EP3994169A1 - Immunoconjugates comprising a mutant interleukin-2 and an anti-cd8 antibody - Google Patents
Immunoconjugates comprising a mutant interleukin-2 and an anti-cd8 antibodyInfo
- Publication number
- EP3994169A1 EP3994169A1 EP20734407.8A EP20734407A EP3994169A1 EP 3994169 A1 EP3994169 A1 EP 3994169A1 EP 20734407 A EP20734407 A EP 20734407A EP 3994169 A1 EP3994169 A1 EP 3994169A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- amino acid
- domain
- immunoconjugate
- polypeptide
- seq
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2815—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD8
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/20—Interleukins [IL]
- A61K38/2013—IL-2
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/6425—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a receptor, e.g. CD4, a cell surface antigen, i.e. not a peptide ligand targeting the antigen, or a cell surface determinant, i.e. a part of the surface of a cell
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- 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
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/54—Interleukins [IL]
- C07K14/55—IL-2
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0684—Cells of the urinary tract or kidneys
- C12N5/0686—Kidney cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
Definitions
- the present invention generally relates to immunoconjugates, particularly immunoconjugates comprising a mutant interleukin-2 polypeptide and an antibody that binds to CD8, having a strong cis-targeting effect on CD8 + cells only.
- the invention relates to polynucleotide molecules encoding the immunoconjugates, and vectors and host cells comprising such polynucleotide molecules.
- the invention further relates to methods for producing the mutant immunoconjugates, pharmaceutical compositions comprising the same, and uses thereof.
- Interleukin-2 also known as T cell growth factor (TCGF)
- TCGF T cell growth factor
- TCGF T cell growth factor
- IL-2 mediates its action by binding to IL-2 receptors (IL-2R), which consist of up to three individual subunits, the different association of which can produce receptor forms that differ in their affinity to IL-2.
- IL-2R IL-2 receptors
- Association of the a (CD25), b (CD122), and g (y c , CD132) subunits results in a trimeric, high-affinity receptor for IL-2.
- Dimeric IL-2 receptor consisting of the b and g subunits is termed intermediate-affinity IL-2R.
- the a subunit forms the monomeric low affinity IL-2 receptor.
- the dimeric intermediate-affinity IL-2 receptor binds IL-2 with approximately 100-fold lower affinity than the trimeric high-affinity receptor
- both the dimeric and the trimeric IL-2 receptor variants are able to transmit signal upon IL-2 binding
- the a-subunit, CD25 is not essential for IL- 2 signalling. It confers high-affinity binding to its receptor, whereas the b subunit, CD 122, and the g-subunit are crucial for signal transduction (Krieg et al., Proc Natl Acad Sci 107, 11906-11 (2010)).
- Trimeric IL-2 receptors including CD25 are expressed by (resting) CD4 + forkhead box P3 (FoxP3) + regulatory T (Treg) cells. They are also transiently induced on conventional activated T cells, whereas in the resting state these cells express only dimeric IL-2 receptors. Treg cells consistently express the highest level of CD25 in vivo (Fontenot et al., Nature Immunol 6, 1142- 51 (2005)).
- IL-2 is synthesized mainly by activated T-cells, in particular CD4 + helper T cells. It stimulates the proliferation and differentiation of T cells, induces the generation of cytotoxic T lymphocytes (CTLs) and the differentiation of peripheral blood lymphocytes to cytotoxic cells and lymphokine-activated killer (LAK) cells, promotes cytokine and cytolytic molecule expression by T cells, facilitates the proliferation and differentiation of B-cells and the synthesis of immunoglobulin by B-cells, and stimulates the generation, proliferation and activation of natural killer (NK) cells (reviewed e.g. in Waldmann, Nat Rev Immunol 6, 595-601 (2009); Olejniczak and Kasprzak, Med Sci Monit 14, RA179-89 (2008); Malek, Annu Rev Immunol 26, 453-79 (2008)).
- CTLs cytotoxic T lymphocytes
- LAK lymphokine-activated killer
- IL-2 lymphocyte populations in vivo and to increase the effector functions of these cells confers antitumor effects to IL-2, making IL-2 immunotherapy an attractive treatment option for certain metastatic cancers. Consequently, high-dose IL-2 treatment has been approved for use in patients with metastatic renal-cell carcinoma and malignant melanoma.
- IL-2 has a dual function in the immune response in that it not only mediates expansion and activity of effector cells, but also is crucially involved in maintaining peripheral immune tolerance.
- AICD IL-2 induced activation-induced cell death
- T cells A major mechanism underlying peripheral self-tolerance is IL-2 induced activation-induced cell death (AICD) in T cells.
- AICD is a process by which fully activated T cells undergo programmed cell death through engagement of cell surface-expressed death receptors such as CD95 (also known as Fas) or the TNF receptor.
- CD95 also known as Fas
- FasL Fas ligand
- TNF tumor necrosis factor
- IL-2 is also involved in the maintenance of peripheral CD4 + CD25 + regulatory T (Treg) cells (Fontenot et al., Nature Immunol 6, 1142-51 (2005); D’Cruz and Klein, Nature Immunol 6, 1152-59 (2005); Maloy and Powrie, Nature Immunol 6, 1171-72 (2005), which are also known as suppressor T cells. They suppress effector T cells from destroying their (self-)target, either through cell-cell contact by inhibiting T cell help and activation, or through release of immunosuppressive cytokines such as IL-10 or TGF-b. Depletion of Treg cells was shown to enhance IL-2 induced anti-tumor immunity (Imai et al., Cancer Sci 98, 416-23 (2007)).
- IL-2 is not optimal for inhibiting tumor growth, because in the presence of IL-2 either the CTLs generated might recognize the tumor as self and undergo AICD or the immune response might be inhibited by IL-2 dependent Treg cells.
- VLS vascular leak syndrome
- VLS Low-dose IL-2 regimens have been tested in patients to avoid VLS, however, at the expense of suboptimal therapeutic results.
- VLS was believed to be caused by the release of proinflammatory cytokines, such as tumor necrosis factor (TNF)-a from IL-2-activated NK cells, however it has recently been shown that IL-2-induced pulmonary edema resulted from direct binding of IL-2 to lung endothelial cells, which expressed low to intermediate levels of functional abg IL-2 receptors (Krieg et al., Proc Nat Acad Sci USA 107, 11906-11 (2010)).
- TNF tumor necrosis factor
- 2007/0036752 have substituted three residues of IL-2 (Asp20Thr, Asn88Arg, and Glnl26Asp) that contribute to affinity for the intermediate-affinity IL-2 receptor to reduce VLS.
- Gillies et al. (WO 2008/0034473) have also mutated the interface of IL-2 with CD25 by amino acid substitution Arg38Trp and Phe42Lys to reduce interaction with CD25 and activation of Treg cells for enhancing efficacy.
- Wittrup et al. (WO 2009/061853) have produced IL-2 mutants that have enhanced affinity to CD25, but do not activate the receptor, thus act as antagonists.
- the mutations introduced were aimed at disrupting the interaction with the b- and/or g-subunit of the receptor.
- a particular mutant IL-2 polypeptide designed to overcome the above-mentioned problems associated with IL-2 immunotherapy (toxicity caused by the induction of VLS, tumor tolerance caused by the induction of AICD, and immunosuppression caused by activation of Treg cells), is described in WO 2012/107417.
- Substitution of the phenylalanine residue at position 42 by alanine, the tyrosine residue at position 45 by alanine and the leucine residue at position 72 of IL-2 by glycine essentially abolishes binding of this mutant IL-2 polypeptide to the a-subunit of the IL-2 receptor (CD25).
- IL-2 immunotherapy may be improved by selectively targeting IL-2 to tumors, e.g. in the form of immunoconjugates comprising an antibody that binds to an antigen expressed on tumor cells.
- immunoconjugates comprising an antibody that binds to an antigen expressed on tumor cells.
- Tumors may be able, however, to escape such targeting by shedding, mutating or downregulating the target antigen of the antibody.
- tumor-targeted IL-2 may not come into optimal contact with effector cells such as cytotoxic T lymphocytes (CTLs), in tumor microenvironments that actively exclude lymphocytes.
- CTLs cytotoxic T lymphocytes
- An approach, which may circumvent the problems of tumor-targeting, is to target IL-2 directly to effector cells, in particular CTLs.
- Ghasemi et al. have described a fusion protein of IL-2 and an NKG2D binding protein (Ghashemi et al., Nat Comm (2016) 7, 12878), for targeting IL-2 to NKG2D-bearing cells such as natural killer (NK) cells.
- NK natural killer
- CD8 + cytotoxic lymphocytes have been consistently reported as having diagnostic and prognostic significance in various cancers.
- Antibodies that bind to CD8 are described e.g. in WO 2019/033043 A2.
- the present invention provides a novel approach of targeting a mutant form of IL-2 with advantageous properties for immunotherapy directly to immune effector cells, such as cytotoxic T lymphocytes, rather than tumor cells.
- Targeting to immune effector cells is achieved by conjugation of the mutant IL-2 molecule to an antibody that binds to CD8.
- the IL-2 mutant used in the present invention has been designed to overcome the problems associated with IL-2 immunotherapy, in particular toxicity caused by the induction of VLS, tumor tolerance caused by the induction of AICD, and immunosuppression caused by activation of Treg cells.
- targeting of the IL-2 mutant to immune effector cells may further increase the preferential activation of CTLs over immunosuppressive Treg cells.
- the antibodies binding to CD8 as disclosed herein have strong cis-targeting effects on CD8 + cells. Thus, they do not interfere with the interaction of the T cell antigen receptor (TCR) and the peptide bound major histocompatibility complex (pMHC). In this respect, the antibodies are non-functional.
- the invention provides an immunoconjugate comprising a mutant interleukin-2 (IL-2) polypetide and an antibody that binds to CD8, wherein the IL-2 polypeptide is a mutant IL-2 polypeptide comprising the amino acid substitutions F42A, Y45A and L72G (numbering relative to the human IL-2 sequence SEQ ID NO: 13).
- IL-2 interleukin-2
- the invention provides an immunoconjugate comprising a mutant interleukin- 2 (IL-2) polypetide and an antibody that binds to CD8, wherein the mutant IL-2 polypeptide is a human IL-2 molecule comprising the amino acid substitutions F42A, Y45A and L72G (numbering relative to the human IL-2 sequence SEQ ID NO: 13); and wherein the antibody comprises (a) a heavy chain variable region (VH) comprising a heavy chain complementary determining region (HCDR) 1 comprising the amino acid sequence of SEQ ID NO: 1, a HCDR 2 comprising the amino acid sequence of SEQ ID NO:2, a HCDR 3 comprising the amino acid sequence of SEQ ID NO:3, and (b) a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 comprising the amino acid sequence of SEQ ID NO:4, a LCDR 2 comprising the amino acid sequence of SEQ ID NO:5, and a LCDR 3 comprising the
- the invention procides an immunoconjugate comprising a mutant interleukin-2 (IL-2) polypetide and an antibody that binds to CD8, wherein the mutant IL-2 polypeptide is a human IL-2 molecule comprising the amino acid substitutions F42A, Y45A and L72G (numbering relative to the human IL-2 sequence SEQ ID NO: 13); and wherein the antibody comprises (a) a heavy chain variable region (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:7, and (b) a light chain variable region (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence of SEQ ID NO:8 or SEQ ID NO:29.
- VH heavy chain variable region
- VL light chain variable region
- the mutant IL-2 polypeptide further comprises the amino acid substitution T3A and/or the amino acid substitution C125A.
- the mutant IL-2 polypeptide comprises the sequence of SEQ ID NO: 14.
- the immunoconjugate comprises not more than one mutant IL-2 polypeptide.
- the antibody comprises an Fc domain composed of a first and a second subunit.
- the Fc domain is an IgG class, particularly an IgGl subclass, Fc domain and/or a human Fc domain.
- the antibody is an IgG class, particularly an IgGl subclass immunoglobulin.
- the Fc domain comprises a modification promoting the association of the first and the second subunit of the Fc domain.
- an amino acid residue in the CH3 domain of the first subunit of the Fc domain an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain of the first subunit which is positionable in a cavity within the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit is positionable.
- the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the second subunit of the Fc domain the tyrosine residue at position 407 is replaced with a valine residue (Y407V) and optionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numberings according to Kabat EU index).
- the mutant IL-2 polypeptide is fused at its amino-terminal amino acid to the carboxy-terminal amino acid of one of the subunits of the Fc domain, particularly the first subunit of the Fc domain, optionally through a linker peptide. 18.
- the linker linker peptide has the amino acid sequence of SEQ ID NO: 15.
- the Fc domain comprises one or more amino acid substitution that reduces binding to an Fc receptor, particularly an Fey receptor, and/or effector function, particularly antibody-dependent cell- mediated cytotoxicity (ADCC).
- said one or more amino acid substitution is at one or more position selected from the group of L234, L235, and P329 (Kabat EU index numbering).
- each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A and P329G (Kabat EU index numbering).
- the immunoconjugate comprises a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 9 or SEQ ID NO: 30, a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 10, and a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 11 or SEQ ID NO: 12.
- the immunoconjugate comprises a polypeptide comprising an amino acid sequence of SEQ ID NO:9 or SEQ ID NO:29, a polypeptide comprising an amino acid sequence of SEQ ID NO: 10, and a polypeptide comprising an amino acid sequence of SEQ ID NO: 11.
- the immunoconjugate comprises a polypeptide comprising an amino acid sequence of SEQ ID NO:9 or SEQ ID NO:29, a polypeptide comprising an amino acid sequence of SEQ ID NO: 10, and a polypeptide comprising an amino acid sequence of SEQ ID NO: 12.
- the immunoconjugate essentially consists of a mutant IL-2 polypeptide and an IgGl immunoglobulin molecule, joined by a linker sequence.
- the invention further provides one or more isolated polynucleotide encoding an immunoconjugate of the invention as described herein, one or more vector, particularly expression vector, comprising said polynucleotide(s) and host cells comprising said polynucleotide(s) or said vector(s).
- the invention further provides a pharmaceutical composition comprising an immunoconjugate of the invention and a pharmaceutically acceptable carrier, and methods of using an immunoconjugate of the invention.
- the invention encompasses an immunoconjugate according to the invention for use as a medicament, and for use in the treatment of a disease.
- said disease is cancer.
- an immunoconjugate according to the invention in the manufacture of a medicament for the treatment of a disease.
- said disease is cancer.
- a method of treating a disease in an individual comprising administering to said individual a therapeutically effective amount of a composition comprising the immunoconjugate according to the invention in a pharmaceutically acceptable form.
- said disease is cancer.
- a method of stimulating the immune system of an individual comprising administering to said individual an effective amount of a composition comprising the immunoconjugate according to the invention in a pharmaceutically acceptable form.
- interleukin-2 refers to any native IL-2 from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated.
- the term encompasses unprocessed IL-2 as well as any form of IL-2 that results from processing in the cell.
- the term also encompasses naturally occurring variants of IL-2, e.g. splice variants or allelic variants.
- the amino acid sequence of an exemplary human IL-2 is shown in SEQ ID NO: 13.
- Unprocessed human IL-2 additionally comprises an N- terminal 20 amino acid signal peptide having the sequence of SEQ ID NO: 19, which is absent in the mature IL-2 molecule.
- IL-2 mutant or "mutant IL-2 polypeptide” as used herein is intended to encompass any mutant forms of various forms of the IL-2 molecule including full-length IL-2, truncated forms of IL-2 and forms where IL-2 is linked to another molecule such as by fusion or chemical conjugation.
- Full-length when used in reference to IL-2 is intended to mean the mature, natural length IL-2 molecule.
- full-length human IL-2 refers to a molecule that has 133 amino acids (see e.g. SEQ ID NO: 13).
- the various forms of IL-2 mutants are characterized in having a at least one amino acid mutation affecting the interaction of IL-2 with CD25.
- an IL-2 mutant may be referred to herein as a mutant IL-2 peptide sequence, a mutant IL-2 polypeptide, a mutant IL-2 protein or a mutant IL-2 analog.
- Designation of various forms of IL-2 is herein made with respect to the sequence shown in SEQ ID NO: 13.
- Various designations may be used herein to indicate the same mutation.
- a mutation from phenylalanine at position 42 to alanine can be indicated as 42A, A42, A42, F42A, or Phe42Ala.
- a“human IL-2 molecule” as used herein is meant an IL-2 molecule comprising an amino acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95% or at least about 96% identical to the human IL-2 sequence of SEQ ID NO: 13. Particularly, the sequence identity is at least about 95%, more particularly at least about 96%.
- the human IL-2 molecule is a full-length IL-2 molecule.
- the term“amino acid mutation” as used herein is meant to encompass amino acid substitutions, deletions, insertions, and modifications.
- amino acid sequence deletions and insertions include amino- and/or carboxy-terminal deletions and insertions of amino acids.
- An example of a terminal deletion is the deletion of the alanine residue in position 1 of full-length human IL-2.
- Preferred amino acid mutations are amino acid substitutions.
- non-conservative amino acid substitutions i.e. replacing one amino acid with another amino acid having different structural and/or chemical properties, are particularly preferred.
- Preferred amino acid substitions include replacing a hydrophobic by a hydrophilic amino acid.
- Amino acid substitutions include replacement by non-naturally occurring amino acids or by naturally occurring amino acid derivatives of the twenty standard amino acids (e.g. 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5 -hydroxy lysine).
- Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis and the like. It is contemplated that methods of altering the side chain group of an amino acid by methods other than genetic engineering, such as chemical modification, may also be useful.
- a“wild-type” form of IL-2 is a form of IL-2 that is otherwise the same as the mutant IL-2 polypeptide except that the wild-type form has a wild-type amino acid at each amino acid position of the mutant IL-2 polypeptide.
- the IL-2 mutant is the full-length IL-2 (i.e. IL-2 not fused or conjugated to any other molecule)
- the wild-type form of this mutant is full-length native IL-2.
- the IL-2 mutant is a fusion between IL-2 and another polypeptide encoded downstream of IL-2 (e.g.
- the wild-type form of this IL-2 mutant is IL-2 with a wild-type amino acid sequence, fused to the same downstream polypeptide. Furthermore, if the IL-2 mutant is a truncated form of IL-2 (the mutated or modified sequence within the non-truncated portion of IL-2) then the wild-type form of this IL-2 mutant is a similarly truncated IL-2 that has a wild-type sequence.
- wild-type encompasses forms of IL-2 comprising one or more amino acid mutation that does not affect IL-2 receptor binding compared to the naturally occurring, native IL-2, such as e.g. a substitution of cysteine at a position corresponding to residue 125 of human IL-2 to alanine.
- wild-type IL-2 for -l i the purpose of the present invention comprises the amino acid substitution C125A (see SEQ ID NO: 20).
- the wild-type IL-2 polypeptide to which the mutant IL-2 polypeptide is compared comprises the amino acid sequence of SEQ ID NO: 13. In other embodiments the wild-type IL-2 polypeptide to which the mutant IL-2 polypeptide is compared comprises the amino acid sequence of SEQ ID NO: 20.
- CD25 or“a-subunit of the IL-2 receptor” as used herein, refers to any native CD25 from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated.
- the term encompasses“full-length”, unprocessed CD25 as well as any form of CD25 that results from processing in the cell.
- the term also encompasses naturally occurring variants of CD25, e.g. splice variants or allelic variants.
- CD25 is human CD25.
- the amino acid sequence of human CD25 is found e.g. in UniProt entry no. P01589 (version 185).
- high-affinity IL-2 receptor refers to the heterotrimeric form of the IL- 2 receptor, consisting of the receptor g-subunit (also known as common cytokine receptor y- subunit, y c , or CD132, see UniProt entry no. P14784 (version 192)), the receptor b-subunit (also known as CD122 or p70, see UniProt entry no. P31785 (version 197)) and the receptor a-subunit (also known as CD25 or p55, see UniProt entry no. P01589 (version 185)).
- the receptor g-subunit also known as common cytokine receptor y- subunit, y c , or CD132, see UniProt entry no. P14784 (version 192)
- the receptor b-subunit also known as CD122 or p70, see UniProt entry no. P31785 (version 197)
- the receptor a-subunit also known as CD25 or p55, see UniProt entry no. P0
- intermediate-affinity IL-2 receptor refers to the IL-2 receptor including only the y- subunit and the b-subunit, without the a-subunit (for a review see e.g. Olejniczak and Kasprzak, Med Sci Monit 14, RA179-189 (2008)).
- Binding affinity refers to intrinsic binding affinity which reflects a 1: 1 interaction between members of a binding pair (e.g., an antigen binding moiety and an antigen, or a receptor and its ligand).
- the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD), which is the ratio of dissociation and association rate constants (k 0 ff and k 0 n, respectively).
- equivalent affinities may comprise different rate constants, as long as the ratio of the rate constants remains the same.
- Affinity can be measured by well established methods known in the art, including those described herein.
- a particular method for measuring affinity is Surface Plasmon Resonance (SPR).
- SPR Surface Plasmon Resonance
- the affinity of the mutant or wild-type IL-2 polypeptide for various forms of the IL-2 receptor can be determined in accordance with the method set forth in the WO 2012/107417 by surface plasmon resonance (SPR), using standard instrumentation such as a BIAcore instrument (GE Healthcare) and receptor subunits such as may be obtained by recombinant expression (see e.g. Shanafelt et al., Nature Biotechnol 18, 1197-1202 (2000)).
- binding affinity of IL-2 mutants for different forms of the IL-2 receptor may be evaluated using cell lines known to express one or the other such form of the receptor. Specific illustrative and exemplary embodiments for measuring binding affinity are described
- Treg cells are characterized by expression of the a-subunit of the IL-2 receptor (CD25) and the transcription factor forkhead box P3 (FOXP3) (Sakaguchi, Annu Rev Immunol 22, 531-62 (2004)) and play a critical role in the induction and maintenance of peripheral self-tolerance to antigens, including those expressed by tumors. Treg cells require IL-2 for their function and development and induction of their suppressive characteristics.
- effector cells refers to a population of lymphocytes that mediate the cytotoxic effects of IL-2. Effector cells include effector T cells such as CD8 + cytotoxic T cells, NK cells, lymphokine-activated killer (LAK) cells and macrophages/monocytes.
- effector T cells such as CD8 + cytotoxic T cells, NK cells, lymphokine-activated killer (LAK) cells and macrophages/monocytes.
- telomere binding is meant that the binding is selective for the antigen and can be discriminated from unwanted or non-specific interactions.
- a specific antigen e.g. CD8
- ELISA enzyme-linked immunosorbent assay
- SPR surface plasmon resonance
- the extent of binding of an antibody to an unrelated protein is less than about 10% of the binding of the antibody to the antigen as measured, e.g., by SPR.
- the antibody comprised in the immunoconjugate described herein specifically binds to CD8.
- polypeptide refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds).
- polypeptide refers to any chain of two or more amino acids, and does not refer to a specific length of the product.
- peptides, dipeptides, tripeptides, oligopeptides, "protein”, “amino acid chain”, or any other term used to refer to a chain of two or more amino acids are included within the definition of "polypeptide”, and the term “polypeptide” may be used instead of, or interchangeably with any of these terms.
- polypeptide is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids.
- a polypeptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It may be generated in any manner, including by chemical synthesis.
- Polypeptides may have a defined three-dimensional structure, although they do not necessarily have such structure. Polypeptides with a defined three-dimensional structure are referred to as folded, and polypeptides which do not possess a defined three- dimensional structure, but rather can adopt a large number of different conformations, and are referred to as unfolded.
- an “isolated” polypeptide or a variant, or derivative thereof is intended a polypeptide that is not in its natural milieu. No particular level of purification is required.
- an isolated polypeptide can be removed from its native or natural environment.
- Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purpose of the invention, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.
- Percent (%) amino acid sequence identity with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or the FASTA program package.
- % amino acid sequence identity values are generated using the ggsearch program of the FASTA package version 36.3.8c or later with a BLOSUM50 comparison matrix.
- the FASTA program package was authored by W. R. Pearson and D. J. Lipman (1988),“Improved Tools for Biological Sequence Analysis”, PNAS 85:2444-2448; W. R. Pearson (1996)“Effective protein sequence comparison” Meth. Enzymol. 266:227- 258; and Pearson et. al.
- Genomics 46:24-36 is publicly available from http://fasta.bioch.virginia.edu/fasta_www2/fasta_down.shtml.
- polynucleotide refers to an isolated nucleic acid molecule or construct, e.g. messenger RNA (mRNA), virally-derived RNA, or plasmid DNA (pDNA).
- mRNA messenger RNA
- pDNA virally-derived RNA
- a polynucleotide may comprise a conventional phosphodiester bond or a non-conventional bond (e.g. an amide bond, such as found in peptide nucleic acids (PNA).
- PNA peptide nucleic acids
- nucleic acid molecule refers to any one or more nucleic acid segments, e.g. DNA or RNA fragments, present in a polynucleotide.
- isolated nucleic acid molecule or polynucleotide is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment.
- a recombinant polynucleotide encoding a polypeptide contained in a vector is considered isolated for the purposes of the present invention.
- Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution.
- An isolated polynucleotide includes a polynucleotide molecule contained in cells that ordinarily contain the polynucleotide molecule, but the polynucleotide molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.
- Isolated RNA molecules include in vivo or in vitro RNA transcripts of the present invention, as well as positive and negative strand forms, and double-stranded forms. Isolated polynucleotides or nucleic acids according to the present invention further include such molecules produced synthetically.
- a polynucleotide or a nucleic acid may be or may include a regulatory element such as a promoter, ribosome binding site, or a transcription terminator.
- isolated polynucleotide (or nucleic acid) encoding [e.g. an immunoconjugate of the invention]” refers to one or more polynucleotide molecules encoding antibody heavy and light chains and/or IL-2 polypeptides (or fragments thereof), including such polynucleotide molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.
- expression cassette refers to a polynucleotide generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a target cell.
- the recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment.
- the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter.
- the expression cassette comprises polynucleotide sequences that encode immunoconjugates of the invention or fragments thereof.
- the term“vector” or "expression vector” refers to a DNA molecule that is used to introduce and direct the expression of a specific gene to which it is operably associated in a cell.
- the term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced.
- the expression vector of the present invention comprises an expression cassette. Expression vectors allow transcription of large amounts of stable mRNA. Once the expression vector is inside the cell, the ribonucleic acid molecule or protein that is encoded by the gene is produced by the cellular transcription and/or translation machinery.
- the expression vector of the invention comprises an expression cassette that comprises polynucleotide sequences that encode immunoconjugates of the invention or fragments thereof.
- host cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
- Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
- a host cell is any type of cellular system that can be used to generate the immunoconjugates of the present invention.
- Host cells include cultured cells, e.g.
- mammalian cultured cells such as HEK cells, CHO cells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, insect cells, and plant cells, to name only a few, but also cells comprised within a transgenic animal, transgenic plant or cultured plant or animal tissue.
- antibody herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, monovalent antibodies (e.g., one- armed antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity, i.e., binding to CD8 (such as a human CD8, a cynomolgous CD8, and/or a rhesus CD8).
- CD8 such as a human CD8, a cynomolgous CD8, and/or a rhesus CD8.
- monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e. the individual antibodies comprised in the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts.
- polyclonal antibody preparations typically include different antibodies directed against different determinants (epitopes)
- each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.
- the modifier“monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
- the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.
- an "isolated” antibody is one which has been separated from a component of its natural environment, i.e. that is not in its natural milieu. No particular level of purification is required.
- an isolated antibody can be removed from its native or natural environment.
- Recombinantly produced antibodies expressed in host cells are considered isolated for the purpose of the invention, as are native or recombinant antibodies which have been separated, fractionated, or partially or substantially purified by any suitable technique. As such, the immunoconjugates of the present invention are isolated.
- an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) methods.
- electrophoretic e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis
- chromatographic e.g., ion exchange or reverse phase HPLC
- antibody fragment refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.
- antibody fragments include but are not limited to Fv, Fab, Fab', Fab’-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g. scFv), and single-domain antibodies.
- scFv single-chain antibody molecules
- Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific.
- Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody.
- a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see e.g. U.S. Patent No. 6,248,516 Bl).
- Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g. E. cob or phage), as described herein.
- immunoglobulin molecule refers to a protein having the structure of a naturally occurring antibody.
- immunoglobulins of the IgG class are heterotetrameric glycoproteins of about 150,000 daltons, composed of two light chains and two heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable domain (VH), also called a variable heavy domain or a heavy chain variable region, followed by three constant domains (CHI, CH2, and CH3), also called a heavy chain constant region.
- each light chain has a variable domain (VL), also called a variable light domain or a light chain variable region, followed by a constant light (CL) domain, also called a light chain constant region.
- VL variable domain
- CL constant light
- the heavy chain of an immunoglobulin may be assigned to one of five types, called a (IgA), d (IgD), e (IgE), g (IgG), or m (IgM), some of which may be further divided into subtypes, e.g. gi (IgGi), yi (IgG2), j3 (IgG3), j4 (IgG4), ai (IgAi) and a2 (IgA2).
- the light chain of an immunoglobulin may be assigned to one of two types, called kappa (K) and lambda (l), based on the amino acid sequence of its constant domain.
- K kappa
- l lambda
- An immunoglobulin essentially consists of two Fab molecules and an Fc domain, linked via the immunoglobulin hinge region.
- an antigen binding domain refers to the part of an antibody that comprises the area which specifically binds to and is complementary to part or all of an antigen.
- An antigen binding domain may be provided by, for example, one or more antibody variable domains (also called antibody variable regions).
- an antigen binding domain comprises an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).
- variable region or“variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen.
- the variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindt et ak, Kuby Immunology, 6 th ed., W.H. Freeman and Co., page 91 (2007).
- a single VH or VL domain may be sufficient to confer antigen-binding specificity.
- Kabat numbering refers to the numbering system set forth by Kabat et ak, Sequences of Proteins of Immunological Interest , 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).
- amino acid positions of all constant regions and domains of the heavy and light chain are numbered according to the Kabat numbering system described in Kabat, et ak, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), referred to as“numbering according to Kabat” or “Kabat numbering” herein.
- Kabat numbering system see pages 647-660 of Kabat, et ak, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)
- CL constant domain
- Kabat EU index numbering system see pages 661-723
- CHI heavy chain constant domains
- hypervariable region refers to each of the regions of an antibody variable domain which are hypervariable in sequence (“complementarity determining regions” or“CDRs”) and/or form structurally defined loops (“hypervariable loops”) and/or contain the antigen-contacting residues (“antigen contacts”).
- CDRs complementarity determining regions
- hypervariable loops form structurally defined loops
- antigen contacts antigen contacts
- antibodies comprise six HVRs; three in the VH (HI, H2, H3), and three in the VL (LI, L2, L3).
- Exemplary HVRs herein include:
- HVR residues and other residues in the variable domain are numbered herein according to Rabat et al., supra.
- FR Framework or "FR” refers to variable domain residues other than hypervariable region (HVR) residues.
- the FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following order in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.
- A“humanized” antibody refers to a chimeric antibody comprising amino acid residues from non human HVRs and amino acid residues from human FRs.
- a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody.
- Such variable domains are referred to herein as“humanized variable region”.
- a humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody.
- some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.
- A“humanized form” of an antibody e.g. of a non-human antibody, refers to an antibody that has undergone humanization.
- Other forms of "humanized antibodies” encompassed by the present invention are those in which the constant region has been additionally modified or changed from that of the original antibody to generate the properties according to the invention, especially in regard to Clq binding and/or Fc receptor (FcR) binding.
- A“human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
- a human antibody is derived from a non human transgenic mammal, for example a mouse, a rat, or a rabbit.
- a human antibody is derived from a hybridoma cell line.
- Antibodies or antibody fragments isolated from human antibody libraries are also considered human antibodies or human antibody fragments herein.
- The“class” of an antibody or immunoglobulin refers to the type of constant domain or constant region possessed by its heavy chain.
- the heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, d, e, g, and m, respectively.
- Fc domain or“Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
- the term includes native sequence Fc regions and variant Fc regions.
- the boundaries of the Fc region of an IgG heavy chain might vary slightly, the human IgG heavy chain Fc region is usually defined to extend from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain.
- antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain.
- an antibody produced by a host cell by expression of a specific nucleic acid molecule encoding a full-length heavy chain may include the full-length heavy chain, or it may include a cleaved variant of the full-length heavy chain (also referred to herein as a“cleaved variant heavy chain”).
- a“cleaved variant heavy chain” also referred to herein as a“cleaved variant heavy chain”.
- the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering according to Kabat EU index). Therefore, the C- terminal lysine (Lys447), or the C-terminal glycine (Gly446) and lysine (K447), of the Fc region may or may not be present.
- a heavy chain including a subunit of an Fc domain as specified herein comprises an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to EU index of Kabat).
- a heavy chain including a subunit of an Fc domain as specified herein, comprised in an immunoconjuate according to the invention comprises an additional C-terminal glycine residue (G446, numbering according to EU index of Kabat).
- Compositions of the invention such as the pharmaceutical compositions described herein, comprise a population of immunoconjugates of the invention.
- the population of immunoconjugates may comprise molecules having a full- length heavy chain and molecules having a cleaved variant heavy chain.
- the population of immunoconjugates may consist of a mixture of molecules having a full-length heavy chain and molecules having a cleaved variant heavy chain, wherein at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the immunoconjugates have a cleaved variant heavy chain.
- a composition comprising a population of immunoconjugates of the invention comprises an immunoconjugate comprising a heavy chain including a subunit of an Fc domain as specified herein with an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to EU index of Kabat).
- a composition comprising a population of immunoconjugates of the invention comprises an immunoconjugate comprising a heavy chain including a subunit of an Fc domain as specified herein with an additional C-terminal glycine residue (G446, numbering according to EU index of Kabat).
- such a composition comprises a population of immunoconjugates comprised of molecules comprising a heavy chain including a subunit of an Fc domain as specified herein; molecules comprising a heavy chain including a subunit of a Fc domain as specified herein with an additional C-terminal glycine residue (G446, numbering according to EU index of Kabat); and molecules comprising a heavy chain including a subunit of an Fc domain as specified herein with an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to EU index of Kabat).
- A“subunit” of an Fc domain as used herein refers to one of the two polypeptides forming the dimeric Fc domain, i.e. a polypeptide comprising C-terminal constant regions of an immunoglobulin heavy chain, capable of stable self-association.
- a subunit of an IgG Fc domain comprises an IgG CH2 and an IgG CH3 constant domain.
- A“modification promoting the association of the first and the second subunit of the Fc domain” is a manipulation of the peptide backbone or the post-translational modifications of an Fc domain subunit that reduces or prevents the association of a polypeptide comprising the Fc domain subunit with an identical polypeptide to form a homodimer.
- a modification promoting association as used herein particularly includes separate modifications made to each of the two Fc domain subunits desired to associate (i.e. the first and the second subunit of the Fc domain), wherein the modifications are complementary to each other so as to promote association of the two Fc domain subunits.
- a modification promoting association may alter the structure or charge of one or both of the Fc domain subunits so as to make their association sterically or electrostatically favorable, respectively.
- (hetero)dimerization occurs between a polypeptide comprising the first Fc domain subunit and a polypeptide comprising the second Fc domain subunit, which might be non-identical in the sense that further components fused to each of the subunits (e.g. antigen binding moieties) are not the same.
- the modification promoting association comprises an amino acid mutation in the Fc domain, specifically an amino acid substitution.
- the modification promoting association comprises a separate amino acid mutation, specifically an amino acid substitution, in each of the two subunits of the Fc domain.
- effector functions when used in reference to antibodies refers to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype.
- antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex- mediated antigen uptake by antigen presenting cells, down regulation of cell surface receptors (e.g. B cell receptor), and B cell activation.
- Antibody-dependent cell-mediated cytotoxicity is an immune mechanism leading to the lysis of antibody-coated target cells by immune effector cells.
- the target cells are cells to which antibodies or derivatives thereof comprising an Fc region specifically bind, generally via the protein part that is N-terminal to the Fc region.
- the term“reduced ADCC” is defined as either a reduction in the number of target cells that are lysed in a given time, at a given concentration of antibody in the medium surrounding the target cells, by the mechanism of ADCC defined above, and/or an increase in the concentration of antibody in the medium surrounding the target cells, required to achieve the lysis of a given number of target cells in a given time, by the mechanism of ADCC.
- the reduction in ADCC is relative to the ADCC mediated by the same antibody produced by the same type of host cells, using the same standard production, purification, formulation and storage methods (which are known to those skilled in the art), but that has not been engineered.
- the reduction in ADCC mediated by an antibody comprising in its Fc domain an amino acid substitution that reduces ADCC is relative to the ADCC mediated by the same antibody without this amino acid substitution in the Fc domain.
- Suitable assays to measure ADCC are well known in the art (see e.g. PCT publication no. WO 2006/082515 or PCT publication no. WO 2012/130831).
- An“activating Fc receptor” is an Fc receptor that following engagement by an Fc domain of an antibody elicits signaling events that stimulate the receptor-bearing cell to perform effector functions.
- Human activating Fc receptors include FcyRIIIa (CD16a), FcyRI (CD64), FcyRIIa (CD32), and FcaRI (CD89).
- engineer engineered, engineering
- engineering includes modifications of the amino acid sequence, of the glycosylation pattern, or of the side chain group of individual amino acids, as well as combinations of these approaches.
- Reduced binding for example reduced binding to an Fc receptor or CD25, refers to a decrease in affinity for the respective interaction, as measured for example by SPR.
- the term includes also reduction of the affinity to zero (or below the detection limit of the analytic method), i.e. complete abolishment of the interaction.
- “increased binding” refers to an increase in binding affinity for the respective interaction.
- immunoconjugate refers to a polypeptide molecule that includes at least one IL-2 molecule and at least one antibody.
- the IL-2 molecule can be joined to the antibody by a variety of interactions and in a variety of configurations as described herein.
- the IL-2 molecule is fused to the antibody via a peptide linker.
- Particular immunoconjugates according to the invention essentially consist of one IL-2 molecule and an antibody joined by one or more linker sequences.
- fused is meant that the components (e.g. an antibody and an IL-2 molecule) are linked by peptide bonds, either directly or via one or more peptide linkers.
- first and second with respect to Fc domain subunits etc., are used for convenience of distinguishing when there is more than one of each type of moiety. Use of these terms is not intended to confer a specific order or orientation of the immunoconjugate unless explicitly so stated.
- an “effective amount” of an agent refers to the amount that is necessary to result in a physiological change in the cell or tissue to which it is administered.
- a “therapeutically effective amount” of an agent refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
- a therapeutically effective amount of an agent for example eliminates, decreases, delays, minimizes or prevents adverse effects of a disease.
- mammals include, but are not limited to, domesticated animals (e.g. cows, sheep, cats, dogs, and horses), primates (e.g. humans and non human primates such as monkeys), rabbits, and rodents (e.g. mice and rats). Particularly, the individual or subject is a human.
- domesticated animals e.g. cows, sheep, cats, dogs, and horses
- primates e.g. humans and non human primates such as monkeys
- rabbits e.g. mice and rats
- rodents e.g. mice and rats
- composition refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the composition would be administered.
- A“pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical composition, other than an active ingredient, which is nontoxic to a subject.
- a pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
- treatment refers to clinical intervention in an attempt to alter the natural course of a disease in the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
- immunoconjugates of the invention are used to delay development of a disease or to slow the progression of a disease.
- the immunoconjugates according to the present invention comprise a mutant IL-2 polypeptide having advantageous properties for immunotherapy.
- pharmacological properties of IL-2 that contribute to toxicity but are not essential for efficacy of IL-2 are eliminated in the mutant IL-2 polypeptide.
- Such mutant IL-2 polypeptides are described in detail in WO 2012/107417, which is incorporated herein by reference in its entirety.
- different forms of the IL-2 receptor consist of different subunits and exhibit different affinities for IL-2.
- the intermediate-affinity IL-2 receptor consisting of the b and g receptor subunits, is expressed on resting effector cells and is sufficient for IL-2 signaling.
- the high-affinity IL-2 receptor is mainly expressed on regulatory T (Treg) cells as well as on activated effector cells where its engagement by IL-2 can promote Treg cell-mediated immunosuppression or activation-induced cell death (AICD), respectively.
- Treg regulatory T
- AICD activation-induced cell death
- reducing or abolishing the affinity of IL-2 to the a- subunit of the IL-2 receptor should reduce IL-2 induced downregulation of effector cell function by regulatory T cells and development of tumor tolerance by the process of AICD.
- maintaining the affinity to the intermediate-affinity IL-2 receptor should preserve the induction of proliferation and activation of effector cells like NK and T cells by IL-2.
- the mutant interleukin-2 (IL-2) polypeptide comprised in the immunoconjugate according to the invention comprises at least one amino acid mutation that abolishes or reduces affinity of the mutant IL-2 polypeptide to the a-subunit of the IL-2 receptor and preserves affinity of the mutant IL-2 polypeptide to the intermediate-affinity IL-2 receptor each compared to a wild-type IL-2 polypeptide.
- Mutants of human IL-2 (hIL-2) with decreased affinity to CD25 may for example be generated by amino acid substitution at amino acid position 35, 38, 42, 43, 45 or 72 or combinations thereof (numbering relative to the human IL-2 sequence SEQ ID NO: 13).
- Exemplary amino acid substitutions include K35E, K35A, R38A, R38E, R38N, R38F, R38S, R38L, R38G, R38Y, R38W, F42L, F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, K43E, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K.
- Particular IL-2 mutants useful in the immunoconjugates of the invention comprise an amino acid mutation at an amino acid position corresponding to residue 42, 45, or 72 of human IL-2, or a combination thereof.
- said amino acid mutation is an amino acid substitution selected from the group of F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K, more specifically an amino acid substitution selected from the group of F42A, Y45A and L72G.
- These mutants exhibit substantially similar binding affinity to the intermediate-affinity IL-2 receptor, and have substantially reduced affinity to the a-subunit of
- useful mutants may include the ability to induce proliferation of IL-2 receptor-bearing T and/or NK cells, the ability to induce IL-2 signaling in IL-2 receptor-bearing T and/or NK cells, the ability to generate interferon (IFN)-Y as a secondary cytokine by NK cells, a reduced ability to induce elaboration of secondary cytokines - particularly IL-10 and TNF-a - by peripheral blood mononuclear cells (PBMCs), a reduced ability to activate regulatory T cells, a reduced ability to induce apoptosis in T cells, and a reduced toxicity profile in vivo.
- IFN interferon
- Particular mutant IL-2 polypeptides useful in the invention comprise three amino acid mutations that abolish or reduce affinity of the mutant IL-2 polypeptide to the a-subunit of the IL-2 receptor but preserve affinity of the mutant IL-2 polypeptide to the intermediate affinity IL-2 receptor.
- said three amino acid mutations are at positions corresponding to residue 42, 45 and 72 of human IL-2.
- said three amino acid mutations are amino acid substitutions.
- said three amino acid mutations are amino acid substitutions selected from the group of F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K.
- said three amino acid mutations are amino acid substitutions F42A, Y45A and L72G (numbering relative to the human IL-2 sequence of SEQ ID NO: 13).
- said amino acid mutation reduces the affinity of the mutant IL-2 polypeptide to the a-subunit of the IL-2 receptor by at least 5 -fold, specifically at least 10-fold, more specifically at least 25-fold.
- the combination of these amino acid mutations may reduce the affinity of the mutant IL-2 polypeptide to the a-subunit of the IL-2 receptor by at least 30-fold, at least 50-fold, or even at least 100-fold.
- said amino acid mutation or combination of amino acid mutations abolishes the affinity of the mutant IL-2 polypeptide to the a-subunit of the IL-2 receptor so that no binding is detectable by surface plasmon resonance.
- Substantially similar binding to the intermediate-affinity receptor i.e. preservation of the affinity of the mutant IL-2 polypeptide to said receptor, is achieved when the IL-2 mutant exhibits greater than about 70% of the affinity of a wild-type form of the IL-2 mutant to the intermediate- affinity IL-2 receptor.
- IL-2 mutants of the invention may exhibit greater than about 80% and even greater than about 90% of such affinity.
- Reduction of the affinity of IL-2 for the a-subunit of the IL-2 receptor in combination with elimination of the O-glycosylation of IL-2 results in an IL-2 protein with improved properties.
- elimination of the O-glycosylation site results in a more homogenous product when the mutant IL-2 polypeptide is expressed in mammalian cells such as CHO or HEK cells.
- the mutant IL-2 polypeptide comprises an additional amino acid mutation which eliminates the O-glycosylation site of IL-2 at a position corresponding to residue 3 of human IL-2.
- said additional amino acid mutation which eliminates the O-glycosylation site of IL-2 at a position corresponding to residue 3 of human IL-2 is an amino acid substitution.
- Exemplary amino acid substitutions include T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, and T3P.
- said additional amino acid mutation is the amino acid substitution T3A.
- the mutant IL-2 polypeptide is essentially a full-length IL-2 molecule. In certain embodiments the mutant IL-2 polypeptide is a human IL-2 molecule. In one embodiment the mutant IL-2 polypeptide comprises the sequence of SEQ ID NO: 13 with at least one amino acid mutation that abolishes or reduces affinity of the mutant IL-2 polypeptide to the a-subunit of the IL-2 receptor but preserve affinity of the mutant IL-2 polypeptide to the intermediate affinity IL-2 receptor, compared to an IL-2 polypeptide comprising SEQ ID NO: 13 without said mutation.
- the mutant IL-2 polypeptide comprises the sequence of SEQ ID NO: 13 with at least one amino acid mutation that abolishes or reduces affinity of the mutant IL-2 polypeptide to the a-subunit of the IL-2 receptor but preserve affinity of the mutant IL-2 polypeptide to the intermediate affinity IL-2 receptor, compared to an IL-2 polypeptide comprising SEQ ID NO: 13 without said mutation.
- the mutant IL-2 polypeptide can elicit one or more of the cellular responses selected from the group consisting of: proliferation in an activated T lymphocyte cell, differentiation in an activated T lymphocyte cell, cytotoxic T cell (CTL) activity, proliferation in an activated B cell, differentiation in an activated B cell, proliferation in a natural killer (NK) cell, differentiation in a NK cell, cytokine secretion by an activated T cell or an NK cell, and NK/lymphocyte activated killer (LAK) antitumor cytotoxicity.
- CTL cytotoxic T cell
- NK natural killer
- LAK NK/lymphocyte activated killer
- the mutant IL-2 polypeptide has a reduced ability to induce IL-2 signaling in regulatory T cells, compared to a wild-type IL-2 polypeptide. In one embodiment the mutant IL- 2 polypeptide induces less activation-induced cell death (AICD) in T cells, compared to a wild- type IL-2 polypeptide. In one embodiment the mutant IL-2 polypeptide has a reduced toxicity profile in vivo, compared to a wild-type IL-2 polypeptide. In one embodiment the mutant IL-2 polypeptide has a prolonged serum half-life, compared to a wild-type IL-2 polypeptide.
- AICD activation-induced cell death
- a particular mutant IL-2 polypeptide useful in the invention comprises four amino acid substitutions at positions corresponding to residues 3, 42, 45 and 72 of human IL-2. Specific amino acid substitutions are T3A, F42A, Y45A and L72G.
- said quadruple mutant IL-2 polypeptide exhibits no detectable binding to CD25, reduced ability to induce apoptosis in T cells, reduced ability to induce IL-2 signaling in Treg cells, and a reduced toxicity profile in vivo. However, it retains ability to activate IL-2 signaling in effector cells, to induce proliferation of effector cells, and to generate IFN-g as a secondary cytokine by NK cells.
- said mutant IL-2 polypeptide has further advantageous properties, such as reduced surface hydrophobicity, good stability, and good expression yield, as described in WO 2012/107417.
- said mutant IL-2 polypeptide also provides a prolonged serum half- life, compared to wild-type IL-2.
- IL-2 mutants useful in the invention in addition to having mutations in the region of IL-2 that forms the interface of IL-2 with CD25 or the glycosylation site, also may have one or more mutations in the amino acid sequence outside these regions. Such additional mutations in human IL-2 may provide additional advantages such as increased expression or stability.
- the cysteine at position 125 may be replaced with a neutral amino acid such as serine, alanine, threonine or valine, yielding C125S IL-2, C125A IL-2, C125T IL-2 or C125V IL-2 respectively, as described in U.S. Patent no. 4,518,584.
- a neutral amino acid such as serine, alanine, threonine or valine
- the IL-2 mutant may include a mutation whereby methionine normally occurring at position 104 of wild-type human IL-2 is replaced by a neutral amino acid such as alanine (see U.S. Patent no.
- the resulting mutants e. g., des-Al M104A IL-2, des-Al Ml 04 A C125S IL-2, M104A IL-2, M104A C125A IL-2, des-Al M104A C125A IL-2, or M104A C125S IL-2 (these and other mutants may be found in U.S. Patent No. 5,116,943 and in Weiger et al., Eur J Biochem 180, 295-300 (1989)) may be used in conjunction with the particular IL-2 mutations of the invention.
- the mutant IL-2 polypeptide comprises an additional amino acid mutation at a position corresponding to residue 125 of human IL-2.
- said additional amino acid mutation is the amino acid substitution C125A.
- the mutant IL-2 polypeptide comprises no more than 12, no more than 11, no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, or no more than 5 amino acid mutations as compared to the corresponding wild-type IL-2 sequence, e.g. the human IL-2 sequence of SEQ ID NO: 13.
- the mutant IL-2 polypeptide comprises no more than 5 amino acid mutations as compared to the corresponding wild-type IL-2 sequence, e.g. the human IL-2 sequence of SEQ ID NO: 13.
- mutant IL-2 polypeptide comprises the sequence of SEQ ID NO: 14. In one embodiment the mutant IL-2 polypeptide consists of the sequence of SEQ ID NO: 14. Immunoconiugates
- Immunoconjugates as described herein comprise an IL-molecule and an antibody. Such immunoconjugates significantly increase the efficacy of IL-2 therapy by directly targeting IL-2 e.g. into a tumor microenvironment.
- an antibody comprised in the immunoconjugate can be a whole antibody or immunoglobulin, or a portion or variant thereof that has a biological function such as antigen specific binding affinity.
- an antibody comprised in an immunoconjugate recognizes a tumor-specific epitope and results in targeting of the immunoconjugate molecule to the tumor site. Therefore, high concentrations of IL-2 can be delivered into the tumor microenvironment, thereby resulting in activation and proliferation of a variety of immune effector cells mentioned herein using a much lower dose of the immunoconjugate than would be required for unconjugated IL-2.
- IL-2 in form of immunoconjugates allows lower doses of the cytokine itself, the potential for undesirable side effects of IL-2 is restricted, and targeting the IL-2 to a specific site in the body by means of an immunoconjugate may also result in a reduction of systemic exposure and thus less side effects than obtained with unconjugated IL-2.
- the increased circulating half- life of an immunoconjugate compared to unconjugated IL-2 contributes to the efficacy of the immunoconjugate.
- IL-2 immunoconjugates may again aggravate potential side effects of the IL-2 molecule: Because of the significantly longer circulating half- life of IL-2 immunoconjugate in the bloodstream relative to unconjugated IL-2, the probability for IL-2 or other portions of the fusion protein molecule to activate components generally present in the vasculature is increased. The same concern applies to other fusion proteins that contain IL-2 fused to another moiety such as Fc or albumin, resulting in an extended half-life of IL-2 in the circulation. Therefore, an immunoconjugate comprising a mutant IL-2 polypeptide as described herein and in WO 2012/107417, with reduced toxicity compared to wild-type forms of IL-2, is particularly advantageous.
- IL-2 directly to immune effector cells rather than tumor cells may be advantageous for IL-2 immunotherapy.
- the invention provides a mutant IL-2 polypeptide as described hereinbefore, and an antibody that binds to CD8.
- the mutant IL-2 polypeptide and the antibody form a fusion protein, i.e. the mutant IL-2 polypeptide shares a peptide bond with the antibody.
- the antibody comprises an Fc domain composed of a first and a second subunit.
- the mutant IL-2 polypeptide is fused at its amino-terminal amino acid to the carboxy -terminal amino acid of one of the subunits of the Fc domain, optionally through a linker peptide.
- the antibody is a full-length antibody.
- the antibody is an immunoglobulin molecule, particularly an IgG class immunoglobulin molecule, more particularly an IgGi subclass immunoglobulin molecule.
- the mutant IL-2 polypeptide shares an amino-terminal peptide bond with one of the immunoglobulin heavy chains.
- the antibody is an antibody fragment.
- the antibody is a Fab molecule or a scFv molecule.
- the antibody is a Fab molecule.
- the antibody is a scFv molecule.
- the immunoconjugate may also comprise more than one antibody. Where more than one antibody is comprised in the immunoconjugate, e.g.
- each antibody can be independently selected from various forms of antibodies and antibody fragments.
- the first antibody can be a Fab molecule and the second antibody can be a scFv molecule.
- each of said first and said second antibodies is a scFv molecule or each of said first and said second antibodies is a Fab molecule.
- each of said first and said second antibodies is a Fab molecule.
- each of said first and said second antibodies binds to CD8.
- immunoconjugate formats are described in PCT publication no. WO 2011/020783, which is incorporated herein by reference in its entirety. These immunoconjugates comprise at least two antibodies.
- the immunoconjugate according to the present invention comprises a mutant IL-2 polypeptide as described herein, and at least a first and a second antibody.
- said first and second antibody are independently selected from the group consisting of an Fv molecule, particularly a scFv molecule, and a Fab molecule.
- said mutant IL-2 polypeptide shares an amino- or carboxy- terminal peptide bond with said first antibody and said second antibody shares an amino- or carboxy-terminal peptide bond with either i) the mutant IL-2 polypeptide or ii) the first antibody.
- the immunoconjugate consists essentially of a mutant IL-2 polypeptide and first and second antibodies, particularly Fab molecules, joined by one or more linker sequences. Such formats have the advantage that they bind with high affinity to the target antigen (CD8), but provide only monomeric binding to the IL-2 receptor, thus avoiding targeting the immunoconjugate to IL-2 receptor bearing immune cells at other locations than the target site.
- a mutant IL-2 polypeptide shares a carboxy-terminal peptide bond with a first antibody, particularly a first Fab molecule, and further shares an amino-terminal peptide bond with a second antibody, particularly a second Fab molecule.
- a first antibody, particularly a first Fab molecule shares a carboxy-terminal peptide bond with a mutant IL-2 polypeptide, and further shares an amino-terminal peptide bond with a second antibody, particularly a second Fab molecule.
- a first antibody shares an amino-terminal peptide bond with a first mutant IL-2 polypeptide, and further shares a carboxy-terminal peptide with a second antibody, particularly a second Fab molecule.
- a mutant IL-2 polypeptide shares a carboxy- terminal peptide bond with a first heavy chain variable region and further shares an amino- terminal peptide bond with a second heavy chain variable region.
- a mutant IL-2 polypeptide shares a carboxy-terminal peptide bond with a first light chain variable region and further shares an amino-terminal peptide bond with a second light chain variable region.
- a first heavy or light chain variable region is joined by a carboxy-terminal peptide bond to a mutant IL-2 polypeptide and is further joined by an amino- terminal peptide bond to a second heavy or light chain variable region.
- a first heavy or light chain variable region is joined by an amino-terminal peptide bond to a mutant IL-2 polypeptide and is further joined by a carboxy-terminal peptide bond to a second heavy or light chain variable region.
- a mutant IL-2 polypeptide shares a carboxy- terminal peptide bond with a first Fab heavy or light chain and further shares an amino-terminal peptide bond with a second Fab heavy or light chain.
- a first Fab heavy or light chain shares a carboxy-terminal peptide bond with a mutant IL-2 polypeptide and further shares an amino-terminal peptide bond with a second Fab heavy or light chain.
- a first Fab heavy or light chain shares an amino-terminal peptide bond with a mutant IL-2 polypeptide and further shares a carboxy-terminal peptide bond with a second Fab heavy or light chain.
- the immunoconjugate comprises a mutant IL-2 polypeptide sharing an amino-terminal peptide bond with one or more scFv molecules and further sharing a carboxy-terminal peptide bond with one or more scFv molecules.
- the immunoconjugates comprise an immunoglobulin molecule as antibody.
- Such immunoconjugate formats are described in WO 2012/146628, which is incorporated herein by reference in its entirety.
- the immunoconjugate comprises a mutant IL-2 polypeptide as described herein and an immunoglobulin molecule that binds to CD8, particularly an IgG molecule, more particularly an IgGi molecule.
- the immunoconjugate comprises not more than one mutant IL-2 polypeptide.
- the immunoglobulin molecule is human.
- the immunoglobulin molecule comprises a human constant region, e.g. a human CHI, CH2, CH3 and/or CL domain.
- the immunoglobulin comprises a human Fc domain, particularly a human IgGi Fc domain.
- the mutant IL-2 polypeptide shares an amino- or carboxy-terminal peptide bond with the immunoglobulin molecule.
- the immunoconjugate essentially consists of a mutant IL-2 polypeptide and an immunoglobulin molecule, particularly an IgG molecule, more particularly an IgGi molecule, joined by one or more linker sequences.
- the mutant IL-2 polypeptide is fused at its amino-terminal amino acid to the carboxy-terminal amino acid of one of the immunoglobulin heavy chains, optionally through a linker peptide.
- the mutant IL-2 polypeptide may be fused to the antibody directly or through a linker peptide, comprising one or more amino acids, typically about 2-20 amino acids.
- Linker peptides are known in the art and are described herein. Suitable, non-immunogenic linker peptides include, for example, (G4S)n, (SG4)n, (G4S)n or G4(SG4)n linker peptides “n” is generally an integer from 1 to 10, typically from 2 to 4.
- the linker peptide has a length of at least 5 amino acids, in one embodiment a length of 5 to 100, in a further embodiment of 10 to 50 amino acids. In a particular embodiment, the linker peptide has a length of 15 amino acids.
- the linker peptide is (G4S)3 (SEQ ID NO: 15).
- the linker peptide has (or consists of) the amino acid sequence of SEQ ID NO: 15.
- the immunoconjugate comprises a mutant IL-2 molecule and an immunoglobulin molecule, particularly an IgGi subclass immunoglobulin molecule, that binds to CD8, wherein the mutant IL-2 molecule is fused at its amino-terminal amino acid to the carboxy- terminal amino acid of one of the immunoglobulin heavy chains through the linker peptide of SEQ ID NO: 15.
- the immunoconjugate comprises a mutant IL-2 molecule and an antibody that binds to CD8, wherein the antibody comprises an Fc domain, particularly a human IgGi Fc domain, composed of a first and a second subunit, and the mutant IL-2 molecule is fused at its amino-terminal amino acid to the carboxy-terminal amino acid of one of the subunits of the Fc domain through the linker peptide of SEQ ID NO: 15.
- the antibody comprised in the immunoconjugate of the invention binds to CD8, particularly human CD8, and is able to direct the mutant IL-2 polypeptide to a target site where CD8is expressed, particularly to a T cell that expresses CD8, for example associated with a tumor.
- CD8 antibodies that may be used in the immunoconjugate of the invention are described in PCT publication WO 2019/033043 A2, which is incorporated herein by reference in its entirety.
- the immunoconjugate of the invention may comprise two or more antibodies, which may bind to the same or to different antigens. In particular embodiments, however, each of these antibodies binds to CD8.
- the antibody comprised in the immunoconjugate of the invention is monospecific.
- the immunoconjugate comprises a single, monospecific antibody, particularly a monospecific immunoglobulin molecule.
- the antibody can be any type of antibody or fragment thereof that retains specific binding to CD8, particularly human CD8.
- Antibody fragments include, but are not limited to, Fv molecules, scFv molecule, Fab molecule, and F(ab')2 molecules. In particular embodiments, however, the antibody is a full-length antibody.
- the antibody comprises an Fc domain, composed of a first and a second subunit.
- the antibody is an immunoglobulin, particularly an IgG class, more particularly an IgGi subclass immunoglobulin.
- the antibody is a monoclonal antibody.
- An anti-CD8 antibody provided herein has one or more of following characteristics: (a) the antibody does not inhibit or stimulate the activation of CD8 + T cells; (b) the antibody does not induce CD8 + T cell proliferation; (c) the antibody does not induce IFNy production; (d) the antibody specifically binds human CD8; (e) the antibody specifically binds rhesus CD8; (f) the antibody specifically binds cynomolgous CD8; (g) the antibody does not bind CD4 + cells; (g) the antibody does not bind CD3 cells; and (h) the antibody does not deplete CD8 + T cells from the circulation.
- Such characteristics can be assessed using well known methods, e.g., methods used in PCT application WO 2019/033043 A2.
- an anti-CD8 antibody is an antibody that binds to CD8 with sufficient affinity and specificity.
- the anti-CD8 antibody binds human CD8 with a Kn of about any one of 1 mM, 100 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM, or 0.001 nM (e.g. 10 8 M or less, e.g. from 10 8 M to 10 13 M, e.g., from 10 9 M to 10 13 M), including any range in between these values.
- the anti-CD8 antibody binds rhesus CD8 with a Kn of about any one of 1 mM, 100 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM, or 0.001 nM (e.g. 10 8 M or less, e.g. from 10 8 M to 10 13 M, e.g., from 10 9 M to 10 13 M), including any range in between these values.
- a Kn of about any one of 1 mM, 100 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM, or 0.001 nM (e.g. 10 8 M or less, e.g. from 10 8 M to 10 13 M, e.g.,
- the anti-CD8 antibody binds cynomolgus CD8 with a Kn of 1 pM, 100 nM, 50 nM, 40 nM, 30 nM, 20nM, 10 nM, 5nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM, or 0.001 nM (e.g. 10 8 M or less, e.g. from 10 8 M to 10 13 M, e.g., from 10 9 M to 10 13 M), including any range in between these values
- the anti-CD8 antibody binds (a) human CD8 with a Kn of about 1 pM, 100 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM, or 0.001 nM (e.g. 10 8 M or less, e.g.
- rhesus CDS with a Kn of about 1 pM, 100 nM, 50 nM, 40 nM, 30nM, 20nM, 10 nM, 5nM, 1 nM, 0.5 nM, 1.1 nM, 0.05 nM, or 0.001 nM (e.g. 10 8 M or less, e.g.
- cynomolgus CD8 with a Kn of about 1 pM, 100 nM, 50 nM, 40 nM, 30 nM, 20nM, 10 nM, 5 nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM, or 0.001 nM (e.g. 10 8 M or less, e.g. from 10 8 M to 10 13 M, e.g., from 10 9 M to 10 13 M), including any range in between these values.
- the Kn of an anti-CD8 antibody provided herein for human CD8, rhesus CD8 and/or cynomolgous CD8 can be determined by any method known in the art, including, but not limited to, e.g., ELISA, fluorescence activated cell sorting (FACS) analysis, radioimmunoprecipitation (RIA), and surface plasmon resonance (SPR).
- the Kn of an anti-CD8 antibody provided herein for human CD8, rhesus CD8 and/or cynomolgous CD8 is determined via SPR.
- the Kn of an anti-CD8 antibody provided herein for human CD8, rhesus CD8 and/or cynomolgous CD8 is determined via F ACS.
- the anti-CD8 antibody provided herein does not bind (e.g. specifically bind) mouse CD8.
- the anti-CD8 antibody does not bind (e.g. specifically bind) rat CD8.
- the anti-CDS antibody does not bind (e.g. specifically bind) to either mouse CD8 or rat CD8, e.g., as determined via SPR and/or FACS.
- the antibody comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: l, a HCDR2 comprising the amino acid sequence of SEQ ID NO:2, a HCDR3 comprising the amino acid sequence of SEQ ID NO:3, a LCDR1 comprising the amino acid sequence of SEQ ID NO:4, a LCDR2 comprising the amino acid sequence of SEQ ID NO:5, and a LCDR3 comprising the amino acid sequence of SEQ ID NO:6.
- the antibody comprises (a) a heavy chain variable region (VH) comprising a HCDR1 comprising the amino acid sequence of SEQ ID NO: l, a HCDR2 comprising the amino acid sequence of SEQ ID NO:2, a HCDR3 comprising the amino acid sequence of SEQ ID NO:3, and (b) a light chain variable region (VL) comprising a LCDR1 comprising the amino acid sequence of SEQ ID NO:4, a LCDR2 comprising the amino acid sequence of SEQ ID NO:5, and a LCDR3 comprising the amino acid sequence of SEQ ID NO:6.
- the heavy and/or light chain variable region is a humanized variable region.
- the heavy and/or light chain variable region comprises human framework regions (FR).
- the antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:7.
- the antibody comprises a light chain variable region (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 8.
- the antibody comprises (a) a heavy chain variable region (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:7, and (b) a light chain variable region (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence of SEQ ID NO: 8.
- VH heavy chain variable region
- VL light chain variable region
- the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 7, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 8.
- the antibody comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: l, a HCDR2 comprising the amino acid sequence of SEQ ID NO:2, a HCDR3 comprising the amino acid sequence of SEQ ID NO:3, a LCDR1 comprising the amino acid sequence of SEQ ID NO:4, a LCDR2 comprising the amino acid sequence of SEQ ID NO:5, and a LCDR3 comprising the amino acid sequence of SEQ ID NO:28.
- the antibody comprises (a) a heavy chain variable region (VH) comprising a HCDR1 comprising the amino acid sequence of SEQ ID NO: l, a HCDR2 comprising the amino acid sequence of SEQ ID NO:2, a HCDR3 comprising the amino acid sequence of SEQ ID NO:3, and (b) a light chain variable region (VL) comprising a LCDR1 comprising the amino acid sequence of SEQ ID NO:4, a LCDR2 comprising the amino acid sequence of SEQ ID NO:5, and a LCDR3 comprising the amino acid sequence of SEQ ID NO:28.
- the heavy and/or light chain variable region is a humanized variable region.
- the heavy and/or light chain variable region comprises human framework regions (FR).
- the antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:7.
- the antibody comprises a light chain variable region (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:29.
- the antibody comprises (a) a heavy chain variable region (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:7, and (b) a light chain variable region (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence of SEQ ID NO:29.
- VH heavy chain variable region
- VL light chain variable region
- the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 7, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 29.
- VH heavy chain variable region
- VL light chain variable region
- the antibody is a humanized antibody.
- the antibody is an immunoglobulin molecule comprising a human constant region, particularly an IgG class immunoglobulin molecule comprising a human CHI, CH2, CH3 and/or CL domain.
- Exemplary sequences of human constant domains are given in SEQ ID NOs 22 and 23 (human kappa and lambda CL domains, respectively) and SEQ ID NO: 24 (human IgGl heavy chain constant domains CH1-CH2-CH3).
- the antibody comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 23, particularly the amino acid sequence of SEQ ID NO: 24.
- the antibody comprises a heavy chain constant region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 24.
- the heavy chain constant region may comprise amino acid mutations in the Fc domain as described herein.
- the antibody comprised in the immunconjugates according to the invention comprises an Fc domain, composed of a first and a second subunit.
- the Fc domain of an antibody consists of a pair of polypeptide chains comprising heavy chain domains of an immunoglobulin molecule.
- the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, each subunit of which comprises the CH2 and CH3 IgG heavy chain constant domains.
- the two subunits of the Fc domain are capable of stable association with each other.
- the immunoconjugate of the invention comprises not more than one Fc domain.
- the Fc domain of the antibody comprised in the immunoconjugate is an IgG Fc domain.
- the Fc domain is an IgGi Fc domain. In another embodiment the Fc domain is an IgG4 Fc domain. In a more specific embodiment, the Fc domain is an IgG4 Fc domain comprising an amino acid substitution at position S228 (Kabat EU index numbering), particularly the amino acid substitution S228P. This amino acid substitution reduces in vivo Fab arm exchange of IgG4 antibodies (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). In a further particular embodiment the Fc domain is a human Fc domain. In an even more particular embodiment, the Fc domain is a human IgGi Fc domain. An exemplary sequence of a human IgGi Fc region is given in SEQ ID NO: 21.
- Immunoconjugate s comprise a mutant IL-2 polypeptide, particularly a single (not more than one) mutant IL-2 polypeptide, fused to one or the other of the two subunits of the Fc domain, thus the two subunits of the Fc domain are typically comprised in two non identical polypeptide chains. Recombinant co-expression of these polypeptides and subsequent dimerization leads to several possible combinations of the two polypeptides. To improve the yield and purity of the immunoconjugate in recombinant production, it will thus be advantageous to introduce in the Fc domain of the antibody a modification promoting the association of the desired polypeptides.
- the Fc domain of the antibody comprised in the immunoconjugate according to the invention comprises a modification promoting the association of the first and the second subunit of the Fc domain.
- the site of most extensive protein-protein interaction between the two subunits of a human IgG Fc domain is in the CH3 domain of the Fc domain.
- said modification is in the CH3 domain of the Fc domain.
- the CH3 domain of the first subunit of the Fc domain and the CH3 domain of the second subunit of the Fc domain are both engineered in a complementary manner so that each CH3 domain (or the heavy chain comprising it) can no longer homodimerize with itself but is forced to heterodimerize with the complementarily engineered other CH3 domain (so that the first and second CH3 domain heterodimerize and no homodimers between the two first or the two second CH3 domains are formed).
- said modification promoting the association of the first and the second subunit of the Fc domain is a so-called“knob-into-hole” modification, comprising a“knob” modification in one of the two subunits of the Fc domain and a“hole” modification in the other one of the two subunits of the Fc domain.
- knob-into-hole technology is described e.g. in US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001).
- the method involves introducing a protuberance (“knob”) at the interface of a first polypeptide and a corresponding cavity (“hole”) in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation.
- Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g. tyrosine or tryptophan).
- Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine).
- an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain of the first subunit which is positionable in a cavity within the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit is positionable.
- amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W).
- amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V).
- the protuberance and cavity can be made by altering the nucleic acid encoding the polypeptides, e.g. by site-specific mutagenesis, or by peptide synthesis.
- the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the CH3 domain of the second subunit of the Fc domain (the“hole” subunit) the tyrosine residue at position 407 is replaced with a valine residue (Y407V).
- the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numberings according to Rabat EU index).
- the serine residue at position 354 is replaced with a cysteine residue (S354C) or the glutamic acid residue at position 356 is replaced with a cysteine residue (E356C) (particularly the serine residue at position 354 is replaced with a cysteine residue), and in the second subunit of the Fc domain additionally the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C) (numberings according to Rabat EU index).
- S354C cysteine residue
- E356C cysteine residue
- Y349C cysteine residue
- the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W
- the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (numbering according to Rabat EU index).
- the second subunit of the Fc domain additionally comprises the amino acid substitutions H435R and Y436F (numbering according to Kabat EU index).
- the mutant IL-2 polypeptide is fused (optionally through a linker peptide) to the first subunit of the Fc domain (comprising the“knob” modification).
- fusion of the mutant IL-2 polypeptide to the knob-containing subunit of the Fc domain will (further) minimize the generation of immunoconjugates comprising two mutant IL-2 polypeptides (steric clash of two knob-containing polypeptides).
- Other techniques of CH3 -modification for enforcing the heterodimerization are contemplated as alternatives according to the invention and are described e.g.
- the heterodimerization approach described in EP 1870459 is used alternatively. This approach is based on the introduction of charged amino acids with opposite charges at specific amino acid positions in the CH3/CH3 domain interface between the two subunits of the Fc domain.
- One preferred embodiment for the antibody comprised in the immunoconjugate of the invention are amino acid mutations R409D; K370E in one of the two CH3 domains (of the Fc domain) and amino acid mutations D399K; E357K in the other one of the CH3 domains of the Fc domain (numbering according to Kabat EU index).
- the antibody comprised in the immunoconjugate of the invention comprises amino acid mutation T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutations T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, and additionally amino acid mutations R409D; K370E in the CH3 domain of the first subunit of the Fc domain and amino acid mutations D399K; E357K in the CH3 domain of the second subunit of the Fc domain (numberings according to Kabat EU index).
- the antibody comprised in the immunoconjugate of the invention comprises amino acid mutations S354C, T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutations Y349C, T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, or said antibody comprises amino acid mutations Y349C, T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutations S354C, T366S, L368A, Y407V in the CH3 domains of the second subunit of the Fc domain and additionally amino acid mutations R409D; K370E in the CH3 domain of the first subunit of the Fc domain and amino acid mutations D399K; E357K in the CH3 domain of the second subunit of the Fc domain (all numberings according to Kabat EU index).
- a first CH3 domain comprises amino acid mutation T366K and a second CH3 domain comprises amino acid mutation L351D (numberings according to Kabat EU index).
- the first CH3 domain comprises further amino acid mutation L351K.
- the second CH3 domain comprises further an amino acid mutation selected from Y349E, Y349D and L368E (preferably L368E) (numberings according to Kabat EU index).
- a first CH3 domain comprises amino acid mutations L351Y, Y407A and a second CH3 domain comprises amino acid mutations T366A, K409F.
- the second CH3 domain comprises a further amino acid mutation at position T411, D399, S400, F405, N390, or K392, e.g.
- T411N, T411R, T411Q, T411K, T411D, T411E or T411W b) D399R, D399W, D399Y or D399K
- S400E, S400D, S400R, or S400K d) F405I, F405M, F405T, F405S, F405V or F405W, e) N390R, N390K or N390D, f) K392V, K392M, K392R, K392L, K392F or K392E (numberings according to Kabat EU index).
- a first CH3 domain comprises amino acid mutations L351Y, Y407A and a second CH3 domain comprises amino acid mutations T366V, K409F.
- a first CH3 domain comprises amino acid mutation Y407A and a second CH3 domain comprises amino acid mutations T366A, K409F.
- the second CH3 domain further comprises amino acid mutations K392E, T411E, D399R and S400R (numberings according to Kabat EU index).
- heterodimerization approach described in WO 2011/143545 is used alternatively, e.g. with the amino acid modification at a position selected from the group consisting of 368 and 409 (numbering according to Kabat EU index).
- a first CH3 domain comprises amino acid mutation T366W and a second CH3 domain comprises amino acid mutation Y407A.
- a first CH3 domain comprises amino acid mutation T366Y and a second CH3 domain comprises amino acid mutation Y407T (numberings according to Kabat EU index).
- the antibody comprised in the immunoconjugate or its Fc domain is of IgG2 subclass and the heterodimerization approach described in WO 2010/129304 is used alternatively.
- a modification promoting association of the first and the second subunit of the Fc domain comprises a modification mediating electrostatic steering effects, e.g. as described in PCT publication WO 2009/089004.
- this method involves replacement of one or more amino acid residues at the interface of the two Fc domain subunits by charged amino acid residues so that homodimer formation becomes electrostatically unfavorable but heterodimerization electrostatically favorable.
- a first CH3 domain comprises amino acid substitution of K392 or N392 with a negatively charged amino acid (e.g. glutamic acid (E), or aspartic acid (D), preferably K392D or N392D) and a second CH3 domain comprises amino acid substitution of D399, E356, D356, or E357 with a positively charged amino acid (e.g. lysine (K) or arginine (R), preferably D399K, E356K, D356K, or E357K, and more preferably D399K and E356K).
- the first CH3 domain further comprises amino acid substitution of K409 or R409 with a negatively charged amino acid (e.g.
- the first CH3 domain further or alternatively comprises amino acid substitution of K439 and/or K370 with a negatively charged amino acid (e.g. glutamic acid (E), or aspartic acid (D)) (all numberings according to Rabat EU index).
- a negatively charged amino acid e.g. glutamic acid (E), or aspartic acid (D)
- a first CH3 domain comprises amino acid mutations K253E, D282K, and K322D and a second CH3 domain comprises amino acid mutations D239K, E240K, and K292D (numberings according to Rabat EU index).
- heterodimerization approach described in WO 2007/110205 can be used alternatively.
- the first subunit of the Fc domain comprises amino acid substitutions R392D and R409D
- the second subunit of the Fc domain comprises amino acid substitutions D356R and D399R (numbering according to Rabat EU index).
- the Fc domain confers to the immunoconjugate favorable pharmacokinetic properties, including a long serum half-life which contributes to good accumulation in the target tissue and a favorable tissue-blood distribution ratio. At the same time it may, however, lead to undesirable targeting of the immunoconjugate to cells expressing Fc receptors rather than to the preferred antigen bearing cells. Moreover, the co-activation of Fc receptor signaling pathways may lead to cytokine release which, in combination with the IL-2 polypeptide and the long half-life of the immunoconjugate, results in excessive activation of cytokine receptors and severe side effects upon systemic administration. In line with this, conventional IgG-IL-2 immunoconjugates have been described to be associated with infusion reactions (see e.g. King et al., J Clin Oncol 22, 4463-4473 (2004)).
- the Fc domain of the antibody comprised in the immunoconjugate according to the invention exhibits reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a native IgGi Fc domain.
- the Fc domain (or the antibody comprising said Fc domain) exhibits less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5% of the binding affinity to an Fc receptor, as compared to a native IgGi Fc domain (or an antibody comprising a native IgGi Fc domain), and/or less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5% of the effector function, as compared to a native IgGi Fc domain domain (or an antibody comprising a native IgGi Fc domain).
- the Fc domain domain (or an antibody comprising said Fc domain) does not substantially bind to an Fc receptor and/or induce effector function.
- the Fc receptor is an Fey receptor.
- the Fc receptor is a human Fc receptor.
- the Fc receptor is an activating Fc receptor.
- the Fc receptor is an activating human Fey receptor, more specifically human FcyRIIIa, FcyRI or FcyRIIa, most specifically human FcyRIIIa.
- the effector function is one or more selected from the group of CDC, ADCC, ADCP, and cytokine secretion. In a particular embodiment the effector function is ADCC.
- the Fc domain domain exhibits substantially similar binding affinity to neonatal Fc receptor (FcRn), as compared to a native IgGi Fc domain domain.
- FcRn neonatal Fc receptor
- Substantially similar binding to FcRn is achieved when the Fc domain (or an antibody comprising said Fc domain) exhibits greater than about 70%, particularly greater than about 80%, more particularly greater than about 90% of the binding affinity of a native IgGi Fc domain (or an antibody comprising a native IgGi Fc domain) to FcRn.
- the Fc domain is engineered to have reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a non-engineered Fc domain.
- the Fc domain of the antibody comprised in the immunoconjugate comprises one or more amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor and/or effector function.
- the same one or more amino acid mutation is present in each of the two subunits of the Fc domain.
- the amino acid mutation reduces the binding affinity of the Fc domain to an Fc receptor.
- the amino acid mutation reduces the binding affinity of the Fc domain to an Fc receptor by at least 2- fold, at least 5-fold, or at least 10-fold.
- the combination of these amino acid mutations may reduce the binding affinity of the Fc domain to an Fc receptor by at least 10-fold, at least 20-fold, or even at least 50-fold.
- the antibody comprising an engineered Fc domain exhibits less than 20%, particularly less than 10%, more particularly less than 5% of the binding affinity to an Fc receptor as compared to an antibody comprising a non-engineered Fc domain.
- the Fc receptor is an Fey receptor.
- the Fc receptor is a human Fc receptor.
- the Fc receptor is an activating Fc receptor.
- the Fc receptor is an activating human Fey receptor, more specifically human FcyRIIIa, FcyRI or FcyRIIa, most specifically human FcyRIIIa.
- binding to each of these receptors is reduced.
- binding affinity to a complement component, specifically binding affinity to Clq is also reduced.
- binding affinity to neonatal Fc receptor (FcRn) is not reduced. Substantially similar binding to FcRn, i.e.
- the Fc domain or an antibody comprising said Fc domain
- the Fc domain, or antibody comprised in the immunoconjugate of the invention comprising said Fc domain may exhibit greater than about 80% and even greater than about 90% of such affinity.
- the Fc domain of the antibody comprised in the immunoconjugate is engineered to have reduced effector function, as compared to a non-engineered Fc domain.
- the reduced effector function can include, but is not limited to, one or more of the following: reduced complement dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling inducing apoptosis, reduced crosslinking of target-bound antibodies, reduced dendritic cell maturation, or reduced T cell priming.
- CDC complement dependent cytotoxicity
- ADCC reduced antibody-dependent cell-mediated cytotoxicity
- ADCP reduced antibody-dependent cellular phagocytosis
- reduced immune complex-mediated antigen uptake by antigen-presenting cells reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling inducing
- the reduced effector function is one or more selected from the group of reduced CDC, reduced ADCC, reduced ADCP, and reduced cytokine secretion. In a particular embodiment the reduced effector function is reduced ADCC. In one embodiment the reduced ADCC is less than 20% of the ADCC induced by a non-engineered Fc domain (or an antibody comprising a non-engineered Fc domain).
- the amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor and/or effector function is an amino acid substitution. In one embodiment the Fc domain comprises an amino acid substitution at a position selected from the group of E233, L234, L235, N297, P331 and P329 (numberings according to Kabat EU index).
- the Fc domain comprises an amino acid substitution at a position selected from the group of L234, L235 and P329 (numberings according to Kabat EU index). In some embodiments the Fc domain comprises the amino acid substitutions L234A and L235A (numberings according to Kabat EU index). In one such embodiment, the Fc domain is an IgGi Fc domain, particularly a human IgGi Fc domain. In one embodiment the Fc domain comprises an amino acid substitution at position P329. In a more specific embodiment the amino acid substitution is P329A or P329G, particularly P329G (numberings according to Kabat EU index).
- the Fc domain comprises an amino acid substitution at position P329 and a further amino acid substitution at a position selected from E233, L234, L235, N297 and P331 (numberings according to Kabat EU index).
- the further amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S.
- the Fc domain comprises amino acid substitutions at positions P329, L234 and L235 (numberings according to Kabat EU index).
- the Fc domain comprises the amino acid mutations L234A, L235A and P329G (“P329G LALA”, “PGLALA” or“LALAPG”).
- each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A and P329G (Kabat EU index numbering), i.e. in each of the first and the second subunit of the Fc domain the leucine residue at position 234 is replaced with an alanine residue (L234A), the leucine residue at position 235 is replaced with an alanine residue (L235A) and the proline residue at position 329 is replaced by a glycine residue (P329G) (numbering according to Kabat EU index).
- the Fc domain is an IgGi Fc domain, particularly a human IgGi Fc domain.
- the Fc domain of the antibody comprised in the immunoconjugate of the invention is an IgG4 Fc domain, particularly a human IgG4 Fc domain.
- the IgG4 Fc domain comprises amino acid substitutions at position S228, specifically the amino acid substitution S228P (numberings according to Kabat EU index).
- the IgG4 Fc domain comprises an amino acid substitution at position L235, specifically the amino acid substitution L235E (numberings according to Kabat EU index).
- the IgG4 Fc domain comprises an amino acid substitution at position P329, specifically the amino acid substitution P329G (numberings according to Kabat EU index).
- the IgG4 Fc domain comprises amino acid substitutions at positions S228, L235 and P329, specifically amino acid substitutions S228P, L235E and P329G (numberings according to Kabat EU index).
- Such IgG4 Fc domain mutants and their Fey receptor binding properties are described in PCT publication no. WO 2012/130831, incorporated herein by reference in its entirety.
- the Fc domain exhibiting reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a native IgGi Fc domain is a human IgGi Fc domain comprising the amino acid substitutions L234A, L235A and optionally P329G, or a human IgG4 Fc domain comprising the amino acid substitutions S228P, L235E and optionally P329G (numberings according to Kabat EU index).
- the Fc domain comprises an amino acid mutation at position N297, particularly an amino acid substitution replacing asparagine by alanine (N297A) or aspartic acid (N297D) (numberings according to Kabat EU index).
- Fc domains with reduced Fc receptor binding and/or effector function also include those with substitution of one or more of Fc domain residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056) (numberings according to Kabat EU index).
- Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called“DANA” Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581).
- Mutant Fc domains can be prepared by amino acid deletion, substitution, insertion or modification using genetic or chemical methods well known in the art. Genetic methods may include site-specific mutagenesis of the encoding DNA sequence, PCR, gene synthesis, and the like. The correct nucleotide changes can be verified for example by sequencing. Binding to Fc receptors can be easily determined e.g. by ELISA, or by Surface Plasmon Resonance (SPR) using standard instrumentation such as a BIAcore instrument (GE Healthcare), and Fc receptors such as may be obtained by recombinant expression. Alternatively, binding affinity of Fc domains or antibodies comprising an Fc domain for Fc receptors may be evaluated using cell lines known to express particular Fc receptors, such as human NK cells expressing Fcyllla receptor.
- Effector function of an Fc domain, or an antibody comprising an Fc domain can be measured by methods known in the art.
- Examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362; Hellstrom et al. Proc Natl Acad Sci USA 83, 7059-7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502 (1985); U.S. Patent No. 5,821,337; Bruggemann et al., J Exp Med 166, 1351-1361 (1987).
- non radioactive assays methods may be employed (see, for example, ACTITM non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox 96 ® non-radioactive cytotoxicity assay (Promega, Madison, WI)).
- Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
- PBMC peripheral blood mononuclear cells
- NK Natural Killer
- ADCC activity of the molecule of interest may be assessed in vivo , e.g. in a animal model such as that disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652- 656 (1998).
- binding of the Fc domain to a complement component, specifically to Clq is reduced.
- said reduced effector function includes reduced CDC.
- Clq binding assays may be carried out to determine whether the Fc domain, or antibody comprising the Fc domain, is able to bind Clq and hence has CDC activity. See e.g., Clq and C3c binding ELISA in WO 2006/029879 and WO 2005/100402.
- a CDC assay may be performed (see, for example, Gazzano- Santoro et al., J Immunol Methods 202, 163 (1996); Cragg et al., Blood 101, 1045-1052 (2003); and Cragg and Glennie, Blood 103, 2738- 2743 (2004)).
- FcRn binding and in vivo clearance/half life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., Int’l. Immunol. 18(12): 1759-1769 (2006); WO 2013/120929).
- the invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to CD8,
- mutant IL-2 polypeptide is a human IL-2 molecule comprising the amino acid substitutions F42A, Y45A and L72G (numbering relative to the human IL-2 sequence SEQ ID NO: 13);
- the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:7, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 8.
- the invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to CD8, wherein the mutant IL-2 polypeptide is a human IL-2 molecule comprising the amino acid substitutions T3A, F42A, Y45A, L72G and C125A (numbering relative to the human IL-2 sequence SEQ ID NO: 13); and wherein the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:7, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 8.
- VH heavy chain variable region
- VL light chain variable region
- the invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to CD8, wherein the mutant IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 14; and wherein the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:7, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 8.
- VH heavy chain variable region
- VL light chain variable region
- the antibody is an IgG class immunoglobulin, comprising a human IgGi Fc domain composed of a first and a second subunit, wherein in the first subunit of the Fc domain the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the second subunit of the Fc domain the tyrosine residue at position 407 is replaced with a valine residue (Y407V) and optionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numberings according to Kabat EU index),
- each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A and P329G (Kabat EU index numbering).
- mutant IL-2 polypeptide may be fused at its amino-terminal amino acid to the carboxy -terminal amino acid of the first subunit of the Fc domain, through a linker peptide of SEQ ID NO: 15.
- the invention provides an immunoconjugate comprising a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 9, a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 10, and a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 11.
- the invention provides an immunoconjugate comprising a polypeptide comprising an amino acid sequence of SEQ ID NO:9, a polypeptide comprising an amino acid sequence of SEQ ID NO: 10, and a polypeptide comprising an amino acid sequence of SEQ ID NO: 11.
- the invention provides an immunoconjugate comprising two polypeptides comprising an amino acid sequence of SEQ ID NO:9, a polypeptide comprising an amino acid sequence of SEQ ID NO: 10, and a polypeptide comprising an amino acid sequence of SEQ ID NO: 11.
- the invention provides an immunoconjugate comprising a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 9, a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 10, and a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 12.
- the invention provides an immunoconjugate comprising a polypeptide comprising an amino acid sequence of SEQ ID NO:9, a polypeptide comprising an amino acid sequence of SEQ ID NO: 10, and a polypeptide comprising an amino acid sequence of SEQ ID NO: 12.
- the invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to CD8, wherein the mutant IL-2 polypeptide is a human IL-2 molecule comprising the amino acid substitutions F42A, Y45A and L72G (numbering relative to the human IL-2 sequence SEQ ID NO: 13); and wherein the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:7, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:29.
- VH heavy chain variable region
- VL light chain variable region
- the invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to CD8, wherein the mutant IL-2 polypeptide is a human IL-2 molecule comprising the amino acid substitutions T3A, F42A, Y45A, L72G and C125A (numbering relative to the human IL-2 sequence SEQ ID NO: 13); and wherein the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:7, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:29.
- VH heavy chain variable region
- VL light chain variable region
- the invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to CD8, wherein the mutant IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 14; and wherein the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:7, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:29.
- VH heavy chain variable region
- VL light chain variable region
- the antibody is an IgG class immunoglobulin, comprising a human IgGl Fc domain composed of a first and a second subunit, wherein in the first subunit of the Fc domain the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the second subunit of the Fc domain the tyrosine residue at position 407 is replaced with a valine residue (Y407V) and optionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numberings according to Kabat EU index), and wherein further each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A and P329G (Kabat EU index numbering).
- the mutant IL-2 polypeptide may be fused at its amino-terminal amino acid
- the invention provides an immunoconjugate comprising a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 30, a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 10, and a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 11.
- the invention provides an immunoconjugate comprising a polypeptide comprising an amino acid sequence of SEQ ID NO:30, a polypeptide comprising an amino acid sequence of SEQ ID NO: 10, and a polypeptide comprising an amino acid sequence of SEQ ID NO: 11.
- the invention provides an immunoconjugate comprising two polypeptides comprising an amino acid sequence of SEQ ID NO:30, a polypeptide comprising an amino acid sequence of SEQ ID NO: 10, and a polypeptide comprising an amino acid sequence of SEQ ID NO: 11.
- the invention provides an immunoconjugate comprising a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 30, a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 10, and a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 12.
- the invention provides an immunoconjugate comprising a polypeptide comprising an amino acid sequence of SEQ ID NO:30, a polypeptide comprising an amino acid sequence of SEQ ID NO: 10, and a polypeptide comprising an amino acid sequence of SEQ ID NO: 12.
- Polynucleotides The invention further provides isolated polynucleotides encoding an immunoconjugate as described herein or a fragment thereof. In some embodiments, said fragment is an antigen binding fragment.
- polynucleotides encoding immunoconjugates of the invention may be expressed as a single polynucleotide that encodes the entire immunoconjugate or as multiple (e.g., two or more) polynucleotides that are co-expressed.
- Polypeptides encoded by polynucleotides that are co expressed may associate through, e.g., disulfide bonds or other means to form a functional immunoconjugate.
- the light chain portion of an antibody may be encoded by a separate polynucleotide from the portion of the immunoconjugate comprising the heavy chain portion of the antibody and the mutant IL-2 polypeptide.
- the heavy chain polypeptides When co-expressed, the heavy chain polypeptides will associate with the light chain polypeptides to form the immunoconjugate.
- the portion of the immunoconjugate comprising one of the two Fc domain subunits and the mutant IL-2 polypeptide could be encoded by a separate polynucleotide from the portion of the immunoconjugate comprising the the other of the two Fc domain subunits.
- the Fc domain subunits will associate to form the Fc domain.
- the isolated polynucleotide encodes the entire immunoconjugate according to the invention as described herein. In other embodiments, the isolated polynucleotide encodes a polypeptide comprised in the immunoconjugate according to the invention as described herein.
- an isolated polynucleotide of the invention encodes the heavy chain of the antibody comprised in the immunoconjugate (e.g. an immunoglobulin heavy chain), and the mutant IL-2 polypeptide.
- an isolated polynucleotide of the invention encodes the light chain of the antibody comprised in the immunoconjugate.
- RNA for example, in the form of messenger RNA (mRNA).
- mRNA messenger RNA
- RNA of the present invention may be single stranded or double stranded.
- Mutant IL-2 polypeptides useful in the invention can be prepared by deletion, substitution, insertion or modification using genetic or chemical methods well known in the art. Genetic methods may include site-specific mutagenesis of the encoding DNA sequence, PCR, gene synthesis, and the like. The correct nucleotide changes can be verified for example by sequencing.
- the nucleotide sequence of native IL-2 has been described by Taniguchi et al. (Nature 302, 305-10 (1983)) and nucleic acid encoding human IL-2 is available from public depositories such as the American Type Culture Collection (Rockville MD).
- the sequence of native human IL-2 is shown in SEQ ID NO: 13. Substitution or insertion may involve natural as well as non-natural amino acid residues.
- Amino acid modification includes well known methods of chemical modification such as the addition of glycosylation sites or carbohydrate attachments, and the like.
- Immunoconjugates of the invention may be obtained, for example, by solid-state peptide synthesis (e.g. Merrifield solid phase synthesis) or recombinant production.
- one or more polynucleotide encoding the immunoconjugate (fragment), e.g., as described above, is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell.
- Such polynucleotide may be readily isolated and sequenced using conventional procedures.
- a vector, preferably an expression vector, comprising one or more of the polynucleotides of the invention is provided.
- the expression vector can be part of a plasmid, virus, or may be a nucleic acid fragment.
- the expression vector includes an expression cassette into which the polynucleotide encoding the immunoconjugate (fragment) (i.e. the coding region) is cloned in operable association with a promoter and/or other transcription or translation control elements.
- a "coding region" is a portion of nucleic acid which consists of codons translated into amino acids.
- a "stop codon" (TAG, TGA, or TAA) is not translated into an amino acid, it may be considered to be part of a coding region, if present, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, 5' and 3' untranslated regions, and the like, are not part of a coding region.
- Two or more coding regions can be present in a single polynucleotide construct, e.g. on a single vector, or in separate polynucleotide constructs, e.g. on separate (different) vectors.
- any vector may contain a single coding region, or may comprise two or more coding regions, e.g.
- a vector of the present invention may encode one or more polypeptides, which are post- or co-translationally separated into the final proteins via proteolytic cleavage.
- a vector, polynucleotide, or nucleic acid of the invention may encode heterologous coding regions, either fused or unfused to a polynucleotide encoding the immunoconjugate of the invention, or variant or derivative thereof.
- Heterologous coding regions include without limitation specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain.
- An operable association is when a coding region for a gene product, e.g.
- a polypeptide is associated with one or more regulatory sequences in such a way as to place expression of the gene product under the influence or control of the regulatory sequence(s).
- Two DNA fragments (such as a polypeptide coding region and a promoter associated therewith) are "operably associated” if induction of promoter function results in the transcription of mRNA encoding the desired gene product and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed.
- a promoter region would be operably associated with a nucleic acid encoding a polypeptide if the promoter was capable of effecting transcription of that nucleic acid.
- the promoter may be a cell-specific promoter that directs substantial transcription of the DNA only in predetermined cells.
- Other transcription control elements besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can be operably associated with the polynucleotide to direct cell-specific transcription.
- Suitable promoters and other transcription control regions are disclosed herein.
- a variety of transcription control regions are known to those skilled in the art. These include, without limitation, transcription control regions, which function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (e.g. the immediate early promoter, in conjunction with intron-A), simian virus 40 (e.g.
- transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit b-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers as well as inducible promoters (e.g. promoters inducible tetracyclins). Similarly, a variety of translation control elements are known to those of ordinary skill in the art.
- the expression cassette may also include other features such as an origin of replication, and/or chromosome integration elements such as retroviral long terminal repeats (LTRs), or adeno-associated viral (AAV) inverted terminal repeats (ITRs).
- LTRs retroviral long terminal repeats
- AAV adeno-associated viral
- Polynucleotide and nucleic acid coding regions of the present invention may be associated with additional coding regions which encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a polynucleotide of the present invention.
- proteins secreted by mammalian cells have a signal peptide or secretory leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated.
- polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the translated polypeptide to produce a secreted or "mature" form of the polypeptide.
- human IL-2 is translated with a 20 amino acid signal sequence at the N-terminus of the polypeptide, which is subsequently cleaved off to produce the mature, 133 amino acid human IL-2.
- the native signal peptide e.g.
- the IL-2 signal peptide or an immunoglobulin heavy chain or light chain signal peptide is used, or a functional derivative of that sequence that retains the ability to direct the secretion of the polypeptide that is operably associated with it.
- a heterologous mammalian signal peptide, or a functional derivative thereof may be used.
- the wild-type leader sequence may be substituted with the leader sequence of human tissue plasminogen activator (TP A) or mouse b-glucuronidase.
- DNA encoding a short protein sequence that could be used to facilitate later purification (e.g. a histidine tag) or assist in labeling the immunoconjugate may be included within or at the ends of the immunoconjugate (fragment) encoding polynucleotide.
- a host cell comprising one or more polynucleotides of the invention.
- a host cell comprising one or more vectors of the invention.
- the polynucleotides and vectors may incorporate any of the features, singly or in combination, described herein in relation to polynucleotides and vectors, respectively.
- a host cell comprises (e.g. has been transformed or transfected with) one or more vector comprising one or more polynucleotide that encodes the immunoconjugate of the invention.
- the term "host cell” refers to any kind of cellular system which can be engineered to generate the immunoconjugates of the invention or fragments thereof.
- Host cells suitable for replicating and for supporting expression of immunoconjugates are well known in the art. Such cells may be transfected or transduced as appropriate with the particular expression vector and large quantities of vector containing cells can be grown for seeding large scale fermenters to obtain sufficient quantities of the immunoconjugate for clinical applications.
- Suitable host cells include prokaryotic microorganisms, such as E. coli, or various eukaryotic cells, such as Chinese hamster ovary cells (CHO), insect cells, or the like.
- polypeptides may be produced in bacteria in particular when glycosylation is not needed. After expression, the polypeptide may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.
- eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for polypeptide-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been“humanized”, resulting in the production of a polypeptide with a partially or fully human glycosylation pattern.
- fungi and yeast strains whose glycosylation pathways have been“humanized”, resulting in the production of a polypeptide with a partially or fully human glycosylation pattern.
- Suitable host cells for the expression of (glycosylated) polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells.
- baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.
- Plant cell cultures can also be utilized as hosts. See e.g. US Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIESTM technology for producing antibodies in transgenic plants).
- Vertebrate cells may also be used as hosts.
- mammalian cell lines that are adapted to grow in suspension may be useful.
- TM4 cells as described, e.g., in Mather, Biol Reprod 23, 243-251 (1980)
- monkey kidney cells CV1
- African green monkey kidney cells VERO-76
- human cervical carcinoma cells HELA
- canine kidney cells MDCK
- buffalo rat liver cells BBL 3 A
- human lung cells W138
- human liver cells Hep G2
- mouse mammary tumor cells MMT 060562
- TRI cells as described, e.g., in Mather et ak, Annals N.Y.
- MRC 5 cells MRC 5 cells
- FS4 cells Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including dhfr CHO cells (Urlaub et ak, Proc Natl Acad Sci USA 77, 4216 (1980)); and myeloma cell lines such as YO, NS0, P3X63 and Sp2/0.
- CHO Chinese hamster ovary
- dhfr CHO cells Urlaub et ak, Proc Natl Acad Sci USA 77, 4216 (1980)
- myeloma cell lines such as YO, NS0, P3X63 and Sp2/0.
- Host cells include cultured cells, e.g., mammalian cultured cells, yeast cells, insect cells, bacterial cells and plant cells, to name only a few, but also cells comprised within a transgenic animal, transgenic plant or cultured plant or animal tissue.
- the host cell is a eukaryotic cell, preferably a mammalian cell, such as a Chinese Hamster Ovary (CHO) cell, a human embryonic kidney (HEK) cell or a lymphoid cell (e.g., Y0, NS0, Sp20 cell).
- CHO Chinese Hamster Ovary
- HEK human embryonic kidney
- a lymphoid cell e.g., Y0, NS0, Sp20 cell.
- Cells expressing a mutant-IL-2 polypeptide fused to either the heavy or the light chain of an antibody may be engineered so as to also express the other of the antibody chains such that the expressed mutant IL-2 fusion product is an antibody that has both a heavy and a light chain.
- a method of producing an immunoconjugate according to the invention comprises culturing a host cell comprising one or more polynucleotide encoding the immunoconjugate, as provided herein, under conditions suitable for expression of the immunoconjugate, and optionally recovering the immunoconjugate from the host cell (or host cell culture medium).
- the mutant IL-2 polypeptide may be genetically fused to the antibody, or may be chemically conjugated to the antibody. Genetic fusion of the IL-2 polypeptide to the antibody can be designed such that the IL-2 sequence is fused directly to the polypeptide or indirectly through a linker sequence. The composition and length of the linker may be determined in accordance with methods well known in the art and may be tested for efficacy. Particular linker peptides are described herein. Additional sequences may also be included to incorporate a cleavage site to separate the individual components of the fusion if desired, for example an endopeptidase recognition sequence.
- an IL-2 fusion protein may also be synthesized chemically using methods of polypeptide synthesis as is well known in the art (e.g. Merrifield solid phase synthesis). Mutant IL-2 polypeptides may be chemically conjugated to other molecules, e.g. antibodies, using well known chemical conjugation methods. Bi-functional cross-linking reagents such as homofunctional and heterofunctional cross-linking reagents well known in the art can be used for this purpose. The type of cross-linking reagent to use depends on the nature of the molecule to be coupled to IL-2 and can readily be identified by those skilled in the art.
- mutant IL-2 and/or the molecule to which it is intended to be conjugated may be chemically derivatized such that the two can be conjugated in a separate reaction as is also well known in the art.
- the immunoconjugates of the invention comprise an antibody. Methods to produce antibodies are well known in the art (see e.g. Harlow and Lane, "Antibodies, a laboratory manual", Cold Spring Harbor Laboratory, 1988). Non-naturally occurring antibodies can be constructed using solid phase-peptide synthesis, can be produced recombinantly (e.g. as described in U.S. patent No. 4,186,567) or can be obtained, for example, by screening combinatorial libraries comprising variable heavy chains and variable light chains (see e.g. U.S. Patent.
- Non limiting antibodies useful in the present invention can be of murine, primate, or human origin. If the immunoconjugate is intended for human use, a chimeric form of antibody may be used wherein the constant regions of the antibody are from a human.
- a humanized or fully human form of the antibody can also be prepared in accordance with methods well known in the art (see e. g. U.S. Patent No. 5,565,332 to Winter). Humanization may be achieved by various methods including, but not limited to (a) grafting the non-human (e.g., donor antibody) CDRs onto human (e.g. recipient antibody) framework and constant regions with or without retention of critical framework residues (e.g.
- Human framework regions that may be used for humanization include but are not limited to: framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151 :2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA , 89:4285 (1992); and Presta et al. J. Immunol ., 151 :2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci.
- Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel, Curr Opin Pharmacol 5, 368-74 (2001) and Lonberg, Curr Opin Immunol 20, 450-459 (2008). Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal’s chromosomes. In such transgenic mice, the endogenous immunoglobulin loci have generally been inactivated.
- Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse- human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, e.g., Kozbor J. Immunol ., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boemer et al., J. Immunol., 147: 86 (1991).) Human antibodies generated via human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006).
- Additional methods include those described, for example, in U.S. Patent No. 7,189,826 (describing production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas).
- Human hybridoma technology Trioma technology
- Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology , 27(3): 185-91 (2005).
- Human antibodies may also be generated by isolation from human antibody libraries, as described herein.
- Antibodies useful in the invention may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. Methods for screening combinatorial libraries are reviewed, e.g., in Lerner et al. in Nature Reviews 16:498-508 (2016). For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, e.g., in Frenzel et al. in mAbs 8: 1177-1194 (2016); Bazan et al. in Human Vaccines and Immunotherapeutics 8:1817-1828 (2012) and Zhao et al. in Critical Reviews in Biotechnology 36:276-289 (2016) as well as in Hoogenboom et al.
- repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al. in Annual Review of Immunology 12: 433-455 (1994).
- Phage typically display antibody fragments, either as single chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high- affinity antibodies to the immunogen without the requirement of constructing hybridomas.
- naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self antigens without any immunization as described by Griffiths et al. in EMBO Journal 12: 725-734 (1993).
- naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro , as described by Hoogenboom and Winter in Journal of Molecular Biology 227: 381-388 (1992).
- Patent publications describing human antibody phage libraries include, for example: US Patent Nos.
- immunoconjugate of the invention may be desirable.
- problems of immunogenicity and short half-life may be improved by conjugation to substantially straight chain polymers such as polyethylene glycol (PEG) or polypropylene glycol (PPG) (see e.g. WO 87/00056).
- PEG polyethylene glycol
- PPG polypropylene glycol
- Immunoconjugates prepared as described herein may be purified by art-known techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like.
- the actual conditions used to purify a particular protein will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity etc., and will be apparent to those having skill in the art.
- affinity chromatography purification an antibody, ligand, receptor or antigen can be used to which the immunoconjugate binds.
- an antibody which specifically binds the mutant IL-2 polypeptide may be used.
- affinity chromatography purification of immunoconjugates of the invention a matrix with protein A or protein G may be used.
- sequential Protein A or G affinity chromatography and size exclusion chromatography can be used to isolate an immunoconjugate essentially as described in the Examples.
- the purity of the immunoconjugate can be determined by any of a variety of well known analytical methods including gel electrophoresis, high pressure liquid chromatography, and the like.
- compositions Compositions, Formulations, and Routes of Administration
- the invention provides pharmaceutical compositions comprising an immunoconjugate as described herein, e.g., for use in any of the below therapeutic methods.
- a pharmaceutical composition comprises any of the immunoconjugates provided herein and a pharmaceutically acceptable carrier.
- a pharmaceutical composition comprises any of the immunoconjugates provided herein and at least one additional therapeutic agent, e.g., as described below.
- an immunoconjugate of the invention in a form suitable for administration in vivo, the method comprising (a) obtaining an immunoconjugate according to the invention, and (b) formulating the immunoconjugate with at least one pharmaceutically acceptable carrier, whereby a preparation of immunoconjugate is formulated for administration in vivo.
- compositions of the present invention comprise a therapeutically effective amount of immunoconjugate dissolved or dispersed in a pharmaceutically acceptable carrier.
- pharmaceutically acceptable refers to molecular entities and compositions that are generally non-toxic to recipients at the dosages and concentrations employed, i.e. do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate.
- the preparation of a pharmaceutical composition that contains immunoconjugate and optionally an additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference.
- compositions are lyophilized formulations or aqueous solutions.
- pharmaceutically acceptable carrier includes any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g.
- antibacterial agents antifungal agents
- isotonic agents absorption delaying agents, salts, preservatives, antioxidants, proteins, drugs, drug stabilizers, polymers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.
- An immunoconjugate of the invention can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration.
- Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g. by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.
- parenteral compositions include those designed for administration by injection, e.g. subcutaneous, intradermal, intralesional, intravenous, intraarterial intramuscular, intrathecal or intraperitoneal injection.
- the immunoconjugates of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer.
- the solution may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
- the immunoconjugates may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
- Sterile injectable solutions are prepared by incorporating the immunoconjugates of the invention in the required amount in the appropriate solvent with various of the other ingredients enumerated below, as required. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and/or the other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsion, the preferred methods of preparation are vacuum-drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered liquid medium thereof.
- the liquid medium should be suitably buffered if necessary and the liquid diluent first rendered isotonic prior to injection with sufficient saline or glucose.
- the composition must be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less that 0.5 ng/mg protein.
- Suitable pharmaceutically acceptable carriers include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides
- Aqueous injection suspensions may contain compounds which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, dextran, or the like.
- the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
- suspensions of the active compounds may be prepared as appropriate oily injection suspensions.
- Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl cleats or triglycerides, or liposomes.
- Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin- microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano particles and nanocapsules) or in macroemulsions.
- colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano particles and nanocapsules
- Sustained-release preparations may be prepared.
- sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, which matrices are in the form of shaped articles, e.g. films, or microcapsules.
- prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin or combinations thereof.
- the immunoconjugates may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
- the immunoconjugates may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
- compositions comprising the immunoconjugates of the invention may be manufactured by means of conventional mixing, dissolving, emulsifying, encapsulating, entrapping or lyophilizing processes.
- Pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the proteins into preparations that can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
- the immunoconjugates may be formulated into a composition in a free acid or base, neutral or salt form.
- Pharmaceutically acceptable salts are salts that substantially retain the biological activity of the free acid or base. These include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine. Pharmaceutical salts tend to be more soluble in aqueous and other protic solvents than are the corresponding free base forms.
- Immunoconjugates of the invention may be used as immunotherapeutic agents, for example in the treatment of cancers.
- immunoconjugates of the invention would be formulated, dosed, and administered in a fashion consistent with good medical practice.
- Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.
- Immunoconjugates of the invention may be particularly useful in treating disease states where stimulation of the immune system of the host is beneficial, in particular conditions where an enhanced cellular immune response is desirable. These may include disease states where the host immune response is insufficient or deficient. Disease states for which the immunoconjugates of the invention may be administered comprise, for example, a tumor or infection where a cellular immune response would be a critical mechanism for specific immunity.
- the immunoconjugates of the invention may be administered per se or in any suitable pharmaceutical composition.
- immunoconjugates of the invention for use as a medicament are provided.
- immunoconjugates of the invention for use in treating a disease are provided.
- immunoconjugates of the invention for use in a method of treatment are provided.
- the invention provides an immunoconjugate as described herein for use in the treatment of a disease in an individual in need thereof.
- the invention provides an immunoconjugate for use in a method of treating an individual having a disease comprising administering to the individual a therapeutically effective amount of the immunoconjugate.
- the disease to be treated is a proliferative disorder.
- the disease is cancer.
- the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., an anti-cancer agent if the disease to be treated is cancer.
- the invention provides an immunoconjugate for use in stimulating the immune system.
- the invention provides an immunoconjugate for use in a method of stimulating the immune system in an individual comprising administering to the individual an effective amount of the immunoconjugate to stimulate the immune system.
- An “individual” according to any of the above embodiments is a mammal, preferably a human.
- “Stimulation of the immune system” according to any of the above embodiments may include any one or more of a general increase in immune function, an increase in T cell function, an increase in B cell function, a restoration of lymphocyte function, an increase in the expression of IL-2 receptors, an increase in T cell responsiveness, an increase in natural killer cell activity or lymphokine-activated killer (LAK) cell activity, and the like.
- LAK lymphokine-activated killer
- the invention provides for the use of an immunconjugate of the invention in the manufacture or preparation of a medicament.
- the medicament is for the treatment of a disease in an individual in need thereof.
- the medicament is for use in a method of treating a disease comprising administering to an individual having the disease a therapeutically effective amount of the medicament.
- the disease to be treated is a proliferative disorder.
- the disease is cancer.
- the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., an anti-cancer agent if the disease to be treated is cancer.
- the medicament is for stimulating the immune system.
- the medicament is for use in a method of stimulating the immune system in an individual comprising administering to the individual an effective amount of the medicament to stimulate the immune system.
- LAK lymphokine- activated killer
- the invention provides a method for treating a disease in an individual.
- the method comprises administering to an individual having such disease a therapeutically effective amount of an immunoconjugate of the invention.
- a composition is administered to said invididual, comprising the immunoconjugate of the invention in a pharmaceutically acceptable form.
- the disease to be treated is a proliferative disorder.
- the disease is cancer.
- the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., an anti-cancer agent if the disease to be treated is cancer.
- the invention provides a method for stimulating the immune system in an individual, comprising administering to the individual an effective amount of an immunoconjugate to stimulate the immune system.
- An“individual” according to any of the above embodiments may be a mammal, preferably a human.
- “Stimulation of the immune system” according to any of the above embodiments may include any one or more of a general increase in immune function, an increase in T cell function, an increase in B cell function, a restoration of lymphocyte function, an increase in the expression of IL-2 receptors, an increase in T cell responsiveness, an increase in natural killer cell activity or lymphokine-activated killer (LAK) cell activity, and the like.
- LAK lymphokine-activated killer
- the disease to be treated is a proliferative disorder, particularly cancer.
- cancers include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer.
- neoplasms located in the: abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic region, and urogenital system. Also included are pre-cancerous conditions or lesions and cancer metastases.
- the cancer is chosen from the group consisting of kidney cancer, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer, head and neck cancer, prostate cancer and bladder cancer.
- the immunoconjugates may not provide a cure but may only provide partial benefit.
- a physiological change having some benefit is also considered therapeutically beneficial.
- an amount of immunoconjugate that provides a physiological change is considered an "effective amount” or a "therapeutically effective amount".
- the subject, patient, or individual in need of treatment is typically a mammal, more specifically a human.
- an effective amount of an immunoconjugate of the invention is administered to a cell. In other embodiments, a therapeutically effective amount of an immunoconjugates of the invention is administered to an individual for the treatment of disease.
- an immunoconjugate of the invention when used alone or in combination with one or more other additional therapeutic agents, will depend on the type of disease to be treated, the route of administration, the body weight of the patient, the type of molecule (e.g. comprising an Fc domain or not), the severity and course of the disease, whether the immunoconjugate is administered for preventive or therapeutic purposes, previous or concurrent therapeutic interventions, the patient's clinical history and response to the immunoconjugate, and the discretion of the attending physician..
- the practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.
- Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.
- the immunoconjugate is suitably administered to the patient at one time or over a series of treatments.
- about 1 pg/kg to 15 mg/kg (e.g. 0.1 mg/kg - 10 mg/kg) of immunoconjugate can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion.
- One typical daily dosage might range from about 1 pg/kg to 100 mg/kg or more, depending on the factors mentioned above.
- the treatment would generally be sustained until a desired suppression of disease symptoms occurs.
- One exemplary dosage of the immunoconjugate would be in the range from about 0.005 mg/kg to about 10 mg/kg.
- a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein.
- a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc. can be administered, based on the numbers described above.
- one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 5.0 mg/kg or 10 mg/kg (or any combination thereof) may be administered to the patient.
- Such doses may be administered intermittently, e.g. every week or every three weeks (e.g. such that the patient receives from about two to about twenty, or e.g. about six doses of the immunoconjugate).
- An initial higher loading dose, followed by one or more lower doses may be administered.
- other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
- the immunoconjugates of the invention will generally be used in an amount effective to achieve the intended purpose.
- the immunoconjugates of the invention, or pharmaceutical compositions thereof are administered or applied in a therapeutically effective amount. Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.
- a therapeutically effective dose can be estimated initially from in vitro assays, such as cell culture assays.
- a dose can then be formulated in animal models to achieve a circulating concentration range that includes the ICso as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.
- Initial dosages can also be estimated from in vivo data, e.g., animal models, using techniques that are well known in the art. One having ordinary skill in the art could readily optimize administration to humans based on animal data. Dosage amount and interval may be adjusted individually to provide plasma levels of the immunoconjugates which are sufficient to maintain therapeutic effect. Usual patient dosages for administration by injection range from about 0.1 to 50 mg/kg/day, typically from about 0.5 to 1 mg/kg/day. Therapeutically effective plasma levels may be achieved by administering multiple doses each day. Levels in plasma may be measured, for example, by HPLC.
- the effective local concentration of the immunoconjugates may not be related to plasma concentration.
- One having skill in the art will be able to optimize therapeutically effective local dosages without undue experimentation.
- a therapeutically effective dose of the immunoconjugates described herein will generally provide therapeutic benefit without causing substantial toxicity.
- Toxicity and therapeutic efficacy of an immunoconjugate can be determined by standard pharmaceutical procedures in cell culture or experimental animals. Cell culture assays and animal studies can be used to determine the LD50 (the dose lethal to 50% of a population) and the ED50 (the dose therapeutically effective in 50% of a population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50/ED50.
- Immunoconjugates that exhibit large therapeutic indices are preferred. In one embodiment, the immunoconjugate according to the present invention exhibits a high therapeutic index.
- the data obtained from cell culture assays and animal studies can be used in formulating a range of dosages suitable for use in humans.
- the dosage lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
- the dosage may vary within this range depending upon a variety of factors, e.g., the dosage form employed, the route of administration utilized, the condition of the subject, and the like.
- the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See, e.g., Fingl et ah, 1975, In: The Pharmacological Basis of Therapeutics, Ch. 1, p. 1, incorporated herein by reference in its entirety).
- the attending physician for patients treated with immunoconjugates of the invention would know how and when to terminate, interrupt, or adjust administration due to toxicity, organ dysfunction, and the like. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity).
- the magnitude of an administered dose in the management of the disorder of interest will vary with the severity of the condition to be treated, with the route of administration, and the like. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency will also vary according to the age, body weight, and response of the individual patient.
- the maximum therapeutic dose of an immunoconjugate comprising a mutant IL-2 polypeptide as described herein may be increased from those used for an immunoconjugate comprising wild- type IL-2.
- an immunoconjugate of the invention may be co administered with at least one additional therapeutic agent.
- therapeutic agent encompasses any agent administered to treat a symptom or disease in an individual in need of such treatment.
- additional therapeutic agent may comprise any active ingredients suitable for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
- an additional therapeutic agent is an immunomodulatory agent, a cytostatic agent, an inhibitor of cell adhesion, a cytotoxic agent, an activator of cell apoptosis, or an agent that increases the sensitivity of cells to apoptotic inducers.
- the additional therapeutic agent is an anti-cancer agent, for example a microtubule disruptor, an antimetabolite, a topoisomerase inhibitor, a DNA intercalator, an alkylating agent, a hormonal therapy, a kinase inhibitor, a receptor antagonist, an activator of tumor cell apoptosis, or an antiangiogenic agent.
- an anti-cancer agent for example a microtubule disruptor, an antimetabolite, a topoisomerase inhibitor, a DNA intercalator, an alkylating agent, a hormonal therapy, a kinase inhibitor, a receptor antagonist, an activator of tumor cell apoptosis, or an antiangiogenic agent.
- Such other agents are suitably present in combination in amounts that are effective for the purpose intended.
- the effective amount of such other agents depends on the amount of immunoconjugate used, the type of disorder or treatment, and other factors discussed above.
- the immunoconjugates are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate.
- Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate compositions), and separate administration, in which case, administration of the immunoconjugate of the invention can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent and/or adjuvant.
- Immunoconjugates of the invention may also be used in combination with radiation therapy.
- an article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of the disorders described above comprises a container and a label or package insert on or associated with the container.
- Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc.
- the containers may be formed from a variety of materials such as glass or plastic.
- the container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
- At least one active agent in the composition is an immunoconjugate of the invention.
- the label or package insert indicates that the composition is used for treating the condition of choice.
- the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises an immunoconjugate of the invention; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent.
- the article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition.
- the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
- BWFI bacteriostatic water for injection
- phosphate-buffered saline such
- FIG. 1A and IB Schematic representation of the IgG-IL-2 immunoconjugate format, comprising mutant IL-2 polypeptide.
- FIG. lA two-armed anti-CD8;
- FIG.2A one-armed anti- CD8.
- Figure 2. Binding of two-armed antiCD8-IL2v (CD8-IL2v TA) and one-armed antiCD8-IL2v (CD8-IL2v OA) in comparison to antiFAP-IL2v (FAP-IL2v) to CD8 T cells within resting PBMCs was determined by flow cytometry. Molecules were detected with a fluorescently labeled anti-human Fc specific secondary antibody.
- CD8 T cells were identified using CD3 and CD8 staining of PBMCs.
- FIG.3A Figure 3A, 3B, 3C and 3D.
- STAT5 phosphorylation in CD8 T cells Fig.3A
- CD4 T cells Fig.3C
- regulatory T cells Fig.3D
- NK cells Fig.3D
- FIG.4C Proliferation of NK cells (Fig.4C), CD4 T cells (Fig.4B) and CD8 T cells (Fig.4C) within PBMCs with CD8-IL2v TA, CD8-IL2v OA and FAP-IL2v was determined by flow cytometry.
- FIG.5A Figure 5A, 5B, 5C and 5D.
- STAT5 phosphorylation in CD8 T cells Fig.5A
- NK cells Fig.5B
- CD4 T cells Fig.5C
- regulatory T cells Fig.5D
- the abbreviation“TA” stands for“two-armed” herein.
- the abbreviation“OA” stands“for one- armed” herein.
- the terms“antiCD8-IL2v TA” and“CD8-IL2v TA” are used interchangeable herein.
- the terms“antiCD8-IL2v OA” and“CD8-IL2v OA” are used interchangeable herein.
- IgG-IL2 molecules were generated by transient transfection of HEK293 EBNA cells.
- the cells are transfected with the corresponding expression vectors in 1 :1 :2 (“vector heavy chain (VH-CH 1 -CH2-CH3 )” :“vector heavy chain (VH-CHl-CH2-CH3-IL2v)”: “vector light chain (VL-CL)”).
- the cells are transfected with the corresponding expression vectors in a 1 : 1 : 1 ratio (“vector heavy chain (VH-CH 1-CH2-CH3)” : “vector heavy chain (CH2-CH3-IL2v)” :“vector light chain (VL-CL)” ).
- VH-CH 1-CH2-CH3 vector heavy chain
- VL-CL vector light chain
- Cells were centrifuged and, medium was replaced by pre-warmed CD CHO medium (Thermo Fisher, Cat N° 10743029).
- Expression vectors were mixed in CD CHO medium, PEI (Polyethylenimine, Polysciences, Inc, Cat N° 23966-1) was added, the solution vortexed and incubated for 10 minutes at room temperature.
- Proteins were purified from filtered cell culture supernatants referring to standard protocols.
- Fc containing proteins were purified from cell culture supernatants by Protein A-affmity chromatography (equilibration buffer: 20 mM sodium citrate, 20 mM sodium phosphate, pH 7.5; elution buffer: 20 mM sodium citrate, 100 mM NaCl, 100 mM glycine pH 3.0). Elution was achieved at pH 3.0 followed by immediate pH neutralization of the sample.
- the protein was concentrated by centrifugation (Millipore Amicon® ULTRA- 15 (Art.Nr. : UFC903096), and aggregated protein was separated from monomeric protein by size exclusion chromatography in 20 mM histidine, 140 mM sodium chloride, pH 6.0.
- concentrations of purified proteins were determined by measuring the absorption at 280 nm using the mass extinction coefficient calculated on the basis of the amino acid sequence according to Pace, et al., Protein Science, 1995, 4, 2411-1423. Purity and molecular weight of the proteins were analyzed by CE-SDS in the presence and absence of a reducing agent using a LabChipGXII (Perkin Elmer).
- Determination of the aggregate content was performed by HPLC chromatography at 25°C using analytical size-exclusion column (TSKgel G3000 SW XL or UP- SW3000) equilibrated in running buffer (25 mM K2HPO4, 125 mM NaCl, 200mM L-arginine monohydrochloride, pH 6.7 or 200 mM KH2PO4, 250 mM KC1 pH 6.2, respectively).
- running buffer 25 mM K2HPO4, 125 mM NaCl, 200mM L-arginine monohydrochloride, pH 6.7 or 200 mM KH2PO4, 250 mM KC1 pH 6.2, respectively.
- antiCD8-IL2v TA consists of two polypeptids with the amino acid sequence of SEQ ID NO:9, one polypeptide with the amino acid sequence of SEQ ID NO: 10 and one polypeptide with the amino acid sequence of SEQ ID NO: 11 (format: A2HK).
- antiCD8-IL2v OA consists of one polypeptide with the amino acid sequence of SEQ ID NO: 9, one polypeptide with the amino acid sequence of SEQ ID NO: 10 and one polypetide with the amino acid sequence of SEQ ID NO: 12 (format: AHK).
- antiCD8(OKT8.vl l)-IL2v TA consists of two polypeptids with the amino acid sequence of SEQ ID NO: 30, one polypeptide with the amino acid sequence of SEQ ID NO: 10 and one polypeptide with the amino acid sequence of SEQ ID NO: 11 (format: A2HK).
- antiCD8(OKT8.vl l)-IL2v TA consists of one polypeptide with the amino acid sequence of SEQ ID NO: 30, one polypeptide with the amino acid sequence of SEQ ID NO: 10 and one polypetide with the amino acid sequence of SEQ ID NO: 12 (format: AHK).
- FACS buffer PBS, 2% FBS, 5 mM EDTA, 0.025% NaN3
- antiCD8-IL2v TA and antiCD8-IL2v OA molecules were evaluated for their capacity to bind to CD8 T cells within PBMCs in comparison to FAP-IL2v (as e.g. disclosed in W02012107417A1, which is incorporated by reference; FAP-IL2v comprises the polypeptides according to SEQ ID NO: 25, 26 and 27).
- FAP-IL2v comprises the polypeptides according to SEQ ID NO: 25, 26 and 27.
- antiCD8-IL2v TA showed very strong binding to CD8 T cells, anti CD8-IL2v OA bound weaker to CD8 T cells but still much stronger than FAP- IL2v.
- Freshly isolated PBMCs from healthy donors were seeded in warm medium (RPMI1640, 10% FCS, 2 mM Glutamine) into a 96 well round bottom plate (200 ⁇ 00 cells/well). The plates were centrifuged at 300 g for 10 min and the supernatant was removed. The cells were re-suspended in
- the cells were washed twice with 150 m ⁇ cold FACS buffer and split in two 96 well round bottom plates and stained each with 20 m ⁇ of the antibody mix I or II for 60 min in the fridge.
- Antibody mix I was used to stain pSTAT5 in CD4 T cells and regulatory T cells and antibody mix II was used to stain pSTAT5 in CD8 T cells and NK cells.
- the cells were washed twice with FACS buffer and re-suspended in 200 m ⁇ FACS buffer containing 2 % PFA per well.
- CD8 T cells CD3 + CD8 +
- NK cells CD3 CD56 +
- CD4 T cells CD4 +
- Tregs CD4 + CD25 + FoxP3 +
- the functional activity of antiCD8-IL2v TA and antiCD8-IL2v OA to induce STAT5 phosphorylation was compared to the functional activity of FAP-IL2v on the different immune cell subsets within PBMCs.
- STAT5 phosphorylation was used as a marker for IL2 receptor (IL2R) activation.
- IL2R IL2 receptor
- antiCD8-IL2v TA and antiCD8-IL2v OA had a much higher potency in inducing STAT5 phosphorylation with antiCD8-IL2v TA being slightly more potent than antiCD8-IL2v OA.
- antiCD8-IL2v TA and antiCD8-IL2v OA were a bit more potent than FAP-IL2v, probably because of a fraction of NK cells that are positive for CD8.
- PBMCs Freshly isolated PBMCs from healthy donors were labeled with CFSE (5(6)-Carboxyfluorescein diacetate N-succinimidyl ester, 21888, Sigma-Aldrich). Briefly 30 million PBMCs were washed once with PBS. In parallel the CSFE stock solution (2 mM in DMSO) was diluted 1 :20 in PBS. PBMCs were resuspended in 30 ml prewarmed PBS, 30 m ⁇ of the CFSE solution was added and the cells were mixed immediately. For an optimal labeling the cells were incubated for 15 min at
- PBMCs were washed twice with FACS buffer before fixing them with 1% PFA in FACS buffer and measuring the fluorescence with a BD Fortessa. Proliferation was determined by measuring CFSE dilution of CD8 T cells (CD3 + CD8 + ), CD4 T cells (CD3 + CD4 + ) and NK cells (CD3 CD56 + ).
- CD8-IL2v and CD8-IL2v OA were tested for their ability to induce proliferation of CD8 T cells, CD4 T cells and NK cells in comparison to FAP-IL2v.
- CD8-IL2v TA and CD8-IL2v OA had similar activity as FAP-IL2v to induce proliferation of CD4 T cells and NK cells.
- both molecules were much more potent in inducing proliferation than FAP-IL2v due to direct targeting of the molecules to these cells via CD8.
- CD8-IL2v TA was about 1300 fold more potent and CD8-IL2v OA was about 200 fold more potent. This is in line with the differences observed in binding and STAT5 phosphorylation on CD8 T cells.
- Freshly isolated PBMCs from healthy donors were seeded in warm medium (RPMI1640, 10% FCS, 2 mM Glutamine) into a 96 well round bottom plate (200 ⁇ 00 cells/well). The plates were centrifuged at 300 g for 10 min and the supernatant was removed. The cells were re-suspended in
- the cells were immediately fixed after stimulation with equal amount of pre-warmed Cytofix buffer (554655, BD Bioscience) for 10 min at 37 °C. Afterwards the plates were centrifuged for 10 min at 300 g and the supernatant was removed. To allow intracellular staining, the cells were permeabilized in 200 m ⁇ Phosflow Perm buffer III (558050, BD Bioscience) for 30 min at 4°C. Then the cells were washed twice with 150 m ⁇ cold FACS buffer and split in two 96 well round bottom plates and stained each with 20 m ⁇ of the antibody mix I or II for 60 min in the fridge.
- Cytofix buffer 554655, BD Bioscience
- Antibody mix I was used to stain pSTAT5 in CD4 T cells and regulatory T cells and antibody mix II was used to stain pSTAT5 in CD8 T cells and NK cells. Afterwards the cells were washed twice with FACS buffer and re-suspended in 200 m ⁇ FACS buffer containing 2 % PFA per well. The analysis was performed using a BD Fortessa flow cytometer gating on CD8 T cells (CD3 + CD8 + ), NK cells (CD3 CD56 + , CD4 T cells (CD4 + ) and Tregs (CD4 + CD25 + FoxP3 + ).
- the functional activity of CD8-IL2v TA and CD8-IL2v OKT8.vl l TA to induce STAT5 phosphorylation was compared to the functional activity of FAP-IL2v on the different immune cell subsets within PBMCs.
- STAT5 phosphorylation was used as a marker for IL2 receptor (IL2R) activation.
- IL2R IL2 receptor
- Regs regulatory T cells
- CD8-IL2v TA and CD8-IL2v OKT8.vl l TA showed a much higher potency in inducing STAT5 phosphorylation but there is no difference in activation between CD8-IL2v TA and CD8-IL2v OKT8.V11 TA.
- NK cells CD8-IL2v TA and CD8-IL2v OKT8.vl l TA were a bit more potent than FAP-IL2v, probably because of a fraction of NK cells that are positive for CD8.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Biomedical Technology (AREA)
- Veterinary Medicine (AREA)
- Biotechnology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Wood Science & Technology (AREA)
- Pharmacology & Pharmacy (AREA)
- Gastroenterology & Hepatology (AREA)
- General Engineering & Computer Science (AREA)
- General Chemical & Material Sciences (AREA)
- Toxicology (AREA)
- Urology & Nephrology (AREA)
- Epidemiology (AREA)
- Microbiology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Cell Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicinal Preparation (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
Description
Claims
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP19183829 | 2019-07-02 | ||
PCT/EP2020/068186 WO2021001289A1 (en) | 2019-07-02 | 2020-06-29 | Immunoconjugates comprising a mutant interleukin-2 and an anti-cd8 antibody |
Publications (1)
Publication Number | Publication Date |
---|---|
EP3994169A1 true EP3994169A1 (en) | 2022-05-11 |
Family
ID=67296960
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP20734407.8A Withdrawn EP3994169A1 (en) | 2019-07-02 | 2020-06-29 | Immunoconjugates comprising a mutant interleukin-2 and an anti-cd8 antibody |
Country Status (6)
Country | Link |
---|---|
EP (1) | EP3994169A1 (en) |
JP (1) | JP2022538139A (en) |
CN (1) | CN114051500A (en) |
AR (1) | AR119338A1 (en) |
TW (1) | TW202115115A (en) |
WO (1) | WO2021001289A1 (en) |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP3648786A4 (en) | 2017-07-03 | 2021-12-15 | Torque Therapeutics, Inc. | Fusion molecules targeting immune regulatory cells and uses thereof |
WO2023004305A1 (en) * | 2021-07-20 | 2023-01-26 | Inhibrx, Inc. | Cd8-targeted modified il-2 polypeptides and uses thereof |
TW202321283A (en) * | 2021-07-20 | 2023-06-01 | 美商英伊布里克斯公司 | Cd8-binding polypeptides and uses thereof |
CN116041539B (en) * | 2022-10-31 | 2023-07-21 | 山东博安生物技术股份有限公司 | IL-2 mutant immunoconjugates |
Family Cites Families (65)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
BR7801185A (en) | 1977-04-18 | 1978-11-28 | Hitachi Metals Ltd | ORNATO ADAPTED TO BE FIXED BY AT LEAST ONE PERMANENT IMA |
US4518584A (en) | 1983-04-15 | 1985-05-21 | Cetus Corporation | Human recombinant interleukin-2 muteins |
US5116943A (en) | 1985-01-18 | 1992-05-26 | Cetus Corporation | Oxidation-resistant muteins of Il-2 and other protein |
DE3676670D1 (en) | 1985-06-26 | 1991-02-07 | Cetus Corp | SOLUBILIZATION OF PROTEINS FOR PHARMACEUTICAL COMPOSITIONS BY POLYMER CONJUGATION. |
US5206344A (en) | 1985-06-26 | 1993-04-27 | Cetus Oncology Corporation | Interleukin-2 muteins and polymer conjugation thereof |
US6548640B1 (en) | 1986-03-27 | 2003-04-15 | Btg International Limited | Altered antibodies |
IL85035A0 (en) | 1987-01-08 | 1988-06-30 | Int Genetic Eng | Polynucleotide molecule,a chimeric antibody with specificity for human b cell surface antigen,a process for the preparation and methods utilizing the same |
ATE102631T1 (en) | 1988-11-11 | 1994-03-15 | Medical Res Council | CLONING OF IMMUNOGLOBULIN SEQUENCES FROM THE VARIABLE DOMAINS. |
DE3920358A1 (en) | 1989-06-22 | 1991-01-17 | Behringwerke Ag | BISPECIFIC AND OLIGO-SPECIFIC, MONO- AND OLIGOVALENT ANTI-BODY CONSTRUCTS, THEIR PRODUCTION AND USE |
US5959177A (en) | 1989-10-27 | 1999-09-28 | The Scripps Research Institute | Transgenic plants expressing assembled secretory antibodies |
US6150584A (en) | 1990-01-12 | 2000-11-21 | Abgenix, Inc. | Human antibodies derived from immunized xenomice |
US6075181A (en) | 1990-01-12 | 2000-06-13 | Abgenix, Inc. | Human antibodies derived from immunized xenomice |
GB9015198D0 (en) | 1990-07-10 | 1990-08-29 | Brien Caroline J O | Binding substance |
US5770429A (en) | 1990-08-29 | 1998-06-23 | Genpharm International, Inc. | Transgenic non-human animals capable of producing heterologous antibodies |
DK0564531T3 (en) | 1990-12-03 | 1998-09-28 | Genentech Inc | Enrichment procedure for variant proteins with altered binding properties |
US5571894A (en) | 1991-02-05 | 1996-11-05 | Ciba-Geigy Corporation | Recombinant antibodies specific for a growth factor receptor |
DE69233254T2 (en) | 1991-06-14 | 2004-09-16 | Genentech, Inc., South San Francisco | Humanized Heregulin antibody |
GB9114948D0 (en) | 1991-07-11 | 1991-08-28 | Pfizer Ltd | Process for preparing sertraline intermediates |
ES2136092T3 (en) | 1991-09-23 | 1999-11-16 | Medical Res Council | PROCEDURES FOR THE PRODUCTION OF HUMANIZED ANTIBODIES. |
US5587458A (en) | 1991-10-07 | 1996-12-24 | Aronex Pharmaceuticals, Inc. | Anti-erbB-2 antibodies, combinations thereof, and therapeutic and diagnostic uses thereof |
DE69333807T2 (en) | 1992-02-06 | 2006-02-02 | Chiron Corp., Emeryville | MARKERS FOR CANCER AND BIOSYNTHETIC BINDEPROTEIN THEREFOR |
US5229109A (en) | 1992-04-14 | 1993-07-20 | Board Of Regents, The University Of Texas System | Low toxicity interleukin-2 analogues for use in immunotherapy |
US5731168A (en) | 1995-03-01 | 1998-03-24 | Genentech, Inc. | Method for making heteromultimeric polypeptides |
US5869046A (en) | 1995-04-14 | 1999-02-09 | Genentech, Inc. | Altered polypeptides with increased half-life |
JP4213224B2 (en) | 1997-05-02 | 2009-01-21 | ジェネンテック,インコーポレーテッド | Method for producing multispecific antibody having heteromultimer and common component |
US6040498A (en) | 1998-08-11 | 2000-03-21 | North Caroline State University | Genetically engineered duckweed |
US6610833B1 (en) | 1997-11-24 | 2003-08-26 | The Institute For Human Genetics And Biochemistry | Monoclonal human natural antibodies |
WO1999029888A1 (en) | 1997-12-05 | 1999-06-17 | The Scripps Research Institute | Humanization of murine antibody |
US6737056B1 (en) | 1999-01-15 | 2004-05-18 | Genentech, Inc. | Polypeptide variants with altered effector function |
US7125978B1 (en) | 1999-10-04 | 2006-10-24 | Medicago Inc. | Promoter for regulating expression of foreign genes |
AU782626B2 (en) | 1999-10-04 | 2005-08-18 | Medicago Inc. | Method for regulating transcription of foreign genes |
US20030180714A1 (en) | 1999-12-15 | 2003-09-25 | Genentech, Inc. | Shotgun scanning |
US6596541B2 (en) | 2000-10-31 | 2003-07-22 | Regeneron Pharmaceuticals, Inc. | Methods of modifying eukaryotic cells |
TWI313299B (en) | 2000-11-30 | 2009-08-11 | Medarex Inc | Transgenic transchromosomal rodents for making human antibodies |
AU2002355955A1 (en) | 2001-08-13 | 2003-03-03 | University Of Southern California | Interleukin-2 mutants with reduced toxicity |
RU2312677C9 (en) | 2001-12-04 | 2008-03-27 | Мерк Патент Гмбх | Immunocytokines possessing modulated selectivity |
CA2488441C (en) | 2002-06-03 | 2015-01-27 | Genentech, Inc. | Synthetic antibody phage libraries |
CA2510003A1 (en) | 2003-01-16 | 2004-08-05 | Genentech, Inc. | Synthetic antibody phage libraries |
WO2005097832A2 (en) | 2004-03-31 | 2005-10-20 | Genentech, Inc. | Humanized anti-tgf-beta antibodies |
US7785903B2 (en) | 2004-04-09 | 2010-08-31 | Genentech, Inc. | Variable domain library and uses |
NZ549872A (en) | 2004-04-13 | 2009-09-25 | Hoffmann La Roche | Anti-P-selectin antibodies |
TWI380996B (en) | 2004-09-17 | 2013-01-01 | Hoffmann La Roche | Anti-ox40l antibodies |
RS59761B1 (en) | 2005-02-07 | 2020-02-28 | Roche Glycart Ag | Antigen binding molecules that bind egfr, vectors encoding same, and uses thereof |
EP3050963B1 (en) | 2005-03-31 | 2019-09-18 | Chugai Seiyaku Kabushiki Kaisha | Process for production of polypeptide by regulation of assembly |
EP2465870A1 (en) | 2005-11-07 | 2012-06-20 | Genentech, Inc. | Binding polypeptides with diversified and consensus VH/VL hypervariable sequences |
WO2007064919A2 (en) | 2005-12-02 | 2007-06-07 | Genentech, Inc. | Binding polypeptides with restricted diversity sequences |
CA2646965C (en) | 2006-03-24 | 2016-06-21 | Jonathan H. Davis | Engineered heterodimeric protein domains |
EP2016101A2 (en) | 2006-05-09 | 2009-01-21 | Genentech, Inc. | Binding polypeptides with optimized scaffolds |
EP2035456A1 (en) | 2006-06-22 | 2009-03-18 | Novo Nordisk A/S | Production of bispecific antibodies |
ES2368868T3 (en) | 2006-09-20 | 2011-11-23 | Mt-Biomethan Gmbh | PROCEDURE AND DEVICE FOR SEPARATION OF METHANE AND CARBON DIOXIDE OF BIOGAS. |
US8906356B2 (en) | 2007-11-05 | 2014-12-09 | Massachusetts Institute Of Technology | Mutant interleukin-2 (IL-2) polypeptides |
EP2235064B1 (en) | 2008-01-07 | 2015-11-25 | Amgen Inc. | Method for making antibody fc-heterodimeric molecules using electrostatic steering effects |
CA2759233C (en) | 2009-04-27 | 2019-07-16 | Oncomed Pharmaceuticals, Inc. | Method for making heteromultimeric molecules |
EP2467165B1 (en) | 2009-08-17 | 2015-01-07 | Roche Glycart AG | Targeted immunoconjugates |
WO2011090762A1 (en) | 2009-12-29 | 2011-07-28 | Emergent Product Development Seattle, Llc | Heterodimer binding proteins and uses thereof |
JP6022444B2 (en) | 2010-05-14 | 2016-11-09 | ライナット ニューロサイエンス コーポレイション | Heterodimeric protein and method for producing and purifying it |
JP6167040B2 (en) | 2010-11-05 | 2017-07-19 | ザイムワークス,インコーポレイテッド | Design of stable heterodimeric antibodies with mutations in the Fc domain |
SI3489255T1 (en) | 2011-02-10 | 2021-11-30 | Roche Glycart Ag | Mutant interleukin-2 polypeptides |
PT2691417T (en) | 2011-03-29 | 2018-10-31 | Roche Glycart Ag | Antibody fc variants |
EA201892619A1 (en) | 2011-04-29 | 2019-04-30 | Роше Гликарт Аг | IMMUNOCONJUGATES CONTAINING INTERLEUKIN-2 MUTANT POLYPETIPS |
US9527927B2 (en) | 2011-12-20 | 2016-12-27 | Medimmune, Llc | Modified polypeptides for bispecific antibody scaffolds |
ES2676031T3 (en) | 2012-02-15 | 2018-07-16 | F. Hoffmann-La Roche Ag | Affinity chromatography based on the Fc receptor |
JP6393255B2 (en) | 2012-04-20 | 2018-09-19 | メルス ナムローゼ フェンノートシャップ | Methods of producing heterodimeric IgG-like molecules, heterodimeric IgG-like molecules, heterodimeric antibodies, recombinant host cells, pharmaceutical compositions, methods of making host cells, and cultures |
EP3648786A4 (en) * | 2017-07-03 | 2021-12-15 | Torque Therapeutics, Inc. | Fusion molecules targeting immune regulatory cells and uses thereof |
EP3665197A2 (en) | 2017-08-11 | 2020-06-17 | H. Hoffnabb-La Roche Ag | Anti-cd8 antibodies and uses thereof |
-
2020
- 2020-06-29 CN CN202080048209.9A patent/CN114051500A/en active Pending
- 2020-06-29 EP EP20734407.8A patent/EP3994169A1/en not_active Withdrawn
- 2020-06-29 TW TW109121834A patent/TW202115115A/en unknown
- 2020-06-29 JP JP2021576948A patent/JP2022538139A/en active Pending
- 2020-06-29 WO PCT/EP2020/068186 patent/WO2021001289A1/en unknown
- 2020-07-02 AR ARP200101876A patent/AR119338A1/en unknown
Also Published As
Publication number | Publication date |
---|---|
AR119338A1 (en) | 2021-12-09 |
JP2022538139A (en) | 2022-08-31 |
CN114051500A (en) | 2022-02-15 |
TW202115115A (en) | 2021-04-16 |
WO2021001289A1 (en) | 2021-01-07 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20230071733A1 (en) | Immunoconjugates | |
US20230134606A1 (en) | Immunoconjugates | |
EP3994169A1 (en) | Immunoconjugates comprising a mutant interleukin-2 and an anti-cd8 antibody | |
US20230192795A1 (en) | Immunoconjugates | |
WO2022148853A1 (en) | Immunoconjugates | |
WO2023062048A1 (en) | Alternative pd1-il7v immunoconjugates for the treatment of cancer | |
AU2022362681A1 (en) | New interleukin-7 immunoconjugates |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: UNKNOWN |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE |
|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE |
|
17P | Request for examination filed |
Effective date: 20220202 |
|
AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR |
|
DAV | Request for validation of the european patent (deleted) | ||
DAX | Request for extension of the european patent (deleted) | ||
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
|
18D | Application deemed to be withdrawn |
Effective date: 20220823 |