ADCC activity, for example, by including the Fc region of IgGl .
  • the disclosure relates to methods for treating conditions associated with cell-surface expressed nucleolin, e.g., cancer, by administration of said antibodies.
  • the disclosure is based, in part, on the surprising discovery that antibodies targeting the N-terminal domain of nucleolin have cytotoxic activity against nucleolin- overexpressing cells, including cancer cells, and that when said antibodies comprise the Fc region of IgGl, the antibodies have effective ADCC activity against cancer cells.
  • antibodies or antigen binding fragments thereof that (i) specifically bind the N-terminal domain of nucleolin; and (ii) promote antibody dependent cellular cytotoxicity (ADCC) towards a cell expressing nucleolin.
  • the antibodies or antigen binding fragments comprise an F3 peptide.
  • the F3 peptide comprises 8-31 contiguous amino acids of F3.
  • the F3 peptide comprises residues 5-14 of F3.
  • the antibodies or antigen binding fragments comprise a variable domain comprising a CDRl, a CDR2, and a CDR3.
  • one or more of CDRl, CDR2, and CDR3 comprises the F3 peptide.
  • the variable domain comprises a CDRl, a CDR2, and a CDR3 comprising the F3 peptide.
  • the variable domain comprises a CDRl, a CDR2, and the F3 peptide substituted for CDR3.
  • the N-terminal domain of nucleolin comprises amino acids 1-283 of nucleolin. In some embodiments, the antibodies or antigen binding fragments specifically bind amino acids 1-283 of the N-terminal domain of nucleolin. In some embodiments, the antibodies or antigen binding fragments specifically bind amino acids 43-51 of the N-terminal domain of nucleolin. In some embodiments, the antibodies or antigen binding fragments specifically bind amino acids 221-233 of the N-terminal domain of nucleolin.
  • the antibodies or antigen binding fragments are human or humanized monoclonal antibodies. In some embodiments, the antibodies are full length antibodies. In some embodiments, the antibodies comprise an IgGl Fc domain. In some embodiments, the antigen binding fragments are Fab, Fab', F(ab')2, Fv, a VHH, or scFv. In some embodiments, the antigen binding fragments comprise scFv-Fc. In some embodiments, the antigen binding fragments comprise VHH-Fc. In some embodiments, the Fc domain is an IgGl Fc domain.
  • the Fc domain comprises one or more modifications to increase ADCC.
  • the one or more modifications comprises one or more of L234Y, S239D, T256A, K290A, K290Y, Y296W, S298A, A330F, A330L, I332E, E333A, K334A, K334V, A339T, E356K, K392D, D339K and K409D.
  • the one or more modifications comprises the level of Fc-bound carbohydrate structure, including, but not limited to, alterations at the level of fructose, galactose, mannose, bisecting sugars and/or sialic acid.
  • the antibodies are bispecific antibodies, and wherein the antibodies specifically bind a molecule present on the surface of immune cells.
  • the molecule present on the surface of immune cells comprises MHC class I or MHC class II proteins, T cell receptors, B cell receptors, CD28, ICOS, TLT2, CD27, CD 137, OX40, HVEM, DR3, NKG2D, TIM-1, TIM-2, DN AM- 1 , CRTAM, CTLA-4, PD-1, PD-L1, PD-L2, CXCR4, CD3, B7-1, B7-2, BTLA, CD 160, LAG-3, TIM-3, TIGIT, LAIR-1, CAR, CD40, GITR, BAFF-R, TACI, BCMA, CD72, CD22, CD96, 2B4, NTB-A, CRACC, Siglec- 3.7.9, KLRG1, NKR-P1A, ILT2, KIR2DL1, KIR2DL2, KIR
  • an isolated nucleic acid or a set of nucleic acids which collectively encode any of the anti-nucleolin antibodies or antibody fragments described herein, a vector or vector set (e.g., expression vectors) that comprise the nucleic acid or the set of nucleic acids, and a host cell or host cell set that comprises the vector or vector set.
  • a vector or vector set e.g., expression vectors
  • Exemplary host cells include, but are not limited to, bacterial cells, yeast cells, insect cells, plant cells, and mammalian cells.
  • compositions comprising any of the anti-nucleolin antibodies or antibody fragments described herein and a pharmaceutically acceptable carrier.
  • Such pharmaceutical compositions can be for use in treating a disease associated with cell-surface localized nucleolin or for treating cancer.
  • provided herein are methods for treating a disease associated with cell- surface localized nucleolin.
  • the method comprises administering a therapeutically effective amount of any of the anti-nucleolin antibodies or antibody fragments described herein to a subject in need thereof.
  • the disease is cancer.
  • the cancer is a solid tumor forming cancer.
  • the subject is administered a treatment for cancer.
  • the treatment for cancer is a chemotherapy, a radiation therapy, or an
  • FIG. 1 is a schematic representation of a cloning process used to generate the novel anti-nucleolin small antibody formats (VHHs), with indication of the restriction sites of Hindlll and Bglll, engrafted sequence and linkers, and the regions of primer hybridization.
  • VHHs novel anti-nucleolin small antibody formats
  • Figures 2A-2J are graphical representations of the parental VHH and the novel anti- nucleolin VHH constructions, highlighting the leader peptide sequence, histidine tag (used for protein purification) and HA tag (used for protein detection) as well as CDR1 and CDR3 regions, in which the nucleolin-binding sequence was grafted.
  • Figures 2A and 2B the nucleotide sequence of the parental VHH and its amino acid sequence are presented, respectively.
  • Figures 2C-2J the nucleotide and amino acid sequences of each one of the four novel anti-nucleolin VHHs are presented.
  • Figures 3A and 3B are graphs showing the binding of 100 pmol of the VHH fragments to human recombinant nucleolin and human recombinant TNF-a, respectively, following 1 h incubation at 37°C, as evaluated by ELISA. The results are from a representative experiment.
  • Figures 4A-4F illustrate the binding of the generated VHH fragments to nucleolin- overexpressing cells, following incubation for 45 min at 4°C, as evaluated by flow cytometry with an anti-HA-FITC antibody.
  • Figures 4A-4C show the binding of anti-nucleolin VHH (aNCL-CDRl , aNCL-CDRl -L, aNCL-CDR3 and aNCL-CDR3-L) to nucleolin-overexpressing MDA-MB-435S ( Figure 4A), MDA-MD-231 ( Figure 4B) and 4T1 ( Figure 4C) cell lines. Parental VHH without the F3 peptide graft was also tested.
  • Figures 4D-4F show competitive inhibition assays, upon pre-incubation of the nucleolin-overexpressing MDA-MB-435S (Figure 4D), MDA-MD-231 ( Figure 4E) and 4T1 ( Figure 4F) cell lines with 75 ⁇ F3 peptide or 1 ⁇ infliximab, for 30 min at 4°C. An additional control, without pre-incubation of a ligand, was also performed. Data represents the mean ⁇ SD of three independent experiments. One-way ANOVA followed by Tukey's Multiple Comparison Test was performed to evaluate the differences in binding among the five VHH fragments (*p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001). For each protein, differences in the competitive inhibition assays were evaluated by Student's t-test (*p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001).
  • Figures 5A-5C are graphs showing the cytotoxicity of the VHH fragments against the nucleolin-overexpressing MDA-MB-435S (Figure 5A), MDA-MD-231 ( Figure 5B) and 4T1 ( Figure 5C) cell lines, upon incubation at 37°C for 72 h, as determined by the 3-(4,5- Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay.
  • Data represents the mean ⁇ SD of at least three independent experiments.
  • One-way ANOVA followed by Tukey's Multiple Comparison Test was performed to evaluate the differences in cytotoxicity among the four anti-nucleolin VHH fragments tested, along with the parental VHH fragment (*p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001).
  • Figures 6A-6B are graphical representations of the nucleic acid ( Figure 6A) and amino acid ( Figure 6B) sequences of anti-nucleolin VHH-Fc, highlighting the IL2 signaling sequence, the anti-nucleolin VHH, and the Fc regions.
  • Figures 7A-7C are graphs showing the cytotoxicity of anti-nucleolin and parental VHH- Fc antibody against the nucleolin-overexpressing MDA-MB-435S ( Figure 7A), MDA-MD-231 ( Figure 7B) and 4T1 ( Figure 7C) cell lines, after an incubation at 37°C for 72 h, as determined by the MTT assay. Data represents the mean ⁇ SD of three independent experiments. Student's t-test was performed to evaluate the differences in cytotoxicity between the proteins (*p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001).
  • Figures 8A and 8B are graphs showing the cytotoxic effect of anti-nucleolin and parental VHH-Fc antibody against the nucleolin-overexpressing MDA-MB-435S cell line, with or without effector cells (PBMCs), as evaluated by cell impedance using xCELLigence technology.
  • the cytotoxic effect of the anti-nucleolin VHH-Fc was assessed relative to the parental VHH-Fc ( Figure 8A) or the anti-nucleolin VHH ( Figure 8B) constructs.
  • the present disclosure in one aspect, relates to the surprising discovery that antibodies targeting the N-terminal domain of nucleolin have cytotoxicity against nucleolin-overexpressing cells, including different sub-populations of tumor cells, such as cancer cells, cancer stem cells and endothelial cells from tumor blood vessels.
  • tumor cells such as cancer cells, cancer stem cells and endothelial cells from tumor blood vessels.
  • the resulting anti-nucleolin antibody triggers ADCC responses against these nucleolin- overexpressing cells.
  • the present disclosure provides antibodies to the N-terminal domain of nucleolin, e.g., antibodies comprising the F3 peptide, and having ADCC activity.
  • the present disclosure provides methods for treating conditions associated with cell- surface expressed nucleolin, e.g., cancer, by administration of antibodies to the N-terminal domain of nucleolin, e.g., antibodies comprising the F3 peptide.
  • Nucleolin is a 76.6 kDa protein involved in the synthesis and maturation of ribosomes.
  • Nucleolin has a 3 -component structure: an N-terminal domain with acidic stretches, which is involved in several protein-protein interactions, and which controls rDNA transcription; a central globular domain, with four RNA-binding domains, which is involved in pre-RNA processing; and a C-terminal domain, constituted by arginine-glycine-glycine repeats, which interacts with ribosomal proteins (Srivastava & Pollard 1999. The FASEB Journal, 13, pp.191 1- 1922).
  • Nucleolin is located mainly in dense fibrillar regions of the nucleolus. Although nucleolin is typically present in the nucleus, including in the nucleolis, expression increases and nucleolin is translocated to the cell surface in highly proliferating cells, such as cancer cells (Srivastava & Pollard 1999). Importantly, this overexpression at the cell surface is also observed in endothelial cells from angiogenic blood vessels, which play a central role in tumor growth and progression (Christian et al. 2003. The Journal of Cell Biology, 163(4), pp.871- 878). As such, cell-surface localized nucleolin serves as a marker for oncogenesis and potentially as a means of targeting cancerous cells.
  • cell surface nucleolin represents less than 10% of nuclear nucleolin (Hovanessian et al. 2010) and undergoes a rapid turnover, with an estimated half-life of less than one hour, compared to more than eight hours in the nucleus (Hovanessian et al. 2010).
  • amino acid sequence of nucleolin is as follows:
  • Nucleolin has previously been targeted with peptides and pseudopeptides.
  • the nucleolin-binding F3 peptide is a 31 -amino acid peptide that binds to the N-terminal domain of nucleolin (Christian et al. 2003 The Journal of Cell Biology, 163(4), pp.871-878).
  • F3 is able to be internalized and has been used as a targeting ligand of nanoparticles aiming at the delivery of small weight drugs, such as doxorubicin (Moura et al. 2012. Breast Cancer Research and Treatment, 133(1), pp.61-73), paclitaxel (Hu et al. 2013.
  • the nucleolin binding- aptamer AS141 has anti-tumorigenic activity (Bates et al. 2009. Experimental and Molecular Pathology, 86(3), pp.151-64), and has also been used as a targeting ligand of nanoparticles containing siRNA (Li et al. 2014. Biomaterials, 35(12), pp.3840-3850) or small weight drugs (Guo et al. 201 1. Biomaterials, 32(31), pp.8010-8020; Z. Li et al. 2012. Biomacromolecules, 13, pp.4257-4263; Song et al. 2013. Molecular Pharmaceutics, 10(10), pp.3555-3563; Latorre et al. 2014. Nanoscale, 6, pp.7436-7442; Ai et al. 2014. Talanta, 1 18, pp.54-60), towards nucleolin-overexpressing cells.
  • N6L Intraperitoneal administration of N6L led to tumor growth inhibition of orthotopic models of breast and prostate cancer, derived from MDA-MB-231 and PC3 cell lines, respectively, as well as to increased survival in A20- and T29-derived lymphoma models when administered by intravenous injection (Destouches et al. 201 1). Inhibition of angiogenesis, was also provided.
  • nucleolin antibodies including antibodies that target the RNA- binding domains of the central globular domain of nucleolin or the C-terminal domain, have not demonstrated ADCC activity. (Palmieri, D. et al., 2015. Proceedings of the National Academy of Sciences of the United States of America, 1 12(30), pp.9418-23; WO201 1062997).
  • NCL3 A rabbit nucleolin-specific antibody (NCL3), which binds the N-terminal domain of nucleolin (Christian et al. 2003. The Journal of Cell Biology, 163(4), pp.871-878), was developed. No ADCC activity was reported.
  • a nucleolin-targeting single-chain Fragment variable (scFv) antibody, 4LB5 has also been developed, against the central domain of the protein. Once again, no ADCC activity was reported (Palmieri et al. 2015).
  • the antibodies presented herein provide for improved anti-cancer cell cytotoxicity by targeting the N-terminal domain of nucleolin and by triggering ADCC activity.
  • ADCC response depends on the availability of a target protein at the cell surface. This determines the extent of antibody binding and the level of binding of the antibody to the Fc receptor of NK cells through the Fc domain, which triggers ADCC. This is supported by the demonstration antibodies engineered against the same target having lower internalization rates have the strongest ADCC response (Yang et al. PLoS ONE, 201 1, 6(6); Vasu et al. 2016 Blood, 127(23), pp.2879-2889). In addition, strategies to block antibody internalization resulted in improved ADCC response (WO2014063205 Al ; Roghanian et al. 2015).
  • N-terminal domain of nucleolin is believed to have greater exposure to the cell surface than the central globular domain of nucleolin or the C-terminal domain. Accordingly, without wishing to be bound by theory, it is believed that anti-nucleolin N-terminal domain antibodies having Fc domains have optimal ADCC activity because of the cell surface exposure.
  • the present disclosure provides antibodies that bind nucleolin, for example, the N- terminal domain of nucleolin.
  • antibody molecule refers to a protein comprising at least one immunoglobulin variable domain sequence.
  • the term antibody molecule includes, for example, full-length, mature antibodies and antigen-binding fragments of an antibody.
  • an antibody molecule can include a heavy (H) chain variable domain sequence (abbreviated herein as VH), and a light (L) chain variable domain sequence (abbreviated herein as VL).
  • an antibody molecule in another example, includes two heavy (H) chain variable domain sequences and two light (L) chain variable domain sequence, thereby forming two antigen binding sites, such as Fab, Fab' , F(ab')2, Fc, Fd, Fd', Fv, VHH, scFv-Fc, VHH-Fc, single chain antibodies (scFv for example), single variable domain antibodies, diabodies (Dab) (bivalent and bispecific), and chimeric (e.g., humanized) antibodies, which may be produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA technologies.
  • These functional antibody fragments retain the ability to selectively bind with their respective antigen or receptor.
  • Antibodies and antibody fragments can be from any class of antibodies including, but not limited to, IgG, IgA, IgM, IgD, and IgE, and from any subclass (e.g., IgGl, IgG2, IgG3, and IgG4) of antibodies.
  • the antibodies can be monoclonal or polyclonal.
  • the antibody can also be a human, humanized, CDR-grafted, or in vitro generated antibody.
  • the antibody can have a heavy chain constant region chosen from, e.g., IgGl, IgG2, IgG3, or IgG4.
  • the antibody can also have a light chain chosen from, e.g., kappa or lambda.
  • the antibody can be a whole antibody.
  • antigen-binding fragments include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a diabody (dAb) fragment, which consists of a VH domain; (vi) a camelid or camelized variable domain; (vii) a single chain Fv (scFv), see e.g., Bird et al.
  • a Fab fragment a monovalent fragment consisting of the VL, VH, CL and CHI domains
  • a F(ab')2 fragment a bivalent fragment comprising two Fab fragments linked by a
  • antibody includes intact molecules as well as functional fragments thereof. Constant regions of the antibodies can be altered, e.g., mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of: Fc receptor binding, antibody
  • glycosylation the number of cysteine residues, effector cell function, or complement function.
  • the antibodies disclosed herein can also be single domain antibodies.
  • Single domain antibodies can include antibodies whose complementary determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies.
  • Single domain antibodies may be any of the art, or any future single domain antibodies.
  • Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, fish, shark, goat, rabbit, and bovine.
  • a single domain antibody is a naturally occurring single domain antibody known as heavy chain antibody devoid of light chains. Such single domain antibodies are disclosed in WO 9404678, for example.
  • VHH variable domain derived from a heavy chain antibody naturally devoid of light chain
  • a VHH or nanobody to distinguish it from the conventional VH of four chain immunoglobulins.
  • Such a VHH molecule can be derived from antibodies raised in Camelidae species, for example in camel, llama, dromedary, alpaca and guanaco. Other species besides Camelidae may produce heavy chain antibodies naturally devoid of light chain; such VHHs are also contemplated.
  • VHH molecules are about 1 Ox smaller than IgG molecules. They are single polypeptides and very stable, resisting extreme pH and temperature conditions. Moreover, they are resistant to the action of proteases which is not the case for conventional antibodies.
  • VHHs in vitro expression of VHHs produces high yield, properly folded functional VHHs.
  • antibodies generated in Camelids will recognize epitopes other than those recognised by antibodies generated in vitro through the use of antibody libraries or via immunisation of mammals other than Camelids (WO 9749805).
  • anti- albumin VHH's may interact in a more efficient way with serum albumin which is known to be a carrier protein.
  • serum albumin which is known to be a carrier protein.
  • serum albumin As a carrier protein some of the epitopes of serum albumin may be inaccessible by bound proteins, peptides and small chemical compounds.
  • the VHH comprises an Fc domain.
  • the Fc comprises CH2 and CH3.
  • the VH and VL regions can be subdivided into regions of hypervariability, termed “complementarity determining regions” (CDR), interspersed with regions that are more conserved, termed “framework regions” (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • the extent of the framework region and CDRs has been precisely defined by a number of methods (see, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917; and the AbM definition used by Oxford Molecular's AbM antibody modeling software.
  • Each VH and VL typically includes three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • an "immunoglobulin variable domain sequence” refers to an amino acid sequence which can form the structure of an immunoglobulin variable domain.
  • the sequence may include all or part of the amino acid sequence of a naturally-occurring variable domain.
  • the sequence may or may not include one, two, or more N- or C-terminal amino acids, or may include other alterations that are compatible with formation of the protein structure.
  • antigen-binding region refers to the part of an antibody molecule that comprises determinants that form an interface that binds to nucleolin, or an epitope thereof.
  • the antigen-binding region typically includes one or more loops (of at least, e.g., four amino acids or amino acid mimics) that form an interface that binds to nucleolin.
  • the antigen-binding region of an antibody molecule includes at least one or two CDRs, or more typically at least three, four, five or six CDRs.
  • monoclonal antibody or “monoclonal antibody composition” as used herein refer to a preparation of antibody molecules of single molecular composition.
  • a monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.
  • a monoclonal antibody can be made by hybridoma technology or by methods that do not use hybridoma technology (e.g., recombinant methods).
  • the antibodies described herein can be murine, rat, human, or any other origin (including chimeric or humanized antibodies). Such antibodies are non-naturally occurring, i.e., would not be produced in an animal without human act (e.g., immunizing such an animal with a desired antigen or fragment thereof).
  • any of the antibodies described herein can be either monoclonal or polyclonal.
  • a “monoclonal antibody” refers to a homogenous antibody population and a “polyclonal antibody” refers to a heterogeneous antibody population. These two terms do not limit the source of an antibody or the manner in which it is made.
  • humanized antibodies refer to forms of non-human (e.g. murine) antibodies that are specific chimeric immunoglobulins, immunoglobulin chains, or antigen-binding fragments thereof that contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity.
  • CDR complementary determining region
  • Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • the humanized antibody may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin.
  • Antibodies may have Fc regions modified as described in WO 99/58572.
  • Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, six) which are altered with respect to the original antibody, which are also termed one or more CDRs "derived from" one or more CDRs from the original antibody.
  • Humanized antibodies may also involve affinity maturation.
  • the antibody described herein is a chimeric antibody, which can include a heavy constant region and a light constant region from a human antibody.
  • Chimeric antibodies refer to antibodies having a variable region or part of variable region from a first species and a constant region from a second species.
  • the variable region of both light and heavy chains mimics the variable regions of antibodies derived from one species of mammals (e.g., a non-human mammal such as mouse, rabbit, and rat), while the constant portions are homologous to the sequences in antibodies derived from another mammal such as human.
  • amino acid modifications can be made in the variable region and/or the constant region.
  • the anti-nucleolin antibodies described herein specifically bind to the corresponding target antigen or an epitope thereof.
  • An antibody that "specifically binds" to an antigen or an epitope is a term well understood in the art. A molecule is said to exhibit “specific binding” if it reacts more frequently, more rapidly, with greater duration and/or with greater affinity with a particular target antigen than it does with alternative targets.
  • An antibody “specifically binds" to a target antigen or epitope if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other substances.
  • an antibody that specifically (or preferentially) binds to an antigen (nucleolin, e.g., the N-terminal domain of nucleolin) or an antigenic epitope therein is an antibody that binds this target antigen with greater affinity, avidity, more readily, and/or with greater duration than it binds to other antigens or other epitopes in the same antigen. It is also understood with this definition that, for example, an antibody that specifically binds to a first target antigen may or may not specifically or preferentially bind to a second target antigen. As such, “specific binding” or “preferential binding” does not necessarily require (although it can include) exclusive binding.
  • an antibody that "specifically binds" to a target antigen or an epitope thereof may not bind to other antigens or other epitopes in the same antigen.
  • the antibodies described herein specifically bind to nucleolin.
  • the antibodies described herein specifically bind to the N-terminal domain of nucleolin, e.g., amino acids 1-283 of nucleolin.
  • the antibodies described herein specifically bind to amino acids 43-51 of the N-terminal domain of nucleolin.
  • the antibodies described herein specifically bind to amino acids 221-233 of the N-terminal domain of nucleolin.
  • the antibody binds nucleolin with sufficient specificity such that is can distinguish nucleolin, in vivo, adequately to yield a treatment that is without off-target effects that would undermine its ability to perform as a therapeutic.
  • an anti-nucleolin antibody as described herein has a suitable binding affinity for the target antigen ⁇ e.g., nucleolin) or antigenic epitopes thereof.
  • binding affinity refers to the apparent association constant or KA.
  • the KA is the reciprocal of the dissociation constant (KD).
  • the anti-nucleolin antibody described herein may have a binding affinity (KD) of at least 10 "5 , 10 "6 , 10 "7 , 10 "8 , 10 "9 , 10 10 M, or lower for the target antigen or antigenic epitope.
  • An increased binding affinity corresponds to a decreased KD.
  • any of the anti-nucleolin antibodies may be further affinity matured to increase the binding affinity of the antibody to the target antigen or antigenic epitope thereof.
  • Binding affinity can be determined by a variety of methods including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (e.g., using a fluorescence assay).
  • Exemplary conditions for evaluating binding affinity are in HBS-P buffer (10 mM HEPES pH7.4, 150 mM NaCl, 0.005% (v/v) Surfactant P20). These techniques can be used to measure the concentration of bound binding protein as a function of target protein concentration.
  • the concentration of bound binding protein [Bound] is generally related to the concentration of free target protein ([Free]) by the following equation:
  • [Bound] [Free]/(Kd+[Free]) It is not always necessary to make an exact determination of KA, though, since sometimes it is sufficient to obtain a quantitative measurement of affinity, e.g., determined using a method such as ELISA or FACS analysis, is proportional to KA, and thus can be used for comparisons, such as determining whether a higher affinity is, e.g., 2-fold higher, to obtain a qualitative measurement of affinity, or to obtain an inference of affinity, e.g., by activity in a functional assay, e.g., an in vitro or in vivo assay.
  • a functional assay e.g., an in vitro or in vivo assay.
  • the anti-nucleolin antibody comprises a F3 peptide.
  • the F3 peptide comprises 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 contiguous amino acids of F3. In some embodiments, the F3 peptide comprises 7- 12, 8-1 1, or 9-10 contiguous amino acids of F3. In some embodiments, the F3 peptide comprises residues 3 to 11, 3 to 12, or 3 to 13 of F3. In some embodiments, the F3 peptide comprises residues 4 to 12, 4 to 13, or 4 to 14 of F3. In some embodiments, the F3 peptide comprises residues 5 to 13, 5 to 14, or 5 to 15 of F3. In some embodiments, the F3 peptide comprises residues 6 to 14, 6 to 15, or 6 to 16 of F3.
  • the F3 peptide comprises residues 7 to 15, 7 to 16, or 7 to 17 of F3. In some embodiments, the F3 peptide comprises residues 5 to 14 of F3. In some embodiments, the F3 peptide comprises the sequence PQRRSARLSA (SEQ ID NO: 7).
  • the F3 peptide is 80, 82, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.9 or 100% identical to the corresponding sequence of F3 (SEQ ID NO: 2).
  • the F3 peptide comprises a sequence with 1, 2, or 3 substitutions relative to the corresponding sequence of F3.
  • the F3 peptide comprises the nucleic acid sequence of SEQ ID NO: 7.
  • the F3 peptide further comprises a linker.
  • the linker comprises the sequence SGGGS (SEQ ID NO: 3).
  • the linker is N-terminal to the sequence of F3 in the F3 peptide.
  • the linker is C- terminal to the sequence of F3 in the F3 peptide.
  • the F3 peptide comprises a linker at both the N-terminus and the C-terminus.
  • the F3 peptide comprises the nucleic acid sequence of SEQ ID NO: 8. In some embodiments, the F3 peptide is 80, 82, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.9 or 100% identical to SEQ ID NO: 8. In some embodiments, the F3 peptide comprises a sequence with 1, 2, or 3 substitutions relative to SEQ ID NO: 8. Any of the anti-nucleolin antibodies described herein may comprise a heavy chain that includes a heavy chain variable region and optionally, a heavy chain constant region.
  • anti-nucleolin antibodies described herein may comprise a light chain that includes a light chain variable region and optionally, a light chain constant region. In some embodiments, anti-nucleolin antibodies described herein may comprise a heavy chain and a light chain. In some embodiments, anti-nucleolin antibodies described herein may comprise a VHH chain that includes a VHH chain variable region and optionally, a Fc domain.
  • the anti-nucleolin antibody comprises a CDRl, a CDR2, and a CDR3. In some embodiments, the anti-nucleolin antibody comprises a heavy chain comprising a heavy chain variable region that comprises a heavy chain CDRl, a heavy chain CDR2, and/or a heavy chain CDR3. In some embodiments, the anti-nucleolin antibody comprises a light chain comprising a light chain variable region that comprises a light chain CDRl, a light chain CDR2, and/or a light chain CDR3. In some embodiments, the anti-nucleolin antibody comprises a VHH comprising a VHH variable region that comprises a VHH CDRl, a VHH CDR2, and/or a VHH CDR3.
  • CDRl, CDR2, and/or CDR3 comprises an F3 peptide. In some embodiments, CDRl comprises an F3 peptide. In some embodiments, CDR2 comprises an F3 peptide. In some embodiments, CDR3 comprises an F3 peptide. In some embodiments, CDRl and CDR2 comprises an F3 peptide. In some embodiments, CDRl and CDR3 comprises an F3 peptide. In some embodiments, CDR2 and CDR3 comprises an F3 peptide. In some embodiments, CDRl, CDR2, and CDR3 comprises an F3 peptide.
  • CDRl, CDR2, and/or CDR3 are substituted for an F3 peptide. In some embodiments, CDRl is substituted for an F3 peptide. In some embodiments, CDR2 is substituted for an F3 peptide. In some embodiments, CDR3 is substituted for an F3 peptide. In some embodiments, CDRl and CDR2 are substituted for an F3 peptide. In some embodiments, CDRl and CDR3 are substituted for an F3 peptide. In some embodiments, CDR2 and CDR3 are substituted for an F3 peptide. In some embodiments, CDRl, CDR2, and CDR3 are substituted for an F3 peptide.
  • CDR1, CDR2, and/or CDR3 comprises or are substituted for an F3 peptide and the CDRs that do not comprise F3 or are not substituted for F3 target an antigen other than nucleolin, e.g., TNF-a.
  • the anti-nucleolin antibody comprises an F3 peptide flanked by FW (framework) 1 and FW2. In some embodiments, the anti-nucleolin antibody comprises an F3 peptide flanked by FW2 and FW3. In some embodiments, the anti-nucleolin antibody comprises an F3 peptide flanked by FW3 and FW4.
  • the CDR1 region comprises the amino acid sequence of SEQ ID NO: 4, 7, or 8.
  • the CDR2 region comprises the amino acid sequence of SEQ ID NO: 5, 7, or 8.
  • the CDR3 region comprises the amino acid sequence of SEQ ID NO: 6, 7, or 8.
  • the variable region of an anti-nucleolin antibody as described herein comprises the amino acid sequence of any of SEQ ID NOs: 15- 18.
  • the variable region of an anti-nucleolin antibody as described herein comprises the nucleic acid sequence of any of SEQ ID NOs: 1 1-14.
  • the anti-nucleolin antibody as described herein comprises the amino acid sequence of SEQ ID NO: 32.
  • the anti-nucleolin antibody as described herein comprises the nucleic acid sequence of SEQ ID NO: 31.
  • the anti-nucleolin antibody comprises chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) identical to any one of the CDR sequence provided by SEQ ID NO 4-8. In some embodiments, the anti-nucleolin antibody comprises a variable region that is at least 80% (e.g., 85%, 90%, 95%, or 98%) identical to the variable region of any of SEQ ID NO: 15-18.
  • Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997.
  • the default parameters of the respective programs e.g., XBLAST and NBLAST.
  • the anti-nucleolin antibody is a functional variant of an antibody comprising a VHH variable region provided by any one of SEQ ID NO: 15- 18.
  • a functional variant can comprise up to 5 (e.g., 4, 3, 2, or 1) amino acid residue variations in one or more of the CDR regions of the antibody that comprise the F3 peptide and binds the same epitope of nucleolin with substantially similar affinity (e.g., having a KD value in the same order).
  • the amino acid residue variations are conservative amino acid residue substitutions.
  • a "conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made.
  • Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York.
  • Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.
  • the heavy chain constant region is responsible for the prolonged serum half-life of the antibody, upon interacting with the neonatal Fc receptor (FcRn), which transports the antibody within and across cells, thus preventing its degradation.
  • the heavy chain constant region also plays a central role in mediating different types of cell death, such as antibody- dependent cell-mediated cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC) (Brekke & Sandlie 2002. Nature Reviews Drug Discovery, 2(1), pp.52-62).
  • the antibody molecule has a heavy chain constant region chosen from, e.g., the heavy chain constant regions of IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE; particularly, chosen from, e.g., the (e.g., human) heavy chain constant regions of IgGl, IgG2, IgG3, and IgG4.
  • the anti-nucleolin antibodies as described herein comprise a portion (e.g., CHI , CH2, CH3, or a combination thereof) of a heavy chain constant region.
  • anti-nucleolin antibodies as described herein comprise Fc, e.g., an IgGl Fc, which is CH2 and CH3 of a heavy chain constant region.
  • the antibody molecule has a light chain constant region chosen from, e.g., the (e.g., human) light chain constant regions of kappa or lambda.
  • the heavy and light chain constant regions can of any suitable origin, e.g., human, mouse, rat, or rabbit.
  • the constant region can be altered, e.g., mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of: Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, and/or complement function).
  • the antibody has effector function and can fix complement. In other embodiments the antibody does not recruit effector cells or fix complement.
  • the antibody constant region is altered in some embodiments.
  • Methods for altering an antibody constant region are known in the art.
  • Antibodies with altered function e.g. altered affinity for an effector ligand, such as FcR on a cell, or the CI component of complement can be produced by replacing at least one amino acid residue in the constant portion of the antibody with a different residue (see e.g., EP 388, 151 Al, U.S. Pat. No. 5,624,821 and U.S. Pat. No. 5,648,260, the contents of all of which are hereby incorporated by reference).
  • Amino acid mutations which stabilize antibody structure such as S228P (EU nomenclature, S241P in Kabat nomenclature) in human IgG4 are also contemplated. Similar type of alterations could be described which if applied to the murine, or other species immunoglobulin would reduce or eliminate these functions.
  • the antibody that specifically binds to nucleolin described herein binds an epitope that comprises the following segments of SEQ ID NO: 1 : residues 1-283, residues 43-51, and/or residues 221-233.
  • the anti-nucleolin antibody molecule can be used alone in unconjugated form, or can be bound to a substance, e.g., a toxin or moiety (e.g., a therapeutic drug; a compound emitting radiation; molecules of plant, fungal, or bacterial origin; or a biological protein or particle.
  • a substance e.g., a toxin or moiety
  • the anti-nucleolin antibody can be coupled to a radioactive isotope such as an ⁇ -, ⁇ -, or ⁇ -emitter, or a ⁇ -and ⁇ -emitter.
  • An antibody molecule can be derivatized or linked to another functional molecule (e.g., another peptide or protein).
  • a "derivatized" antibody molecule is one that has been modified.
  • Methods of derivatization include but are not limited to the addition of a fluorescent moiety, a radionucleotide, a toxin, an enzyme or an affinity ligand such as biotin. Accordingly, the antibody molecules are intended to include derivatized and otherwise modified forms of the antibodies described herein, including immunoadhesion molecules.
  • an antibody molecule can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a diabody), a detectable agent, a toxin, a pharmaceutical agent, and/or a protein or peptide that can mediate association of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).
  • another antibody e.g., a bispecific antibody or a diabody
  • detectable agent e.g., a toxin, a pharmaceutical agent, and/or a protein or peptide that can mediate association of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).
  • Exemplary fluorescent detectable agents include fluorescein, fluorescein isothiocyanate, rhodamine, 5 dimethylamine-l-napthalenesulfonyl chloride, phycoerythrin and the like.
  • An antibody may also be derivatized with detectable enzymes, such as alkaline phosphatase, horseradish peroxidase, ⁇ - galactosidase, acetylcholinesterase, glucose oxidase and the like.
  • detectable enzymes such as alkaline phosphatase, horseradish peroxidase, ⁇ - galactosidase, acetylcholinesterase, glucose oxidase and the like.
  • detectable enzymes such as alkaline phosphatase, horseradish peroxidase, ⁇ - galactosidase, acetylcholinesterase, glucose oxidase and the like.
  • an antibody is derivatized with a detectable enzyme, it is detected by adding additional reagents that the enzyme uses to produce a detectable reaction product.
  • the detectable agent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine leads to a
  • an antibody may be derivatized with biotin, and detected through indirect measurement of avidin or streptavidin binding.
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; and examples of bioluminescent materials include luciferase, luciferin, and aequorin.
  • Labeled antibody molecule can be used, for example, diagnostically and/or experimentally in a number of contexts, including (i) to isolate a predetermined antigen by standard techniques, such as affinity chromatography or immunoprecipitation; (ii) to detect a predetermined antigen (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the protein; (iii) to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen.
  • standard techniques such as affinity chromatography or immunoprecipitation
  • detect a predetermined antigen e.g., in a cellular lysate or cell supernatant
  • a predetermined antigen e.g., in a cellular lysate or cell supernatant
  • Some types of derivatized antibody molecule are produced by crosslinking two or more antibodies (of the same type or of different types, e.g., to create bispecific antibodies).
  • Suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate).
  • Such linkers are available from Pierce Chemical Company, Rockford, IL.
  • an antibody or antigen fragment thereof, as is described herein, is bispecific.
  • the bispecific antibody or antigen fragment specifically binds nucleolin and a second antigen.
  • the second antigen is a molecule present on the surface of immune cells.
  • the molecule present on the surface of immune cells comprises MHC class I or MHC class II proteins, T cell receptors, B cell receptors, CD28, ICOS, TLT2, CD27, CD 137, OX40, HVEM, DR3, NKG2D, TIM- 1, TIM-2, DNAM-1, CRTAM, CTLA-4, PD-1, PD-Ll, PD-L2, CXCR4, CD3, B7-1, B7-2, BTLA, CD160, LAG-3, TIM-3, TIGIT, LAIR- 1, CAR, CD40, GITR, BAFF-R, TACI, BCMA, CD72, CD22, CD96, 2B4, NTB-A, CRACC, Siglec-3.7.9, KLRG1, NKR-P1A, ILT2, KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, KIR3DL2, CD94-NKG2A, CEACAMl, Plexin-Al, Plex
  • An antibody molecule may be conjugated to another molecular entity, typically a label or a therapeutic (e.g., immunomodulatory, immunostimularoty, cytotoxic, or cytostatic) agent or moiety.
  • Radioactive isotopes can be used in diagnostic or therapeutic applications. Radioactive isotopes that can be coupled to the anti-nucleolin antibodies include, but are not limited to ⁇ -, ⁇ -, or ⁇ -emitters, or ⁇ -and ⁇ -emitters.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • FcyRIIa and FcYRIIIa/CD16a are low-affinity activating receptors for IgGl Fc and are expressed by subpopulations of NK cells, macrophages, and T cells. Polymorphisms in these genes augment the affinity of the IgGl Fc region towards the receptor and correlate with better clinical responses, when compared with the responses in the cohort without the polymorphisms.
  • non-Hodgkin lymphoma patients presenting with a mutation at position 158 of FcyRIIIa show a complete response to rituximab and the Fc receptor has stronger binding to the Fc region relative to wild-type (Cartron et al. 2002. Blood, 99(3), pp.754-758; Paiva et al. 2008. Cancer Genetics and
  • IgG3 presents the longest hinge, thus being the subclass with the most flexible hinge region between Fabs and Fc region.
  • the flexibility of the hinge region decreases, subsequently, in the following order: IgGl, IgG4 and IgG2. Based on this, IgGl and IgG3 are more prone to trigger immune functions. IgGl has the strongest ADCC activity, and IgG3 has the strongest CDC capacity. However, other factors besides the hinge region flexibility, rule the effectiveness and extent of responses of this nature.
  • CDC effects are regulated by the presence of membrane -bound complement inhibitory proteins, whereas ADCC responses are affected, among other factors, by the antibody affinity and the antigen density (Velders et al. 1998. British Journal of Cancer, 78(4), pp.478 ⁇ 183; Tang et al. 2007. The Journal of Immunology, 179(5), pp.2815-2823; Gancz & Fishelson 2009. Molecular Immunology, 46(14), pp.2794-2800; M. Li et al. 2012. Cellular and Molecular Immunology, 9(1), pp.54-61).
  • IgG3 is the least stable, with a serum half- life of 7 days.
  • IgG2 or IgG4 are the common choices when immune responses arising from release of pro-inflammatory cytokines are undesirable, as in inflammatory and autoimmune disorders.
  • IgGl is the subclass of preference.
  • VHH-Fc anti-nucleolin fusion proteins
  • the epitope recognized by the structure herein reported may detect nucleolin regions that do not trigger endocytosis of the
  • the Fc region of the antibody or antibody fragment contains one or more substations selected from L234Y, S239D, T256A, K290A, K290Y, Y296W, S298A, A330F, A330L, I332E, E333A, K334A, K334V, A339T, E356K, K392D, D339K and K409D. In some embodiments, said substitutions increase the ADCC effect.
  • the level of Fc-bound carbohydrate chains including, but not limited to, alterations at the level of fructose, galactose, mannose, bisecting sugars and/or sialic acid is altered. In some embodiments, said alterations increase the ADCC effect.
  • the Fc portion of the antibody or antibody fragment is coupled to a cytotoxic drug, e.g., auristatin, maytansinoid, calicheamicin, duocarmycin, amatoxin or pyrrolobenzodiazepine.
  • a cytotoxic drug e.g., auristatin, maytansinoid, calicheamicin, duocarmycin, amatoxin or pyrrolobenzodiazepine.
  • said coupling increases the ADCC effect.
  • an anti-nucleolin antibody described herein has cytotoxicity towards a cell expressing nucleolin. In some embodiments, an anti-nucleolin antibody described herein has ADCC towards a cell expressing nucleolin.
  • an anti-nucleolin antibody described herein has ADCC towards a cell expressing nucleolin sufficient to cause 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% cell death.
  • an anti-nucleolin antibody described herein has ADCC towards a cell expressing nucleolin sufficient to cause 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, 200%, 300%, 400%, or 500% more cell death than a control antibody.
  • a control antibody an antibody to a target other than nucleolin, e.g., TNF-a.
  • an anti-nucleolin antibody described herein with an Fc region has ADCC towards a cell expressing nucleolin sufficient to cause 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, 200%, 300%, 400%, or 500% more cell death than the same antibody without an Fc region.
  • Preparation of anti-nucleolin antibodies Antibodies capable of binding nucleolin as described herein can be made by any method known in the art. See, for example, Harlow and Lane, (1998) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.
  • antibodies specific to a target antigen can be made by the conventional hybridoma technology.
  • the full- length target antigen or a fragment thereof, optionally coupled to a carrier protein such as KLH, can be used to immunize a host animal for generating antibodies binding to that antigen.
  • the route and schedule of immunization of the host animal are generally in keeping with established and conventional techniques for antibody stimulation and production, as further described herein. General techniques for production of mouse, humanized, and human antibodies are known in the art and are described herein.
  • any mammalian subject including humans or antibody producing cells therefrom can be manipulated to serve as the basis for production of mammalian, including human hybridoma cell lines.
  • the host animal is inoculated intraperitoneally, intramuscularly, orally, subcutaneously, intraplantar, and/or intradermally with an amount of immunogen, including as described herein.
  • Hybridomas can be prepared from the lymphocytes and immortalized myeloma cells using the general somatic cell hybridization technique of Kohler, B. and Milstein, C. (1975) Nature 256:495-497 or as modified by Buck, D. W., et al., In Vitro, 18:377-381 (1982).
  • X63-Ag8.653 available myeloma lines, including but not limited to X63-Ag8.653 and those from the Salk Institute, Cell Distribution Center, San Diego, Calif, USA, may be used in the hybridization.
  • the technique involves fusing myeloma cells and lymphoid cells using a fusogen such as polyethylene glycol, or by electrical means well known to those skilled in the art.
  • a fusogen such as polyethylene glycol
  • the cells are separated from the fusion medium and grown in a selective growth medium, such as hypoxanthine-aminopterin-thymidine (HAT) medium, to eliminate unhybridized parent cells.
  • HAT hypoxanthine-aminopterin-thymidine
  • Any of the media described herein, supplemented with or without serum, can be used for culturing hybridomas that secrete monoclonal antibodies.
  • EBV immortalized B cells may be used to produce the anti-nucleolin monoclonal antibodies described herein.
  • the hybridomas are expanded and subcloned, if desired, and supernatants are assayed for anti-immunogen activity by conventional
  • Hybridomas that may be used as source of antibodies encompass all derivatives, progeny cells of the parent hybridomas that produce monoclonal antibodies capable of interfering with cell-surface localized nucleolin activity. Hybridomas that produce such antibodies may be grown in vitro or in vivo using known procedures.
  • the monoclonal antibodies may be isolated from the culture media or body fluids, by conventional immunoglobulin purification procedures such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography, and ultrafiltration, if desired.
  • Undesired activity if present, can be removed, for example, by running the preparation over adsorbents made of the immunogen attached to a solid phase and eluting or releasing the desired antibodies off the immunogen.
  • an antibody (monoclonal or polyclonal) of interest may be sequenced and the polynucleotide sequence may then be cloned into a vector for expression or propagation.
  • the sequence encoding the antibody of interest may be maintained in vector in a host cell and the host cell can then be expanded and frozen for future use.
  • the polynucleotide sequence may be used for genetic manipulation to "humanize” the antibody or to improve the affinity (affinity maturation), or other characteristics of the antibody.
  • the constant region may be engineered to more resemble human constant regions to avoid immune response if the antibody is used in clinical trials and treatments in humans.
  • Fully human antibodies can be obtained by using commercially available mice that have been engineered to express specific human immunoglobulin proteins.
  • Transgenic animals that are designed to produce a more desirable (e.g., fully human antibodies) or more robust immune response may also be used for generation of humanized or human antibodies. Examples of such technology are Xenomouse R TM from Amgen, Inc. (Fremont, Calif.) and HuMAb-Mouse R TM and TC MouseTM from Medarex, Inc. (Princeton, N.J.).
  • antibodies may be made recombinantly by phage display or yeast technology. See, for example, U.S. Pat. Nos.
  • Antigen-binding fragments of an intact antibody can be prepared via routine methods.
  • F(ab')2 fragments can be produced by pepsin digestion of an antibody molecule, and Fab fragments that can be generated by reducing the disulfide bridges of F(ab')2 fragments.
  • DNA encoding a monoclonal antibodies specific to a target antigen can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibodies).
  • the hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into one or more expression vectors, which are then transfected into host cells such as E.
  • the DNA can then be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences, Morrison et al., (1984) Proc. Nat. Acad. Sci. 81 :6851 , or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non- immunoglobulin polypeptide.
  • chimeric antibodies such as “chimeric” or “hybrid” antibodies
  • Techniques developed for the production of “chimeric antibodies” are well known in the art. See, e.g., Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81, 6851; Neuberger et al. (1984) Nature 312, 604; and Takeda et al. (1984) Nature 314:452.
  • variable regions of VH and VL of a parent non-human antibody are subjected to three-dimensional molecular modeling analysis following methods known in the art.
  • framework amino acid residues predicted to be important for the formation of the correct CDR structures are identified using the same molecular modeling analysis.
  • human VH and VL chains having amino acid sequences that are homologous to those of the parent non-human antibody are identified from any antibody gene database using the parent VH and VL sequences as search queries. Human VH and VL acceptor genes are then selected.
  • the CDR regions within the selected human acceptor genes can be replaced with the CDR regions from the parent non-human antibody or functional variants thereof.
  • residues within the framework regions of the parent chain that are predicted to be important in interacting with the CDR regions can be used to substitute for the corresponding residues in the human acceptor genes.
  • a single-chain antibody can be prepared via recombinant technology by linking a nucleotide sequence coding for a heavy chain variable region and a nucleotide sequence coding for a light chain variable region.
  • a flexible linker is incorporated between the two variable regions.
  • techniques described for the production of single chain antibodies can be adapted to produce a phage or yeast scFv library and scFv clones specific to a nucleolin can be identified from the library following routine procedures. Positive clones can be subjected to further screening to identify those that inhibit cell surface-localized nucleolin activity.
  • Antibodies obtained following a method known in the art and described herein can be characterized using methods well known in the art. For example, one method is to identify the epitope to which the antigen binds, or "epitope mapping.” There are many methods known in the art for mapping and characterizing the location of epitopes on proteins, including solving the crystal structure of an antibody-antigen complex, competition assays, gene fragment expression assays, and synthetic peptide -based assays, as described, for example, in Chapter 1 1 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1999. In an additional example, epitope mapping can be used to determine the sequence to which an antibody binds.
  • the epitope can be a linear epitope, i.e., contained in a single stretch of amino acids, or a conformational epitope formed by a three-dimensional interaction of amino acids that may not necessarily be contained in a single stretch (primary structure linear sequence).
  • Peptides of varying lengths e.g., at least 4-6 amino acids long
  • the epitope to which the antibody binds can be determined in a systematic screening by using overlapping peptides derived from the target antigen sequence and determining binding by the antibody.
  • the open reading frame encoding the target antigen is fragmented either randomly or by specific genetic constructions and the reactivity of the expressed fragments of the antigen with the antibody to be tested is determined.
  • the gene fragments may, for example, be produced by PCR and then transcribed and translated into protein in vitro, in the presence of radioactive amino acids. The binding of the antibody to the radioactive ly labeled antigen fragments is then determined by immunoprecipitation and gel electrophoresis. Certain epitopes can also be identified by using large libraries of random peptide sequences displayed on the surface of phage particles (phage libraries). Alternatively, a defined library of overlapping peptide fragments can be tested for binding to the test antibody in simple binding assays.
  • mutagenesis of an antigen binding domain can be performed to identify residues required, sufficient, and/or necessary for epitope binding.
  • domain swapping experiments can be performed using a mutant of a target antigen in which various fragments of the nucleolin polypeptide have been replaced (swapped) with sequences from a closely related, but antigenically distinct protein. By assessing binding of the antibody to the mutant nucleolin, the importance of the particular antigen fragment to antibody binding can be assessed.
  • competition assays can be performed using other antibodies known to bind to the same antigen to determine whether an antibody binds to the same epitope as the other antibodies. Competition assays are well known to those of skill in the art.
  • an anti-nucleolin antibody is prepared by recombinant technology as exemplified below.
  • Nucleic acids encoding the heavy and light chain of an anti-nucleolin antibody as described herein can be cloned into one expression vector, each nucleotide sequence being in operable linkage to a suitable promoter.
  • each of the nucleotide sequences encoding the heavy chain and light chain is in operable linkage to a distinct prompter.
  • nucleotide sequences encoding the heavy chain and the light chain can be in operable linkage with a single promoter, such that both heavy and light chains are expressed from the same promoter.
  • an internal ribosomal entry site IVS
  • nucleic acids encoding the VHH chain of an anti-nucleolin antibody can be cloned into an expression vector under a suitable promoter.
  • the nucleotide sequences encoding the heavy and light chains of the antibody are cloned into two vectors, which can be introduced into the same or different cells.
  • the two chains are expressed in different cells, each of them can be isolated from the host cells expressing such and the isolated heavy chains and light chains can be mixed and incubated under suitable conditions allowing for the formation of the antibody.
  • a nucleic acid sequence encoding one or all chains of an antibody can be cloned into a suitable expression vector in operable linkage with a suitable promoter using methods known in the art.
  • the nucleotide sequence and vector can be contacted, under suitable conditions, with a restriction enzyme to create complementary ends on each molecule that can pair with each other and be joined together with a ligase.
  • synthetic nucleic acid linkers can be ligated to the termini of a gene. These synthetic linkers contain nucleic acid sequences that correspond to a particular restriction site in the vector. The selection of expression vectors/promoter would depend on the type of host cells for use in producing the antibodies.
  • promoters can be used for expression of the antibodies described herein, including, but not limited to, cytomegalovirus (CMV) intermediate early promoter, a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR, HTLV- 1 LTR, the simian virus 40 (SV40) early promoter, E. coli lac UV5 promoter, and the herpes simplex tk virus promoter.
  • CMV cytomegalovirus
  • a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR, HTLV- 1 LTR
  • SV40 simian virus 40
  • E. coli lac UV5 promoter E. coli lac UV5 promoter
  • herpes simplex tk virus promoter the herpes simplex tk virus promoter.
  • Regulatable promoters can also be used.
  • Such regulatable promoters include those using the lac repressor from E. coli as a transcription modulator to regulate transcription from lac operator-bearing mammalian cell promoters [Brown, M. et al., Cell, 49:603-612 (1987)], those using the tetracycline repressor (tetR) [Gossen, M., and Bujard, H., Proc. Natl. Acad. Sci. USA 89:5547-5551 (1992); Yao, F. et al., Human Gene Therapy, 9: 1939-1950 (1998); Shockelt, P., et al., Proc. Natl. Acad. Sci.
  • Regulatable promoters that include a repressor with the operon can be used.
  • the lac repressor from E. coli can function as a transcriptional modulator to regulate transcription from lac operator-bearing mammalian cell promoters [M. Brown et al., Cell, 49:603-612 (1987)]; Gossen and Bujard (1992); [M. Gossen et al., Natl. Acad. Sci.
  • tetracycline repressor tetR
  • VP 16 transcription activator
  • tetO-bearing minimal promoter derived from the human cytomegalovirus (hCMV) major immediate-early promoter to create a tetR-tet operator system to control gene expression in mammalian cells.
  • hCMV human cytomegalovirus
  • a tetracycline inducible switch is used.
  • the tetracycline repressor (tetR) alone, rather than the tetR-mammalian cell transcription factor fusion derivatives can function as potent trans-modulator to regulate gene expression in mammalian cells when the tetracycline operator is properly positioned downstream for the TATA element of the CMVIE promoter (Yao et al., Human Gene Therapy).
  • tetracycline inducible switch is that it does not require the use of a tetracycline repressor-mammalian cells transactivator or repressor fusion protein, which in some instances can be toxic to cells (Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992); Shockett et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)), to achieve its regulatable effects.
  • the vector can contain, for example, some or all of the following: a selectable marker gene, such as the neomycin gene for selection of stable or transient transfectants in mammalian cells; enhancer/promoter sequences from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; SV40 polyoma origins of replication and ColEl for proper episomal replication; internal ribosome binding sites (IRESes), versatile multiple cloning sites; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA.
  • a selectable marker gene such as the neomycin gene for selection of stable or transient transfectants in mammalian cells
  • enhancer/promoter sequences from the immediate early gene of human CMV for high levels of transcription
  • transcription termination and RNA processing signals from SV40 for mRNA stability SV40 polyoma origins of replication and ColEl for proper episomal replication
  • One or more vectors comprising nucleic acids encoding any of the antibodies may be introduced into suitable host cells for producing the antibodies.
  • the host cells can be cultured under suitable conditions for expression of the antibody or any polypeptide chain thereof.
  • Such antibodies or polypeptide chains thereof can be recovered by the cultured cells (e.g., from the cells or the culture supernatant) via a conventional method, e.g., affinity purification.
  • polypeptide chains of the antibody can be incubated under suitable conditions for a suitable period of time allowing for production of the antibody.
  • methods for preparing an antibody described herein involve a recombinant expression vector that encodes both the heavy chain and the light chain of an anti- nucleolin antibody, or a VHH chain of an anti-nucleolin antibody, as also described herein.
  • the recombinant expression vector can be introduced into a suitable host cell (e.g., a dhfr- CHO cell) by a conventional method, e.g., calcium phosphate -mediated transfection.
  • Positive transformant host cells can be selected and cultured under suitable conditions allowing for the expression of the polypeptide chain(s) that form the antibody, which can be recovered from the cells or from the culture medium.
  • the antibody comprises heavy and light chains
  • the two chains recovered from the host cells can be incubated under suitable conditions allowing for the formation of the antibody.
  • two recombinant expression vectors are provided, one encoding the heavy chain of the anti-nucleolin antibody and the other encoding the light chain of the anti- nucleolin antibody.
  • Both of the two recombinant expression vectors can be introduced into a suitable host cell (e.g., dhfr- CHO cell) by a conventional method, e.g., calcium phosphate - mediated transfection.
  • each of the expression vectors can be introduced into a suitable host cells. Positive transformants can be selected and cultured under suitable conditions allowing for the expression of the polypeptide chains of the antibody.
  • the antibody produced therein can be recovered from the host cells or from the culture medium.
  • the polypeptide chains can be recovered from the host cells or from the culture medium and then incubated under suitable conditions allowing for formation of the antibody.
  • the two expression vectors are introduced into different host cells, each of them can be recovered from the corresponding host cells or from the corresponding culture media. The two polypeptide chains can then be incubated under suitable conditions for formation of the antibody.
  • Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recovery of the antibodies from the culture medium.
  • some antibodies can be isolated by affinity chromatography with a Protein A or Protein G coupled matrix.
  • nucleic acids encoding the heavy chain, the light chain, or both of an anti- nucleolin antibody as described herein vectors (e.g., expression vectors) containing such; and host cells comprising the vectors are within the scope of the present disclosure.
  • the antibodies, as well as the encoding nucleic acids or nucleic acid sets, vectors comprising such, or host cells comprising the vectors, as described herein can be mixed with a pharmaceutically acceptable carrier (excipient) to form a pharmaceutical composition for use in treating a target disease.
  • a pharmaceutically acceptable carrier excipient
  • “Acceptable” means that the carrier must be compatible with the active ingredient of the composition (and preferably, capable of stabilizing the active ingredient) and not deleterious to the subject to be treated.
  • compositions to be used in the present methods can comprise pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions.
  • pharmaceutically acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations used, and may comprise buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride;
  • hexamethonium chloride benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol;
  • polypeptides such as serum albumin, gelatin, or immunoglobulins
  • hydrophilic polymers such as polyvinylpyrrolidone
  • amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine
  • chelating agents such as EDTA
  • sugars such as sucrose, mannitol, trehalose or sorbitol
  • salt- forming counter-ions such as sodium
  • metal complexes e.g. Zn-protein complexes
  • non-ionic surfactants such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG).
  • the pharmaceutical composition described herein comprises liposomes containing the antibodies (or the encoding nucleic acids) which can be prepared by methods known in the art, such as described in Epstein, et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang, et al., Proc. Natl. Acad. Sci. USA 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.
  • Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG- derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.
  • PEG-PE PEG- derivatized phosphatidylethanolamine
  • the antibodies, or the encoding nucleic acid(s), may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • macroemulsions for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules.
  • sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No.
  • copolymers of L-glutamic acid and 7 ethyl-L-glutamate copolymers of L-glutamic acid and 7 ethyl-L-glutamate, non-degradable ethylene -vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid- glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3- hydroxybutyric acid.
  • LUPRON DEPOTTM injectable microspheres composed of lactic acid- glycolic acid copolymer and leuprolide acetate
  • sucrose acetate isobutyrate sucrose acetate isobutyrate
  • poly-D-(-)-3- hydroxybutyric acid poly-D-(-)-3- hydroxybutyric acid.
  • compositions to be used for in vivo administration must be sterile. This is readily accomplished by, for example, filtration through sterile filtration membranes.
  • Therapeutic antibody compositions are generally placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • compositions described herein can be in unit dosage forms such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories, for oral, parenteral or rectal administration, or administration by inhalation or insufflation.
  • the principal active ingredient can be mixed with a pharmaceutical carrier, e.g., conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g. , water, to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention, or a non-toxic pharmaceutically acceptable salt thereof.
  • a pharmaceutical carrier e.g., conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g. , water, to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention, or a non-toxic pharmaceutically acceptable salt thereof.
  • the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules.
  • the tablets or pills of the novel composition can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action.
  • the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former.
  • the two components can be separated by an enteric layer that serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release.
  • enteric layers or coatings such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.
  • Suitable surface-active agents include, in particular, non-ionic agents, such as polyoxyethylenesorbitans (e.g., TweenTM 20, 40, 60, 80 or 85) and other sorbitans (e.g., SpanTM 20, 40, 60, 80 or 85).
  • Compositions with a surface-active agent will conveniently comprise between 0.05 and 5% surface- active agent, and can be between 0.1 and 2.5%. It will be appreciated that other ingredients may be added, for example mannitol or other pharmaceutically acceptable vehicles, if necessary.
  • Suitable emulsions may be prepared using commercially available fat emulsions, such as IntralipidTM, LiposynTM, InfonutrolTM, LipofundinTM and LipiphysanTM.
  • the active ingredient may be either dissolved in a pre-mixed emulsion composition or alternatively it may be dissolved in an oil (e.g. , soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or almond oil) and an emulsion formed upon mixing with a phospholipid (e.g. egg phospholipids, soybean phospholipids or soybean lecithin) and water.
  • an oil e.g. , soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or almond oil
  • a phospholipid e.g. egg phospholipids, soybean phospholipids or soybean lecithin
  • other ingredients may be added, for example glycerol or glucose, to adjust the tonicity of the emul
  • Suitable emulsions will typically contain up to 20% oil, for example, between 5 and 20%.
  • the fat emulsion can comprise fat droplets between 0.1 and 1.0 .im, particularly 0.1 and 0.5 .im, and have a pH in the range of 5.5 to 8.0.
  • the emulsion compositions can be those prepared by mixing an antibody with
  • IntralipidTM or the components thereof (soybean oil, egg phospholipids, glycerol and water).
  • compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders.
  • the liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as set out above.
  • the compositions are administered by the oral or nasal respiratory route for local or systemic effect.
  • compositions in preferably sterile pharmaceutically acceptable solvents may be nebulised by use of gases. Nebulised solutions may be breathed directly from the nebulising device or the nebulising device may be attached to a face mask, tent or intermittent positive pressure breathing machine. Solution, suspension or powder compositions may be administered, preferably orally or nasally, from devices which deliver the formulation in an appropriate manner.
  • any of the antibodies, as well as the encoding nucleic acids or nucleic acid sets, vectors comprising such, or host cells comprising the vectors, described herein are useful for treating a disease or disorder associated with cell-surface localized nucleolin, including cancer.
  • an effective amount of the antibody described herein can be administered to a subject (e.g., a human) in need of the treatment via a suitable route, such as intravenous administration, e.g. , as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, inhalation or topical routes.
  • nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers are useful for administration.
  • Liquid formulations can be directly nebulized and lyophilized powder can be nebulized after reconstitution.
  • the antibodies as described herein can be aerosolized using a fluorocarbon formulation and a metered dose inhaler, or inhaled as a lyophilized and milled powder.
  • the antibodies described herein are administered by intravenous administration.
  • the subject to be treated by the methods described herein can be a mammal, more preferably a human. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice and rats.
  • a human subject who needs the treatment may be a human patient having, at risk for, or suspected of having a target disease/disorder, such as cancer.
  • a subject having a target disease or disorder can be identified by routine medical examination, e.g., laboratory tests, organ functional tests, CT scans, or ultrasounds.
  • a subject suspected of having any of such target disease/disorder might show one or more symptoms of the disease/disorder.
  • a subject at risk for the disease/disorder can be a subject having one or more of the risk factors for that disease/disorder.
  • the methods and compositions described herein may be used to treat any disease or disorder associated with cell-surface localized nucleolin.
  • the target disease is cancer.
  • Cancers include but are not limited to: Oral: buccal cavity, lip, tongue, mouth, pharynx; Cardiac: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratoma;
  • Lung non-small cell lung cancer (NSCLC), small cell lung cancer, bronchogenic carcinoma (squamous cell or epidermoid, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma; Gastrointestinal
  • leiomyosarcoma lymphoma
  • stomach carcinoma, lymphoma, leiomyosarcoma
  • pancreas ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel or small intestines (adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel or large intestines (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma), rectal, colon, colon-rectum, colorectal; Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor [nephroblastoma], lymphoma, leukemia), bladder and
  • hepatoma hepatocellular carcinoma
  • cholangiocarcinoma hepatoblastoma
  • angiosarcoma hepatocellular adenoma
  • hemangioma hemangioma
  • biliary passages Bone: osteogenic sarcoma
  • nerveous system skull (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), head and neck cancer, meninges (meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma [pinealoma],
  • cystadenocarcinoma mucinous cystadenocarcinoma, unclassified carcinoma] granulosa-thecal cell tumors, Sertoll-Leydig cell tumors, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal
  • rhabdomyosarcoma fallopian tubes (carcinoma), breast
  • Hematologic blood (myeloid leukemia [acute and chronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignant lymphoma] hairy cell; lymphoid disorders; Skin:
  • Thyroid gland papillary thyroid carcinoma, follicular thyroid carcinoma; medullary thyroid carcinoma, multiple endocrine neoplasia type 2A, multiple endocrine neoplasia type 2B, familial medullary thyroid cancer, pheochromocytoma, paraganglioma; and Adrenal glands:
  • an effective amount refers to the amount of each active agent required to confer therapeutic effect on the subject, either alone or in combination with one or more other active agents.
  • the therapeutic effect is reduced cell-surface localized nucleolin. Determination of whether an amount of the antibody achieved the therapeutic effect would be evident to one of skill in the art. Effective amounts vary, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment.
  • Empirical considerations such as the half-life, generally will contribute to the determination of the dosage.
  • antibodies that are compatible with the human immune system such as humanized antibodies or fully human antibodies, may be used to prolong half-life of the antibody and to prevent the antibody being attacked by the host's immune system.
  • Frequency of administration may be determined and adjusted over the course of therapy, and is generally, but not necessarily, based on treatment and/or suppression and/or amelioration and/or delay of a target disease/disorder.
  • sustained continuous release formulations of an antibody may be appropriate.
  • formulations and devices for achieving sustained release are known in the art.
  • dosages for an antibody as described herein may be determined empirically in individuals who have been given one or more administration(s) of the antibody. Individuals are given incremental dosages of the antagonist. To assess efficacy of the antagonist, an indicator of the disease/disorder can be followed.
  • the appropriate dosage of an antibody as described herein will depend on the specific antibody, antibodies, and/or non-antibody peptide (or compositions thereof) employed, the type and severity of the disease/disorder, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antagonist, and the discretion of the attending physician.
  • the clinician will administer an antibody, until a dosage is reached that achieves the desired result.
  • the desired result is a decrease the severity of cancer.
  • Administration of one or more antibodies can be continuous or intermittent, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners.
  • the administration of an antibody may be essentially continuous over a preselected period of time or may be in a series of spaced dose, e.g., either before, during, or after developing a target disease or disorder.
  • treating refers to the application or administration of a composition including one or more active agents to a subject, who has a target disease or disorder, a symptom of the disease/disorder, or a predisposition toward the disease/disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disorder, the symptom of the disease, or the predisposition toward the disease or disorder.
  • Alleviating a target disease/disorder includes delaying the development or progression of the disease, or reducing disease severity. Alleviating the disease does not necessarily require curative results.
  • "delaying" the development of a target disease or disorder means to defer, hinder, slow, retard, stabilize, and/or postpone progression of the disease. This delay can be of varying lengths of time, depending on the history of the disease and/or individuals being treated.
  • a method that "delays" or alleviates the development of a disease, or delays the onset of the disease is a method that reduces probability of developing one or more symptoms of the disease in a given time frame and/or reduces extent of the symptoms in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a number of subjects sufficient to give a statistically significant result.
  • “Development” or “progression” of a disease means initial manifestations and/or ensuing progression of the disease. Development of the disease can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that may be undetectable. For purpose of this disclosure, development or progression refers to the biological course of the symptoms. “Development” includes occurrence, recurrence, and onset. As used herein "onset” or “occurrence” of a target disease or disorder includes initial onset and/or recurrence.
  • the antibodies described herein are administered to a subject in need of the treatment at an amount sufficient to inhibit the activity of the target antigen by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater) in vivo. In other embodiments, the antibodies are administered in an amount effective in reducing the activity level of a target antigen by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater).
  • compositions can be administered via other conventional routes, e.g., administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.
  • parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques.
  • injectable depot routes of administration such as using 1-, 3-, or 6-month depot injectable or biodegradable materials and methods.
  • the pharmaceutical composition is administered intraocularly or intravitreally.
  • compositions may contain various carriers such as vegetable oils,
  • water soluble antibodies can be administered by the drip method, whereby a pharmaceutical formulation containing the antibody and a physiologically acceptable excipient is infused.
  • Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer's solution or other suitable excipients.
  • Intramuscular preparations e.g., a sterile formulation of a suitable soluble salt form of the antibody
  • a pharmaceutical excipient such as Water-for-Injection, 0.9% saline, or 5% glucose solution.
  • an antibody is administered via site-specific or targeted local delivery techniques.
  • site-specific or targeted local delivery techniques include various implantable depot sources of the antibody or local delivery catheters, such as infusion catheters, an indwelling catheter, or a needle catheter, synthetic grafts, adventitial wraps, shunts and stents or other implantable devices, site specific carriers, direct injection, or direct application. See, e.g., PCT Publication No. WO 00/5321 1 and U.S. Pat. No. 5,981,568.
  • Targeted delivery of therapeutic compositions containing an antisense polynucleotide, expression vector, or subgenomic polynucleotides can also be used.
  • Receptor-mediated DNA delivery techniques are described in, for example, Findeis et al., Trends Biotechnol. (1993) 1 1 :202; Chiou et al., Gene Therapeutics: Methods And Applications Of Direct Gene Transfer (J. A. Wolff, ed.) (1994); Wu et al., J. Biol. Chem. (1988) 263:621 ; Wu et al., J. Biol. Chem. (1994) 269:542; Zenke et al., Proc. Natl. Acad. Sci. USA (1990) 87:3655; Wu et al., J. Biol. Chem. (1991) 266:338.
  • the therapeutic polynucleotides and polypeptides described herein can be delivered using gene delivery vehicles.
  • the gene delivery vehicle can be of viral or non-viral origin (see generally, Jolly, Cancer Gene Therapy (1994) 1 :51; Kimura, Human Gene Therapy (1994) 5:845; Connelly, Human Gene Therapy (1995) 1 : 185; and Kaplitt, Nature Genetics (1994)
  • Expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters and/or enhancers. Expression of the coding sequence can be either constitutive or regulated.
  • Viral-based vectors for delivery of a desired polynucleotide and expression in a desired cell are well known in the art.
  • Exemplary viral-based vehicles include, but are not limited to, recombinant retroviruses (see, e.g., PCT Publication Nos. WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; WO 93/1 1230; WO 93/10218; WO 91/02805; U.S. Pat. Nos.
  • alphavirus- based vectors e.g., Sindbis virus vectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)
  • AAV adeno-associated virus
  • WO 94/12649 WO 93/03769; WO 93/19191 ; WO 94/28938; WO 95/1 1984 and WO 95/00655).
  • Administration of DNA linked to killed adenovirus as described in Curiel, Hum. Gene Ther. (1992) 3: 147 can also be employed.
  • Non- viral delivery vehicles and methods can also be employed, including, but not limited to, polycationic condensed DNA linked or unlinked to killed adenovirus alone (see, e.g., Curiel, Hum. Gene Ther. (1992) 3: 147); ligand-linked DNA (see, e.g., Wu, J. Biol. Chem. (1989) 264: 16985); eukaryotic cell delivery vehicles cells (see, e.g., U.S. Pat. No. 5,814,482; PCT Publication Nos. WO 95/07994; WO 96/17072; WO 95/30763; and WO 97/42338) and nucleic charge neutralization or fusion with cell membranes. Naked DNA can also be employed.
  • Exemplary naked DNA introduction methods are described in PCT Publication No. WO 90/1 1092 and U.S. Pat. No. 5,580,859.
  • Liposomes that can act as gene delivery vehicles are described in U.S. Pat. No. 5,422,120; PCT Publication Nos. WO 95/13796; WO 94/23697; WO 91/14445; and EP Patent No. 0524968. Additional approaches are described in Philip, Mol. Cell. Biol. (1994) 14:2411, and in Woffendin, Proc. Natl. Acad. Sci. (1994) 91 : 1581.
  • the particular dosage regimen i.e., dose, timing and repetition, used in the method described herein will depend on the particular subject and that subject's medical history.
  • more than one antibody, or a combination of an antibody and another suitable therapeutic agent may be administered to a subject in need of the treatment.
  • the antibody can also be used in conjunction with other agents that serve to enhance and/or complement the effectiveness of the agents.
  • Treatment efficacy for a target disease/disorder can be assessed by methods well-known in the art.
  • the anti-nucleolin antibody or antibody fragment described herein is administered in conjunction with additional cancer therapy.
  • the anti-nucleolin antibody or antibody fragment is administered to a subject concurrently with one or more additional therapies (either simultaneously or separately but in close proximity), prior to, or after administration of one or more additional therapies.
  • the anti-nucleolin antibody or antibody fragment is conjugated to one or more additional therapies.
  • an additional cancer therapy comprises a chemotherapeutic agent.
  • Chemotherapeutic agents include, for example, including alkylating agents, anthracyclines, cytoskeletal disruptors (Taxanes), epothilones, histone deacetylase inhibitors, inhibitors of topoisomerase I, inhibitors of topoisomerase II, kinase inhibitors, nucleotide analogs and precursor analogs, peptide antibiotics, platinum-based agents, retinoids, vinca alkaloids and derivatives thereof.
  • Non-limiting examples include: (i) anti-angiogenic agents (e.g., TNP-470, platelet factor 4, thrombospondin- 1, tissue inhibitors of metalloproteases (TIMP1 and TIMP2), prolactin (16-Kd fragment), angiostatin (38-Kd fragment of plasminogen), endostatin, bFGF soluble receptor, transforming growth factor beta, interferon alpha, soluble KDR and FLT- 1 receptors, placental proliferin-related protein, as well as those listed by Carmeliet and Jain (2000)); (ii) a VEGF antagonist or a VEGF receptor antagonist such as anti-VEGF antibodies, VEGF variants, soluble VEGF receptor fragments, aptamers capable of blocking VEGF or VEGFR, neutralizing anti-VEGFR antibodies, inhibitors of VEGFR tyrosine kinases and any combinations thereof; and (iii) chemotherapeutic compounds such as, e.g., pyr
  • actinomycin D daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycin, plicamycin (mithramycin) and mitomycin
  • enzymes L-asparaginase which systemically metabolizes L-asparagine and deprives cells which do not have the capacity to synthesize their own asparagine
  • antiplatelet agents antiproliferative/antimitotic alkylating agents such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethylenimines and methylmelamines (hexamethylmelamine and thiotepa), alkyl sulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs, streptozocin), trazenes- dacarbazinine (D
  • mitochondrial dysfunction inducers and caspase activators; and chromatin disruptors.
  • an additional cancer therapy comprises external radiation therapy and internal radiation therapy (also called brachytherapy).
  • Energy sources for external radiation therapy include x-rays, gamma rays and particle beams, energy sources used in internal radiation include radioactive iodine (iodine 125 or iodinel31), strontium89, or radioisotopes of phosphorous, palladium, cesium, indium, phosphate, or cobalt. Methods of administering radiation therapy are well known to those of skill in the art.
  • an additional cancer therapy comprises an immunotherapy.
  • Cancer immunotherapy is the use of the immune system to reject cancer.
  • the main premise is stimulating the subject's immune system to attack the tumor cells that are responsible for the disease. This can be either through immunization of the subject, in which case the subject's own immune system is rendered to recognize tumor cells as targets to be destroyed, or through the administration of therapeutics, such as antibodies, as drugs, in which case the subject's immune system is recruited to destroy tumor cells by the therapeutic agents.
  • Cancer immunotherapy includes an antibody-based therapy and cytokine-based therapy.
  • a number of therapeutic monoclonal antibodies have been approved by the FDA for use in humans, and more are underway.
  • the FDA-approved monoclonal antibodies for cancer immunotherapy include antibodies against CD52, CD33, CD20, ErbB2, vascular endothelial growth factor and epidermal growth factor receptor. These and other antibodies targeting one or more cancer-associated antigen are thus suitable for use in a combination therapy to be administered in conjunction with an anti-nucleolin antibody or antibody fragment.
  • Examples of monoclonal antibodies approved by the FDA for cancer therapy include, without limitation: Rituximab (available as RituxanTM), Trastuzumab (available as HerceptinTM), Alemtuzumab (available as Campath-IHTM), Cetuximab (available as ErbituxTM), Bevacizumab (available as AvastinTM), Panitumumab (available as VectibixTM), Gemtuzumab ozogamicin (available as MylotargTM), Ibritumomab tiuxetan (available as ZevalinTM), Tositumomab (available as BexxarTM), Ipilimumab (available as YervoyTM), Ofatunumab (available as ArzerraTM), Daclizumab (available as ZinbrytaTM), Nivolumab (available as OpdivoTM), and Pembrolizumab (available as KeytrudaTM).
  • Rituximab available as RituxanTM
  • Examples of monoclonal antibodies currently undergoing human clinical testing for cancer therapy in the United States include, without limitation: WX-G250 (available as RencarexTM), Zanolimumab (available as HuMax-CD4), chl4.18, Zalutumumab (available as HuMax-EGFr), Oregovomab (available as B43.13, OvalRexTM), Edrecolomab (available as IGN-101, PanorexTM), 131I-chTNT-I/B (available as CotaraTM), Pemtumomab (available as R-1549, TheragynTM), Lintuzumab (available as SGN-33), Labetuzumab (available as hMN14, CEAcideTM), Catumaxomab (available as RemovabTM), CNTO 328 (available as cCLB8), 3F8, 177Lu-J591 , Nimotuzumab, SGN-30, Ticilimumab (available as
  • Cancer immunotherapy also includes a cytokine-based therapy.
  • the cytokine-based cancer therapy utilizes one or more cytokines that modulate a subject's immune response.
  • cytokines useful in cancer treatment include interferon-a (IFN-a), interleukin-2 (IL-2), Granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-12 (IL-12).
  • a second PCR was carried out to overlap the two obtained sequences (i.e., the first sequence being from the beginning of the VHH to the end of the grafted CDR(1 or 3) and the second sequence being from the beginning of the grafted CDR(1 or 3) to the end of the VHH).
  • PCR conditions were the same as for the first PCR.
  • the parental VHH nucleic acid and amino acid sequence are shown in SEQ ID NO: 9- 10, respectively.
  • the nucleic acid and amino acid sequences of the novel anti-nucleolin VHH constructs are shown in SEQ ID NO: 1 1-14 and 15-18, respectively.
  • the amino acid sequences of the CDRs are shown in Table 3.
  • Binding of the generated anti-nucleolin VHH fragments to human nucleolin and to human TNF-a proteins was assessed by enzyme-linked immunosorbent assay (ELISA). Plates were first coated with 100 ng of human nucleolin or 200 ng of human TNF-a in carbonate buffer (50 mM sodium carbonate, pH 9.6), at 4°C, overnight.
  • ELISA enzyme-linked immunosorbent assay
  • Nonspecific binding sites were then blocked with 3% (w/v) bovine serum albumin (BSA) in phosphate buffer saline (PBS, 137 mM NaCl, 2.7 mM KC1, 10 mM Na 2 HP0 4 , 1.8 mM KH 2 P0 4 , pH 7.4) for 1 h, at 37°C, washed once with PBS and further incubated with 100 pmol of each VHH construct (diluted in 1%, w/v, BSA in PBS), for 1 h at 37°C.
  • BSA bovine serum albumin
  • Human MDA-MB-435S cancer cells, human MDA-MB 231 cells, and mouse 4T1 breast cancer cells (ATCC, USA) were maintained in RPMI-1640 (Lonza, Switzerland), supplemented with 10%) (v/v) heat-inactivated fetal bovine serum (FBS, HyClone, USA), 2 mM of L- glutamine (Lonza, Switzerland) and 1% (v/v) Pen/Strep/Fungizone solution (HyClone, USA), at 37°C in a humidified atmosphere of 5% CO2.
  • nucleolin-overexpressing cells previously treated with dissociation buffer were incubated with 10, 100 or 1000 nM VHH protein at 4°C for 45 min. After washing, a second incubation with anti-HA-FITC antibody (Y- 11 sc-805, Santa Cruz Biotechnology, USA) was performed at room temperature for 30 min. Cells were again washed, fixed and analyzed by flow cytometry (Guava easyCyte 5HT, Merck Millipore, USA), using the InCyte software module (Merck Millipore, USA).
  • aNCL- CDR3 VHH and aNCL-CDR3-L VHH were compared with aNCL-CDRl VHH, aNCL-CDRl-L VHH or the parental VHH (p ⁇ 0.05, compared to CDR1 -grafted VHHs at 4 ⁇ and p ⁇ 0.01 or p ⁇ 0.001, respectively, compared to aNCL-CDRl VHH; p ⁇ 0.001 compared to aNCL-CDRl -L VHH; p ⁇ 0.01 or pO.001 , respectively, compared to parental VHH at 8 ⁇ ).
  • CDR1- and CDR3 -grafted anti-nucleolin VHHs showed a decrease of cell viability that was 60% and 40% (at 4 ⁇ ) or 55% and 20% (at 8 ⁇ ) lower, respectively, than that observed against MDA-MB- 435 S cancer cells. These results are consistent with the reduced binding levels observed with MDA-MB-231 cells relative to MDA-MB-435S cells. Parental VHH also reduced cell viability (down to 60%), suggesting a bispecific effect of the anti-nucleolin VHHs in these cells as in MDA-MB-435S cells (Figure 5C).
  • CDR3 was cloned into a pFuse vector for construction of a fusion protein (aNCL- VHH-Fc) ( Figures 6A-6B).
  • This vector incorporates an IL2 signal sequence that causes secretion of the protein to the extracellular medium.
  • the parental VHH was also cloned into this vector to generate the parental VHH-Fc antibody.
  • DNA of the parental and aNCL-CDR3 VHH was digested from the pT7 vector using Ncol and Bglll (Thermo Scientific, USA). Ligation of the digested fragments in the pFuse vector and subsequent transformation of JM109 bacteria were performed as described in Example 1.
  • HEK293T cells were then transfected, by the calcium phosphate method, with a positive clone of each sequence for protein expression (Graham & Van der Eb 1973. Virology, 52, pp.456-467; Jordan et al. 1996. Nucleic Acids Research, 24(4), pp.596-601).
  • HEK293T cells were maintained in Dulbecco's Modified Eagle Medium (DMEM, Lonza, Switzerland), supplemented with 10% (v/v) heat-inactivated FBS (HyClone, USA), 2 mM of L-glutamine (Lonza, Switzerland) and 1% (v/v) Penicillin/Streptomycin/Fungiezone solution (HyClone, USA).
  • the anti-nucleolin VHH-Fc antibody led to a 1.7- or a 1.5-fold decrease in viability of MDA-MB-435S or MDA- MB-231 cells, respectively, relative to the parental VHH-Fc antibody.
  • the parental VHH-Fc antibody did not alter the viability of 4T1 cells but decreased the viability of MDA-MB-435S and MDA- MB-231 cells to 53% and 64%, respectively.
  • Example 7 ADCC of anti-nucleolin VHH-Fc against nucleolin-expressing cancer cells
  • PBMCs used as effector cells, were isolated from buffy coat harvested from four healthy donors by a Ficoll-Paque PLUS density gradient (GE Healthcare, UK). Following culture in RPMI-1640 (supplemented as described in Example 3) at 37°C in a humidified atmosphere of 5% CO2, PBMCs were stored at -80°C in freezing medium (10% v/v dimethyl sulfoxide in FBS) until use.
  • PBMCs When needed, PBMCs were thawed, resuspended in culture medium and incubated overnight at 37°C in a humidified atmosphere of 5% CO2.
  • 7500 MDA-MB-435S adherent cancer cells were incubated on a RTCA 96-well plate for 24 h and then incubated with PBMCs at a final 5: 1 or 10:1 effector/target cell ratio and 25 nM of anti-nucleolin VHH-Fc. Cancer cells incubated only with effector cells or antibody were included as additional controls.
  • ADCC is an Fc-dependent mechanism
  • MDA-MB-435S cells were also incubated with the VHH counterpart of the VHH-Fc antibodies. As the VHH-Fc antibodies are dimeric, the VHH fragments were added in a concentration of 50 nM.
  • the anti-nucleolin VHH-Fc antibody enabled higher cell death than the parental VHH-Fc (Figure 8A). This effect was partly dependent on the presence of the Fc region, as the anti- nucleolin VHH protein did not trigger the same level of cancer cell death in the presence of PBMCs ( Figure 8B). These results confirmed that the anti-nucleolin VHH-Fc antibody was able to trigger ADCC. This was a nucleolin-specific effect, as there were no differences in cell death between parental VHH and parental VHH-Fc, in the presence of PBMCs ( Figure 8C). The ADCC effect of the anti-nucleolin VHH-Fc was observed using PBMCs harvested from four donors (Table 4), showing different levels of ADCC activity.
  • the anti-nucleolin VHH-Fc protein Upon PBMC incubation, and depending on the donor, the anti-nucleolin VHH-Fc protein enabled a 1.6- to 2.2-fold increase in cell death, relative to the parental VHH-Fc and a 1.3- to 2.1 -fold increase when compared to the anti-nucleolin VHH counterpart. Therefore, regardless of the PBMC origin, an Fc-dependent, nucleolin-specific effect has been shown, demonstrating an ADCC effect of the anti-nucleolin VHH-Fc antibody.
  • Table 4 summarizes the cytotoxicity of anti-nucleolin and parental VHH-Fc or anti- nucleolin VHH constructs in the presence of PBMCs harvested from four different donors (values indicate percentage of cell death). Differences in the ADCC capacity among the proteins tested, were evaluated with repeated measures ANOVA, followed by Tukey's Multiple 5 Comparison Test.
  • amino acid sequence coded for by SEQ ID NO: 33 is SEQ ID NO: 5
  • KKTAI AI AVALAG FATVAQAAQVQLQESG G G LVQP GG SLR LSCAASSG G GS PQ R RSAR LSASG G GSWF RQAPG KE R E FVAR I YWSSG NTYYADSV KG R FAIS RD I AK N TVD LTM N N LE P E DTAVYYCAAR D G I PTS RSV ESYN YWG QGTQVTVSSG QAGQH H H H H H H GAYPYDVP DYAS SEQ ID NO: 18 - QLNCL-CDR3-L Amino acid sequence M KKTAI AI AVALAG FATVAQAAQVQLQESG G G LVQP GG SLR LSCAASG RTFS D HS GYTYTI GW F RQAPG K E R E FVAR I YWSSG NTYYADSVKG R FAIS R D I AKNTVD LTM N N LE P E DTAVYYCAA SG
  • inventive embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed.
  • inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein.
  • a reference to "A and/or B", when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
  • the phrase "at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements.
  • This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified.
  • At least one of A and B can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

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Abstract

L'invention concerne des anticorps anti-nucléoline et des méthodes d'utilisation de ceux-ci pour traiter des maladies associées à la nucléoline localisée à la surface de cellules, notamment le cancer.
PCT/IB2018/055471 2017-07-21 2018-07-23 Anticorps anti-nucléoline WO2019016784A1 (fr)

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* Cited by examiner, † Cited by third party
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CN111875708A (zh) * 2020-05-28 2020-11-03 浙江康佰裕生物科技有限公司 一种嵌合抗原受体t淋巴细胞及其在制备治疗实体肿瘤的产品中的应用
US11186636B2 (en) 2017-04-21 2021-11-30 Amgen Inc. Anti-human TREM2 antibodies and uses thereof
CN113980139A (zh) * 2021-09-10 2022-01-28 钦元再生医学(珠海)有限公司 自分泌TREM2 scFv的嵌合抗原受体细胞及其制备方法和应用
WO2024043227A1 (fr) * 2022-08-23 2024-02-29 小野薬品工業株式会社 Anticorps bispécifique

Citations (49)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3773919A (en) 1969-10-23 1973-11-20 Du Pont Polylactide-drug mixtures
US4485045A (en) 1981-07-06 1984-11-27 Research Corporation Synthetic phosphatidyl cholines useful in forming liposomes
US4544545A (en) 1983-06-20 1985-10-01 Trustees University Of Massachusetts Liposomes containing modified cholesterol for organ targeting
WO1987004462A1 (fr) 1986-01-23 1987-07-30 Celltech Limited Sequences d'adn recombinant, vecteurs les contenant et procede d'utilisation de ces sequences
US4704692A (en) 1986-09-02 1987-11-03 Ladner Robert C Computer based system and method for determining and displaying possible chemical structures for converting double- or multiple-chain polypeptides to single-chain polypeptides
GB2200651A (en) 1987-02-07 1988-08-10 Al Sumidaie Ayad Mohamed Khala A method of obtaining a retrovirus-containing fraction from retrovirus-containing cells
US4777127A (en) 1985-09-30 1988-10-11 Labsystems Oy Human retrovirus-related products and methods of diagnosing and treating conditions associated with said retrovirus
EP0345242A2 (fr) 1988-06-03 1989-12-06 Smithkline Biologicals S.A. Expression de protéines gag de rétrovirus dans les cellules eucaryotes
WO1990007936A1 (fr) 1989-01-23 1990-07-26 Chiron Corporation Therapies de recombinaison pour infections et troubles hyperproliferatifs
US4946778A (en) 1987-09-21 1990-08-07 Genex Corporation Single polypeptide chain binding molecules
WO1990011092A1 (fr) 1989-03-21 1990-10-04 Vical, Inc. Expression de sequences de polynucleotides exogenes chez un vertebre
WO1991002805A2 (fr) 1989-08-18 1991-03-07 Viagene, Inc. Retrovirus de recombinaison apportant des constructions de vecteur a des cellules cibles
US5013556A (en) 1989-10-20 1991-05-07 Liposome Technology, Inc. Liposomes with enhanced circulation time
WO1991014445A1 (fr) 1990-03-21 1991-10-03 Research Development Foundation Liposomes heterovesiculaires
WO1993003769A1 (fr) 1991-08-20 1993-03-04 THE UNITED STATES OF AMERICA, represented by THE SECRETARY, DEPARTEMENT OF HEALTH AND HUMAN SERVICES Transfert induit par adenovirus de genes vers la voie gastro-intestinale
WO1993010218A1 (fr) 1991-11-14 1993-05-27 The United States Government As Represented By The Secretary Of The Department Of Health And Human Services Vecteurs comprenant des genes etrangers et des marqueurs selectifs negatifs
WO1993011230A1 (fr) 1991-12-02 1993-06-10 Dynal As Cellule souche modifiee de mammifere bloquant la replication virale
US5219740A (en) 1987-02-13 1993-06-15 Fred Hutchinson Cancer Research Center Retroviral gene transfer into diploid fibroblasts for gene therapy
WO1993019191A1 (fr) 1992-03-16 1993-09-30 Centre National De La Recherche Scientifique Adenovirus recombinants defectifs exprimant des cytokines pour traitement antitumoral
WO1993025234A1 (fr) 1992-06-08 1993-12-23 The Regents Of The University Of California Procedes et compositions permettant de cibler des tissus specifiques
WO1993025698A1 (fr) 1992-06-10 1993-12-23 The United States Government As Represented By The Particules vecteurs resistantes a l'inactivation par le serum humain
WO1994003622A1 (fr) 1992-07-31 1994-02-17 Imperial College Of Science, Technology & Medicine Vecteurs retroviraux du type d, bases sur le virus du singe mason-pfizer
WO1994004678A1 (fr) 1992-08-21 1994-03-03 Casterman Cecile Immunoglobulines exemptes de chaines legeres
WO1994012649A2 (fr) 1992-12-03 1994-06-09 Genzyme Corporation Therapie genique de la fibrose kystique
WO1994023697A1 (fr) 1993-04-22 1994-10-27 Depotech Corporation Liposomes de cyclodextrine encapsulant des composes pharmacologiques et leurs procedes d'utilisation
WO1994028938A1 (fr) 1993-06-07 1994-12-22 The Regents Of The University Of Michigan Vecteurs d'adenovirus pour therapie genique
WO1995000655A1 (fr) 1993-06-24 1995-01-05 Mc Master University Vecteurs a base d'adenovirus destines a la therapie genique
WO1995007994A2 (fr) 1993-09-15 1995-03-23 Viagene, Inc. Vecteurs composes d'alphavirus recombinants
WO1995011984A2 (fr) 1993-10-25 1995-05-04 Canji, Inc. Vecteur recombinant d'adenovirus et procedes d'utilisation
WO1995013796A1 (fr) 1993-11-16 1995-05-26 Depotech Corporation Vesicules a taux controle de liberation des principes actifs
US5422120A (en) 1988-05-30 1995-06-06 Depotech Corporation Heterovesicular liposomes
WO1995030763A2 (fr) 1994-05-09 1995-11-16 Chiron Viagene, Inc. Vecteurs retroviraux a taux de recombinaison reduit
WO1996017072A2 (fr) 1994-11-30 1996-06-06 Chiron Viagene, Inc. Vecteurs d'alphavirus de recombinaison
US5565332A (en) 1991-09-23 1996-10-15 Medical Research Council Production of chimeric antibodies - a combinatorial approach
US5580717A (en) 1990-05-01 1996-12-03 Affymax Technologies N.V. Recombinant library screening methods
US5580859A (en) 1989-03-21 1996-12-03 Vical Incorporated Delivery of exogenous DNA sequences in a mammal
US5624821A (en) 1987-03-18 1997-04-29 Scotgen Biopharmaceuticals Incorporated Antibodies with altered effector functions
WO1997042338A1 (fr) 1996-05-06 1997-11-13 Chiron Corporation Vecteurs retroviraux sans croisement
WO1997049805A2 (fr) 1996-06-27 1997-12-31 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Molecules de reconnaissance ayant une interaction specifique avec le site actif ou la fissure d'une molecule cible
US5733743A (en) 1992-03-24 1998-03-31 Cambridge Antibody Technology Limited Methods for producing members of specific binding pairs
US5814482A (en) 1993-09-15 1998-09-29 Dubensky, Jr.; Thomas W. Eukaryotic layered vector initiation systems
US5981568A (en) 1993-01-28 1999-11-09 Neorx Corporation Therapeutic inhibitor of vascular smooth muscle cells
WO1999058572A1 (fr) 1998-05-08 1999-11-18 Cambridge University Technical Services Limited Molecules de liaison derivees d'immunoglobulines ne declenchant pas de lyse dependante du complement
WO2000053211A2 (fr) 1999-03-09 2000-09-14 University Of Southern California Methode de stimulation de la proliferation de myocytes et de la reparation des tissus myocardiques
US6265150B1 (en) 1995-06-07 2001-07-24 Becton Dickinson & Company Phage antibodies
WO2009142525A2 (fr) * 2008-05-22 2009-11-26 Universidade De Coimbra Administration ciblée à destination d'affections et troubles humains
WO2011062997A2 (fr) 2009-11-17 2011-05-26 Musc Foundation For Research Development Anticorps monoclonaux humains pour nucléoline humaine
WO2014063205A1 (fr) 2012-10-26 2014-05-01 The University Of Queensland Utilisation d'inhibiteurs de l'endocytose et d'anticorps pour une thérapie anticancéreuse
WO2017011411A1 (fr) * 2015-07-10 2017-01-19 Ohio State Innovation Foundation Méthodes et compositions en rapport avec des immunoagents recombinants contre la nucléoline

Patent Citations (51)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3773919A (en) 1969-10-23 1973-11-20 Du Pont Polylactide-drug mixtures
US4485045A (en) 1981-07-06 1984-11-27 Research Corporation Synthetic phosphatidyl cholines useful in forming liposomes
US4544545A (en) 1983-06-20 1985-10-01 Trustees University Of Massachusetts Liposomes containing modified cholesterol for organ targeting
US4777127A (en) 1985-09-30 1988-10-11 Labsystems Oy Human retrovirus-related products and methods of diagnosing and treating conditions associated with said retrovirus
WO1987004462A1 (fr) 1986-01-23 1987-07-30 Celltech Limited Sequences d'adn recombinant, vecteurs les contenant et procede d'utilisation de ces sequences
US4704692A (en) 1986-09-02 1987-11-03 Ladner Robert C Computer based system and method for determining and displaying possible chemical structures for converting double- or multiple-chain polypeptides to single-chain polypeptides
GB2200651A (en) 1987-02-07 1988-08-10 Al Sumidaie Ayad Mohamed Khala A method of obtaining a retrovirus-containing fraction from retrovirus-containing cells
US5219740A (en) 1987-02-13 1993-06-15 Fred Hutchinson Cancer Research Center Retroviral gene transfer into diploid fibroblasts for gene therapy
US5648260A (en) 1987-03-18 1997-07-15 Scotgen Biopharmaceuticals Incorporated DNA encoding antibodies with altered effector functions
US5624821A (en) 1987-03-18 1997-04-29 Scotgen Biopharmaceuticals Incorporated Antibodies with altered effector functions
US4946778A (en) 1987-09-21 1990-08-07 Genex Corporation Single polypeptide chain binding molecules
US5422120A (en) 1988-05-30 1995-06-06 Depotech Corporation Heterovesicular liposomes
EP0345242A2 (fr) 1988-06-03 1989-12-06 Smithkline Biologicals S.A. Expression de protéines gag de rétrovirus dans les cellules eucaryotes
WO1990007936A1 (fr) 1989-01-23 1990-07-26 Chiron Corporation Therapies de recombinaison pour infections et troubles hyperproliferatifs
WO1990011092A1 (fr) 1989-03-21 1990-10-04 Vical, Inc. Expression de sequences de polynucleotides exogenes chez un vertebre
US5580859A (en) 1989-03-21 1996-12-03 Vical Incorporated Delivery of exogenous DNA sequences in a mammal
WO1991002805A2 (fr) 1989-08-18 1991-03-07 Viagene, Inc. Retrovirus de recombinaison apportant des constructions de vecteur a des cellules cibles
US5013556A (en) 1989-10-20 1991-05-07 Liposome Technology, Inc. Liposomes with enhanced circulation time
EP0524968A1 (fr) 1990-03-21 1993-02-03 Res Dev Foundation Liposomes heterovesiculaires.
WO1991014445A1 (fr) 1990-03-21 1991-10-03 Research Development Foundation Liposomes heterovesiculaires
US5580717A (en) 1990-05-01 1996-12-03 Affymax Technologies N.V. Recombinant library screening methods
WO1993003769A1 (fr) 1991-08-20 1993-03-04 THE UNITED STATES OF AMERICA, represented by THE SECRETARY, DEPARTEMENT OF HEALTH AND HUMAN SERVICES Transfert induit par adenovirus de genes vers la voie gastro-intestinale
US5565332A (en) 1991-09-23 1996-10-15 Medical Research Council Production of chimeric antibodies - a combinatorial approach
WO1993010218A1 (fr) 1991-11-14 1993-05-27 The United States Government As Represented By The Secretary Of The Department Of Health And Human Services Vecteurs comprenant des genes etrangers et des marqueurs selectifs negatifs
WO1993011230A1 (fr) 1991-12-02 1993-06-10 Dynal As Cellule souche modifiee de mammifere bloquant la replication virale
WO1993019191A1 (fr) 1992-03-16 1993-09-30 Centre National De La Recherche Scientifique Adenovirus recombinants defectifs exprimant des cytokines pour traitement antitumoral
US5733743A (en) 1992-03-24 1998-03-31 Cambridge Antibody Technology Limited Methods for producing members of specific binding pairs
WO1993025234A1 (fr) 1992-06-08 1993-12-23 The Regents Of The University Of California Procedes et compositions permettant de cibler des tissus specifiques
WO1993025698A1 (fr) 1992-06-10 1993-12-23 The United States Government As Represented By The Particules vecteurs resistantes a l'inactivation par le serum humain
WO1994003622A1 (fr) 1992-07-31 1994-02-17 Imperial College Of Science, Technology & Medicine Vecteurs retroviraux du type d, bases sur le virus du singe mason-pfizer
WO1994004678A1 (fr) 1992-08-21 1994-03-03 Casterman Cecile Immunoglobulines exemptes de chaines legeres
WO1994012649A2 (fr) 1992-12-03 1994-06-09 Genzyme Corporation Therapie genique de la fibrose kystique
US5981568A (en) 1993-01-28 1999-11-09 Neorx Corporation Therapeutic inhibitor of vascular smooth muscle cells
WO1994023697A1 (fr) 1993-04-22 1994-10-27 Depotech Corporation Liposomes de cyclodextrine encapsulant des composes pharmacologiques et leurs procedes d'utilisation
WO1994028938A1 (fr) 1993-06-07 1994-12-22 The Regents Of The University Of Michigan Vecteurs d'adenovirus pour therapie genique
WO1995000655A1 (fr) 1993-06-24 1995-01-05 Mc Master University Vecteurs a base d'adenovirus destines a la therapie genique
WO1995007994A2 (fr) 1993-09-15 1995-03-23 Viagene, Inc. Vecteurs composes d'alphavirus recombinants
US5814482A (en) 1993-09-15 1998-09-29 Dubensky, Jr.; Thomas W. Eukaryotic layered vector initiation systems
WO1995011984A2 (fr) 1993-10-25 1995-05-04 Canji, Inc. Vecteur recombinant d'adenovirus et procedes d'utilisation
WO1995013796A1 (fr) 1993-11-16 1995-05-26 Depotech Corporation Vesicules a taux controle de liberation des principes actifs
WO1995030763A2 (fr) 1994-05-09 1995-11-16 Chiron Viagene, Inc. Vecteurs retroviraux a taux de recombinaison reduit
WO1996017072A2 (fr) 1994-11-30 1996-06-06 Chiron Viagene, Inc. Vecteurs d'alphavirus de recombinaison
US6265150B1 (en) 1995-06-07 2001-07-24 Becton Dickinson & Company Phage antibodies
WO1997042338A1 (fr) 1996-05-06 1997-11-13 Chiron Corporation Vecteurs retroviraux sans croisement
WO1997049805A2 (fr) 1996-06-27 1997-12-31 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Molecules de reconnaissance ayant une interaction specifique avec le site actif ou la fissure d'une molecule cible
WO1999058572A1 (fr) 1998-05-08 1999-11-18 Cambridge University Technical Services Limited Molecules de liaison derivees d'immunoglobulines ne declenchant pas de lyse dependante du complement
WO2000053211A2 (fr) 1999-03-09 2000-09-14 University Of Southern California Methode de stimulation de la proliferation de myocytes et de la reparation des tissus myocardiques
WO2009142525A2 (fr) * 2008-05-22 2009-11-26 Universidade De Coimbra Administration ciblée à destination d'affections et troubles humains
WO2011062997A2 (fr) 2009-11-17 2011-05-26 Musc Foundation For Research Development Anticorps monoclonaux humains pour nucléoline humaine
WO2014063205A1 (fr) 2012-10-26 2014-05-01 The University Of Queensland Utilisation d'inhibiteurs de l'endocytose et d'anticorps pour une thérapie anticancéreuse
WO2017011411A1 (fr) * 2015-07-10 2017-01-19 Ohio State Innovation Foundation Méthodes et compositions en rapport avec des immunoagents recombinants contre la nucléoline

Non-Patent Citations (123)

* Cited by examiner, † Cited by third party
Title
"Animal Cell Culture", 1987
"Antibodies: a practical approach", 1988, IRL PRESS
"Cell and Tissue Culture: Laboratory Procedures", August 1993, J. WILEY AND SONS
"Cell Biology: A Laboratory Notebook", 1998, ACADEMIC PRESS
"Current Protocols in Immunology", 1991
"Current Protocols in Molecular Biology", 1987
"Current Protocols in Molecular Biology", JOHN WILEY & SONS, INC.
"Gene T ransfer Vectors for Mammalian Cells", 1987
"Handbook of Experimental Immunology"
"Methods in Enzymology", ACADEMIC PRESS, INC.
"Methods in Molecular Biology", HUMANA PRESS
"Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
"Monoclonal antibodies: a practical approach", 2000, OXFORD UNIVERSITY PRESS
"Oligonucleotide Synthesis", 1984
"PCR: The Polymerase Chain Reaction", 1994
"Short Protocols in Molecular Biology", 1999, WILEY AND SONS
"The Antibodies", 1995, HARWOOD ACADEMIC PUBLISHERS
"Using antibodies: a laboratory manual", 1999, COLD SPRING HARBOR LABORATORY PRESS
AI ET AL., TALANTA, vol. 118, 2014, pages 54 - 60
ALTSCHUL ET AL., J. MOL. BIOL., vol. 215, 1990, pages 403 - 10
ALTSCHUL ET AL., NUCLEIC ACIDS RES., vol. 25, no. 17, 1997, pages 3389 - 3402
ALTSCHUL, PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 5873 - 77
BALCA-SILVA J ET AL: "Nucleolin overexpression in glioblastoma stem-like cells enables targeted intracellular delivery and improves cytotoxicity", FEBS JOURNAL, WILEY-BLACKWELL PUBLISHING LTD, GB, vol. 283, no. Suppl. 1, Sp. Iss. SI, 31 August 2016 (2016-08-31), pages 298, XP009509683, ISSN: 1742-464X *
BATES ET AL., EXPERIMENTAL AND MOLECULAR PATHOLOGY, vol. 86, no. 3, 2009, pages 151 - 64
BIBEAU ET AL., J OURNAL OF CLINICAL ONCOLOGY, vol. 27, no. 7, 2009, pages 1122 - 1129
BIRD ET AL., SCIENCE, vol. 242, 1988, pages 423 - 426
BIRTALAN ET AL., MOLECULAR BIOSYSTEMS, vol. 6, 2010, pages 1186 - 1194
BREKKE; SANDLIE, NATURE REVIEWS DRUG DISCOVERY, vol. 2, no. 1, 2002, pages 52 - 62
BROWN, M. ET AL., CELL, vol. 49, 1987, pages 603 - 612
BUCK, D. W. ET AL., IN VITRO, vol. 18, 1982, pages 377 - 381
C. A. J ANEWAY; P. TRAVERS: "Immunobiology", 1997
CARTRON ET AL., BLOOD, vol. 99, no. 3, 2002, pages 754 - 758
CHIOU ET AL., GENE THERAPEUTICS: METHODS AND APPLICATIONS OF DIRECT GENE TRANSFER, 1994
CHOTHIA, C. ET AL., J. MOL. BIOL., vol. 196, 1987, pages 901 - 917
CHRISTIAN ET AL., THE JOURNAL OF CELL BIOLOGY, vol. 163, no. 4, 2003, pages 871 - 878
CHRISTIAN ET AL., THEJ OURNAL OF CELL BIOLOGY, vol. 163, no. 4, 2003, pages 871 - 878
CHRISTIAN S ET AL: "Nucleolin expressed at the cell surface is a marker of endothelial cells in angiogenic blood vessels", THE JOURNAL OF CELL BIOLOGY : JCB, THE ROCKEFELLER UNIVERSITY PRESS, US, vol. 163, no. 4, 24 November 2003 (2003-11-24), pages 871 - 878, XP002533863, ISSN: 0021-9525, DOI: 10.1083/JCB.200304132 *
CONNELLY, HUMAN GENE THERAPY, vol. 1, 1995, pages 185
CORNEL I SSEN ET AL., EJ NMMI RESEARCH, vol. 2, 2012, pages 9
CURIEL, HUM. GENE THER., vol. 3, 1992, pages 147
CURIEL, HUM. GENETHER., vol. 3, 1992, pages 147
DARIO PALMIERI ET AL: "Human anti-nucleolin recombinant immunoagent for cancer therapy", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 112, no. 30, 13 July 2015 (2015-07-13), US, pages 9418 - 9423, XP055421179, ISSN: 0027-8424, DOI: 10.1073/pnas.1507087112 *
DESTOUCHES ET AL., CANCER RESEARCH, vol. 71, no. 9, 2011, pages 3296 - 3305
DESTOUCHES ET AL., PLOS ONE, vol. 3, no. 6, 2008, pages e2518
EPSTEIN ET AL., PROC. NATL. ACAD. SCI. USA, vol. 82, 1985, pages 3688
FELLOUSE ET AL., PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 101, no. 34, 2004, pages 12467 - 12472
FINDEIS ET AL., TRENDS B I OTECHNOL., vol. 11, 1993, pages 202
GANCZ; FISHELSON, MOLECULAR IMMUNOLOGY, vol. 46, no. 14, 2009, pages 2794 - 2800
GOMES-DA-SILVA ET AL., INTERNATIONAL J OURNAL OF PHARMACEUTICS, vol. 434, no. 1 -2, 2012, pages 9 - 19
GOMES-DA-SILVA ET AL., NANOMEDICINE, vol. 8, no. 9, 2013, pages 1397 - 413
GOSSEN ET AL., NATL. ACAD. SCI. USA, vol. 89, 1992, pages 5547 - 5551
GOSSEN, M.; BUJARD, H., PROC. NATL. ACAD. SCI. USA, vol. 89, 1992, pages 5547 - 5551
GRAHAM; VANDER EB, VIROLOGY, vol. 52, 1973, pages 456 - 467
GREGORIO A ET AL: "Targeting cell-surface nucleolin in metastatic breast cancer", EUROPEAN JOURNAL OF CANCER, ELSEVIER, AMSTERDAM, NL, vol. 50, no. Suppl. 5, 30 June 2014 (2014-06-30), pages S180, XP009509681, ISSN: 0959-8049, DOI: 10.1016/S0959-8049(14)50657-0 *
GRIGORY A. EFIMOV ET AL: "Cell-type-restricted anti-cytokine therapy: TNF inhibition from one pathogenic source", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 113, no. 11, 2 March 2016 (2016-03-02), US, pages 3006 - 3011, XP055463138, ISSN: 0027-8424, DOI: 10.1073/pnas.1520175113 *
GUO ET AL., BIOMATERIALS, vol. 32, no. 31, 2011, pages 8010 - 8020
HARLOW; LANE: "Antibodies: A Laboratory Manual", 1998, C OL D SPRING H ARBOR L ABORATORY
HARLOW; LANE: "Using Antibodies, a Laboratory Manua", 1999, COLD SPRING HARBOR LABORATORY PRESS
HEATHER J. CHALFIN ET AL: "Nucleolin Staining May Aid in the Identification of Circulating Prostate Cancer Cells", CLINICAL GENITOURINARY CANCER, vol. 15, no. 3, 1 June 2017 (2017-06-01), US, pages e477 - e481, XP055526829, ISSN: 1558-7673, DOI: 10.1016/j.clgc.2016.12.004 *
HU ET AL., BIOMATERIALS, vol. 34, 2013, pages 1135 - 1145
HUSTON ET AL., PROC. NATL. A CAD. SCI. USA, vol. 85, 1988, pages 5879 - 5883
HWANG ET AL., PROC. NATL. ACAD. SCI. USA, vol. 77, 1980, pages 4030
J. P. MATHER; P. E. ROBERTS: "Introduction to Cell and Tissue Culture", 1998, PLENUM PRESS
JOLLY, CANCER GENE THERAPY, vol. 1, 1994, pages 51
JORDAN ET AL., NUCLEIC ACIDS RESEARCH, vol. 24, no. 4, 1996, pages 596 - 601
KABAT, E. A. ET AL.: "Proteins of Immunological Interest", 1991, U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES, NIH PUBLICATION
KAPLITT, NATURE GENETICS, vol. 6, 1994, pages 148
KARLIN; ALTSCHUL, PROC. NATL. ACAD. SCI. USA, vol. 87, 1990, pages 2264 - 68
KIMURA, HUMAN GENE THERAPY, vol. 5, 1994, pages 845
KOHLER, B.; M I I STEI N, C., NATURE, vol. 256, 1975, pages 495 - 497
LATORRE ET AL., NANOSCALE, vol. 6, 2014, pages 7436 - 7442
LI ET AL., BIOMATERIALS, vol. 35, no. 12, 2014, pages 3840 - 3850
M. BROWN ET AL., CELL, vol. 49, 1987, pages 603 - 612
M. GOSSEN ET AL., NATL. ACAD. SCI. USA, vol. 89, 1992, pages 5547 - 5551
M. LI ET AL., CELLULAR AND MOLECULAR IMMUNOLOGY, vol. 9, no. 1, 2012, pages 54 - 61
MAXIM A. NOSENKO ET AL: "VHH-Based Bispecific Antibodies Targeting Cytokine Production", FRONTIERS IN IMMUNOLOGY, vol. 8, 1 January 2017 (2017-01-01), pages 1073, XP055528297, DOI: 10.3389/fimmu.2017.01073 *
MCCAFFERTY ET AL., NATURE, vol. 348, 1990, pages 552 - 553
MORRISON ET AL., PROC. NAT. ACAD. SCI., vol. 81, 1984, pages 6851
MORRISON ET AL., PROC. NATL. ACAD. SCI. USA, vol. 81, 1984, pages 6851
MOSMANN, J OURNAL OF IMMUNOLOGICAL METHODS, vol. 65, no. 1-2, 1983, pages 55 - 63
MOURA ET AL., BREAST CANCER RESEARCH AND TREATMENT, vol. 133, no. 1, 2012, pages 61 - 73
MUSOLINO ET AL., JOURNAL OF CLINICAL ONCOLOGY, vol. 26, no. 11, 2008, pages 1789 - 1796
NEUBERGER ET AL., NATURE, vol. 312, 1984, pages 604
P. FINCH: "Antibodies", 1997
PAIVAETAL., CANCER GENETICS AND CYTOGENETICS, vol. 183, no. 1, 2008, pages 35 - 40
PALMIERI, D. ET AL., PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF A MERICA, vol. 112, no. 30, 2015, pages 9418 - 23
PHILIP, MOL. CELL. BIOL., vol. 14, 1994, pages 2411
PORKKA ET AL., PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 99, no. 11, 2002, pages 7444 - 7449
QUEEN ET AL., PROC. NATL. ACAD. SCI. USA, vol. 86, 1989, pages 10029 - 10033
REDDY ET AL., CLINICAL CANCER RESEARCH, vol. 12, 2006, pages 6677 - 6686
REMI NGTON: "The Science and Practice of Pharmacy", 2000, LIPPINCOTT WILLIAMS AND WILKINS
REMINGTON: "The Science and Practice of Pharmacy", 2000, LIPPINCOTT WILLIAMS AND WILKINS
REMINGTON: "The Science and Practice of Pharmacy", 2000, MACK PUBLISHING
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR PRESS
SCOTT ET AL., NATURE REVIEWS. CANCER, vol. 12, no. 4, 2012, pages 278 - 87
SHOCKELT, P ET AL., PROC. NATL. ACAD. SCI. USA, vol. 92, 1995, pages 6522 - 6526
SHOCKETT ET AL., PROC. NATL. ACAD. SCI. USA, vol. 92, 1995, pages 6522 - 6526
SOFIA ROMANO ET AL: "Anticancer activity and antibody-dependent cell-mediated cytotoxicity of novel anti-nucleolin antibodies", SCIENTIFIC REPORTS, vol. 8, no. 1, 10 May 2018 (2018-05-10), XP055526782, DOI: 10.1038/s41598-018-25816-8 *
SONG ET AL., MOLECULAR PHARMACEUTIC, vol. 10, no. 10, 2013, pages 3555 - 3563
SRIVASTAVA; POLLARD, THE FASE B J OURNAL, vol. 13, 1999, pages 1911 - 1922
TAKEDA ET AL., NATURE, vol. 314, 1984, pages 452
TAMURA ET AL., ANNALS OF ONCOLOGY, vol. 22, no. 6, 2011, pages 1302 - 1307
TANG ET AL., THEJOURNAL OF IMMUNOLOGY, vol. 179, no. 5, 2007, pages 2815 - 2823
TAYLOR ET AL., CANCER IMMUNOL RES, vol. 3, no. 5, 2015, pages 567 - 574
TORRE ET AL., A CANCER J OURNAL FOR CLINICIANS, vol. 65, no. 2, 2015, pages 87 - 108
V ASU ET AL., BLOOD, vol. 127, no. 23, 2016, pages 2879 - 2889
V ELDERS ET AL., BRITISHJ OURNAL OF CANCER, vol. 78, no. 4, 1998, pages 478 - 483
VALERIO-FERNANDES A ET AL: "Targeting nucleolin in lung cancer - an emerging strategy to overcome stroma-mediated anti-VEGF resistance", EUROPEAN JOURNAL OF CANCER, ELSEVIER, AMSTERDAM, NL, vol. 50, no. Suppl. 5, 30 June 2014 (2014-06-30), pages S145, XP009509682, ISSN: 0959-8049, DOI: 10.1016/S0959-8049(14)50533-3 *
VALERIO-FERNANDES ANGELA ET AL: "Targeting nucleolin: A potential strategy to overcome stroma-mediated bevacizumab resistance in lung cancer", CANCER RESEARCH, & 102ND ANNUAL MEETING OF THE AMERICAN-ASSOCIATION-FOR-CANCER-RESEARCH (AACR); ORLANDO, FL, USA; APRIL 02 -06, 2011, vol. 74, no. 19, Suppl. S, 30 September 2014 (2014-09-30), pages 3003, XP009509684, ISSN: 0008-5472, DOI: 10.1158/1538-7445.AM2014-3003 *
VERA MOURA ET AL: "Targeted and intracellular triggered delivery of therapeutics to cancer cells and the tumor microenvironment: impact on the treatment of breast cancer", BREAST CANCER RESEARCH AND TREATMENT, KLUWER ACADEMIC PUBLISHERS, BO, vol. 133, no. 1, 30 July 2011 (2011-07-30), pages 61 - 73, XP035047622, ISSN: 1573-7217, DOI: 10.1007/S10549-011-1688-7 *
WEINER ET AL., NATURE REVIEWS. IMMUNOLOGY, vol. 10, no. 5, 2010, pages 317 - 27
WINER ET AL., CANCER RESEARCH, vol. 70, no. 21, 2010, pages 8674 - 8683
WINTER ET AL., ANNU. REV. IMMUNOL., vol. 12, 1994, pages 433 - 455
WOFFENDIN, PROC. NATL. ACAD. SCI., vol. 91, 1994, pages 1581
WU ET AL., J. BIOL. CHEM, vol. 263, 1988, pages 621
WU ET AL., J. BIOL. CHEM, vol. 269, 1994, pages 542
WU ET AL., J. BIOL. CHEM., vol. 266, 1991, pages 338
WU, J. BIOL. CHEM, vol. 264, 1989, pages 16985
Y ANG ET AL., PLOS ONE, vol. 6, 2011, pages 6
Y AO ET AL., HUMAN GENE THERAPY
YAO, F. ET AL., HUMAN GENE THERAPY, vol. 9, 1998, pages 1939 - 1950
Z. LI ET AL., BIOMACROMOLECULES, vol. 13, 2012, pages 4257 - 4263
ZENKE ET AL., PROC. NATL. ACAD. SCI. USA, vol. 87, 1990, pages 3655

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* Cited by examiner, † Cited by third party
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US11186636B2 (en) 2017-04-21 2021-11-30 Amgen Inc. Anti-human TREM2 antibodies and uses thereof
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