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WO2022214664A1 - Mutant amélioré d'interféron-gamma - Google Patents

Mutant amélioré d'interféron-gamma Download PDF

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WO2022214664A1
WO2022214664A1 PCT/EP2022/059451 EP2022059451W WO2022214664A1 WO 2022214664 A1 WO2022214664 A1 WO 2022214664A1 EP 2022059451 W EP2022059451 W EP 2022059451W WO 2022214664 A1 WO2022214664 A1 WO 2022214664A1
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seq
ifny
antibody
bifunctional molecule
amino acid
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Roberto DE LUCA
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Philogen SpA
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Philogen SpA
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • C07K14/555—Interferons [IFN]
    • C07K14/57—IFN-gamma
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61K2039/507—Comprising a combination of two or more separate 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
    • C07K2317/00—Immunoglobulins specific features
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Definitions

  • the present application relates to an improved interferon gamma mutant and conjugates thereof for the treatment of disease.
  • Interferon-gamma is a pleiotropic cytokine that plays a central role in promoting both innate and adaptive mechanisms host defence. It has an heterogenous C-terminus with its most predominant active human form being 138 amino acid in length.
  • IFNy is able to inhibit tumour cell growth by stimulating CD8+ T cells, NK cells and macrophages (Berraondo et al. 2018).
  • Recombinant IFNy (ACTIMMUNETM) is approved for the treatment of infections associated with chronic granulomatous disease.
  • systemic toxicity and short serum half-life have limited the systemic use of this cytokine.
  • the antibody-mediated targeted delivery of anti-cancer cytokines “immunocytokines” to the tumor microenvironment represents a well-known strategy to maximise the concentration of the cytokine at the site of disease while sparing toxicity in the healthy organs.
  • an antibody-cytokine fusion protein consisting of the L19 antibody (specific to the alternatively spliced extra domain B of Fibronectin “EDB”) in scFv format fused to a murine IFNy variant has been previously described (Ebbinghaus et ah, 2015). Biodistribution studies on immunocompetent, nude, or interferon-gamma-receptor (IFNyR) knockout mice showed that the targeting ability of the L19-IFNy immunocytokine is influenced by the number of IFNyRs expressed in the mouse.
  • IFNyR interferon-gamma-receptor
  • Another immunocytokine based on the F8 antibody (specific to the alternatively spliced extra domain A of Fibronectin “EDA”) in diabody format fused to murine IFNy did not exhibit the expected preferential localization on tumors in vivo indicating a receptor-trapping mechanism (Hemmerle et al, 2014).
  • IFNy variants with reduced biological activity and receptor affinity may be used for the targeted delivery of the payload.
  • WO20 18/077893 discloses a heavy-chain-only antibody (VHH) specific for human CD20 conjugated to a number of human IFND ⁇ variants with reduced affinity for IFNDR whose biological activity is restored by attachment with a targeting moiety.
  • VHH heavy-chain-only antibody
  • a chimeric protein comprising (a) a modified IFN-g, said modified IFN-g having, in one embodiment, a truncation at the C terminus of 14, 15 or 16 amino acid residues, (b) one or more targeting moieties, which bind to antigens or receptors of interest
  • Huyghe et al. developed a truncated version of murine IFNy fused to a VHH specific for murine CD20 (6).
  • the protein showed reduced biological activity in an in vitro cell-based assay.
  • FIG. 1 Size exclusion chromatography profile of L19-IFNy-“KRG”.
  • B SDS-PAGE analysis under non-reducing (NR) and reducing (R) conditions.
  • Figure 2 Shows the MS analysis of L 19-IFNy-“KRG” and of L19-IFNy-“KR”.
  • Figures 2.1 and 2.2 show the N-terminal MS analysis confirming the correct sequence for both conjugates.
  • Figures 2.3 and 2.4 show the C-terminal analysis indicating respectively that L I 9-IFNy-“KRG” - but not L I 9-IFNy-“KR” - maintains the correct sequence.
  • Figure 2.5 shows a further confirmation about the identity of the y-ions annotated in the MS2 spectrum in the L19-IFNy-“KRG”.
  • MS3 spectrum 150-850 m/z
  • C-terminal y9 ion PAAKTGKKRG1+ 885.5 m/z
  • B- and y-ions series match with the MS-identified peaks, thus confirming the correct amino acid sequence at the C- terminus.
  • a selected representative C-terminal y-ion (y91+) was selected for additional confirmation by MS3 : the matching of the b and y-ions series generated by HCD fragmentation in MS3 validated the correct aminoacidic composition of the C-terminus.
  • Figure 3 (A) shows the binding properties of L19-IFNy-“WT” compared to L I 9-IFNy-“KRG” and L19-IFNy-“KR” to IFNy-receptor 1 (Rl) as measured by Surface Plasmon Resonance.
  • Figure 3 (B) shows the binding of L19-IFNy-“KRG” to the target antigen (EDB) recognized by the L19 antibody as measured by Surface Plasmon Resonance.
  • Figure 3 (C) shows the binding properties of L19-IFNy-“WT” compared to L19-IFNy- “TRUNC” and to IFNy-receptor 1 (Rl) as measured by Surface Plasmon Resonance.
  • Figure 4 (A) shows the proliferation of IFNy- responsive cells upon stimulation with L19-IFNy- “WT” and L19-IFNy-“KRG” when the target antigen is not coated on the test plates.
  • Figure 4 (B) shows the same experiment when the target antigen is coated on the test plates.
  • Figure 4 (C) shows the production of IL6 by IFNy- responsive cells upon stimulation with L19- IFNy-“WT” and L19-IFNy-“KRG” when the cognate antibody binds its target and when it does not.
  • Figure 5. Shows the amino acid sequences of (i) the wild type full sequence wildtype of IFNy (SEQ ID NO 3), (ii) the truncated version “TRUNC” (SEQ ID NO 51) of IFNy (iii) the truncated version “KR” of IFNy (SEQ ID NO 2) and (iv) the mutated IFNy “KRG” (SEQ ID NO 1) of the invention.
  • Figure 6 Shows Quantitative biodistrubution analysis of radioiodinated L19-hIFNy “KRG” and L19-mIFNy “KRG” in immunocompetent mice bearing F9 teratocarcinoma tumors.
  • Figure 7 Shows the pharmacokinetic analysis in cynomolgus monkey of L19-IFNy “KRG”. Pharmacokinetics were evaluated in one animal per group injected once at O.lmg/kg (upper panel) or 0.5mg/kg (bottom panel). Blood samples were collected before dosing and at 2, 10, 20, 30, 60, 120 and 240 minutes after treatment. The figure shows a nice linear profile.
  • Figure 8. Shows in panel (A) the therapeutic performance of L19-mIFNy“KRG” alone and in combination with a mouse anti -PD- 1 antibody; in panel (B) the therapeutic performance of L19-mIFNy“KRG” alone and in combination with a mouse anti-LAG3 antibody; in panel (C) the therapeutic performance of L19-mIFNy“KRG” in WEHI-164 tumor bearing mice.
  • Figure 9 Shows in panel (A) the body weight change of L19-mIFNy “KRG” alone and in combination with a mouse anti -PD- 1 antibody; in panel (B) the body weight change of LI 9- mlFNy “KRG” alone and in combination with a mouse anti-LAG3 antibody; in panel (C) body weight change of L19-mIFNy “KRG” in WEHI-164 tumor bearing mice.
  • conjugates which include a novel mutated IFNy sequence have a superior resistance to proteolysis of its C-terminus. At the same time such conjugates have a reduced affinity for IFNyR and a biological activity which is restored upon binding of the antibody to its target antigen.
  • a bifunctional molecule comprising:
  • a modified IFNy protein comprising, relative to the amino acid sequence of the IFNy wildtype (SEQ ID NO 3), a) the substitution K130G, and optionally b) a C-terminal truncation of between > 1 and ⁇ 8 amino acid residues, and
  • a bifunctional molecule comprising:
  • a modified IFNy protein comprising, relative to the amino acid sequence of the IFNy wildtype (SEQ ID NO 3), c) the substitution K130G, and d) a C-terminal truncation of between > 1 and ⁇ 8 amino acid residues, and (ii) a protein binder specific for a given target.
  • the respective variant having the K130G substitution is called IFNy-KRG herein.
  • the following plot shows an alignment between one embodiment of IFNy-KRG (SEQ ID NO 1) relative to the amino acid sequence of the wildtype (IFNy-WT) (SEQ ID NO 3).
  • the following plot shows an alignment between one embodiment of IFNy-KRG (SEQ ID NO 1) relative to the amino acid sequence of a prior art mutant (IFNy-KR) (SEQ ID NO 2) that lacks 9 amino acid residues relative to the wildtype.
  • IFNy-KR prior art mutant
  • the following plot shows an alignment between one embodiment of IFNy-KRG (SEQ ID NO 1) relative to the amino acid sequence of a prior art mutant (IFNy-TRUNC) (SEQ ID NO 51) that lacks 11 amino acid residues relative to the wildtype.
  • the inventors have shown that, relative to IFNy-WT and IFNy-KR, the new variant having the K130G substitution (IFNy-KRG) has improved properties. This finding, supported by the experiments disclosed herein, is surprising in view of the seemingly minor modification, K130G.
  • WO20 18077893 discussed in the introduction discloses a heavy-chain-only antibody (VHH) specific for human CD20 conjugated to a number of human IFNy variants with reduced affinity for IFNyR whose biological activity is restored by attachment with a targeting moiety.
  • VHH heavy-chain-only antibody
  • This document discloses, inter alia, a truncated variant of IFNy wildtype, as SEQ ID NO 950, which is identical to the above discussed IFNy-KR, so not identical with IFNy-KRG.
  • the ⁇ FNy-“KRG” variant comprises an additional “G” residue that is not encompassed in the wildtype sequence, which renders IFNy- KRG novel over that piece of prior art.
  • IFNy will be fused to an antibody via its N terminus. This is for example shown in SEQ ID NO 16, 28, 39 and 50 of the present disclosure.
  • the inventors have shown that the new C-terminal “KRG” variant confers superiority in maintaining the correct size (in light of its improved resistance to proteolysis) over the closest prior art IFNy-“KR”, as documented by the MS analysis of Example 2. This feature is highly advantageous for a therapeutic protein.
  • an heterogenous preparation as for IFNy-“KR” may potentially create issues both in terms of immunogenicity and of batch to batch reproducibility.
  • the modified IFNy protein comprises the amino acid sequence of SEQ ID NO 1. According to one embodiment, the modified IFNy protein consists of the amino acid sequence of SEQ ID NO 1.
  • the modified IFNy protein is conjugated to at least one chain of the protein binder by a peptide linker.
  • the linker consists of a short GS stretch ((GS) n with n between > 1 and ⁇ 5) that replaces the C terminal K residue that is common to CH3 of IgG4 heavy chains.
  • the protein binder binds to a target associated to neoplastic growth and/or angiogenesis.
  • the protein binder binds to at least one of
  • the protein binders can for example comprise sequences of the antibody L19 (EDB of fibronectin), of the antibody F8 (EDA of fibronectin), of the antibody F16 (Tenascin C) or of the antibody XE114 (Carbonic Anhydrase IX).
  • Fibronectin (UniProt: P02751) is a high-molecular weight ( ⁇ 440kDa) glycoprotein of the extracellular matrix that binds to membrane-spanning receptor proteins called integrins. Similar to integrins, fibronectin binds extracellular matrix components such as collagen, fibrin, and heparan sulfate proteoglycans (e.g. syndecans).
  • Fibronectin has been implicated in carcinoma development. In lung carcinoma, fibronectin expression is increased, especially in non-small cell lung carcinoma. The adhesion of lung carcinoma cells to fibronectin enhances tumorigenicity and confers resistance to apoptosis- inducing chemotherapeutic agents. Fibronectin may promote lung tumor growth/survival and resistance to therapy, and has been discussed to represent a novel target for the development of new anticancer drugs.
  • Fibronectin exists as a protein dimer, consisting of two nearly identical monomers linked by a pair of disulfide bonds.
  • the fibronectin protein is produced from a single gene, but alternative splicing of its precursor mRNA, produced from a single copy fibronectin gene, occurs at three sites coding for the EDA, EDB and IIICS domains and results in the creation of several isoforms.
  • Fibronectin isoforms comprising the EDA or EDB domains are known as oncofetal forms due to their importance in embryonic development and their restricted presence in normal adult tissues. These isoforms are also recognized as important markers of angiogenesis, a crucial physiological process in development and required by tumor cells in cancer progression.
  • ED- B fibronectin as well as ED-A fibronectin are expressed in tumor tissues, particularly in breast carcinomas, brain tumors, lymphoma cells, and prostate cancers.
  • Tenascins including Tenascin C, are extracellular matrix glycoproteins. They are abundant in the extracellular matrix of developing vertebrate embryos and they reappear around healing wounds and in the stroma of some tumors.
  • Carbonic anhydrase IX (CA9/CA IX) is an enzyme that in humans is encoded by the CA9 gene. It is one of the 14 carbonic anhydrase isoforms found in humans and is a transmembrane dimeric metalloenzyme with an extracellular active site that facilitates acid secretion in the gastrointestinal tract.
  • CA IX is overexpressed in many types of cancer including clear cell renal cell carcinoma (RCC) as well as carcinomas of the cervix, breast and lung where it promotes tumor growth by enhancing tumor acidosis.
  • the protein binder is an antibody, or a target binding fragment or derivative thereof.
  • Whole antibodies include IgA, IgD, IgE, IgG or IgM.
  • the whole antibody is IgG. More preferably the whole antibody is an IgGl or IgG4.
  • Antigen-binding fragments of whole antibodies include (i) the Fab fragment consisting of VL, VH, CL and CHI domains; (ii) the Fd fragment consisting of the VH and CHI domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward et al. (1989) Nature 341, 544-546; McCafferty et al., (1990) Nature, 348, 552- 554; Holt et al.
  • Fv, scFv or diabody molecules may be stabilized by the incorporation of disulphide bridges linking the VH and VL domains (Reiter et al. (1996), Nature Biotech, 14, 1239-1245).
  • Minibodies comprising a scFv joined to a CH3 domain may also be made (Hu et al. (1996), Cancer Res., 56(13):3055-61).
  • binding fragments are Fab’, which differs from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CHI domain, including one or more cysteines from the antibody hinge region, and Fab’-SH, which is a Fab’ fragment in which the cysteine residue(s) of the constant domains bear a free thiol group.
  • a single chain Fv may be comprised within a mini-immunoglobulin or small immunoprotein (SIP), e.g. as described in (Li et al., (1997), Protein Engineering, 10: 731-736).
  • SIP small immunoprotein
  • a SIP may comprise an scFv molecule fused to the CH4 domain of the human IgE secretory isoform IgE-S2 (e S2 -CH4; Batista et al., (1996), J. Exp. Med., 184: 2197-205) forming a homo- dimeric mini-immunoglobulin antibody molecule.
  • the VH and VL domains of the antibody are preferably linked by a 14 to 20 amino acid linker.
  • the VH and VL domains may be linked by an amino acid linker which is 14, 15, 16, 17, 18, 19, or 20 amino acid in length. Suitable linker sequences are known in the art.
  • Diabodies are multimers of polypeptides, each polypeptide comprising a first domain comprising a binding region of an immunoglobulin light chain and a second domain comprising a binding region of an immunoglobulin heavy chain, the two domains being linked (e.g. by a peptide linker) but unable to associate with each other to form an antigen-binding site: antigen binding sites are formed by the association of the first domain of one polypeptide within the multimer with the second domain of another polypeptide within the multimer (WO94/13804; Holliger and Winter, 1997; Holliger et al., 1993).
  • VH heavy chain variable domain
  • VL light chain variable domain
  • the VH and VL domains are preferably linked by a 5 to 12 amino acid linker.
  • a diabody comprises two VH-VL molecules which associate to form a dimer.
  • the VH and VL domains of each VH-VL molecule are preferably linked by a 5 to 12 amino acid linker.
  • the VH and VL domains may be linked by an amino acid linker which is 5, 6, 7, 8, 9, 10, 11, or 12 amino acid in length.
  • the amino acid linker is 5 amino acids in length. Suitable linker sequences are known in the art.
  • the VL domain of the scFv antibody is preferably linked to the CH4 domain of human IgE (Batista et al., (1996), J. Exp. Med., 184: 2197-205) via a 2 to 20 amino acid linker, more preferably a 2 to 10 amino acid linker. Suitable linker sequences are known in the art.
  • VH-VL VH-VL domains
  • VH-VL VH-VL
  • each of the VH and VL domains within a set is connected by a short or ‘non-flexible’ peptide linker.
  • This type of peptide linker sequence is not long enough to allow pairing of the VH and VL domains within the set.
  • a short or ‘non flexible’ peptide linker is around 5 amino acids.
  • the two sets of VH and VL domains are connected as a single-chain by a long or ‘flexible’ peptide linker.
  • This type of peptide linker sequence is long enough to allow pairing of the VH and VL domains of the first set with the complementary VH and VL domains of the second set.
  • a long or ‘flexible’ linker is 15 to 20 amino acids.
  • an “antigen-binding site” describes the part of the single-chain diabody which comprises the area which specifically binds to and is complementary to part or all of an antigen. Where an antigen is large, the single-chain diabody may only bind to a particular part of the antigen, which part is termed an epitope.
  • the antigen-binding sites of the single-chain diabody may be identical or different but preferably are identical. Each of the antigen-binding sites in the single-chain diabody may bind the same antigen or epitope. This can be achieved by providing two identical antigen-binding sites such as two identical VH-VL domain pairs, or by providing two different antigen-binding sites, for example comprising different VH and VL domains, which nevertheless both bind the same antigen or epitope.
  • the single-chain diabody may be bispecific. By ‘bispecific”, we mean that each of the antigen-binding sites binds a different antigen.
  • two antigen-binding sites may bind two different antigens mentioned herein, e.g. two different antigens of the (extracellular matrix), or two different domains of a particular antigen.
  • the single-chain diabody may bind their target.
  • the binding may be specific.
  • the term "specific” may be used to refer to the situation in which one member of a specific binding pair will not show any significant binding to molecules other than its specific binding partner(s).
  • the term is also applicable where e.g. an antigen-binding site is specific for a particular epitope that is carried by a number of antigens, in which case the single-chain diabody carrying the antigen-binding site will be able to bind to the various antigens carrying the epitope.
  • the antibody molecule comprises or consists of a single chain Fv, a small immunoprotein, a diabody, a single-chain diabody or a (whole) IgG molecule.
  • the protein binder a comprises a set of heavy chain/light chain complementarity determining regions (CDR) comprised in the heavy chain/light variable domain sequence pair set forth in the following pairs of SEQ ID NOs:
  • • 40 and 41 b) comprises a set of heavy chain/light chain complementarity determining regions (CDR) comprising the following SEQ ID NOs, in the order (HCDR1; HCDR2; HCDR3; LCDR1; LCDR2 and LCDR3)
  • c) comprises the heavy chain/light chain complementarity determining regions (CDR) of b), with the proviso that at least one of the CDRs has up to 3 amino acid substitutions relative to the respective SEQ ID NOs, and/or d) comprises the heavy chain/light chain complementarity determining regions (CDR) of b) or c), with the proviso that at least one of the CDRs has a sequence identity of > 66 % to the respective SEQ ID NOs, wherein the CDRs are embedded in a suitable protein framework so as to be capable to bind to their target with sufficient binding affinity.
  • CDR or “complementarity determining region” is intended to mean the non-conti guous antigen combining sites found within the variable region of both heavy and light chain polypeptides. These particular regions have been described by Kabat et al. (1977), Kabat et al. (1991), Chothia et al. (1987) and MacCallum et al., (1996) where the definitions include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or grafted antibodies or variants thereof is intended to be within the scope of the term as defined and used herein.
  • variable region framework when used in reference to an antibody variable region is entered to mean all amino acid residues outside the CDR regions within the variable region of an antibody. Therefore, a variable region framework is between about 100-120 amino acids in length but is intended to reference only those amino acids outside of the CDRs.
  • KD is the equilibrium dissociation constant, a ratio of k 0ff /k 0n , between the antibody or fragment and its antigen.
  • KD and affinity are inversely related.
  • the KD value relates to the concentration of antibody or fragment (the amount of antibody or fragment needed for a particular experiment) and so the lower the KD value (lower concentration) and thus the higher the affinity of the binding domain.
  • the following table shows typical KD ranges of monoclonal antibodies
  • the antibody or fragment has up to 2 amino acid substitutions, and more preferably up to 1 amino acid substitution.
  • At least one of the CDRs of the antibody or fragment has a sequence identity of > 67 %; > 68 %; > 69 %; > 70 %; > 71 %; > 72 %; > 73 %; > 74 %; > 75 %; > 76 %; > 77 %; > 78 %; > 79 %; > 80 %; > 81 %; > 82 %; > 83 %; > 84 %; > 85 %; > 86 %; > 87 %; > 88 %; > 89 %; > 90 %; > 91 %; > 92 %; > 93 %; > 94 %; > 95 %; > 96 %; > 97 %; > 98 %; > 99 %, and most preferably 100 % to the respective SEQ ID NO.
  • Percentage of sequence identity is determined by comparing two optimally aligned biosequences (amino acid sequences or polynucleotide sequences) over a comparison window, wherein the portion of the corresponding sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence, which does not comprise additions or deletions, for optimal alignment of the two sequences.
  • the percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same sequences.
  • Two sequences are “substantially identical” if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity over a specified region, or, when not specified, over the entire sequence of a reference sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
  • polypeptides that are substantially identical to the polypeptides exemplified herein.
  • identity or substantial identity can exist over a region that is at least 5, 10, 15 or 20 amino acids in length, optionally at least about 25, 30, 35, 40, 50, 75 or 100 amino acids in length, optionally at least about 150, 200 or 250 amino acids in length, or over the full length of the reference sequence.
  • shorter amino acid sequences e.g ., amino acid sequences of 20 or fewer amino acids
  • substantial identity exists when one or two amino acid residues are conservatively substituted, according to the conservative substitutions defined herein.
  • At least one of the CDRs has been subject to CDR sequence modification, including
  • Affinity maturation in the process by which the affinity of a given antibody is increased in vitro is based on the principles of mutation and selection. It has successfully been used to optimize antibodies, antibody fragments or other peptide molecules like antibody mimetics. Random mutations inside the CDRs are introduced using radiation, chemical mutagens or error-prone PCR. In addition, the genetic diversity can be increased by chain shuffling. Two or three rounds of mutation and selection using display methods like phage display usually results in antibody fragments with affinities in the low nanomolar range. For principles see Eylenstein et al. (2016) or US20050169925A1, the content of which is incorporated herein by reference for enablement purposes.
  • Engineered antibodies contain murine-sequence derived CDR regions that have been engrafted, along with any necessary framework back-mutations, into sequence-derived V regions. Hence, the CDRs themselves can cause immunogenic reactions when the humanized antibody is administered to a patient. Methods of reducing immunogenicity caused by CDRs are disclosed in Harding et al. (2010), or US2014227251A1, the content of which is incorporated herein by reference for enablement purposes.
  • the protein binder comprises a) the heavy chain/light chain variable domain (HCVD/LCVD) pairs set forth in the following pairs of SEQ ID NOs:
  • the HCVD has a sequence identity of > 80 % to the respective SEQ ID NO, and/or
  • the LCVD has a sequence identity of > 80 % to the respective SEQ ID NO, c) the heavy chain/light chain variable domains (VD) pairs of a) or b), with the proviso that at least one of the HCVD or LCVD has up to 10 amino acid substitutions relative to the respective SEQ ID NO, said antibody or fragment still being capable to bind to the respective target.
  • variable domain when used in reference to an antibody or a heavy or light chain thereof is intended to mean the portion of an antibody which confers antigen binding onto the molecule and which is not the constant region.
  • the term is intended to include functional fragments thereof which maintain some of all of the binding function of the whole variable region.
  • Variable region binding fragments include, for example, functional fragments such as Fab, F(ab)2, Fv, single chain Fv (scFv) and the like. Such functional fragments are well known to those skilled in the art. Accordingly, the use of these terms in describing functional fragments of a heteromeric variable region is intended to correspond to the definitions well known to those skilled in the art. Such terms are described in, for example, Huston et ah, (1993) or Pliickthun and Skerra (1990).
  • the HCVD and/or LCVD has a sequence identity of > 81 %; > 82 %; > 83 %; > 84 %; > 85 %; > 86 %; > 87 %; > 88 %; > 89 %; > 90 %; > 91 %; > 92 %; > 93 %; > 94 %; > 95 %; > 96 %; > 97 %; > 98 %; > 99 %; or most preferably 100 % to the respective SEQ ID NO.
  • At least one amino acid substitution in the antibody or fragment is a conservative amino acid substitution.
  • a “conservative amino acid substitution”, as used herein, has a smaller effect on antibody function than a non-conservative substitution. Although there are many ways to classify amino acids, they are often sorted into six main groups on the basis of their structure and the general chemical characteristics of their R groups.
  • a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Familie of amino acid residues having similar side chains have been defined in the art. These families include amino acids with
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine.
  • amino acid side chain families can also occur across amino acid side chain families, such as when substituting an asparagine for aspartic acid in order to modify the charge of a peptide.
  • Conservative changes can further include substitution of chemically homologous non- natural amino acids (i.e. a synthetic non-natural hydrophobic amino acid in place of leucine, a synthetic non-natural aromatic amino acid in place of tryptophan).
  • the antibody or fragment has at least a target binding affinity of > 50 % to the respective target compared to that of the antibody or fragment according to the above description.
  • binding affinity is intended to mean the strength of a binding interaction and therefore includes both the actual binding affinity as well as the apparent binding affinity.
  • the actual binding affinity is a ratio of the association rate over the disassociation rate. Therefore, conferring or optimizing binding affinity includes altering either or both of these components to achieve the desired level of binding affinity.
  • the apparent affinity can include, for example, the avidity of the interaction.
  • a bivalent heteromeric variable region binding fragment can exhibit altered or optimized binding affinity due to its valency.
  • a suitable method for measuring the affinity of a binding agent is through surface plasmon resonance (SPR).
  • SPR surface plasmon resonance
  • This method is based on the phenomenon which occurs when surface plasmon waves are excited at a metal/liquid interface. Light is directed at, and reflected from, the side of the surface not in contact with sample, and SPR causes a reduction in the reflected light intensity at a specific combination of angle and wavelength. Biomolecular binding events cause changes in the refractive index at the surface layer, which are detected as changes in the SPR signal.
  • the binding event can be either binding association or disassociation between a receptor- ligand pair.
  • the changes in refractive index can be measured essentially instantaneously and therefore allows for determination of the individual components of an affinity constant. More specifically, the method enables accurate measurements of association rates (k on ) and disassociation rates (k 0ff ).
  • Measurements of k on and k 0ff values can be advantageous because they can identify altered variable regions or optimized variable regions that are therapeutically more efficacious.
  • an altered variable region, or heteromeric binding fragment thereof can be more efficacious because it has, for example, a higher k on valued compared to variable regions and heteromeric binding fragments that exhibit similar binding affinity.
  • Increased efficacy is conferred because molecules with higher k on values can specifically bind and inhibit their target at a faster rate.
  • a molecule of the invention can be more efficacious because it exhibits a lower k 0ff value compared to molecules having similar binding affinity.
  • Another suitable method for measuring the affinity of a binding agent is through surface is by FACS/scatchard analysis.
  • said target binding affinity is > 51%, > 52%, > 53%, > 54%, > 55%, > 56%, > 57%,
  • the antibody or fragment a) competes for binding to the respective target with, or b) binds to essentially the same, or the same, region as, the antibody or fragment according to the above description.
  • the term "competes for binding” is used in reference to one of the antibodies defined by the sequences as above, meaning that the actual antibody or fragment as an activity which binds to the same target, or target epitope or domain or subdomain, as does said sequence defined antibody or fragment, and is a variant of the latter.
  • the efficiency e.g., kinetics or thermodynamics
  • the equilibrium binding constant for binding to the substrate may be different for the two antibodies.
  • Such competition for binding can be suitably measured with a competitive binding assay.
  • assays are disclosed in Finco et al. 2011, the content of which is incorporated herein by reference for enablement purposes, and their meaning for interpretation of a patent claim is disclosed in Deng et al. 2018, the content of which is incorporated herein by reference for enablement purposes.
  • the bifunctional molecule according to the above description comprises, optionally consists of, a pair of amino acid sequences according to
  • the bifunctional molecule comprises a protein binder comprising SEQ ID NO 30, with the proviso that the residue at position 88 of the VL domain of SEQ ID NO 30 is Gin and/or the residue at position 90 of the VL domain of SEQ ID NO 30 is Ser.
  • a nucleic acid that encodes for at least one chain of a bifunctional molecule according to the above description.
  • a nucleic acid or a pair of nucleic acids, is provided which encodes for the heavy chain and the light chain, respectively, of the binding agent, in case the latter is a monoclonal antibody having a heteromeric structure of at least one light chain and one heavy chain.
  • the nucleic acid can also be used for pharmaceutic purposes.
  • the nucleic acid can be an RNA molecule, or an RNA derivative comprising, e.g., modified nucleotides, like pseudouridine (Y) or N-l Methyl Pseudouridine (m 1 Y) to provide stability and reduce immunogenicity (see, e.g., US8278036 and US9428535, the contents of which are incorporated herein for enablement purposes).
  • the RNA comprises the most GC-rich codon is selected to provide stability and reduce immunogenicity (see e.g. EP1392341 the content of which is incorporated herein for enablement purposes).
  • the mRNA can for example be delivered in suitable liposomes and comprises either specific sequences or modified uridine nucleosides to avoid immune responses and/or improve folding and translation efficiency, sometimes comprising cap modifications at the 5’- and/or 3’ terminus to target them to specific cell types.
  • the nucleic acid can likewise be a DNA molecule.
  • the molecule can be a cDNA that is optionally integrated into a suitable vector, e.g., an attenuated, non pathogenic virus, or is provided as one or more plasmids.
  • plasmids can for example be administered to a patient by means of an electroporation device as e.g. disclosed in patent EP3397337B1, the content of which is incorporated herein for enablement purposes.
  • a pharmaceutical composition comprising a bifunctional molecule or a nucleic acid according to the above description is provided, and optionally one or more pharmaceutically acceptable excipients.
  • a pharmaceutical composition or combination comprising a bifunctional molecule or a nucleic acid or a pharmaceutical composition according to the above description is provided together with one or more additional therapeutically active compounds.
  • Such pharmaceutical combination can consist of two or more separate entities (one comprising the bifunctional molecule or nucleic acid and one or more others comprising the one or more additional therapeutically active compounds). These entities are administered to a subject simultaneously or subsequently, in all conceivable orders.
  • said combination or composition comprises, as therapeutically active compound, at least one checkpoint inhibitor.
  • a checkpoint inhibitor is a therapeutic entity that targets immune checkpoints, i.e., key regulators of the immune system that when stimulated can dampen the immune response to an immunologic stimulus. Some cancers can protect themselves from attack by stimulating immune checkpoint targets.
  • Checkpoint therapy can block inhibitory checkpoints, restoring immune system function.
  • the first anti-cancer drug targeting an immune checkpoint was ipilimumab, a CTLA4 blocker approved in the United States in 2011.
  • the immune checkpoint inhibitor is a binder, inhibitor or antagonist of at least one of CTLA-4, PD-1, PD-L1, LAG 3, TIM3, 0X40, and/or TIGIT.
  • the immune checkpoint inhibitor is PD-1, PD-L1 or LAG 3.
  • the binder, inhibitor or antagonist is an antibody, or a target binding fragment or derivative thereof.
  • the antibody is at least one selected from the group consisting of Ipilimumab (anti-CTLA-4), Nivolumab (anti-PD-1), Pembrolizumab (anti-PD-1), Cemiplimab (anti-PD-1 ),Spartalizumab (anti-PD-1), Atezolizumab (anti-PD-Ll), Avelumab (anti-PD-Ll), Durvalumab (anti-PD-Ll), Etigilimab (anti-TIGIT), BGB-A1217 (anti-TIGIT) BMS-986207 (anti-TIGIT), AB154 (anti-TIGIT) ASP8374 (anti-TIGIT), MK 7684 (anti- TIGIT), and/or Tiragolumab (anti-TIGIT).
  • Ipilimumab anti-CTLA-4
  • Nivolumab anti-PD-1
  • Pembrolizumab anti-PD-1
  • Cemiplimab anti-PD-1
  • said combination or composition comprises, as therapeutically active compound, at least one chemotherapeutic agent.
  • the chemotherapeutic agent is at least one selected from the group consisting of DNA replication inhibitors (e.g. Capecitabine, Fluorouracil), anti metabolites (e.g. Gemcitabine), antimicrotubule agent (taxane e.g. Abraxane, Paclitaxel), anthracyclines (e.g. Doxorubicin), purine analogs (e.g. dacarbazine), alkylating agents (e.g. Lomustine, Temozolomide), Vinka alkaloids (e.g. Vinblastine), Kinase inhibitors (e.g. Bortezomib), platinum-containing antineoplastic agents (e.g.
  • DNA replication inhibitors e.g. Capecitabine, Fluorouracil
  • anti metabolites e.g. Gemcitabine
  • antimicrotubule agent e.g. Abraxane, Paclitaxel
  • anthracyclines e.g. Doxorubicin
  • FOLFOX is composed of Fluorouracil, Oxaliplatin and Folinic Acid
  • FOLFIRI is composed of Fluorouracil, Irinotecan and Folinic Acid.
  • the chemotherapeutic agent may be selected from the group consisting of FOLFOX, FOLFIRI or Capecitabine when the treatment is for the treatment of colorectal cancer.
  • the chemotherapeutic agent may be selected from the group consisting of FOLFOX, FOLFIRI or the combination of Gemcitabine and Abraxane when the treatment is for the treatment of pancreatic cancer.
  • the chemotherapeutic agent may be selected from the group consisting of Doxorubicin or dacarbazine when the treatment is for the treatment of soft-tissue sarcoma.
  • the chemotherapeutic agent may be selected from the group consisting of Lomustine or Temozolomide when the treatment is for the treatment of glioblastoma.
  • the chemotherapeutic agent may be selected from the group consisting of Paclitaxel, Vinblastine or Bortezomib.
  • a bifunctional molecule or a nucleic acid or a (pharmaceutical) composition or combination is provided (for the manufacture of a medicament) in the treatment of a human or animal subject
  • brackets are deemed absent
  • EPC2000 EPC2000
  • a method for treating a human or animal subject suffering from, being diagnosed for, or being at risk of, developing a neoplastic disease, or preventing such subject from such condition comprises administration, to the human or animal subject, of a bifunctional molecule or a composition or combination according to the above description, in a therapeutically sufficient dose
  • the bifunctional molecule is administered to a human or animal subject in combination with a therapeutically active compound, at least one checkpoint inhibitor or chemotherapeutic agent
  • the therapeutically active compound may be administered to the human or animal subject concurrently with, sequentially to, or separately from the administration of the bifunctional molecule.
  • the therapeutically active compound is at least a chemotherapeutic agent and is administered separately from the bifunctional molecule
  • the chemotherapeutic agent may be first administered to the human or animal subject followed by the administration of the the bifunctional molecule.
  • the bifunctional molecule e.g. L19-IFNg“KRG”
  • the bifunctional molecule e.g. L19-IFNg“KRG”
  • the checkpoint inhibitor e.g. Anti-PDLl, anti-PD-1 or anti-LAG3
  • a therapeutic kit of parts comprising: a) a bifunctional molecule or a nucleic acid or a pharmaceutical composition or a combination composition according to the above description b) an apparatus for administering the composition, composition or combination, and c) optionally, instructions for use.
  • the three IFNy conjugates were cloned and expressed according to the protocols below.
  • the gene encoding for human interferon gamma was amplified into a mammalian expression vector using BamHI and Notl restriction enzymes.
  • RD-1 CTCTGGGTGGATCCCAAGACCCCTATGTGAAAGAAGCCGAGAA
  • the gene encoding for human interferon gamma was amplified into a mammalian expression vector using BamHI and Notl restriction enzymes.
  • RD-1 CTCTGGGTGGATCCCAAGACCCCTATGTGAAAGAAGCCGAGAA
  • the PCR product was digested with BamHI and Notl and ligated into a vector digested with same enzymes.
  • the gene encoding for human interferon gamma was amplified into a mammalian expression vector using BamHI and Notl restriction enzymes.
  • RD-1 CTCTGGGTGGATCCCAAGACCCCTATGTGAAAGAAGCCGAGAA
  • RD-3 ATAGTTTAGCGGCCGCATTCTTATTCACCCTGTCTTAGCAGCAGGAGACA.
  • the PCR product was digested with BamHI and Notl and ligated into a vector digested with same enzymes.
  • the gene encoding for human interferon gamma was amplified into a mammalian expression vector using BamHI and Notl restriction enzymes.
  • RD-1 CTCTGGGTGGATCCCAAGACCCCTATGTGAAAGAAGCCGAGAA
  • RD-5 ATAGTTTAGCGGCCGCTTAGCCCCGTTTCCCTGTCTTAGCAGCAG.
  • the PCR product was digested with BamHI and Notl and ligated into a vector digested with same enzymes.
  • TGE transient gene expression
  • a conjugate which included the novel mutated IFNy-KRG sequence was compared with an analogue conjugate carrying the prior art variant IFNy-KR.
  • the eluate from Ci 8 purification was adjusted to 48 % CH3CN / 0.2 % HCOOH and then directly injected into a Q Exactive mass spectrometer (Thermo Scientific) applying an in-source CID (Collision Induced Dissociation) offset voltage of 40 eV for the recording of the intact mass.
  • the in-source CID offset voltage was then increased to 80 eV to induce fragmentation of the full L19-IFNy “WT”.
  • the resulting MS2 spectra were screened for the presence of b ions, which contain N- terminal sequence information and for y ions, which contain C-terminal sequence information.
  • MS2 and MS3 spectra were manually annotated allowing a mass error of 10 ppm.
  • Conjugates including (i) the novel IFNy-KRG mutant (SEQ ID NO 1) (ii) the full sequence IFNy-WT (SEQ ID NO 3) (iii) the prior art IFNy-KR (SEQ ID NO 2) and (iv) the truncated forms “TRUNC” of IFNy were analyzed for their binding to IFNy-receptorl (IFNy-Rl).
  • Receptor affinity measurements were performed by surface plasmon resonance using BIAcore XI 00 instrument using an IFNy-Rl coated CM5 chip. Samples were injected at 500nM. Regeneration of the chip was performed by lOmM HC1.
  • Antigen affinity measurements were performed by surface plasmon resonance using BIAcore XI 00 instrument using an EDB coated CM5 chip. Samples were injected as serial dilutions, in a concentration range from 15.625nM to lOOOnM. Regeneration of the chip was performed by 50mM NaOH.
  • Wild type IFNy-“WT” conjugate antibody strongly binds to IFNy-Rl indicating a possible trapping mechanism and/or toxicity mediated by the receptor.
  • the novel IFNy-“KRG” conjugates showed a weak affinity for IFNy-Rl while the closest prior art IFNy-“KR” conjugate did not bind IFNy-Rl because of its degradation at the C-terminus ( Figure 3).
  • the weak affinity of the IFNy-“KRG” conjugate for the IFNy-Rl indicates that such recognition - albeit low - does not correspond to a total C-terminus degradation.
  • a conjugate which included the novel mutated ⁇ FNy-“KRG” sequence was compared with an analogue wild type -IFNy conjugate “WT” to monitor its biological activity when the antibody was “on” and “off’ its target antigen.
  • the biological activity of the IFNy conjugates was determined by their ability to induce proliferation of THP-1 cells, which are stimulated by IFNy.
  • Cells were seeded in 96-well plates (wells were pre-coated either with lOOnM EDB or with PBS for lh at 37°C) in the culture medium supplemented with varying concentrations of the conjugates. After incubation at 37°C for 72 hours, cell proliferation was determined with Cell Titer Aqueous One Solution.
  • L19-IFNy-“KRG” The biological activity of L19-IFNy-“KRG” was further evidenced by its ability to induce Interleukin 6 (IL6) production by THP-1 cells.
  • IL6 Interleukin 6
  • Cells were seeded in 96-well plates (wells were pre-coated either with lOOnM EDB or with PBS for lh at 37°C) in the culture medium supplemented with lOOnM of fusion protein. After incubation at 37°C for 18 hours, supernatant was collected and IL6 levels were measured by sandwich ELISA with anti-IL6 antibodies.
  • IL6 a cytokine induced by IFNY
  • IFNy- “WT” conjugate with or without coating the plates with the target antigen
  • IFNy- “KRG” conjugate can induce IL6 production only when the antibody binds the antigen coated on the plates ( Figure 4C).
  • the ⁇ FNy-“KRG” mutant conjugate can induce a production of IL6 which is comparable to the IFNy-“WT” conjugate when the antibody of the conjugates binds to its target.
  • the IL6 released by the THP-1 cells when the antibody of the ⁇ FNy-“KRG” mutant conjugate does not bind the target was similar to the negative control (no conjugate added to the cells).
  • L19-(human)IFNY“KRG” and L19-(murine)IFNY”KRG selectively localize to neoplastic lesions with excellent tumor-to-organ ratios at 48 hours post injection (Figure 6).
  • the higher uptake for the human IFNy compared to the murine counterpart could be explained since human IFNY does not cross-react with the murine IFNY receptor.
  • L19-(human)IFNY-“KRG” Two different doses of L19-(human)IFNY-“KRG” were injected i.v. in cynomolgus monkeys to monitor its pharmacokinetic behaviour in non-human primates.
  • the low dose treatment was administered at O.lmg/kg, the high dose treatment was administered at 0.5mg/kg.
  • Individual dose volumes were calculated based on the most recent body weight recorded.
  • Blood samples of 0.6 mL each were collected at approximately the following 8 time points: before dosing and at 2, 10, 20, 30, 60, 120 and 240 minutes after treatment. Blood samples were allowed to clot in tubes for maximum 60 minutes at room temperature then spun down by centrifugation (10 minutes 2300 g, +4°C). Serum aliquots: for each sample two aliquots of at about 100 pL collected in labelled secondary tubes and stored in a freezer at -80°C ⁇ 10°C pending analysis. Fusion protein concentrations in serum were assessed by AlphaLISA. Briefly, Streptavidin Donor Beads were coated with biotinylated antigen (EDB). Acceptor Beads coated with an anti-IFNy antibody were used for detection.
  • EDB biotinylated antigen
  • L19-mIFNy “KRG” The therapeutic potential of L19-mIFNy “KRG” was tested alone and in combination with other therapeutic modalities.
  • L19- mIFNy“KRG” and KSF-mIFNy “KRG” (where KSF is an irrelevant anti-lysozyme antibody used as a negative control) were dissolved in PBS (pH: 7.0) and administered at a dose of 20 pg/mouse every 72 hours for three times.
  • a saline group was included as control.
  • Anti-PD-1 and anti-LAG3 were dissolved in PBS (pH:7.4) and administered at a dose of 200 pg/mouse every 72 hours for three times.
  • immune check-point inhibitors were injected 24h after the immunocytokine.

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Abstract

La présente invention se rapporte à des conjugués comprenant un mutant d'un interféron-gamma (IFNγ) et un élément de liaison spécifique tel qu'une molécule d'anticorps. La molécule d'anticorps se lie de préférence à un antigène associé à la croissance néoplasique et/ou à l'angiogenèse, tel que l'extra-domaine A (EDA) et l'extra-domaine B (EDB) de la fibronectine. Les conjugués peuvent être utilisés, par exemple, dans le traitement du cancer.
PCT/EP2022/059451 2021-04-09 2022-04-08 Mutant amélioré d'interféron-gamma Ceased WO2022214664A1 (fr)

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