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WO2017118613A1 - Anticorps bispécifiques ciblant cd73 humain - Google Patents

Anticorps bispécifiques ciblant cd73 humain Download PDF

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WO2017118613A1
WO2017118613A1 PCT/EP2017/050003 EP2017050003W WO2017118613A1 WO 2017118613 A1 WO2017118613 A1 WO 2017118613A1 EP 2017050003 W EP2017050003 W EP 2017050003W WO 2017118613 A1 WO2017118613 A1 WO 2017118613A1
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cancer
bispecific antibody
antibodies
antibody
binding site
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Henrik Ditzel
Odd Lilleng GAMMELGAARD
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Syddansk Universitet
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Syddansk Universitet
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell

Definitions

  • the present invention relates to a bispecific antibody targeting CD73.
  • the present invention relates to a bispecific antibody targeting different epitopes on CD73 or a bispecific antibody targeting an epitope on CD73 and an epitope on a different antigen.
  • CD73 is a 70 kDa GPI-anchored 5'-ecto-nucleotidase, which is overexpressed in several types of cancers, including triple-negative breast cancer (TNBC).
  • TNBC triple-negative breast cancer
  • the enzymatic activity of CD73 is known to dephosphorylate adenosine
  • AMP monophosphate
  • Overexpression of CD73 results in accumulation of adenosine in the tumour microenvironment, which suppress immune surveillance and induce angiogenesis and resistance towards chemotherapy. Increased levels of adenosine do also raise the migratory potential of cancer cells thereby enhancing metastasis. Additionally, non- enzymatic functions of CD73 affecting adhesion and migration have also been reported, which - when blocked - protect against spontaneous metastasis formation.
  • the mouse anti-human CD73 antibody called mlE9 is known to inhibit the enzymatic activity of CD73, but not to protect against experimental metastasis formation.
  • the mouse anti-human CD73 antibody called mAD2 does only weakly inhibit the enzymatic activity of CD73, but does protect against both spontaneous and experimental metastasis formation by a mechanism, which includes clustering and internalization of CD73 (Terp et al. Anti-Human CD73 Monoclonal Antibody Inhibits Metastasis Formation in Human Breast Cancer by Inducing Clustering and Internalization of CD73 Expressed on the Surface of Cancer Cells. The Journal of Immunology, 2013, 191 : 4165-4173).
  • Cancer targets such as CD73 may be overexpressed in cancerous tissue, but are not exclusively expressed in the diseased tissue. Antibodies targeting these cancer antigens will conseq uently also affect healthy tissue to some degree, potentially causing adverse side effects.
  • variable region of m l E9 and mAD2 were cloned onto a wild type or a HexaBody-mutated human IgG l backbone, respectively.
  • ADCC antibody dependent cell mediated cytotoxicity
  • the enzymatic inhibitory arm needs a secondary active arm to function in a non-static environment (like in the human body) .
  • the secondary arm can be designed so that the bispecific antibody only affects cells, which express both antigens.
  • dual-epitope targeting enhanced the potency of both internalization and ADCC as well as efficiency of drug- delivery.
  • Bispecific antibodies have several advantageous compared to e.g. a combination of two independent antibodies :
  • - Bispecific antibodies comprise both epitopes on one antibody, which may reduce production cost.
  • Bispecific antibodies can be designed with high cell-type specificity, as both targeting epitopes has to be present on the cell type for hig h avidity binding to occur, which is critical for delivery of cytotoxic agents
  • an object of the present invention relates to the provision of an alternative antibody for binding to CD73 with one or both arms.
  • one aspect of the invention relates to a bispecific antibody, which binds to CD73, the antibody comprising
  • an antigen binding site which binds specifically to CD73 and promotes internalization of CD73 (when CD73 is bound on a cell surface);
  • Another aspect of the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the bispecific antibody according to the invention and a
  • Figure 1 shows the effect of bispecific antibodies, combination of antibodies and individual monospecific antibodies, respectively, on the catalytic activity of CD73.
  • Figure 2 shows the effect of bispecific antibodies, combination of antibodies and individual monospecific antibodies, respectively, on internalization of CD73.
  • Figure 3 shows the ADCC activity of bispecific antibodies, combination of antibodies and individual monospecific antibodies.
  • Figure 4 shows the survival of cancer cells (MDA-MB-468) upon treatment with bispecific antibodies, combination of antibodies and individual monospecific antibodies used as vehicles for Duostatin3-delivery.
  • FIG. 5 shows the survival of cancer cells (M4A4) upon treatment with bispecific antibodies, combination of antibodies and individual monospecific antibodies used as vehicles for Duostatin3-delivery.
  • Figure 6 shows the survival of cancer cells (M4A4) upon short exposure treatment with bispecific antibodies, combination of antibodies and individual monospecific antibodies used as vehicles for Duostatin3-delivery.
  • Figures 7-8 show antibodies inhibiting the enzymatic activity of CD73 under various circumstances. Only antibody formats which include at least one lE9-fab arm significantly inhibits the enzymatic activity of CD73. Importantly, the functional monovalent (lE9xbl2) do inhibits the enzymatic activity, but is easily washed away, indicating that it will not stick to cells in vivo (exert inhibition). Both functional bivalent antibodies comprising at least one 1E9 arm (1E9 and AD2xlE9) significantly and potently inhibits the enzymatic activity on all cells lines and circumstances investigated. Bonferroni one-way ANOVA was used to calculate stastistical significant differences relative to bl2 treated cells, * P ⁇ 0.0005, ** P ⁇ 0.0001.
  • Figure 9 shows the saturated antibody deposition on two cancer cell lines (A375 and MDA-MB-231-Luc2).
  • Dual-epitope targeting strategies AD2xlE9 and AD2 + 1E9 deposits more antibody on the surface of cancer cells compared to the parental antibodies AD2 and 1E9, which again deposits more antibody than their functionally monovalent counterparts (AD2xbl2 and lE9xbl2).
  • the bispecific AD2xlE9 antibody consistently outperforms the combination of AD2 and 1E9 suggesting that the bispecific AD2xlE9 may exert higher antibody effector function activity.
  • an antibody is a protein that specifically binds a
  • Antibodies may particularly stem from the immune system of e.g. mammals, and may be directed towards antigens related to foreign bodies.
  • An antibody is an intact immunoglobulin having two light and two heavy chains.
  • a single isolated antibody or fragment may be originating from the non- limiting list of a polyclonal antibody, a monoclonal antibody, a synthetic antibody, a recombinant antibody, a chimeric antibody, a heterochimeric antibody, or a humanized antibody.
  • the term antibody is used both to refer to a homogeneous molecular mixture, or a mixture such as a serum product made up of a plurality of different molecular entities.
  • a monoclonal antibody is an antibody produced by a single clone of cells, such as a single clone of hybridoma cells.
  • a monoclonal antibody is therefore a single pure homogeneous type of antibody. All monoclonal antibodies produced from the same clone are identical and have the same antigen specificity.
  • the monoclonal antibodies herein specifically include “chimeric” antibodies
  • immunoglobulins in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.
  • chimeric antibodies are antibodies whose light and heavy chain genes have been constructed, typically by genetic engineering, from antibody variable and constant region genes belonging to different species.
  • antigen binding site refers to the portion of the antibody, which binds the antigen.
  • the antigen-binding site is found in the variable region, Fv, within the Fab region of an antibody.
  • Said variable region comprises a hypervariable region/complementarity determining region (CDR) which determines the specificity of the antibody against a specific antigen.
  • CDR hypervariable region/complementarity determining region
  • first antigen binding site and second antigen binding site refers to two individual antigen-binding sites of a single antibody.
  • a bispecific antibody refers to an antibody, which comprises specificity against two different antigens or epitopes.
  • the bispecific functionality enables the bispecific antibody to interfere with multiple surface receptors or ligands associated with processes or diseases, such as cancer, proliferation or inflammation.
  • the two epitopes may be present in the same molecule (such as a cell surface protein, such as CD73), be present on two different molecules, being present on the same cell, or the epitopes being present in two different molecules not present on the same cell.
  • the last option may be relevant for the case where one of the binding arms of the bispecific antibody is recruiting (has affinity for) e.g. an immune cell, a carbohydrate or a drug.
  • the immune cell is a T-cell, such as a CD8-positve T-cell.
  • a vector refers to an artificially constructed plasmid or a virus that may be used for genetic engineering, such as introduction of specific genes into a target cell and expression of proteins encoded by these genes.
  • epitope refers to the antigenic determinant recognized by the CDRs of the antibody.
  • epitope refers to that portion of any molecule capable of being recognized by, and bound by, an antibody.
  • epitopes consist of chemically active surface groupings of molecules, for example, amino acids and/or sugar side chains, and have specific three-dimensional structural characteristics as well as specific charge characteristics.
  • sequence identity is a measure of identity between proteins at the amino acid level and a measure of identity between nucleic acids at nucleotide level.
  • the protein sequence identity may be determined by comparing the amino acid sequence in a given position in each sequence when the sequences are aligned.
  • nucleic acid sequence identity may be determined by comparing the nucleotide sequence in a given position in each sequence when the sequences are aligned. To determine the percentage identity of two nucleic acid sequences or of two amino acids, the sequences are aligned for optimal comparison purposes (e.g ., gaps may be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared .
  • the two seq uences are the same length .
  • Gapped BLAST may be utilised .
  • PSI-Blast may be used to perform an iterated search, which detects distant relationships between molecules.
  • the default parameters of the respective programs may be used . See http ://www. ncbi . nlm . nih .gov.
  • sequence identity may be calculated after the sequences have been aligned e.g . by the BLAST program in the EMBL database (www.ncbi . nlm .gov/cgi-bin/BLAST) .
  • the default settings with respect to e.g . "scoring matrix” and "gap penalty” may be used for alig nment.
  • the BLASTN and PSI BLAST default settings may be advantageous.
  • the percentage identity between two sequences may be determined using techniques similar to those described above, with or without allowing gaps. In calculating percentage identity, only exact matches are counted . Active arm
  • an "active arm” refers to a variable region of a single arm of a bispecific antibody, which binds an epitope (e.g. on the same target cell as the other arm of the bispecific antibody).
  • the second arm would be said to be active if it targets a different epitope on CD73 (or e.g. a different antigen on the same target cell).
  • bl2 constitutes an inactive arm, because its antigen is not found on the target cancer cell.
  • inhibitor of catalytic activity refers to a decrease in the catalytic activity of CD73.
  • the catalytic activity of CD73 is determined by a colorimetric measurement of the turnover of AMP to adenosine and inorganic phosphate as described in example 2.
  • antigen internalization refers to the action in which CD73 localized on the cell surface is being absorbed into the cell by an internalization process, such as receptor-mediated endocytosis. Antigen internalization is measured by incubating target cells of the present invention at either 4 or 37 °C and subsequently stain remaining surface-bound CD73 with an antibody with a fluorophore attached directed against the antibodies of the present invention, as described in example 3.
  • CD73 also known as 5'-nucleotidase (5'-NT) or ecto-5'- nucleotidase, is an enzyme that in humans are encoded by the NT5E gene.
  • CD73 converts AMP to adenosine and inorganic phosphate.
  • P-L1 Programmed Death Ligand 1
  • CD274 also known as CD274 or B7-H1
  • B7-H1 is a protein encoded by the CD274 gene.
  • the multiple mechanisms of action and high activity of the bispecific antibody make it feasible to investigate the efficiency of dual-epitope targeting of CD73 in combination with other therapeutic compounds.
  • the present invention relates to bispecific antibodies binding to at least CD73.
  • a first aspect of the invention relates to an (isolated) bispecific antibody, which binds to (human) CD73, the antibody comprising
  • an antigen binding site which binds specifically to CD73 and promotes internalization of CD73 (when CD73 is bound on a cell surface);
  • a second antigen binding site (2) which is different from the first antigen binding site (1A or IB).
  • the invention relates to a bispecific antibody, which binds to (human) CD73, the antibody comprising a first antigen binding site (1), which is an antigen binding site (1), which binds specifically to CD73; and
  • a second antigen binding site (2) which is different from the first antigen binding site (1).
  • such antibodies have several advantages compared to a mixture of two individual antibodies comprising the same antige binding sites, such as higher specificity.
  • the antibody is isolated .
  • the CD73 is human CD73, since it is the most relevant seen from a medical point of view.
  • the antibody should have a binding site promoting enzymatic inhibition of CD73,
  • said first antigen binding site ( 1) specifically binds to (human) CD73 and inhibits the catalytic activity of CD73.
  • the first binding site corresponds to ( 1A) .
  • the mouse anti-human CD73 antibody called m lE9 is known to inhibit the enzymatic activity of CD73, but not to protect against experimental metastasis formation (Terp et al . Anti-Human CD73 Monoclonal Antibody Inhibits Metastasis Formation in Human Breast Cancer by Inducing Clustering and Internalization of CD73 Expressed on the Surface of Cancer Cells. The Journal of Immunology, 2013, 191 : 4165-4173) .
  • the antibody should have a binding site promoting just that.
  • said first antigen binding site ( 1) specifically binds to (human) CD73 and promotes antigen internalization of CD73 (when CD73 is bound on a cell surface) .
  • the first binding site corresponds to ( I B) .
  • the mouse anti-human CD73 antibody called mAD2 does only weakly inhibit the enzymatic activity of CD73, but does protect against both spontaneous and experimental metastasis formation by a
  • said first antigen binding site ( 1) specifically binds to (human) CD73 and promotes antigen internalization of CD73 (when CD73 is bound on a cell surface)
  • said second antigen binding site (2) specifically binds to (human) CD73 and inhibits the catalytic activity of CD73.
  • the antibodies of the invention may be particular relevant when the two antigen binding sites, target two epitopes (such as cell surface proteins) present on the same cell (such as cancer cells). These epitopes may be on the same protein or different proteins.
  • the epitope for said second antigen binding site (2) is present on a cell also expressing CD73, preferably on the cell surface.
  • the antigen for the second antigen binding site is on a cell surface protein .
  • the epitope for said second antigen binding site (2) is present on a cell, preferably present on a cell also expressing CD73.
  • the antigen for the second antigen-binding site is a protein, carbohydrate or chemical substance.
  • said second antigen binding site (2) specifically binds to PD-L1, PD-L2, DR4, DR5, Tissue-factor, GITR, CTLA-4, CD137, TIM3, LAG3, CD40, EGFR, HER2, CD25, CD4, or CD39.
  • PD-L1 PD-L2, DR4, DR5
  • Tissue-factor GITR, CTLA-4, CD137, TIM3, LAG3, CD40, EGFR, HER2, CD25, CD4, or CD39.
  • PD-L1 PD-L1.
  • the epitope for the first antigen binding site (1) and the epitope for the second antigen-binding site (2) are on a cancer cell, preferably the same cancer cell .
  • the epitope for the first antigen binding site (1) and the epitope for the second antigen-binding site (2) are on a single cell, such as a cancer cell .
  • the antigen binding sites may also be defined by specific sequences derived from the antibodies m lE9 and mAD2,
  • the first antigen binding site (1) is selected from any of SEQ ID NO : 1-4 OR a sequence having at least 90% sequence identity to any of SEQ ID NO : 1-4, such as at least 95% sequence identity, such as at least 97% sequence identity, or such as at least 99% sequence identity.
  • the second antigen binding site (2) is selected from SEQ ID NO : 1-4 OR a sequence having at least 90% sequence identity to SEQ ID NO : 1-4 such as at least 95% sequence identity, such as at least 97% sequence identity, or such as at least 99% sequence identity,
  • said second antigen binding site (2) is different from the first antigen binding site (1), and optionally, with the proviso that when the first antigen binding site is SEQ ID NO: 1, the second antigen binding site is not SEQ ID NO : 2 (and vice versa), and when the first antigen binding site is SEQ ID NO: 3, the second antigen binding site is not SEQ ID NO : 4 (and vice versa).
  • the first antigen-binding site (1) is the variable region/light chain from the mouse anti-human CD73 antibody mAD2.
  • the second antigen-binding site (2) is the variable region/light chain from the mouse anti-human CD73 antibody mlE9.
  • bispecific antibody comprises a human backbone, such as an IgGl backbone or a hexabody backbone.
  • the antibodies of the invention comprise a human Fc region from any isotype, either IgG, IgM, IgA, IgE, or IgD.
  • the Fc region is from human IgG, such as IgGl, IgG2, IgG3, or IgG4.
  • the bispecific antibody is selected from the group consisting of monoclonal antibodies, conjugates, non-immunoglubulins, fusion proteins, polyclonal antibodies, antibodies wherein the heavy chain and the light chain are connected by a flexible linker, Fv molecules, Fab fragments, Fab' fragments, F(ab') 2 molecules, fully human antibodies, humanized antibodies, and chimeric antibodies.
  • said bispecific antibody comprises at least two antigen binding sites positioned on different polypeptide chain, coupled through a linker, such as one or more disulfide bridges. It may however also be possible to have the antigen binding sites are positioned on a single polypeptide chain.
  • bispecific antibody comprises more than one binding site for each epitope.
  • the bispecific antibody comprises one or more first antigen binding sites (1), such as 2-6, such as 2-4, or such as 2-3 or such as 2 first antigen binding sites (1).
  • the bispecific antibody comprises one or more second antigen binding sites (2), such as 2-6, such as 2-4, or such as 2-3 or such as 2 second antigen binding sites (2).
  • the antibody only comprises one binding site for each epitope.
  • the bispecific antibody is a monoclonal bispecific antibody, or a biclonal bispecific antibody.
  • the antibody of the invention may be further coupled to a drug.
  • the antibody is further coupled to a drug. In example 5, such coupling is shown.
  • the drug may be coupled to the antibody in different ways.
  • the drug in an
  • the coupling between the antibody and the drug is selected from the group consisting of a linker, a covalent coupling, a non-covalent coupling, and a ionic coupling, preferably, a covalent coupling.
  • the drug is selected from the group consisting of anticancer drugs, radionuclides, tubulin inhibitors, DNA alkylating agents, small-molecule drugs, Maytansines, Auristatins, Duostatins, Calicheamicins, anthracyclines, taxanes, and derivatives thereof. In example 5, a duostatin is tested.
  • the antibodies of the invention may be encoded by one or more nucleic acid molecules.
  • an aspect of the invention relates to one or more isolated nucleic acid molecules encoding a bispecific antibody according to the invention, preferably one nucleic acid.
  • nucleic acids according to the invention may be positioned in a vector.
  • a further aspect of the invention relates to one or more vectors comprising the one or more nucleic acid molecules according to the invention, preferably one vector.
  • nucleic acids and vectors according to the invention may be positioned in a cell line.
  • a further aspect relates to a cell line comprising the one or more vectors according and/or the one or more isolated nucleic acid molecules according to the invention.
  • the invention also relates to (pharmaceutical) compositions.
  • a (pharmaceutical) composition comprising the bispecific antibody according to the invention and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is for use in the treatment of mammals, such as humans.
  • the invention relates to a composition according to the invention for use as a medicament.
  • the invention relates to a composition according to the invention for use as a medicament in the treatment of cancer.
  • the cancer is selected from the group consisting of bladder cancer, leukemia, glioma, glioblastoma, melanoma, ovarian cancer, thyroid cancer, esophageal cancer, gastric cancer, colon cancer, prostate cancer, breast cancer, head and neck cancer, and cancer exosomes.
  • the cancer is breast cancer.
  • the breast cancer is triple-negative breast cancer (TNBC) .
  • the invention relates to a method of inhibiting growth of a tumor in a subject, comprising administering to the subject a therapeutically effective amount of the bispecific antibody according to the invention and/or a composition according to the invention .
  • Bispecific antibodies was produced as described in Labrijn et al. ("Controlled Fab- arm exchange for the generation of stable bispecific IgGl. ", Nature protocols. 2014;9(10) : 2450-63). Briefly, CH3-domain matched antibodies were mixed 1 : 1 in a 15 ml tube, and the volume adjusted by addition of PBS. 2-mercaptoethylamine was added to the antibody mixture (75 mM final concentration) and the mixture was incubated for 5 minutes at room temperature while shaking . The mixture was subsequently incubated at 31 °C for 5 hours.
  • 3xl0 5 cells (MDA-MB-231 or MDA-MB-468) were resuspended in media (DMEM AQmedia (SIGMA, D0819) supplemented with 10 % FBS (Sigma-Aldrich, F7524) and 1 % Penicillin-Streptomycin (Sigma, P0781-100 ml)) supplemented with 10 Mg/ml of antibody as indicated in Figure 1.
  • Cells were incubated for 1 hour at 37 °C and were subsequently washed x3 in TBS and resuspended in 500 ⁇ _ glycine buffer (75 mM glycine, 5 mM MgSC , pH 7.4) with and without 0.2 mM AMP.
  • the antibodies AD2 and 1E9 would be functional when removing one of the binding arms.
  • the results showed that their capability to inhibit the catalytic activity of CD73 was significantly weakened when one of the binding arms were substituted with a non-functional arm (bl2) (see Figure 1, lanes AD2xbl2 DB and lE9xbl2 DB compared to lanes AD2 and 1E9 respectively).
  • bl2 non-functional arm
  • the bispecific antibody AD2xlE9 was constructed.
  • the bispecific antibody inhibits the catalytic activity of CD73 to a similar level as the combination (AD2 + 1E9 (1 : 1)). This is surprising taken into account the poor inhibition levels of the AD2 and 1E9 antibodies having one binding site substituted. Overall, the example demonstrates that if both arms of the antibody are active, then a single anti-CD73 arm is sufficient to inhibit the enzyme activity. This important finding is independent of the backbone, as both the wild type and HexaBodyTM mutated backbone are equally effective.
  • Example 3 promotion of internalization of CD73 mediated by bispecific antibodies
  • bispecific antibodies may e.g. course conformational alterations, thereby abolishing the intended binding effect. Similar, the removal of one functional arm of an antibody may also abolish or at minimize the binding effect of the antibody.
  • the present example was intended to elucidate the effect of altering one arm of the binding arms of the AD2 and 1E9 antibodies, especially in respect of promotion of internalization of CD73.
  • 2xl0 5 cells (MDA-MB-231 or MDA-MB-468) were resuspended in media (DMEM AQmedia (SIGMA, D0819) supplemented with 10 % FBS (Sigma-Aldrich, F7524) and 1 % Penicillin-Streptomycin (Sigma, P0781-100 ml)) supplemented with 10 g/ml antibody as indicated in Figure 2.
  • Cells were incubated for 90 minutes at either 4°C or 37°C, after which plates were washed x4 in ice cold PBS. Cells were subsequently stained with Alexa Fluor 488 goat anti-human IgG (Life
  • the bispecific antibody (AD2xlE9 DB) is a potent promotor of internalization of 15 CD73 ( Figure 2). This important finding is independent of the backbone, as both the wild type and HexaBodyTM mutated backbone are equally effective.
  • PBMCs Peripheral blood mononuclear cells
  • PBMCs were added at a target effector ratio of 1 : 100, and the plate was incubated at 37 °C for 4 hours. The supernatant was transferred to tubes and 35 measured for radiation. Cytotoxicity was calculated by subtracting the values in the absence of PBMCs relative to max release (cells incubated with 10 % Triton-X 100). The data represent average values of 3 wells per condition.
  • the EC50 values of AD2 and the dual-epitope targeting strategies are very low (between 0.1-2 ng/ml, Figure 3). Additionally, when cancer cells are fully saturated, the dual-epitope targeting results in very high levels of cancer cell death (approximately 80 %), which may be critical for efficient tumour
  • the dual-epitope targeting is therefore a potent effector of antibody dependent cell mediated cytotoxicity (ADCC).
  • ADCC antibody dependent cell mediated cytotoxicity
  • 5xl0 3 cells (MDA-MB-468, M4A4) was seeded in flat-bottomed 96 well plates, and incubated for 4 hours for attachment. Antibodies diluted in media supplemented with DuoStatin3 (final concentration 1 Mg/ml) was added. The plates were incubated for either 2 hours or 4 days at 37 °C. In the first case, Duostatin3 containing media was removed after two hours and fresh media added . The cells were subsequently incubated for 4 days at 37 °C. On day four cells were added 15 ⁇ Cell titer GLO (Promega) and incubated for 90 minutes at 37 °C. 100 ⁇ of supernatant was transferred to a white bottomed plate and luminescence was measured.
  • DuoStatin3 final concentration 1 Mg/ml
  • both dual-epitope targeting strategies are significantly more potent than the parental antibodies, which are significantly more potent than the functional monovalent antibodies.
  • These important findings indicate a synergetic effect of dual-epitope targeting, both when treated as a combination and a bispecific antibody.
  • the bispecific format ensures that both fab-arms reaches the same target and thereby avoid complications with bio-distribution.
  • the bispecific AD2xlE9 antibody was the only antibody, which killed a substantial fraction of cancer cells upon short toxin exposure. This difference in internalization kinetics may be very relevant in a physiological setting were antibodies are more prone to be washed away and thus need to exerts its function quickly.
  • Example 6 Bispecific antibodies with a first arm targeting CD73 and a second arm a different antigen on the same cell
  • Bispecific antibodies targeting CD73 with one arm and another antigen on the same cell with the other arm can be designed. Such an antibody do only attach strongly to cells which simultaneously express both antigens, and would thus be more cancer specific, while still blocking the cancer promoting functions of CD73 and the other antigen.
  • An example could be a bispecific antibody comprising the anti-CD73 arm from 1E9 and a PDL-1 targeting arm on the other. Such an antibody would only recognize cancer cells and antigen presenting cells, but no vital organs, and thus allow for much more aggressive dosing . Furthermore, the immune-releasing mechanism would only be exerted locally in the tumour, which may limit autoimmune complications.
  • Bispecific antibodies which target CD73 and other antigens, can be produced to potently inhibit several cancer promoting functions with enhanced specificity. This is supported by the fact that lE9xbl2 do inhibit the enzymatic activity of CD73 (bivalent binding is not necessary) but when the cells are washed lE9xbl2 detach from the cells ( Figures 7-8). Both of the antibodies 1E9 and AD2xlE9 bind bivalently to the cell and exert its anticancer functions even after extensive washing . Thus, the anticancer activity can be completely rescued when the other arm are functional.
  • Example 7 Antibody deposition
  • the amount of antibody being deposited on a cell controls the highest antibody effector functions activity possible. We therefore investigated and compared the antibody deposition capacity of each anti-CD73 antibody.
  • Cells (A375 or MDA-MB-213-Luc2+) were harvested by 5 mM EDTA treatment for 20 minutes. Cells were subsequently washed in media and seeded 1.5E5 cells/well in a 96 well plate. The plate was cooled on ice, added serial diluted antibodies as indicated in the figures and incubated for 30 minutes. Upon incubation cells were washed and stained with an Alexa Flour 488 labelled anti-human IgG (Life

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  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

La présente invention concerne un anticorps bispécifique ciblant CD73. En particulier, la présente invention concerne un anticorps bispécifique ciblant des épitopes différents sur CD73 ou un anticorps bispécifique ciblant un épitope sur CD73 et un épitope sur un antigène différent.
PCT/EP2017/050003 2016-01-08 2017-01-02 Anticorps bispécifiques ciblant cd73 humain Ceased WO2017118613A1 (fr)

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WO2019173692A3 (fr) * 2018-03-09 2019-11-14 Agenus Inc. Anticorps anti-cd73 et leurs procédés d'utilisation
EP3481869A4 (fr) * 2016-07-11 2020-02-26 Corvus Pharmaceuticals, Inc. Anticorps anti-cd73
WO2020146795A1 (fr) 2019-01-11 2020-07-16 Omeros Corporation Procédés et compositions pour le traitement du cancer
WO2021097223A2 (fr) 2019-11-15 2021-05-20 Genzyme Corporation Anticorps cd73 biparatopique
WO2021227307A1 (fr) * 2020-05-12 2021-11-18 普米斯生物技术(珠海)有限公司 Anticorps anti-cd73 et son utilisation
US11299550B2 (en) 2018-03-09 2022-04-12 Phanes Therapeutics, Inc. Anti-CD73 antibodies and uses thereof
WO2023201267A1 (fr) 2022-04-13 2023-10-19 Gilead Sciences, Inc. Polythérapie pour le traitement de cancers exprimant trop-2
WO2023236971A1 (fr) * 2022-06-08 2023-12-14 上海华奥泰生物药业股份有限公司 Thérapie de cocktail d'anticorps cd73
JP2023554326A (ja) * 2020-12-11 2023-12-27 上海華奥泰生物藥業股▲フン▼有限公司 Ｃｄ７３の抗原結合蛋白及びその使用
WO2024040195A1 (fr) 2022-08-17 2024-02-22 Capstan Therapeutics, Inc. Conditionnement pour l'ingénierie de cellules immunitaires in vivo
WO2024199468A1 (fr) * 2023-03-29 2024-10-03 中山康方生物医药有限公司 Anticorps bispécifique, composition pharmaceutique et utilisation
WO2024249954A1 (fr) 2023-05-31 2024-12-05 Capstan Therapeutics, Inc. Formulations et compositions de nanoparticules lipidiques
US12187806B2 (en) 2022-03-04 2025-01-07 Development Center For Biotechnology Anti-CD73 antibodies and use thereof
WO2025076113A1 (fr) 2023-10-05 2025-04-10 Capstan Therapeutics, Inc. Lipides cationiques ionisables à espacement conservé et nanoparticules lipidiques
WO2025076127A1 (fr) 2023-10-05 2025-04-10 Capstan Therapeutics, Inc. Lipides cationiques ionisables contraints et nanoparticules lipidiques
WO2025137640A1 (fr) 2023-12-22 2025-06-26 Gilead Sciences, Inc. Inhibiteurs azaspiro de wrn
WO2025179294A2 (fr) 2024-02-22 2025-08-28 Capstan Therapeutics, Inc. Amplification d'ingénierie immunitaire
WO2025217454A2 (fr) 2024-04-11 2025-10-16 Capstan Therapeutics, Inc. Lipides cationiques ionisables et nanoparticules lipidiques
WO2025217452A1 (fr) 2024-04-11 2025-10-16 Capstan Therapeutics, Inc. Lipides cationiques ionisables contraints et nanoparticules lipidiques
WO2025233264A1 (fr) 2024-05-07 2025-11-13 Innate Pharma Utilisation d'agents bloquant le cd73 en association avec des engageurs de lymphocytes t anti-cd20 x cd3

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US12351644B2 (en) 2016-07-11 2025-07-08 Corvus Pharmaceuticals, Inc. Anti-CD73 antibodies
EP3481869A4 (fr) * 2016-07-11 2020-02-26 Corvus Pharmaceuticals, Inc. Anticorps anti-cd73
JP7319992B2 (ja) 2018-03-09 2023-08-02 アジェナス インコーポレイテッド 抗ｃｄ７３抗体およびそれらの使用方法
WO2019173692A3 (fr) * 2018-03-09 2019-11-14 Agenus Inc. Anticorps anti-cd73 et leurs procédés d'utilisation
CN112074535B (zh) * 2018-03-09 2024-10-11 艾吉纳斯公司 抗cd73抗体及其使用方法
JP2021515559A (ja) * 2018-03-09 2021-06-24 アジェナス インコーポレイテッド 抗ｃｄ７３抗体およびそれらの使用方法
TWI823906B (zh) * 2018-03-09 2023-12-01 美商艾吉納斯公司 抗-cd73 抗體及其使用方法
CN112074535A (zh) * 2018-03-09 2020-12-11 艾吉纳斯公司 抗cd73抗体及其使用方法
US11299550B2 (en) 2018-03-09 2022-04-12 Phanes Therapeutics, Inc. Anti-CD73 antibodies and uses thereof
AU2019231791B2 (en) * 2018-03-09 2022-08-11 Agenus Inc. Anti-CD73 antibodies and methods of use thereof
US11692042B2 (en) 2018-03-09 2023-07-04 Agenus Inc. Anti-CD73 antibodies and methods of use thereof
WO2020146795A1 (fr) 2019-01-11 2020-07-16 Omeros Corporation Procédés et compositions pour le traitement du cancer
CN115003385A (zh) * 2019-11-15 2022-09-02 建新公司 双互补位cd73抗体
WO2021097223A2 (fr) 2019-11-15 2021-05-20 Genzyme Corporation Anticorps cd73 biparatopique
WO2021227306A1 (fr) * 2020-05-12 2021-11-18 普米斯生物技术(珠海)有限公司 Anticorps anti-cd73 et son utilisation
WO2021227307A1 (fr) * 2020-05-12 2021-11-18 普米斯生物技术(珠海)有限公司 Anticorps anti-cd73 et son utilisation
JP2023554326A (ja) * 2020-12-11 2023-12-27 上海華奥泰生物藥業股▲フン▼有限公司 Ｃｄ７３の抗原結合蛋白及びその使用
EP4261224A4 (fr) * 2020-12-11 2025-04-23 Shanghai Huaota Biopharmaceutical Co., Ltd. Protéine de liaison à l'antigène cd73 et son application
US12187806B2 (en) 2022-03-04 2025-01-07 Development Center For Biotechnology Anti-CD73 antibodies and use thereof
WO2023201267A1 (fr) 2022-04-13 2023-10-19 Gilead Sciences, Inc. Polythérapie pour le traitement de cancers exprimant trop-2
WO2023236971A1 (fr) * 2022-06-08 2023-12-14 上海华奥泰生物药业股份有限公司 Thérapie de cocktail d'anticorps cd73
WO2024040194A1 (fr) 2022-08-17 2024-02-22 Capstan Therapeutics, Inc. Conditionnement pour l'ingénierie de cellules immunitaires in vivo
WO2024040195A1 (fr) 2022-08-17 2024-02-22 Capstan Therapeutics, Inc. Conditionnement pour l'ingénierie de cellules immunitaires in vivo
WO2024199468A1 (fr) * 2023-03-29 2024-10-03 中山康方生物医药有限公司 Anticorps bispécifique, composition pharmaceutique et utilisation
WO2024249954A1 (fr) 2023-05-31 2024-12-05 Capstan Therapeutics, Inc. Formulations et compositions de nanoparticules lipidiques
WO2025076113A1 (fr) 2023-10-05 2025-04-10 Capstan Therapeutics, Inc. Lipides cationiques ionisables à espacement conservé et nanoparticules lipidiques
WO2025076127A1 (fr) 2023-10-05 2025-04-10 Capstan Therapeutics, Inc. Lipides cationiques ionisables contraints et nanoparticules lipidiques
WO2025137640A1 (fr) 2023-12-22 2025-06-26 Gilead Sciences, Inc. Inhibiteurs azaspiro de wrn
WO2025179294A2 (fr) 2024-02-22 2025-08-28 Capstan Therapeutics, Inc. Amplification d'ingénierie immunitaire
WO2025217454A2 (fr) 2024-04-11 2025-10-16 Capstan Therapeutics, Inc. Lipides cationiques ionisables et nanoparticules lipidiques
WO2025217452A1 (fr) 2024-04-11 2025-10-16 Capstan Therapeutics, Inc. Lipides cationiques ionisables contraints et nanoparticules lipidiques
WO2025233264A1 (fr) 2024-05-07 2025-11-13 Innate Pharma Utilisation d'agents bloquant le cd73 en association avec des engageurs de lymphocytes t anti-cd20 x cd3

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